404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 vol. 8 no. 1 january–april 2020 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ copyright © 2020, ijtid, issn 2085-1103 research article prognostic factors of severe dengue infections in children senja baiduri1,dominicus husada2, dwiyanti puspitasari3, leny kartina4, parwati setiono basuki5, ismoedijanto6 department of child health, faculty of medicin, universitas airlangga – rsud dr. soetomo, surabaya rsud dr. soewandhie, surabaya a corresponding author: dominicushusada@yahoo.com received: 26th december 2018; revised: 28th december 2018; accepted: 26th december2019 abstract the incidence of dengue fever increase annually and can increase morbidity and mortality. dengue fever is mosquitoborne disease and caused by one of four serotype dengue viruses. severe dengue is characterized either by plasma leakage, fl uid accumulation, respiratory distress, severe bleeding, or organ impairment. mortality and serious morbidity of dengue were caused by several factors including the late recognition of the disease and the changing of clinical signs and symptoms. understanding the prognostic factors in severe dengue will give early warning to physician thus decreasing the morbidity and mortality, and also improving the treatment and disease management. the aim of this study was to analyze the prognostic factors of severe dengue infection in children. this study was observational cohort study in children (2 months-18 years) with dengue infection according to who 2009 criteria which admitted in soetomo and soewandhie hospital surabaya. analysis with univariate, bivariate and multivariate with ibm spss statistic 17. all patients were confi rmed by serologic marker (ns-1 or igm/igg dengue). clinical and laboratory examination such as complete blood count, aspartate aminotransferase (ast), alanine aminotransferase (alt), albumin, and both partial trombocite time and activated partial trombosit time (ptt and appt) were analyzed comparing nonsevere dengue and severe dengue patients. there were 40 subjects innonsevere and 27 subjects with severe dengue infection. on bivariate analysis, there were signifi cant diff erences of nutritional status, abdominal pain, petechiae, pleural eff usion, leukopenia, thrombocytopenia, hypoalbuminemia, history of transfusion, increasing ast > 3x, prolonged ppt and aptt between severe and nonsevere dengue group. after multivariate analyzed, the prognostic factors of severe dengue were overweight/obesity (p=0.003, rr 94), vomiting (p=0.02, rr 13.3), hepatomegaly (p=0.01, rr=69.4), and prolonged aptt (p=0.005, rr=43.25). in conclusion, overweight/obesity, vomiting, hepatomegaly, and prolonged aptt were prognostic factors in severe dengue infection in children. those factors should be monitored closely in order to reduce the mortality and serious morbidity. keywords: severe dengue, dengue infection, increased aptt, overweight/obesity, hepatomegaly abstrak dengue merupakan penyakit virus yang disebabkan oleh satu dari empat serotipe virus dengue dan ditularkan oleh nyamuk. kasus dengue berat berdasarkan kriteria who 2009 di defi nisikan sebagai dengue dengan satu atau lebih kondisi berikut; kebocoran plasma yang menyebabkan syok (dengue syok) dan atau akumulasi cairan dengan distres nafas, perdarahan berat dan yang ketiga adalah keterlibatan organ. diagnosa dini bermanfaat menurunkan morbiditas dan mortalitas, managemen klinis, surveilans dan control penyakit serta menurunkan durasi rawat inap. penelitian ini menganalisis faktor prognosis infeksi dengue berat pada anak. kohortobservasional pada pasien usia 2 bulan-18 tahun dengan infeksi dengue berdasarkan kriteria who 2009 yang mrs ataupun di poliklinik rawat jalan di rsud dr. soetomo dan rsud soewandhie surabaya. analisis data dilakukan dengan univariat, bivariate dan multivariate menggunakan ibm spss statistic 17. semua pasien terkonfi rmasi dengan pemeriksaan serologis (ns-1 atau igm/igg dengue). data klinis dan laboratorium (darah lengkap, ast, alt, albumin, aptt dan ppt) dianalisis untuk membandingkan antara kelompok dengue tidak berat dan dengue berat. sebanyak 40 subyek pada kelompok infeksi dengue tidak berat dan 27 subyek pada kelompok dengue berat yang memenuhi corresponding author. e-mail: dominicushusada@yahoo.com 44 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 44–54 copyright © 2020, ijtid, issn 2085-1103 kriteria inklusi. didapatkan perbedaan yang bermakna berdasarkan analisis bivariate pada variabel overweight/ obesitas, nyeri perut, efusi pleura, hepatomegali, leukopeni, trombositopenia, hipoalbuminemia, ast meningkat>3x, ppt dan aptt meningkat serta riwayat transfusi. overweight/obesitas (p=0.003, 95% rr 94), muntah (p=0.02, rr 13.3), hepatomegali (p=0.01, rr=69.4), dan pemanjangan aptt (p=0.005, rr=43.25) merupakan faktor prognosis infeksi dengue berat berdasarkan analisis multivariat. status nutrisi, muntah, hepatomegali dan pemanjangan aptt merupakan faktor prognostik infeksi dengue berat pada anak. monitoring terhadap factor tersebut perlu dilakukan untuk menurunkan mortalitas dan morbiditas. kata kunci: dengue berat, infeksi dengue, peningkatan aptt, overweight/obesitas, hepatomegaly how to cite: baiduri, senja. husada, dominicus. puspitasari, dwiyanti. kartina, leny. basuki, parwati setiono. ismoedijanto, ismoedijanto. prognostic factors of severe dengue infections in children. indonesian journal of tropical and infectious disease, [s.l.], v.8, n.1, p.211-222 jan. 2020. issn 2085-1103. available at: https://ejournal.unair.ac.id/ ijtid/article/view/10721. date accessed: 09 dec. 2019. doi: http://dx.doi.org/10.20474/ijtid.v8i1.10721 introduction the worldwide prevalence of dengue fever is estimated 50 -100 billion and dengue hemorrhagic fever about 250.000-500.000.1 incidence of dhf over the past 45 years in indonesia increased rapidly.2 dengue fever is mosquito-borne disease and caused by one of four serotype dengue viruses. this four serotype are dengue virus serotype-1 (denv-1), serotype-2 (denv-2), serotype-3 (denv-3), and serotype-4 (denv-4). dengue infection is characterized by fever and constitutional symptoms to hemorrhagic manifestations and shock, or dengue hemorrhagic fever/dengue shock syndrome (dhf/dss). the most serious spectrum of this disease, severe dengue, is characterized either by plasma leakage, fluid accumulation, respiratory distress, severe bleeding, or organ impairment.1,3 clinical manifestations of dengue fever are the expression of host and viral factors, some acquired, others intrinsic to the individual. the virulence of the virus and the flavivirus infection history, age, gender and genotype of the host can determine to severity of the disease.4 the warning signs in dengue usage was proposed for early detection of potentially severe cases for timely treatment, to avoid unnecessary hospitalizations, and to decrease the case fatality of the disease.1,5 early prediction of severe dengue in patients without any warning signs who might later develop severe dengue is very important to give the best supportive.6 patients should be monitored by health care providers for temperature pattern, volume of fluid intake and losses, urine output (volume and frequency), warning signs, haematocrit, and white blood cell and platelet counts.1 the rapidly expanding global footprint of dengue is a public health challenge with an economic burden that is currently unmet by licensed vaccines, specific therapeutic agents, or efficient vectorcontrol strategies.3 there are several signs and symptoms called warning signs that can be used to predict severe dengue, hence recognizing the warning signs is important for successful clinical management. warning signs include abdominal pain, evidence of fluid accumulation, hepatomegaly and increases in hematocrit accompanied by a fall in the platelet count.2 the benefit of prompt diagnosis is decreasing morbidity and mortality, improving treatment and surveillance, and also enhancing disease management.7,8 early diagnosis of dengue infection can improved by algorithms using early clinical indicators. indicator addition of severe plasma leakage to who definition led to increase the sensitivity using white blood cells (wbc), ast, platelet count and age.9 a retropsective study by nguyen et al, reported the final prognostic model included history of vomiting, platelet count, ast level and ns1 rapid test.10 we were conducted prospective study to evaluation prognostic factors in severe dengue infection in children. 45senja baiduri, et al.: prognostic factors of severe dengue infections in children copyright © 2020, ijtid, issn 2085-1103 materials and methods this was a cohort observational study. the study population was the patient in the pediatric outpatient clinic and pediatric emergency department at dr. soetomo hospital and soewandhie hospital in surabaya. the minimal sample requirements were 26 based on the formula by lemeshow. subject eligible between two months until 18 years old with fever ≥ 3 days and probable dengue infection symptoms such as headache, nausea-vomiting, petechiae, arthralgia, and retro-orbital pain were included. patients were assessed as severe dengue and nonsevere dengue based on who 2009 guideline and positively serology marker such as igm or antigen non structural-1 (ns-1). the who 2009 guideline mentioned the severe dengue as either by plasma leakage, fluid accumulation, respiratory distress, severe bleeding, or organ impairment.5 the exclusion criteria were a congenital anomaly, malignancy, autoimmune and immunodeficiency disease because the clinical signs and symptoms, and the laboratory test results of those diseases can mimic or influence the clinical and laboratory pictures of severe dengue patients. we also performed chest x ray examination to distinguish pleural effusion when patients admission. nutritional status was assessed by bmi cdc growth chart 2000 for patients 2-18 years old and who 2007b for patient below than 2 years old. the data were analyzed by the statistical program for social science software (spss) ibm spss windows statistic 17.0. chi-square test was used to assess the categorical data and logistic regression carried out to evaluate multivariate analysis. in this study, laboratory examination was carried out from the material of venous blood samples by using hematology analyzer sysmex xn1000 and celldyne ruby sysmex cs2100 to analyze complete blood count, siemens dimension to analyze albumin, ast and alt. while sysmex cs2100 to analyzed ppt and aptt. for repeated tests, we use the worst results before the severe condition. this study was approved by both health research ethical committee of dr. soetomo and soewandhie hospital (document no. 640/panke.kke/ xi/2017 date: november 13th 2017-november 13th 2018 and no. 070/12334/436.8.6/2018 date : mei 23rd 2018). results and discussion during the eight-month period of the study, a total of 67 patients with dengue infection met the inclusion criteria, in which 27 and 40 were diagnosed as severe dengue and non-severe dengue infection, respectively. all patients were confirmed by serologic marker (ns-1 or igm/ igg dengue). all subjects were carried out anamnesis, physical examination, and laboratory. clinical and laboratory examination (complete blood count, ast, alt, albumin, aptt, and ppt) were analyzed comparing non-severe dengue and severe dengue patients. figure 1. 7 patients were excluded : 3 patients with blood disorders 4 patients with congenital heart disease 74 patients with dengue infection 67 patients met inclusion criteria 64 patients in dr. soetomo hospital 40 patients with non severe dengue 27 patients with severe dengue death : 4 patients life : 63 patients figure 1. flow diagram of subject recruitment. 46 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 44–54 copyright © 2020, ijtid, issn 2085-1103 table 1. baseline characteristic characteristics groups p value severe dengue n=27 (%) non severe dengue n=40 (%) age,(year) ≤ 5 years 4 (14.8) 7(17.5) 0.77 >5 years 23 (82.5) 33 (82.5) gender, n(%) male 14 (43.8) 18 (56.3) 0.76 female 13 (37.1) 22 (62.9) referral, n(%) yes 18 (66.7) 17 (42.5) 0.052 no 9 (33.3) 23 (57.5) nutritional state non overweight/obesity 12 (24.0) 38 (76.0) <0.001* overweight/obesity 15(88.2) 2 (11.8) doi (day of illness)(day) 2-6 2-6 los (length of stay)(day) duration 1-11 (day) 2-8 (hari) outcome life death 23 (85.2) 4 (14.8) 40 (100) 0 (0) 0.012* describe row diagram of subject recruitment in this study. characteristics of 67 research subjects can be seen in table 1. on bivariate analysis, there were significant differences of thrombocytopenia, hypoalbuminemia, history of transfusion, increasing ast>3x, nutritional status, abdominal pain, petechiae, pleural effusion, leucopenia, prolonged ppt and aptt between severe and non-severe dengue groups. after multivariate analyzed, the prognostic factors of severe dengue were overweight/obesity (p=0.003, rr 94), vomiting (p=0.02, rr 13.3), hepatomegaly (p=0.01, rr=69.4), dan prolonged aptt (p=0.005, rr=43.25). the baseline characteristics of children with dengue infection and controls in this study were very similar except nutritional status and outcome. age ≤ 5 years in severe dengue were 14.8% and 82.5% in subject more than 5 years old (>5 years). non severe dengue were more common in subject > 5 years old (82.5%) than subjects with age ≤ 5 years (17.5%). male to female proportion in severe dengue were 43.8% and 37.1% , non severe dengue were 56.3% and 62.9% respectively. referral subjects with severe dengue were 66.7% and non referral were 33.3%. while more than half of subjects with non severe dengue (57.3%) came to soetomo hospital by itself and 42.5% were referral from other hospital. nutritional status was statistic significantly in bivariate and multivariate analysis (rr 2.93, 95% ci 2.18-6.20) whereas the previous study by maron et al and yulianto et al and ledika et al, excess nutrition does not appear to be a risk factor for severe dengue infection.11,12,13 in addition, normal nutritional status had negative correlation with dhf and dss.14 however, metaanalysis and systematic review recently enroled 15 studies from 2000 until 2016 reported obesity as a risk factor of severity in children with dengue infection (or = 1.38; 95% ci:1.10, 1.73).15 in addition, recent study shown obese patients with dengue infection possess many clinical parameters suggestive of more severe clinical manifestations.16 study of obesity in severe dengue infection still rare. leptin is a major mediator of the altered immune balance in the obese individuals and has been shown to promote macrophage phagocytosis, increase secretion of pro-inflammatory cytokines and modulate the adaptive immune system. elevated leptin and socs3 levels correlates with a decreased type 47senja baiduri, et al.: prognostic factors of severe dengue infections in children copyright © 2020, ijtid, issn 2085-1103 1 interferon response, which serves as a crucial innate immune system activator in stimulating an antiviral state.17 severe dengue group in this study had a prolonged length of stay (1-11 days) than nonsevere dengue group (2-8 days). the mortality rate in this study was 5.9% in all subjects and 50% were severe dengue patients with obesity, while other study by patrayusha et al18 reported mortality rate was 6.25% and 1.03% in mishra et al 19 mortality of dhf or dss estimated 40-50% in pitfall management. however who was stated management properly can save lives and mortality rates from more than 20% to less than 1%.1 the proportion of severe dengue and non-severe dengue with vomiting were 88.9% and 70% respectively. vomiting more common in dss and expanded dengue syndrome than non-severe dengue with frequent variously range 3-5x/day. in the previous study was reported the prevalence of vomiting symptom was higher in severe dengue group than dengue infection/dengue infection with warning sign group.20 persistent vomiting is one warning sign according to who 2009.1 ledika et al held study in patients with severe dengue showed persistent vomiting had correlated with severe dengue.13 meta analysis study by zhang et al was reported nausea-vomiting, as the predictor of severe dengue in children. vomiting was often found in dengue patients, especially in children. vomiting could cause fluid imbalance and also difficulty in assessing the hydration state of the patient.21 in present study, abdominal pain in severe dengue was 85% while in non severe dengue groups about 45%. despite statistic significantly from bivariate analysis (p= 0.002, rr 3.6) however from the regression logistic shown unsignificantly. abdominal pain is one of warning sign in dengue infection and epigastrium pain is sign of dengue hemorrhagic fever.1 meta analysis study by zhang et al was stated abdominal pain could predict of severe dengue infection.21 the mechanism of abdominal pain in dengue infection was unknown. gupta et al was reported the most common specific cause of acute abdominal pain was acute hepatitis,22 previously shabir et al was reported proportion of abdominal pain was 32% and liver involvement was the common cause of abdominal pain in dengue fever.23 in present study, bleeding manifestation presented with epistaxis, ptechie, melena and hematemesis. in addition, proportion of torniquet test were very similar in severe dengue and non severe dengue group (92.6% and 100%). melena in severe dengue group was 14.48% and 2.5% in non severe dengue group. both of bivariate and multivariate analysis revealed statistic unsignificantly (p= 0.16 and 0.14 respectively in table. 2 and table. 3 ). epistaksis found in 3 patients with severe dengue (11.1%) and 7 patients with non severe dengue (17.5%) while ptechie more common in severe dengue than non severe dengue patients (66.7% and 35%). epistaxis and ptechie occurred in 3-5 days of illness. whereas, hematemesis occurred in 2 patients with severe dengue (7.4%) and 1 patients with non severe dengue (2.5%). statistic unsignificantly noticed in bivariate analysis ( p= 0.73 in table. 2). bleeding (hematemesis or melena) occurred in 5-7 day of illness. melena range from 50cc1000cc and leading to hemodinamic imbalance. two patients with severe dengue required whole blood transfusion. in this study, massif bleeding and profound shock due to hematemesis and melena leading to mortality in two patient with severe dengue. in this study, transfusion administration in 9 patients with severe dengue. whole blood and prc were given in patients with bleeding and hemodinamic disturbance with previous colloid and crystaloid administration. ffp was given in patients with prolonged aptt and bleeding manifestation (hematemesis and melena). all of them accompanied with trombocytophenia (<50000/μl) and 7 patients with increasing of ast (>200-12186 u/l). five patients with decreasing of hemoglobine and hematocrit also accompanied prolonged shock. all of subject with transfusion were severe dengue group. the most common spontaneous bleeding sign in dengue infection was ptechie. prathyusha et al was shown that ptechie occurred in 70% in children with dengue infection.18 while branco et al was reported that epistaxis, hemoptisis and persistent vomiting associated with mortality 48 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 44–54 copyright © 2020, ijtid, issn 2085-1103 in children with dengue infection.24 zhang was reported that patients with bleeding after denv infection had approximately a 14-fold increased risk for progression into severe dengue (including dhf and dss). according to this meta-analysis, the two kinds of gastrointestinal bleeding that strongly predicted severe dengue were hematemesis and melena.21 otherwise, torniquet test and petechiae not significant associated with severe dengue. a positive tourniquet test in febrile phase increases the probability of dengue but indistinguishable between severe and non severe dengue case.1 previously, pongpan et al was reported thrombocytopenia (≤50.000 mm3) as prognostic factor severe dengue in children,10 in addition some studies by ledika et al13 and yulianto et al12 were reported thrombocytopenia as risk factor severe dengue in children. this result similar with present study and statistic signifi cantly on bivariate analysis (p=0.001, rr 3.9, ci 2.06-7.72). however from the multivariate analysis reveal unsignificantly (p= 0.87, rr 0.46, ci 0.001-291) (table 3). hemoglobine, and hematocrite were statistic unsignifi cantly (p= 0.17, rr 1.4, ci 0.75-2.6 and p= 0.74, rr 0.83 ci 0.46-1.5 respectively). the proportion of thrombocytopenia, leukopeni, hemoglobine and increase of hematocrit were 70.3%, 40.7%, 62.9% and 62.9% in severe dengue group. while in non severe dengue group the proportion were 15%, 72.5%, 50% and 70% respectively (table. 2). otherwise, leukopeni was common in non severe dengue group. ho et al was reported the most notable laboratory fi nding included thrombocytopenia, leukopeni, prolonged aptt and elevation of serum aminotransferase.25 according to who, leukopeni is common in early phase of fever.25 ledika at al in cross sectional study reported leukocyte ≥5000/mm3 in early admission associated to severe dengue in children.13 pongpan at el was also reported white cell count > 5000/μl as prognostic factor in severe dengue.10 hepatomegaly in this study was 92.6% in severe dengue group and statistic significantly in bivariate (rr 37.18, 95% ci: 3.6-352 ) and multivariate analysis (rr 1.97, 95% ci = 3.1-47.2). this was similar to the previous study by jagadiskumar, held in 110 children with dengue viral infection accompanied with liver involvement reported hepatomegaly was 79%26 and the most common symptom while roy et al 2013 reported 120 subjects with dengue virus infection and proportion of liver involvement was 80.8%.27 enlargement of the liver (hepatomegaly) is observed at some stage of the illness in 90%-98% of children. the frequency varies with time and/or the observer.29 several study reported hepatomegaly > 2 cm in defervescence phase as prognostic factor severe dengue in children.12,13 pongpan was reported in dengue severity score, hepatomegaly had the highest score of predictive severe dengue in children (or 12.31, 95% ci = 8.84–17.15, p<0.001) than other variable e.x hematocrit, age>6 years, platelets ≤50000 μl, wbc>5000μl and systolic blood pressure <90 mmhg.10 liver dysfunction is one of the atypical forms of clinical manifestation in the dengue infection. hepatomegaly is one of liver involvement in dengue infection and most commonly in children than adult patients. clinical evidence of liver involvement in dengue infections includes the presence of hepatomegaly and increased serum liver enzymes.28 hepatomegaly is frequent and more common in patients with dengue with shock than in those with df.29 currently, the exact mechanism by which the host immunity damages liver is unknown. in a recent report, liver from fatal cases of dengue hemorrhagic fever (dhf) exhibited high expression of tlr2, tlr3, il6, and granzyme b also presented inos, il18, and tgfβ in inflammatory infiltrate, indicating their possible involvement in the physiopathology.30 however ferreira et al was reported cxcl10/ ip10 elevation was associated with painful hepatomegaly, and both il10 and cxcl10/ ip10 were associated with liver disorders in children.31 in this study, hipoalbuminemia was defined if albumin level <3.5 g/dl. hipoalbuminemia in severe dengue group was 66.7% and 12.5% in non severe dengue group. twelve (12 patients) with hipoalbuminemia and history of shock and 5 patients with liver involvement. 49senja baiduri, et al.: prognostic factors of severe dengue infections in children copyright © 2020, ijtid, issn 2085-1103 bivariate analysis revealed statistic significantly (p=0.001, rr 3.8, ci 2.05-7.21), on the contrary, multivariate regression revealed unsignificantly (p=0.22, rr3.5, ci 0.47-26.54). hipoproteinemia can be found in critical phase and correlated with plasma leakage. previous study by suwarto et al in adult patients was reported the lowest albumin concentration at the critical phase was ≤ 3.49 g/dl.32 according to who, hypoproteinemia/ albuminaemia was a common finding as a consequence of plasma leakage in critical phase. a significantly decreased serum albumin >0.5 gm/dl from baseline or <3.5 gm % is indirect evidence of plasma leakage.29 the serum albumin level was lower in the serious group based on pone et al33 and elling et al.34 pone et al used cutoff albumin level < 3 g/dl and serious dengue disease was defined as occurrence of death, or table 2. prognostic factors based on bivariate analysis prognostic factors groups p value rr 95% cisevere dengue n 27 (%) non severe dengue n 40(%) abdominal pain 23 (85) 18 (45) 0.002* 3.6 1.4-9.3 nausea 24 (88.9) 28 (70) 0.13 0.788 0.62-1.0 vomiting 18(66.7) 18 (45) 0.13 1.72 0.98-3.26 epistaxis 3 (11.1) 7(17.5) 0.71 0.71 0.69-2.54 melena 4 (14.8) 1 (2.5) 0.16 2.16 1.25-3.7 hematemesis 2 (7.4) 1 (2.5) 0.73 1.7 0.7-4.0 ptechie 18 (66.7) 14 (35) 0.022* 2.17 1.15-4.15 rumple leede 26 (96.2) 40 (100) 0.84 0.39 0.29-0.53 pleural effusion 23 (85) 13 (32.5) <0,001* 4.95 1.9-12.7 hepatomegaly 25 (92.6) 9 (22.5) <0,001* 12.1 3.1-47.2 hemoglobin 17 (62.90 20 (50) 0.13 1.4 0.75-2.6 leukopeni (<5000/mm3) 11 (40.7) 29 (72.5) 0.019* 1.7 1.09-2.9 increase of hematocrit 17 (62.9) 28 (70) 0.74 0.83 0.46-1.5 thrombocytopenia (≤50.000/μl) 19 (70.3) 6 (15) <0,001* 3.9 2.06-7.72 hypoalbuminemia (< 3.5 g/dl) 18 (66.7) 5 (12.5) <0,001* 3.8 2.05-7.21 ast > 3x 20 (74) 16 (40) 0.013* 2.46 1.2-5.03 alt >3x 15 (55.6) 23 (57.5) 0.87 0.9 0.53-1.7 increase of ppt 7 (25.9) 2 (5) 0.036* 2.27 1.4-3.7 increase of apt 24 (88.9) 12 (30) <0,001* 6.9 2.3-20.6 secondary dengue infection 8 (29.6) 14 (3.5) 0.64 1.18 0.58-2.4 transfusion 9 (33.3) 0(0) <0,001* 3.2 2.19-4.7 * p significantly <0.05*,chi squaretest the use amines, inotrop, colloids, mechanical ventilation, non invasive mechanical ventilation or hemodialisis.34 ferreira et al was found involvement of inflammatory cytokine cxcl10/ ip10 and il10 in plasma leakage was shown since hypoalbuminemia was associated with both factor levels. pleural and pericardial effusions and ascites were detected frequently in more severe patients.31 in present study, pleural effusion more common in severe dengue (85%) than in non severe dengue group 32.5% with chi square revealed statistic significantly (p=0.001, rr 4.95, ci 1.9-12.7, table 2.) even though unsignificantly based on multivariate analysis. this study was showed that increase of aptt in severe dengue more than non-severe dengue group with proportion are 88.9%. increasing of aptt range from 1.5x until more than 100 50 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 44–54 copyright © 2020, ijtid, issn 2085-1103 table 3. prognostics factors based on multivariate analysis prognostic factors b p rr 95% ci hepatomegaly 2.77 0.01* 69.4 2.18-287.4 increase of aptt 3.42 0.005* 43.25 2.6-699 obesity/overweight 4.5.3 0,003* 94 4.47-1989 vomiting 2.59 0.02* 13.3 1.5-118.8 leucopenia -29.95 0.9 000 0.000 ast >3x 0.000 abdominal pain 2.87 0.27 17.68 0.09-3221 melena -2.65 0.14 0.071 0.002-2.28 albumin <3.5g/dl 1.26 0.22 3.5 0.47-26.54 pleural effusion -0.56 0.72 0.57 0.028-11.73 increase of ppt -1.6 0.25 4.9 0.3-77.3 transfusion 30.09 1 0.99 ~ hb -1.34 0.42 0.26 0.009-7.23 ptechie 2.27 0.16 9.7 0.42-226 vomiting -2.36 0.31 0.095 0.001-8.81 thrombocytopenia(≤ 50.000/μl) -7.8 0.81 0.46 0.001-291 constant -6.95 0.001* 0.001 * p significantly <0.05*,chi squaretest seconds from the normal level. twenty patients (72.2%) with aptt elevation acompanied with hepatomegaly. chi-square analysis revealed statistic significantly (rr 6.9 95% ci 2.3-20.6) (table 2) and also multivariate analysis carried out with logistic regression as prognostic factor of severe dengue in children (rr 43.25, 95% ci: 2.6-699) (table 3). its similar to study held by mishra et al, rise in aptt/pt also depicts severity of disease.19 patients with severe dengue may have coagulation abnormalities, but these are usually not sufficient to cause major bleeding. when major bleeding does occur, it is almost always associated with profound shock since this, in combination with thrombocytopaenia, hypoxia and acidosis, can lead to multiple organ failure and advanced disseminated intravascular coagulation.1 prolongation of aptt in acute phase correlates with the severity of infection and can be made as early indicator dss/dhf.35 plasma leakage in dengue patients also directly related to aptt level.36 previously, budastra et al found that there was significant relationship between prolonged aptt during early stages of dhf with bleeding manifestation at the later stage of disease.37 coagulophaty can be induced by hepatitis viral infection due to decreasing of coagulation factors. this can be caused by either down regulation of the synthesis of specific factors or by increased consumption of specific factors. an analysis of the linear correlation and regression between the levels of aspartate aminotransferase (ast)/ alanine aminotransferase (alt) and aptt shows a strong association between ast/alt elevation and aptt prolongation in dhf patients. dysfunction of the damaged liver might be responsible for the decreased synthesis of specific factors in the intrinsic pathway.35 in this study, increased of aptt also accompanied with ast elevation (72.2%) and alt elevation (44.4%). while from bivariate analysis was significantly, however from multivariate logistic regression insignificantly. another hypothesis of coagulopathy is ns-1 protein excreted during early stage infection will binding to prothrombin may inhibit its activation.36 chuang et al was suggested that molecular mimicry between denv and coagulation factors can induce the production of 51senja baiduri, et al.: prognostic factors of severe dengue infections in children copyright © 2020, ijtid, issn 2085-1103 auto antibodies with biological effects similar to those of antithrombin antibodi/atas found in dengue patients. these coagulation-factor crossreactive anti-denv antibodies can interfere with the balance of coagulation and fibrinolysis.38 in this presents study, elevation ast>3x in severe dengue groups and non severe dengue group were 74% and 40% respectively. both of elevation of alt >3x found in severe and non severe dengue groups (55.6% and 57.5%). world health organization defined ast or alt 1000 units/liter (u/l) as a severe dengue.1 roy et al was reported liver dysfunction more common in subjects accompanied with warning sign. elevation of alt 84.4% and ast 93.7% in group with warning sign also elevation of alt 94.5% and ast 95.5% in severe dengue group.26 while lee at al reported ast and alt elevation might not distinguish from severe dengue with non severe dengue infection.39 however fernando et al reported liver function tests done at earlier dates might not reflect the extent of of liver involvement in acute dengue infection. the highest ast level were seen on day 6 of illness and both ast level were significantly higher in severe dengue patients.40 in this study ast and alt were performed in 48 hours in early admission suggest the result were statistic unsignificantly. the limitations of this study are width confidence interval due to sample limitation. this condition caused by dengue infection commonly occurred in rainy season and rarely to be found in other season so that impacted small number subjects obtained. outbreaks of dengue disease often occur in most tropical countries around the world, with close to 75% of the global population exposed to the disease living in the asia-pacific region.41 in most disease endemic areas dengue transmission has a definite seasonality, but the reasons for the seasonal patterns are not fully understood. the amount of rainfall is the single most important factor for dengue virus transmission, since this condition is most suitable for mosquitoes to lay their eggs and for the humans and mosquito to come into contact.42 this study was conducted with cohort observational which fever ≥ 3 days as inclusion criteria hence subjects came with various phase of illness. conclusion in conclusion, overweight/obesity, vomiting, hepatomegaly, and prolonged aptt were prognostic factors in severe dengue infection in children. considering these factors for awareness of severe dengue in patients with dengue virus infection. clinicians should emphasize the monitoring of these factors for early detection of serious dengue state. acknowledgements we sincerely thank all patients of dr. soetomo and soewandhi hospital for the participation in this research. we thank to dr. budiono for great statistical analysis. conflict of interest the authors declare that there is no conflict of interest for this research. references 1. who. dengue, guidelines for diagnosis, treatment, prevention and control. genewa. 2009:1-160. 2. karyanti mr, kusriastuti r, hadinegoro sr, rovers mm, heesterbeek h, hoes aw, et al. the changing incidence of dengue haemorrhagic fever in indonesia: a 45-year registry-based analysis. bmc infect dis. 2014;26:1-7. 3. simons cp, farrar jj, chau nv, wills b. dengue. n engl j med 2012;366:1423-32. 4. whitehorn j, simmons cp. the pathogenesis of dengue. vaccine. 2011;29(42):7221-8. 5. gutierrez g, gresh l, petrez ma, elizondo d, aviles w and kuan g, et al. evaluation of the diagnostic utility of the traditional and revised who dengue case definitions. plos negl trop dis. 2013;7:e2385. 6. john dv, lin ys, guey perng gc. biomarkers of severe dengue disease – a review. j biomed sci. 2015;22:83 7. nguyen mt, nguyen vv, nguyen th, ho tn, ha mt and ta vt , et al. an evidence-based algorithm for early prognosis of severe dengue in the outpatient setting. clin infect dis. 2017 mar 1;64(5):656-63. 52 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 44–54 copyright © 2020, ijtid, issn 2085-1103 8. potts ja, gibbon rv, rothman al, srikiatkhachorn a, thomas sj, supradish po et al. prediction of dengue disease severity among pediatric thai patients using early clinical laboratory indicators. plos one negl trop dis. 2010 august 3;4(8):e769. 9. tuan nm, nhan ht chau nv, hung nt, tuan hm, tram tv, et al. sensitivity and specificity of a novel classifier for the early diagnosis of dengue. plos negl trop dis. 2015 apr 2; 9(4):e0003638. 10. pongpan s, wisitong a, tawichasri c, patumond j, namwongprom s. development of dengue infection severity score. isrn pediatrics, 2013;2(1):12-8. 11. marón gm, clará w, diddle jw, pleités eb, miller l, macdonald g, adderson ee. et al. association between nutritional status and severity of dengue infection in children in el salvador. am j trop med hyg. 2010 feb ;82(2):324–9 12. yulianto a, laksono is, juffri m. faktor prognosis derajat keparahan infeksi dengue. sari pediatri. 2016;18(3):198-203. 13. ledika ma, setiabudi d, dhamayanti m. association between clinical profiles and severe dengue iinfection in children in developing country. american journal epidemiology and infectious disease. 2015;3(3):45-9. 14. trang ntht, long np, hue ttm, hung lp, trung td , dinh dn, et al. association between nutritional status and dengue infection: a systematic review and meta-analysis. bmc infec dis. 2016 apr 20;16:172. 15. zulkipli ms, dahlui m, jamil n, wai hvc, bulgiba a, rampai s, et al.the association between obesity and dengue severity among pediatric patients: a systematic review and metanalysis. plos negl trop dis. 2018 feb 7;12(2):1-22. 16. tan vpk, ngim cf, lee ez, ramadas a, pong ly , hassan sy, et al. the association between obesity and dengue virus (denv) infection in hospitalised patients. plos one. 2018 jul 17; 13(7): e0200698. w18. prathyusha cv, rao ms, sudarsini p, rao kum. clinico-haematological profile and outcome of dengue fever in children. int j curr microbiol app sci. 2013;2(10):338-46. 19. mishra s, ramanathan r, agarwalla s. clinical profile of dengue fever in children: a study from southern odisha, india. scientifica. 2016:1-6. 20. vuong nl, manh dh, mai nt, phuc lh, luong vt, quan vd et al. criteria of “persistent vomiting” in the who 2009 warning signs for dengue case classification. 2016;44:14 21. zhang h, zou yp, peng hj, zhang xh, zhou fy, liu zh, chen gx. predictive symptoms and signs of severe dengue disease for patients with dengue fever: a metaanalysis. biomed research int. 2014 july1;1-10. 22. gupta bk, nehera hr, parmar s, meena sl, gajraj s et al. acute abdomen presentation in dengue fever during recent outbreak. j acute disease. 2017;6(5):198-204. 23. sabbir b, qadir h, shafi f, mahboob f. acute abdominal pain in dengue fever. pjmhs. 2012;6:155-8. 24. branco md, luna ej, braga junior ll, oliviera rv, rios lt, silva md et al. risk factors associated with death in brazilian children with severe dengue: a casecontrol study. clinics. 2014;69(1):55-60. 25. ho ts, wang sm, lin ys, liu cc. clinical and predictive markers for acute for dengue infection. j biomed science. 2013;20:75. 26. jagadishkumar k; jain p, manjunath vg, umesh l. hepatic involvement in dengue fever in children. iran j pediatr. 2011; 22(2):231-6. 27. roy a, sarkar d, chakraborty s, chaudhuri j, ghosh p, chakraborty s. profile of hepatic involvement by dengue virus in dengue infected children. n am j med sci. 2013; 5(8):480-5. 28. samanta j, sharma v. dengue and its effect in liver. world j clin cases. 2016 feb 16;3(2):125-31. 29. who. comprehensive guideline for prevention dan control dengue and dengue hemorrhagic fever. new delhi: who, 2011.pp.1-212. 30. pagliari c, quaresma ja, fernandes er., stegun fw, brasil ra, de andrade jr hf, et al. immunopathogenesis of dengue hemorrhagic fever: contribution to the study of human liver lesions. j. med. virol. 2014;86:1193–7. 31. ferreira ra, de oliveira sa, gandini m et al. circulating cytokines and chemokines associated with plasma leakage and hepatic dysfunction in brazilian children with dengue fever. acta trop. 2015;149:138-47. 32. suwarto s, nainggolan l, sinto r, effendi b, ibrahim e, suryamin m, r. sasmono t. dengue score: a proposed diagnostic predictor for pleural effusion and/or ascites in adults with dengue infection. bmc infectious diseases (2016) 16:322. 33. pone sh, hokerberg yhm, de oliviera r, daumas rp, pone tm, da silva pone mv, brasil p, et al. clinical and laboratory signs associated to serious dengue disease in hospitalized childre. j pediatr (rio j). 2016;92(5):464-471. 34. elling r, henneke p, hatz c, hufnagal m. dengue fever in children: where are we now? pediatr infect dis j. 2013;32:1020-2. 35. lei hy, huang kj, lin ys, liu hs, liu cc. immunopathogenesis of dengue hemorrhagic fever. am j infect dis. 2008;4:1-9. 36. chuang yc, lin ys, liu cc, et al. factors contributing to the disturbance of coagulation and fibrinolysis in dengue virus infection. j formos med assoc. 2012;112(1):12-7. 37. budastra in, arhana bnp, mudita ib. plasma prothrombin time and activated partial thromboplastin time as predictors of bleeding manifestation during dengue hemorrhagic fever. paediatr indones. 2009;49(2):69-74. 38. chuang yc, lin ys, liu hs, yeh tm. molecular mimicry between dengue virus and coagulation 53senja baiduri, et al.: prognostic factors of severe dengue infections in children copyright © 2020, ijtid, issn 2085-1103 factors induces antibodies to inhibit thrombin activity and enhance fibrinolysis. j virol. 2014 ;88(23): 13759–68. 39. lee lk, gan vc, lee vj, tan as, leo ys, lye dc et al. clinical relevance and discriminatory value of elevated liver aminotransferase levels for dengue severity. plos one. 20126(6):1676. 40. fernando s, wijewickrama a, gomes l, punchihewa ct, madusanka sd, dissanayake h, et al. patterns and causes of liver involvement in acute dengue infection. bmc infect dis. 2016 16:319. 41. world health organization, global strategy for dengue prevention and control 2012-2020. geneva, switzerland, 2012 42. chanprasopcha p, pongsumpu p, ming tang i. effect of rainfall for the dynamical transmission model of the dengue disease in thailand. comput math methods in med. 2017 august 8:1-17 �0 vol. 2. no. 1 january–march 2011 the role of polysaccharide krestin from coriolus versicolor mushroom on immunoglobulin isotype of mice which infected by mycobacterium tuberculosis adita ayu permanasari1,2, sri puji astuti wahyuningsih1, win darmanto1 1 department of biology, faculty of science and technology, airlangga university 2 institute of tropical disease, airlangga university abstract this research was aimed to determine the role of polysaccharide krestin (psk) with different timing on levels and types of mice immunoglobulin (ig) isotype which infected by mycobacterium tuberculosis. this research used 30 adult female mice of mus musculus strain, polysaccharide krestin was isolated from coriolus versicolor mushroom, and for infection used mycobacterium tuberculosis h37rv (atcc 27294 t) strain. provision of polysaccharide krestin was done over 7 consecutive days via gavage. mycobacterium tuberculosis infection was done 2 times with an interval of 1 week via intraperitoneal. immunoglobulin isotype serums were analyzed using the elisa test and the results were analyzed descriptively through the color reaction and od values. the result showed the highest levels of immunoglobulin was found in the provision of psk before and after mycobacterium tuberculosis infection with total 6.280 of od ig isotype. immunoglobulin isotype dominant was igm with lambda light chain. the conclusion of this research was psk increased mice ig isotype levels at the time of provision before, after or before and after infection mycobacterium tuberculosis. ig isotype which was formed i.e. igm, iga, igg2b, igg3, igg2a, igg1 with kappa and lambda light chain. key words: polysaccharide krestin, mycobacterium tuberculosis, immunoglobulin isotype introduction tuberculosis (tb) is still become a serious problem in the world [12].this bacteria is divided into extracellular and intracellular bacteria[2]. specific response against extracellular bacteria with produce antibodies by b cells. while in response against intracellular bacteria, the response that happens is the cellular immune response (t cell) [7]. however, intracellular bacteria can induce the development of t cells into th1 cell phenotype then also can stimulate antibody production by b cells [5]. in the early formation of immunoglobulin molecules (antibodies) by b cells is stimulated by antigen [9]. in mice, the class of immunoglobulin (ig) based on the h-chain (heavy chain) consists of igm, igg, iga, igd, and ige. in mice, igg consists of four subclasses i.e. igg1, igg2a, igg2b, and igg3 [23]. in addition, there are 2 types of lchain (light chain), namely kappa (κ) and lambda (λ) [19]. some researchers use the immunomodulator as an adjunctive therapy for tuberculosis [18]. coriolus versicolor is a mushroom that commonly used in the treatment of disease. various active components are isolated from this mushroom, both taking from fruiting bodies or culture mycelium. active components that are important in the treatment are polysaccharide krestin (psk) and polysaccharide peptide (psp). both psk and psp consist of active compounds named β-glucan [16]. beta (β)-glucan plays a role to activate macrophages and stimulate b cells in the process of antibodies production [3]. beta glucan increase the production of important cytokines there is interleukin-2 (il-2) which stimulates the differentiation of b cells which are active [29] then the active b cell differentiation into plasma cells (clones plasma) which can produce immunoglobulin [30]. looking at the capabilities of the psk on the modulation of immune responses and saw its consumed in a long time in the community without significant side effects, the researcher wanted to investigate how the levels and kinds of immunoglobulin isotype of mice which infected by mycobacterium tuberculosis on providing psk with ��permanasari et al.: the role of polysaccharide krestin different timing. enzyme-linked immunosorbent assay (elisa) became selected test for measuring the levels and kinds of immunoglobulin isotype related to the specificity of antigen[7]. method stage in psk isolation from coriolus versicolor coarse powder of 200 g coriolus versicolor is added with water as much as 3 l and is heated at a temperature of 80–98° c for 2 3 hours. do extraction twice more with the addition of 2 l of water on the residue, the results obtained in form of supernatant from the three times extractions are ± 2 l [10]. mushroom extract solution is filtered using whatman no 41 filter and then its liofilisasi supernatant (for 150 ml for ± 24 hours). precipitation mushroom powder extracts using ammonium sulfate 90% and then dialysis using nitrocellulose membranes for 24 hours [11]. stage of making psk solution 3.5 g of ammonium sulfate is added with 50 ml aquades and 1 g of mushroom powder mixed into one. stirer solution for 2 h at 4° c and then centrifuged 9000 rpm for 12 min at 4° c. take the sediment and added 12 ml saline. polysaccharide concentration is measured using the phenol-sulfuric acid assay. dose of psk that used is 500 μg[29]. stage of provisioning psk and mycobacterium tuberculosis infection thirty animals are divided become 6 groups as follows table 1. treatment group group provision of psk on 1st-7th day mycobacterium tuberculosis infection on 8th and 15th day provision of psk on 23th-30th day i _ _ _ ii + _ + iii _ + _ iv + + _ v _ + + vi + + + description: (+) indicates treatment (-) indicates no treatment, were given only aquades i : as a control, have given only aquades ii : as a positive control, provision of psk only iii : as a negative control, mycobacterium tuberculosis infection only iv : provision of psk before infection with mycobacterium tuberculosis v : provision of psk after infection with mycobacterium tuberculosis vi : provision of psk before and after infection with mycobacterium tuberculosis mice infected with 0.5 mc farland or equivalent to 1.5× ×108 cfu/ml bacteria intraperitoneally. stage of analysis ig isotype serum ig isotype (igg1, igg2a, igg2b, igg3, iga, ig m, kappa and lambda chains) were analyzed with pierce rapid elisa kit mouse mab isotyping. the reading of od values by using elisa reader at a wavelength of 450 nm[4]. result table 2. od values of ig isotype kinds of ig optical density (od) values of ig k (k+) (k-) p1 p2 p3 igg1 igg2a igg2b igg3 iga igm kappa lambda 0,247 0,338 0,705 0,414 0,322 0,909 0,438 0,584 0,393 0,400 0,972 0,780 0,968 1,286 0,909 1,068 0,304 0,426 0,908 0,573 0,953 1,349 0,806 1,100 0,652 0,697 1,029 1,000 1,108 1,335 1,119 1,112 0,479 0,661 0,986 0,654 1,015 1,420 0,945 1,180 0,755 0,872 1,041 1,047 1,155 1,410 1,077 1,169 figure 1. e l i s a t e s t f o r d e t e r m i n i n g t h e k i n d s a n d l e v e l s o f i m m u n o g l o b u l i n description: k (1a-1h) control, k (+) (2a-2h) positive control is provision of psk only, k (-) (3a-3h) negative control with mycobacterium tuberculosis infection only, p1 (4a-4h) provision of psk before infection with mycobacterium tuberculosis, p2 (5a-5h) providing psk after infection with mycobacterium tuberculosis, p3 (6a6h) providing psk before and after infection with mycobacterium tuberculosis. �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 30-33 discussion in serum (k) although there has been color reaction, but not so striking as in serum (k+). this is because the control (k) not be immunized previously with antigens, which meant there was no previous contact with antigens. color reaction that occurred probably due to the presence of natural antibodies in the body of mice whose concentration is low. in (k+) provision of psk only, it has od values higher than (k). high concentration of immunoglobulin appropriate with the statement of bellanti (1993), that the potential immunomodulator can increase or make higher levels of certain responses as a whole. according vetvicka et al. (2002), beta-glucan is known to increase the production of lymphocytes. in (k-) od value is higher than (k). this is because mycobaterium tuberculosis can not make invasion of the immune system so it does not decrease the immune response. according todar (2009), mycobaterium tuberculosis can be multiply after 7–21 days early after infection and abbas et al. (2000) states that the maximum antibody in the primary response can be detected in the third week after immunization. kresno (2001) states that levels of antibody reduced later and generally only a few can be detected on 4–5 weeks after exposure. tuberculosis bacterial population are divided into extracellular and intracellular bacteria[2]. immunoglobulin which produced by b cells is the major protective immune component for extracellular bacteria that can serves to get rid of microbes and neutralize the toxin [5]. in the fight against intracellular bacteria there are 2 types of reaction are occurred, i.e. the first is killing of intracellular bacteria by macrophages activated through phagocytes in which the activation of macrophages occurs through cytokines, especially ifn-g, produced by t cells. the second way is with lysis of infected cells by cd8+ t cells. intracellular bacterial protein can stimulate cd4+ t cells (through mhc class ii antigens complex) or cd8+ t cells (through mhc class i antigens complex). intracellular bacteria induce t cell development into th1 cell phenotype, because these bacteria stimulates the production of il-12 by macrophages, and ifn-g by nk cells, both types of these cytokines promote the development of th1 cells (cd4+). on the other hand th1 cells produce ifn-g which activate macrophages to produce roi and enzymes that can kill bacteria. ifn-g also stimulate immunoglobulin isotype production by b lymphocytes [19]. in (k-) has a lower od value than the p1, p2 and p3. this shows psk has a role as immunostimulator. this is consistent with the statement of cui and chisti (2003), kidd (2000), and vetvicka et al. (2002), that the psk is immunostimulator or imunopotensiator. polysaccharide krestin contains 34–35% carbohydrate (91–93% glucan) [10]. beta (β)-glucan is known for stimulate the immune system [24], according to hong et al. (2004), beta (β)-glucan present in the gut then make contact with macrophages that exist in the intestinal wall which is assisted by m cells (microfold) that are specialized cells and found in the ileum. m cells will take glucan through pinositosis and took it through the intestinal wall where some cells such as macrophages, t cells, b cells and other immune cells have been waiting. beta(β)-glucan which phagocytosis by macrophages would be degraded into fragments, and then transported to a bone marrow where fragments-glucan degradation results will be released. according to chan et al., (2009), these fragments were arrested by the complement receptor (cr3) which located at the cell surface of granulocytes, monocytes, and dendritic cells. these cells with antibodies then activated. beta (β)-glucan will bind to macrophage on the cr3 receptor, it is combination receptor that has two binding regions. the first area is responsible for binding the type of complement, a soluble blood protein called c3 (or ic3b). c3 will be attached to the specific antibodies then bind to the targeted pathogen and do opsonisasi. the second area in cr3 binding to carbohydrate receptors on cells of yeast or fungus (psk) that allows macrophages to recognize yeast as ”nonself” [14]. from the second signal of psk, it can help the process of phagocytosis of macrophages in tuberculosis infection. the highest of od value was found for the igm isotype in all treatment groups. immunization of mycobacterium tuberculosis in live cell form and are conducted twice within an interval of one week makes the immune responses which occured is still primary immune response. according bellanti (1993), antibodies can be detected after 10 to 14 days after injection of bacterial cells. the first meeting with the bacteria will raise primary immune response. immune response which raised by imunogen is dominated by igm. od or absorbance values with the second highest concentration in the (k-), p1, p2 and p3 is iga. high concentration of iga in serum according to the statement baratawidjaja (2006) which states that high iga levels in serum will be found in respiratory and gastrointestinal infections, like tuberculosis. this is supported by frank (1995) which states that iga has functions in early antiviral and antibacterial defense by preventing bacterial adhesion to the mucous membranes. subclass igg2b has a higher concentration may be caused by its ability to bind antigens with a form of protein. according to scott et al. (1990), igg2a, and igg2b in mice with igg1, and igg3 of human have similarities in their ability to embed complement and protein antigens. polysaccharide krestin (psk) is a complex polysaccharide binding protein [21] and mycobacterium tuberculosis is a bacterium which contains several proteins that bind to lipids [15]. so, they make subclass of igg26 has a higher concentration. according to scott et al. (1990), igg3 of mice and igg2 humans have similarity in recognition of carbohydrate epitopes. the existence of higher enough concentration of igg3 indicated that psk take a role in increasing the types ��permanasari et al.: the role of polysaccharide krestin of that immunoglobulin. according to robinson (1995), beta (β)-glucan is a natural polysaccharide derivatives which having 7-10 monosaccharide units that are classified into the oligosaccharide. monosaccharide of psk consists of glucose (74.6%), mannose (15,5%) xylose (4.8% of), galactose (2.7% of), and high fructose (2.4% of) [28]. igg2a and igg1 subclass had the lowest concentration in the serum of treatment group, this probably occurred because igg1 more capable of binding mast cells[25]. immunoglobulin light chains are divided into two types, namely kappa light (κ) and lambda (λ) chains. according to tizard (1987) and bellanti (1993), the ratio between the kappa and lambda light chains are highly variable among species and their combinations are normally present in each individual. ig isotype highest with total 6.280 is founded on providing psk before and after infection with mycobacterium tuberculosis. this indicates the role of providing psk with different times on ig isotype of mice which infected by mycobacterium tuberculosis. provision of psk before infection with mycobacterium tuberculosis has function as prevention (preventive) that encourage to increase the number of lymphocytes formation then increases levels of immunoglobulin more optimally, so that levels of immunoglobulin against mycobacterium tuberculosis infection will further increases and will be further improved with the provision of psk after mycobacterium tuberculosis infection as a treatment (curative). polysaccharide krestin is expected to prepare and boost immunity against disease that will enter the body. pietro (2003) states that β-glucan is more effective for prevention (preventive) and treatment (curative) of diseases in related with immune system durability. references 1. abbas, a. k., lichtman, a. h., dan j. s. pober, 2000, cellular and molecular immunology, 2’th edition, w. b saunders company, united states of america. 2. alsagaff, h., dan a. mukty, 2002, dasar-dasar ilmu penyakit paru, airlangga university press, surabaya, hal 73–108. 3. anonimus, 2008, immune-stimulant, http//technicalservice. wordpress.com/2008/06 /02 /immune-stimulant, diakses tanggal 4 mei 2009. 4. anonimus, 2009, instructions pierce rapid elisa mouse mab isotyping kit, www. thermo.com/pierce, diakses tanggal 5 oktober 2009. 5. baratawidjaja, k.g, 2006, imunologi dasar, edisi 7, penerbit fakultas kedokteran universitas indonesia, jakarta. 6. bellanti, j.a, 1993, imunologi iii, edisi bahasa indonesia, gadjah mada university press, yogyakarta. 7. burgess, g.w., 1995, teknologi elisa : dalam dignosis dan penelitian, cetakan pertama, gadjah mada university press, yogyakarta. 8. chan, g. c., wing k. c., dan daniel m., 2009, the effects of β-glucan on human immune and cancer cells, journal hemato oncology, 2: 25. 9. cruse. j.m, dan r.e. lewis, 2004, atlas of immunology, second edition, crc press, boca raton london. 10. cui, j dan chisti, y, 2003, polysaccharopeptides of coriolus vercicolor : physological activity, uses, and production, elsevier science, biotechnology advance 21, 109–122. 11. cui, j., goh, k.t., archer, r.h., dan sigh, h., 2007, characterisation and bioactivity of protein-bound polysaccharide from submerged culture fermentation of coriolus versicolor wr-74 and atcc20545, journal of industrial microbiology and biotechnology 34, 393–402. 12. dye, c., scheele s., dolin p., pathania v, dan reviglione m., 1999, global burden of tuberculosis, estimate incident, prevelence, and mortality by country. jama 282: 677–686. 13. frank, c. l., 1995, toksikologi dasar, asas, organ sasaran, dan penilaian resiko, edisi ke-2, penerjemah edi nugroho, indonesia university press, jakarta. 14. hong, f., jun y., jarek t. b., daniel j., richard d., gary r., pei x. x., nai k., cheung dan gordon d. ross, 2004, mechanism by which orally administered beta-1,3-glucans enhance the tumoricidal activity of antitumor monoclonal antibodies in murine tumor models, journal immunology, 173: 797–806. 15. jawetz, e., elnick, j. l, adelberg, e.a, butel, j. s, dan ornston l. n., 1996, mikrobiologi kedokteran, penerbit egc, jakarta. 16. keitges, j., 2002, coriolus versicolor, http: //www. geocities. com / gaiasgate / mono graphs / coriolus.htm, diakses tanggal 28 juni 2009. 17. kidd, p. m., 2000, the use of mushroom glucans and proteoglycans in cancer treatment, alternative medicine review, vol 5 no 1. 18. koendhori, e.b, 2008, peran ethanol extract proposis terhadap produksi interveron g il 10 dan tgf β1 serta kerusakan jaringan paru pada mencit yang diinfeksi dengan mycobacterium tuberculosis,. disertasi program pascasarjana, universitas airlangga. 19. kresno, s. b, 2001, imunology: diagnosa dan prosedur laboratorium, edisi iv, cetakan ke 1 (dengan perbaikan), balai penelitian fkni, jakarta. 20. ningsih, w. c. p., 2008, pengaruh imunisasi protein membran spermatozoa kelinci (oryctolagus cuniculus) pada macam imunoglobulin mencit (mus musculus), skripsi, jurusan biologi, fakultas sains dan teknologi, universitas airlangga. 21. ooi, v.e, dan liu, f., 2000, a review of pharmacological activities of mushroom polysaccharides, international journal medicinal mushroom : 196–206. 22. pietro p., 2003, composition for preventif and or treatment of lipid metabolism disorders and allergic form, http://freepatentonlin. com/20030017999.html, diakses tanggal 19 januari 2010. 23. rantam, f.a., 2003, metode imunologi, penerbit airlangga universiti press, surabaya, hal: 1–9. 24. robinson, t., 1995, kandungan organik tumbuhan tinggi, penerbit itb, bandung 25. scott, m. g., d. e. briles dan m.h. nahm, 1990, selective igg subclass expression: biologic, clinical, and functional aspect, pathology, 161–183. 26. tizard, i., 1987, pengantar imunologi veteriner, penerbit airlangga university press, surabaya. 27. todar, k., 2009, todar’s online textbox of bacteriology. university of wiscosin madison depertment of baceriology. 28. tsukagoshi, s., hashimoto, y., fujii, g., kobayashi, h., nomoto dan orita k, 1994, krestin (psk), cancer treatment reviews 11: 131–155. 29. vetvicka, v., kiyomi t., rosemade m., paulin b., bill k., dan gary o., 2002, orally-administered yeast β-1,3 glucan prophylactically protects against anthrax infection and cancer in mice, journal american nutraceutical assosiation, vol 5 no 2. 30. weir, d.r., 1990, segi pratis imunologi, binarupa aksara, jakarta, hal 1–54. copyright © 2020, ijtid, issn 2085-1103 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 8 no. 1 january–april 2020 research article mcp-1 levels and atypical lymphocytes in early fever of dengue virus infection with non-structural protein 1 (ns-1) antigen test in dr. darsono hospital, pacitan indah agustinaningrum1,5, jusak nugraha2,4,a hartono kahar3,4 1master of immunology, postgraduate school, universitas airlangga, surabaya, indonesia 2clinical pathology, faculty of medicine and institute of tropical disease, universitas airlangga 3clinical pathology, faculty of medicine, universitas airlangga 4clinical pathology dr soetomo hospital, surabaya, indonesia 5medical technologist dr darsono hospital, pacitan, east java corresponding author: jusak.nugraha@yahoo.com received: 8th april 2019; revised: 18th june 2016; accepted: 2nd january 2020 abstract dengue infection caused by denv and transmitted by mosquitoes aedes aegypti and aedes albopictus is a major health problem in the world, including indonesia. clinical manifestations of dengue infection are very widely, from asymptomatic until dengue shock syndrome (dss). denv will attack macrophages and dendritic cells (dc) and replicate them. monocytes are macrophages in the blood (± 10% leukocytes). macrophages produce cytokines and chemokines such as monocyte chemotactic protein-1 (mcp-1)/ccl2. the monocytes that are infected with denv will express mcp-1, which will increase the permeability of vascular endothelial cells so that they have a risk of developing dhf/dss. macrophages and dc secrete ns1 proteins, which are the co-factors that are needed for viral replication and can be detected in the early phase of fever. the increased mcp-1 levels in dengue infection followed by an increase in the number of atypical lymphocytes indicate the arrival of macrophages and monocytes to the site of inflammation which triggers proliferation rather than lymphocytes. this is an observational analytical study with a cross-sectional design to determine the mcp-1 level in dengue infection patients with 1st until the 4th day of fever and the presence of a typical lymphocytes. dengue infection was determined by rapid tests ns1 positive or negative and mcp-1 levels were measured using by elisa sandwich method.mcp-1 level of sixty patients dengue infection ns-1 rapid positive or negative with 2nd until 4rt fever were significantly higher than healthy subjects (420.263 ± 158,496 vs 29, 475 ± 23.443;p=0.000), but there was no significant difference in subjects with df, dhf or dss (436,47 ± 225,59 vs 422,77 ± 170,55 vs 448,50 ± 117,39; p =0.844). a typically lymphocytes differs significantly in healthy subjects than subjects infected with denv an average of 2% (p= 0,000). in conclusion, this shows the arrival of macrophages and monocytes to the site of inflammation, which triggers the proliferation of lymphocytes. keywords: mcp-1, atypical lymphocytes, ns-1, hematology parameter, pacitan abstrak infeksi dengue disebabkan denv ditularkan oleh nyamuk aedes aegypti dan aedes albopictus yang masalah kesehatan utama di dunia, termasuk indonesia. manifestasi klinik infeksi dengue sangat bervariasi, dapat asimptomatik sampai dengue shock syndrome (dss). denv akan menyerang makrofag juga sel dendritik (dc) dan akan bereplikasi. monosit merupakan makrofag dalam darah (±10% leukosit). makrofag memproduksi sitokin dan kemokin seperti mcp 1. mcp-1 terekspresi oleh monosit terinfeksi denv yang dapat meningkatkan permeabilitas sel endotel vaskular sehingga memiliki risiko mengalami dhf/ dss. makrofag dan dc mengeluarkan corresponding author. e-mail: jusak.nugraha@yahoo.com protein ns1 merupakan co-factor yang dibutuhkan untuk replikasi virus dan dapat dideteksi pada fase awal demam. mailto:jusak.nugraha@yahoo.com mailto:jusak.nugraha@yahoo.com copyright © 2020, ijtid, issn 2085-1103 30 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 30–43 adanya peningkatan kadar mcp-1 pada infeksi dengue diikuti dengan peningkatan jumlah limfosit biru menunjukkan datangnya makrofag dan monosit ke tempat terjadinya inflamasi yang memicu proliferasi daripada limfosit. penelitian ini merupakan penelitian observasional analitik dengan desain potong lintang untuk mengetahui kadar mcp-1 pada subyek terinfeksi dengue dengan ns-1 positif pada hari demam ke 1-4 dan adanya limfosit plasma biru. subyek penelitian adalah pasien dewasa terinfeksi dengue dengan hasil rapid tes ns-1 positif dan negatif. kadar mcp-1 pada serum/ plasma diukur menggunakan metode elisa sandwich. subyek penelitian terdiri 50 pasien infeksi dengue dengan ns-1 rapid tes positif dengan demam hari kedua sampai hari keempat memiliki kadar mcp-1 lebih tinggi dibandingkan subyek sehat (420.263 ± 158,496 vs 29, 475 ± 23.443;p=0.000), dan tidak ada perbedaan bermakna kadar mcp-1 subyek dengan df, dhf dan dss (436,47 ± 225,59 vs 422,77 ± 170,55 vs 448,50 ± 117,39; p =0.844). limfosit plasma biru berbeda bermakna pada subyek sehat dengan subyek terinfeksi denv rata-rata 2% (p= 0,000). kesimpulannya, hal ini menunjukkan datangnya makrofag dan monosit ke tempat terjadinya inflamasi yang memicu proliferasi daripada limfosit pada infeksi dengue. kata kunci: mcp-1, limfosit plasma biru, ns-1, hematology parameter, pacitan how to cite: agustiningrum, indah; nugraha, jusak; kahar, hartono. mcp-1 levels and atypical lymphocytes in early fever of dengue virus infection with non-structural protein 1 (ns-1) antigen test in dr darsono hospital, pacitan. indonesian journal of tropical and infectious disease, [s.l.], v. 8, n. 1, p. 30-43, mar. 2020. issn 23560991. available at: <https://e-journal.unair.ac.id/ijtid/article/view/12696/9922>. date accessed: 04 apr. 2020. doi:http://dx.doi.org/10.20473/ijtid.v8i1.12696. introduction dengue virus (denv) is a flaviviridae family and the flavivirus genus. denv is a positive rna virus that contains envelopes with genomes ~10.7 kb in four serotypes (denv1-4). denv infection is an acute disease caused by one of the four viral serotypes of the genus flavivirus, flaviviridae family, transmitted by mosquito bites aedes aegypti and aedes albopictus1,2 and in some cases developed until dengue hemorrhagic fever (dengue hemorrhagic fever/ dhf) or even until dengue shock syndrome (dss).3 dengue virus infection cause is asymptomatic or symptomatic infections, from undifferentiated fever, dengue fever (df), dengue hemorrhagic fever (dhf), dengue shock syndrome (dss) and expanded dengue syndrome.4,5 dengue disease may manifest with gastrointestinal symptoms which may make diagnosis and treatment difficult and wrong.6 in indonesia, there were 68,407 cases of dengue hemorrhagic fever in 2017 compared to 2016 with 204,171 cases with the highest cases in the provinces of west java, central java, and east java. the highest mortality dengue in 2017 in the east java province with the death rate or case fatality rate (cfr) of 2017 dhf by 1.3%. 7 pacitan in 2016 there were 1,338 dhf sufferers with a population of 552,327 dhf ( incidence rate = ir) morbidity amounted to 242.3 per 100,000 population and there was 1 death from dengue fever.8 this prospective cohort study in west java provides several important findings on the epidemiology of dengue virus infections in adults living in an endemic area. first, the dengue virus is a major etiology of febrile illness (12.4%) in adults in bandung, west java, indonesia.9 denv nonstructural protein (ns)-1 is a diagnostic marker to early detection of denv compared to serological tests because it is detected in serum patients infected with denv as early as one day after the appearance of day post onset symptoms (dpo). dpo up to 18 at a concentration of ns-1 up to 50 μg/ml.10 the ns-1 is a glycoprotein with two glycosylation sites that are conserved among flaviviruses. it is synthesized in the er as a hydrophilic monomer but exists as a more hydrophobic homodimer. the ns-1 dimer is transported to the golgi apparatus where it undergoes carbohydrate trimming. the role of ns-1 in virus replication is unknown but is believed to facilitate viral infection and denv pathogenesis. ns-1 is in addition secreted from infected cells (sns-1) and has been shown to be immunologically important.1 during natural dengue infection in humans, the mosquito delivers virus in skin epithelium where it infects and replicates in the cells of mononuclear lineage like monocytes, dendritic cells, macrophages, and langerhans cells.11 dengue virus later attached to monocytes through the receptor factor and into monocytes. in this https://e-journal.unair.ac.id/ijtid/article/view/12696/9922 http://dx.doi.org/10.20473/ijtid.v8i1.12696 copyright © 2020, ijtid, issn 2085-1103 indah agustinaningrum, et al.: cp-1 levels and atypical lymphocytes 31 situation, there is an afferent mechanism in which the virus has been developed through unification and attachment of several gene segments and receptor factors are developed. furthermore, efferent mechanisms occur, namely monocytes containing distributed viruses.12 these infected monocytes carry the virus to the lymph nodes where it replicates resulting in viremia followed by systemic infect ions of the liver, lungs, and spleen.11 the migration of these infected cells around the lymphatic system triggers the production of cytokines and the recruitment of other immune cells. these include monocytes and macrophages, which are the primar y target of infection and the main site of denv replication.13 two mechanisms of immunity are considered responsible the first occurrence of dengue hemorrhagic fever is a nonneutralizing antibodies produced from previous infections believed to increase viral replication by connecting the virion with the fc receptor on the surface of the target cell, which then carries it into the cytoplasm, thereby increasing the number of infected cells, the number of virus particles entering each cell and the release of cytokines and other vasoactive mediators. second, cd8+ t reactive memory cells can attack monocytes and macrophages that express viral epitopes on their surface, triggering an explosive inflammatory response.14 dengue infection induces the overexpression of many chemokines and cytokines in monocytes, such as tumor necrosis factor (tnf)-α, ifn-γ, il-1β, il-8, il-12, macrophage inflammatory protein (mip)-1α, mcp-1/ ccl(chemokine(c-c mo t if) ligand)2, and rantes (regulat ed upon activation, normal t-cell expressed and secreted). mcp-1 levels in the plasma of df and dhf patients were increased significantly.15 monocyte chemoattractant protein(mcp)-1/ ccl2 is chemo k ine which regu lat es t he movement of monocytes/macrophages.16 the production of mcp-1 and monokine induced by gamma interferon (mig) from monocytes or macrophages could be induced by interferon (ifn)-γ upon denv infection in order to recruit more leucocytes to the site of infection for viral clearance.17 denv-infected monocytes enhance functional regulation of caspase-1 mrna and activation of procaspase-1 in late response to infection responsible for excretion of interleukin(il)-1β and pyroptosis from denv infected monocytes. late activation of caspase-1 in monocytes infected with denv can contribute to pro-inflammatory results that may play a role in the immunopathogenesis of dengue.18 mcp-1 causes openings of tight endothelial cell connections in vitro19 and expression of vegf-induced mcp-1 in vascular endothelial cells increases changes in endothelial permeability in vivo.20 recombinant (rh)mcp-1 and mcp-1 containing condit ioned media from denv infected monocytes increased the permeability of vascular endothelial cells and also clarified that mcp-1 but not vegf in denv-infected monocyte culture media increased endothelial permeability.19 mcp-1/ ccl2 s elain recruit and direct the movement of leukocytes also may affect t-cell and ccl2 enhances the secretion of il-4 by tcells. other chemokines and their receptors will be associated with specific t-helper cell responses.21 ccl2, -7, -8, and-13 (mcp -1 to -4) are strong chemotactic factors for colonization of inflamed target tissues not only for t-cells but also for nk cells and immature dendritic cells. in virus ifn-γ infection produced locally at the site of infection by th1 effector cells or nk cells, it is responsible for the formation of chemokines cxcl10 and cxcl9, and this then dances ccytotoxic effector t cells or cells expressing cxcr3.22 t-cells may be the so urce of at yp ica l lymphocytes for their activation and proliferation of t-cells that are seen in most of the viral infection. it is possible that atypical lymphocytes can also represent cellmediat ed immu ne responses host for dengue virus.23 the aim of this study based on the levels of mcp-1 and the ns1 protein in the serum or plasma from whole blood with k2edta (dipotassium ethylene diamine tetra acetate) anticoagulant of denv infection patient and control healthy control, where increasing the level of mcp-1 from healthy people can not describe the possibility of prognosis more severe dengue copyright © 2020, ijtid, issn 2085-1103 32 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 30–43 hemorrhagic fever (dhf) or dengue shock syndrome (dss). denv infection increased mcp-1 levels will increase monocyte activation towards the inflammatory area and the presence of atypical lymphocytes in denv infection. materials and methods study population this study was done in dr. darsono pacitan hospital and faculty of medicine laboratory, gajah mada university, yogyakarta in february 2019. this study included 60 patients with dengue fever and 10 healthy people as the controls who were selected from dr. darsono hospital, pacitan indonesia. patients who were selected were in accordance with the criteria of inclusion in patients with clinical symptoms had on set 1st until 4th fever day and ages limit 18 55 years. patients who did not enter the inclusion and exclusion criteria were excluded from the study. the group control with 10 healthy subjects with comparable age characteristics, no positive history of dengue infection, no fever for 1 month before the study and cbc levels within normal limits. the clinical disease severity was classified according to the 2011 world health organization (who) dengue diagnostic criteria. in patients with fever where the sufferer experiences with a fever between 39-40°c and headache or lasting 5-7 days in the majority of cases and the other common symptoms include anorexia were classified as df. the dhf grade i if there are plasma leakage obtained tourniquet test positive and thrombocytopenia equal to 100.000/m³ and hematocrit greater than 20% of the baseline. if there is spontaneous bleeding, is dhf grade ii, and if hypotension and the patient are nervous, is dhf grade iii, and dhf grade iv infection dss that is shock was defined as having cold clammy skin, along with a narrowing of pulse pressure of 20 mmhg4. the aim of this study was to determine the relationship between mcp1 and atypical lymphocytes levels to the risk of dhf / dss. the difference between mcp-1 and atypical lymphocyte from a patient with ds or dhf and dss by counting the mean of sd. ethics statement the ethical agreement was obtained from the ethical review committee faculty of dentistry, universitas airlangga. all research subjects used informed consent. blood samples the blood sample of the study was taken from patients on fever day 1st-4th, accommodated in 2 types of tubes namely tubes 3ml without anticoagulants and tubes 2ml with k2-edta anticoagulants. to obtain a blood sample serum in a tube without anticoagulants, it is waiting to clot for 20-30 minutes and centrifuges 5-15 minutes 1500-3000 rpm, then the serum is separated in a tube sample of 1 ml each. blood samples in tubes with anticoagulants were thoroughly examined and blood smears were made which were colored with wright's stain for atypical lymphocyte examination.24,25 to get plasma blood samples in tubes with anticoagulants rotated 5-15 minutes 1500-3000 rpm, then plasma was separated in a tube sample of 1 ml each. samples are stored at -20°c (stabilized 1 month) before mcp-1(insert kit ) is examined and the ns-1 examination of the rapid test method was immediately carried out at that time. laboratory diagnosis the examination of ns-1 on the subject serum was using dengue early rapid tests panbio with the ict (immunochromatography test) method with results expressed in positive or negative (insert kit). examination of atypical lymphocyte is counting in 100 leukocytes on a blood smear stained by wright's and the results expressed in percent (%)23. examination of mcp-1 levels with the sandwich-elisa method from the elabscience kit according to the insert kit procedure and the results are expressed in pg/ml.26 statistical analysis in this study, we analyzed mcp-1 levels, atypical lymphocytes and ns1 in patients with copyright © 2020, ijtid, issn 2085-1103 indah agustinaningrum, et al.: cp-1 levels and atypical lymphocytes 33 dengue fever are determined clinically and then use the mann-whitney test and kruskal–wallis test to see the difference between dengue infection with fever on fever day 14 and healthy subject. quantitative variables not following a normal distribution such as mcp-1 levels and atypical lymphocytes were compared using a non-parametric test (kruskal-wallis test) to see the difference between dengue fever (df), dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss). statistic package for social sciences (spss) was used for data entry, processing and statistical analysis at the end of the study. p-values less than 0.05 were considered significant.9 results and discussion study population this study was 60 patient and 10 healthy subjects as control without dengue infection from various ages, 60 patient has infected dengue with 10 patient ns-1 negative and 50 patient ns-1 positives. table 1 gives the age and gender distribution of the participants and fever day when was sampling. the age grouping used was based on the ministry of health (2009), grouping between the ages of 17-35 years and 36-55 years. the majority of dengue infection are between the age group of table 1. age and gender distribution of the participants (n = 70) 17 to 35 years (50%), and at the age of 36-55 years is 3,3%. patient dengue infection with the ns-1 positive at the age of 17-35 years was 50% (55% males) and patients with dengue infection with the ns-1 negative at the age of 36-55 years 7% ( 5% males), the males were found more than females. there various fever days at sampling, 2nd fever day until 4rd. ns-1 test ns-1 was examined in the serum of dengue infected patients performed on the condition of subjects with a fever between 2nd until 4th. the method used for the ns-1 examination uses the panbio rapid test. of the 60 serum samples of dengue fever patients, 50 ns1 samples were positive and 10 ns-1 samples were negative. from positive ns-1 sufferers sampling on 2nd feverday = 4 (6.7%), 3rd fever day = 20 (33.3%), 4th fever day=16 (26.7%) and in patients with ns-1 negative sampling at fever day 2nd=1 (1.7%), 3rd= 5 (8.3%), and 4th= 4 (6.7%). mcp-1 levels mcp-1 levels examined in serum or plasma of patients with sampling table 1 gives time at 2nd-day fever until 4th-day fever. table 2 gives the mcp-1 levels in patients dengue infection increased significantly (p = 0,000 ; p<0.05) from healthy subjects, in healthy subjects the average emission value mcp-1 levels is 29.475 ± 23.443 pg/ml and in samples infected with dengue ns-1 negative average 471,290 pg/ml higher than patients ns-1 positive who averaged 420.262 pg/ml, but mcp-1 levels were no significant difference in 60 patients dengue-infected with ns-1 positive and ns-1 negative. table 2. mean difference of mcp-1 level in research subjects sampling (33.3%) day 4: 16 (26.7%) day 3: 5 (8.3%) day 4: 4 dengue ns-1 (-) 10 471,290 ± 266,386 (6.7%) mann-whitney test control ns-1 post (+) ns-1 neg (-) n (%) n (%) n (%) age 17-35 8 30 (50%) 3 ( 5 %) 36-55 2 20 (33%) 7 ( 11.7 %) gender man 6 33 (55%) 5 (8, 3 %) women 4 17 (28.3%) 5 (8, 3 %) fever day at the 0 day 2: 4 (6.7%) day 3:20 day 2: 1 (1.7%) sampel n average mcp-1 level (pg / ml average ± sb p healthy subjects 10 29,475 ± 23,443 <0.05 dengue ns-1 (+) 50 420,263 ± 158,496 copyright © 2020, ijtid, issn 2085-1103 34 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 30–43 table 3. mean difference of mcp-1 level based on fever day table 5. differences in mean atypical lymphocytes in research subjects sampel average p variable sample atypical lymphocyte p n mcp-1 level (pg / ml average ± sb n % (in 100 leucocytes) average ± sb 2nd day fever 6 503,869 ± 149,632 = 0.562 healthy subjects 10 not found / zero <0,005 3rd day fever 30 428,165 ± 195,446 p > 0.05 dengue ns-1 (+) 50 3 ± 2 4th day fever 24 410,744 ± 165,490 dengue ns-1 (-) 10 1 ± 1 kruskal–wallis test table 4. mean differences in mcp-1 levels based on the diagnosis of dengue infection patients variable n (%) average p mcp-1 level average ± sb df 15 ( 436,467 ± 225,585 =0,884 25%) dhf 39 ( 422,770 ± 170,548,803 > 0.05 65%) dss 6 ( 10%) 448,499 ± 117,391 mann-whitney test mcp-1 levels tend to increase in all patients with clinical dengue disease and also the presence of atypical lymphocyte with an average of 2% in 100 leucocytes ( p = 0,000), but there is no significant difference in sufferers of dengue infection with ns1 positive and ns1 negative (pmcp-1 =0, 744; p lpb=1,000) (ρ> 0.05). table 5 gives the highest atypical lymphocyte number was 6% of patient ns-1 positive and the highest mcp-1 level was found in patients with kruskal–wallis test table 3 gives the average mcp-1 levels were examined by sampling time when 2nd fever days until 4th no significant differences p= 0,562 (p>0,005). table 4 gives the average mcp-1 levels were examined by clinical development of patients both negative ns-1 781,494 pg/ml. table 6. differences in mcp-1 levels, hematology variables and fever day when sampling the dengue infection severity variable infection dengue p df dhf dss df, dhf and dss are no significant differences ( p = 0.884) ( p > 0.005). mcp-1 levels 436,47 ± 225,59 422,77 ± 170,55 448,50 ± 117,39 0,844 atypical lymphocyte hemoglobin 13,8 ± 1,48 13,9 ± 1,87 13,7 ± 2,60 0,836 atypical lymphocyte examined in blood smear of dengue infected patients with sampling hct 39,5± 4,1 39,4 ± 38,7 ± 5,7 0,706 4,66 plt 171±93 136±62 94 ± 37 0,131 time 2nd fever day until 4th. the percentage of atypical lymphocyte in patients with dengue % monosit 7,42 ± 2,37 8,25 ± 3,32 8,43 ± 3,45 0,915 infection increased significantly ( p <0.05) from the percentage of atypical lymphocyte healthy subjects as controls not found atypical lymphocyte, the average levels atypical lymphocyte patient dengue-infected with ns-1 positive is equal to 3 ± 2% in 100 leucocytes and the samples were patient dengue-infected with ns-1 negative average 1 ± 1% in 100 leucocytes. % lymphocyte atypical lympocyte feverday on the sampling time 27,0±13,2 21,2±10,4 25,4±13,4 0,122 2±1 2±1 3±1 0,313 3±1(2-4) 3±1(2-4) 3±1(2-4) 0,579 kruskal–wallis test copyright © 2020, ijtid, issn 2085-1103 indah agustinaningrum, et al.: cp-1 levels and atypical lymphocytes 35 hematology parameters table 6 gives the laboratory investigations are evaluated in our study, the finding shows that hemoglobin levels, hematocrit, monocyte, and lymphocyte were not a significant difference from healthy subjects. platelet level in 1st until 4th every day decreased from healthy subjects. leukopenia was mainly found in ns-1 seropositive patients. the hematology result of this study differs from the kauser study in 2014.27 discussion dengue fever can be caused by one of four distinct dengue virus (denv) serotypes that cocirculate in many parts of the world. they're suggesting that infection to denv does not provide lifelong immunit y and a person can be infected with the same virus.28 the importance of plasma leakage as a key feature of dhf facilitated the development of clinical management guidelines that successfully reduced dengue-related morbidity and mortality. recognition of the predominant infection of monocytic cells, the increased risk for dhf associated with the circulation of multiple denv serotypes and secondary denv infections and the association of dhf with enhanced cytokine production in vivo guided development of disease models, diagnostic tests and candidate therapeutics.2 proinflammatory cytokines were secreted to initiate the inflammation and to control the denv replication especially at the early stage of infection. however, dysregulation of these cytokines was also considered an important reason in dengue pathogenesis, especially in dhf and dss.29 the occurrence of dhf/dss is thought to result from a complex interplay between the virus, host genetics, and host immune factors.30 denv infection of dcs resulted in ccl2, ccl3, and ccl4 expression, cytokines il-6, tnfα, and ifn-γ and chemokines ccl2, ccl3, and ccl4 have been associated with disease severity, endothelial dysfunction, and vasodilation.31 this study showed that in patients with dengue ns-1 test and obtained from 60 samples of patients with dengue fever found 50 patients with ns-1 positive and 10 patients with ns-1 negative, it homogeneous sample in this study is done by limiting the sampling time of patients with fever day 1-4. some studies have found that ns-1 antigen levels, especially during days 4–8 of illness, were lower in patients with more severe forms of illness.20 denv nonstructural protein 1 (ns-1) is a unique diagnostic marker for early detection of denv compared to serological tests (i.e., anti-denv igm) because it is detected in the serum of denv-infected patients as early as one day post onset of symptoms (dpo) to 18 dpo at ns-1 concentration up to 50 μg/ml and it is a confirmatory test. this elisa was sensitive and specific to denv-4 with no cross-reactivity to other three denv (1–3) serotypes and other heterologous flaviviruses.10 the secondary infection with a different serotype occurs, the immune response can lead to the presentation of dengue fever is more severe in some cases.32 the incubation period ranges from 3 to 14 days and symptoms usually develop between 4 and 7 days after vector bites.33 dengue infection is usually confirmed by viral genomic rna identification, antigen, or the antibodies it causes. an antigen detection test based on ns-1 detection has been used to detect viral ns-1 proteins released from dengue that infect and appear early in the bloodstream. rapid tests such as the ns-1 enzyme-linked immunosorbent assay (elisa) are commercially available for denv with relat ively good sensitivity and specificity.10 clinical diagnosis of dengue can be challenging, depending largely on what stage in the infection process a patient presents. depending on the geographic region of the world, there can be a number of disease-causing pathogens or disease states that can mimic the disease spectrum arising from dengue infection. in the early stages of clinical disease, dengue can present as a mild undifferentiated “flu-like” fever with symptoms similar to those of other diseases such as influenza, measles, zika, chikungunya, yellow fever, and malaria.34 copyright © 2020, ijtid, issn 2085-1103 36 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 30–43 dengue infection is usually confirmed by viral genomic rna identification, antigen, or the antibodies it causes. an antigen detection test based on ns-1 detection has been used to detect viral ns-1 protein released from dengue that infects cells and appears early in the bloodstream. ns-1 rapid detection tests or enzyme-linked immunosorbent assay (elisa) is available commercially for denv was sensitive and specific to denv-4 with no cross-reactivity to other three denv (1–3) serotypes and other heterologous flaviviruses.10 ordering a dengue ns-1 antigen assay within the first week of symptom onset is one of the initial investigations reco mmended by the ministry of healt h, singapore, for dengue infections.35 ns1 test detects at the same time as viral rna and before an antibody response, so the time to do the best examination is day 0 to day 4 and can be detected before the decrease in platelets. our study included several patients with negative ns-1 results when examined using the panbio rapid test. in, patients with 1-4 day dengue fever with ns-1 positive showed that there was dengue infection. conversely, dengue fever patients with negative ns-1 did not rule out dengue infection but ns1 was detected at a low level that caused false negatives and further examination was needed. to detect viral proteins, a sufficient level of virus is needed, whereas in the initial stage there is not enough virus, but if it takes samples after the appearance of antibodies, the level of the dengue virus will also decrease.34 the ns-1 protein itself is secreted from infected cells and is found in serum at detectable levels that overlap with peak viremia (and rna detection). these ns-1 levels also coincide with the onset of detectable igm in acute primary cases and igg in acute nonprimary cases. it has been found that elevated levels of serum ns-1 directly indicate increased viral burden and further establish a positive correlation between viremia and ns-1 profiles.36 the rapid tests and qrt-pcr had high sensitivity for dengue diagnosis. both tests correlated well with the serological diagnosis for case definition (positive ns-1 elisa) and the overall performance of the method was satisfactory when compared with ns-1 elisa.37 immunochromatography based rapid diagnostic tests (rdts) can detect ns-1 dengue antigen because they may provide a rapid (poct) point-of-care test in high specificity.38 the advantage of the ns-1 antigen rapid test for dengue diagnosis has been widely documented. although the who has recommended the ns-1 rapid test as one of the diagnostic tests for dengue infection, the use of the test is still limited due to its high cost and low sensitivity. in general, the result of the ns1 antigen rapid test should be carefully interpreted because its accuracy can change over the course of illness following the dynamics of viral antigen and antibody levels. therefore, clinical information of patients must be considered along with the ns1 test result.39 dengue fever is a disease that is difficult to treat at the clinical level, mostly because of the late manifestation of severe disease in some patients.36 early in the acute febrile period of the disease, dengue fever presents with the same clinical symptoms as primary dengue. later, during defervescence, patients can rapidly deteriorate, progressing to hemorrhage with or without a vascular leak. during this period, patients can experience bleeding, thrombocytopenia with <100.000 platelets/μl, ascites, pleural effusion, increased hematocrit concentrations, severe abdominal pain, restlessness, vomiting, and sudden reduction in temperature with profuse perspiration and adynamia.34 the sensitivity of ns-1 detection depends on the technique used and whether the sample corresponds to a primary or secondary infection. in primary infection, the sensitivity can exceed 90%, while in secondary infection, the sensitivity is lower and ranges from 60% to 80%. 40,41,42 a longer duration of ns-1 antigenemia than that of viremia in primary denv infection makes the ns-1 detection method an advantage over denv nucleic acid detection technique for dengue diagnosis during the acute phase of infection. in secondary denv infect ion, however, a decreased sensitivity of the ns-1 ag strip test was observed.43 the roles of denv ns-1 antigen and lipid mediators such as (platelet-activating factor) paf in causing vascular leak are emerging denv copyright © 2020, ijtid, issn 2085-1103 indah agustinaningrum, et al.: cp-1 levels and atypical lymphocytes 37 ns-1 are likely to be helpful in reducing disease pathogenesis due to ns-1, drugs that block paf receptors or the pathways in which paf is generated may be helpful in the treatment of acute illness.20 paf was previously found to be an important contributor to vascular leak and paf receptor blockade was found to inhibit the effects of acute dengue sera on the expression of the tight junction protein zo-1, and in the reduction of trans-endothelial resistance.15 endocytosed denv like particles has been proven by ultrastructural analysis of platelets from patients with dengue. in vitro studies identified the mechanisms of denv binding and internalization by platelets requiring dc sign and heparan sulfate proteoglycans for viral attachment. when isolated platelets are infected with denv in vitro, positiveand negative-sense viral rna, as well as denv ns-1, accumulate in platelets, indicating replication and translation of viral genome.44 although thrombocytopenia is more common in dhf than df, a significant fraction o f df patients also develop thrombocytopenia. thrombocytopenia is not an early indicator for dhf as the platelet counts during the early febrile phase of df and dhf are not significantly different. as such, platelet counts serve as a monitoring tool for disease progression rather than an early indicator of severe disease. platelet counts are rarely low enough to cause spontaneous hemorrhage in dhf patients but may contribute to the hemorrhagic tendency in cases complicated with plasma leakage and shock.2 mcp-1/ccl2 dengue hemorrhagic fever has unique pathogenesis which is a consequence of the evolution of the virus into four different serotypes. denv infects a variety of cell types in vitro including epithelial cells, endothelial cells, hepatocytes, muscle cells, dendrit ic cells, monocytes, and mast cells.45 the pathological basis of dengue fever lies in a complex series of immunological responses resulting in a rapid increase in the levels of cytokine and other chemical mediators that are central to the severe manifestations of dengue hemorrhagic fever, such as plasma leakage, shock, and bleeding.46 primary dengue infection causes unpleasant but rarely fatal, diseases such as influenza resulting from the temporary release of proinflammatory cytokines from monocytes and macrophages that are infected with the virus. a pathogenic role for an aberrant inflammasome and monocyte activation in the development of the severe form of dengue disease.47 chemokine plays an important role in the immune respo nse, ccl2, -7, -8, and -13 (mcp-1 to -4) are strong chemotactic factors for colonizing target tissue that is inflamed not only for t cells. chemokines are cytokines that stimulate the migration of cells that are attracted to the sites with a higher concentration of ligands. 22 chemokines share the common function of attracting leukocytes to sites of an inflammatory or immune response. a standardized nomenclature in which chemokines were given numerical names, like the interleukins and the chemokine receptors, was proposed more than a decade ago and is now widely used.48 the monocyte chemoattractant protein-1 (mcp-1) is secreted from macrophages, monocytes, endothelial cells, epithelial cells, and fibroblasts after stimulation with microbial products or cytokines, primarily attracts monocytes and t cells.49 the inflammatory response against denv is believed to play an important role in its pathogenesis. the different manifestations between mild and severe dengue pat ient s indicate that inflammatory response may differ substantially. many studies have demonstrated that levels of inflammation mediators such as tnf-α, ifn-γ, ip-10, il-8 are elevated in dengue patients and higher levels in severe cases.50 rothman and colleagues reviewed how innate and adaptive immune responses contribute to promoting severe dhf manifestations, also reviewing specific cytokines and chemokines, tnf-α, vascular endothelial growth factors (vegf-a), il-6, il-10, il-8, il-8, ccl2 and cxcl10, and how they promote the production of clinical presentation dhf cellular and molecular contributor cytokine storm but will likely on copyright © 2020, ijtid, issn 2085-1103 38 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 30–43 targeting single soluble mediators and focus over common in the inflammatory cascade.51 peaks of pro-inflammatory cytokines and peaks of anti-inflammatory cytokines coexist. mcp-1 is a potent monocytes chemoattractant and has been reported that increased levels correlated with severe dengue symptoms. the results in this study also proved that inflammation in severe dengue patients resolved differently from mild cases.50 oliveira et al showed expression profiles of 36 cytokines and chemokines in 44 acute serum samples acute-phase df patients (n=25), dhf (n=19) and healthy controls (n=6). all 36 proteins were expressed at a higher level in patients infected with denv compared to healthy controls.52 huang et al 2018 in his study that the ccl2 cx cl9, ip-10, cxcl11, il-8, and il 10 serum levels were significantly higher in the group of patients infected denv during the first two weeks compared to the control group.29 the majority of denv-infected patients make a full recovery after the febrile period and do not enter the critical phase of the disease. however, patients that do enter the critical phase may develop warning signs that indicate increased capillary permeabilit y leading to plasma leakage. generally, patients worsen at the time of defervescence (from illness day 4th) when their temperature drops to 37.5°c–38°c, and it is during this period that early symptoms of vascular leakage may be seen.34 the initial prediction of severe dengue in patients without warning signs who can later develop severe dhf is very important to choose the right intensive supportive therapy. severe responses to dengue include activation and apoptosis of t-cells and b-cells, cytokine storm, hematological disorders, and complement activation. cytokines, complement and other unknown factors can temporarily work on the endothelium and change the normal fluid barrier function of endothelial cells and cause plasma leakage. the elevated levels of cytokine in severe dengue make them good predictors of the severity of dengue fever. cytokine estimation at presentation can provide us a clue whether a patient is likely to develop severe manifestations of dengue or not.46 differences in levels of cytokines and chemokines were found when sera/plasma samples from dengue hemorrhagic fever (dhf) and dengue fever (df) patients were compared.53,54 there has been limited evidence of endothelial injury measured as apoptosis or structural changes. these findings together with the transient nature of plasma leakage and rapid recovery suggest that transient perturbation of vascular barrier integrity is the main mechanism underlying plasma leakage in dhf and that the activation of endothelial cells and the coagulation system is likely mediated by cytokines produced by the innate and adaptive immune system. in vitro studies demonstrated the production of antiviral and proinflammatory cytokines by these cells when exposed to denv. these cytokines include type i ifns and chemotactic factors such as migration inhibition factor (mif), monocyte chemotactic factor (mcp), and il-8. infection of dendritic cells by denv also induces the production of mmp-2 and mmp-9 which may facilitate the migration of dendritic cells to the local lymph nodes where virus further replicates and subsequently enters the circulation. most studies to date have described the production of th1 cytokines and minimal pro duct io n of th2 cytokines by denv specific t-cells. il-17 and il-21 secretion by denv specific tcells is just beginning to be described and therefore the roles of these cytokines in dengue pathogenesis are currently unknown.45 malagive et al 2018 have found that il-10, il-1β, monocyte chemoattractant protein (mcp)-1 and il-8 levels were associated with severe dengue and that monocytes were likely to be the predominant source of il-10. apart from interaction with monocytes, platelets are also known to contribute to vascular permeability due to the production of il-1β by platelet microparticles. other mediators that are known to cause vascular leak include bradykinins, complement proteins c3a and c5a, il-33, fibrin products, prostaglandins e2, f2a, and d2.15 hemorrhagic manifestation in dengue virus infection patients are not common and within mild to severe. skin hemorrhage, including petechiae and purpura, are the most common, copyright © 2020, ijtid, issn 2085-1103 indah agustinaningrum, et al.: cp-1 levels and atypical lymphocytes 39 along with gum bleeding, epistaxis, menorrhagia, and gastrointestinal bleeding.55 atypical lymphocytes the atypical lymphocyte is a non-malignant leukocyte seen in the peripheral blood. it is a reactive lymphocyte of lymphoid origin and produced in a variety of disorders. it appears to be a nonspecific response to stress from a variety of stimuli. a small lymphocyte becomes larger in size and capable of dividing.56 atypical lymphocytes or reactive lymphocytes on peripheral blood smear in dengue which the morphology of reactive lymphocytes often reported.57 in this study, it was found that subjects with dengue infection with ns-1 positive and with ns-1 negative percentage of atypical lymphocyte 2-6%. but there is no difference in patients who subsequently experience df, dhf or dss. cardinal and joseph alba showed in the philippines that are 155 confirmed cases of dengue fever, a total of 137 (88.4%) patients had atypical lymphocytes and 18 (11.6%) were found to be negative. the positive and negative predictive values of atypical lymphocytes were 86.2% and 86.9%, respectively. however, no differences were noted when the proportion of atypical lymphocytes was compared across all dengue severity. lymphocytes plasma blue or atypical lymphocytes are predictors were significantly dengue based on an analysis logistic regression showed that the risk of patients with atypical lymphocytes was 41.16 times higher for dengue than those who did not have atypical lymphocytes.58 in the 2007 jampangern study also found a significant increase in the absolute number of atypical lymphocytes on the incubation day and one day after incubation during acute dengue virus infection, especially in dhf patients. of the 49 dengue hemorrhagic fever (dhf), 25 dengue fever (df), and 26 dengue fever (dfs) cases. atypical 10% or higher lymphocyte count is a good indicator of dengue infection (50% sensitivity and 86% specificity).23 a disease is atypical as a special hematological finding in patients with dengue fever, and although it is not a classic specific finding of the disease, their concentration is significantly higher in these patients, especially in the form of the severe disease. there may be a relationship between the presence of atypical lymphocytes and dengue virus infection, but the intensity and usefulness of these findings require further study and analysis.59 patients who have > 300 cells/μl absolute atypical lymphocytes can be used to predict the development of severe dengue because patients with severe dengue have a greater level of absolute atypical lymphocytes than patients with dengue fever who do not severe. this finding is similar to previous. after a secondary dengue infection, atypical lymphocytes could indicate an augmented immune response attempting to control the spread of dengue-infected cells. simultaneously, these antibodies could enhance the entry of the dengue virus into macrophages and dendritic cells whereupon the virus would replicate. previous reports have also indicated that patients with higher dengue viremia have higher disease severity.60 the presence of atypical lymphocytes is due to the t-cell activation should be considered as a useful screening parameter for dengue infection.61 conclusion in conclusion, this study analyzed mcp-1 levels and at ypical lymphocytes inpat ient dengue-infected which detecting use ns-1 rapid test. the correlation mcp-1 levels and atypical lymphocytes were found an increase in mcp-1 levels was followed by an increase of atypical lymphocytes. this shows the arrival of macrophages and monocytes to the site of inflammation which triggers proliferation rather than lymphocytes. until now, biomarkers cannot act as predictors of cytokine storm in dengue infection patients. from the results of this study, there were no significant different parameters between df, dhf, and dss. copyright © 2020, ijtid, issn 2085-1103 40 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 30–43 acknowledgements the author would like to thank, dr darsono hospital, pacitan; prof. dr. jusak nugraha and dr. hartono kahar for guidance in this study, farid alfaraby for mcp-1 same technician, atlm dr darsono laboratory for pool sample activity in this study and my family for everything. conflict of interest the authors declare that there is no conflict of interest for this research. references 1. tuiskunen bäck a, lundkvist å. dengue viruses – an overview. infect ecol epidemiol. 2013;3(1):19839. doi:10.3402/iee.v3i0.19839 2. rothman al. dengue virus. london new york: springer heidelberg dordrecht london new york; 2010. doi:10.1007/978-3-642-02215-9 3. diamond ms, pierson tc. molecular insight into dengue virus pathogenesis and its implications for disease control. cell. 2015;162(3):488-492. doi:10.1016/j.cell.2015.07.005 4. who. comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. revised and expanded edition.; 2011. doi:10.1017/ cbo9781107415324.004 5. goura kudesia, wreghitt t. clinical and diagnostic virology. united states of america by cambridge university press, new york states of america by cambridge university press, new york: cambridge university press; 2009. 6. ebrahimi m, abadi a, bashizadeh -fa khar h, fahimi4 e. dengue fever in iran. a case report. acta medica mediterr. 2016;32(specialissue5):2025-2027. doi:10.17795/zjrms-9953.case 7. dinas kesehatan propinsi jawa timur. profil kesehatan propinsi jawa timur 2017. nucleic acids res. 2017;34(11):e77-e77. 8. dinas kesehatan kabupaten pacitan. profil kesehatan kabupaten pacitan tahun 2016. profil kesehatan tahun 2016. 2016;(031). depkespacitan. 9. melody s. goodman. biostatistics for clinical and public health research. first published 2018 by routledge 711 third avenue, new york, ny 10017; 2018. 10. gelanew t, hunsperger e. devel opm ent and characterization of serotype-specific monoclonal antibodies against the dengue virus-4 (denv-4) non structural protein (ns1). virol j. 2018;15(1):1-12. doi:10.1186/s12985-018-0925-7 11. khetarpal n, khanna i. dengue fever: causes, complications, and vaccine strategies. j immunol res. 2016;2016(3):1-14. doi:10.1155/2016/6803098 12. lardo s, soesatyo mhne, juffrie, umniyati sr. the worsening factors of dengue hemorrhagic fever (dhf) based on cohort study with nested case-control in a tertiary hospital. iop conf ser earth environ sci. 2018;125(1). doi:10.1088/1755-1315/125/1/012011 13. whitehorn j. the pathogenesis and clinical management of dengue. london sch hyg trop med. 2015:207. doi:10.17037/pubs.02373944 14. richman d. clinical virology. third edit. (edition f, ed.). washington, dc 20036-2904, usa; 2017. 15. malavige gn, wijewickrama a, fernando s, et al. a preliminary study on efficacy of rupatadine for the treatment of acute dengue infection. sci reports | 83857 | doi101038/s41598-018-22285-x. 2018;8(1):1-14. doi:10.1038/s41598-018-22285-x 16. deshmane sl, kremlev s, amini s, sawaya be. monocyte chemoattractant protein-1 (mcp-1): an overview. j interf cytokine res. 2009;29(6):313-326. doi:10.1089/jir.2008.0027 17. ching cl. the regulations of cytokines and chemokines in dengue virus-infected patients.(2011). 18. tan ty, chu jjh. dengue virus-infected human monocytes trigger late activation of caspase-1, which mediates pro-inflammatory il-1β secretion and pyroptosis. j gen virol. 2013;94(part10):2215-2220. doi:10.1099/vir.0.055277-0 19. ahmad r, al-roub a, kochumon s, et al. the synergy between palmitate and tnf-α for ccl2 production is dependent on the trif/irf3 pathway: implications for metabolic inflammation. j immunol. 2018:ji1701552. doi:10.4049/jimmunol.1701552 20. malavige gn, ogg gs. pathogenesis of vascular leak in dengue virus in fecti on. immunology. 2017;151(3):261-269. doi:10.1111/imm.12748 21. gao x, wen y, wang j, et al. delayed and highly specific antibody response to nonstructural protein 1 (ns1) revealed during natural human zikv infection by ns1-based capture elisa. bmc infect dis. 2018;18(1):1-7. doi:10.1186/s12879-018-3173-y 22. dembic z. the role of cytokines in disease related to immune response. mica haley copyright © 2015 elsevier inc. all rights reserved.; 2015. doi:10.1016/ b978-0-12-419998-9.01001-4 23. jampangern w, vongthoung k, jittmittraphap a, et al. characterization of atypical lymphocytes and immunophenotypes of l ymphocyt es in patients with dengue virus infection. asian pacific j allergy immunol. 2007;25(1):27-36. 24. departemen kesehatan republik indonesia 2008. pedoman praktik laboratorium kesehatan yang benar. 2008:34-52. copyright © 2020, ijtid, issn 2085-1103 indah agustinaningrum, et al.: cp-1 levels and atypical lymphocytes 41 25. kemenkes ri. cara penyelenggaraan laboratorium klinik yang baik. permenkes no 43 tahun 2013. 2013:44-67. 26. watson j. the laser guidebook second edition. vol 26.; 2002. doi:10.1016/0030-3992(94)90101-5 27. mm k, gp k, m r, et al. a study of clinical and laboratory profile of dengue fever in tertiary care hospital in central karnataka, india. glob j med res. 2014;14(5 version 1.0):7-12. 28. raj kumar patro a, mohanty s, prusty bk, et al. cytokine signature associated with disease severity in dengue. viruses. 2019;11(1):1-12. doi:10.3390/ v11010034 29. huang j, liang w, chen s, et al. serum cytokine profiles in patients with dengue fever at the acute infection phase. dis markers. 2018;2018: 1-8. doi:10.1155/2018/8403937 30. malavige gn, huang lc, salimi m, gomes l, jayaratne sd, ogg gs. cellular and cytokine correlates of severe dengue infection. plos one. 2012;7(11). doi:10.1371/ journal.pone.0050387 31. sprokholt jk, kaptein tm, van hamme jl, overmars rj, gringhuis si, geijtenbeek tbh. rig-i− like receptor triggering by dengue virus drives dendritic cell immune activation and th1 differentiation. j immunol. 2017;198(12):4764-4771. doi:10.4049/ jimmunol.1602121 32. megawati d, masyeni s, yohan b, et al. dengue in bali: clinical characteristics and genetic diversity of circulating dengue viruses. plos negl trop dis. 2017;11(5). doi:10.1371/journal.pntd.0005483 33. fukusumi m, arashiro t, arima y, et al. dengue sentinel traveler surveillance: monthly and yearly notification trends among japanese travelers, 2006-2014. plos negl trop dis. 2016;10(8):2006-2014. doi:10.1371/ journal.pntd.0004924 34. muller da, depelsenaire aci, young pr. clinical and laboratory diagnosis of dengue virus infection. j infect dis. 2017;215(suppl 2):s89-s95. doi:10.1093/infdis/ jiw649 35. chan hby, how ch, ng cwm. definitive tests for dengue fever: when and which should i use? singapore med j. 2017;58(11):632-635. doi:10.11622/ smedj.2017100 36. ambrose jh, sekaran sd, azizan a. dengue virus ns1 protein as a diagnostic marker: commercially available elisa and comparison to qrt-pcr and serological diagnostic assays currently used by the state of florida. j trop med. 2017;2017:1-6. doi:10.1155/2017/8072491 37. mat jusoh tna, shueb rh. performance evaluation of commercial dengue diagnostic tests for early detection of dengue in clinical samples. j trop med. 2017;2017:1-4. doi:10.1155/2017/4687182 38. huits r, soentjens p, maniewski-kelner u, et al. clinical utility of the nonstructural 1 antigen rapid diagnostic test in the management of dengue in returning travelers with fever. open forum infect dis. 2017;4(1):1-6. doi:10.1093/oid/ofw273 39. sa-ngamuang c, haddawy p, luvira v, piyaphanee w, iamsirithaworn s, lawpoolsri s. accuracy of dengue clinical diagnosis with and without ns1 antigen rapid test: comparison between human and bayesian network model decision. plos negl trop dis. 2018;12(6):1-14. doi:10.1371/journal.pntd.0006573 40. guzman mg, halstead sb, artsob h, et al. dengue: a continuing global threat. nat rev microbiol. 2010;8(12):s7-s16. doi:10.1038/nrmicro2460 41. hunsperger ea, muñoz-jordán j, beltran m, et al. performance of dengue diagnostic tests in a single-specimen diagnostic algorithm. j infect dis. 2016;214(6):836-844. doi:10.1093/infdis/jiw103 42. palomares-reyes c, silva-caso w, del valle lj, et al. dengue diagnosis in an endemic area of peru: clinical characteristics and positive frequencies by rt-pcr and serology for ns1, igm, and igg. int j infect dis. 2019;81:31-37. doi:10.1016/j.ijid.2019.01.022 43. teoh bt, sam ss, tan kk, et al. the use of ns1 rapid diagnostic test and qrt-pcr to complement igm elisa for improved dengue diagnosis from single specimen. sci rep. 2016;6(june):1-8. doi:10.1038/ srep27663 44. hottz ed, bozza fa, bozza pt. platelets in immune response to virus and immunopathology of viral infections. front med. 2018;5(may). doi:10.3389/ fmed.2018.00121 45. srikiatkhachorn a, mathew a, rothman al. immune mediated cytokine storm and its role in severe dengue. 2017;39(5):1-10. doi:10.1109/embc.2016.7590696. upper 46. sehrawat p, biswas a, kumar p, et al. mediterranean journal of hematology and infectious diseases role of cytokines as molecular marker of dengue severity. mediterr j hematol infect dis. 2018;10(101):2-6. doi:10.4084/mjhid.2018.023 47. yong yk, tan hy, jen sh, et al. aberrant monocyte responses predict and characterize dengue virus infection in individuals with severe disease. j transl med. 2017;15(1). doi:10.1186/s12967-017-1226-4 48. detrick b, schmitz jl, hamilton rg. manual of molecular and clinical laboratory immunology.; 2016. 49. güneş h, mete r, aydin m, et al. relationship among mif, mcp-1, viral loads, and hbs ag levels in chronic hepatitis b patients. turkish j med sci. 2015;45(3):634 637. doi:10.3906/sag-1401-171 50. zhao h, zhang fc, zhu q, et al. epidemiological and virological characterizations of the 2014 dengue outbreak in guangzhou, china. plos one. 2016;11(6):1 10. doi:10.1371/journal.pone.0156548 51. teijaro jr. cytokine storms in infectious diseases. semin immunopathol. 2017;39(5):501-503. doi:10.1007/ s00281-017-0640-2 copyright © 2020, ijtid, issn 2085-1103 42 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 30–43 52. oliveira afc da s, teixeira rr, oliveira as de, souza apm de, silva ml da, paula so de. potential antivirals: natural products targeting replication enzymes of dengue and chikungunya viruses. molecules. 2017;22(3):505. doi:10.3390/molecules22030505 53. hernández si de la c, nelson hnp-g, flores h, et al. primary dengue virus infections induce differential cytokine production in mexican patients. mem inst oswaldo cruz. 2016;111(3):161-167. doi:10.1590/0074 02760150359 54. soo k-m, khalid b, ching s-m, tham cl, basir r, chee h-y. meta-analysis of biomarkers for severe dengue infections. peerj. 2017;5:e3589. doi:10.7717/ peerj.3589 55. soegijanto s, sari dw, yamanaka a, kotaki t, kameoka m, konishi e. awareness of using ringer lactat solution in dengue virus infection cases could induce severity. indones j trop infect dis. 2017;4(4):35. doi:10.20473/ijtid.v4i4.231 56. simon mw. the atypical lymphocyte. int pediatr. 2003;18(1):20-22. 57. shetty a, kasukurti p, vijaya c, jayalakshmi vj. original article the reactive lymphocyte : a morphological indicator of platelet counts in dengue seropostive patients. 2016;(15). 58. avegail m cardinal, alba vj. national surveillance for influenza and influenza like illness in qatar, j a n ua r yâ € “d e c em ber 2015: an an a l ysi s of sentinel surveillance systems. j infect dis ther. 2017;05(03):4172. doi:10.4172/2332-0877-c1-027 59. rey-caro la, villar-centeno l angel. atypical lymphocytes in dengue: role in diagnosis and prognosis of disease. a systematic review of literature. rev ciencias la salud. 2012;10(3):323-335. 60. thanachartwet v, oer-areemitr n, chamnanchanunt s, et al. identification of clinical factors associated with severe dengue among thai adults: a prospective study. bmc infect dis. 2015;15(1):1-11. doi:10.1186/ s12879-015-1150-2 61. yunus ym. morphological features analysis in pathogenic dengue infection as an alternative screening method. int j acad res bus soc sci 2017, vol 7, no 2 issn 2222-6990. 2017;7(2):801-811. doi:10.6007/ijarbss/v7-i2/2716 introduction materials and methods ethics statement blood samples laboratory diagnosis statistical analysis results and discussion ns-1 test mcp-1 levels atypical lymphocyte hematology parameters discussion conclusion acknowledgements conflict of interest references ijtid vol 8 no 3 sept-dec 2020.indd vol. 8 no. 3 september–december 2020 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 research report correlation of nutritional status with hookworm and strongyloides stercoralis infection in children under five years in kokar public health center, alor regency, east nusa tenggara benaya y. onesiforus1, indra e. lalangpuling2, mahardika a.wijayanti.3, e. elsa herdiana murhandarwati4,*, 1 study program of medical laboratory technology of mangunwijaya catholic polytechnic , semarang, 50135, indonesia 2 health polytechnic ministry of health of republic indonesia of manado, manado 55281, indonesia 3,4 department of parasitology, faculty of medicine, universitas gadjah mada, yogyakarta 55281, indonesia received: 29th march 2019; revised: 6th april 2020; accepted: 6th october 2020 abstract malnutrition can reduce immune response particularly in cytokine (il-4, il-5, il-10) production and immune eff ector (eosinophil, ige, and mast cell), thus increasing the probability of intestinal nematode infection. through this study, intestinal nematode infections occurred among children under fi ve years, at diff erent nutrition status, in kokar public health center, alor regency, east nusa tenggara was captured. hookworm and strongyloides stercoralis were studied as both of them have devastating impacts compare to other helminthes compare to other helminths. this study is a crosssectional study with a quote sampling technique. as many as 238 children, aged 12-59 months living in kokar’s public health center area, alor regency were recruited in this study i.e. 7.7% severely underweight, 19.2% underweight, 70.5% normal and 2.6% overweight. data were collected in august october 2016. hookworm and s. stercoralis infection were determined from collected fecal samples of all subjects using either baermann test, koga agar plate (kap), or haradamori culture method. the prevalence of hookworm and s. stercoralis infection was 8.82%, and 0,42%. correlation between nutritional status and hookworm infection were analyzed by mann-whitney test with p value = 0.54 (p > 0.05). prevalence of hookworm and s. stercoralis among children under fi ve years in kokar were 8.82% and 0.42%. there was no signifi cant correlation between nutritional status with hookworm infection prevalence. keywords: hookworm infection prevalence, nutritional status, kokar, alor regency abstrak malnutrisi dapat menyebabkan penurunan pada sistem imun terutama pada produksi sitokin (il-4, il-5, il-10) dan kinerja sel efektor pada respon imun (eosinofi l, ige, dan sel mast). gangguan ini dapat berakibat pada meningkatnya risiko infeksi nematoda usus. selain malnutrisi, faktor perilaku tidak higienis juga dapat meningkatkan risiko infeksi nematode usus. infeksi nematoda usus pada balita dapat menyebabkan gangguan pertumbuhan dan perkembangan. nematoda usus yang menyerang anak-anak usia balita umumnya disebabkan oleh soil-transmitted helminth (sth), diantaranya adalah hookworm dan strongyloides stercoralis. studi ini bertujuan untuk mengetahui hubungan status gizi terhadap prevalensi infeksi pada balita di puskesmas kokar, kabupaten alor. rancangan penelitian bersifat cross-sectional, dengan teknik quote sampling. sampel 238 balita di puskesmas kokar, kabupaten alor. pengumpulan data pada bulan agustus oktober 2016. subjek dan orangtua yang memenuhi kriteria penelitian diwawancara menggunakan panduan kuisioner. sampel feses dikumpulkan dan diperiksa jenis infeksi menggunakan metode uji baermann, koga agar plate (kap), dan haradamori. prevalensi infeksi hookworm dan s. stercoralis pada balita di puskesmas kokar adalah 8,82% dan 0,42%. korelasi antara status gizi dan infeksi hookworm dianalisis menggunakan mann-whitney test didapatkan nilai p = 0,54 (> 0,05). perilaku tidak higienis tidak memiliki korelasi terhadap infeksi hookworm. prevalensi infeksi hookworm dan s. stercoralis pada balita di kokar adalah 8,82% dan 0,42%. tidak ada * corresponding author: benayayamin@gmail.com 138 indonesian journal of tropical and infectious disease, vol. 8 no. 3 september–december 2020: 137–143 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 hubungan bermakna antara status gizi balita dan prevalensi infeksi hookworm. tidak ada korelasi antara perilaku tidak higienis dengan prevalensi infeksi hookworm. kata kunci: prevalensi infeksi hookworm, status gizi balita, perilaku, kokar, alor how to cite: onesiforus, by. lalangpuling, ie.,wijayanti, ma., murhandarwati, eeh. correlation of nutritional status with hookworm and strongyloides stercoralis infection in children under five years in kokar public health center, alor regency, east nusa tenggara. indonesian journal of tropical and infectious disease, 8(3), 137–143. high prevalence of sth helminthiasis in children, such as: in jayapura (50%), central mollucas regency (99.4%), padang (51.3%), nangroe aceh darussalam (59.2%), east nusa tenggara (27.7%), and west kalimantan (26.2%)9,10,11. in 2013, the province of east nusa tenggara ranked second highest in malnourished children in indonesia with a percentage of 11.50%2. the numbers are escalating in the work area of kokar’s public health center, with a percentage of severeweight (6.00%), underweight (12.70%), normal weight (77.80%), and overweight (3.50%). such condition had put kokar to ranked third highest in percentage of severe-weight and underweight children after apui and kabir in alor regency. kokar selected as study area due to complete category of nutritional status and sufficient facilities related to this study (electricity). this study focused on finding correlation between nutritional status with hookworm and s. stercoralis infection. this study also try review about s. stercoralis prevalence due to lack of strongyloidiasis data in indonesia until present time which perhaps due to diffi culty in performing s. stercoralis detection method such as baermann and koga agar plate (kap) tests. results of this study might be useful for further action was taken by stakeholders and local government. materials and methods a cross-sectional study with nutritional status as an independent variable and hookworm and s. stercoralis infection as a dependent variable. the reachable population of this study were children under 5 years for in kokar totaling 631 people. through slovin formula12, total samples in this study determined as 245 children of 12-59 months old. samples were selected from population based introduction nutritional status is a health condition of children under five that is measured by age, body weight (bw) and body height (bh)1. nutritional status could be infl uenced by nutrition intake in food, parenting style, children’s health services, environmental health, economic factors, sociocultural factors, and parents' educational factors (knowledge). in 2012, the prevalence of severe-weight and underweight children in indonesia is at 19.60% spread across all levels of the community's economy2. malnutrition may lead to increasing risk of intestinal nematode infection. the relationship of malnutrition to intestinal nematode infection o c c u r s t h r o u g h t w o p a t h w a y s , n a m e l y : malnutrition causes an increased risk of infection and helminthiasis is caused malnutrition3,4. increased risk of infection is probably due to reduced production of cytokines (il-4, il-5, il10) and eff ectors cell performance on immune (eosinophils, ige, and mast cells)5-7. thus low level of il-4 is suggested to increase the probability of sth infection due to less immune system to prevent helminth infection. this study focused on hookworm and s. stercoralis infection as it may cause anemia and malabsorption syndrome due to haemorrhagic and desquamation of intestinal epithelium respectively8. briefl y, both infections may lead to a growth disturbance in children. approximately 1.4 billion of the world's population are estimated infected with soil tr a n s m i t t e d h e l m i n t h s ( s t h ) w i t h t h e highest number of cases occurring in developing countries. the prevalence of sth infection in indonesia is ranging low to high, with the highest prevalence occurs usually in remote or underdeveloped areas. study reports showed a 139benaya y. onesiforus, et al.: correlation of nutritional status with hookworm and strongyloides stercoralis copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 on inclusion and exclusion criteria as follows. inclusion criteria include: (1) children aged 12 to 59 months and (2) children did not consume the anthelmintic drugs for the last 4 months. exclusion criteria include: (1) feces contaminated by dirt, water, and urine, (2) feces were given for more than 24 hours after defecate, (3) children’s parent did not approve inform consent. nutritional status data were secondary data based on kartu menuju sehat/ children growth chart (kms/cgc) obtained from kokar health center. nutritional status data based on anthropometric measurements of body weight (kg) for age (month). subjects recruited in four categories nutritional status, in line with the proportion in the population, i.e. 7.7% severely underweight, 19.2% underweight, 70.5% normal and 2.6% overweight. data collection data collection was held in august october 2016 in the working area of kokar public health, alor barat laut district, east nusa tenggara province. children’s parent are given an explanation of how to collect and store stool samples e.g. stool sample collected must be fresh (no more than 24 hours) without any contamination from water, soil and urine, and the respondent was not given any anthelmintic treatment in the last four months. stool samples collected in stool containers. after accommodated, the sample was submitted the public health center staff and researcher will retrieve afterward. each stool sample were examined using 3 diagnostic methods: baermann test, koga agar plate (kap), and harada-mori culture methods to identify the presence or absence of infection. baermann method (bm) in this study, the baermann method composed of tea strainer, plastic funnel, gauze bandages, hose and hose clamp. each tea strainers placed on funnel mouth with gauze bandage on strainer. warm water used to check whether there is a bubble in the hose. part of fresh feces (5 gram), wrapped with gauze bandages, then placed on strainer. a 40w bulb lighted at the bottom of hose for 2-3 hours. the fi ltrate poured as many as 15 ml, centrifuged at 2,500 rpm for at least 5 minutes. the supernatant discarded fast, the sediment examined under light microscope with a magnifi cation of 100 and 400 times13. koga agar plate (kap) kap method started with the making of agar medium consist of 15 gram of agar-agar gepulvert, merck, art.1615; 5 gram of bacto-liver, difco, control 763182; 10 gram of tryptone peptone, difco, 211705, and 5 gram solid nacl merck, 1.06404.1000. all the ingredients were dissolved with 1 liter of hot aquades. agar poured into petri dish as much as 10 ml. a 5 gram of feces were placed in the center of agar, and given an identifi cation code number. the covered dishes incubated at room temperature for 48 hours. after 48 hours, agar medium cleaned with 10 ml of sodium acetate-acetic-formalin (saf). the saf solution retrieved then, poured into a centrifuge tube, and centrifuged at 2,500 rpm for 5 minutes. the supernatant discarded fast, the sediment examined under a light microscope with a magnifi cation of 100 and 400 times13, 14. harada-mori culture method (hm) feces (0.5-1 gram) smeared in the center of the fi lter paper. smeared fi lter paper entered in a plastic bag fi lled with ±5 ml water, with a record the smeared section that is not submerged in the water. plastic bag attached with paper clips and hung up for 7 days at room temperature, avoid exposure to direct sunlight. water retrieved from the plastic bags by way of cut and placed in a centrifuge tube. the volume of water added up to 15 ml before centrifuged at a speed of 2,500 rpm for 5 minutes. the supernatant was discarded fast, the sediment examined under a light microscope with a magnifi cation of 100 and 400 times15. data analysis results were analyzed statistically and descriptively. descriptive analysis was meant to determine the frequency distribution of measurement results independent variables and 140 indonesian journal of tropical and infectious disease, vol. 8 no. 3 september–december 2020: 137–143 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 the dependent variable. correlation between hookworm and s. stercoralis infection with nutritional status were analyzed by mannwhitney’s test. ethical considerations the study was approved by the ethics committee for medical and health research, medical faculty, gadjah mada university with reference number ref: ke/fk/892/ec/2016. result and discussion one of the criteria for children to participate in this study did not anthelmintic drugs for the last 4 months due to sth reinfection processes. reinfection time varies on the helminth species, a. lumbricoides takes 2 months, t. trichiura takes 3 months, and hookworm takes 35 days to reach sexually mature inside the human body8,16. another condition is that the feces should not be contaminated by dirt, water, and urine. contamination by dirt may lead to false positive due to contamination by hookworm rhabditiform larva live in soil. condition of sample should not be contaminated by water and urine due to fecal sample in this study were used together with lalangpuling17 which used kato-katz method. contamination by water and urine can lead feces become inconvenient to be mold in kato-katz due to feces low consistency. table 1 shows that from total of 238 children under 5 years fecal samples, 21 were found positive in hookworm infection and only 1 subject found positive in s. stercoralis infection. table 1 also shows that the kap method has a better sensitivity with success rate (effi cacy) 68.18% of detect hookworm infection compares to harada mori method with success rate of 50%. this result agrees with reiss et al18 which found that kap were superior to harada mori methods in hookworm larvae detection. baermann test were the only method to reach its goal to detect s. stercoralis infection, with only on 1 subject. examination in multiple diagnostic methods needed to reach a true prevalence due to absence of a gold standard for s. stercoralis detection14. filariform larvae which found in harada mori method identifi ed as necator americanus from its appearance of gap between esophagus and intestinum31 (see figure 1). table 1. identifi cation of fecal samples from children under 5 years in kokar public health center total infection (n =22) strongyloides stercoralis n(%) hookworm n(%) kap 15 (68.18%) harada mori 11 (50 %) baermann test 1 (4.54%) kap + harada mori 21 (95.45%) figure 2. rhabditiform larvae tracks in koga agar plate figure 1. necator americanus fi lariform larvae, 400 x magnifi cation 141benaya y. onesiforus, et al.: correlation of nutritional status with hookworm and strongyloides stercoralis copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 a study of hookworm claims that kap is better than harada-mori in hookworm detection due to diversity in stool weight requirement in both methods, which also found in this study that harada-mori requires a lesser than 5 gram of faeces18. other study held in the same period and with the same subjects reveals that there were only a light infection of hookworm (1-1999 epg) in children under 5 years in kokar17. a low concentration in a few stools can lead to the decrease in the probability of hookworm eggs to be carried in fi lter paper of harada-mori culture method. one of kap method’s results is a larva track in agar surface (see figure 2), though not all positive kap were found with larva tracks on the agar (21). this occurrence also happen in this study the tracks might be made by rhabditiform larvae and become obvious due to growing of bacterial colonies that grow along path19,20. in kap with larva tracks, it is advised to use a dissecting microscope to search for larvae directly. compare to another study of hookworm and s. stercoralis in other regions of indonesia and from other countries, the prevalence of hookworm and s. stercoralis in this study are considered low. many studies of hookworm infection in many regions in indonesia found a higher prevalence like in jayapura (14.3%) and in maluku tengah regency (56.8%)9,10. prevalence of s. stercoralis are found higher in bali (1.6%)21. this study found that the prevalence of hookworm and s. stercoralis infection obtained in this study is at 8.82% and 0.42% respectively (see table 2). hookworm infection occurs in almost all categories of respondents, except in overweight children. the highest score was in children under fi ve years with normal nutritional status (7.14%). strongyloides stercoralis infection only found in 1 child from overall 238 children. the low prevalence of hookworm might be due to mda (mass drug administration) with dec (diethylcarbamizine) and albendazole as an anthelmintic drug for a brugian fi lariasis and sth infections. the program was held in alor regency from 2002 to 2007, with a target of alor citizens from 3-50 years old. in post evaluation, 3 years after the program was held, hookworm (n. americanus) prevalence showed a 75% decrease, from 28% become 7%12. a study by pion et al.22 and supali et al.23 found that fi lariasis treatment for 12 months with ivermectin and albendazole could diminish sth infection to 91%. this low prevalence of infection could lead to low risk of transmission from parents to their children. children under 5 years with normal status have the highest score, both in percentage and quantity of hookworm infection (see table 3). the relationship between nutritional status and hookworm infection were analyzed using mann whitney test with p-value = 0.54, which shows that nutritional status has no correlation with table 2. distribution of hookworm and s. stercoralis cases in children under 5 years old based on age and nutritional status in kokar public health center, alor regency characteristic of respondent hookworm n(%) s. stercoralis n(%) n age (month) 12-23 months 4 (1.68) 0 84 24-59 months 17 (7.14) 1 (0.42) 154 nutritional status severe-weight 1 (0.42) 0 18 underweight 3 (1.26) 0 46 normal weight 17 (7.14) 1 (0.42) 168 overweight 0 0 6 total 21 (8.82) 1 (0.42) 22 (9.24) table 3. correlation between variable of nutritional status and hookworm infection in children under 5 years in kokar public health center, alor regency, august-october 2016 nutritional status, n (%) hookworm infection (n=238) p negative (n=217) positive (n=21) total (n=238) severe weight 17 (7.14) 1 (0.42) 18 (7.56) 0.54 underweight 43 (18.07) 3 (1.26) 46 (19.33) normal 151 (63.44) 17 (7.14) 168 (70.58) overweight 6 (2.52) 0 (0) 6 (2.52) 142 indonesian journal of tropical and infectious disease, vol. 8 no. 3 september–december 2020: 137–143 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 hookworm infection. this study was in line with another study of elementary school children in purus, padang11. the absence of correlation might be due to the adequacy of protein requirements. this condition leads to body capable in normal production of il-4, which is the main cytokine in ige production. ige antibody is an adaptive immunity response against helminth infection5,24. other reason might be due to other confounding variables such as children’s behavior. a study by alemu et al.25 shows that hookworm infection has an association with children’s habit of shoe wearing. outdoor activities are the main cause of hookworm infection due to the practice of open defecation in society14,26-28. therefore playing with soil and walking barefooted in outdoor activities may lead children to increase the risk probability of fi lariform infection. in addition to behavior, social-economic status may also have an eff ect on sth infection. in kokar most of children’s parents are farmer (71,4%) with low monthly household income (95,3%)17. some studies reveal that socialeconomic status such as family income were signifi cant risk factors for sth infection23,29. our results do not display a correlation between nutritional status and s. stercoralis infection due to quantity of subjects with positive infection were too few, therefore cannot be used as a reference for data processing. conclusion our result found that the prevalence of hookworm and s. stercoralis among children under 5 years old in centre of public health in kokar, alor regency, is 8.82% and 0.42% respectively. there was no correlation between nutritional status and hookworm infection among children under 5 years. this study recommended collaborating kap and harada-mori to detect the presence of hookworm infections as it is able to increase the detection level up to 95.45%. conflict of interest there is no confl ict of interest of this study. acknowledgement the authors are grateful for cooperation of head and all staff of centre of public health in kokar, and to all local authorities that facilitated this study. references 1. ministry of health of republic of indonesia. standar anthropometri penilaian status gizi anak. jakarta : direktorat jendral bina gizi dan kesehatan ibu dan anak; 2011. 2. riset kesehatan dasar (riskesdas) 2013. jakarta: health ministry of republic of indonesia; 2013. 3. scrimshaw np and san giovanni jp. synergism of nutrition, infection, and immunity: a overview. am j clin nutr. 1997; 66(2):464s-77s 4. gutierrez-jimenez j, torres-sanchez mgc, fajardomartinez lp, schlie-guzman ma, luna-cazares lm, gonzalez-esquinca ar, et al. original article malnutrition and the presence of intestinal parasites in children from the poorest municipalities of mexico. journal infect dev ctries. 2013; 7(10): 741–747 5. ing r, su z, scott me, koski kg. suppressed t helper 2 immunity and prolonged survival of a nematode parasite in protein-malnourished mice. pnas. 2000; 97(13):7078-7083 6. schaible ue and kaufmann she. malnutrition and infection: complex mechanisms and global impact. plos med. 2007; 4(5):e115. 7. franca tgd, ishikawa llw, zorzella-pezavento sfg, chiuso-minicucci f, da cunha mlrs, sartori a. 2009. impact of malnutrition on immunity and infection. j venom anim toxins incl trop dis. 2009; 15(3):374379 8. paniker ckj and ghosh s. paniker’s textbook of parasitology. 7th ed. new delhi : jaypee brothers medical publishers (p) ltd; 2013. 9. martila, sandy s, paembonan n. hubungan higiene perorangan dengan kejadian kecacingan pada murid sd negeri abe pantai jayapura. plasma. 2015; 1(pt 2) : 87-96. 10. ndona mv. 2015. hubungan pengetahuan, kondisi lingkungan, dan sosial ekonomi dengan infeksi soil transmitted helminth (sth) pada anak usia sekolah dasar di kecamatan salahutu dan leihitu di kabupaten maluku tengah, provinsi maluku [thesis]. yogyakarta : univ gadjah mada; 2015 143benaya y. onesiforus, et al.: correlation of nutritional status with hookworm and strongyloides stercoralis copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 11. renanti r, rusjdi sr, elmatris sy. hubungan infeksi soil transmitted helminth dengan status gizi pada murid sdn 29 purus padang. jurnal kesehatan andalas. 2015; 4(2). 12. notoatmodjo s. metodologi penelitian kesehatan (edisi revisi). jakarta: penerbit rineka cipta; 2005 13. knopp s, mgeni af, khamis is, steinmann p, stothard jr, rollinson, d, et al. diagnosis of soil-transmitted helminths in the era of preventive chemotherapy: eff ect of multiple stool sampling and use of diff erent diagnostic techniques. plos negl trop dis. 2008; 2(11):e331 14. khieu v, schar f, marti h, sayasone s, duong s, muth s, et al. diagnosis, treatment and risk factors of strongyloides stercoralis in schoolchildren in cambodia. plos negl trop dis. 2013; 7: e2035. 15. vaden sl, knoll js, smith jr fwk, tilley lp. blackwell’s five-minute veterinary consult : laboratry test and diagnostic procedures : canine and feline. lowa : wiley & sons publishing ltd; 2009. 16. chiodini p, moody ah, manser dw. atlas of medical helmintology and protozoologi. 4th edition. london : churchhill livingstone, 2001 17. lalangpuling ie. hubungan infeksi sth dengan status gizi dan anemia pada balita di puskesmas kokar, kabupaten alor; 2017 ma 634-649; manado : seminar nasional tahun, 2018 18. reiss d, harrison lm, bungiro r, cappello m. short report: an agar plate method for culturing hookworm larvae: analysis of growth kinetics ana infectivity compared with standard coproculture techniques. am. j. trop. med. hyg. 2007; 77(6):1087–1090 19. steinmann p, zhou xn, du zw, jiang jy, wang lb, wang xz, et al. occurrence of strongyloides stercoralis in yunnan province, china, and comparison of diagnostic methods. plos negl trop dis 2007; 1: e75 20. arakaki t, iwanaga m, kinjo f, saito a, asato r, ikeshiro t. effi cacy of agar-plate culture in detection of strongyloides stercoralis infection. j. parasitol 76 1990; (3):25-428 21. widjana dp and sutisna p. the prevalence of strongyloides stercoralis infection in rural population of bali: a preliminary study. med j indonesia. 2001; 10(3):174-177. 22. pion sds, chesnais cb, bopda j, louya f, fischer pu, majewski ac. the impact of two semiannual treatments with albendazole alone on lymphatic filariasis and soil-transmitted helminth infection : a community-based study in the republic of congo. am. j. trop. med. hyg. 2015; 92(5):959-966 23. supali t, djuardi y, bradley m, noordin r, ruckert p, fischer pu. impact of six rounds of mass drug administration on brugian filariasis and soiltransmitted helminth infections in eastern indonesia. plos negl trop dis. 2013; 7 (12): e2586 24. abbas ak, lichtman ah, pillai s. cellular and molecular immunology 7th ed. philadelphia: elsevier saunders, 2012 25. alemu a, atnafu a, addis z, shiferaw y, teklu t, mathewos b, et al. soil transmitted helminths and schistosoma mansoni infections among school children in zarima town, northwest ethiopia. bmc infectious diseases. 2011; 11:189 26. walana w, aidoo enk, tay sck., prevalence of hookworm infection: a retrospective study in kumasi. asian pac j trop biomed. 2014; 4(suppl 1):158-161. 27. walkers m, hall a, basanez, mg. individual predisposition, household clustering and risk factors for human infection with ascaris lumbricoides: new epidemiological insights. plos negl trop dis. 2011; 5(4): e1047. 28. forrer a, vounatsou p, sayasone s, vonghachac, y, bouakhasith d, utzinger, et al. risk profi ling of hookworm infection and intensity in southern lao people’s democratic republic using bayesian models. plos negl trop dis. 2015; 9(3):e0003486. 29. texeira jc and heller i. impact of water supply, domiciliary water reservoirs and sewage on faecoorally transmitted parasitic disease in children residing in poor area in juiz de for a, brazil. epidemiol. infect. 2006; 134:694-698 30. al-delaimy ak, al-mekhlafi hm, lim ya, nasr na., sady h, atroosh wa. developing and evaluating health education learning package (help) to control soil-transmitted helminth 31. stiles. necator americanus. in: chatterjee, k.d. parasitology : protozoology & helminthology in relation to clinical medicine 13th ed. new delhi : cbs publishers & distributors pvt. ltd, 2009 ijtid vol 7 no 6 may-august 2019.indd 144 vol. 7 no. 6 september-december 2019 incidence of dengue hemorrhagic fever (dhf) in semarang coastal area: epidemiology descriptive case and bionomic vector okti t.d retnaningrum1, martini martini2a, mursid raharjo3 1,2 department of epidemiology, postgraduate school, university of diponegoro, indonesia. 3 department of health environment, faculty of public health, university of diponegoro, indonesia. a corresponding author: martini@live.undip.ac.id abstract semarang utara sub-district is located on the coast of the java sea. the coastal area is characterized by high salt content on both the ground and the water compare to other areas. the high salt content environment should have limited the breeding of dengue hemorrhagic fever (dhf) vectors; yet, quite high incidents of dhf cases are reported taken place in semarang coastal area. the aim of this study was to describe the epidemiology of dhf incidence, characteristic of cases, and bionomics vector in the coastal area of semarang utara sub-district. this study was applied descriptive observational design to analyze samples consisting of 62 dengue cases and 184 houses. the research variables consisted of coordinate of dhf cases, water salinity, house index (hi), container index (ci), and aedes species. data were processed using spss in a bivariate manner; while, mapping was analyzed spatially using arcgis 10.3. a total of 184 houses were surveyed and 55 cases of dhf were identified. most cases occurred in 6 -16 year age group (47.3%), water salinity ranged from 2-3%, indicating that the water in the coastal area tended to be brackish water. the results of the pearson correlation test showed that there was no relationship between hi and incidence of dhf in semarang utara sub-district. aedes aegypti was identified in a positive container, otherwise aedes albopictus was not found. dhf cases mostly occurred in school age groups, and were distributed in all villages near or far from the beach. dhf vector could breed in areas with little brackish water, so that dengue transmission might occur in this area. keywords: dhf, aedes aegypti, aedes albopictus, bionomic vector, semarang beach abstrak kecamatan semarang utara terletak di pantai laut jawa. kondisi daerah pantai dicirikan dengan kandungan garam baik di tanah dan air menjadi lebih tinggi dibandingkan area lain. lingkungan dengan kadar garam yang tinggi dapat membatasi perkembangbiakan dari vektor demam berdarah dengue (dbd), namun laporan kasus dbd di wilayah pantai semarang selalu ada dengan insiden yang cukup tinggi. penelitian ini bertujuan untuk mendeskripsikan epidemiologi kejadian dbd di wilayah pesisir kecamatan semarang utara, karakteristik responden serta vektor bionomik. penelitian ini menggunakan desain deskriptif observasional. sampel penelitian sebanyak 62 kasus dbd dan 184 rumah sekitar kasus. variabel penelitian meliputi koordinat kasus dbd, salinitas air, house index (hi), container index (ci) dan spesies aedes. data diolah menggunakan spss secara bivariat, sedangkan pemetaan dianalisis spasial menggunakan arcgis 10.3. sebanyak 55 kasus dbd teridentifikasi dan 184 rumah telah disurvei. sebagian besar kasus dalam kelompok usia 6-16 tahun (47,3%). salinitas air berkisar 2-3 ‰, tingkat salinitas ini menunjukkan air di wilayah pantai cenderung dikategorikan air payau. hasil uji pearson correlation menunjukkan tidak ada hubungan antara hi dengan incidence rate (ir) dbd di kecamatan semarang utara. aedes aegypti teridentifikasi dalam kontainer yang positif sebaliknya tidak ditemukan aedes albopictus. kasus dbd sebagian besar terjadi pada kelompok usia sekolah, dan terdistribusi di semua kelurahan baik dekat atau jauh dari pantai. vektor dbd dapat berkembangbiak di wilayah yang airnya sedikit payau, sehingga penularan dbd dapat terjadi di wilayah ini. kata kunci: dbd, aedes aegypti, aedes albopictus, bionomik vector, pantai semarang research report 145martini, et al.: incidence of dengue hemorrhagic fever introduction dengue hemorrhagic fever (dhf) is a disease caused by dengue virus and can be transmitted through the bite of aedes aegypti or aedes albopictus mosquitoes.1 factors that play an important role in the transmission of dengue virus infection are humans, intermediary vectors, and environment.1 in addition, factors of population density, rainfall, humidity, wind speed, air temperature, and altitude can also affect the rapid spread of dengue transmission.2 dhf is a contagious disease that becomes a health problem in the city of semarang. based on the health profile data of semarang city, the incidence of dengue fever in semarang city tends to be fluctuated with high numbers. the incidence of dhf in semarang city decreased to 18.14 per 100,000 population3,4,5,6,7 in 2016 and 2017 due to the changes in the operational definition of dengue cases as of october 1, 2016. currently, dhf case is defined as a case with dengue fever (df) symptoms followed by an increase in hematocrit ≥ 20% without taking into account the results of serological examination.7 however, these conditions do not eliminate the risk of dengue disease occurrence in the city of semarang because of the population of dhf vector, ae. aegypti, still not fully maintained and controlled. ae. aegypti mosquito is the main vector of dengue disease. theoretically, ae. aegypti mosquitoes reproduce in clear water that does not touch soil. however, the results of recent studies suggest that the ae. aegypti larvae are able to survive in the clear water from precipitated water in the ditch.8 in addition, the growth of ae. aegypti also depends on the chemical conditions of the environment. ae. aegypti mosquitoes can survive in containers containing water with normal ph ranging from 5.8 to 8.6 and water with salinity concentration of 0-0.7%. the most recent research conducted in brazil was showed that ae. aegypti is able to adapt to certain salinity conditions in littoral, coastal, and highland areas.9 meanwhile, data generated from experimental studies in semarang city was showed that ae. aegypti can develop both in various water ph conditions from ph 4 to ph 10 and in water salinity ranging from 0% to 6%.8 north semarang sub-district, one of the areas in semarang city lying on the coast of the java sea, has high salt content in both soil and water compare to other regions. this condition should have made semarang utara sub-district free from dhf endemic because high na cl concentrations resulted in an imbalance between larval body fluid and medium brood fluid. the difference in osmosis pressure causes the mortality of larvae on instar ii.8 however, data from the semarang city health office showed that some villages in the semarang utara sub-district have been categorized as dhf endemic areas. north semarang sub-district borders with the java sea in the north, with semarang tengah sub-district in the south, with semarang timur sub-district in the east, and with semarang barat sub-district in the west. semarang utara sub-district consists of 9 villages, 89 sub villages, and 708 neighborhoods. within 10.9 km2, in 2014, the population density was 11,272 per million, and the number of household was 32,000 each of which had 4 family members, even one house might dwell more than one households; one of the most populated are in semarang. given this situation, research needs to be done to describe epidemiological conditions of the incidence of dhf from the perspective of the characteristics of the people, of the place, and of the time. the characteristic of the people was described by age, occupation, and history of the patient’s activity before being diagnosed to be exposed to dhf. meanwhile, as environmental factors are important to detect the presence of dhf vectors, vector density was observed. in addition, the characteristic of the time studied was related to climate at the time the dhf occurred in patients. therefore, the aim of this study was to describe the epidemiology of the incidence of dhf in the coastal area of semarang, based on characteristic and behavior of cases, also bionomic of the vector. method and material this study took place in the coastal area in semarang utara sub-district of central java province and was conducted in 2017. the population was those who exposed to dhf and dengue shock syndrome (dss) as many as 62 patients. total sampling method was used to describe the semarang utara sub-district. as this study described the epidemiological conditions of the incidence of dengue in the semarang utara sub-district, larvae survey was used to determine the density of vectors in a particular region. the number of houses surveyed was calculated using purposive sampling method. the theory used to determine the number of houses was within the mosquito fly distance is ± 100 meters; therefore, it was estimated that mosquitoes could transmit the dengue virus at a radius of 100 meters around the sufferer. as a result, surveyed larvae was carried out on 4 houses around the patient’s home, either from the north, east, west, or south and the number of houses to be surveyed was 248 houses. the cases of dhf was diagnosed by medical team in hospital and supported by clinical laboratory test. research variables measured in this study were age, occupation, illness history, behavior to eradicating mosquitoes nest (emn), type of mosquito breeding place, water source, container location, water salinity, distance from the shoreline, water salinity was measured by refracto meter equipment. house index (hi), as well as container index (ci). hi is a percentage of houses identified positive larvae per total of examined houses. ci is a percentage of containers identified positive larvae per total of examined containers. bivariate analysis was carried out in testing hi; while, pearson/rank spearman correlation test was used to test the incidence of dhf, all of which were analyzed using arcgis version 10.3 software. the data analyzed was exhibited the 146 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 144–149 coordinates of dhf cases and hi as well as the urban ci where the cases were located. furthermore, the distance of the house of dengue cases to the north semarang coastline were also described. result and discussion the incidence of dhf in the coastal area of north semarang sub-district semarang city health office reported 62 cases to be diagnosed both dhf and dss in semarang utara subdistrict in 2017. in the field, 55 cases of dhf were found; while, 7 other cases were not found as the sufferers had been moved to another place. table 1. characteristics of dhf cases in north semarang sub-district in 2017 characteristics frequency (n= 55) percentage (%) 1. age (year) a. 1-4 b. 5-9 c. 10-14 d. 15-44 e. > 45 7 10 14 17 7 12.7 18.2 25.5 30.9 12.7 2. occupation a. jobless b. labor c. trader d. retired/housewife 40 1 9 5 72.7 1.8 9 9.1 3. education a. not yet school b. not finish elementary school c. elementary school d. secondary school e. high school/ vocational high school f. university 11 1 23 8 8 4 20 1.8 41.8 14.5 14.5 7.3 4. home distance from coastline a. ≤ 100 meter 5 9.1 a. > 100 meter 50 90.9 based on table 1. dhf cases in semarang utara sub-district were exposed to people aged from 2 years to 88 years, with the most age being 11 years (9.1%). the largest age group exposed (30.9%) is the age group of 15-44 years. as many as 72.7% of the people suffering from dengue cases in semarang utara sub-district have not worked, 41.8% complete elementary schools or are taking elementary education. case history of dhf was obtained through an in-depth interview to 55 respondents of the dhf patients or their family using open questions about activities carried out by dhf patients before being diagnosed with dhf. the result of the interview was showed that there were patients carrying out activities outside semarang utara sub-district before being diagnosed with dhf, whether they were traveling outside the district or out of town more than two days. in addition, there were patients who mostly spend their daily activities outside the semarang utara sub-district due to work or school. the fact also was showed that there were school-age who previously did not exposed to; yet, after being contacted with their friends who suffered from dhf, they started to be infected. from the distance of the house to coastline, 5 sufferers’ houses were standing right to the coastline. in general, the distance of the house to the coastline ranging from 0 meter to 2,844 meters with 1,311.42 meters in average. behavior to eradicating mosquito nests (psn) in north semarang sub-district to find out the behavior to eradicating mosquito nests (psn), interviews were conducted with 55 respondents whose families were dhf sufferers. table 2. behavior of eradicating mosquito nests (psn) behavior yes percentage (%) no percentage (%) covering water container inside the house 9 16.4 46 83.6 covering water container outside the house 49 89.1 6 10.9 routinely draining water container 41 74.5 14 25.5 brushing water container 25 45.5 30 54.5 disposing of used goods 47 85.5 8 14.5 recycling of used goods 5 9.1 50 90.9 using insect repellent 47 85.5 8 14.5 using bed nets 29 52.7 26 47.3 using abate powder 8 14.5 47 85.5 maintaining fish larvae eaters 8 14.5 47 85.5 window/ventilation 45 81.8 10 18.2 enough lighting 31 56.4 24 43.6 hanging clothes 23 41.8 32 58.2 another family hung clothes 23 41.8 32 58.2 based on table 2, the results of the interview showed that 90.9% of respondents did not recycle used goods, and 85.5% did not use abate powder. these habits might increase the risk of dengue vector mosquitoes to explode. , 147martini, et al.: incidence of dengue hemorrhagic fever most of the water container was made from plastic (41.8% respondents), and 61.8% respondents used nontap water; instead, they are used water from deep well water, dug well water, and supplied water from tanah mas housing complex. as much as 50.9% of the water samples taken contained 2 % salt which was categorized as brackish water (the category of water according to the salt content in a row, namely water < 0.5 -; 0.5-30 %; and > 30 % is fresh water, brackish water, and salt water).10 table 3. characteristics and conditions of water container characteristic frequency percentage (%) 1. container material (n=55) · plastic · ceramic · cement 23 20 12 41.8 36.4 21.8 2. water source (n=55) · non tap water · tap water 34 21 61.8 38.2 3. salinity (n=5) · 0 ‰ · 1 ‰ · 2 ‰ · 3 ‰ 11 28 13 3 20.0 50.9 23.6 5.5 4. house index (n=184) · low (hi < 10%) · high (hi ≥ 10%) 6 villages 3 villages 5. container index · low (ci < 5%) · high (ci ≥ 5%) 6 villages 3 villages based on table 3, to determine the density of dengue mosquito vector in each village in north semarang sub-district, 184 houses scattered in all villages were surveyed. the results of hi and ci were grouped into high and low categories according to who provisions.11,12 there were 3 villages with low hi and ci namely bulu lor, plombokan, and purwosari villages; while, the other 6 villages had high hi and ci, namely tanjungmas, dadapsari, kuningan, bandarharjo, panggung kidul, and panggung lor. figure 1 shows villages with the value of the container index (ci) in each village in the sub-district of north semarang. there are 3 villages with ci in the low category (ci < 5%), namely bulu lor, plombokan, and purwosari; while, the other 6 villages have high category of ci values (≥ 5% ci), namely tanjungmas, dadapsari, kuningan, bandarharjo, panggung kidul, and panggung lor. figure 1 shows villages with the value of the container index (ci) and hi in each village in the sub-district of semarang utara. there are 3 villages with ci in the low category (ci < 5%), namely bulu lor, plombokan, and purwosari; while, the other 6 villages have high category of ci values (≥ 5% ci), namely tanjungmas, dadapsari, kuningan, bandarharjo, panggung kidul, and panggung lor. discussion there were five homes of dhf sufferers in north semarang sub-district standing right on the coastline. of the five houses, two houses were identified to be positive for having mosquito larvae. the closer distance between the house and the beach allows the mixing of ground water with seawater to make the region's water source brackish.14 if the salt content of a water source is high, mosquito larvae will not be able to develop.11,15 however, the fact showed that mosquito larvae were found in that region. after analyzing the salt content using refractometer, it was found that the salinity ranged from 2-3 ‰. water is categorized as brackish water if it exceeds 0.5‰, so it can be concluded that the ae. aegypti mosquito could live in brackish water.11,15 other research finding reported that there was a significant change in ion transportation by anal papillae of the mosquito larvae living in the salt water.16,17 the morphological and physiological changing showed that aedes aegypti mosquito had been able to adapt to the case distribution of dhf with larvae density figure 1. hi, ci, and dengue cases in north semarang subdistrict 148 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 144–149 changing environment of the breeding place, especially the one having high content of salt. through in-depth interviews, it was identified that the water source used by residents in tanjungmas area came from 1-2 main sources channeled by pipes to homes. the deep well water source was used by most of the residents. water from the deep well should not have had salt or fresh water. however, in reality there had been a change in the quality of fresh groundwater to brackish in deep wells in the north semarang region. this phenomenon was related to seawater intrusion or ancient salt dissolution trapped in sediment when rock sedimentation took place.14 increased levels of seawater could increase the number of mosquitoes tolerant to salt content and allow mosquito vectors adaptation that were not tolerant to salt content to be tolerant to brackish water. this explained the increase in dengue cases in the coastal areas. the increased population living in the coastal area is predicted to be 134 people/ km2 by 2050.11,15,18 therefore, if the control of vectors in coastal areas was not implemented properly, there would be an increase in dengue cases in that particular areas. an area is considered to be at high risk for dengue transmission if the container index is ≥ 5% and the house index is ≥ 10%,19 based on which villages of tanjungmas, dadapsari, kuningan, bandarharjo, panggung kidul dan panggung lor were at high risk to dhf spreading. as shown in figure 1, there was a tendency for dhf cases to be in the villages with high hi and ci. however, the results of the correlation test showed that the hi value did not correlate to the incidence of dhf in north semarang sub-district. this proved that the hi value was not the main risk factor for the spread of dhf in the north semarang sub-district. this finding was in line with the research result conducted in sendangmulyo village of semarang city.20 the interviews with patients concluded that the patients were infected with dhf after traveling outside the sub-district. therefore, the spread of dhf in north semarang sub-district did not originally come from inside of the sub-district but also from outside of the sub-district area; therefore, the high and low hi values did not affect the incidence of dhf. conclusion dhf cases in the coastal areas of north semarang occur in all villages areas both in and off the coast. dhf cases mostly occur in the 6-16 years age group, namely school age. the incidence of dhf is not related to monthly rainfall. the distribution of dengue cases in the north semarang sub-district is not related to high population density, house index, and container index. aedes aegypti can live and breed in the coastal areas; even though, the water sources used show higher salt content or tend to be brackish. no aedes albopictus is found at the coastal location. survival aedes aegypti can affect the transmission of dhf in the coastal areas of north semarang. no exception in controlling dhf and its vector, coastal communities also need to carry out actively in psn activities in their area. conflict of interest there is no conflict of interest occurred in this study, both among researchers, and communities. this research has obtained permission from the kesbangpolinmas office, the health office, as well as the sub-district to rt units to carry out the research. respondents involved in the interview were provided informed consent to get their approval. acknowledgement this research could run well because of the help of various parties; therefore, we would like to extend our appreciation to undip graduate director for the approval of the research topic, semarang city health office which provided data on dhf sufferers, as well as respondents who participated in this study. references 1. gede, wempi. kontainer larva aedes sp di desa saung naga kabupaten oban komesing ulu sumatra selatan tahun 2012”, jurnal aspirator, 2013; 5(1):16-22 2. kementrian kesehatan ri. demam berdarah dengue, situasi 2011 dibanding 2010. jakarta: kementrian kesehatan ri, 2011. 3. dinas kesehatan kota semarang. profil kesehatan kota semarang tahun 2012. semarang: dinkes kota semarang, 2013 4. dinas kesehatan kota semarang. profil kesehatan kota semarang tahun 2013. semarang: dinkes kota semarang, 2014 5. dinas kesehatan kota semarang. profil kesehatan kota semarang tahun 2014. semarang: dinkes kota semarang, 2015 6. dinas kesehatan kota semarang. profil kesehatan kota semarang tahun 2015. semarang: dinkes kota semarang, 2016 7. dinas kesehatan kota semarang. data hews dbd tahun 2017. semarang: dinkes kota semarang, 2017 8. anggraini ts, cahyati wh. perkembangan aedes aegypti pada berbagai ph air dan salinitas air. higeia journal of public health research and development. 2017; 1(3): 1-10. 9. arduino mb, mucci lf, serpa lln. effect of salinity on the behaviour of aedes aegypti populations from coast and plateau of southheasternn brazil. journal vector borne disease.2015; 52: 79-87. 10. ramasamy r, surendran sn, jude pj, et. al. larval development of aedes aegypti and aedes albopictus in peri-urban brackish water and its implications for transmission of arboviral disease. plos neglected tropical disease.2011; 5(11). 11. who. comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. india: regional office if southe-east asia. 2011 12. who. dengue and severe dengue. 2014. available from : http:// www.who.int/mediacentre/factsheets/fs117/en/ 13. bakti h, naily w, lubis rf, et.al. penjejak keluaran air tanah lepas pantai (kalp) di pantai utara semarang dan sekitarnya dengan radon. riset geologi dan pertambangan. 2014; 24(1) : 43-51 14. jude pj, tharmasegaram t, sivasubramaniyam g, et.al. salinitytoerant larvae of mosquito vectors in the tropical coast of jaffna, sri 149martini, et al.: incidence of dengue hemorrhagic fever lanka and the effect of salinity on the toxicity of bacillus thuringiensis to aedes aegypti larvae. parasites & vectors 5, 2012: 269 15. clark t.m, bradley t.j. malpihian tubules of larval aedes aegypti are hormonally stimulated by 5-hydroxytryptamine in response to increased salinity. insect biochemistry and physiology. 1997; 34(2):123-141 16. donini a, gaidhu m.p, strasberg dr, et.al. changing salinity induces alterations in hemolymph ion concentration and na+ and cltransport kinetics of the anal papillae in the larval mosquito, aedes aegypti. the journal of experimental biology. 2007; 210: 983-992 17. united nation environment programme (2007) global programme of action for protection of the marine environment from land-based activities 2007: physical alteration and destruction of habitats. unep. nairobi, kenya. available: http://gpa.unep.org/content. html?id=199&ln = 6 18. joharina as, widiarti. kepadatan larva nyamuk sebagai indikator penularan demam berdarah dengue di daerah endemis di jawa timur. jurnal vektor penyakit. 2014; 8(2): 33-4. 19. saraswati ld, martini m. hubungan kepadatan jentik dengan penyakit dbd di kelurahan sendangmulyo kota semarang melalui pendekatan analisis spasial. jurnal kesmas indonesia. 2012; 5(1): 52-64. 20. saraswati ld, martini m. hubungan kepadatan jentik dengan penyakit dbd di kelurahan sendangmulyo kota semarang melalui pendekatan analisis spasial. jurnal kesmas indonesia 5(1), 2012: 52-64. ijtid vol 6 no 2 mei-agustus 2016_edit.indd 34 vol. 6. no. 2 mei–agustus 2016 case report kerion type of tinea capitis treated with double pulse dose terbinafine tinea capitis treated with double pulse dose terbinafine franky chandra1a, risa miliawati nh1, lies marlysa r1 1 dermato-mycology division, department of dermatology and venereology, faculty of medicine, universitas padjadjaran-dr. hasan sadikin general hospital, bandung, indonesia. a corresponding author: franky_chandra_87@yahoo.co.id abstract background: tinea capitis is a common dermatophyte infection affecting hair and skin which always requires systemic treatment to get a clinical and mycologic cure, preventing relapse, and infection spread. griseofulvin has been the antifungal therapy of choice for tinea capitis, but it often requires higher doses and a longer duration than recommended. thus, effective alternative antifungal with good oral tolerability and shorter course of treatment are therefore required. the objective of this report is to evaluate the effectiveness of double pulse dose terbinafine for tinea capitis alternative therapy. method: a case of kerion type of tinea capitis in a two-year-old girl was reported. diagnosis was established based on clinical manifestations of alopecia, presented as erythematous macule with pustules, hemorrhagic crusts, and scales on the scalp, accompanied with occipital lymphadenopathy. fungal culture showed growth of microsporum canis (m. canis) colonies. patient was treated with doubled pulse dose terbinafine 125 mg/day and 2% ketoconazole shampoo for two months. result: clinical improvements were found on 35th day of follow up, while mycologic cure was achieved on 60th day of follow up. tolerability was excellent and no side effects observed. conclusion: double pulse dose terbinafine is effective for kerion type of tinea capitis key words: double pulse dose, kerion, m. canis, terbinafine, tinea capitis abstrak latar belakang: tinea kapitis merupakan infeksi jamur pada folikel rambut dan kulit yang membutuhkan terapi sistemik untuk mencapai kesembuhan klinis dan mikologis, mencegah kekambuhan, dan penyebaran infeksi. griseofulvin merupakan terapi pilihan untuk tinea kapitis. namun, griseofulvin seringkali membutuhkan dosis lebih tinggi dan durasi pengobatan lebih lama dari yang direkomendasikan. oleh karena itu, terapi oral antijamur alternatif yang efektif dengan toleransi baik dan jangka pengobatan lebih pendek sangat diperlukan. laporan kasus ini bertujuan untuk mengevaluasi efektivitas terbinafin sebagai terapi alternatif untuk tinea kapitis. metode: dilaporkan satu kasus tinea kapitis tipe kerion pada anak perempuan berusia dua tahun. diagnosis ditegakkan berdasarkan gambaran klinis alopesia dengan permukaan kulit kepala berambut berupa makula eritema dengan pustula, krusta sanguinolenta, dan skuama, disertai limfadenopati oksipital. kultur jamur menunjukkan pertumbuhan koloni microsporum canis (m. canis). pasien mendapat terapi dengan terbinafin dosis denyut ganda 125 mg/hari dan sampo ketokonazol 2 % selama dua bulan. hasil: perbaikan klinis tampak pada hari ke-35, sedangkan kesembuhan mikologis didapatkan pada pengamatan hari ke-60. terbinafin dapat ditoleransi dengan baik tanpa ada efek samping yang terjadi. kesimpulan: terbinafin dosis denyut ganda efektif untuk tinea kapitis tipe kerion. kata kunci: dosis denyut ganda, kerion, m. canis, terbinafin, tinea kapitis 35chandra, et al.: kerion type of tinea capitis treated introduction tinea capitis is a common dermatophyte infection affecting hair and skin which frequently caused by trichophyton and microsporum species.1,2,3 human, animal, and fomite (i.e. object or article of clothing or dish that may be contaminated with infectious organism and serve in their transmission) contact spread are potential sources of infection.4,5 clinical appearance of tinea capitis may varied, including inflammatory and noninflammatory type.6,7,8 kerion is one of tinea capitis type2,5 which represents its inflammatory form.2,3 griseofulvin has been the gold standard for tinea capitis since the late 1950s.2 griseofulvin recommended duration for tinea capitis is 6-12 weeks2,5 or until the patient tests negative for fungi.2 the increased failure rate necessitating higher doses and longer treatment course required that will increases the risk of nonadherence3 has lead to consideration of newer antifungal agents.7 terbinafine is an allylamine antifungal agent 9,10,11 which has been approved by food and drugs association (fda) as tinea capitis alternative therapy in children aged two years 12 or older.8,12,13 side effects of terbinafine are uncommon2,13 and include gastrointestinal symptoms, rashes, and headache.2 terbinafine daily dose is 62.5 mg/day for children weighing less than 20 kg, 125 mg for weighing 20–40 kg, and 250 mg for those weighing more than 40 kg.9,10,14 the pulse therapy consisted of one week treatment duration followed by three week period without treatment.11 double dose administration in this case is twice standard dose that is given to children based on the body weight. this report will describe a case of kerion type of tinea capitis in a twoyear-old girl, weighs 16 kg, treated with doubled pulse dose terbinafine 125 mg/day for two months. case a two-year-old girl was taken by her parents to dermato-mycology division, department of dermatology and venereology, dr. hasan sadikin general hospital, bandung, indonesia with the chief complaint of alopecia on occipital area, presented as erythematous macule with pustules and pruritus. history of outside activities and frequent contact with soil were denied, but history of contact with cat which appeared to have skin problem was admitted. patient took bath twice a day using water from the well, liquid soap, and shampoo. patient also used personal towel, comb, and rarely used hat. previous similar history was denied. on physical examination, there was one cm in diameter of occipital lymphadenopathy, rubbery, and nontender on palpation. on the right occipital scalp area, there was an erythematous macule, 6x7cm, irregular-shape, clear border alopecia, with pustules, hemorrhagic crust, and scales on the skin surface. dermatological state figure 1. lesion on occipital area of the scalp. note the 6x7 cm solitary area with irregular-shape, clear border alopecia, with erythematous macule, pustules, hemorrhagic crust, and scales on the skin surface. figure 2. direct microscopic examination of hair and skin scraping from scalp lesion using koh 20 % + blueblack parker® ink solution revealed no hyphae nor spores on identification. were identified. figure 3. wood’s lamp examination showed no fluorescency direct microscopic examination of hair and skin scraping from scalp lesion using potassium hydroxide (koh) 20 % added with blue-black parker® ink solution revealed no hyphae nor spores. direct microscopic examination from pustule on the scalp lesion using gram 36 indonesian journal of tropical and infectious disease, vol. 6. no.2 mei–agustus 2016: 34−38 staining demonstrated epithelial cells, polymorphonuclear (pmn) cells, and gram positive cocci. wood’s lamp examination showed no fluorescence. fungal culture revealed microsporum canis (m. canis) growth. in this case, patient was treated with terbinafine 125 mg/day for one week-followed by three drug-free-weeks, topical ketoconazole 2% shampoo applied on scalp 3x/ week, cetirizine 1x1/2 tea spoon/day, and amoxycillin clavulanic acid 3 x ½ teaspoon. clinical improvements were seen as erythema on scalp and itching were decreasing after one month of therapy. treatment was well tolerated with patient has no experienced any side effect along the therapy regimen. normal hair growth over the alopecia area has begun and the culture gave negative result on day-60. discussion tinea capitis commonly affects children 9,15 aged less than 12 years old with a peak incidence at 3–7 years old.16 in this report, the patient is a two-year-old girl. on the basis of host preference and natural habitat, the fungal causes may be anthropophilic, zoophilic, or geophilic.2 the source for most tinea capitis infections in children and infants are human and animal.2,5 the patient in this case had a contact history with cat and no similar complaint in her family. thus, it can be speculated that this infection spread is zoophilic. kerion is an inflammatory type of tinea capitis 2,3 with painful mass 6 that can be accompanied by malaise,3 fever,2,3 and occipital lymphadenopathy.2 kerion lesion may take form as nodules2 with induration,3 pustular mass,2,3,8 and vesicles3 infected scalp may be inflammed with pustule eruptions,2 but secondary infection may also exist.12 kerion diagnosis in children is often delayed, especially when pustular symptoms are misdiagnosed as bacterial infection. on physical examination, bacterial folliculitis may mimic kerion with tender lesion of erythematous plaques associated with pus. however, carbuncle rarely causes alopecia,17 because the infection does not reach the hair bulb.18 delay in diagnosis and/or improper treatment may lead to complication and infection to other individuals.5 patient’s history taking and physical examination supported the diagnosis of kerion. the patient in this case had clear border alopecia, irregular shape, presented as erythematous macule, and itch, accompanied with pustules, hemorrhagic crust, and scales. wood’s lamp and microscopic examinations demonstrated negative results. somehow, fungal identification through culture is necessary to establish the diagnosis and etiology of tinea capitis.2,3,5 wood’s lamp examination may help diagnosing tinea capitis, but it has poor sensitivity.3 microscopic examination of inflammatory type tinea capitis may also give negative result.9,16 thus, fungal culture for identification of tinea capitis etiology should be done.3,19 if the clinical index of suspicion is high, therapy should be initiated after the culture specimen is obtained because it take time for confirming the culture results to establish the diagnosis.8 the result of patient’s fungal culture showed m. canis growth. based on the result, we can conclude the diagnosis and etiology of this patient is kerion type of tinea capitis which is caused by m. canis. tinea capitis requires systemic treatment because topical antifungal could not penetrate to the deepest part of the hair follicle 2,20 nor eradicate the infection. furthermore, the use of topical antifungal treatment alone may contribute to develop a carriers and cause transmission, since symptoms and clinical signs are minimal, but mycologic cure has not been achieved.20 adjunctive topical therapies have been shown to decrease the viable spores responsible for the figure 4. lesion on 35th day: note the improvement with decreased erythema and normal hair growth in almost all area of the scalp skin surface. patient felt no more itchy at these moment. figure 5. lesion on 60th day of follow up 37chandra, et al.: kerion type of tinea capitis treated disease contagiousness, reinfection, and may shorten the duration of therapy courses. ketoconazole shampoo should be applied three times weekly until the patient is clinically and mycologically cured. 21 some considerations in systemic therapy administration for tinea capitis are high efficacy level of treatment, low relapse rate, time and cost effectiveness, as well as safety. griseofulvin is considered to be the treatment of choice for tinea capitis10 for its effectiveness and safety to dermatophyte infection. somehow its main disadvantage is the long duration of treatment required which may lead to reduced compliance.2 griseofulvin therapy duration which is recommended for tinea capitis is 6-12 weeks or until clinical and mycologic cure are achieved.2 also, griseofulvin requires continuous administration because it has low affinity for keratin.22 terbinafine offer a shorter therapy duration and a less variable absorption compared to griseofulvin. terbinafine absorption is not altered when taken with food, so the administration is easier compared to other systemic antifungals, such as griseofulvin and itrakonazole, which should be given with food.15 terbinafine is also very lipophilic and keratinophilic, so it can be distributed to adipose, epidermis, dermis,2 hair,2,10 nail,2 and it can persist until one month after the treatment was stopped.23 terbinafine also has less side effect compared to griseofulvin5 and safe, including in pregnancy. moreover, terbinafine rarely interacts with other medication.24 since its availability in 1991, terbinafine has been approved for the management of tinea capitis in many countries including australia, new zealand, china, japan, holland, and india.15 terbinafine has fungicidal effect to dermatophytes since it inhibits squalene epoxidase2,24 that leads to a decrease in ergosterol which is an essential component of fungal cell membranes.15,24 panagiotidou et al.14 studied the efficacy and tolerability of terbinafine use for eight weeks in children with tinea capitis caused by m. canis. in that study, highest mycologic cure rate (97,1 %) was gained in dosage use of 7-12,5 mg/kg/day, followed by cure in 91,3% patients with dosage between 6-7 mg/kg/day, and 2,7 % in patients with dosage 3,3-6 mg/kg/day. koumantaki et al.25 experimented on terbinafine dosage for tinea capitis and stated that oral terbinafine should be given at a daily dose according to body weight: 125 mg for patients weight 10-25 kg and 250 mg/day for patients weight > 25 kg. terbinafine can be administered in pulsed and continued dose. an advantage of terbinafine pulse therapy for tinea capitis over the continuous regi men is that it allows the physician to individualize the treatment schedules so that just sufficient therapy is administered to gain a cure. the decision to give a second or third pulse of terbinafine was based on the clinical appearance of the lesion prior to the time-point at which the next pulse was due.11 patient in this case was treated with terbinafine 125 mg/day for one week and followed by 3 drug-free-weeks, ketoconazole 2% shampoo applied 3x/week, cetirizine 1x1/2 tea spoon/day to decrease itching, and amoxycillin clavulanic acid 3 x ½ teaspoon for the secondary bacterial infection. clinical improvement were seen as erythema and itching were decreasing after one month of therapy. more over, normal hair growth over the alopecia area has begun and the culture result came out negative on follow up day-60, so therapy can be discontinued. tinea capitis is not life-threatening, but kerion type of tinea capitis may cause scarring and permanent alopecia.2 treatment of the animal source of infection is the effort should be made in tinea capitis case.3 in conclusion, we feel that as regard griseofulvin to remain the antifungal drug of choice in tinea capitis, terbinafine may constitute an alternative drug which is well tolerated and safe. reference 1. schieke sm, garg a. superficial fungal infection. in: goldsmith la, katz si, gilchrest ba, paller as, wolff k, leffel da, editor. fitzpatrick’s dermatology in general medicine. 8th ed. new york:mcgraw-hill;2012. p. 2277-97. 2. bennassar a, grimalt r. management of tinea capitis in childhood. clin cosm invest dermatol. 2010;3:89-98. 3. mohrenschlager m, seidl hp, ring j, abeck d. pediatric tinea capitis recognition and management. am j clin dermatol. 2005;6(4):20313. 4. larralde m, gomar b, boggio p, abad me, pagotto b. neonatal kerion celsi: report of three cases. pediatr dermatol. 2010;27(4):361-3. 5. michaels b, rosso jqd. tinea capitis in infants: recognition, evaluation, and management suggestions. j clin aesthet dermatol. 2012;5(2):49–59. 6. elewski eb. clinical diagnosis of common scalp disorder. j invest dermatol symp proc. 2005;10:190-3. 7. pomeranz aj, sabnis ss. tinea capitis epidemiology, diagnosis and management strategies. pediatr drugs. 2002:4(12):779-83. 8. oliver mm. tinea capitis: diagnostic criteria and treatment options. dermatol nurs. 2009:21(8):281-5. 9. aste n, pau m. tinea capitis caused by microsporum canis treated with terbinafine. mycoses. 2004;47:428-30. 10. chan yc, friedlander sf. new treatment for tinea capitis. curr opin infect dis. 2004;17:97-103. 11. gupta ak, adam p. terbinafine pulse therapy is effective in tinea capitis. pediatr dermatol. 1998;15(1):56-8. 12. andrews md, burns m, common tinea infections in children. am fam physician. 2008;77(10):1415-20. 13. horii ka. terbinafine vs griseofulvin for tinea capitis. aap grand rounds. 2008;20;49-50. 14. panagiotidou dd, eremondi thk. efficacy and tolerability of 8 weeks treatment with terbinafine in children with tinea capitis caused by microsporum canis: a comparison of three doses. j eur acad dermatol venereol. 2004;18:155-9. 15. gupta ak, adamiak a, cooper ea. the efficacy and safety of terbinafine in children. j eur acad dermatol venereol. 2003:17;62740. 16. patel ga, schwartz ra. tinea capitis: still an unsolved problem? mycoses. 2009;54:183-8. 17. kelly b. superficial fungal infections. pediatr rev. 2012;33(4):2237. 18. mihić ll, barisic f, bulat v, buljan m, situm m, bradic l, mihić j. differential diagnosis of the scalp hair folliculitis. acta clin croat. 2011;50:395-402. 38 indonesian journal of tropical and infectious disease, vol. 6. no.2 mei–agustus 2016: 34−38 19. gonzález u, seaton t, bergus g, jacobson j,martínez-monzón c. systemic antifungal therapy for tinea capitis in children. cochrane database of systematic reviews 2007;4:1-73 20. fuller lc, child fj, midgley g, higgins em. diagnosis and management of scalp ringworm. br med j. 2003;236:539-41. 21. kakourou t, uksal u. guidelines for the management of tinea capitis in children. pediatr dermatol. 2010;27(3):226-8. 22. alvarez ms, silverberg nb. tinea capitis. cutis. 2006;78:189-96. 23. newland jg, abdel-rahman sm. update on terbinafine with a focus on dermatophytoses. clin cosm invest dermatol. 2009:2;49-63. 24. graham lvd, elewski be. recent updates in oral terbinafine: its use in onychomycosis and tinea capitis in the us.mycoses.2011;54: 679-85. ijtid vol 3 no 1 jan-maret 2012.indd 30 vol. 3. no. 1 january–march 2012 the uveitis – periodontal disease connection in pregnancy: controversy between myth and reality widyawati sutedjo1, chiquita prahasanthi1, daniel haryono utomo2 1 department of periodontology, airlangga university 2 dental clinic faculty of dentistry, airlangga university abstract background: recently, it had been recognized that oral infection, especially periodontal disease are potential contributing factors to a variety of systemic diseases, such as cardiovascular and cerebrovascular diseases, pregnancy problem, diabetes mellitus type 2, etc. however, the adverse effect of periodontal disease toward uveitis still not clearly understood especially if happens during pregnancy. interestingly, in indonesia, there is still a myth that pregnant women should not get any dental treatment, therefore, it may deteriorate periodontal disease during pregnancy. purpose: to explain the possible connection between periodontal disease and uveitis and increase the awareness of these problems during pregnancy that could be understood by doctor and laymen. reviews: literatures revealed that dental infection can caused uveitis via metastatic spread of toxin and inflammatory mediators. additionaly, more recent investigation reported that the neural system may also stimulated by oral infection. in the orofacial regions there's trigeminal nerve complex that also related to the orbital region, thus may also involved in the uveitis pathogenesis. the effects of periodonto pathogens toxins toward immunocompetent cell and nerves had also been reported by researcher. moreover, pregnant women are more susceptible to periodontal disease, therefore maintaining oral hygiene and dental monitoring is a mandatory. conclusion: in woman who susceptible to uveitis, periodontal disease may exacerbate the symptoms especially in pregnancy. therefore simple explanation about connection of oral infection-systemic diseases especially in pregnancy should be widespread among indonesian people. key words: periodontal disease, uveitis connection, indonesian, myth introduction in recent years there has been a reawakening of the dangers of oral infections and their potential disastrous effects on systemic health. gingivitis and periodontitis is the potential sources of this oral infection. in modern dentistry by performing a treatment called scalling root planning, curretage or assisted drainage treatment (adt) must surely be one of the prime candidates for this reassessment. as dentists we are indoctrinated that it is better to keep the oral hygiene to prevent from any other diseases by teaching how to keep the oral hygiene. historically, periodontal disease was regarded as an infection caused by bacterial species that colonize the periodontal pocket. microbial products trigger the release of proinflammatory cytokines and host derived enzymes, the excessive and/or dysregulated production may results in tissue breakdown. the impact of microbial products such as lipopolysaccharide (lps) on induction of immune responses, toll like receptor (tlr) signaling and cytokine networks is crucial to inflammatory changes that develop in the tissues. elevated levels of tissue-destructive enzymes such as matrix metalloproteinase's (mmps) and proinflammatory cytokines can be detected in the gingival crevicular fluid (gcf) and saliva of patients with periodontitis. the pathogenic inflammatory mechanisms may lead to the development and progression of disease.1 a possible correlation of focal infection with uveitis could be predicted regarding to an object observation of a phenomenon that related to the uveitissymptoms. literature review 31sutedjo, et al.: the uveitis – periodontal disease connection in pregnancy periodontal treatment that had been conducted to a patient suffered from symptoms uveitis was able to relief all of the symptoms. a 30 year old female patient come to the clinic. she is a housewife and suffered from several symptoms such as headache, neck pain and spasm, eye redness; blurring of vision; watery; pain and sensitivity to light. the illnesses started 1 year earlier and the treatment and medications had already been conducted by general practitioner and ophthalmologist. her doctor said that she got the uveitis. there were a lot of prescribed drugs such as, medison (corticosteroid) and sandimun (corticosteroid). but she is not getting better and she come to the dentist. from physical examination, despite her moonface (because of the usage of corticosteroid long term), extra oral were normal, intra orally there were a lot of calculus deposits and gingivitis noted in all regions. probing revealed that deep periodontal pockets (5 mm) existed in left and right posterior teeth maxilla and right posterior teeth mandible especially over m1 and m2. no caries was found. periodontal treatment in several literatures were able to reduce or eliminate several symptoms such as headache, sinusitis, fatigue, muscle pain or spasms.2,3,4 the same result also occurred in this patient, who had no more headache, redness of the eye, blurring of vision and everything were normal again. the purpose of this article review is to reveal the possibility of the periodontal disease involvement in the etiopathogenesis of uveitis, based on the remarkable result of periodontal treatment to a patient suffered from uveitis. however, further researches should be done to support the validity of this successful clinical evidence-based case treatment. literature reviews what is uveitis? uveitis is an inflammation of the uveal tract, the middle layer between the sclera and the retina is called the uvea. the uvea contains many of the blood vessels which nourish the eye. inflammation of the uvea can affect the cornea, the retina, the sclera, and other vital parts of the eye. uveitis can also be related to diseases in other parts of the body, such as arthritis or may be caused by infectious agents (e.g., pneumocystis carinii), may be idiopathic (e.g., sarcoidosis), or may be autoimmune in origin (sympathetic ophthalmia).5 the symptoms of uveitis include light sensitivity, blurring of vision, pain, redness of the eye and headache. 5nevertheless, there are several theories related to the etiopathogenesis of headache, such as the increase of proinflammatory cytokines level,6,7,8 no6 involvement of the trigeminal nerve (v2) associated with the sphenopalatine ganglion (spg)9,10 and the "neurogenic switching" mechanism.11 systemic effects of periodontal disease. in abundant literatures reported the effect of periodontal diseases to systemic diseases such as cerebrovascular, cardiovascular diseases, diabetes mellitus type 2, etc. several researchers also revealed the effect of periodontopathic bacteria part i.e. lipopolysaccharides, fimbriae, whole bacteria to systemic condition including allergy. according to a research by utomo in 2009, by injection lps1435/1450 porphyromonas gingivalis (pg lps1435/1450) with low dosage on a gingival sulcus maxillawistar rat. on the 14th day, had found the increases of mrna sp and cgrp in the bronkus.12 moreover on the research by abd el-aleem et al., 2004 who injected salmonella typhimurium intragingival on the papil interdental between first and second molars of wistar rat mandible. on examination with the hybridizatio in situ on days 3, 7 and 10 found an increase level of sp and cgrp mrna in various branches of the n. trigeminal, namely n. mandible, n. maxilla and n. ophthalmicus. in the study of lps used and injected in the upper jaw pg lps1435/1450. 13 and it is possible that periodontal disease can cause uveitis. host immune response and periodontal disease is a common, complex, inflammatory disease characterized by the destruction of tooth-supporting soft and hard tissues of the periodontium, including alveolar bone and periodontal ligament (pdl). although the inflammation is initiated by bacteria, the tissue breakdown events that lead to the clinical signs of disease result from the host inflammatory response that develops to combat the challenge presented by the subgingival biofilm.1 discussion researches done by li et al. revealed the possibility of the relationship between oral focal infection and non-oral diseases. metastatic spread of infection from oral cavity which may be done in several ways were shown in table 1 (li et al, 2000).14 one of the systemic effects of infection is sickness behavior; it refers to the coordinated set of behavioral changes that develop in sick individuals during an infection. at the molecular level, these changes are due to the effects of local proinflammatory cytokines such as interleukin1β (il-1β) and tumor necrosis factor-α (tnf-α) which may also affected the brain if produced in sufficient concentration.16,17 the cytokine-induced sickness behavior symptoms such as fatigue, malaise, headache, sleep disturbances, inability to concentrate and other symptoms are due to the brain action of pro-inflammatory cytokines7,17 and nitric oxide (no)which is produced by inflammation and infection.6 in addition, cfs is closely related with cytokine-induced sickness behavior.16,17 there is a possibility that uveitis also related to cytokine-induced behavior. bacterial endotoxins (lipopolysaccharides, lps) are part of outer cell wall of gram-negative bacteria. lipopolysaccharide challenge upregulates the expression of endothelial cells adhesion 32 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 30−34 molecules-1 and stimulate the release of high levels proinflammatory mediators by macrophages or monocytes such as il-1β, il-6, tnf-α, prostaglandin e2 (pge2)14,18 and no.18 other effects are mast cell degranulation19 and indirectly stimulate afferent nerve endings.20 in order to recognize the effect of stress to immune response, the study of psychoneuroimmunology should also be understood.21 stress consisted of stress perception and stress response.22 stress, mediated by cns, activates the hypothalamic-pituitary-adrenal axis (hpa-axis) and increases the cortisol secretion.16,21,23 at the same time, stress also activates the sympathetic-adrenal medullary axis (sam-axis) to produce more catecholamines (noradrenalin and adrenalin).21 upon stressful condition, high-stress perception individuals also produce il-1β, tnf-α and il-6 that significantly higher compared to low-perception individuals. 24 pro-inflammatory cytokines are also capable of stimulating glucorticoid synthesis through the hpa axis.16,21,23 interleukin-6 which is also elevated by stress and adrenaline25 is a potential stimulator of hpa axis resulting in cortisol secretion to help control the inflammation.16 unfortunately, high cortisol level depresses immune function.21 in this patient who had uveitis, the stress in his work was suspected as the main trigger of the existing symptoms. stress impaired body defense reaction to local infection. altered mood and emotional condition may be involved in the periodontal disease, stress is suggested to affect periodontal health by increasing thelevel il-1β, tnf-α and il-6.25 as a consequence of unsuccessful elimination of oral focal infection, in this case periodontal infection, may perpetuate the systemic infection and the cytokine inducedsickness behavior did not come to an end. these never ending sickness behavior may be related to the debilitating symptoms.16 oral inflammation may propagate to distant targets could be through the interplay of immunogenic and neurogenic inflammation.20 interplay between immunogenic and neurogenic inflammation is termed "neurogenic switching".9,26 immunogenic inflammation may initiated by mast cell degranulation which induced by antigens, bacteria, proteoglycans, lps, neuropeptides (i.e. substance p, sp), chemokines, calcium ionophores and physical factors.27 degranulated mast cells release histamine and tryptase which may stimulate neurogenic inflammation by binding to a protease activated receptor (par) in afferent nerve fibers.20 additionally, pro-inflammatory cytokines and no released by lps-induced macrophage or monocytes, and bradykinin from damaged tissue are able to stimulate neuropeptides release from local afferent sensory fibers in the periodontal tissue. stimulated nerve fibers release neuropeptides i.e sp, calcitonin gene-related peptide (cgrp), vasoactive intestinal peptides (vip) and neuropeptide y (npy).20 there was a plausible explanation regarding to the instant disappearing of the symptoms which related to the oozed blood that occurred during the periodontal treatment. it was supposed to be an assisted drainage to the existing pro-inflammatory mediators (cytokines, pge2, bradykinin, no) in the periodontal disease which then may immediately "cut off" the neurogenic switching mechanism.4 there are several theories related to the etiopathogenesis of headache, such as the increase of pro-inflammatory cytokines level,6,7,8 no6; involvement of the trigeminal nerve (v2) associated with the sphenopalatine ganglion (spg)9,10 and the "neurogenic switching" mechanism.11 headache symptoms in this case which accompanied by neck pain or spasm suffered by the patient according to several literatures are diagnosed as migraine.28,29 activated primary afferent neurons of trigeminal nerve sends impulses via trigeminus nucleus caudalis which acts as sensory relay center. neck pain may resulted from the excitation of trigeminus nucleus caudalis which may extend to dorsal horn for stimulation of c2, c3 and c4.28 periodontal ligament in the maxilla is also innervated by v2. stimulated c fibers from maxillary periodontal ligaments (v2) may antidromically release sp and cgrp, this mechanism is proposed to be the etiology of sinusitis and migraine.9,10 therefore, through the neurogenic switching mechanism20, periodontal inflammation may also directly affects sinus inflammation (mucosa and artery) through the neuropeptides release of sp and cgrp by afferent nerve of nasal mucosa via the sphenopalatine ganglion.9 pathway for oral infection possible nonoral disease metastatic infection from oral cavity via transient bacteremia........................ subacute infective endocarditis, acute bacterial myocarditis, brain abscess, cavernous sinus thrombosis, sinusitis, lung abscess/infection, ludwig's angina, orbital cellulitis, skin ulcer, osteomyelitis, prosthetic joint infection metastatic injury from circulation of oral microbial toxins........................ cerebral infarction, acute myocardial infarction, abnormal pregnancy outcome, persistent pyrexia, idiopathic trigeminal neuralgia, toxic shock syndrome, systemic granulocytic cell defect, chronic meningitis metastatic inflammation caused by immunological injury from oral organism........................ behcet's syndrome, chronic urticaria, uveitis, inflammatory bowel disease, crohn's disease (adapted from li et al., 200014) 33sutedjo, et al.: the uveitis – periodontal disease connection in pregnancy the trigeminovascular reflex, which is related to intracranial arterial vasodilatation due to increase no concentration or inflammation is a normal mechanism. neurons of the first division of trigeminal nerve (v1) reported this condition to the trigeminal sensory nucleus. however, in certain individuals with elevated sympathetic tone or pre-sensitized afferent nerves may trigger headache.9 conclusions periodontal disease is the source of lps, proinflammatory mediators14 including pge2, no and bradykinin18 that were able to lower pain threshold of the afferent nerve fibers of the trigeminal nerve30 (figure 1). the release of gingipains r, a proteolytic enzyme from p gingivalis which triggers decreasedof blood flow, especially in micr ovasculatures,gingipains r in the bloodstream can active factor ix, factor x, prothrombin, and c reactive protein, thus promoting a thrombotic tendency through the release of thrombin, subsequent platelet aggregation, conversion of fibrinogen to fibrin and intravascular clot formation.14 visual disturbances such as blurred vision and posterior uveitis,14 may be induced by proinflammatory cytokines or lps originated from the periodontal infection via the blood stream.14 another possibility is by neurogenic switchig mechanism related to afferent nerves of v i (ophthalmic division of trigeminal nerve).33,34 palpitation may be caused by noradrenaline or adrenaline, released in the state of stress to stimulate the body defense system, especially increase of heart rate and force heart contraction.35 the instant relief of headache, improve of eyesight and other symptoms after scaling procedures may be caused by decreasing of the "neurogenic switching" mechanism. the oozed blood during scaling should contain pro-inflammatory mediators, bacteria and lps which may directly "cut off" the "neurogenic switching" mechanism.4 gradual remission of pain and spasm in muscles should be caused from the diminish of hyperalgesia and sensitization of afferent nerve fibers which formerly caused by high concentration of pge2, bradykinin and no. this review article base on an evidence based case of patient suffered from uveitis according to the patient's medical history and examined by a dental practitioner. further studies with the true uveitis should be done in collaboration with competent medical practitioners and comprehensive medical diagnostic procedures. based on the remarkable result of the periodontal treatment and supported by literature reviews in case reported, it is concluded that a correlation oral focal infection, especially periodontal disease with uveitis symptoms should be exist. further investigation should be done about the etiopathogenesis of periodontal – systemic related illnesses and increase the multidisciplinary approach in the scope of dentistry and general medicine to explore new interrelated cases. references 1. preshaw p. etiologi of periodontal diseases. in: newman mg, takei hh, klokkevold pr, carranza fa, editors. clinical periodontology. 11thed. st. louis: saunders, 2012: 193. 2. utomo h. elimination of oral focal infection: a new solution in chronic fatigue syndrome management? majalah kedokteran gigi surabaya; 2005: 38(4): 169–72. 3. utomo h and prahasanti c. periodontal disease in headache and menstrual pains.lustrum fkg universitas gadjah mada; 2005: 14: 101–6. 4. utomo h. sensitization of the sphenopalatine ganglion by periodontal inflammation: a possible etiology for headache and sinusitis in children. majalah kedokteran gigi fkg universitas airlangga; 2006: 39(2): 63–7. 5. goldstein da, pyatetsky d, tessler hh. classification, symptoms, and signs of uveitis. in: tasman w, jaeger ea, eds. duane's ophthalmology. 15th ed. philadelphia, pa: lippincott williams & wilkins; 2009: chap 32. 6. stirparo g, zicari a, favilla m, lipari m and martelletti p. linked activation of nitric oxide synthase and cyclooxygenase in peripheral monocytes of asymptomatic migraine without aura patients. cephalalgia;2000: 20(2): 100–6. 7. dantzer r. cytokine-induced sickness behavior: where do we stand? brain behaviour and immunity;2001: 15: 7–24. 8. jeong hj, hong sh and nam yc. effect of acupuncture in inflammatory cytokine production in patients with chronic headache. american journal of chinesemedicine; 2003: 31(6): 945–54. 9. boyd jp. pathophysiology of migraine: and rationale for a targeted approach of prevention. available on line at url http://www. drjimboyd.com. accessed, sept, 25, 2011. 10. okeson jp. bell's orofacial pain. 6th ed. carol stream. quintessence pub. 2005. p. 52–3. figure 1. pathogenetic model for uveitis and the relationship with periodontal disease (adapted from furman et al., 2005). 34 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 30−34 11. cady rk and schreiber cp. sinus headache or migraine. neurology; 2002: 58; s10–4. 12. utomo h.immunoneuromodulatory mechanism of the "assisted drainage" therapy towards allergic reaction of rat induced by lipopolysacharide from porphyromonas gingivalis. experimental study in rat subjects. dissertation. postgraduate program airlangga university. surabaya. 2009: p. 218–9. 13. abd el-aleem sa, begonia m, aza m and donaldson lf. sensory neuropeptide mrna up-regulation is bilateral in periodontitis in the rat: a possible neurogenic component to symmetrical periodontal disease. eur j neurosci; 2004: 19: 650–9. 14. li xj, kolltveit km, tronstad l and olsen i. systemic diseases caused by oral infection. clinical microbiological reviews. 2000: 13(4): 547–58. 15. newman mg. the normal periodontium. in: newman mg, takei hh, klokkevold pr, carranza fa, editors. clinical periodontology. 11thed. st. louis: saunders, 2012: 11. 16. licinio j and frost p (2000). the neuroimmune-endocrine axis: pathophysiological implications for the central nervous system cytokines and hypothalamus-pituitary-adrenal hormone dynamics. brazillian journal of medical and biological research 33: 1141– 8. 17. kiecolt-glaser jk and glaser r. depression and immune function: central pathways to morbidity and mortality. journal of psychosomatic research. 2002; 53: 873–76. 18. madianos pn, bobetsis ya and kinane df. generation of inflammatory stimuli: how bacteria set up inflammatory responses in the gingiva. journal of clinical periodontolology. 2005; 32(s6): 57–71 19. supajatura v, ushio h, nakao a, akira s, okumura k, ra c and ogawa h (2002). differential responses of mast cell toll-like receptors 2 and 4 in allergy and innate immunity. journal of clinical investigation 109: 1351–9. 20. lundy w and linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. critical review of oral biology and medicine; 2004 15(2): 82–98. 21. padgett da and glaser r. how stress influence the immune response. trends in immunology; 2003: 24: 444–8 22. putra st and asnar es. perkembangan konsep stres dan penggunaannya dalam paradigma psikoneuroimunologi. in: psikoneuroimunologi kedokteran 1st ed. ed putra, s.t. surabaya. gramik. (in indonesian language). 2005. p.12 23. roitt im and delves pj (2001). essential immunology. 10th ed.. london. blackwell science. pp 214–216 24. kamma jj, giannopoulou c, vasdekis vds and mombelli a (2004). cytokine profile in gingival crevicular fluid of aggressive periodontitis: influence of smoking and stress. journal clinical periodontology. 31: 894–902. 25. shapira l, wilensky a and kinane df (2005). effect of genetic variability on the inflammatory response to periodontal infection. journal of clinical periodontology 32 (s6): 72–86 26. meggs wj. neurogenic switching: a hypothesis for a mechanism for shifting the site of inflammation in allergy and chemical sensitivity. environ health perspect 1997; 105: s2 1–10 27. walsh lj (2003). mast cells and oral inflammation. critical reviews of oral biology andlmedicine 14(3): 188–98. 28. green mw. diagnosing and treating migraine: low tech diagnosis, high-tech treatment. available online at url: http://www.ama-assn. org/ama1/pub/upload/mm/31/24pres-green.pdf accesed september 26, 2011. 29. unger j, cady rk and farmer-cady k (2005). understanding migraine: pathophysiology and presentation. available online at url http://www.femalepatient.com. accessed september 25, 2011 30. kidd bl and urban la (2001). mechanisms of inflammatory pain. british journal of anaesthesia, 87(1): 3–11 31. klinghardt dk. the sphenopalatine ganglion (spg) and environmental sensitivity. lecture on 23rd annual international symposium on man and his environment. june 9-12, 2005. dallas texas. available online at url http://www.naturaltherapy.com. accessed september 26, 2011 32. white g and de paolis m (2005). uveitis. available online at url. http://www.allaboutvision.com. accessed september 27, 2011 33. herndon m. uveitic glaucoma. available online at url http://www. emedicine.co./opht/intraocular_pressure.htm. accessed september 26, 2011. 34. yang p, de vos af and kijlstra a (1998). interferon gamma immunoreactivity in iris nerve fibres during endotoxin induced uveitis in the rat. british journal ofophthalmology 82: 695–9. 35. sherwood l (2005). fundamentals of physiology: a human perspective.3rd ed. belmont. thomson brooks/cole. p. 133–5. 36. weigand la, myers ac, meeker s, undem bj. mast cell-cholinergic nerve interaction in mouse airway. j physiol 2009; 587(13): 3355–62 vol. 7 ● no. 3 sept–dec 2018 e issn 2356 0991 p issn 2085 1103 e-journal.unair.ac.id/index.php/ijtid indexed by: blastocystis and other intestinal parasites infections in elementary school children in dukuh village, karangasem district, bali rna isolation of dengue virus type 1 with different precipitation solvents: dimethyl sulfoxide, acetone, and ethanol 70% preliminary study of wuchereria bancrofti l3 larvae detection in culex quinquefasciatus as vector potential of filariasis in endemic area of south tangerang, by utilizing pcr assay for l3-activated cuticlin transcript mrna gene and tph-1 gene the risk factors for drug induced hepatitis in pulmonary tuberculosis patients in dr. soetomo hospital potency of luteolin with solid lipid nanoparticle (sln)-polyethylene glycol (peg) modification for artemisinin-resistant plasmodium falciparum infection determinant factors of drop out (do) among multi drugs resistance tuberculosis (mdr tb) patients at jakarta province in 2011 to 2015 e issn 2356 0991 p issn 2085 1103volume 7 number 3 september–december 2018 editorial team of indonesian journal of tropical and infectious disease editor in chief prihartini widiyanti, indonesia editorial board mark alan graber, united states kazufumi shimizu, japan masanori kameoka, japan hak hotta, japan fumihiko kawamoto, japan nasronudin nasronudin, indonesia maria inge lusida, indonesia puruhito puruhito, indonesia indropo agusni, indonesia retno handajani, indonesia kuntaman kuntaman, indonesia soegeng soegijanto, indonesia bambang prajogo, indonesia ni nyoman sri budayanti, indonesia achmad fuad hafid, indonesia tri wibawa, indonesia irwanto irwanto, indonesia marcellino rudyanto, indonesia yulis setiya dewi, indonesia laura navika yamani, indonesia secretariat zakaria pamoengkas widi listiyas rini secretariat office publishing unit of indonesian journal of tropical and infectious disease, institute of tropical disease universitas airlangga kampus c, jalan mulyorejo surabaya 60115, jawa timur – indonesia. phone 62-31-5992445-46 faximile 62-31-5992445 e-mail: ijtid@itd.unair.ac.id homepage: e-journal.unair.ac.id/index.php/ijtid contents page printed by: universitas airlangga press. (rk 449/10.18/aup). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, fax. (031) 5992248. e-mail: adm@aup.unair.ac.id volume 7 number 3 september–december 2018 e issn 2356 0991 p issn 2085 1103 1. blastocystis and other intestinal parasites infections in elementary school children in dukuh village, karangasem district, bali ni luh putu eka diarthini, i kadek swastika, luh ariwati, rahmadany isyaputri, moh. yasin fitri n, sri hidajati, sukmawati basuki ................................................................... 57–61 2. rna isolation of dengue virus type 1 with different precipitation solvents: dimethyl sulfoxide, acetone, and ethanol 70% a n i s a m a h a r a n i , t e g u h h a r i s u c i p t o , h a r s a s i s e t y a w a t i , s i t i c h u r r o t i n , ilham harlan amarullah, puspa wardhani, aryati, shuhai ueda, soegeng soegijanto ......... 62–66 3. preliminary study of wuchereria bancrofti l3 larvae detection in culex quinquefasciatus as vector potential of filariasis in endemic area of south tangerang, by utilizing pcr assay for l3-activated cuticlin transcript mrna gene and tph-1 gene silvia fitrina nasution, chris adhiyanto, evi indahwati ............................................................ 67–72 4. the risk factors for drug induced hepatitis in pulmonary tuberculosis patients in dr. soetomo hospital soedarsono, sari mandayani, kinasih prayuni, rika yuliwulandari ........................................ 73–79 5. potency of luteolin with solid lipid nanoparticle (sln)-polyethylene glycol (peg) modification for artemisinin-resistant plasmodium falciparum infection william kamarullah, erika indrajaya, janice emmanuella ...................................................... 80–86 6. determinant factors of drop out (do) among multi drugs resistance tuberculosis (mdr tb) patients at jakarta province in 2011 to 2015 sitti farihatun, putri bungsu ......................................................................................................... 87–92 63 vol. 6. no. 3 september–december 2016 research report betle leaf essential oil for hemophiliac patients and its antibacterial effects on mycobacterium tuberculosis teguh hari sucipto1a, nourmalasari aisyah2, puji lestari2, harsasi setyawati2 1 institute of tropical disease, universitas airlangga 2 department of chemistry, faculty of science and technology, universitas airlangga a corresponding author: teguhharisucipto@yahoo.co.id abstract betle leaf (piper betle l.) is a medicinal plant. it contains essential oil and shows various biological activities, such as antibacterial, anticoagulant, etc. it is further reported to have low anticoagulant activities; thus, it is highly potential as a candidate for coagulant drug. coagulant is used to prevent bleeding for patients with blood clotting disorders like hemophilia. in indonesia, 1,236 people were reported with hemophilia. the standard parameters of anticoagulant activity are the freezing period and the compound concentrations. the purpose of this study was to determine the effect of betle leaf’s essential oil on blood coagulation in patients with factor viii and ix of blood plasma disorders. the isolation of essential oil is conducted through steam distillation method with two kinds of solvents, namely distilled water and n-hexane. the obtained n-hexane extract is then separated from the liquid-liquid extraction and rotary evaporator. essential oil is diluted with citrate plasma solution. the test results of blood clots increase as the concentration of essential oils increase. the results are recorded as such: essential oils ½ times dilution of 99.67 seconds; ¼ times dilution of 127 seconds; 1/8 times dilution of 179 seconds; and 1/16 times dilution of 242.67 seconds. the test above proves that the piper betle extract possesses a coagulant activity. the ethanol extract contained in the piper betle could stimulate clotting in the blood cells. it is caused by the increase of blood plasma concentration which further escalate the plasma fluid into the blood cells. based on this study, the activity of mycobacterium tuberculosis can be obstructed by betle leaf in ½ times dilution. the extract significantly reduces acid which accelerates bacteria development. keywords: betle leaf, liquid-liquid extraction, blood clotting, coagulant, anti-mycobacterium tuberculosis abstrak daun sirih (piper betle l.) merupakan tanaman obat. daun sirih terdapat kandungan minyak atsiri dan menunjukkan berbagai aktivitas biologi, diantaranya adalah antibakteri, antikoagulan, dan lain sebagainya. di indonesia, jumlah penderita hemofilia dilaporkan 1.236 orang. koagulan digunakan untuk mencegah pendarahan pada penderitan kelainan pembekuan darah seperti hemofilia. daun sirih dilaporkan memiliki aktivitas antikoagulan rendah, sehingga sangat potensial untuk kandidat obat koagulan. parameter standar untuk aktifitas antikoagulan adalah waktu pembekuan dan konsentrasi senyawa. tujuan dari penelitian ini adalah mengetahui pengaruh minyak atsiri daun sirih terhadap pembekuan darah pada penderita kelainan faktor viii dan ix plasma darah. isolasi minyak atsiri dilakukan dengan metode destilasi uap menggunakan dua macam pelarut yaitu aquades dan n-heksana. ekstrak n-heksana yang diperoleh dipisahkan dengan ekstraksi cair-cair dan rotary evaporator. minyak atsiri didilusi dengan larutan plasma sitrat. hasil uji pembekuan darah minyak atsiri meningkat seiring konsentrasi minyak atsiri yaitu pengenceran 1/2 kali 99.67 detik; pengenceran 1/4 kali 127 detik; pengenceran 1/8 kali 179 detik; dan pengenceran 1/16 kali 242.67 detik. pengujian di atas menunjukkan bahwa ekstrak piper betle memiliki aktivitas koagulan. ekstrak etanol yang terkandung dalam piper betle dapat menyebabkan pembekuan dalam sel-sel darah. hal ini disebabkan konsentrasi plasma darah naik, yang meningkatkan cairan plasma ke dalam sel darah. berdasarkan penelitian ini, aktivitas mycobacterium tuberculosis dapat dihambat oleh ekstrak daun sirih pada pengenceran 1/2 kali. ekstrak secara signifikan mengurangi sifat asam yang dapat mempercepat perkembangan bakteri. kata kunci : daun sirih, ekstraksi cair-cair, pembekuan darah, koagulan, anti-mycobacterium tuberculos 64 indonesian journal of tropical and infectious disease, vol. 6. no. 3 september–december 2016: 63-67 material and method betle leaf’s essential oil extraction betle leaf’s essential oil is isolated using steam distillation technique. prior to the first steam distillation, the betle leaves are cut into small pieces to facilitate the distillation and insulate the essential oil inside the betle leaf. solvents are used for the distillation. time required to isolate the essential oil is about 2 hours until the solution in the distillation equipment condenser becomes colorless. the color indicates that the essential oil has been all isolated. isolation is separated between water phase and organic phase using liquid-liquid extraction with an organic n-hexane solvent. the extraction is performed 5 times to perfectly separate the essential oil in the water. the essential oil will be mixed with the n-hexane solvent. the last phase of separation is conducted using a rotary evaporator. the essential oil in n-hexane is separated using the principle of boiling point. the heating process is carried out at approximately 60 to 70°c. n-hexane’s boiling point is recorded at 63°c in which it is still in the form of gas; while the essential oil remains in liquid form due to the extremely high boiling point of the volatile oil. the heating process produces two products, namely n-hexane and pure essential oil of betle leaf. the essential oil obtained in this isolation is 4.5 ml with a percentage of 0.9%. this is because the properties of essential oil is volatile, thus, it reduces their products. volatile chemical compounds have a high vapor pressure at ordinary room temperature.6 extract dilutions extract dilutions is conducted using pz solution (saline), because this solution is deemed to have the same osmotic pressure with the fluid contained in human body. dilution is done by extracting the essential oil of betle leaf as much as 1 cc, added with 1 cc solution of pz, then ½ times dilution of the extract concentrated essential oil is obtained. a quarter times, 1/8 times, and 1/16 times dilutions are also conducted to determine the most effective dilution to speed up blood clotting.6 separation of plasma from red blood twenty-five cc blood from normal individual is mixed with 3.8% sodium citrate in 9:1 ratio; then, the mixture is put in a tube of blood plasma and made sure it is perfectly blended. the mixture is centrifuged for about 30 minutes at 1500 rpm. tubes are excluded from clinical centrifuges. at the top of the tube, there is clear yellowish liquid; while at the bottom, red sediment can be seen. the clear liquid, which is called citrate plasma, is then extracted and stored in a refrigerator. introduction indonesia has a tropical climate suitable to grow various medicinal plants, one of them is betle leaf (piper betle l.).1 indonesian people who live in rural areas particularly use betle leaf to cure various diseases. a part of the leaf is mainly used for some health treatments, such as nosebleed (epistaxis). the leaf is rolled up and put into one’s nostrils.2 moreover, betle leaves can also be used as a mouthwash. a dried betle leaf can also be used as a traditional medicine, such as cough medicine, drugs, or eye wounds. betle is a chemical plant which consists of saponins, flavonoids, polyphenols, and essential oil.3 there is an increase usage of natural materials through a large scale of fabrication. the use of traditional medicine is considered having fewer side effects compared with the chemical drugs and more affordable.4 modern drug is widely believed to cause spasm of the bile duct sphincter and impede bile flow; whereas the effect on renal development receives less attention.5 meanwhile, hemophilia is a hereditary disorder which is heavily associated with a deficiency or an abnormality of biological factor viii and factor ix in blood plasma.6 this genetic disorder affects many people. in indonesia, the number of hemophiliac was reported at 1,236 people. the contents of the essential oil in a betle plant are chavicol, eugenol, cineol, and carvacrol. essential oils functions as antibacterial, antioxidant, antifungal, anti-ulcerogenic, anti-amoebic, anti-inflammatory, antifilarial, anti-microbial, anti-fertility, anti-hyperglycemic, anti-dermatophytid, anti-naceptive, and radioprotective properties.1 tuberculosis (tb), which is caused by mycobacterium tuberculosis, is a highly infectious disease. its morbidity and mortality continue being a cause of concern. there has been a substantial increase in these last decades in the investigation of medicinal plants to find out their biological efficacies for the treatment of various disorder. in the field of anti-tb agents, several studies on potential medicinal plants have been reported from various parts of the world. piper nigrum extract, a combination of acetone and ethanol extracts of 50 μg/ml each, was effectively tested against anti-mycobacterium tuberculosis.7 the antibacterial activity from the plant is caused by secondary metabolic compounds with phenolic compounds. this study chooses betle plant leaves as the research object, as it is often used as nosebleed cure.8 this study uses piper betle l. species from jajar village, kediri district, east java, indonesia. on the description above, the researchers look for a new solution by leveraging the existing knowledge which increases the potential betle leaf’s essential oil extracts (piper betle l.) on hemophiliac patients. the purpose of this study is to determine the effects of betle leaf’s essential oil on hemophiliac patients in vitro using clotting time method and study of anti-mycobacterium tuberculosis activity. 65sucipto, et al.: betle leaf essential oil for hemophiliac patients figure 1. citrate plasma figure 2. control solution figure 3. dilution of essential oil with citrate plasma control solution citrate plasma of 0.8 ml for each blood group is mixed with 0.2 cc pz solution, then they are shaken and left for some time to mix. next, 0.2 cc and 0.2 cc plus cacl2 are taken for control solution until the first fiber is formed. the fiber is in the form of white threads called fibrin. blood coagulation experiment using the essential oil of betle leaf the 0.8 cc citrate plasma solution is added to 0.2 cc betle leaf’s essential oil extract. after making sure it is blended well, 0.2 cc from the mixed solution is added to 0.2 cc cacl2. the researchers then observe and record the freezing time. the same procedures are performed to ½ times, ¼ times, 1/8 times, and 1/16 times dilutions. citrate plasma uses all blood types. the experiments are performed at 37°c. anti-mycobacterium tuberculosis test mycobacterium tuberculosis refers to the strain h37rv. the preparation for medium 7h10 was dissolved with aquadest, then autoclaved in 121°c for 10 minutes. medium 7h10 is added with essential oil and incubated for 4-3 weeks at 37°c in a co2 incubator. the tested essential oil concentrations consist of ½ times dilution, ¼ times dilution, 1/8 times dilution, and 1/16 times dilution. result and discussion blood plasma is the most important [object] in this research, because it consists of plasma proteins which have a big impact on blood clotting. the blood plasma was separated by a mixture of blood centrifuged between normal and 3.8% sodium citrate. this study uses the normal blood group b. therefore, sodium citrate anticoagulant of 3.8%, which slows down clotting process, is added to make the normal blood to have the same nature with the blood with clotting factor disorders. although both citrate and heparin are used as anticoagulants during apheresis, citrate is preferred for most exchange procedures because of its safety and effectiveness.9 the 9:1 (blood to sodium citrate 3.8%) ratio is used for the anticoagulants as an ideal comparison, because the anticoagulation in a greater portion of blood clotting takes longer processing time.10 the plasma from the reaction above is called citrate plasma (figure the control solution is used as a comparison to the blood clotting process using betle leaf’s essential oil. a control solution is considered as successfully made if the color is brownish yellow (figure 2). blood clotting test is conducted by mixing the essential oil dilution with plasma citrate in four different reaction tubes. a solution of cacl2 is then added into the mixture. the fourth solution is formed in yellow-brown color with different intensities of concentration. the color intensity for the solution concentration of essential oil with ½ times dilution is higher or more concentrated than the essential oil with 1/16 times dilution. this solution results in the same color with the earlier control solution (figure 3-4). blood clotting mechanism cannot be shown directly in this study, as this study is only conducted in vitro; however, there are some visual data obtained. cacl2 solution acts as the activation for prothrombin. the study is conducted at 37°c, matching the temperature of human body. the result of blood clotting test is showed in table 1. anova test is then performed to analyze the results. a significant difference between four conditions of essential oil is obtained (f-count is recorded at 232.69, greater than the f-table at 4:07). 66 indonesian journal of tropical and infectious disease, vol. 6. no. 3 september–december 2016: 63-67 figure 4. freezing blood test with essential oil table 1. the results of blood clotting in seconds concentration of essential oil time (second) 1/2 times 99.67 1/4 times 127 1/8 times 179 1/16 times 242.67 figure 5. curve blood clotting in betle leaf’s essential oil 1/16 times 1/8 times ¼ times ½ times figure 6. culture of mycobacterium tuberculosis containing essential oil from piper betle l. extract possesses the coagulant activity. blood clotting is a complex procedure which involved numerous factors in the plasma and tissues. both intrinsic and extrinsic pathways play vital roles. inhibitors of the blood coagulation affect some factors in blood (figure 5).15 the chemical components of betle leaf’s essential oil are monoterpenes, sesquiterpenes, alcohols, esters, aldehydes, and phenols.16 according to tangkery in 2013, ethanol causes clotting in the blood cells to stick to each other; however, the red blood cells, or erythrocytes, no longer have any forms, because the cell’s wall has been destroyed. it is caused by the increase of blood plasma concentration which further escalates plasma fluid into the blood cells.11 anti-mycobacterium tuberculosis test amidst the emerging drug resistance in infectious diseases field, the use of medicinal plants provides an alternative therapy. unfortunately, there are limited report on the anti-mycobacterium tuberculosis in indonesian medicinal plants. essential oil from piper betle l. was shown to have anti-mycobacterium tuberculosis activity (figure 6). the essential oil with 1/2 times dilution concentration demonstrates an inhibitory activity against mycobacterium tuberculosis. it proves its effectiveness for the inhibited activity of mycobacterium tuberculosis. some drops of fungal are shown in the ½ times dilution bottle, however, there are no bacteria showed. mycobacterium tuberculosis and fungal are shown in ¼ times dilution, 1/8 times dilution and 1/16 times dilution. non-active essential oil and activity of mycobacterium tuberculosis are demonstrated in the lower concentration of the essential oil. meanwhile, piperine is an active compound in piper belte l. extract. in the literature, piperine of 1.0 and 10 μg/ ml showed an up-regulation of ifn-γ and il-2 production in mycobacterium tuberculosis. an effective immunestimulant can complement the host cellular immune response by specifically inducing the type 1 (th-1) response.17 in according to table 1, the frozen blood from each donor was shown in time difference. normal blood clotting occurs from 3 to 18 minutes.11 blood clots occurred because plasma protein prothrombin is changed into thrombin. thrombin is an enzyme which catalyzes the forming of fibrinogen. it is a soluble protein which changes into fibrin. in a few second, fibrin polymerized a mesh which is composed by some fibrin threads. the thread runs to every direction and forms a net which catches blood element and forms a clot.12 based on blood clotting curve of the betle leaf’s essential oil, it can be concluded that the higher the concentration is, the faster the blood clotting process takes place.13 the test results of blood clots increase as the concentration of essential oil increases. the results are as follows: essential oils ½ times dilution for 99.67 seconds; ¼ times dilution for 127 seconds; 1/8 times dilution for 179 seconds; and 1/16 times dilution for 242.67 seconds. this research successfully demonstrates that the essential oil of betle leaf can be used as blood clotting. the betle leaf is known to have antibacterial activity. staphylococcus aureus’ activity was inhibited by betle leaf in 200 mg/ml concentration. the extract is found to significantly reduce acid production of the bacteria.14 the test above indicates that the piper betle blood clotting test is conducted by mixing the essential oil dilution with plasma citrate in four different reaction tubes. a solution of cacl2 is then added into the mixture. the fourth solution is formed in yellow-brown color with different intensities of concentration. the color intensity for the solution concentration of essential oil with ½ times dilution is higher or more concentrated than the essential oil with 1/16 times dilution. this solution results in the same color with the earlier control solution. figure 3. dilution of essential oil with citrate plasma blood clotting mechanism cannot be shown directly in this study, as this study is only conducted in vitro; however, there are some visual data obtained. cacl2 solution acts as the activation for prothrombin. the study is conducted at 37°c, matching the temperature of human body. figure 4. freezing blood test with essential oil this study illustrates the average time (in seconds) for blood clotting. table 1. the results of blood clotting in seconds concentration of essential oil time (second) 1/2 times 99.67 1/4 times 127 67sucipto, et al.: betle leaf essential oil for hemophiliac patients this regard, the key cytokine in mice and humans seems to be gamma interferon (inf-γ) which activates bactericidal effector mechanisms in the mycobacterial host cell, the macrophage.18 piperine (1 mg/kg) in mice which are infected with mycobacterium tuberculosis activates the differentiation of the t cells into th-1 sub-population (cd4+/cd8+ subsets).17 protective immunity against mycobacterium tuberculosis requires the generation of cell-mediated immunity. the secretion of th-1 cytokines by antigen-specific t cells plays an important role in protective granuloma formation and stimulates the antimicrobial activity of the infected macrophages.19 conclusion the dilution of the betle leaf’s essential oil extraction at ½ dilution have the most rapid blood clotting effectiveness for hemophilia treatment in vitro. it is caused by the capability of ethanol compound in the betle leaf’s extract to yield clotting in blood cells. the blood clot further increases blood plasma concentration and plasma fluid in blood cells. plasma fluid is an important component for blood clot factor, because it contains prothrombin and fibrinogen. betle leaf extract provides anti-infection activity, mainly against mycobacterium tuberculosis. acknowledgment this study was supported by direktorat jenderal pendidikan tinggi (dikti) indonesian republic, indonesian red cross society in surabaya, department of chemistry, faculty of science and technology, universitas airlangga and institute of tropical disease universitas airlangga references 1. sripradha s. betel leaf – the green gold. j pharm sci res. 2014;6(1):36–7. 2. a tp, farida y. total phenolic, flavonoids content and antioxidant activity of the ethanolic extract of betel leaf (piper betle l.). in: total phenolic, flavonoids content and antioxidant activity of the ethanolic extract of betel leaf (piper betle l). jakarta: the international conference in nanotechnology; 2013. p. 1–4. 3. ovedoff d. kapita selekta kedokteran. in: kapita selekta kedokteran. binarupa aksara; 2002. 4. oktora l, kumala r. pemanfaatan obat tradisional dan keamanannya. maj ilmu kefarmasian. 2006;iii(1):1–7. 5. schreuder mf, bueters rr, huigen mc, russel fgm, masereeuw r, van den heuvel lp. effect of drugs on renal development. clin j am soc nephrol. 2011 jan 1;6(1):212–7. 6. ramström s. clotting time analysis of citrated blood samples is strongly affected by the tube used for blood sampling. blood coagul fibrinolysis. 2005 sep;16(6):447–52. 7. birdi t, d’souza d, tolani m, daswani p, nair v, tetali p, et al. assessment of the activity of selected indian medicinal plants against mycobacterium tuberculosis: a preliminary screening using the microplate alamar blue assay. european j med plants. 2012 jan 10;2(4):308–23. 8. tedjasulaksana r. ekstrak etil asetat dan etanol daun sirih (piper betle l.) dapat memperpendek waktu pendarahan mencit (mus musculus). j kesehat gigi. 2013;1(1):32–9. 9. lee g, arepally gm. anticoagulation techniques in apheresis: from heparin to citrate and beyond. j clin apher. 2012;27(3):117–25. 10. félix-silva j, souza t, camara rbbg, cabral b, silva-júnior aa, rebecchi imm, et al. in vitro anticoagulant and antioxidant activities of jatropha gossypiifolia l. (euphorbiaceae) leaves aiming therapeutical applications. bmc complement altern med. 2014 dec 20;14(1):405. 11. rossi c, holbrook m, james sh, mabel and d. medical and forensic aspects of blood clot formation in the presence of saliva – a preliminary studytle. j bloodstain pattern anal. 2012;28(3):3–12. 12. tangkery rab, paransa ds, rumengan a. uji aktivitas antikoagulan ekstrak mangrove aegiceras corniculatum. j pesisir dan laut trop. 2013;1:7–14. 13. miranda cas, cardoso mg, mansanares me, gomes ms, marcussi s. preliminary assessment of hedychium coronarium essential oil on fibrinogenolytic and coagulant activity induced by bothrops and lachesis snake venoms. j venom anim toxins incl trop dis. 2014;20(1):39. 14. nalina t, rahim zha. the crude aqueous extract of piper betle l. and its antibacterial effect towards streptococcus mutans. am j biochem biotechnol. 2007 jan 1;3(1):10–5. 15. jesonbabu j, spandana n, reddy ms. a bioactive compound from piper betel with anticoagulant activity. int j pharm pharm sci. 2012;4(3):2012. 16. satyal p, setzer wn. chemical composition and biological activities of nepalese piper betle l . ijpha. 2012;1(2):23–6. 17. sharma s, kalia np, suden p, chauhan ps, kumar m, ram ab, et al. protective efficacy of piperine against mycobacterium tuberculosis. tuberculosis (edinb). 2014 jul;94(4):389–96. 18. holten-andersen l, doherty tm, korsholm ks, andersen p. combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and synthetic mycobacterial cord factor as an efficient adjuvant for tuberculosis subunit vaccines. infect immun. 2004 mar;72(3):1608–17. 19. quiroga mf, jurado jo, martínez gj, pasquinelli v, musella rm, abbate e, et al. cross-talk between cd31 and the signaling lymphocytic activation molecule-associated protein during interferon gamma production against mycobacterium tuberculosis. j infect dis. 2007 nov 1;196(9):1369–78. vol. 8 no. 1 january-april 2020 copyright © 2020, ijtid, issn 2085-1103 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ research report sensitivity of erythromycin against toxigenic strain of corynebacterium diphtheriae alif mutahhar1, dwiyanti puspitasari2, dominicus husada2, leny kartina2, parwati setiono basuki2, ismoedijanto moejito2 1medical study program, faculty of medicine, universitas airlangga, surabaya, east java, indonesia 2division of tropical and infectious disease, child health department, dr. soetomo general hospital/ faculty of medicine, universitas airlangga, surabaya, east java, indonesia corresponding author: dwiyanti-p@fk.unair.ac.id received: 25th january 2019; revised: 28th january 2019; accepted: 2nd january 2020 abstract diphtheria is an acute infection disease caused by corynebacterium diphtheriae. it remains a problem in indonesia in a recent several years especially in east java province, which suff ered from an outbreak of diphtheria in 2011. erythromycin is the second line antibiotics therapy for diphteria if the patient is allergic to penicillin, also serving as a prophylactic and carrier therapy for contact diphtheria. erythromycin has been used for diphtheria for a very long time, but there is little recent data on its sensitivity against c. diphtheriae. the purpose of this study is to identify whether erythromycin still has a strong antibacterial activity against corynebacterium diphtheriae by invitro test. this was a descriptive study which observed the sensitivity pattern of erythromycin against corynebacterium diphtheriae using the epsilometer test (etest) as a diff usion technique. samples used in this study were 30 isolates of toxigenic corynebacterium diphtheriae strain mitis and gravis at the center for health laboratory (bblk) surabaya obtained during 2011 until 2014. we retrieved the data based on gender, age, and districts of patients for each of the samples then analyzed them descriptively. in this study, a sensitivity test of 30 toxigenic corynebacterium diphtheriae isolates revealed that 27 (90%) were sensitive to erythromycin (average minimum inhibitory concentration/ mic) <0.016 μg/ml and all were strain mitis, while 3 (10%) had intermediate sensitivity with mic 1 μg/ml (all were strain gravis). no resistance result was found from the sensitivity test. according to the result, we conclude that erythromycin still has a strong antibacterial activity against corynebacterium diphtheriae. keywords: c. diphtheriae, erythromycin, sensitivity, epsilometer test abstrak difteri merupakan penyakit infeksi akut yang disebabkan oleh bakteri corynebacterium diphtheriae. difteri masih menjadi masalah di indonesia dalam beberapa tahun terakhir ini terutama di wilayah provinsi jawa timur yang mengalami kejadian luar biasa (klb) difteri pada tahun 2011. eritromisin merupakan antibiotik pilihan kedua bila pasien mengalami alergi terhadap penisilin dalam penanganan difteri, selain itu juga digunakan sebagai terapi karier dan profi laksis kontak difteri. eritromisin telah digunakan dalam terapi difteri sejak zaman dahulu, namun tidak banyak data publikasi terkini mengenai sensitivitas eritromisin terhadap bakteri c. diphtheriae. tujuan dari penelitian ini adalah untuk mengetahui apakah eritromisin masih memiliki aktivitas antibakteri yang kuat terhadap corynebacterium diphtheriae secara uji invitro. penelitian ini menggunakan desain deskriptif yang mengamati pola sensitivitas eritromisin terhadap corynebacterium diphtheriae dengan menggunakan teknik difusi epsilometer (etest) eritromisin untuk uji sensitivitas. sampel dari penelitian ini adalah 30 isolat corynebacterium diphtheriae strain mitis dan gravis yang bersifat toksigenik yang terdapat di balai besar laboratorium kesehatan (bblk) surabaya dalam rentang waktu sejak 2011 hingga 2014. karakteristik sampel yang dihimpun kemudian dikelompokkan berdasarkan jenis kelamin, usia, dan asal daerah pasien dari masing-masing isola kemudian dianalisis secara deskriptif. hasil penelitian menunjukkan uji sensitivitas eritromisin terhadap 30 isolat corresponding author. e-mail: dwiyanti-p@fk.unair.ac.id 25alif mutahhar, et al.: sensitivity of erythromycin against toxigenic strain copyright © 2020, ijtid, issn 2085-1103 corynebacterium diphtheriae, diantaranya 27 isolat (90%) bersifat sensitif dengan rata-rata konsentrasi hambat minimal (khm) <0,016 μg/ml dan semuanya merupakan strain mitis, sementara 3 isolat (10 %) memiliki sensitivitas intermediet dengan khm 1 μg/ml dan semuanya merupakan strain gravis. tidak ditemukan hasil resisten dalam uji sensitivitas ini. berdasarkan hasil tersebut, dapat disimpulkan bahwa eritromisin masih memiliki aktivitas antibakteri yang kuat terhadap corynebacterium diphtheriae. kata kunci: c. diphtheriae, eritromisin, sensitivitas, tes epsilometer. how to cite: mutahhar, alif. puspitasari, dwiyanti. husada, dominicus. kartina, leny. basuki, parwati setiono. moedjito, ismoedijanto. sensitivity of erythromycin against toxigenic strain of corynebacterium diphtheriae. indonesian journal of tropical and infectious disease, [s.l.], v.8, n.1, p.182-189, jan. 2020. issn 2085-1103. available at: https://ejournal. unair.ac.id/ijtid/article/view/11654 . date accessed: 09 dec. 2019. doi: h p://dx.doi.org/10.20474/ij d.v8i1.11654 introduction diphtheria is an acute infection caused by corynebacterium diphtheriae transmitted to humans through respiratory droplets by coughing or sneezing. it can also be transmitted through contaminated clothes after skin diphtheria lesion. the usual symptoms and signs are fever, pain in swallowing, weakness, anda greyish-thick pseudomembrane formed by the growth of bacteria, toxin, necrosis in underlying tissues, and host immunity response. the toxin produced is called diphtheria toxin and is disseminated through the bloodstream, causing systemic infection and organ damage.1-2 there are four strains of corynebacterium diphtheriae, namely mitis, gravis, intermedius, and belfanti, which differ from each other according to biological and chemical tests.3 diphtheria is also one of the world’s vaccinepreventable diseases but today still poses a problem in several parts of the world4. according to who in 2012, indonesia had the second highest prevalence of diphtheria, with 1192 cases.5 up until october 2012, the number of cases of diphtheria in east java was as many as 710 and situbondo district had the highest prevalence, with 113 cases and 7 deaths from it.6 the first-line antibiotic used for diphtheria is penicillin due to its bactericidal action compared to erythromycin as a bacteriostatic7,8. unfortunately, in indonesia penicillin is only available in the form of an injection that has to be given intramuscularly, which is uncomfortable. oral erythromycinisan alternative antibiotic for those who are hypersensitive to penicillin, and as a prophylactic treatment. the secondary use of erythromycin is for the eradication of corynebacterium diphtheriae.9,10 erythromycin has been widely used in daily treatment for other respiratory infections for a very long time, raising questions about its sensitivity against c. diphtheria. there are very few recent studies or data about the sensitivity of erythromycin against corynebacterium diphtheriae, especially in east java province, indonesia. therefore, we conduct a study to identify whether erythromycin still has an antibacterial activity against corynebacterium diphtheriae. materials and methods this is a descriptive study to observe the sensitivity pattern of erythromycin against corynebacterium diphtheriae. the samples are retrieved from 216 c. diphtheria isolates collected during diphtheria outbreaks in east java province between 2011 until 2014, stored at the center for health laboratory (bblk), surabaya, as the national referral laboratory for diphtheria in indonesia. the isolates came from several districts and cities in east java province, and consist of c. diphtheria strains mitis and gravis. we used the stratified sampling method to determine 30 isolates of corynebacterium diphtheriae as the sample size. we divided the total population (216 isolates) into groups based on their district/ city of origin, then we proportionally counted the number of samples needed from each district group based on their incidence rate: bangkalan 18 (60%), jember 5 (16.7%), bondowoso 4 (13.3%), 26 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 24–29 copyright © 2020, ijtid, issn 2085-1103 banyuwangi 1 (3.3%), bojonegoro 1 (3.3%), and tuban 1 (3.3%). isolates from each district/group were then simply chosen randomly. the inclusion criterion was toxigenic isolates determined by elek tests,11 while the exclusion criterion was isolates that did not grow in agar medium and showed negative in nitrate and glucose tests. the sensitivity of erythromycin was tested by the epsilometer test (etest) as a diffusion technique.12 the result was interpreted based on the erythromycin mic in accordance with the clinical laboratory standard and institute (clsi), where mic ≤ 0.5 μg/ml is sensitive, mic = 1 μg/ml is intermediate, and mic ≥ 2 μg/ ml is resistant.13 the number of isolates showing sensitive, intermediate, or resistant results were explained descriptively. the study was approved by the medical research ethics commission of the faculty of medicine, universitas airlangga no:191/ec/kepk/fkua/2014. results and discussion the characteristics of patients with positive corynebacterium diphtheriae cultures used in this study are shown in table 1. from the 30 isolates tested, 53.3% were obtained from patients aged ≥ 15 years and the highest prevalence came from bangkalan district (60%). this was different from the data based on east java health office in 2012, which showed that diphtheria cases in the < 15 years age group were more prevalent and that most cases of diphtheria came from situbondo district.6 based on the sex distribution, 66.7% of isolates were obtained from female patients. a study by volzke in germany showed that women without toxoid immunization had four times the risk of suffering from diphtheria compared to non-immunized men.14the study by nath et al. in india showed a different result, however, and from 60 cases of diphtheria reported, males were affected more than females, with figures of 53.33% and 46.67% respectively.1 meanwhile, the majority c. diphtheriae strain found was c. diphtheriae mitis (90%),while strain gravis accounted for 10%, and neither the intermedius nor the belfanti strain was found. table 1. characteristics of patients with positive corynebacterium diphtheriae culture in this study no category frequency % 1 age (year) < 15 14 46.7 ≥ 15 16 53.3 2 sex male 10 33.3 female 20 66.7 3 origin bangkalan 18 60 jember 5 16.7 bondowoso 4 13.3 banyuwangi 1 3.3 bojonegoro 1 3.3 tuban 1 3.3 4 strain mitis 27 90 gravis 3 10 5 sensitivity of erythomycin sensitive 27 90 intermediate 3 10 resistant 0 0 the results of the present study are shown in table 2, which makes clear the significant finding of 90% sensitive results with an average mic <0.016 μg/ml (mic ≤0.5 μg/ml was sensitive) and 10% intermediate results (mic 1 μg/ml was intermediate). no resistance was found (mic ≥ 2 μg/ml). clinically, we should increase the dose therapy of erythromycin for the management of diphtheria based on the invitro intermediate results in order to eradicate corynebacterium diphtheriae. although the strain mitis has much greater prevalence than gravis (90% mitis and 10% gravis), it can be treated by a normal dose of erythromycin based on its sensitivity result to corynebacterium diphtheriae (strain mitis 100% sensitive, gravis 100% intermediate). few studies have been conducted recently on the sensitivity of antibiotics against corynebacterium diphtheriae and, of these, several studies are outdated because the number of cases of diphtheria has declined significantly in recent years due to good immunization coverage and surveillance 27alif mutahhar, et al.: sensitivity of erythromycin against toxigenic strain copyright © 2020, ijtid, issn 2085-1103 table 2. sensitivity of erythromycin against corynebacterium diphtheriae by epsilometer test (etest) no gender age origin strain mic (μg/ml) interpretation 1 male 40 bangkalan mitis < 0.016 sensitive 2 male 5 bangkalan mitis < 0.016 sensitive 3 female 4 bangkalan mitis 0.016 sensitive 4 male 4 bangkalan mitis < 0.016 sensitive 5 female 20 bangkalan mitis 0.016 sensitive 6 female 6 bangkalan mitis 0.016 sensitive 7 female 23 bangkalan mitis < 0.016 sensitive 8 male 6 bangkalan mitis 0.016 sensitive 9 female 12 bangkalan mitis < 0.016 sensitive 10 male 6 bangkalan mitis < 0.016 sensitive 11 female 37 bangkalan mitis < 0.016 sensitive 12 female 17 bangkalan mitis < 0.016 sensitive 13 female 7 bangkalan mitis < 0.016 sensitive 14 female 25 bangkalan mitis 0.016 sensitive 15 male 15 bangkalan mitis 0.016 sensitive 16 male 18 bangkalan mitis < 0.016 sensitive 17 female 36 bangkalan mitis < 0.016 sensitive 18 female 6 bangkalan mitis < 0.016 sensitive 19 female 18 banyuwangi mitis < 0.016 sensitive 20 female 13 bojonegoro mitis < 0.016 sensitive 21 female 13 tuban mitis < 0.016 sensitive 22 female 12 bondowoso mitis < 0.016 sensitive 23 female 16 bondowoso mitis < 0.016 sensitive 24 female 29 bondowoso gravis 1 intermediate 25 male 9 bondowoso mitis < 0.016 sensitive 26 female 20 jember mitis < 0.016 sensitive 27 female 52 jember mitis < 0.016 sensitive 28 male 17 jember mitis < 0.016 sensitive 29 male 11 jember gravis 1 intermediate 30 female 16 jember gravis 1 intermediate systems, especially in well developed countries. the result of the current study was similar to several previous studies. gordon (1970)in texas, usa, used the dilution technique of sensitivity against corynebacterium diphtheriae and showed that all of the 14 toxigenic isolates were sensitive, with mic 0.01 μg/ml.16 mclaughlin (1971) in atlanta, usa, used erythromycin 15 μg ina disk diffusion technique for a sensitivity test against corynebacterium diphtheriae and showed that 136 isolates between 1969–1970 were sensitive, with a mean mic of approximately 40 mm (sensitive ≥ 23 mm).17 the study by rockhill et al. in jakarta, indonesia (1980), also using the disk diffusion technique with erythromycin 15 μg, showed that 133 isolates were all sensitive to erythromycin.18 engler et al.(2000) in england also found that 405 of 410 isolates were sensitive, with mic 0.026 μg/ml, and 5 others were resistant with mic of 2–4 μ/ml.19 another study from barraud et al. in france using the etest method showed the susceptibility of many antibiotics including erythromycin against 46 isolates of corynebacterium diphtheriae biovar mitis in the period from 1993 to 2010.20 a study in russia by chagina et al. using the etest method showed that of 664 isolates between 1987–2013, most of them turned out to be sensitive to all antibacterial preparation, although 0.4–0.6% were intermediate and 4–4.4% were resistant to macrolide.21 28 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 24–29 copyright © 2020, ijtid, issn 2085-1103 conclusion erythromycin still has a strong antibacterial activity against corynebacterium diphtheriae(90% sensitive, 10% intermediate). the use of erythromycin for the management of diphtheria, especially for those who have penicillin allergy, or as a prophylactic treatment is recommended. acknowledgements i sincerely thank the faculty of medicine, universitas airlangga and our teachers for their guidance to do this research. i also want to express my gratitude to the staff members of the center for health laboratory, surabaya for their help and providing me with an opportunity to carry out this research. conflict of interest the authors declare that there is no conflict of interest for this research. references 1. centers for disease control and prevention. diphtheria [monograph online]. atlanta, us department of health and human services; 2013. [cited 2013 june 22]. available from: cdc. 2. vyas jm, zieve d, black b, slon s, wang n. diphtheria symptoms and treatment [monograph online]. adam healths solutions; 2013. [cited 2013 may 17]. available from: pubmed. 3. h e a l t h p r o t e c t i o n a g e n c y . i d e n t i f i c a t i o n o f corynebacterium spp. [monograph online] uk standards for microbiology investigations; 2011. [cited 2013 june 23]. available from: hpa. 4. white nj, hien tt. manson’s tropical diseases. 21st ed. 2009. saunders. london: elsevier. p 1133-1137. 5. world health organization. diphtheria reported cases [monograph online]. geneva, who vaccinepreventable diseases monitoring global summary; 2014. [cited 2015 jan 8]. available from: who. 6. health office of east java province. kegiatan sub pin difteri sebagai bagian penanggulangan klb difteri di jawa timur [monograph online]. surabaya, health office of east java province site; 2012. [cited 2015 jan 8]. available from: health office of east java province. 7. indonesian pediatric society. child infection and tropical diseases handbook. 2nd ed. 2008. jakarta:ips. p. 312-321. 8. kanoh s, rubin bk. mechanisms of action and clinical application of macrolides as immunomodulatory medications. clinical microbiology reviews. 2010 jul;23(3):590-615. 9. benes j, dzupova o. treating diphtheria in the 21st century. klinicka mikrobiologie a infekcni lekarstvi. 2013 dec;19(4):112-4. 10. katzung bg, masters sb,trevor aj. basic and clinical pharmacology. 11th ed. san francisco:mcgraw hill professional; 2009. p. 1024-25. 11. neal se, efstratiou a. international external quality assurance for laboratory diagnosis of diphtheria. journal of clinical microbiology. 2009 dec;47(12):4037-4042. 12. vading m, samuelsen o, haldorsen b, sundsfjord as, giske cg. comparison of disk diffusion, etest, and vitek2 for detection of carbapenemase-producing klebsiella pneumoniae with the eucast and clsi breakpoint systems. clinical microbiology and infection. 2011 may;17(5):668-74. 13. clinical and laboratory standards institute. methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria; proposed guideline. us: m45-p; 2007. vol. 25(26). 14. volzke h, kloker km, kramer a, guertier l, doren m, baumeister se, et al. susceptibility to diphtheria in adults: prevalence and relationship to gender and social variables. clinical microbiology and infection. 2006 oct;12(10):961-7. 15. nath b, mahanta tg. investigation of an outbreak of diphtheria in borborooah block of dibrugarh district, assam. indian journal of community medicine. 2010 jul;35(3):436-438. 16. gordon rc, yow md, clark dj, stephenson wb. in vitro susceptibility of corynebacterium diphtheriae to thirteen antibiotics. applied microbiology. 1971 mar;21(3):548-549. 17. barraud o, badell e, denis f, guiso n, ploy mc. antimicrobial drug resistance in corynebacterium diphtheriae mitis. emerging infectious diseases. 2011 nov;17(11):2078-2080. 18. chagina ia, borisova o, mel’nikov vg, ivashinnikova ga, pimenova as, donskikh ee, et al. sensitivity of corynebacterium diphtheriae strains to antibacterial preparations. zhurnal mikrobiologii epidemiologii i immunobiologii. 2014 jul-aug;(4):8-13. 19. sariadji k, sunarno, puspandari n, sembiring m. antibiotic susceptibility pattern of corynebacterium diphtheriae isolated from outbreaks in indonesia 2010-2015. the indonesian biomedical journal. 2018 apr; 10(1):51-55 20. husada d, soegianto sdp, kurniawati is, hendrata ap, irawan e, kartika l, et al. first-line antibiotic susceptibility pattern of toxigenic cortynebacterium diphtheriae in indonesia. bmb infectious disease. 2019 dec;19(1):1049. vol. 8 no. 1 january–april 2020 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ copyright © 2020, ijtid, issn 2085-1103 original article clinical and hemoglobin profile of malaria patients in karitas hospital, southwest sumba district, indonesia during 2017 alvin johan1*, audrey natalia2, william djauhari3, rambu farah effendi4 1 watu kawula healthcare center, southwest sumba 2 karitas hospital, southwest sumba 3 amahai healthcare center, central maluku 4 department of internal medicine, waikabubak regional general hospital, west sumba received: 1st january 2019; revised: 22nd october 2019; accepted 2nd january 2020 abstract malaria infections in high endemic areas are not pathognomonic and often show non-specifi c symptoms. the southwest sumba district is a high endemic area of malaria with the annual parasite incidence (api) of 14.48‰. the research conducted in this area was to identify the clinical and hemoglobin profi le of malaria patients and to obtain comprehensive information on the clinical characteristics of malaria in a high endemic area of southwest sumba district. this is a descriptive cross-sectional study. the data was obtained from the medical record of malaria patients between january 1st and december 31st, 2017 in karitas hospital, southwest sumba district. inclusion criteria were patients with asexual stages of plasmodium spp. on their giemsa-stained thick and thin peripheral blood smears examination. exclusion criteria were malaria patients with coexisting diseases and who had taken medication before admitted to the hospital. the total number of patients was 322 patients, 50.6% of the subjects were ≥ 15 years old and 59.3% were male. among 322 patients, 133 subjects were treated as inpatients. the result shows that most infection was caused by a single infection of p. falciparum. the most common clinical symptom was fever (98.4%), followed by headache, vomiting, cough, and nausea. the most common physical fi nding was the axillary temperature of > 37.5°c (87.6%) followed by anemic conjunctiva and hepatomegaly, which was mostly found in pediatric patients. the number of patients with hemoglobin level ≤ 10 g/dl was 129. the mcv <80 fl was found in 79% of patients with anemia. severe malaria was found in 116 subjects in this study according to severe malaria criteria set by the indonesian ministry of health. study results were consistent with other existing studies from other high endemic areas in east nusa tenggara province. keywords: malaria, plasmodium, clinical profile, hemoglobin, east nusa tenggara abstrak infeksi malaria di daerah endemis tinggi seringkali tidak khas dengan keluhan klinis tidak spesifi k. kabupaten sumba barat daya merupakan daerah endemis tinggi malaria dengan annual parasite incidence (api) sebesar 14.48‰. penelitian yang dilakukan pada daerah ini untuk mengidentifi kasi profi l klinis dan hemoglobin pasien malaria dan memperoleh informasi komprehensif mengenai karakteristik klinis malaria di kabupaten sumba barat daya yang merupakan daerah endemis tinggi. penelitian ini adalah penelitian deskriptif dengan metode potong lintang. data diambil dari rekam medis pasien malaria dari tanggal 1 januari sampai dengan 31 desember 2017 di rumah sakit karitas, kabupaten sumba barat daya. kriteria inklusi adalah pasien yang melakukan pemeriksaan hapus darah tepi tebal tipis dengan pewarnaan giemsa dan ditemukan stadium aseksual plasmodium spp. kriteria eksklusi adalah pasien malaria dengan penyakit penyerta dan pasien yang sudah meminum obat sebelum datang ke rumah sakit. total pasien berjumlah 322 pasien, 50.6% termasuk dalam kelompok usia ≥ 15 tahun dan 59.3% berjenis kelamin laki-laki. dari 322 pasien, 133 pasien dirawat inap. hasil penelitian menunjukkan kebanyakan infeksi malaria disebabkan oleh infeksi tunggal p. falciparum. gejala klinis yang paling sering ditemukan adalah demam (98.4%), diikuti oleh sakit kepala, muntah, batuk, dan mual. hasil pemeriksaan fi sik corresponding author. e-mail: alvinjohanmd@gmail.com 2 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 1–8 copyright © 2020, ijtid, issn 2085-1103 yang paling banyak ditemukan adalah suhu aksila > 37.5°c (87.6%) diikuti oleh konjungtiva anemis dan hepatomegali yang kebanyakan ditemukan pada pasien pediatrik. sebanyak 129 pasien memiliki kadar hemoglobin ≤ 10 g/dl. kadar mcv < 80 fl ditemukan pada 79% pasien dengan anemia. malaria berat ditemukan pada 116 subjek dalam penelitian ini berdasarkan kriteria dari kementerian kesehatan indonesia. hasil penelitian konsisten dengan penelitian lain di daerah endemis tinggi di provinsi nusa tenggara timur. kata kunci: malaria, plasmodium, profil klinis, hemoglobin, nusa tenggara timur how to cite: johan, alvin. natalia, audrey. djauhari, william. eff endi, rambu farah. clinical and hemoglobin profi le of malaria patients in karitas hospital, southwest sumba district, indonesia during 2017. indonesian journal of tropical and infectious disease, [s.l.], v. 8, n. 1, p. 167-174, jan. 2020. issn 2085-1103. available at: https://e-journal.unair.ac.id/ ijtid/article/view/13454. date accessed: 09 dec. 2019. doi: http://dx.doi.org/10.20474/ijtid.v8i1.13454 introduction malaria is a disease caused by a plasmodium spp. infection and transmitted through anopheles spp. mosquito bites.1,2 in 2015, there were 214 million estimated cases of malaria worldwide, with the highest prevalence in countries with a tropical climate such as africa, south america, and southeast asia.2,3 classical symptoms of malaria include acute paroxysmal fever followed by shivering and excessive sweating. different types of plasmodium spp. may cause different fever patterns. infection of plasmodium falciparum and plasmodium knowlesi may cause intermittent or continuous fever, plasmodium vivax and plasmodium ovale may cause 2days interval paroxysmal fever, while plasmodium malaria infection may cause 3-days intermittent fever. these classical symptoms often were found in the non-immune population from non-endemic areas.1,2 other non-classical symptoms such as headache, nausea, vomiting, diarrhea and muscle pain can also be found. these non-specific symptoms were often found in the population of high endemic areas regardless of high blood parasite density. several cases also show that patients with high parasite density maybe asymptomatic.1,4 the endemicity of malaria is determined by the number of annual parasite incidence (api), which determined by the number of morbidity per 1,000 populations at risk of infection. the endemicity of malaria can be divided into 4 categories, high endemic areas with api > 5‰, moderate endemic areas with api 1-5‰, low endemic areas with api 0-1‰ and non-endemic areas with api 0‰.5 national health profile of east nusa tenggara province from the year of 2015 showed there were 4,622 malaria cases from 319,119 populations at risk in southwest sumba district, with api of 14.48‰.6 the research on the clinical profile of malaria in high endemic areas, especially in east nusa tenggara province was lacked. within the last 5 years, there were only two researches conducted by mau, et al from central sumba district in 20147 and junardi, et al from belu district in 2017.8 mau, et al7 did not discuss physical findings in malaria patients, while junardi, et al8 only focused on the infection of p. falciparum. this research focused on demographical data, history of clinical symptoms, finding of clinical signs and hemoglobin level to obtain comprehensive information on clinical characteristics of malaria in a high endemic area of southwest sumba district. materials and methods this research was a descriptive study with cross-sectional methodology. the data were collected from medical records of patients with a microscopic diagnosis of malaria between january 1st and december 31st, 2017 in karitas hospital. ethical clearance was issued by the medical committee of karitas hospital numbered 008/dir.bpip.e/rsk/vi/208. inclusion criteria were patients with available giemsa-stained thick and thin peripheral blood smears examination and positive containing asexual stages of plasmodium spp.2 exclusion criteria were malaria patients with 3alvin johan, et al.: clinical and hemoglobin profile of malaria patients copyright © 2020, ijtid, issn 2085-1103 coexisting diseases or who have taken medication before admitted to the hospital. a total number of 490 malaria patients involved in this study. however, 168 subjects out of them were then excluded. one hundred out of 168 subjects were due to having a coexisting disease, while 68 subjects had taken medication before admission. therefore, the total number of 322 subjects were used in this study. the density of the parasite was determined by a semi-quantitative or plus-system.9,10 the data were then analyzed using cross-tabulation on the ibm spss statistic 24 software. results and discussion basic characteristics of subjects the basic characteristics of 322 subjects were described in table 1. table 1. basic characteristics of subjects characteristics n (%) demographic characteristics gender male 191 (59.3) female 131 (40.7) age (years), median (range) 15 (0.79) age group 0 – <6 years 73 (22.7) 6 – <15 years 86 (26.7) ≥15 years 163 (50.6) clinical characteristics severe malaria infection 116 (36.0) uncomplicated malaria infection 206 (64.0) admission status outpatient 189 (58.7) inpatient 133 (41.3) based on the age group, malaria subjects were categorized into three age groups (0 – <6 years; 6 – < 15 years; ≥ 15 years). the majority of malaria-infected subjects were ≥ 15 years old. most malaria research in indonesia found that ≥ 15 years old age group was more vulnerable to malaria infection11–15 probably due to the frequent outdoor activity of this age group. the exophagic and exophilic behavior of anopheles indeed play a role in higher malaria infection in this age group.14,16 microscopic-based diagnosis the results of microscopic examination of giemsa-stained thick and thin peripheral blood smears showed that 265 (82.3%) out of 322 patients were infected with p. falciparum, 43 (13.4%) were infected with p. vivax and 3 (0.9%) were infected with p. malaria (table 2). mixed infection was found in 11 (3.4%) subjects, where 10 patients were infected with p. falciparum and p. vivax. one patient was infected with p. falciparum, p. vivax, and p. malaria. these results were consistent with purba, et al16 that concluded malaria cases in east nusa tenggara province were mainly caused by p. falciparum and p. vivax. a low rate of p. vivax infection in an area indicates successful management of malaria cases because the hypnozoites that withstand inside the liver were well treated.16 infections of p. ovale and p. knowlesi were not found in our study. species of p. ovale often found in west africa.2,3 while p. knowlesi infections occurred in the west borneo, where macaca fascicularis and macaca nemestrina are the main host.17 parasite density parasite density was measured using a semiquantitative method on giemsa-stained thick table 2. parasite density based on plasmodium species parasite density plasmodium spp. [n (%)] total n = 322p. falciparum n = 265 p. vivax n = 43 p. malariae n = 3 mixed infection n = 11 + 35 (13.2) 8 (18.6) 1 (33.3) 1 (9.1) 45 (14.0) ++ 24 (9.1) 7 (16.3) 1 (33.3) 4 (36.4) 36 (11.1) +++ 48 (18.1) 15 (34.9) 1 (33.3) 2 (18.2) 66 (20.6) ++++ 158 (59.6) 13 (30.2) 0 (0.0) 4 (36.4) 175 (54.3) 4 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 1–8 copyright © 2020, ijtid, issn 2085-1103 and thin peripheral blood smears. this method indicated the degree of infection, (+) for 1-10 asexual parasites per 100 thick film fields, (++) for 11-100 asexual parasites per 100 thick film fields, (+++) for 110 asexual parasites per single thick film field, and (++++) for > 10 asexual parasites per single thick film field.9,10 distribution of parasite density based on plasmodium species is available in table 2. clinical symptoms the clinical symptoms were summarized in table 3. fever was observed in almost all (98.4%) of malaria subjects. this finding is consistent with previous studies by mau, et al7 and junardi, et al,8 where 96.8% and 100% of table 3. clinical symptoms based on plasmodium species system symptoms plasmodium species [n (%)] total n = 322p. falciparum n = 265 p. vivax n = 43 p. malaria n = 3 mixed infection n = 11 general respiratory fever 261 (98.5) 42 (97.7) 3 (100) 11 (100) 317 (98.4) cough 64 (24.2) 12 (27.9) 1 (33.3) 2 (18.2) 79 (24.5) runny nose 33 (12.5) 5 (11.6) 1 (33.3) 2 (18.2) 41 (12.7) breathlessnessa 3 (1.1) 2 (4.7) 0 (0) 0 (0) 5 (1.6) sore throat 7 (2.6) 1 (2.3) 0 (0) 0 (0) 8 (2.5) gastrointestinal nausea 56 (21.1) 6 (14) 1(33.3) 3 (27.3) 66 (20.5) vomiting 99 (37.4) 6 (14) 2 (66.7) 3 (27.3) 110 (34.2) epigastric pain 47 (17.7) 4 (9.3) 0 (0) 0 (0) 51 (15.8) anorexia 50 (18.9) 7 (16.3) 2 (66.7) 3 (27.3) 62 (19.3) diarrhea 16 (6) 1 (2.3) 0 (0) 0 (0) 17 (5.3) constipation 2 (0.8) 0 (0) 0 (0) 0 (0) 2 (0.6) abdominal painb 20 (7.5) 2 (4.7) 0 (0) 0 (0) 22 (6.8) neurology headache 111 (41.9) 19 (44.2) 1 (33.3) 2 (18.2) 133 (41.3) unconscious 18 (6.8) 0 (0) 0 (0) 0 (0) 18 (5.6) seizure 1x 4 (1.5) 2 (4.7) 0 (0) 0 (0) 6 (1.9) recurrent seizure 12 (4.5) 1 (2.3) 0 (0) 1 (9.1) 14 (4.3) behavioral change 7 (2.6) 0 (0) 0 (0) 0 (0) 7 (2.2) hematology lethargy 34 (12.8) 7 (16.3) 1 (33.3) 1 (9.1) 43 (13.4) pale 24 (9.1) 1 (2.3) 0 (0) 0 (0) 25 (7.8) ictericc 1 (0.4) 0 (0) 0 (0) 0 (0) 1 (0.3) bleedingd 1 (0.4) 0 (0) 0 (0) 0 (0) 1 (0.3) musculo-skeletal muscle pain 11 (4.2) 7 (16.3) 0 (0) 0 (0) 18 (5.6) joint pain 7 (2.6) 1 (2.3) 0 (0) 0 (0) 8 (2.5) back pain 9 (3.4) 3 (7) 0 (0) 0 (0) 12 (3.7) a: all patients had spo2 of > 95% b: all other regions of abdomen beside the epigastric region c: icteric on conjunctiva or skin d: the only bleeding manifestation found in this study was anterior epistaxis malaria patients respectively underwent fever.7,8 fever is the most common symptom of malaria infection especially in high endemic areas of malaria, therefore, patient with fever leads to clinical suspicion of malaria infection.1,2 malaria toxin called glycosylphosphatidylinositol (gpi) and hemozoin are released when schizonts burst. the toxins trigger the immune system to release pyrogenic pro-inflammatory cytokines such as tumor necrosis factor (tnf)-α, interleukin (il)-1 and il-6.18 headache was the most common presenting symptom in malaria subjects after fever, this finding is consistent with other studies by mau, et al7 and purwanto, et al14 in indonesia, herrera, et al19 in colombia, and deshwal, et al20 in 5alvin johan, et al.: clinical and hemoglobin profile of malaria patients copyright © 2020, ijtid, issn 2085-1103 india. headache in malaria infection has an acute onset with non-specific pain distribution.21 pro-inflammatory cytokines such as tnf-α and il-6 are believed to play an important role in the pathogenesis of headaches.2,17,22 pain intensity and frequency of headache between cerebral malaria and non-severe malaria infection is clinically indistinguishable.21 two studies by muddaiah, et al 23 and sonawane, et al24 from india showed a higher incidence of nausea and vomiting than a headache in malaria subjects. the study by junardi, et al8 also found the most common symptoms in malaria subjects were nausea and vomiting, followed by headache, with an incidence of 67.7% and 50.7% respectively. these findings indicated that geographically different endemic areas of malaria are resulting in different profiles of malaria symptoms. body responses to malaria toxins are believed to cause nausea. vomiting has been associated with high parasite density in malaria subjects.25 anstey, et al26 in australia reported cough from malaria patients with an incidence of 36% in p. falciparum-infected subjects and 53% in p. vivax-infected subjects. the cough was not observed by subjects before malaria infection. the cough was described as non-productive and mostly found on subjects who smoked cigarettes. auscultation and chest x-ray performed on malaria subjects with cough revealed no abnormalities. the cough is thought to be caused by increased activity of intravascular monocytes in the lungs which lead to subclinical endothelial dysfunction.26 the suspicion of malaria infection should not be dismissed in patients presenting with fever and cough in a high endemic area. it is difficult to distinguish whether the cough is caused by malaria or other respiratory viruses. testing for malaria is important in patients presenting with flu-like symptoms at health centers in high endemic area.25 physical findings meaningful physical findings found in subjects are listed in table 4. in all cases of the glasgow coma scale (gcs) <11, p. falciparum was found on giemsa-stained thick and thin peripheral blood smear. hepatomegaly (26.8%) was more prevalent in malaria subjects in karitas hospital than splenomegaly (15.1%). this finding was in contrast with the study by purwanto, et al14 that found hepatomegaly (15.4%) was less prevalent than splenomegaly (27.4%). this discrepancy between the two studies was likely due to the difference in demographic characteristics of the subjects. most subjects in purwanto, et al14 study was in 31-40 years old age group and just 0.6% of the subjects aged < 10 years old.14 children with malaria infection are more likely to develop hepatomegaly.2 hepatomegaly is caused by an inflammatory reaction and usually non-tender on palpation.27,28 histopathology observation of hepatic portal system in a patient with severe malaria showed increased activity of nuclear factor-kappa b p65 (nf-κbp65), followed by kupffer cells and lymphocytes apoptosis.29 malarial hepatopathy is a term used to described hepatic dysfunction in patients with severe malaria as shown by increased serum bilirubin and transaminase enzymes. co-infection of hepatitis viruses and exposure to hepatotoxic substances must be excluded to confirm the table 4. physical findings based on plasmodium species physical findings plasmodium species [n (%)] total n = 322p. falciparum n = 265 p. vivax n = 43 p. malaria n = 3 mixed infection n = 11 axillary temperature > 37.5°c 233 (87.9) 35 (81.4) 3 (100) 11 (100) 282 (87.6) hepatomegaly 71 (26.8) 5 (11.6) 0 (0) 0 (0) 76 (23.6) splenomegaly 40 (15.1) 6 (14) 1 (33.3) 0 (0) 47 (14.5) glasgow coma scale <11 15 (5.7) 0 (0) 0 (0) 1 (9.1) 16 (5) icteric sclera 15 (5.7) 1 (2.3) 0 (0) 1 (9.1) 17 (5.3) anemic conjunctiva 82 (30.9) 2 (4.7) 2 (66.7) 2 (18.2) 88 (27.3) 6 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 1–8 copyright © 2020, ijtid, issn 2085-1103 diagnosis of malarial hepatopathy in malaria subjects. splenomegaly is caused by increased erythrocytes destruction, there is also increased activity of mature lymphocytes attacking the erythrocytes, leading to hypertrophy.27 decreased consciousness may indicate central nervous system involvements in malaria infection. however, not all unconscious patients should be treated as having severe malaria. who 2015 criteria of severe malaria use gcs < 11 as the cut-off for clinical diagnosis of severe malaria. comatose patients who were tested positive for p. falciparum infection are diagnosed with cerebral malaria which has a worse prognosis.2,9,22,30 erythrocytes infected by p. falciparum express plasmodium falciparum erythrocyte membrane adhesive protein 1 (pfemp1) which will bond with intracellular adhesion molecule 1 (icam-1) on neurovascular endothelium, causing sequestration that leads to diffuse symmetrical encephalopathy.2,30 the icteric sclera was found in 5.3% of subjects in this study, similar to the study by purwanto, et al14 which found icteric sclera in 3.4% of subjects. the icteric sclera is caused by abnormal deposition of bilirubin in the sclera. according to who 2015 criteria, malaria patient with total serum bilirubin > 3 mg/dl is considered to have severe malaria infection.1,2 icteric sclera was noticeable only in 68% of patients with total serum bilirubin >3.1 mg/dl in one study.27 a routine check of the total of serum bilirubin in malaria subject is suggested, even when sclera appears anicteric on initial examination. anemic conjunctiva indicates oxyhemoglobin deficiency in conjunctiva capillaries, a common finding in patients with anemia.27 hemoglobin profile hemoglobin profile was observed in 270 (83.9%) out of 322 subjects as shown in table 5. anemia, which defined as hemoglobin level ≤ 10 g/dl was found in 129 subjects, only anemia, which defined as hemoglobin level ≤ 10 g/dl was found in 129 subjects, only 55.8% of them appeared to have anemic conjunctiva on initial examination. from 85 subjects whose conjunctiva appear anemic on physical examination, 84.7% was confirmed to have anemia from blood count. literature showed that anemic conjunctiva has sensitivity and specificity of 25-62% and 82-97% respectively for the diagnosis of anemia.27 according to the indonesian ministry of health1, severe malaria in malaria cases in a high endemic area was determined by hemoglobin level < 5 g/dl in children and < 7 g/dl in adults. acute malaria infection usually causes normochromic normocytic anemia by causing hemolysis of plasmodium-infected erythrocytes, a c c e l e r a t e d r e m o v a l b y t h e s p l e e n , a n d dyserythropoietic.2,31,32 in this study, mean corpuscular volume (mcv) < 80 fl were found in 102 (79%) subjects with anemia. the high number of microcytic anemia in this study reflected the chronicity of malaria infection in the area. the underlying medical conditions such as iron deficiency, hemoglobinopathy, or chronic diseases may also cause microcytic anemia.32,33 further tests should be done to confirm the etiology of anemia in these subjects and cannot be done due to limited laboratory equipment. severe malaria based on the clinical symptoms, physical findings and laboratory findings, 116 (36%) table 5. hemoglobin profile based on plasmodium species hemoglobin (g/dl) plasmodium species [n(%)] total n = 270p. falciparum n = 225 p. vivax n = 31 p. malaria n = 3 mixed infection n = 11 >10 111 (49.3) 22 (71.0) 0 (0) 8 (72.7) 141 (52.2) 5 – 10 96 (42.7) 8 (25.8) 3 (100) 3 (27.3) 110 (40.7) <5 18 (8.0) 1 (3.2) 0 (0) 0 (0) 19 (7.0) 7alvin johan, et al.: clinical and hemoglobin profile of malaria patients copyright © 2020, ijtid, issn 2085-1103 subjects in this study had met the criteria of severe malaria infection as stated by indonesian ministry of health.1 majority (68.1%) of subjects with severe malaria were from <15 years old age group. p. falciparum was found in 87.9% of subjects with severe malaria. six out of 116 subjects with severe malaria infection were deceased. all of the deceased subjects had p. falciparum infection with a parasite density of ++++. study limitations this descriptive study has some limitations. medical records used in this study did not reflect all clinical symptoms underwent by malaria patients, probably did not ask by the physician during history taking or some information was not documented. physical findings may vary between examiners as it was determined by examiner’s experiences. another limitation is ancillary tests that are not routinely done in all malaria patients, so the true incidence of severe malaria based on the indonesian ministry of health criteria may be higher than reported in this study. conclusion research for clinical and hemoglobin profiles of malaria especially in high endemic areas has been lacking. this is important as malaria infection in high endemic areas is often not pathognomonic. this study in karitas hospital, southwest sumba district which was a high endemic area of malaria showed similar results with previous studies in other areas of east nusa tenggara province within the last 5 years. our study revealed that most of the malaria subjects in southwest sumba district were infected by p. falciparum. fever was the highest presenting clinical symptom and physical finding in malaria subjects. the most common clinical symptoms following fever were headache, vomiting, cough, and nausea. anemia and hepatomegaly were the most common physical findings following fever. hemoglobin profile of malaria patients in karitas hospital showed that anemia was found in less than half of subjects. most of the anemic subjects had microcytic anemia. severe malaria was found mostly in p. falciparum infection, and all the death of patients was due to p. falciparum infection. acknowledgements the authors thank to the director of karitas hospital for allowing us to conduct this study by using the hospital’s medical records. conflict of interest there was no conflict of interest for this research. references 1. samad i, theodora m, mulyani ps, editors. buku saku penatalaksanaan kasus malaria. jakarta: ditjen pencegahan dan pengendalian penyakit kementerian kesehatan ri; 2017. 2. white nj, breman jg. malaria. in: kasper dl, fauci as, hauser s, et al, editors, harrison’s principles of internal medicine, 19th ed. new york: the mcgraw-hill companies, inc.; 2015. 3. phillips ma, burrows jn, manyando c, van huijsduijnen rh, van voorhis wc, wells tnc. malaria. nat rev dis primer. 2017 aug 3;3:17050. 4. liwan a. diagnosis dan penatalaksanaan malaria tanpa komplikasi pada anak. cermin dunia kedokteran; 2015. 5. anastasia h, nurjana m, jastul. validitas gejala klinis sebagai indikator untuk memprediksi kasus malaria di indonesia. media litbangkes. 2013 dec;23(4):149-57. 6. mete k, hala k. profil kesehatan provinsi nusa tenggara timur tahun 2015. dinas kesehatan provinsi nusa tenggara timur; 2015. 7. mau f, sopi ib. kesesuaian gejala klinis malaria dengan parasitemia positif di wilayah puskesmas wairasa kabupaten sumba tengah provinsi nusa tenggara timur. media peneliti dan pengemb kesehat. 2014;24(2):75–80. 8. junardi rb, somia ka. karakteristik klinis malaria tropika pada pasien rawat inap di rumah sakit umum daerah mgr. gabriel manek, svd atambua periode september 2013 februari 2014. e-j med. 2017 jul;6(7). 9. pedoman teknis pemeriksaan parasit malaria. direktorat jenderal pencegahan dan pengendalian 8 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 1–8 copyright © 2020, ijtid, issn 2085-1103 penyakit kementerian kesehatan republik indonesia; 2017. 10. world health organization. manual of basic techniques for a health laboratory. 2nd ed. geneva:world health organization.2003;p182. 11. irawan h, merry ms, wuryaningsih ns, baskoro t. profil hematologik berdasarkan jenis plasmodium pada pasien malaria rawat inap di rsk lindimara, sumba timur. berk ilm kedokt duta wacana. 2017 apr;2(2):394–401. 12. dwithania m, irawati n, rasyid r. insiden malaria di puskesmas sungai durian dan puskesmas talawi kota sawahlunto bulan oktober 2011 sampai februari 2012. j kesehat andalas. 2013;2(2):76–9. 13. gusra t, irawati n, sulastri d. gambaran penyakit malaria di puskesmas tarusan dan puskesmas balai selasa kabupaten pesisir selatan periode januari-maret 2013. j kesehat andalas. 2014;3(2). 14. purwanto ds, ottay ri. profil penyakit malaria pada penderita rawat inap di rumah sakit umum daerah kota bitung. j biomedik. 2011;3(3). 15. nababan r, umniyati sr. faktor lingkungan dan malaria yang memengaruhi kasus malaria di daerah endemis tertinggi di jawa tengah: analisis sistem informasi geografis. ber kedokt masy. 34(1):11–8. 16. purba ie, hadi uk, hakim l. analisis pengendalian malaria di provinsi nusa tenggara timur dan rencana strategis untuk mencapai eliminasi malaria. spirakel. 2017 feb 9;8(2). 17. bartoloni a, zammarchi l. clinical aspects of uncomplicated and severe malaria. mediterr j hematol infect dis. 2012 may ;4(1):2012-026. 18. oakley ms, gerald n, mccutchan tf, aravind l, kumar s. clinical and molecular aspects of malaria fever. trends parasitol. 2011 oct;27(10):442–9. 19. arévalo-herrera m, lopez-perez m, medina l, moreno a, gutierrez jb, herrera s. clinical profile of plasmodium falciparum and plasmodium vivax infections in low and unstable malaria transmission settings of colombia. malar j. 2015;14(1):154. 20. deshwal r. clinical and laboratory profile of hospitalized malarial patients: an agra-based study. j assoc physicians india. 2016;64:44. 21. wiwanitkit v. headache and malaria: a brief review. acta neurol taiwan. 2009;18(1):56–9. 22. john cc. malaria (plasmodium). in: kliegman r, nelson we, editors. nelson textbook of pediatrics, 20th ed. philadelphia, pa: elsevier, saunders; 2016. 23. muddaiah m, prakash ps. a study of clinical profile of malaria in a tertiary referral centre in south canara. j vector borne dis. 2006 mar;43:29–33. 24. sonawane vb, kotrashetti v, malhotra r. comparison of clinical profile and severity of p. falciparum and p. vivax malaria in a tertiary care hospital of navi mumbai, india: a descriptive study. jmscr; 2017. 25. martins ac, araújo fm, braga cb, guimaraes mg, nogueira r, arruda ra, et al. clustering symptoms of non-severe malaria in semi-immune amazonian patients. peerj. 2015;3:e1325. 26. anstey nm, jacups sp, cain t, pearson t, ziesing pj, fisher da, et al. pulmonary manifestations of uncomplicated falciparum and vivax malaria: cough, small airways obstruction, impaired gas transfer, and increased pulmonary phagocytic activity. j infect dis. 2002;185(9):1326–34. 27. dennis m, bowen wt, cho l. mechanisms of clinical signs. australia: elsevier australia; 2016. 28. tarafder bk, islam mt, roshed mm, siddique mab, hossain am, sarker kd. vivax malaria presenting with fever and tender hepatomegaly. faridpur med coll j. 11(2):90–2. 29. viriyavejakul p, khachonsaksumet v, punsawad c. liver changes in severe plasmodium falciparum malaria: histopathology, apoptosis and nuclear factor kappa b expression. malar j. 2014;13(1):106. 30. abhijeet m, kanjaniindira ps. a clinical profile of cerebral malaria with p. falciparum infection. ijsr. 2017; 6(5):729-32. 31. castelli f, sulis g, caligaris s. the relationship between anaemia and malaria: apparently simple, yet controversial. trans r soc trop med hyg. 2014 apr ;108(4):181–2. 32. quintero jp, siqueira am, tobón a, blair s, moreno a, arévalo-herrera m, et al. malaria-related anaemia: a latin american perspective. mem inst oswaldo cruz. 2011 aug;106 suppl 1:91–104. 33. donker ae, raymakers ra, vlasveld lt, barneveld t van, terink r, dors n, et al. practice guidelines for the diagnosis and management of microcytic anemias due to genetic disorders of iron metabolism or heme synthesis. blood j. 2014 jun;123(25):3873-86. ijtid vol 3 no 1 jan-maret 2012.indd 10 vol. 3. no. 1 january–march 2012 catheter duration and the risk of sepsis in premature babies with umbilical vein catheters hartojo1, martono tri utomo2 1 division of neonatology, department of child health, husada utama hospital 2 department of child health, dr. soetomo general hospital abstract umbilical catheters are frequently required in the management of severely ill premature babies. the risk of complications may increase with duration of uvc use. objective: to determine whether the risk of central line-associated bloodstream infections (clabsis) and sepsis remained constant over the duration of umbilical vein catheters (uvcs) in high-risk premature neonates. methods: retrospective analysis. the data were collected from the medical record of high risk premature neonates who had a uvc placed in neonatal care unit of husada utama hospital between april 1st 2008 to april 30th 2011 with purposive sampling. catheter duration was observed before and after 14 days on placement. blood and uvc culture was performed to establish the risk of cla-bsis and sepsis. chi-square and logistic regression analysis were performed in the laboratorium data. result: a total 44 high risk premature babies with uvcs were enrolled (sepsis group: n = 23 and non sepsis group: n = 21). baseline demographics were similar between the groups. 15 babies in sepsis group have uvcs duration > 14 days, and 8 babies have uvcs < 14 days (p = 0.533). days of uvc < 14 days show uvcs culture performance in 11 babies with positive evidence, blood culture performance shows negative in 21 babies (p = 0.516). days of uvc >14 days show blood culture performance in 11 babies with positive evidence, uvcs culture performance is negative in 18 babies (p = 0.456). burkholderia cepacia and klebsiella pneumonia mostly appeared in blood culture performance. 25% of uvc culture performance shows pseudomonas aeroginosa. conclusions: the catheter duration have no significant difference in risk of sepsis in premature babies with umbilical vein catheters. key words: premature babies sepsis – days of uvc – cla-bsis introduction umbilical catheters are frequently required in the management of severely ill neonates.1.2 umbilicalvein catheters (uvcs) can be used for intravenous administration of parenteral nutrition, hypertonic solutions, blood products, and medication. umbilical-artery catheters (uacs) can be used for blood sampling and continuous monitoring of blood pressure. however, the advantages of umbilical catheters must be carefully balanced against the potential risks. several life threatening complications have been associated with the use of umbilical catheters including catheter-related infections, intestinal necrosis, thrombosis, cardiac arrhythmias, myocardial perforation, as well as pleural and pericardial effusion.1.2.3 according to the literature, mechanical adverse events occur in 5 to 19% of patients with a uvc, infectious adverse events in 5 to 26% and thrombosis in 2 to 26%.4.5 the incidence of neonatal sepsis is approximately 1 to 10 cases per 1000 live births and 1 per 250 live premature births. the incidence rates of neonatal infection in several referral hospitals in indonesia is approximately 8.76%–30.29% with the mortality rate is 11.56%–49.9%. the incidence rates of neonatal sepsis in several referrals hospital in indonesia is 1.5%–3.72% with the mortality rate is 37.09%–80%.6 because the risk of complications may increase with duration of use, uvcs are often removed after relatively short periods and replaced with percutaneous central venous catheters (pcvcs) for maintenance of long-term fluid and nutritional status.2.7 on the basis of these limited data, the centers for disease control and prevention research report 11hartojo: catheter duration and the risk of sepsis in premature babies currently recommend use of uvcs be limited to 14 days. in a retrospective review of 230 infants with birth weights 1251 g who were admitted to our nicu and required a uvc and/or pcvc, the apparent proportion of catheters remaining infection free at 20 days (the time at which the last uvc was removed) was 89% for uvcs and 73% for pcvcs.8 uvcs comprise a large proportion of central lines inserted in the nicu. central line–associated bloodstream infections (cla-bsis) can complicate piccs. an estimated 80 000 cla-bsis occur in the united states every year. the mortality rate for these cla-bsis remains unclear, but recent studies demonstrated a range of 4% to 20%. cla-bsi extends patient length of stay by an average of 7 days, and the attributable cost is $3700 to $29 000 per infection.3.5.8 in this study, we prospectively examined catheterrelated bacteremia and associated sepsis complications in long-term use of uvcs. methods subjects the study was retrospectively done at nicu of husada utama hospital, conducted for 3 years (april 1st 2008 until april 30th 2011). the premature infants with birth weights less than 2000 g who had a uvc placed on nicu admission were eligible for the study. infants who required a uvc for exchange transfusion, infants with gastrointestinal abnormalities including gastroschisis and omphalocele, or infants with congenital heart disease with intra cardiac shunting were excluded. the parents or legal guardians of the patients gave informed consent before enrollment. umbilical catheterization placement of a uvc was attempted in infants < 2000 g on admission to the nicu. either a single or double lumen catheter (3.5f diameter, polyurethane 1270 catheter; vygon healthcare, gloucestershire, uk) was inserted under sterile conditions. a double-lumen uvc was used if it was technically possible to place one. care of the catheters was standardized. catheters were attached to transducers, was changed every 24 hours if the infused concentration of dextrose was >12.5 g/l and every 72 hours for concentrations of dextrose <12.5 g/l. all uvcs had continuous infusion of solutions in the main port. both infusion and flush solutions contained heparin (1.0 iu/ml for infants >1000 g and 0.5 iu/ml for infants <1000 g or on total parenteral nutrition). all catheter connections were checked hourly to guard against any disconnection. catheter placement was confirmed with a chest and abdominal radiograph. the catheter placement was adjusted to place the catheter tip at the inferior vena cava/right atrial junction. we had confirmed the depth of catheter tips using antero-posterior chest x-rays. ideal position of the uvc was defined as the catheter tip being visible between the 9th and 10th thoracic vertebrae on a chest x-rays. catheters were sutured in place into the umbilical cord, and tape was then used to secure the catheter to the infant's abdomen. blood and tips uvc tips cultures blood culture test was performed in premature babies with suspected sepsis based on clinical symptoms, complete blood test and crp using vitex method. whole blood (0.3–1.0 ml) was placed in sterile isolator tubes and transported to the microbiology laboratory. blood was streaked onto blood and chocolate agar plates and then incubated for 5 days under aerobic conditions. uvc tips cultures were placed in an automated reader (bactalert; biomerieux). any positive or potentially positive cultures were gram-stained, streaked on to blood and/or chocolate agar plates (depending on the likely pathogen), and incubated under aerobic conditions. organisms were isolated by either culture system identified with standard microbiologic techniques. definitions clinical sepsis the definition of infection included symptomatology (eg, temperature instability, increased ventilator settings, increased apnea, bradycardia or desaturations, feeding intolerance, lethargy, or blood pressure instability) and either a single positive blood culture for prospectively defined definite pathogens or multiple positive cultures (≥ 2 within 48 hours) for other organisms from usually sterile site(s) (blood, catheter tip, urine, or cerebrospinal fluid, with at least 1 positive culture from the blood).1.9.10 crbsi bacteremia/fungemia in a patient with an intravascular catheter with at least 1 positive blood culture obtained from a peripheral vein, clinical manifestations of infections (fever, chills, and/or hypotension), and no apparent source for the bsi except the catheter. one of the following should be present: a positive semi quantitative (>15 cfu/catheter segment) or quantitative (>103 cfu/catheter segment catheter) culture whereby the same organism (species and antibiogram) is isolated from the catheter segment and peripheral blood; simultaneous quantitative blood cultures with a >5:1 ratio cvc versus peripheral; differential period of cvc culture versus peripheral blood culture positivity of >2 hours.8.11.12 statistical analysis data are presented in distribution tabulation and data analysis was performed with a computer assisted statistical package (spss ver. 12.0). descriptive analysis of catheter duration and risk of sepsis, uvcs and blood culture of the patient were calculated. chi-square analysis and logistic regression were performed in the laboratorium data. 12 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 10−14 results data from april 1st 2008 until april 30th 2011 revealed the premature babies with uvcs were 44 samples. all of them were eligible for analysis, 23 in sepsis group and 21 in no sepsis group. the characteristics of the sample are listed in table 1. table 1. characteristic of high risk premature neonates who had a uvc placed in nicu parameters sepsis no sepsis p n = 23 (%) n = 21 (%) gender .121 female 7 (38.9) 11 (61.1) male 16 (61.5) 10 (38.5) birth weight (g) 1428.3 (sd 324.33) 1450.0 (sd 321.71) .52 gestational age .467 < 30 week's 9 (50.3) 7 (43.8) > 30 week's 14 (50) 14 (50) apgar score .322 ≤ 6 8 (61.5) 5 (38.5) > 6 15(48.4) 16 (51.6) mechanical ventilator .068 yes 18 (62.1) 11 (37.9) no (n-cpap) 5 (33.3) 10 (66.7) table 1 shows that the results have no significant difference based on the gender, birth weight, gestational age, apgar score in premature babies with sepsis risk treated in nicu husada utama hospital. however baby with mechanical ventilator shows to have higher risk compared with n-cpap. in this case: 18 premature babies with mechanical ventilator and 5 with n-cpap affected by sepsis. babies with apgar score less than 6 during the labor have higher risk affected by sepsis, on the other hand apgar score more than 6 shows lower risk. table 2. catheter duration and the risk of sepsis in premature babies days of uvc sepsis positive n(%) negative n(%) < 14 days 15 (53.6) 13 (46.4) > 14 days 8 (50) 8 (50) chi square x2test p = 0.533 table 2 shows that days of uvc have no significant difference in the risk of sepsis in premature babies treated in nicu husada utama hospital. in this study 15 babies with days of uvc less than 14 days were in the risk of sepsis. 8 babies with days of uvc more than 14 days were in the risk of sepsis. table 3. catheter duration and the risk of cla-bsis days of uvc uvc culture blood culture + – + – < 14 days 11 (68.8) 5 (31.2) 7 (25) 21 (75) > 14 days 10 (35.7) 18 (64.3) 11 (68.8) 5 (31.2) chi square x2 test p = 0.516; p = 0.456 table 3 shows that days of uvc have no significant difference in uvc and blood culture result in premature babies treated in nicu husada utama hospital. days of uvc less than 14 days show uvc culture performance in 11 babies suspected sepsis is positive, in fact blood culture performance shows negative in 21 babies. days of uvc more than 14 days show blood culture performance in 11 babies with blood culture positive evidence, even though uvc culture performance is negative in 18 babies. table 4. pathogens that caused cla-bsi in neonates with uvcs in premature babies microorganisms blood culture uvc culture n % n % acinetobacter baumanii 1 2.3 5 11.4 burkholderia cepacia 4 9.1 4 9.1 candida albicans 1 2.3 0 0 enterobacter asburie 1 2.3 0 0 klebsiella pneumoniae 4 9.1 3 6.8 escherichia coli 0 0 4 9.1 pseudomonas aeroginosa 0 0 11 25 enterobacter cloacae 0 0 2 14.5 stenotrophomonas maltophila 1 2.3 0 0 no organism growth 12 27.3 15 34.1 table 4 shows the types of microorganism appeared in blood and uvc culture in 44 premature babies treated in nicu husada utama hospital. burkholderia cepacia and klebsiella pneumonia mostly appeared in blood culture performance. 25% of uvc culture performance shows pseudomonas aeroginosa. of 23 babies suspected sepsis 12 babies show no organism growth on blood culture performance while 15 babies show no organism growth on uvc culture performance. discussion cla-bsis are a common cause of morbidity and mortality among neonates.1.3.6.10 several factors have been 13hartojo: catheter duration and the risk of sepsis in premature babies shown to contribute to the pathogenesis of nosocomial clabsi. host-related risk factors include age, immunologic immaturity, and severity of underlying disease.12.13 in this study shows that gestational age < 30 weeks and mechanical ventilator have contributed the risk of cla-bsi. the risk profiles of a long term uvc to a long-term pcvc have seldom been compared. on the basis of these limited data, the centers for disease control and prevention currently recommend use of uvcs be limited to 14 days. however, a survey of nursery directors revealed that some nicus leave uvcs in place for a longer period of time.7.14.15 the limited data available after 14 days in this study suggest the possibility of increased infection. duration of catheter > 14 days show 11 babies with blood culture positive evidence, although not statistically significant, would have potential clinical significance if it were to be substantiated. migration of skin organisms at the insertion site into the umbilical catheter tract with colonization of the catheter tip is the most common route of infection for centrally inserted, shortterm catheters.4.8.9 contamination of the catheter hub contributes substantially to intraluminal colonization of long-term catheters. occasionally, catheters might become hematogenously seeded from another focus of infection. rarely, infusate contamination leads to crbsi.1.16 important pathogenic determinants of catheter-related infection are 1) the material of which the device is made and 2) the intrinsic virulence factors of the infecting organism.8.9.11 in vitro studies demonstrate that catheters made of polyvinyl chloride or polyethylene are likely less resistant to the adherence of microorganisms than are catheters made of teflon, silicone elastomer, or polyurethane.9.13.15 some catheter materials also have surface irregularities that enhance the microbial adherence of certain species (eg, coagulase-negative staphylococci, acinetobacter calcoaceticus, and pseudomonas aeruginosa); catheters made of these materials are especially vulnerable to microbial colonization and subsequent infection.4.7.8 additionally, certain catheter materials are more thrombogenic than others, a characteristic that also might predispose to catheter colonization and catheter-related infection. this association has led to emphasis on preventing catheter-related thrombus as an additional mechanism for reducing crbsi.17 one study reports that the incident rate of picc related sepsis is between 2 and 21%. this study suggests that the lower incidence of infection in piccs, when compared to other uvcs, might be related to the low concentration of bacteria in peripheral areas (50 to 100 colonies of bacteria per cm2 of skin) when compared to the thorax (1,000 to 10,000 colonies of bacteria per cm2 of skin).18 the literature shows that there are microorganisms more prevalent in catheter-related primary sepsis. the grampositive cocci are responsible for 65% of infections, while the most prevalent are the staphylococcus epidermidis (31%) and the staphylococcus aureus (14%). the gramnegative bacilli account for 30% of infections and the most prevalent are the pseudomonas sp (7%) and the escherichia coli (6%). infection by candida sp is responsible for the remaining 5% of catheter-related infections.1.2.19 coagulase negative staphylococcus was the dominant infection (55.6%) within the first 2 weeks, whereas gram negative bacteria were dominant pathogens (58.3%) after the first 2 weeks.20.21 however, the most frequent microorganism isolated in cultures in this study was the pseudomonas aeroginosa. to avoid contaminating central venous catheters, several measures should be implemented in their insertion and maintenance.10.11 central catheter insertion, whether it is a picc or a uvc, should be aseptic and include measures of barrier precaution such as wearing a cap, mask, sterile gown, sterile gloves and drapes. it is recommended to wash hands with chlorhexidine detergent or alcohol gel before and after contacting with the catheter during uvc maintenance. the dressing has to be changed every seven days or when it is wet or for other reasons taken off, change taps, equipment and extensions every 72 hours and the equipment for parenteral nutrition should be changed every 24 hours, always swabbing the connections and taps of the catheter with 10% concentration of alcohol before handling them.2.6.11.15 adverse events in central catheters were frequent in neonatal populations, both for piccs and in uvcs. the most prevalent adverse event in piccs was catheter occlusion, while clinical sepsis prevailed in uvcs.8.21 piccs presented a higher frequency of mechanical adverse events, especially catheter occlusion and rupture. however, its use presented very low rates of catheterrelated infections; these rates are similar or less than those reported in the literature.20 therefore, we assert that picc is a safe means for parenteral administration in the neonatal population due to the low risk of infection found in this study and in the literature. the use of uvcs resulted in a lower rate of mechanical adverse events: occlusions or ruptures were not found in this catheter in this study. however, the rates of infectious adverse events related to this catheter are the most prevalent.20.21 several limitations should be considered when interpreting our data. we conducted the study on a large cohort of patients over a 3-year period, because our unit has low incidence of cla-bsi. several confounding factors still persist in this study. underlying disease, duration of mechanical ventilator, nosocomial infection were the risk of sepsis in premature babies with uvcs. conclusions the catheter duration have no significant difference in risk of sepsis in premature babies with umbilical vein catheters. burkholderia cepacia and klebsiella pneumonia mostly appeared in blood culture performance. 25% of uvc culture performance shows pseudomonas aeroginosa. 14 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 10−14 references 1. franceschi at, cunha lc. adverse events related to the use of central venous catheters in hospitalized newborns. rev. latinoam. 2010; 18(2): 196–202. 2. o'hara mb, buzzard cj, reubens l, mcdermott mp, digrazio w et.al. a randomized trial comparing long-term and short-term use of umbilical venous catheters in premature infants with birth weights of less than 1251 grams. pediatrics. 2006; 118(1): e25–5. 3. verheij gh, pas ab, witlox rs, wintjens ve, walther fj, and lopriore e. poor accuracy of methods currently used to determine umbilical catheter insertion length. int j pediatr. 2010; 102(4): 1–6. 4. o'grady np, alexander m, dellinger ep, gerberding jl, heard so, maki d et.al. guidelines for the prevention of intravascular catheterrelated infections. pediatrics 2002; 110(5): e51–74. 5. sengupta a, lehmann c, west md, perl tm, milstone am. catheter duration and risk of cla-bsi in neonates with piccs pediatrics. 2010; 125: 648–53. 6. stoll bj, hansen n, fanaroff aa, wright ll, carlo wa, ehrenkranz ra et.al. late-onset sepsis in very low birth weight neonates: the experience of the nichd neonatal research network pediatrics. 2002; 110: 285–94. 7. zahedpasha, kacho ma, hajiahmadi m, haghshenas m. procalcitonin as a marker of neonatal sepsis. iran j pediatr. 2009; 19(2): 117– 22. 8. khilnani p, deopujari s, carcillo j. recent advances in sepsis and septic shock. indian j pediatr. 2008; 75 (8): 821–30. 9. pereira sm, cardoso mh, figuexeds al, mattos h, rozembaum r, ferreira vi, portinho ma et al. sepsis-related mortality of very low birth weight brazilian infants: the role of pseudomonas aeruginosa. int j pediatr. 2009; 6(2): 28–36. 10. tiffany k, burke b, odoms c, oelberg dg. current practice regarding the enteral feeding of high-risk newborns with umbilical catheters in situ. pediatrics 2003; 112(1): 20–6. 11. mermel la, allon m, bouza e. clinical practice guidelines for the diagnosis and management of intravascular catheterrelated infection: 2009 update by the infectious diseases society of america. clin infect dis. 2009; 49(1): 1–45. 12. rubinson l, diette gb. best practices for insertion of central venous catheters in intensive-care units to prevent catheterrelated bloodstream infections. j lab clin med. 2004; 143(1): 5–13. 13. kitterman ja, phibbs rh, tooley wh. catheterization of umbilical vessels in newborn infants. pediatr clin north am. 1970; 17: 895–912. 14. johns aw, kitchen wh, leslie dw. complications of umbilical vessel catheters. med j aust. 1972; 2(15): 810–15. 15. mer m, duse ag, galpin js, richards ga. central venous catheterization: a prospective, randomized, double blind study. clin appl thromb hemost. 2009; 15(1): 19–26. 16. mahieu lm, de muynck ao, leven mm, de dooy jj, goossens hj, van reempts pj. risk factors for central vascular catheterassociated bloodstream infections among patients in a neonatal intensive care unit. j hosp infect. 2001; 48(2): 108–16. 17. safdar n, maki dg. risk of catheter-related bloodstream infection with peripherally inserted central venous catheters used in hospitalized patients. chest. 2005; 128(2): 489–95. 18. ramritu p, halton k, cook d, whitby m, graves n. catheter-related bloodstream infections in intensive care units: a systematic review with meta-analysis. j adv nurs. 2008; 62(1): 3–21. 19. eyer s, brummitt c, crossley k, siegel r, cerra f. catheter-related sepsis: prospective, randomized study of three methods of long-term catheter maintenance. crit care med. 1990; 18(10): 1073–9. 20. linares j, sitges-serra a, garau j, perez jl, martin r. pathogenesis of catheter sepsis: a prospective study with quantitative and semi quantitative cultures of catheter hub and segments. j clin microbiol. 1985; 21: 357–60. 21. cronin wa, germanson tp, donowitz lg. intravascular catheter colonization and related bloodstream infection in critically ill neonates. infect control hosp epidemiol. 1990; 11: 301–8. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 100 vol. 5. no. 4 january–april 2015 literature review pathogenesis, diagnostic and management of toxoplasmosis irma yuliawati,1 nasronudin1,2 1 tropical and infectious disease division-department of internal medicine, dr. soetomo general hospital-faculty of medicine, universitas airlangga, indonesia 2 institute of tropical disease, universitas airlangga, surabaya, indonesia abstract toxoplasma gondii is an obligate intracellular parasite of protozoa groups, can infect humans and all warm-blooded animals, are found in almost all locations around the world. infection generally occurs orally through the consumption of animal products that are not perfectly cooked infected oocyst, parasite containing foods in the form of bradyzoite, contact with cat’s feces containing oocysts or vertical transmission occurring through hematogenous placenta. toxoplamosis can occur in acute or chronic. it divided into five categories, namely, toxoplasmosis in patients immunocompetent, toxoplasmosis in pregnancy, congenital toxoplasmosis, toxoplasmosis in immunocompromised patients and ocular toxoplasmosis. in each category of clinical manifestations of toxoplasmosis are often non-specific. methods of diagnosis and interpretation are often different for each category. toxoplasmosis can be diagnosed through a series of tests such as serology, pcr, histology parasites and parasite isolation. treatment management of this disease requires a long time. therapy depends on the category of infections as well as individual therapeutic response. the combination of pyrimethamine with sulfadiazine is the drug choice for toxoplasmosis. key words: toxoplasma gondii, toxoplasmosis diagnostic, toxoplasmosis management, pcr, parasite abstrak toxoplasma gondii merupakan parasit intraseluler obligat dari kelompok protozoa yang dapat menginfeksi manusia dan seluruh hewan berdarah panas yang ditemukan hampir di seluruh dunia. pada umumnya infeksi tersebar secara oral melalui konsumsi produk hewani terinfeksi ookista yang tidak dimasak sempurna, makanan mengandung parasit dalam bentuk bradizoit, kontak secara langsung dengan kotoran kucing mengandung ookista ataupun terjadi transmisi vertikal melalui plasenta hematogen. toksoplasma dapat terjadi secara akut maupun kronik. toksoplasma terbagi menjadi 5 kategori yaitu toksoplasmosis pada pasien imunokompeten, toksoplasma pada masa kehamilan, toksoplasma kongenital, toksoplasma pada pasien imunokompromais dan toksoplasma okuler. pada setiap kategori manifestasi klinik toksoplasma sering tidak spesifik. metode diagnosa dan interpretasi seringkali berbeda untuk setiap kategori. diagnosa toksoplasma dapat dirumuskan melalui beberapa seri pengujian seperti serologi, pcr, parasit histologi dan isolasi parasit. penatalaksanaan perlakuan terhadap penyakit ini membutuhkan waktu yang lama. proses terapi bergantung pada kategori infeksi seperti halnya terapi respon individual. kombinasi pyrimethamine dengan sulfadiazine adalah pilihan obat untuk toksoplasma. kata kunci: toxoplasma gondii, diagnosa toksoplasma, penatalaksanaan toksoplasma, pcr, parasit introduction toxoplasmosis is a zoonosis disease causing by toxoplasma gondii.1,2 toxoplasma gondii was founded by nicola and manceaux in 1908 on lymphatic and liver of ctenodactylus gondii in tunisia africa and in a rabbit in brazil.3 toxoplamosis spread around the worldwide and mostly without symptoms. generally, infection happen orally from consume animal product that infected oocyst and not cook properly, food that contain parasit like bradyzoite, contact with cat’s feces that contain oocyst or vertical spreading in a hematogen from placenta.4,5,6 101yuliawati and nasronudin: pathogenesis, diagnostic and management of toxoplasmosis immunocompromised condition such as aids object, ferocity and tissue transplanted resipien have high risk of toxoplasma infection. build this disease diagnostic in clinic and laboratory is very important to determine therapy and prognosis plan. it is depend on the knowledge about epidemiology, pathogenesis and clinical manifestation.3 epidemiologi toxoplasma gondii almost can found in worldwide and has been infected more than 50% human population in the world.2,4 about 10–15% inhabitant in united states shown the positive result in serology check up.7 seropositif in hivaids patients estimate about 10–45%.1,2 checkup result of igm and igg anti toxoplasma in indonesia, human about 2–63%, cat 35–73%, pig 11–36%, goat 11–61%, dog 75% and the other livestock under 10%.1 ethiology toxoplasma gondii is a parasite obligate intracellular, there are three type, tachyzoite (proliferative form), cyst (contain bradyzoite) and oocyst (contain spozoite).4,6 tachyzoite form look like sickle moon with pointed point, and the other point about rounded. length 4–8 micron, width 2–4 micron, has membrane cell and one nucleus in center. cyst formed in host cell if tachyzoite who splits have formed a wall. a cyst has varying size, there is a small that only contain some bradyzoite and there is a 200 micron contain about 3000 bradyzoite. cyst in host body can found in lifetime especially in brain, heart muscle and striated muscle. constitute rested stage from t. gondii.1 oocyst has the shape ovale, 11–14 × 9–11 micron. oocyst has a wall, contain one sporoblast that split into two sporoblast. in the next development, both sporoblast forming wall and being sporocyst. every sporocyst contain four spozoite that having size about 8×2 micron.1,4 life cycle and the transmission way toxoplasma gondii has two life cycles. sexual cycle happen on cat as definitive host, while asexual cycle happen in other mamalia (include in human) and various bird strain.1,2 this life cycle consist of three forms, tachyzoite and bradyzoite that forming in host mediator and oocyst stage that forming in definitive host epithelial gut cell. parasite invades erythrocytes then forming microgamete and macrogamete. zygot or oocyst that produced then come out with feces. oocyst undergo meiosis outside cat’s body. oocyst endure for many years in moist condition.2 then oocyst consumed by host mediator and forming tachyzoite inside digestion track that causing acute infection.4 acute infection can be cronic if tachyzoite change into bradyzoite. bradyzoite go into host tissue (brain, heart, muscle and retina) and stay in there for host lifetime in dorman condition.2,4 the changes of tachyzoite stage into bradyzoite depend on multiplication speed, ph, area temperature and the existence of anti mitochondria nitric oxide (no) in host body. if human consume meat or dringking water that contaminate with oocyst so bradiizoit or spozoite that resistance with acid ph and enzyme digestive will reach gut, invaded epithelial cell and after several hours change into tachyzoite.4 pathogenesis and immune response toxoplasmosis can take an acute or chronic. acute infection is associated with proliferative forms (tachyzoite), whereas chronic infections associated with tissue cyst forms. during the acute process, tachyzoite invades all cells in the body except host nucleated cells such as red blood cells.4,6 tachyzoite enters the host cell via active penetration into the host plasmalemma or by phagocytosis. parasites adhere to micronema are able to recognize and target cells, produce enzymes to mature rhoptries parasitophorus vacuoles.5 in vitro replication of intracellular tachyzoite occur every 6-9 hours. having collected 64–128 parasites in each cell the parasite will be out to infect neighboring cells. with the host immune system, can turn into a subpopulation tachyzoite bradyzoite.4 macrophages, nk cells, fibroblasts, epithelial cells and endothelial cells become activated by t.gondii infection in the host body, so it can be inhibited parasite proliferation. non-specific immune response depends on the ability of il 12 produced by macrophages and dendritic cells to stimulate nk cells produce ifn γ. tnf α also increases the ability of il 12 to induce nk cells to produce ifn γ. ifn -γ inhibit the replication of the parasite because it induces macrophages to release nitric oxide (no), which kills the parasite. ifn -γ also increases the activity of indoleamine 2,3 dioxygenase that destroys tryptophan which is a substance necessary for the growth of the parasite.6 these parasites will induce immunity 4 types of t cells, namely cell-mediated immune response as t.gondii are intracellular parasites.6 il 12 produced by macrophages also strengthen the work of cd4 + cells producing ifn γ in. cd8 + cells also induces the release of ifn γ, interferon γ (ifn γ) plays a role in cyst formation by inhibiting replication in macrophages tachyzoite mice and induce antigen specific for bradyzoite. the humoral immune system has a small role in the fight against toxoplasmosis but is of significant importance in the diagnosis of toxoplasmosis in humans. antibodies produced by the humoral immune system is able to kill extracellular t.gondii in and through the activities of its complement can inhibit parasite multiplication.6 102 indonesian journal of tropical and infectious disease, vol. 5. no. 4 january–april 2015: 100–106 p a t h o g e n e s i s o f t o x o p l a s m o s i s i n t h e immunocompromised host such as hiv aids patients is influenced by many things, among others, a decrease in cd4 + cell count, the failure of production of il 12, il 2 and ifn γ and cytotoxic activity of t – limphocyte is declining. cells infected with the hiv virus to inhibit the formation of il 12 and ifn γ, leaving them vulnerable to infection toxoplasmosis.8 levels of ifn -γ usually decrease in patients with aids and it could lead to reactivation of chronic toxoplasmosis.4 the diagnosis of toxoplasmosis the diagnosis of toxoplasmosis can be established through a series of tests such as serology, polymerase chain reaction (pcr), histological examination of the parasite (imunoperoksidase) and the isolation of the parasite.9 serology test the combination of serology is often necessary to determine whether the patient is really infected or not and to determine the acute or chronic infection lasts. the panel of serological tests or toxoplasma serological profile (tsp) includes sabin fieldman dye test (dt), double sandwich igm enzyme linked immunosorbent assay (elisa), elisa iga, ige elisa and aglutination test (ac/hs test).9 igg can be checked by engineering sabin fieldman dt (gold standard), indirect fluorescent antibody (ifa) or elisa. igg appeared in the first 1–2 weeks of infection and usually can last for years or a lifetime.9 however, in immunocompromised patients igg levels can not be detected.10 igg positive indicates that the patient has been exposed by t.gondii but can not indicate whether the newly infected patients or long-term infection.11 igg avidity has been widely used as additional tests to determine if ongoing infection is acute or chronic. high avidity igg titer indicates that the infection lasts approximately 4 months earlier while a low titer indicates acute infection.11,12 igm can be examined by the technique of double sandwich elisa, ifa and immunosorbent agglutination assay (isaga). igm appeared soon after infection and disappears within a few months.11 in some cases igm can be detected for> 12 years, therefore the serum igm positive results still need other tests to determine whether the infection is acute or chronic lasted9,13. the sensitivity and specificity of serology varies greatly depending on the lab and the techniques used. a study comparing 6 of tests igm elisa found that sensitivity ranged from 93–100 %, and a specificity of 99.1 % 77,5.14 iga was detected in acute infection in adults and congenital infection. iga can exist for approximately 1 year. in the examination of congenital toxoplasmosis infection is more sensitive iga. ige was detected by elisa in acute infections in adults and congenital infection and serve as additional tests to identify acute infection.9,13 tests ac / hs uses two antigen preparations, namely methanol -fixed tachyzoites (ac antigen) indicating acute infection and formalin -fixed tachyzoites (hs antigen) that indicates chronic infection. the ratio of the ac and hs ratio may indicate acute results, equivalence or nonreactive.9 serologic tests for toxoplasmosis in immunocompromised patients often do not provide a diagnosis for igg levels in these patients is often low or even undetectable, whereas for the igm test is often negative. examination of antigen in the circulation of patients with aids have been investigated but have low sensitivity.10,14 a definitive diagnosis can be established if the formation tachyzoite obtained on biopsy results.15 pcr pcr could detect dna t.gondii in brain tissue, cerebrospinal fluid, amniotic fluid, aqueous humor and vitreous fluid and bronchoalveolar lavage (bal).9 in patients with toxoplasmic encephalitis sensitivity of pcr in the csf of approximately 50-60 %, a specificity of approximately 100 %. pcr on blood samples had a low sensitivity.8 histology examination immunoperoxsidase staining technique can show tachyzoite formation in tissue sections or infected body fluids. multiple tissue cysts with necrotic inflammation surrounding areas can indicate the presence of an acute infection or reactivation of latent infection. this examination is not routinely performed.2,9 isolation of t. gondii a definitive diagnosis of toxoplasmosis can be established by isolation of the parasite from the body fluids (blood, csf, bal) or tissue biopsy. this examination is not practical because of the culture of the sample takes approximately 6 months.8 categories of toxoplasmosis for clinical purposes, toxoplasmosis is divided into five categories, namely (1) toxoplasmosis in patients immunocompetent, (2) toxoplasmosis in pregnancy, (3) congenital toxoplasmosis, (4) toxoplasmosis in immunocompromised patients, (5) ocular toxoplasmosis.9 1) toxoplasmosis in immunocompetent patients 1. clinical manifestation only 10–20% of toxoplasmosis in children and adults who have symptoms.2 in immunocompetent patients with toxoplasmosis often without symptoms or only mild symptoms and provide non-specific as fever, enlarged lymph nodes, myalgia, stiff neck, painful swallowing or abdominal pain.6,9 2. examination supporting examination of igm and igg performed for initial evaluation on suspicion of toxoplasmosis. parallel examination performed 3–4 weeks after the first examination. results of igm and igg were negative excluding the diagnosis of toxoplasmosis. acute 103yuliawati and nasronudin: pathogenesis, diagnostic and management of toxoplasmosis infection occurs when there is an increase in titer of more than 4 -fold compared to titers at baseline examination. examination of the panel such as toxoplasma serological profile (tsp) or igg avidity to distinguish whether the infection to occur acute or chronic.9 3. management treatment is not necessary in cases of asymptomatic except in children < 5 years.2 only immunocompetent patients who have symptoms are treated. pyrimethamine were given 100 mg loading dose, then 25–50 mg / day in combination with sulfadiazine 2–4 g / day in divided doses 4 times / day for 2–3 weeks or can also be combined with clindamycin 300 mg 4 times / day for 6 weeks. sulfadiazine and clindamycin can be replaced with azithromycin 500 mg / day or 750 mg atovaquone 2 times / day. another alternative that can be given is trimethoprim (tmp) of 10 mg / kg / day, sulfamethoxazole (smx) 50 mg / kg / day for 4 weeks.7 2) toxoplasmosis in immunocompromised patient 1. clinical manifestation in the immunocompromised host such as patients with aids, hematologic malignancies, bone marrow transplant recipients, solid organ transplant (including the heart, liver, liver, kidney), toxoplasmosis can cause encephalitis, meningoencephalitis, myocarditis, and pneumonitis.6,9,17 the incidence of toxoplasmosis in allogenic transplant recipients was 40%, the mortality rate reaches 60–90%. cns infections occur in 5–10% of transplant recipients.15,17 toxoplasmic encephalitis (te) is the most frequent manifestations in immunocompromised patients.9 in 58–89% of cases occur in sub-acute clinical manifestations in the form of focal neurologic abnormalities, in 15–25% of cases with more severe clinical manifestations of seizures and cerebral hemorrhage. other clinical manifestations such as loss of consciousness, meningismus, cerebellar signs, neuropsychiatric disorders, dementia, agitation.2 in hiv patients the risk of cns infection associated with cd4 levels, higher risk in those who only have the number of cd4 + < 200 cells / mm3.15,18 in some studies noted that for every decrease in cd4 + cells by 50 cells will increase the risk of te by 30%, but in the era of haart (highly active antiretroviral therapy) as the current risk and mortality te decreased due to the improvement of the immune system.18 toxoplasmosis in aids patients can also attack the lungs, eyes and other organs. pulmonary toxoplasmosis (pneumonitis) occurred mainly in patients with advanced aids clinical manifestesi include fever, dyspnea, and cough and is often difficult to distinguish from jeroveci pneumocystic pneumonia. the mortality rate ranges from 35%.19 2. examination supporting reactivation of chronic infection is the most frequent cause of toxoplasmosis in immunocompromised patients. igm and igg titer increased in reactivation.8 nonetheless serum antitoxoplasma igm and igg were negative does not automatically exclude the diagnosis of toxoplasmosis.15 isolation of parasites from the blood, infected body fluids, bal fluid is a definite diagnosis of toxoplasmosis infection. other tests that may be done include pcr assay to detect dna t.gondii in the blood or body fluids.2,9 ct scan or mri should be performed on suspicion of cns involvement in t.gondii infection. overview lesions of multiple ring -enhance support the diagnosis of toxoplasmosis.9 3. management toxoplasmosis therapy in hiv aids patients were divided into 2 acute treatment and maintenance therapy. acute therapy is given for at least 3 weeks and can be given for 6 weeks if complete response does not occur, the next required maintenance therapy to prevent relapse.8 primary prophylaxis is recommended in hiv seropositive aids where the number of cd4 + < 100 / mm3 or patients with cd4 < 200 / mm3 were accompanied by opportunistic infections and malignancies. regimens used can be given tmp smx (trimethoprim sulfamethoxazole).8 the dose of tmp smx is one double strength tablet (ds) (160 mg trimethoprim, 800 mg sulfamethoxazole) 2 times / day (14 ds tablets / week).20 in acute infections may be given a combination of pyrimethamine and sulfadiazine. this regimen is the standard regimen for the treatment of te. pyrimethamine initial dose of 200 mg / day next 50-75 mg / day plus sulfadiazine 4–8 g / day for 6 weeks then referred to a lifelong suppressive therapy or to improve the immune system.7,8 in some of the studies mentioned combination of pyrimethamine clindamycin and trimethoprim sulfamethoxazole as effective as the use of a combination of pyrimethamine – sulfadiazine.7 clindamycin can be given at a dose of 600 mg po / iv, 4 times / day for 3–6 weeks. the dosage for suppressive therapy 300–450 mg po every 6–8 hours.2,21 the combination of atovaquone with pyrimethamine or sulfadiazine also provide high effectiveness. these drugs are able to eliminate bradyzoite in experimental animals. can be administered at a dose of 750 mg (5 ml) po when eating for 21 days.2,21 in some studies this regimen gives good results on the clinical and radiological picture of 77% within 6 weeks of treatment and recurrence rate of 5% in the maintenance period.8 maintenance therapy (secondary prophylaxis) can be started after completion of therapy in the acute phase is given, which used the same regimen as in the acute phase but with a half dose.8 primary prophylaxis can be stopped if the cd4 count after the use of antiretroviral (arv) increased > 200 / mm3 were settled for approximately 3 months, with an examination of the amount of virus negative.8,22 secondary prophylaxis was stopped if the patient had 104 indonesian journal of tropical and infectious disease, vol. 5. no. 4 january–april 2015: 100–106 undergone treatment of acute and showed clinical improvement is characterized by loss of the signs and symptoms of toxoplasmosis and improvement of the immune system after treatment with haart are characterized by increased cd4 + > 200 / mm3 were settled for for about 6 months.8,22 3) congenital toxoplasmosis 1. clinical manifestation cases of congenital toxoplasmosis have been reported in indonesia. lazuardi et al (1989) reported t.gondii antibodies in 44.6% of children with mental retardation, 44.6 % in children with ocular lesions and 9.5% in children with common symptoms.1 the risk and severity of congenital toxoplasmosis symptoms more severe if infection occurs early in pregnancy.23 classic triad of congenital toxoplasmosis is chorioretinitis, hydrocephalus, and intracranial calcification. the involvement of neurological and ocular systems often arise later if not found at the time of birth. seizures, mental retardation, and rigidity is the common sequelae.2 2. examination improving igm positive is strong evidence of congenital infection, but a negative igm does not exclude the diagnosis. serum iga is more sensitive for detecting congenital toxoplasmosis than igm.9 when symptoms and serological evidence of toxoplasmosis is detected during pregnancy, infection of the fetus can already be enforced by igm detection and isolation of parasites from fetal blood or amniotic fluid at 18 weeks of gestation. examination before 20 weeks gestation is difficult to enforce because of the immunological response of the fetus is still low. pcr on amniotic fluid can more accurately diagnose infection in the fetus before 20 weeks gestation.9 the sensitivity of this test is 64% with a negative predictive value of 87.8%, specificity and positive predictive value of 100%.9 antenatal ultrasound can identify abnormalities in the fetus is infected. approximately 36% of fetuses with abnormalities can be identified. abnormalities that can be found are bilaterally symmetrical ventricular dilatation, intracranial calcification, increased placental thickness, hepatomegaly and ascites.9 3. management in newborns with toxoplasmosis, can be given a combination of pyrimethamine 1 mg / kg per day for 2 months followed by 1 mg / kg every 2 days for 10 months, sulfadiazine 50 mg / kg body weight per day, as well as folic acid 5–10 mg 3 times week to prevent the side effects of pyrimethamine2. in addition to the provision of drugs are also required regular follow-up. a complete blood count 1–2 times per week to daily dosing of pyrimethamine and 1–2 times per month for the dosing of pyrimethamine performed every 2 days to monitor the toxic effects of the drug. also required a complete pediatric examination, including ophthalmologic examination every 3 months until the age of 18 months and then once a year, as well as neurological examination every 3–6 months to 1 year of age.2 4) ocular toxoplasmosis 1. clinical manifestation toxoplasmic chorioretinitis can occur because of congenital or postnatally acquired infection. infection occurs in 2/1000 pregnancies america, with an average of transplacental infection ≤50%.25 seventy percent of infants with congenital infection showed a scar on korioretina.24 symptoms include blurred vision, scotoma, fotofobi and pain. of ophthalmology examination obtained focal necrotizing retinitis formation that resembles a yellowish white cotton, with unclear boundaries. in congenital infection are often bilateral lesions in infections acquired while generally unilateral.2 2. examination improving serologic tests are often unhelpful because the diagnosis is often obtained with the igg titers were low, often undetectable igm. increased levels of igg 4 times the initial levels within 4 weeks showed primary infection. other tests that can be done is the amplification of parasite dna from the aqueous or vitreous humor.9 3. management treatment depends on several factors such as the location of lesions, degree of inflammation, the threat of blindness and immune status of the patient. if the infection is not on the optic disc and macula and is only accompanied by mild inflammation, treatment is not required.10 pyrimethamine most effective for this infection, given the loading dose of 25 mg 3 times / day followed by 25 mg / day. this drug should be combined with sulfadiazine with further loading dose of 2 g 1 g 4 times / day. therapy is done for 6-12 weeks. treatment response was indicated by the disappearance of a yellowish white spot on the retina, the vitreous becomes clear and atrophic scars korioretina being demarcated. another drug option is clindamycin 300 mg 3-4 times / day for 3-4 weeks, then 150 mg four times / day for the next 3-4 weeks. spiramycin is the drug most commonly used and has the least amount of side effects among other drug options, can be administered in a dose of 1 g 2 times / day.11 5) toxoplasmosis in pregnancy 1. clinical manifestation most pregnant women with acute acquired infection do not experience specific symptoms. some have symptoms of malaise, subfebris, lymphadenopathy. the frequency of vertical transmission to the fetus increased with increasing gestational age.25 2. examination improving examination of igg and igm should ideally be done in the first trimester of pregnancy. serum igg and igm 105yuliawati and nasronudin: pathogenesis, diagnostic and management of toxoplasmosis negative by showing that pregnant women not infected, face further investigation performed during pregnancy to anticipate the occurrence of seroconversion.24 on the positive results of igg but negative igm in pregnancy < 18 weeks showed an infection occurred in the past, while in gestation > 18 weeks of this result is difficult to interpret whether the infection is acute or chronic lasted so avidity required examination. in the results were negative but igg positive igm examination should be repeated in 1–3 weeks later, if the result remains the same mean positive igm has no clinical significance, whereas in case of seroconversion of igg becomes positive which indicates that the infection occurs during pregnancy so that the fetus is at high risk affected by congenital toxoplasmosis.24 on examination of the igg and igm positive follow-up examination to confirm acute or chronic infections such indispensable avidity test.24 high avidity igg indicates that infection occurred > 16 weeks in advance, so that the examination in the first trimester of pregnancy showed an infection occurs before conception reduces the risk of transmission and the risk of fetal defects is low.23 3. management s p i r a m y c i n i s d r u g o f c h o i c e f o r m a t e r n a l toxoplasmosis. dose of 3 g / day po in divided doses 24 times / day for 3 weeks, stopped for 2 weeks and then repeated the cycle of 5 weekly during pregnancy.2,24 if pcr positive amniotic fluid regimens should be replaced with pyrimethamine 50 mg / day and sulfadiazine 3 g / day in 2–3 divided doses for 3 weeks interspersed with the provision of spiramycin 1 g 3 times / day for 3 weeks or can be given pyrimethamine 25 mg / day and sulfadiazine 4 g / day in divided doses 2-4 times / day was given until delivery.7 prevention prevention of toxoplasmosis can be made by cooking the meat until done, wash your hands thoroughly after handling raw meat, wash vegetables and fruits before eating, wash clean kitchen equipment after use, pregnant women should wear gloves when gardening and wash hands afterwards, avoid contact with cat feces, the primary and secondary prophylaxis should be administered to patients with aids.7 prognosis in immunocompromised patients reactivation of chronic toxoplasmosis are common. suppressive therapy and improving the immune system may reduce the risk of recurrent infection. infants with ocular toxoplasmosis acquired have a good prognosis and in the next four years have the same development as uninfected infants. immunocompetent patients have a good prognosis, lymphadenopathy and other symptoms disappear within a few weeks after infection.7 summary methods of diagnosis and interpretation are often different for each category. the diagnosis of toxoplasmosis can be established through a series of tests such as serology, pcr, histology parasites and parasite isolation. management the treatment of this disease requires a long time. therapy depends on the category of infections as well as individual therapeutic response. the combination of pyrimethamine with sulfadiazine is the drug of choice for toxoplasmosis. references 1. chahaya (2003). epidemiologi “toxoplasma gondii”. bagian kesehatan lingkungan fakultaskesehatan masyarakat universitas sumatera utara, hlm 1–13. 2. hokelek m (2009). toxoplasmosis. available at: http://www. emedicine.medscape.com/article/229969. accessed: february 6, 2010 3. nicolle c & manceaux l. (1908). sur une infection a corps de leishman (ou organismes voisins) du gondi. c r seances acad. sci., 147: 763–766. 4. yellita (2004). mekanisme interaksi toxoplasma gondii dengan sel host. pengantar falsafah sains institut pertanian bogor, hal 1–12 5. demar m, ajzenberg d, maubon d, djossou f, panchoe d, punwasi d (2007). fatal outbreak of human toxoplamosis along the mahoni river epidemiological, clinical, and parasitological aspects. clin infect dis, 45: e88–95. 6. waree p (2008). toxoplamosis pathogenesis and immune respone. thammasat medical journal, 8: 487–95. 7. becker j, singh d, sinert rh (2010). toxoplasmosis. available at: http://www.emedicine.medscape.com/article/787505. accessed on october 28, 2010 8. subauste c (2006). toxoplamosis and hiv in hiv insite knowledge base chapter. ucsf hiv insite, pp 1–13. 9. montoya jg (2002). laboratory diagnosis of toxoplasma gondii infection and toxoplamosis. j infect dis, 185: s73–82. 10. mechain b, garin yj, camel jd, gangneun fr, derouin f (2000). lack of utility of specific immunoglogulin g antibody avidity for serodiagnosis of reactivated toxoplamosis in immunocompromise patients. clin diagn lab immunol, 7: 703–05. 11. montoya jg, liesenfeld o (2004). toxoplasmosis. lancet, 363: 1965–76. 12. marcolino p, silva da, leser pg, camargo me, mineo jr (2000). molecular markers in acute and chronic phases of human toxoplamosis: determination of immunoglobulin g avidity by western blotting. clin diagn lab immunol, 7: 384–89 13. jarreau p (2010). serological response to parasitic and fungal infections in clinical immunology, serology a laboratory perspective, eds. stevens cd, fa davis company usa, pp 328–40. 14. wilson m, schantz pm, nutman p, tsang vc (2002). clinical imunoparasitology in manual of clinical laboratory immunology 6th ed. eds rose nr, hamilton rg, asm press washington dc, pp 547–57. 15. walker m, zunt jr (2005). parasitic central nervous system infections in imunocompromised hosts. clin infect dis, 40: 1005–15. 16. hidalgo hf, bulabois ce, pinchart mp, hamidfar r, garban f (2008). diagnosis of toxoplasmois after allogenic stem cell transplantation: results of dna detection and serological techniques. clin infect dis, 49: e9–15 106 indonesian journal of tropical and infectious disease, vol. 5. no. 4 january–april 2015: 100–106 17. belanger f, derouin f, keros lg, meyer l (1999). incidence and risk factor of toxoplamosis in a cohort of human immunodeficiency virus-infected patients 1988-1995. clin infect dis, 575–81. 18. antinori a, larussa d, cingolani a, lorenzini p, bossolasco s, finazzi mg (2004). prevalence, associated factors, and prognostic determinants of aids related toxoplasmic encephalitis in the era of advanced highly active antiretroviral therapy. clin infect dis, 39: 1681–91. 19. ribera e, sola af, juste c, rovira a, romero fj, gil la, ruiz i (1999). comparison of high and low dose of trimethoprimsulfamethoxazole for primary prevention of toxoplasmic encephalitis in human immunodeficiency virus-infected patients. clin infect dis, 29: 1461–6. 20. djakovic od, milenkovic v, nikolic a, bobic b, grujic j (2002). efficacy of atovaquone combined with clindamycin against murine infection with a cystogenic (me49) strain of toxoplasma gondii. j antimicrob chemother, 50: 981–987. 21. kaplan je, holmes kh, masur h (2002). guideline for preventing opportunistic infections among hiv-infected persons recommendation of the u.s. public health service and the infectious diseases society of america. mmwr recomm, 51: 1–53. 22. lazuardi s, srisasi g, ismael s, hendarto sk, soctomenggolo (1989). toksoplasmosis congenital. mki1989; 39: 464–72. 23. ajzenberg d, cogne n, paris l, bessieres mh, thulliez pfilliseti d (2002). genotype of 86 toxoplasma gondii isolates associated with human congenital toxoplamosis, and correlation with clinical findings. j infect dis, 186: 684–9. 24. montoya jg, remington js (2008). management of toxoplasma gondii infection during pregnancy. clin infect dis, 47: 554–66. 25. yamamoto jh, vallochi al, silveira c, filho jk, nussenblatt rb, neto ec (2000). discrimination between patients with acquired toxoplamosis and congenital toxoplamosis on the basis of the immune response to parasite antigens. j infect dis, 181: 2018–22. this journal is a peer-reviewed journal established to promote the recognition of emerging and reemerging diseases specifically in indonesia, south east asia, other tropical countries and around the world, and to improve the understanding of factors involved in disease emergence, prevention, and elimination. the journal is intended for scientists, clinicians, and professionals in infectious diseases and related sciences. we welcome contributions from infectious disease specialists in academia, industry, clinical practice, public health, and pharmacy, as well as from specialists in economics, social sciences, and other disciplines. for information on manuscript categories and suitability of proposed articles 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form and email to ijtid@itd.unair.ac.id) ijtid vol 6 no 1 jan-april 2016_revisi.indd 24 vol. 6. no. 1 january–april 2016 cytotoxicity of justicia gendarussa burm f. leaf extracts on molt-4 cell prihartini widiyantia,b,1, bambang prajogoc, ni putu ermi hikmawantic a faculty of science and technology, universitas airlangga, surabaya, east java, indonesia b institute of tropical disease (itd), universitas airlangga, surabaya, east java, indonesia c department of pharmacognosy, faculty of pharmacy, universitas airlangga, surabaya, east java, indonesia corresponding author: drwidiyanti@yahoo.com abstract justicia gendarussa burm f. (acanthaceae) is known for its activity as a male contraceptive and anti-hiv properties. the present study was designed to evaluate extracts of j. gendarussa for cytotoxicity activity against molt-4 cells. the cytotoxic activity of the fractionated-extract and 70% ethanol extracts of j. gendarussa leaves on molt-4 cells were evaluated using a wst-1 assay. the treatment cells, control cells without treatment and control media were also tested in duplicate. the absorbance was measured at a wavelength of 450 nm using a microplate absorbance reader (bio-rad). the average absorbance measures formazan produced by viable cells that metabolize the wst-1 reagent. then the data was analyzed with regression analysis microsoft excel 2007 program to determine the concentration with 50% cell viability (50% cytotoxicity concentration, cc50). the cc50 values of the fractionated-extract and 70% ethanol extract of j. gendarussa leaves were 94 μg/ml and 78 μg/ml, respectively. the cytotoxicity of fractionated-extract and 70% ethanol extract of j. gendarussa leaves were not significantly different (p > 0.05). it can be concluded that the fractionated-extract and 70% ethanol extract of j. gendarussa leaves are not toxic to molt-4 cells. key words: cytotoxicity; justicia gendarussa burm.f; molt-4 cell; wst-1 assay, anti hiv abstrak justicia gendarussa burm f. (acanthaceae) dikenal untuk aktivitasnya sebagai konstrasepsi pria dan bersifat anti-hiv. studi ini dirancang untuk mengevaluasi ekstrak j. gendarussa untuk aktivitas sitotoksisitas terhadap sel molt-4. aktivitas sitotoksisitas dari esktrak terfraksinasi dan ekstrak entanol 70% daun j. gendarussa pada sel molt-4 dievaluasi menggunakan sebuah uji wst-1. sel dengan perlakuan, sel control tanpa perlakuan serta kontrol media juga diuji berulang. nilai absorbansi diukur pada panjang gelombang 450nm menggunakan microplate absorbance reader (bio-rad). nilai absorbansi rata-rata mengukur formazan yang dihasilkan oleh sel yang bermetabolisis denga reage wst-1. kemudian data dianalisis menggunakan analisis regresi program microsoft excel 2007 untuk menentukan konsentrasi viabilitas sel 50% (50% cytotoxicity concentration, cc50). nilai cc50 dari ekstrak terfraksinasi dan ekstrak entanol 70% daun j. gendarussa adalah 94 μg/ml dan 78 μg/ml, secara berturut-turut. sitotoksisitas dari ekstrak terfraksinasi dan ekstrak entanol 70% daun j. gendarussa tidak jauh berbeda gendarussa (p > 0,05). dapat disimpulkan bahwa ekstrak terfraksinasi dan ekstrak entanol 70% daun j. gendarussa tidak beracun untuk sel molt-4. kata kunci: sitotoksisitas, justicia gendarussa burm.f, sel molt-4, uji wst-1 research report introduction justicia gendarussa burm f. (acanthaceae) leaves are often used in traditional medicine to treat fever, headache, rheumatism, myalgia, respiratory disorders, and back pain.1 j. gendarussa is also used in papua as a male contraceptive. a pre-clinical study of an alkaloidfree 70% ethanol extract of j. gendarussa leaf extract has confirmed male contraceptive activity.2 the 70% ethanol leaf extract (with alkaloids and without alkaloids) from j. 25widiyanti, et al.: cytotoxicity of justicia gendarussa burm f. leaf extracts on molt-4 cell gendarussa also have hiv reverse transcriptase enzyme inhibition activity.3 studies on the in vitro and in vivo toxicity of j. gendarussa leaf extract were previously performed. the administration of a water extract of j. gendarussa leaves in male rabbits did not affect liver and renal function.4 the 60% ethanol and water fraction of the ethanol extract of j. gendarussa leaves were non-toxic in acute toxicity and teratogenic tests. cytotoxicity in human normal lymphocytes cells of the water fraction from the ethanol extract of j. gendarussa leaves had a cc50 of 3215.7 μg/ml. 5 cytotoxicity activities of the methanol extract of j. gendarussa leaves obtained from 4 locations in malaysia (regions of muar, skundal, batu pahat, and pulai) against human cancer cells lines such as ht-29 (colon adenocarcinoma), hela cells (cervix adenocarcinoma), and the bxpc-3 cells (epitheloid cervix adenocarcinoma), as well as mda-mb-468 and mdamb-231 cells (breast cancer cells) using a mtt reagent colorimetric method were also reported as non toxic with cc50 values greater than 41 μg/ml. however, the methanol extract of j. gendarussa leaves from the mersing region was toxic to bxpc-3 cells, hela cells, mda-mb-468 and mda-mb-231 cells with the cc50 values of 16 μg/ml, 5 μg/ml, 23 μg/ml, and 40 μg/ml respectively.6,7 the leaves of j. gendarussa plants contain a substituted aromatic amine,8 flavonoid glycosides including gendarusin a and b2, and justidrusamide alkaloids a, b, c, and d.9 male contraceptive activity has been attributed to gendarusin a and b isolated from the n-butanol fraction of j. gendarussa leaves.2 flavonoids are antioxidants which can protect cells from oxidative stress. flavonoid compounds from j. gendarussa also serve as a natural resource of anti-hiv therapy for aids subjects by inhibiting hiv reverse transcriptase.10,11 however, in high concentrations, flavonoids and other polyphenols can also be cytotoxic, causing increased mitochondria permeability, secretion of cytochrome c, capsase activation, increased p53 and p21 levels, depressed bcl-2, apoptosis induction, and cell necrosis.12, 13, 14, 15,16,17 alkaloids also have pharmacologic activities useful in the treatment of disease18 but may also be toxic to man. thus, cytotoxicity testing against molt-4 cells was performed to evaluate the relative toxicity potential using a fractionated-extract (alkaloid-free) and a 70% ethanol extract of j. gendarussa leaves to assess the safety of j. gendarussa leaf extracts used in preliminary male contraceptive clinical trials. materials and methods materials plants justicia gendarussa burm f. leaves used in this study were obtained from a cultivated crop in trawas, mojokerto, east java-indonesia. the medicinal plants were identified by department of pharmacognosy and phytochemistry, faculty of pharmacy, universitas airlangga, surabaya. 70% pharmaceutical grade ethanol, pro hplc methanol (merck), sterile water for injection, aquadest (pure water) from crc-eird (itd surabaya), rpmi1640 media (gibco), natrium bicarbonate (merck), fetal bovine serum (fbs) (gibco) (inactivated at 56 °c for 30 min), reagent(4-3 (4-iodophenyl)-2-(4-niitrophenyl)-2d5-tetrazolio]-1,3-benzen disulfonate) (wst-1)(roche), dimethyl sulfoxide (dmso) (sigma), and nitrocellulose 0.2 μm membrane filter (whatman). cells molt-4 cells clone#8 (human t lymphocytes cancer cells line) were obtained from the bio-safety level-3 facility crc-erid, itd, surabaya. molt-4 cells were cultured on rpmi-1640 media, with 10% fbs and kept in ccf t25 at a temperature of 37 °c in a 5% co2 incubator (sanyo). methods preparation of sample j. gendarussa leaves powder was divided into 2 fractions, a leaf powder with releasing alkaloids and a leaf powder with non-releasing alkaloids. both powders were extracted using 70% ethanol during 324 hours in macerator and the filtrate obtained evaporated using a rotary evaporator (buchi). the two extracts were dried at 50 °c until fractionated with 70% ethanol extract (alkaloid-free; 2.4% w/w) and 70% ethanol extract (10.8% w/w). a stock solution was made by dissolving 100 mg of each extract in 1000 μl dmso and then diluted using rpmi-1640 medium with 10% fbs to obtain various concentrations (7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0; and 1000.0 μg/ ml) for each trial extract. the concentration of dmso used was less than 1% which does not affect viability.19 detection of flavonoid in j. gendarussa leaf extract the content of gendarusin a, the major flavonoid in j. gendarussa leaves, was analysed by a waters hplc (agilent 1100, reverse phase novapack® column c-18 3.9150 mm using a water:methanol (30:70) eluent with a flow rate of 1 ml/min, and a uv detector at 254 nm wavelength). cytotoxicity assay cytotoxicity of the extracts on molt-4 cells was measured using a colorimetric method with wst-1 reagent (roche). briefly, 50 μl molt-4 cells (1105cells/well) were plated in each well on a 96-well microplate. 50 μl of extract at various concentrations were also added to each well, and incubated for 72 hr at 37 °c in a 5% co2 incubator. the treatment cells, control cells without treatment and control media were also tested in duplicate. total volume in each well was 100 μl. after 72 hr of incubation, 10 μl wst-1 reagents was added into each well and incubated 26 indonesian journal of tropical and infectious disease, vol. 6. no. 1 january–april 2016: 24−28 for an additional 2 hrs at 37 °c in a 5% co2 incubator. the absorbance was measured at a wavelength of 450 nm using a microplate absorbance reader (bio-rad). the average absorbance measures formazan produced by viable cells that metabolize the wst-1 reagent. the percentage cell viability was determined by the equation below: statistical analysis the collected data was analyzed with regression analysis microsoft excel 2007 program to determine the concentration with 50% cell viability (50% cytotoxicity concentration, cc50). the comparison of cytotoxicity activities of both extracts were tested using a paired t-test analysis (microsoft excel 2007 program). the difference was considered to be significant if the probability was p < 0.05. results and discussion the leaf extracts of j. gendarussa tested had low toxicity to molt-4 cells with decreased molt-4 cell viability with increasing extract concentrations (table 1). based on regression analysis, the cc50 of the 70% ethanol extract was 78 μg/ml, while fractionated-70% ethanol extract was 94 μg/m. table 1. cytotoxicity test of 70% ethanol extract and fractionated-70% ethanol extract to molt-4 cells incubated for 72 hr extract concentration (μg/ml) cells viabilities (%) ± sd 70% ethanol extract fractionated-70% ethanol extract 7.8 101.4 ± 2.9 100.4 ± 1.2 15.6 91.3 ± 4.9 89.8 ± 3.8 31.3 72.2 ± 8.8 81.1 ± 12.1 62.5 51.2 ± 1.3 67.7 ± 6.1 125.0 35.1 ± 6.1 37.4 ± 2.5 250.0 15.1 ± 6.1 22.5 ± 0.6 500.0 8.9 ± 5.6 7.3 ± 0.9 1000.0 3.2 ± 11.8 4.7 ± 0.9 a comparison of extract cytotoxicity activity was performed using a paired t-test analysis based on the percentage of cell viability for each treatment. based on the t-test, t = -1.786, t table (0.05) = ± 2.365, the significance value is 0.117 or 11.7% which is larger than 0.05 or 5%. based on t-test results, there was no significant difference for cytotoxicity activitiy on molt-4 cells between the fractionated-70% ethanol extract and the 70% ethanol extract of j. gendarussa leaves (p > 0.05). hplc chromatograms show gendarusin a content in the fractionated-70% ethanol extract (fig. 1b) and the 70% ethanol extract (fig. 1c) as a major flavonoid component of j. gendarussa leaves. fractionated-70% ethanol extract and 70% ethanol extract of j. gendarussa leaves contain 0.53% and 0.95% of gendarusin a, respectively. cytotoxicity activity were evaluated to identify the relative toxicity of fractionated-70% ethanol extract and 70% ethanol extracts from j. gendarussa leaves to molt-4 human t-lymphocytes line cells using a wst-1 test. the test is based on the reduction of tetrazolium sodium salt (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2h-5-tetrazolio]1,3-benzendisulfonate) (water-soluble tetrazolium salt, wst-1) by a succinate-tetrazolium reductase system of the mitochondria respiratory chain. this enzyme system is only active in viable cells. the wst-1 reduction process produces soluble formazan with a bright colour. absorbance measurements of formazan are directly related to cell viability.20,21 a gendarusin a b c gendarusin a gendarusin a gendarusin a figure 1. chromatogram profile of (a) gendarusin a 12.8 μg/ml solution; (b) gendarusin a in 5000.0 μg/ml of the fractionated-70% ethanol extract of j. gendarussa leaves; and (c) gendarusin a in 5000.0 μg/ml of the 70% ethanol extract of j. gendarussa leaves. the cytotoxicity activity of the extract was measured as the concentration of extract that reduces cell viability or cell growth by 50% (cc50). the cytotoxicity levels were based on previous studies where cc50 values less than 20 μg/ml were considered as cytotoxic, 21–40 μg/ml as low cytotoxicity, and over 41 μg/ml as not cytotoxic.22,23,24 27widiyanti, et al.: cytotoxicity of justicia gendarussa burm f. leaf extracts on molt-4 cell using this criteria, fractionated-70% ethanol extract (cc50 -93 μg/ml) and 70% ethanol extract (cc50-78 μg/ml) of j. gendarussa leaves are considered non-cytotoxic to molt-4 cells. the cytotoxicity activity of fractionated-70% ethanol extract and 70% ethanol extract of j. gendarussa leaves at various concentrations on molt-4 cell viability are found in fig 2. the reduction of cell viability is reflected by the inhibition of cell growth related to the suppression of cell proliferation activities so that the total number of dividing or living cells are decreased. various cell signalling activities involved in protein expression of the programmed cells death such as bid, bax, and bcl-2 are likely to be activated when cell lines are exposed to active compounds contained in the extract25 causing an increase in programmed cell death (apoptosis).26 testing by using tetrazolium only measures the formation of formazan (which is related to mitochondria living cell activities) but it is not able to determine the cause of cell death.27 there was no statistical difference between the two extracts despite having twice the amount of gendarusin a in the extract which indicates that gendarusin a probably does not significantly contribute to the cytotoxicity of the extract. the alkaloids present in the 70% ethanol extract probably contributed to the cytotoxicity but also did not result in a significant difference between the alkaloid free and the alkaloid containing extracts. figure 2. comparison of the effect of various concentrations of fractionated-70% ethanol extract and 70% ethanol extract of j. gendarussa leaves on molt-4 cell viability with a 72 hr incubation period. conslusions fractionated-70% ethanol extract and 70% ethanol extract of j. gendarussa leaves are relatively non-cytotoxic to molt-4 cells with no significant difference of cytotoxicity between the fractionated-70% ethanol extract and 70% ethanol extract (p > 0.05). acknowledgements the authors would like to thank the collaborative research center for emerging and reemerging infectious disease (crc-erid), institute of tropical disease (itd), airlangga university, surabaya for bio-safety level-3 facilities. references 1. ratnasooriya wd, deraniyagala sa, dehigaspitiya dc. antinoceptive activity and toxicological study of aqueous leaf extract of justicia gendarussa burm f. in rats. pharmacogn mag, 3, 2007: 145–155. 2. prajogo b, guliet d, queiroz f, wolfernder j-l, cholies n, aucky h, hostettmann k. isolation of male antifertility compound in n-butanol fraction of justicia gendarussa burm f. leaves. folia medica indonesiana, 45 (1) 2009: 28–31. 3. prajogo b, widiyanti p, and riza h. effect of ethanolic extract of justicia gendarussa burm f. against activity of reverse transcriptase hiv enzyme in vitro. jurnal bahan alam indonesia, 8 (6) 2014: 384–388. 4. prajogo b, ifadotunnikmah f, febriyanti ap, jusak n. efek fase air daun gandarusa (justicia gendarussa burm.f) pada fungsi hati dan fungsi ginjal kelinci jantan (uji toksisitas fase air daun gandarusa sebagai bahan kontrasepsi pria). veterinaria medika, 1(3) 2008: 79–82. 5. prajogo b. autentik tanaman justicia gendarussa burm f. sebagai bahan baku obat kontrasepsi pria. surabaya: airlangga university press dengan lp3 unair; 2014. 6. ayob z, samad aa, bohari spm. cytotoxicity activities in local justicia gendarussa crude extracts against human cell lines. jurnalteknologi, 64 (2) 2013: 45–52. 7. ayob z, bohari spm, samad aa, jamil s. cytotoxicity activities in against breast cancer cell of local justicia gendarussa crude extracts. evidance-based complementary and alternative medicine, 2014: 1–12. 8. chakravarty ak, dastiar ppg, and pakrashi sc. simple aromatic amines from justicia gendarussa 13c nmr spectra of the bases and their analogues. tetrahedron, 18(12) 1982: 1797–1802. 9. kiren y, deguchi j, hirasawa y, morita h, prajogo, b.justidrusamides a-d, new 2-aminobenzyl alcohol derivatives from justicia gendarussa. journal of natural medicines; 2014. 10. veljkovic v, mouscadet j-f, veljkovic n, glisic s, debyser z. simple criterion for selection of flavonoid compounds with antihiv activity. bioorganic and medicinal chemistry letters, 17, 2007: 1226–1232. 11. ko y-j, oh h-j, ahn h-m, kang h-j, kim j-h, ko, yh. flavonoids as potential inhiibitors of retroviral enzymes. j. korean soc. appl. biol. chem, 52 (4) 2009: 321–326. 12. bolton jl, trush ma, penning tm, dryhurst g, monks tj. role of quinones in toxicology. chem res toxicol, 13 2000: 135–160. 13. inayat-hussain sh, winski sl, ross d. different involvement of caspase in hydroquinone-induced apoptosis in human leucemic hl-60 and jurcat cells. toxicolapplipharmacol, 175 2001: 95–103. 14. morin d, barthelemy s, zini r, labidalle s, tillement, jp. curcumin induces the mitochondrial permeability transition pore by membrane protein thiol oxidation. febslett, 495, 2005: 131–136. 15. salvi m, brunati am, clari g, toninello a. interaction of genistein with the mitochondrial electron transport chain results in the opening of the membrane transition pore. biochim biophys acta,1556, 2005: 187–156. 28 indonesian journal of tropical and infectious disease, vol. 6. no. 1 january–april 2016: 24−28 16. shen sc, ko ch, tseng sw, tsai sh, chen yc. structurally related antitumor effects of flavanones in vitro and in vivo: involvement of caspase 3 activation, p21 gene expression, and reactive oxygen species production. toxicol appl pharmacol, 197, 2004: 84–95. 17. lee mh, dan dw, hyon sh, park, jc. apoptosis of human fibrosarcoma ht-1080 cell by epigallocathecin-3-o-gallate via induction of p53 and caspase as well as supression of bcl-2 and phosphorylated nuclear factor-κb. apoptosis, 16, 2011: 75–85. 18. harborne jb. metode fitokimia: penuntun cara modern menganalisis tumbuhan, diterjemahkan oleh padmawinata, k., dan soediro, i. bandung: penerbit itb; 1987. 19. awah fm, uzoegwu pn, ifeonu p, oyugi jo, rutherford j, yao x, fehrmann f, fowke kr, eze mo. free radical scavenging activity, phenolic contents and cytotoxicity of selected nigerian medicinal plants. food chemistry. 2012: 131: 1279–1286. 20. berridge mv, herst pm, tan as. tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. biotechnology annual review, 11 2005: 127–151. 21. rampersad sn. multiple applications of alamar blue as an indicator of metabolic function and cellular health in cell viability bioassays. sensors, 12, 2012: 12347–12360. 22. geran, greenberg, macdonald, schumacher, abbott. protocol for screening chemical agent and natural products against animal tumors and other biological systems. cancer chemotherapy reports, 3 1972: 1–103. 23. mohamed sm, ali am, rahmani m, dhaliwal js, yusoff k. apoptotic and neurotic cell death manifestations in leukemic cell treated with methylgerambulin a sulphone from glycosmiscalcicola. journal biochemistry molecular biology and biopysiology, 4, 2000: 253–261. 24. rohaya, manaf a, daud, norhadiani, khozirah, nordin. antioxidant, radical-scavenging, anti-inflammatory, cytotoxic and antibacterial activities of methanolic extracts of some hedyotis species. life sciences. 76, 2005: 1953–1964. 25. singh r. interaction and cytotoxicity of compounds with human cell lines. rom. j. biochem, 51 (1) 2014: 57–74. 26. astuti e, pranowo d, puspitasari sd. cytotoxicity of phaleriamacro carpa (scheff.) boerl. fruit meat and seed ethanol extract to mononuclear perifer normal cell of human body. indo j chem, 6 (2) 2006: 212–218. 27. paul a. manjula. cytotoxic and antiproliferative activity of indian medicinal plant in cancer cell. international journal of science and research, 3 (6) 2014: 88–93 indonesian journal of tropical and infectious disease this journal is a peer-reviewed journal established to promote the recognition of emerging and reemerging diseases spesifically in indonesia, south east asia, other tropical countries and around the world, and to improve the understanding of factors involved in disease emergence, prevention, and elimination. the journal is intended for scientists, clinicians and professionals in infectious diseases and related sciences. we welcome contributions from infectious disease specialists in academia, industry, 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ijtid@itd.unair.ac.id 83 vol. 5. no. 4 january–april 2015 research report a n a l y s i s o n s e c o n d a r y i n f e c t i o n t r i g g e r i n g microorganisms in hiv/aids patients as a model for policy control retno pudji rahayu,1,2 nasronudin,1,3 retno indrawati,1,2 prihartini widiyanti,1,4 bimo dwi lukito,1,3 ferdiansyah,1,3 siti qomariyah khairunisa,1 adiana m,1 tomohiro kotaki5 1 institute of tropical disease, universitas airlangga, surabaya, indonesia, 2 faculty of dentistry, universitas airlangga, surabaya, indonesia, 3 faculty of medicine, universitas airlangga, surabaya, indonesia, 4 faculty of science and technology, universitas airlangga, surabaya, indonesia. 5 collaborating research center emerging reemerging infectious disease universitas airlangga, surabaya, indonesia kobe university, japan abstract hiv infection is associated with immune-compromised and rising in opportunistic infection (secondary infection). therefore, the number of mortality caused by hiv/aids is increasing. the use of arv and development of hiv/aids management are expected to suppress the progress of hiv infection into aids and, therefore, the mortality can be diminished, while in fact most of the patients eventually suffer from aids due to secondary infection that commonly causes death. there should be a management by analysing microorganisms that trigger secondary infection. the method of this study was observational descriptive with cross sectional design. hiv infected blood samples were using elisa antibody (igg and igm) and polymerase chain reaction (pcr) on laboratory test. the result showed correlation between hiv/aids severity and the amount and types of secondary infection. the most common secondary infections were toxoplasm (96.77%), hepatitis c (22.58%), tuberculosis (19.35%), and hepatitis b (3.22%). other less frequent secondary infections, which were quite difficult to diagnose and not commonly found in indonesia, were west nile virus (25.81%), japanese encephalitis virus (3.22%), and enterovirus (3.22%). due to mdgs (millenium development goals) target and the results above, researchers are highly demanded to contribute in decreasing mortality related to aids through early detection of secondary infection, including type of infection which have not been commonly found in indonesia, such as west nile virus and nipah virus. the discovery of secondary infection in this study was not enough to suppress the occurrence of infection in hiv/aids patients. antimicrobes and good nutrition are required. moreover, there should be either a primary or secondary prophylaxis to prevent secondary infection that raises the number of mortality and morbidity of hiv/aids patients. the result of this study was to meet the target of mdgs by establishing new policies in handling hiv/aids infections and have potential as model for policy control in hiv/aids. key words: microorganisms, secondary infection, hiv/aids, model, policy control abstrak infeksi hiv berkaitan dengan immune-compromised dan peningkatan infeksi oportunis (infeksi sekunder). oleh karena itu angka kematian yang disebabkan hiv/aids semakin meningkat. penggunaan arv dan pengembangan penatalaksanaan hiv/aids diharapkan dapat menekan perkembangan hiv menjadi aids. oleh karena itu tingkat kematian pun dapat berkurang, meskipun pada kenyataannya mayoritas pasien pada akhirnya mengidap aids karena infeksi sekunder yang umumnya mengakibatkan kematian. diperlukan adanya sebuah penatalaksanaan dengan menganalisa mikroorganisme yang memicu terjadinya infeksi sekunder. metode yang digunakan pada kajian ini merupakan pengamatan deskriptif dengan desain bagi silang. sampel darah yang terinfeksi hiv dilakukan uji laboratorium menggunakan antibodi elisa (igg dan igm) serta polymerase chain reaction (pcr). hasil penelitian menunjukkan adanya korelasi antara tingkat keparahan hiv/aids dengan jumlah dan jenis infeksi sekunder. infeksi sekunder yang paling umum terjadi ialah toksoplasma (96.77%), hepatitis c (22.58%), tuberkulosis (19.35%) dan hepatitis b (3.22%). infeksi sekunder lainnya dengan frekuensi lebih rendah yang jarang ditemui di indonesia saat ini adalah virus west nile (25.81%), virus japanese encephalitis (3.22%) and enterovirus (3.22%). berdasarkan target millenium development goals (mdg) dan hasil penelitian tersebut di atas, peneliti sangat 84 indonesian journal of tropical and infectious disease, vol. 5. no. 4 january–april 2015: 83-89 dituntut untuk berkontribusi dalam menurunkan tingkat kematian yang berkaitan dengan aids melalui deteksi dini infeksi sekunder, termasuk jenis infeksi yang belum lazim ditemui di indonesia seperti virus west nile dan virus nipah . penelitian infeksi sekunder dalam kajian ini belum cukup untuk menekan terjadinya infeksi pada pasien hiv/aids. antimikroba dan gizi yang baik sangat diperlukan. selain itu diperlukan adanya profilaksi baik primer maupun sekunder untuk mencegah infeki sekunder yang dapat meningkatkan angka kematian dan morbiditas pasien hiv/aids. hasil dari kajian ini adalah untuk memenuhi target mdgs dengan mengadakan kebijakan baru dalam penanganan infeksi hiv/aids dan berpotensi sebagai model untuk kebijakan kontrol pada hiv/aids. kata kunci: mikroorganisme, infeksi sekunder, hiv/aids, model, kebijakan kontrol introduction hiv infection is associated with decreased endurance and increased incidence of opportunistic infections that in a given period of time raises a set of disease called acquired immunodeficiency syndrome (aids).1 human immunodeficiency virus (hiv) remains a global health problem, including in indonesia. world health organization (who) reported that 2001 up to 58 million people worldwide have been infected with hiv, while in indonesia until 2009 there were an estimated 186,000 hivpositive people. the death rate from hiv/aids infection is reported quite high. until 2000 it was reported that there were 22 million deaths related to hiv/aids.2 indonesia was ranked first in the transmission of new cases of hiv and aids in asia. data from the ministry of health said there were 15,372 new hiv cases and 3541 new aids cases in january to september 2012. majority of the patients were male in productive age. the highest transmission is through sexual contact, followed by needles and drug users, and it is reported that the number of patients is increasing sharply compared to ten years ago. along with increased capacity for early detection, screening programs and increased public awareness of hiv disease, we will find more new cases. the area with the highest number of new cases is dki jakarta, followed by papua and east java.3 currently, with the developing management of hiv/aids infection and increasingly widespread use of antiretroviral drugs, the progression of hiv infection to aids and death from aids should be reduced. in fact, most of the patients fell into aids as a result of the emergence of secondary infections (opportunistic infections) that often leads the patients to death.1 in the decline of immune status, especially when the cd4 cells less than 200 cells/ml, a variety of microorganisms such as bacteria, viruses, protozoa and fungal infections also appear tend to be easy to grow and reproduce, causing secondary infections in the body of the people with hiv/aids.4 the lower the cd4 cell count, the more types of microorganisms involved in secondary infection of hiv/aids. fungal infections can occur simultaneously with bacterial infections, viruses and protozoa.2,5 the main problem faced by people with hiv/ aids is an opportunistic infection caused by a secondary infection.6 the more advanced the severity of hiv/aids, the more increasing the potential incidence of secondary infections and death. analysis of microorganisms triggering the secondary infection, as is often seen in people with hiv/aids and other viruses, is associated with cd4 count and viral load. some secondary infections include cmv (cytomegalovirus), mycobacterium tuberculosis, west nile virus, hepatitis b and c virus, and candida sp.7,8 it was not clear whether any people with hiv/aids will be infected by all these microorganisms. patients with infections are often followed by clinical conditions, such as malnutrition and wasting syndrome, which also will result in a decrease in cd4 t lymphocytes count. the condition results in a decrease of t lymphocytes count in patients susceptible to the incidence of secondary infections (opportunistic infections), such as hepatitis c, hepatitis b, hepatitis c, cmv, toxoplasmosis, japanese encephalitis, west nile virus, nipah virus, all of which can be detected with cd4 and hiv rna viral load. in hiv patients with secondary infections the increase of hiv progression is taking place. therefore, hiv management policies is including promotion, prevention of secondary and tertiary infections, and complete therapy in accordance with the mdgs 2014, which comprises the absence of hiv-related deaths, the absence of new infections and the absence of discrimination. this study aims to analyze how far the correlation between hiv/aids severity with the involvement of the type and number of secondary infections. the results are expected to be a new policy on hiv/aids, thereby supporting the achievement of the mdg targets in the field of infectious diseases of hiv/aids is zero new infections, zero discrimination, and zero aids-related deaths. from the laboratory results can be seen how far the relationship between the severity of hiv/aids with the involvement of the type and number of secondary infections. based on the analysis of microorganisms triggers the secondary infection, according mdgs (millennium development goals) in the field of infectious diseases hiv/aids, the results of this study are expected to contribute in lowering aids deaths through early detection of secondary infection, including an infection that has not been commonly detected in indonesia, west nile virus infection and nipah virus. the results of this study are also expected to be a new policy on hiv/aids, thus supporting the achievement of the mdg targets, and can generate a new reference in the information and health sciences in the form of a journal. 85rahayu rp, et al.: analysis on secondary infection-triggering microorganisms in hiv/aids patients methods this study was a descriptive observational using cross-sectional design. blood samples were taken from hiv-infected patients in hospital universitas airlangga (rsua), and infectious disease intermediate care unit (unit perawatan intermediet penyakit infeksi, upipi) dr soetomo hospital, then we conducted laboratory tests to determine secondary infections experienced by the patients. from the laboratory results, we could see how far the relationship between the severity of hiv/aids with the involvement of the type and number of secondary infections. the laboratory tests were carried out at the institute of tropical diseases (itd), universitas airlangga. the study was conducted for three months. the population in this study was hiv-positive patients who have received antiretroviral therapy, whereas the samples in this study were part of the whole object under study who met the inclusion criteria. criteria for inclusion in this study were as follows: willingness to involve, hiv-positive, and has received antiretroviral therapy. to obtain accurate results, this research was conducted using antibody (igg and igm) elisa and pcr. results & discusion the number of patients included in this study was 31 patients. the mean age of patients in this study was 35.06 ± 11.20 years, with the youngest two years old and the oldest 54 years of age. the highest number of the patients in age group of 31–45 years was 22 patients (70.96%), the least in the age group of < 16 years was 2 patients (6.46%), whereas the age group of 16-30 years was 3 patients (9.67%), and 46–60 years was 4 patients (12.91%) (figure 1). figure 1. hiv patients distribution by age group. characteristics of the subjects by sex showed that the males were 15 patients (48.38%) and females 16 patients (51.62%). a total of 26 (83.87%) patients had married and 5 (16.13%) unmarried (figure 2). characteristics of study subjects based on tribes revealed javanese of 27 (87.12%), arabic 1 (3.22%), chinese 1 (3.22%), banjarese 1 (3.22%), and tetungs 1 (3.22%). figure 2. distribution of hiv patients by sex and marital status. in this study, hiv/aids transmission through sex was 21 patients (67.74%), through idu (intravenous drug users) was 8 (25.81%), and through vertical mother to child transmission (mtct) was 2 patients (6.45%) (figure 3). figure 3. distribution of hiv by route of transmission. a total of 29 (93.54%) patients had received antiretroviral drug therapy and 2 (6.46%), while the rest had not received (figure 4). figure 4. distribution of hiv patients by antiretroviral therapy. according to the length of antiretroviral therapy, 7 (24.14%) of the patients had received antiretroviral therapy for < 1 year, 11 (37.94%) patients for 1–3 years, 6 (20.68%) patients for 3–5 years and 5 (17.24%) patients for > 5 years (figure 5). 86 indonesian journal of tropical and infectious disease, vol. 5. no. 4 january–april 2015: 83-89 figure 5. distribution of hiv patients by the length of antiretroviral therapy. based on hiv/aids clinical stage according to who in 2010, this study found that 13 (41.94%) of the patients were at stage i, 11 (35.48%) patients at stage ii, 7 (22.58%) patients at stage iii and there were no patients at stage iv (figure 6). figure 6. distribution of patients based on who’s clinical stage of hiv/aids. in this study, most patients with undetected hiv viral load test results were 23 (74.19%) patients. results of viral load < 4 ´ 102 copies/ml were in 2 (6.45%) patients and viral load > 4 ́ 102 copies/ml were in 5 (16.12%) patients (figure 7). figure 7. patients distribution by hiv viral load. secondary infection in subjects research in this study we performed examination of secondary infections in people with hiv/aids. the results showed that 6 patients (19.35%) of the patients were with secondary infection of tuberculosis of 7 (22,58%) patients of the patients had secondary infection of hepatitis c, 1 (3.22%) of the patients had secondary infection of hepatitis b, 30 (96.77%) with secondary infections toxoplasma, 8 (25.81%) with west nile virus, 1 (3.22%) patients with japanese encephalitis virus, 2 (6.45%) patients with enteroviruses, and 1 (3.22%) patients with secondary infections of dengue virus. there were no patients with secondary infection of cytomegalovirus (figure 8). figure 8. distribution of secondary infection in the subjects. secondary infection in the subjects by hiv/aids clinical stage in this study, we performed examination on secondary infections in people with hiv/aids. based on the clinical stage according to the who in 2010 the secondary infections appeared on stage i was tuberculosis of 3 patients (23.07%), hepatitis c of 4 patients (30.76%), west nile virus of 2 patients (15.38%), and toxoplasma of 12 patients (92.31%). in stage ii the secondary infections were tuberculosis of 3 patients (27.27%), hepatitis c of 1 patients (9.09%), hepatitis b of 1 patients (9.09%), west nile virus of 6 patients (54.55%), japanese encephalitis virus of 1 patients (9.09%), enterovirus of 1 patients (9.09%), dengue virus of 1 patients (9.09%) and toxoplasma of 11 patients (100%). whereas, stage ii the secondary infections were hepatitis c of 2 patients (28.57%), enterovirus of 1 patients (14.28%) and toxoplasma of 7 patients (100%).7 figure 9. distribution of secondary infection by hiv/aids clinical stage. 87rahayu rp, et al.: analysis on secondary infection-triggering microorganisms in hiv/aids patients secondary infection in the subjects by hiv viral load we carried out examination of secondary infections in people with hiv/aids. based on hiv viral load, the secondary infections in patients with hiv viral load < 4 ́ 102 copies/ml was toxoplasma of 2 (100%). patients with hiv viral load > 4 ´ 102 copies/ml the secondary infections were tuberculosis in 2 (40.0%), hepatitis c 2 (40.0 patients, west nile virus of 1 patients (20.0%), japanese encephalitis virus of 1 patients (20.0%), and enterovirus by 1 patients (20.0%), and toxoplasma of 4 patients (80.0%). whereas, in patients with undetectable hiv viral load, the secondary infections were tuberculosis in 4 (17.39%) patients, hepatitis c 5 (21.73%), hepatitis b 1 (4.34%), west nile virus 7 (30.43%), enterovirus 1 (4.34%), dengue virus 1 (4.34%) and toxoplasma in 3 (100%) (figure 10). figure 10. distribution of secondary infection by hiv viral load. characteristics of the study population in this study, the subjects were patients with hiv/ aids of 31 patients. the mean age of the patients was 35.06 ± 11.20 years, with the youngest 2 years old and the oldest 54 years of age. the age distribution of the patients showed those of < 16 years were 6.46%, 16–30 years were 9.67%, 31–45 years were 70.96%, and aged 46–60 years were 12.91%. the proportion of males was 48.38% and females 51.52%, mostly (83.87%) were married and 16.13% unmarried. this figure showed that the incidence of hiv/aids infections is more common in reproductive age, which is along with the data from the ministry of health of indonesia in 2011 that the highest percentage of hiv/aids was at the age of 20–29 years with a ratio of men and women 3:1. indonesia ranked first in the transmission of new hiv and aids cases in asia. data report of the directorate general of pp & pl, the ministry of health, mentioned that there were 15,372 new cases and 3541 new cases of aids in january to september 2012. the majority of sufferers were of childbearing age and men. highest transmission was through sexual contact followed through needles of drug users. the number increased significantly when compared to ten years ago, along with an increase in the ability of the government to detect, to carry out screening programs and increase public awareness of hiv disease, then there will be more new cases to be found. areas with highest number of new cases are jakarta, followed by papua and east java. since 1999 a new phenomenon in hiv/aids dispersion occurred, that was the predisposition of transmission through blood contact, especially among intravenous drug users (idus). transmission in idus occurs rapidly due to the use of shared needles. in 2000 there was a significant increase in the spread of hiv pandemic among sex workers in indonesia (indonesian ministry of health, 2011). in this study, sexual transmission of hiv/aids was 67.74%, through idu (intravenous drug users) was 25.81%, and through mother-to-child vertical transmission or mtct (mother to child transmission) was 6.45%. this indicates that the highest incidence rates of hiv/aids transmission was through unhealthy sexual relationships. the discovery of antiretroviral drugs (arvs) in 1996 led to a revolution in the treatment of plwha (people living with hiv and aids). although antiretroviral therapy has not been able to cure the disease and the presence of major challenges in terms of side effects of drugs and the incidence of chronic resistance to antiretroviral drugs, such therapy can dramatically reduce mortality and morbidity, and improve the quality of life of people living with hiv. currently hiv/aids has been accepted as a disease that can be controlled and no longer considered a dread disease.2 in this study showen that antiretroviral therapy has been widely used in patients with hiv/aids in indonesia, and with quite a number of study subjects who had received antiretroviral therapy for more than 5 years showed the role of arvs in increasing the life expectancy of people with hiv/aids. the findings in this research indicated that the majority of the study subjects were at an early stage (stage i and ii) of hiv/aids infection. undetectable viral load results indicated that the use of antiretroviral therapy in the majority of study subjects could control and suppress hiv/aids progress and improve the quality of life of the patients. this is in accordance with the policy on hiv/aids in indonesia, which includes 4 pillars, all of which are aimed at bringing about a paradigm of zero new infection, zero aids-related death and zero discrimination:9,10 (1) prevention: includes prevention of hiv transmission through sexual behavior and syringe, prevention in prisons and detention centers, prevention of mother-to-child transmission (pmtct), prevention of transmission among sex workers and others, (2) maintenance and support treatment (pdp): includes the strengthening and development of health services, prevention and treatment of opportunistic infections, arv treatment, as well as support and education, training people living with hiv. pdp program is primarily intended to reduce morbidity and hospitalization, mortality related to hiv-aids and improve the quality of life of people living with hiv, (3) mitigation of the impact of psychosocial and economic support, (4) creation of a conducive environment (creating the enabling environment) which includes institutional strengthening and management, program management and policy alignment. with growing hiv/aids management infection and increasingly widespread use of antiretroviral drugs, 88 indonesian journal of tropical and infectious disease, vol. 5. no. 4 january–april 2015: 83-89 progression of hiv infection to aids and death from aids should have been suppressed. in fact, most of the patients fell into the emergence of aids as a result of secondary infections that often lead to death. declining cd4 cell count to some extent (< 200 cells/mm3) will open up opportunities for a secondary infection. the more advanced severity of hiv/aids, the more increase the potential incidence of secondary infection and death.1 based on figure 8, there were no patients with secondary infection of cytomegalovirus. in this study, the encountered secondary infections were mostly toxoplasma, as many as 96.77%. high toxoplasma infection in hiv/aids is related to the deterioration of the immune system.11,12,13 the parasite toxoplasma gondii can reactivate again when cd4 lymphocyte count decreases to below 100 cells/mcl. the incidence of toxoplasma seroprevalence in a group of non-hiv individuals and groups of individuals with hiv/aids is almost the same, which is about 10–40%. in the united states, 67% of people with hiv/aids have positive toxoplasma antibodies. however, the possibility of reactivation is 30% higher in people with hiv/aids.11 secondary infection of tuberculosis in this study was found to be 19.35%. this finding is in line with secondary tuberculosis infection data on hiv/aids. tuberculosis is a secondary infection most often found in people with hiv and is the largest cause of morbidity and mortality in hiv infection in the world. more than 11 million hiv infections is accompanied with tb.14,15,16 thirty percent of is the cause of death in people with hiv is tb.17 data in upipi dr. soetomo hospital showed that manifestations of aids due to secondary infection of pulmonary tb reaches 25–83%.17 hepatitis b and hepatitis c are blood-borne diseases, together with hiv transmission. both are secondary infections commonly found in people living with hiv who are injecting drug users (idus). coinfection of hepatitis c and hiv among injecting drug users were 40–90%, whereas coinfection of hepatitis b and hiv in sexual transmission was 77%.18 in this study, secondary infection of 3.22% with hepatitis b and hepatitis c was 22.58%. this is because the transmission of hiv infection in the study was largely through sexual transmission (67.74%) and through injecting drug use (idu) (25.81%). another finding in this study was the secondary infection that is rare and often undiagnosed in indonesia, such as japanese encephalitis virus, west nile virus (wnv), enterovirus, and dengue viruses.19 in this study, secondary infection of west nile virus was found to be 25.81%, which is quite a high figure for a rare viral infection and rarely diagnosed in indonesia. wnv infection is a viral infection that is transmitted through mosquito bites, self-limited with mild symptoms such as flu-like syndrome that can occur more severe in hiv co-infection with neurological manifestations such as meningoencephalitis. there has been no report on the epidemiological data of wnv and hiv coinfection rate. only in the united states some cases of wnv and hiv co-infection was reported with manifestations of severe encephalitis.20 as wnv, japanese encephalitis virus (jev) is also a coinfection virus that can be found in hiv. often found in asian countries including indonesia, jev is a flavivirus transmitted by mosquito bite with severe neurological manifestations of encephalitis and high mortality rate up to 60%.21 in this study, a secondary infection of jev was found to be 3.22%. although the data on jev findings is low, these findings need attention because of the limitations of the study that was only in surabaya (which is a reference to eastern indonesia). thus the molecular epidemiological studies are necessary to get the database on jev infections that accompany hiv/aids so that the mortality rate of patients with hiv/aids can be prevented early. enterovirus is a virus that is identified as one of the causes meningoencephalitis in patients with hiv. neurological deficits often appear along with a decrease in cd4 cell counts. more common in children, enteroviruses are often associated with complaints of diarrhea in people with hiv.22 dengue virus has been reported to coinfect with hiv. with the decline in immune status in hiv and high infection rates in dengue endemic areas, the incidence of co-infection becomes possible.23 there have been no reports of dengue and hiv coinfection rate, but in this study, the rate was found to be 3.22%. the findings of secondary infection in this study showed arvs alone is not sufficient to reduce the incidence of secondary infection in hiv/aids, so that it requires antimicrobial therapy and adequate nutritional support. there should also be a primary or secondary prophylactic measures to combat secondary infections that can increase mortality and morbidity of patients with hiv/aids. primary prophylaxis is given to prevent an infection that has never been suffered, while secondary prophylaxis is a treatment given to prevent the repetition of an infection which never been suffered before. for primary prophylaxis we can give cotrimoxazole tablets of 960 mg/day single dose for 2 years, while for secondary prophylaxis the treatment was given in accordance with arising secondary infections. conclusion toxoplasma (96.77%) which is the most common secondary infection is higher than other infection. the benefits of this research to the patients is that they know the type and number of secondary infections associated with the severity of hiv/aids suffered, so they may immediately seek treatment in order to have better prognosis. by proving relationships between hiv/aids severity and the involvement of microorganisms in hiv/ aids secondary infection, we can take strategic policy to reduce the transmission rate of secondary infections and related deaths. 89rahayu rp, et al.: analysis on secondary infection-triggering microorganisms in hiv/aids patients acknowledgement thanks to the directorate of research and community service, the directorate general of higher education, ministry of education and culture, over the funding that has been awarded for the continuation of this research. references 1. nasronudin. 2007. penatalaksanaan koinfeksi penderita hiv. dalam: hiv & aids pendekatan biologi molekuler, klinis dan sosial. ed. barakbah j, dkk. surabaya, aup, 177–183. 2. world health organization. 2010. recommendation for hiv/ tuberculosis coinfection. in: antiretroviral therapy for hiv infection in adults and adolescent: recommendation for a public health approach. geneva, switzerland, 58–63. 3. kemenkes ri. 2011. terapi arv pada koinfeksi tuberculosis. dalam: pedoman nasional tata laksana klinis infeksi hiv dan terapi antiretroviral pada orang dewasa. jakarta. kementerian kesehatan ri, 30–31. 4. benson ca, kaplan je, masur h, 2004. treating opportunistic infections among hiv-exposed andinfected children: recommendations from cdc, the national institutes of health, and the infectious diseases society of america. mmwr. 5. sester m, giehl c, mcnerney r, kampmann b, walzl g, cuchi p, wingfield c, lange c, 2010. challenges and perspectives for improved management of hiv/mycobacterium tuberculosis coinfection. eur respir j, 36, 1242–47. 6. cakraborty n, 2008. current trends of opportunistic infection among hiv seropositive patients from eastern india. jpn. j infect dis, 61, 49–53. 7. retnowati e, 2008. koinfeksi hiv dan hepatitis b patogenesis dan diagnosis. dalam: simposium nasional penyakit tropik-infeksi dan hiv&aids, surabaya 22–23 maret 2008, hlm. 149–156. 8. amor sandra, 2009. manson’s tropical diseases: virus infections of the central nervous systems. saunders elsevier: china. p. 859–63. 9. alter jm. 2011. viral hepatitis c. in: tropical infectious diseases principles, pathogens & practice. edinburgh, p. 427–432. 10. astari l, sawitri, yunia eka safitri. 2009. viral load pada infeksi hiv. available from http://journal.unair.ac.id/filerpdf/viral.pdf 11. heller hm. 2012. toxoplasmosis in hiv infected patients. uptodate. 12. hoffmann c, ernst e, meyer p, 2008. evolving characteristics of toxoplasmosis in patients infected with human immunodeficiency virus-1: clinical course and toxoplasma gondii-specific immune responses. j clin microbiol infect. 708–712. 13. karimi k, wheat lj, connolly p. 2002. diferences in histoplasmosis in patients with acquired immunodeficiency syndrome in the united states and brazil. j infect dis: 1655–60. 14. haskins jl. 2009. human immunodeficiency virus (hiv) and mycobacterium tuberculosis: a collaboration to kill. african j microbiol res, 3(13), 1029–35. 15. lawn sd, butera st, shinnick tm. 2002. tuberculosis unleashed: the impact of human immunodeficiency virus infection on the host granulomatous response to mycobacterium tuberculosis. microbes and infection, 4, 635–46. 16. pawlowski a, jansson m, skold m, rottenberg me, kallenius g. 2012. tuberculosis and hiv co-infection. plos pathog, 8(2), 1–5. 17. pawlowski a, jansson m, skold m, rottenberg me, kallenius g. 2012. tuberculosis and hiv co-infection. plos pathog, 8(2), 1–5. 18. dore g, sazadeuzs j. 2003. coinfection hiv and hepatitis virus. australian society for hiv medicine. 19. dyer jr, edis rh, french ma. 1998. enterovirus associated neurological disease in hiv-1 infected man. j neurovirol. 4(5), 569–71. 20. josekuty j, yeh r, mathew s, ene a, ramessar n, trinidad j. 2013. atypical presentation of west nile virus in a newly diagnosed hiv patient in new york. j clin microbiol, 51(4), 1307–1311. 21. cdc, 2010 japenese encephalitis. mmwr, 59(1), www. cdc.gov/ mwr. 22. who, 2007. manualfor the laboratory diagnosis of japanese encephalitis virus. available from http://www.who.int/immunization_ monitoring/manual_lab_diagnosis_je. 23. siong wc, ching th, jong gc, fang cs, sin ly, 2008. dengue infections in hiv patients. southeast asian j trop med pub health, 39(2), 260–68. vol. 8 no. 3 september–december 2020 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ research report genexpert mtb/rif and mycobacterium tuberculosis sputum culture in establishing the diagnosis of pulmonary tuberculosis and rifampicin resistance in suspected childhood pulmonary tuberculosis in soetomo hospital berlian beatrix rarome1, nurul aisah2, retno asih setyoningrum3a, ni made mertaniasih4 1,3 division of respirology, department of child health, faculty of medicine, universitas airlangga / dr. soetomo hospital, surabaya 2 medical school, universitas airlangga, surabaya 4 department of microbiology, faculty of medicine, universitas airlangga / dr. soetomo hospital, surabaya received: 2nd october 2019; revised: 8th september 2020; accepted: 9th september 2020 abstract the diagnosis of childhood tuberculosis remains a challenge worldwide. the genexpert mtb/rif test, a rapid mycobacteria tuberculosis diagnostic tool, was recommended for use in children. no pediatric studies of genexpert mtb/rif assessing pulmonary tuberculosis within a hospital setting has been done in indonesia. we evaluated the performance of the genexpert mtb/rif test compared with sputum culture on lowenstein-jensen (lj) for the diagnosis of childhood pulmonary tuberculosis. this study was conducted in pediatric respirology inpatient and outpatient dr. soetomo hospital, a tertiary care facility in surabaya between june and august 2015 with a cross-sectional design. we consecutively enrolled 27 children aged 3 months to 14 years who had history of close contact with adult tuberculosis patients and showed symptoms of pulmonary tuberculosis. sputum collection was performed by induced sputum and three examination methods were performed (microscopic, genexpert mtb/rif and sputum culture) simultaneously followed by a drug sensitivity test for specimens detected with mtb growth. the genexpert mtb/rif test had a sensitivity of 100% (95% ci 100-100) and a specifi city of 95% (95% ci 85-100). the positive predictive value for diagnosing pulmonary tb was 89% (95% ci 68-100), the negative predictive value was 100% (95% ci 100-100) and positive likelihood ratio was 20 (95% ci 2.82-128). the genexpert mtb/rif test on one sputum sample rapidly and correctly identifi ed all children with culture-confi rmed pulmonary tuberculosis with high specifi city. similar results were obtained between genexpert mtb/rif and sputum culture based on age groups and clinical manifestations. rifampicin resistance were both detected in genexpert mtb/rif and mtb sensitivity test. keywords: childhood pulmonary tuberculosis; sensitivity, specifi city; genexpert mtb/rif abstrak menegakkan diagnosis tuberkulosis (tb) pada anak sampai saat ini masih sulit dikerjakan. genexpert mtb/rif adalah suatu metode diagnostik baru yang dapat mengidentifi kasi mycobacterium tuberculosis (mtb) dengan cepat. walaupun metode ini telah direkomendasikan pada anak-anak, namun penelitian tentang genexpert mtb/rif dalam mendiagnosis tb paru anak di lingkungan rumah sakit (rs) belum pernah dikerjakan di indonesia. kami membandingkan hasil pemeriksaan genexpert mtb/rif dengan kultur dahak mtb pada media lowenstein jensen (lj) dalam menegakkan diagnosis tb paru pada anak yang diduga tb paru. penelitian ini dilakukan di poli dan bangsal respirologi anak rsud dr. soetomo antara juni sampai agustus 2015 secara cross sectional. dengan sampling konsekutif mengumpulkan 27 anak usia 3 bulan sampai 14 tahun yang mempunyai kontak erat dengan penderita tb dewasa dan menunjukkan gejala tb paru. pada setiap anak dilakukan pengambilan dahak dengan cara induksi dahak kemudian dilakukan tiga metode pemeriksaan sekaligus yaitu secara mikroskopis, genexpert mtb/rif dan kultur yang dilanjutkan dengan uji kepekaan mtb bagi spesimen yang terdeteksi ada pertumbuhan mtb. sensitivitas genexpert * corresponding author: retnosoedijo@yahoo.co.id 153berlian beatrix rarome, et al.: genexpert mtb/rif and mycobacterium tuberculosis sputum culture copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 mtb/rif adalah 100% (95% ci 100-100) dan spesifi sitas 95% (95% ci 85-100). nilai duga positif genexpert mtb/rif adalah 89% (95% ci 68-100), sedangkan nilai duga negatifnya adalah 100% (95% ci 100100) dan likelihood positive nya adalah 20 (95%ci 2,82-128). genexpert mtb/rif mampu mendeteksi semua spesimen yang terdeteksi positif mtb oleh kultur dahak mtb namun dalam waktu yang lebih singkat dan dengan spesifi sitas yang tinggi. kesepadanan hasil antara genexpert mtb/rif dan kultur dahak didapatkan berdasarkan kelompok umur dan manifestasi klinis tb. selain dalam mendeteksi resistensi rifampicin, genexpert mtb/rif memberikan hasil yang sama dengan uji kepekaan mtb. kata kunci: tuberkulosis paru anak, sensitivitas, spesifi sitas, genexpert mtb/rif how to cite: genexpert mtb/rif and mycobacterium tuberculosis sputum culture in establishing the diagnosis of pulmonary tuberculosis and rifampicin resistance in suspected childhood pulmonary tuberculosis in soetomo hospital. rarome, bb. aisah, n. setyoningrum, ra. mertaniasih, nm. indonesian journal of tropical and infectious disease, 8(3), 152–162. introduction diffi culty to diagnose tuberculosis in children can lead to under and over diagnosis of tb which can cause higher morbidity and mortality. confi rming the diagnosis of childhood tb is a major challenge. however, research on childhood tuberculosis in relation to better diagnostics is often neglected because of technical diffi culties, such as the slow growth in culture, the diffi culty of obtaining specimens, and the diverse and relatively nonspecifi c clinical presentation of tb in this age group. while the classic presentation of childhood tb is prolonged cough and weight loss, hiv infection, with its chronic pulmonary manifestations and wasting, may confound the diagnosis of childhood tb. these diffi culties are worsened by the increased incidence of multiple drug resistance.1,2 therefore, early diagnosis of tb in children is very important in order to control the incidence of tb disease. tuberculosis remains a major problem for the health of mankind. in 2012, the estimated incidence of tb cases were 8.6 million cases / year and 58% of these cases occur in southeast asia and the western pacifi c. approximately 50-60% of children living with adult pulmonary tuberculosis (ptb) patients who have positive acid-fast bacilli (afb) sputum results will be infected with tb as well and about 10% of them will get tb disease.3 world health organization (who) in 2012 estimated that there were 530,000 new cases of tb in children with a mortality rate of 74,000.4 indonesian tb data in 2012 showed the proportion of tb cases in children among all tb cases was 8.2%.5 the diagnostic approaches that exist today are less sensitive. although conventional examination with a microscope has a high positive predictive value for detecting mycobacterium tuberculosis (mtb), its sensitivity is low. examination using media culture with lowenstein-jensen (lj) is still the gold standard for diagnosis but this test is diffi cult and requires a long time (± 6-9 weeks) to get the results with positive results obtained only in 10% 15% culture examination.6,7 polymerase chain reaction (pcr) test provides high sensitivity by multiplying deoxyribonucleic acid (dna) of bacteria, and has been extensively evaluated in order to detect the dna of mtb. genexpert mtb/rif is an integrated and automated test with molecular approaches. sample preparation, amplifi cation and detection is done automatically by pcr. genexpert mtb/rif is able to detect mtb as well as diagnosing resistance to rifampicin. the results will be obtained in less than 2 hours.8,9,10,11 in december 2010, who has encouraged the use of genexpert mtb/rif as a tool for the diagnosis of tb due to high sensitivity and specifi city but studies on the use of genexpert mtb/rif in children are still rare.11,12 the aim of this study is to compare the genexpert mtb/ rif with mtb sputum culture examination in the diagnosis of ptb and rifampicin resistance in children with suspected ptb in dr. soetomo hospital surabaya. 154 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 3 september–december 2020: 152–160 materials & methods this study was analytical observational to compare the genexpert mtb/rif assay with mtb sputum culture for detection of pulmonary tuberculosis and rifampicin resistance in new pediatric inpatients and outpatients at the department of pediatric and child health, dr. soetomo hospital, surabaya, indonesia, a tertiary referral center. this study was approved by the research ethics committee of dr. soetomo hospital surabaya. the parents of all study participants provided written informed consent. between june 2015 and august 2015, new outpatients and inpatients children, aged 3 months old 14 years old, with the diagnosis of suspected tuberculosis were eligible for enrollment in the study. a patient with suspected tuberculosis was defined as having a symptom and risk factor screening (one or more of fi ve factors: tuberculosis contact, cough for more than 3 weeks, weight loss, malnutrition, or fever for more than 2 weeks with unknown origin) according to the indonesian national tb program. patients were excluded if they were deemed to have a poor prognosis, congenital heart defects, severe congenital abnormalities, acute hemodynamic disturbances (hypotension, shock, heart failure, decreased consciousness), critical illness (sepsis, renal failure, impaired liver function severe), have received tb treatment > 1 month and patients with hiv infection or if parents or guardians refused the informed consent. procedure of sample collection started with history taking and physical examination taken when administered. the clinical manifestations observed in this study were in accordance with tb scores commonly used in indonesia, including a history of close contact with adult tb patients, the results of tst, fever ≥ 2 weeks are not unexplained, coughing ≥ 3 weeks, enlarged neck lymph nodes, inguinal and axillary, nutritional status, swelling of bones/joints and chest x-rays. sputum samples were collected from children who could expectorate. in children who could not spontaneously expectorate, sputums were collected by induced sputum procedure. smear microscopy, culture with lj media and genexpert mtb/rif were done simultaneously on all samples as described previously. cultures were classified as negative when no growth were detected after 8 weeks of incubation. contaminated samples were retreated and recultured, and excluded if still contaminated. drug susceptibility testing was done on lj media. sputum was added to the genexpert mtb/rif sample reagent in a 1:1 ratio (1 ml of sputum to 1 ml of the sample reagent). two ml of this mixture was added to the genexpert mtb/rif cartridge and run in the machine in accordance with manufacturer’s instructions. all clinical and laboratory data were compiled in databases. selected variables were exported to spss (version 21) for analysis. comparisons of genexpert mtb/rif assay and sputum culture assay were done with pearson χ² or fishers exact test. the sensitivity, specifi city, and predictive values of the assays with 95% cis were calculated. the equivalence between genexpert mtb/rif assay and sputum culture were analyzed with mcnemar test and kappa. all statistical tests were two-sided with alpha of 5%. results twenty seven children were recruited and had sputum for analysis. table 1 shows about half (51.9 %) of the children enrolled were younger than 5 years. the most common clinical figure 1. operational framework 155berlian beatrix rarome, et al.: genexpert mtb/rif and mycobacterium tuberculosis sputum culture copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 manifestation reported was fever with no apparent cause lasting more than 2 weeks. eighteen subjects (66.7%) showed positive tst and 9 (33.3%) had negative result. molecular examination with genexpert mtb/rif provides the highest positive results of 33.3%, followed by microbiological culture (29.7%) and microscopic examination (22.2%) (table 2). there was no false negative results for genexpert mtb/rif examination. genexpert mtb/rif correctly identifi ed all 9 rifampicin-sensitive on specimen analysis (table 3). all 8 lj culture positive specimens were also analyzed with the lj drug-sensitivity test and none were rifampicin resistance. therefore mtb drug sensitivity cannot be analyzed statistically since the lj results on solid media were equivalent with the results by the genexpert mtb/rif (table 3). table 4 shows that only 5 (27.8%) of 18 patients with positive tst results were confi rmed positive by genexpert mtb/rif, whereas 13 (72.2%) others showed negative results by genexpert mtb/rif. there were 4 (44.4%) of 9 children who showed negative tst result, but confi rmed positive on genexpert mtb/rif, and 5 (55.5%) of 9 children showed a negative result on both tst and genexpert mtb/rif. table 5 shows the equivalence results of genexpert mtb/rif and mtb sputum culture in the age group ≥ 5 years old as many as 12 samples. there is only one sample which showed a positive result on genexpert mtb/rif but confi rmed table 2. sputum results sputum examination n (%) mtb culture positive 8 (29.7) mtb culture negative 19 (70.3) genexpert mtb/rif positive 9 (33.3) genexpert mtb/rif negative 18 (66.7) smear microscopic positive 6 (22.2) smear microscopic negative 21 (77.8) table 1. baseline characteristics characteristics n(%) age < 5 years old 14 (51.9) age ≥ 5 years old 13 (48.1) gender male 14 (51.9) gender female 13 (48.1) contact identifi ed 18 (66,7) contact not identifi ed 9 (33,3) contact mdr 5 (18.5) contact non mdr 12 (44.4) scar bcg present 21 (77,8) scar bcg none 6 (22,2) cough > 3 weeks 18 (66.7) fever > 2 weeks 19 (70.4) lymph node enlargement 9 (33.3) nutritional status normal 12 (44.4) poor 10 (37.0) malnutrition 5 (18.5) tst positive 18 (66.7) tst negative 9 (33.3) bone destruction 1 (3.7) chest x-ray suggestive tb 16 (59.3) chest x-ray not suggestive tb 11 (40.7) tb score ≥ 6 23 (85.1) tb score < 6 4 (14.8) table 3. drug sensitivity test of positive results (8 lj culture and 9 genexpert mtb/rif) rifampicin sensitivity test n (%) drug sensitivity test with lj rifampicin sensitive (+) 8 (100) rifampicin resistance 0 (0) inh sensitive 7 (87.5) inh resistance 1 (12.5) etambutol sensitive 8 (100) etambutol resistance 0 (0) streptomycin sensitive 7 (87.5) streptomycin resistance 1(12.5) genexpert mtb/rif rifampicin sensitive (+) 9 (100) rifampicin resistance 0 (0) table 4. tst result vs genexpert mtb/rif genexpert mtb/rif total positive (%) negative (%) tst positive 5 (27,8) 13 (72,2) 18 negative 4 (44,4) 5 (55,5) 9 total 9 18 27 156 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 3 september–december 2020: 152–160 negative on mtb sputum culture. mcnemar test shows no signifi cant diff erence. kappa test results shows signifi cant reliability between the results of the genexpert mtb/rif with mtb sputum culture mtb in the age group ≥ 5 years. the agreement between genexpert mtb/rif and mtb sputum culture examination was 92.4%. table 6 shows the total positive clinical tb assessment in 17 (63.0%) of 27 children. there were 9 (53.0%) clinical tb children who have positive results of genexpert mtb/ rif. all children with clinically negative tb showed negative result on genexpert mtb/rif. mcnemar test showed no signifi cant diff erence between the results of genexpert mtb/rif and mtb sputum culture with positive clinical manifestations of tb. kappa test showed significant equivalence between genexpert mtb/rif and mtb sputum culture with positive clinically manifestation of tb. this study showed that genexpert mtb/ rif had 100 % sensitivity (95% ci 100-100), specifi city of 95% (95% ci 85-100), ppv 89% (95%ci 68-100), npv 100% (95% ci 100-100), lr +20 (95% ci 2.82-128), lr 0. discussion in our study, more than 50% of samples had a history of close contact to adult patients with positive afb smear, cough > 3 weeks, fever of unknown origin > 2 weeks, positive tst results and x-rays which showed ptb process. more than 50% of the sample had a value of tb score > 6. positive tst results were not always found in children suspected of pulmonary tb. in our study, positive tst results obtained in 66.67% of the samples, of which only 55.5% of the samples were genexpert mtb/rif positive. sekadde et al (2013) and nicol et al (2011) obtained positive results only at ± 30% of the samples13,14, while nataprawira et al (2001) get positive tst results only in 9.7% of children who had close contacts with adult tb patients or adults suspected of having tb in bandung.15 there are several factors that infl uence the results of tst, such as malnutrition which eff ect on phagocytosis, cellular immunity and cytokine production.16 malnutrition leads to lymphoid tissue atrophy, thus aff ecting the development, diff erentiation and cause a decrease in lymphocytes. moderate and severe malnutrition lead to decreasing delayed-type hypersensitivity reactions and recall process.17 the positive results of microscopic and molecular examination in this study is quite high when compared to previous studies. giang et al reported positive results of genexpert mtb/ rif on 8.6% of samples,18 nicol et al reported 12.8%,14 sekadde et al reported 14%,13 singh et al and nhu et al reported a respective 16.9% and 16.2%.19,20 this condition is likely due to several factors, such as the number of mtb in children (paucibacillary) and sputum production capabilities that are lacking in children. diff erent inclusion criteria with previous studies may also cause these diff erences. in our study, most of the sample had more than 4 clinical manifestations as well, while these other studies established inclusion criteria of children aged ≤ 14 years with at least 2 clinical manifestations of cough ≥ 2 weeks and one of the symptoms of weight table 5. genexpert mtb/rif vs mtb sputum culture in age group genexpert mtb/rif mtb culture agreement (%) mcnemar kappa (+) (-) < 5 years old (+) 2 0 100 p=1,000 1,000 p=0,00(-) 0 12 ≥ 5 years old (+) 6 1 92,4 p=1,000 0,847 p=0,002(-) 0 6 table 6. genexpert mtb/rif vs mtb sputum culture in clinical tb group genexpert mtb/rif mtb culture agreement (%) mcnemar kappa (+) (-) clinical tb (+) 8 1 94,2 1,00 0,883 p=0,000(-) 0 8 non clinical tb (+) 0 0 100 (-) 0 10 157berlian beatrix rarome, et al.: genexpert mtb/rif and mycobacterium tuberculosis sputum culture copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 loss or fever ≥ 2 weeks with unknown origin, or a history of contact with adult tb patients, or a positive result on tst or positive x-ray for tb process.13,14,20 microscopic examination (olympus ch-20, olympus corp., japan; 1000x magnifi cation) is able to detect 66.7% of specimens with positive genexpert mtb/rif and 75% of specimens positive by mtb sputum culture. positive smear cases were found mainly in the > 5 years old age group. this occurs because children > 5 years old or adolescents have pathological features of "adult-type" tb that is not paucibacillary with more bacilli accumulated and generally give more positive results on microscopic examination.21,22 previous studies reported the same result. marlowe et al in the us collected 217 sputum specimens and showed that microscopic examination is able to detect 73% of genexpert mtb/rif positive results. lawn et al (2011) only reported 45% positive smear result of tb cases. this can be explained due to the colony of microscopic detection capabilities is less sensitive than the other two examination modalities. the detection capability of the colony smear microscopy is 5x103 to 5x104 bacilli/ml, the detection capability of genexpert mtb rif is 102-107 cfu/ml and the culture detection capability is 10-100 cfu/ ml.12,23 however, microscopic examination is not specifi c to diagnose tb because there are several other bacteria that are resistant to acid staining which are rhodococcus spp, nocardia spp, legionella micdadei, cysts and isospora of crytopsoridium spp, that will give a false-positive smear result.24 in this study, the sensitivity of genexpert mtb/rif is 100% and specifi city was 94.7%. there is one genexpert mtb/rif positive result that is not detected by mtb sputum culture. pcr concept used in genexpert mtb/rif sequence all of mtb dna without the capability to detect the viability of the mtb. genexpert mtb/rif false-positive may result from patients who had been treated as well. systematic reviews conducted by who in 2013 among 13 studies involving 2,603 participants mention pooled sensitivity of genexpert mtb/ rif tb was 66% (95% ci 52-77) and pooled specifi city was 98%.25 meta-analysis conducted in 2012 of 18 studies involving 10,224 specimens reported sensitivity of genexpert mtb/rif amounted to 90.4% (95% ci 89.2 to 91.4) and specifi city of 98.4% (95% ci 98-98, 7).26 recent meta-analysis of the ability of genexpert mtb/ rif in the diagnosis of childhood ptb reported pooled sensitivity of 62% (95% ci 51-73) and a pooled specifi city of 98% (95% ci 97-99).27 bates et al reported no signifi cant diff erences between specimens derived from sputum or liquid gastric washings in the genexpert mtb/ rif examination and concluded the use of liquid gastric washings can replace sputum specimens if they are not available.28 in the study conducted by nhu et al and singh et al, stored sputum specimens were used instead of fresh sputum specimens.19,20 in a sputum that was kept frozen and then thawed, the dna will be damaged and aff ect the viscosity of sputum, thus giving bias.29 performing genexpert mtb/rif examination twice on one specimen reportedly do not increase the rate of case detection. repeated examination of genexpert mtb/rif will increase the cost, even though there are still other supporting diagnostic examination. bblk surabaya’s policy is to do single sputum genexpert mtb/rif examination for each patient.14,20 the existence of genexpert mtb/rif machines is not widely available in primary and secondary health facilities, therefore sekkade et al conducted a study in uganda and analyze the clinical characteristics associated with genexpert mtb/rif positive results. it is intended to help health workers in limited medical care facilities to predict the likelihood of tb in a suspected tb children. researchers reported some characteristics of the sample that has a tendency to get positive result of genexpert mtb/rif, such as age group of > 5 years, a positive tst result and a positive tb contacts.13 all sputum specimens with positive result of genexpert mtb/rif and mtb sputum culture show sensitive result to rifampicin in this study. a study by carriquiry et al on 130 patients aged > 18 years in peru in 2012 reported 100% (95% ci 61100) and 91% (95% ci 88.7 to 100) for sensitivity and specifi city respectively. predictive result were 158 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 3 september–december 2020: 152–160 66.7% (95% ci 35.4 -87.9) and 100% (95% ci 88.7 to 100) for ppv and npv respectively.29 some researchers have assessed the ability of genexpert mtb/rif in detecting mtb with rifampicin resistance, but the samples were too small and therefore cannot be assessed.14,20,30 tuberculosis is more progressive and fatal in children aged < 5 years old, while those aged ≥ 5 years old was associated with disease progression being "adult-type tb". other than that this age group is the most common group of contracting tb in countries with high tb prevalence. this type of "adult-type tb" has the potential to cause extensive damage to lung parenchyma due to calcifi cation and formation of cavities, and this age group is potentially infectious to the community.21 in our study, the statistical test shows significant equivalence between genexpert mtb/rif and mtb sputum cultures in both age groups. this means that genexpert mtb/rif can be used interchangeably with mtb sputum culture to diagnose ptb in both age groups if there is no mtb sputum culture examination facilities. beside, molecular methods with genexpert mtb/rif also gives advantage of reading the results quickly (± 2 hours) so clinical decisions to initiate tb treatment can be accelerated. sekadde et al and nhu et al reported the same result for sensitivity and specifi city of genexpert mtb/rif for > 5 years old age group higher than group age < 5 years old.19,20 in our study, statistical analysis suggested there is a link between clinical manifestations and genexpert mtb/rif or mtb sputum culture results. there is equivalence between genexpert mtb/rif results and mtb culture result in the clinical tb group. genexpert mtb/rif and mtb sputum cultures had lower sensitivity in diagnosing tb children clinically than molecularly. this is because children naturally had paucibacillary mtb although demonstrated clinical manifestations of tb and have symptoms improvement after treatment. symptoms of tb in children are not specifi c and more than 50% of children with tb are asymptomatic. children with tb exhibiting clinical symptoms mostly will experience lung disorders, while 25% 35% have extrapulmonary disorders. systemic disorders such as fever, night sweats, anorexia may also occur. the most common clinical symptoms are cough, body fatigue and weight loss. specifi city of clinical symptoms depends on the tightness of operational defi nitions used. however there is no cut-off for clinical symptoms that have been validated until now.22,31,32 the diagnosis of ptb in children cannot be established by clinical symptoms alone. laboratory tests need to be done in children with or without clinical symptoms of ptb. however, the negative results of bacteriological examination does not exclude the possibility of tb disease.22 patients aged < 5 years, with a positive tst result and history of close contact with adult tb patients but do not show symptoms of ptb, were given inh prophylaxis of 7-15 mg / kg / day, once daily for 6 months, while patients that show symptoms of ptb were given tb drugs according to standard procedures.33 conclusion genexpert mtb/rif has a good sensitivity and specifi city to diagnose pulmonary tuberculosis (ptb) in children which give parallel results with mtb sputum culture methods in aiding the diagnosis of ptb in children aged ≥ 3 months old 14 years old with suspected ptb. conflict of interest there is no confl ict of interest of this study. acknowledgement the author sincerely thank all members of pediatric department and staff in dr. soetomo hospital for their permission, facilities and fi nancial support. references 1. cuevas le, browning r, bossuyt p, casenghi m, cotton mf, cruz at, et al. evaluation of tuberculosis 159berlian beatrix rarome, et al.: genexpert mtb/rif and mycobacterium tuberculosis sputum culture copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 diagnostics in children: 2. methodological issues for conducting and reporting research evaluations of tuberculosis diagnostics for iintrathoracic tuberculosis in children. consensus from an expert panel. journal of infectious diseases 2012;205:209-15. 2. edwards dj, kitetele f, rie av. agreement between clinical scoring systems used for the diagnosis of pediatric tuberculosis in the hiv era. int j tuberc lung dis 2007;11:263-9. 3. starke jr. tuberculosis ( mycobacterium tuberculosis). in: kilegman rm, stanton bf, schor nf, geme jws, behrman re, editors. nelson textbook of pediatrics 19th edition. philadelphia: elsevier, 2011;996-1011. 4. who. automated real-time nucleic acid amplifi cation technology for rapid and simultanous detection of tuberculosis and rifampicin resistance: xpert mtb/rif assay for the diagnosis of pulmonary and extrapulmonary tb in adults and children. policy update. 2013;23-33 5. kemenkes ri. petunjuk teknis manajemen tb anak. 2013;2-79. 6. marais bj, pai m. new approaches and emerging technologies in the diagnosis of childhood tuberculosis. paediatric respiratory reviews 2007;8:124-33. 7. kulkarni s, singh p, memon a, et al. an in-house multiplex pcr test for the detection of mycobacterium tuberculosis, its validation & comparison with a single target tb-pcr kit. indian j med res 2012;135:78894. 8. caws m, wilson sm, clough c, drobniewski f. role of is6110-targeted pcr, culture, biochemical, clinical, and immunological criteria for diagnosis of tuberculous meningitis. j clin microbiol 2000;38:3150-5. 9. narayanan s, parandaman v, narayanan pr, venkatesan p, girish c, mahadevan s, et al. evaluation of pcr using trc4 and is6110 primers in detection of tuberculous meningitis. j clin microbiol 2001;39:2006-8. 10. chakravorty s, tyagi js. novel multipurpose methodology for detection of mycobacteria in pulmonary and extrapulmonary specimens by smear microscopy, culture, and pcr. j clin microbiol 2005;43:2697-702. 11. who. rapid implementation of the xpert mtbrrif diagnostic test.technical and operational 'how-to' practical considerations. geneva,switzerland: world health organization 2011;5-25 12. lawn sd, nicol mp. xpert®mtb/rif assay: development, evaluation and implementation of a new rapid molecular diagnostic for tuberculosis and rifampicin resistance. future microbiol 2011;6:106782. 13. sekadde mp, wobudeya e, joloba ml, et al. evaluation of the xpert mtb/rif test for the diagnosis of childhood pulmonary tuberculosis in uganda: a cross-sectional diagnostik study. bmc infect dis 2013;13:133-41. 14. nicol mp, workman l, isaacs w, et al. accuracy of the xpert mtb/rif test for the diagnosis of pulmonary tuberculosis in children admitted to hospital in cape town, south africa: a descriptive study. the lancet 2011:1-6. 15. nataprawira hmd, kartasasmita cb, rosmayudi o, et al. diagnosis of pediatric tuberculosis using the indonesian national concencus for pediatric tuberculosis. pediatr indones 2001;41:185-90 16. chandra rk. nutrition and the immune system : an introduction. am j clin nutr 1997;66:460-3 17. cunningham-rundles s, moon a, mcneeley df. malnutrition and host defense. in nutrition in pediatrics. 4th ed. hamilton, ontario, canada: bc decker inc, 2008:261-72 18. giang dc, duong tn, ha dtm,et al. prospective evaluation of genexpert for the diagnosis of hivnegatif pediatric tb cases. bmc infect dis 2015;15:7080 19. singh s, singh a, prajapati s, et al. xpert mtb/ rif assay can be used on archived gastric aspirate and induced sputum samples for sensitif diagnosis of pediatric tuberculosis. bmc microbiol 2015;15:191201 20. nhu ntq, ha dtm, anh nd, et al. evaluation of xpert mtbrif and mods assay for the diagnosis of pediatric tuberculosis. bmc infectious diseases 2013;13:31-40. 21. marais bj, gie rp, schaaf hs, et al the natural history of childhood intra-thoracic tuberculosis: a critical review of literature from the pre-chemotherapy era. int j tuberc lung dis 2004;8:392-402 22. who. guidance for national tuberculosis programmes on the management of tuberculosis in children. 2014;2:21-74 23. marlowe em, novak-weekley sm, cumpio j, et al. evaluation of the cepheid xpert mtb/rif assay for direct detection of mycobacterium tuberculosis complex in respiratory spesimens. j clin microbiol 2011;49:1621-3. 24. kemenkes ri. petunjuk teknis pemeriksaan biakan, identifikasi,dan uji kepekaan mycobacterium tuberculosis pada media padat. 2012:10-44 25. who. automated real-time nucleic acid amplifi cation technology for rapid and simultanous detection of tuberculosis and rifampicin resistance: xpert mtb/rif assay for the diagnosis of pulmonary and extrapulmonary tb in adults and children. policy update. 2013:23-33 26. chang k, lu w, wang j, et al. rapid and eff ective diagnosis of tuberculosis and rifampisin resistance with xpert mtb/rif assay: a meta-analysis. j infect 2012;64: 580-8. 27. detjen ak, keil t, roll s, et al. interferon-g release assays improve the diagnosis of tuberculosis and nontuberculous mycobacterial disease in children in a 160 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 3 september–december 2020: 152–160 country with a low incidence of tuberculosis. clinical infectious diseases 2007;45:322-28. 28. bates m, o’grady j, maeurer m, et al. assessment of the xpert mtb/rif assay for diagnosis of tuberculosis with gastric lavage aspirates in children in sub-saharan africa: a prospective descriptive study. the lancet infect dis 2013;13:36-42. 29. carriquiry g, otero l, gonzalez-lagos e, et al. a diagnostik accuracy study of xpert mtb/rif in hiv positif patients with high clinical suspicion of pulmonary tuberculosis in lima,peru. plos one 2012;7:1-7. 30. theron g, peter j, zyl-smit r, et al. evaluation of the xpert mtb/rif assay for the diagnosis of pulmonary tuberculosis in a high hiv prevalence setting. am j respir crit care med 2011;184:132-40. 31. hesseling ac, schaaf hs, gie rp, et al. a critical review of diagnostic approaches used in the diagnosis of childhood tuberculosis. int j tuberc lung dis 2002;6:1038-45. 32. shingadia d, burgner d. mycobacterial infections. in: taussig lm, landau li, souef pnl, martinez fd, morgan wj, sly pd, editors. pediatric respiratory medicine 2nd edition. philadelphia: elsevier 2008:597614. 33. kemenkes ri. petunjuk teknis manajemen tb anak. 2013:2-79. vol. 8 no. 1 january-april 2020 copyright © 2020, ijtid, issn 2085-1103 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ research report relationship of non structural antigen 1 (ns1) to clinical signs, symptoms and routine blood examination dengue suspected acivrida mega charisma stikes rumah sakit anwar medika corresponding author: acie.vrida@gmail.com received: 8th november 2018; revised: 6th december 2018; accepted: 14th january 2020 abstract early diagnosis of dengue infection is important due to very rapid disease patophysiology because late diagnostic can be fatal to the patient, remembered the journey of the disease is very rapid. currently was a non-structural 1 dengue antigen (ns1) examination which can detect detect dengue viral infections earlier, even on the fi rst day of fever. however, not all health care centers have adequate laboratory facilities for ns1 examination. clinical symptoms and signs as well as a routine blood test are indicators that become basic of diagnosis in health care facilities with limited facilities. this study aims are to determine the relationship of ns1 examination to clinical sign and symptoms and ns1 examination as well as the result of routine blood tests in patients suspected dengue infection. this research was used observational analytic method with cross sectional approach. the research was conducted in clinic laboratory and inpatient clinic room of vita medika kepung kediri from november 2017 to february 2018.the number of research samples of 30 people was determined by the consecutive sampling technique. ns1 examination was done by using rapid immunochromatography test method with mono kits. routine blood examination was done by using micros 60. the reason for using chi square the test is because the two variables studied are variables with data in the form of a categorical scale chi square test on relationship between clinical sign and symptoms examination of dengue with the results of ns1 examination obtained p= 0,310 (p > 0,005), while the results of chi square test on the relationship of routine blood examination results haemoglobine levels, amount of leucocyte, platelet count and of ns1 examination result obtained p value in a row p = 0,235 (p > 0,05), p = 0,013(p < 0,05), p = 0,028(p < 0,05) dan p = 0,132 (p > 0,05). there was a signifi cant correlation between leucocyte count and platelet count to ns1 antigen exanimation result, but there was no correlation between clinical signs and symptoms of dengue patients, haemoglobine level and haematocryt value on ns1 antigen examination result. keywords: dengue hemorragic fever, routine blood count , ns1 antigent dengue abstrak penegakkan diagnosis infeksi dengue sejak dini sangat penting karena keterlambatan diagnosa dapat berakibat fatal bagi penderita , mengingat perjalanan penyakit ini yang sangat cepat. saat ini telah dikembangkan suatu pemeriksaan terhadap antigen non struktural-1 dengue (ns1) yang dapat mendeteksi infeksi virus dengue dengan lebih awal bahkan pada hari pertama onset demam. akan tetapi, tidak semua pusat layanan kesehatan, memiliki fasilitas laboratorium yang memadai untuk pemeriksaan ns1. tanda dan gejala klinis serta pemeriksaan darah rutin merupakan indikator yang menjadi dasar diagnosis pada pusat layanan kesehatan dengan fasilitas yang terbatas. penelitian ini bertujuan untuk mengetahui hubungan hasil pemeriksaan antigen ns1 terhadap gejala , tanda klinis dan hasil pemeriksaan darah rutin pada pasien terduga infeksi dengue. penelitian ini menggunakan metode analitik observasional dengan pendekatan cross sectional. penelitian dilakukan di laboratorium klinik dan ruang rawat inap klinik rawat inap vita medika kepung kediri pada bulan november tahun 2017 – februari tahun 2018. jumlah sampel penelitian 30 orang ditentukan dengan teknik consecutive sampling. pemeriksaan antigen ns1 dilakukan menggunakan metode rapid immunochromatography test dengan kit mono. pemeriksaan darah rutin dilakukan menggunakan micros 60. uji chi square mengenai hubungan antara gejala dan corresponding author. e-mail: acie.vrida@gmail.com 67acivrida mega charisma, et al.: relationship of non structural antigen 1 (ns1) copyright © 2020, ijtid, issn 2085-1103 tanda klinis dengue dengan hasil pemeriksaan ns1 diperoleh nilai p = 0,310 (p > 0,005) sedangkan hasil uji chi square mengenai hubungan hasil pemeriksaan darah rutin yaitu kadar hemoglobin , jumlah lekosit , jumlah trombosit dan nilai hematokrit terhadap hasil pemeriksaan ns1 didapatkan nilai p berturut – turut p = 0,235 (p > 0,05) , p = 0,013(p < 0,05) , p = 0,028(p < 0,05) dan p = 0,132 (p > 0,05). terdapat hubungan yang bermakna antara jumlah lekosit dan jumlah trombosit terhadap hasil pemeriksaan antigen ns1, namun tidak terdapat hubungan antara tanda dan gejala klinis pasien dengue , kadar hemoglobin dan nilai hematokrit terhadap hasil pemeriksaan ns1. kata kunci: dengue, ns1,pemeriksaan darah rutin how to cite: charisma, acivrida mega. relationship of non structural antigen 1 (ns1) to clinical signs, symptoms and routine blood examination dengue suspected . indonesian journal of tropical and infectious disease, [s.l.], v.8, n.1, p.235-245 jan. 2020. issn 2085-1103. available at: https://ejournal.unair.ac.id/ijtid/article/view/10382. date accessed: 09 dec. 2019. doi: http://dx.doi.org/10.20474/ijtid.v8i1.10382 introduction dengue infection is the most common disease the tropical and subtropical district, especially southeast asia, central america, america and carribian. the natural object of dengue is human, the agent is dengue virus that is included to family of flaviridae and flavivirus genus, contains of 4 serotipes for instance den-1, den-2, den-3 and den -41. the disease was transmitted to human through infected mosquito’s bite, mainly aedes aegypti mosquito and ae. albopictus 2 that is nearly found in entire of indonesia.1 usually, dengue patients were experienced the fever phase for 2-7 days, followed by critical phase for 2-3 days. the critical phase and occurs when patient has not in fever anymore, they in the phase patient will be at risk to get shock if do not get adequate treatment.2 on dengue fever, after incubation intrinsic moment for 4-6 days, appears non-specific clinical symptom, constitutional symptom, headache, backache and malaise. dengue bleed fever is indicated with two or more clinical manifestation as follows: headache, retro orbital ache, rash, antralgya and mialgya, bleeding manifestation (positive tourniquet test, petekie), leukopenia and positive dengue serology examination. onset dengue is usually marked by high fever, headache and flushing.3 in general, the diagnosis of dengue is difficult to enforce in the first few days of illness because the symptoms that appear are not specific and difficult to distinguish from other infectious diseases.4 diagnosis of dengue infection only based on clinical syndromes which cannot be fully trusted, so the diagnosis needs to be confirmed using laboratory tests. laboratory tests that can be done to diagnose dengue infection include: virus isolation, detection of viral nucleic acids, detection of viral antigens, tests based on immunological responses (anti-dengue igm and igg), and hematological parameter analysis.5 hematologic parameters that are routinely examined to screen suspected patients with dengue fever are through examination of hemoglobin levels, leucocyte counts, hematocrit values, platelet counts, and peripheral blood smears to see the presence of relative lymphocytosis and blue plasma lymphocyte.6 nowadays ns1 antigen examination has been developed to detect the presence of dengue virus infection in the acute phase, where in various studies it has been shown that ns1 is superior in sensitivity than viral culture and polymerase chain reaction (pcr) examination as well as antidengue igm and igg antibodies. the bag-specific ns1 antigen 100% is as high as the gold standard for viral culture or pcr.7 dengue virus has two types of proteins, namely structural envelope (e) proteins, matrix (m) and capsid (c)) and nonstructural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, ns5) . 8,23 protein e, pr m protein and ns1 protein has antigenic properties.9 nonstructural protein ns1 in the dengue virus is a glycoprotein measuring 46-50 kilodalton expressed on infected host cells both membrane associated (mns1) and secreted (sns1) and not part of the virion structure component.10 ns1 is produced by all flaviviruses and plays an important role in the process of 68 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 67–78 copyright © 2020, ijtid, issn 2085-1103 replication and survival of the virus.11 ns1 acts as an important immunogen in dengue infection and plays a role in protecting against diseases, especially in secondary infections where anti-csf antibodies are found in the patient’s serum. pathogenesis of dengue infection.12 in addition to the viral bond with antibodies, ns1 antigens also play a role in plasma leakage and bleeding in dengue infections. ns1 antigen will bind to specific antibodies resulting in vascular endothelial cell apoptosis and activation of the complement system which contributes to plasma leakage and platelet lysis.13 however, ns1 examination is rarely done in health laboratories, especially in rural areas, this is one of them because the examination price is quite expensive. routine blood tests which include hemoglobin examination, leukocytes, platelets and hematocrit are determinants in diagnosing other than clinical symptoms.14 based on the background above, in this study the researchers intend to find out whether or not there is a relationship between ns1 examination results and clinical signs and symptoms as well as routine blood examination results which include hemoglobin level, leukocyte count, platelet count and hematocrit value in patients suspected of dengue infection. materials and methods this type of research is an observational analytic study with a cross sectional approach. this study aims to determine the relationship between ns1 antigen examination results on clinical symptoms, platelet count and hematocrit values in patients suspected of dengue infection clinic vita medika kepung, kediri regency. the research was conducted in november 2017 february 2018 in the clinical laboratory section and inpatient vita medika kepung clinic in kediri regency. the sample used in this study were patients with suspected dengue infection at the vita medika kepung clinic in kediri regency in november 2017 february 2018 with inclusion criteria as stated bellow male and female sex, age 0-10, 11-20, 21-30, >30 year,4 (with total of 30 subjects for all classifications), illness duration at hospital admission <5 days from the onset of fever, no other illness indications, not consuming any medicine which suppress spinal cord, complete medical record, no blood deviation, willing to be research subject. the sampling technique in this study was consecutive sampling. at consecutive sampling, all subjects who arrived and met the selection criteria were included in the study until the required number of subjects were met. this consecutive sampling is the best type of non-probability sampling and is the easiest method. most clinical studies (including clinical trials) use this technique to select subjects. the number of samples was determined a formula. exclusion criteria in this study were patients with suspected dengue with a long illness since the onset of fever for more than 5 days, patients who were taking medications that suppressed bone marrow, patients who had a history of blood disorders, patients with other coincidental diseases, such as typhoid fever, patients with indications of other infectious diseases, such as respiratory infections, urinary tract infections and gastrointestinal infections, incomplete medical records, patients with symptoms and signs of shock, and unwilling to become respondents in the inform consent. the independent variable (independent variable) in this study is the results of nonstructural antigen 1 (ns1). the dependent variable in this study is the clinical signs and symptoms of dengue infection, hemoglobin level, leukocyte count, platelet count and hematocrit value. clinical symptoms referred to in this study are fever, which is accompanied by at least 2 of the following symptoms: headache, retroorbital pain, myalgia, arthralgia, rash, and bleeding manifestations such as petechiae, positive tourniquet test, and spontaneous bleeding. data processing including the examination of the completeness and clarity of the data, assigning code to each variable data, entering data in the spss program (statistical program for social science), and checking back to ensure that the data has been cleared of errors. data analysis consisted of univariate and bivariate analysis. the 69acivrida mega charisma, et al.: relationship of non structural antigen 1 (ns1) copyright © 2020, ijtid, issn 2085-1103 statistical test used in this study is the chi square test. the reason for using the test is because the two variables studied are variables with data in the form of a categorical scale. if the chi square test does not meet the requirements (the expected count value is less than 5> 20%), then use the fisher exact test for 2x2 tables, kolmogorov smirnov test for 2x2 tables. results and discussion in this part, we provide the research result, it consists of 12 tables. table 1 was showed that in this study it was found that according to gender the number of male respondents were more than female respondents with a ratio of 1.14: 1. these results are in line with the results of research conducted by libraty6,8,24 who get more male sufferers than women with ratio of 2.2: 1, as well as in the research conducted by mayer et al, 15 the number of male respondents was more than women with a ratio of 3: 2 and research by juranah21 in 2011. production of anti inflammation sitocyn in female was more abundant, therefore, female who get dengue infection give unclear clinical complaint and are rarely to be hospitalized or clinic.8 in women the production of anti-inflammatory cytokines is greater, so that women infected with dengue provide clinical complaints that are less clear and rarely treated in hospitals or clinics.2 this is also confirmed by soedarmono et al17 who stated that the xx chromosome in women has a role in managing immunoglobulin production table 1. characteristics of research subject characteristics n % gender male 16 53,3 female 14 46,7 range of age (years old) 0 10 15 50 11 20 8 26,7 21 – 30 3 10 >30 4 13,3 total 30 100 adv: n=frequency quantitatively. but halstead et al18 research shows that there is no difference between the response of infection in women and men. based on the age in this study found the youngest respondents in this study were 3 years and the oldest 38 years, the highest percentage of 15 (50%) respondents were children aged <10 years, followed by respondents with the age group 11-20 years as many as 8 (26.7 %). the results of this study were supported by a statement from the caribbean epidemiology center in 2000, which stated that the most epidemiology of dengue patients was in children and young adults. age is one of the factors that influence sensitivity to dengue virus infection.18 study was conducted in kuba which showed that age had an important role for the emergence of clinical symptoms in the form of plasma leakage.19 table 2 is showed that from 30 respondents as many as 25 (83.3%) experienced clinical symptoms of dengue, namely fever (in this case selected respondents were those who had fever 1 4 days), headaches, joint pain, nausea without signs of bleeding and 5 (16, 7%) of the respondents accompanied by a sign of bleeding which is positive for rumple leed (rl) examination. these results indicate that often the initial clinical symptoms of dengue infection are not typical, as evidenced by the variation in clinical symptoms experienced by respondents. from 25 respondents who do not show bleeding sign, 14 of them (56%) has positive ns1 antigen and 11 of them (44%) has negative ns1 antigen. while in 5 respondents who show bleeding signs, there are 3 respondents (60%) has positive ns1 antigen and 2 respondents (40%) has negative ns1 antigen. chi square test result between respondents clinical symptom when table 2. distribution of clinical signs and symptoms of patients suspected of dengue infection during admission clinical symptoms and signs of dengue (fever, arthritis, headache, nausea) n % without bleeding signs 25 83,3 with bleeding signs 5 13,3 adv: n=frequency 70 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 67–78 copyright © 2020, ijtid, issn 2085-1103 admission toward ns1 antigen checking result obtain p value = 0.310 (p > 0.05) means that there is no correlation between signs and clinical symptoms and ns1 antigen checking result. this result is in line with research muhamad12 in 2007 who claims that there is no correlation between symptoms and clinical signs with ns1 antigen checking result with p value = 0.115 (p > 0.05). table 3 was described that at the time of admission from 30 respondents there were 7 (23.3%) respondents had hb <normal, 23 (76.7%) respondents had hb levels within the normal range, and no (0%) respondents had hb levels> normal. in this study the determination of normal values is distinguished by the sex of the respondents. chi square result between hb and ns1 antigen checking result obtains p value = 0.235 (p > 0.05) which has meaning that there is no correlation between hb and ns1 antigen checking result. this result is in line with research10 conducted in 2016 which found that there is no correlation between hb of patients suspected with dengue infection and ns1 antigen checking result with p value = 0.483 (p > 0.05). table 4 was showed that in this study, at the time of admission, 22 (73.3%) respondents had leukocyte counts <4,000 cells / mm3 (<normal), and as many as 8 (26.7%) respondents had leukocytes between 4,000 10,000 cells / mm3 (within normal limit) and no respondent experienced an increase in leukocyte count. table 3. distribution of hemoglobin levels in patients suspected of dengue infection during admission hemoglobin levels (g/dl) n % male < 13,5 7 23,3 13,5 – 18,0 9 30,0 > 18,0 0 0 female < 11,5 0 0 11,5 – 16,0 14 46,7 > 16,0 0 0 total 30 100 advt: n=frequency table 4. distribution of patient leukocytes suspected of dengue infection during admission leukocyte count (sel/mm3) n % < 4.000 22 73,3 4.000 – 10.000 8 26,7 >10.000 0 0 total 30 100 adv: n=frequency table 5. distribution of patient’s platelet number suspected of dengue infection during admission platelet count (sel/mm3) n % <100.000 13 43,3 >100.000 17 56,7 total 30 100 adv: n=frequency this shows a tendency to decrease the number of leukocytes in the early phase of dengue infection. chi square result between leucosite amount and ns1 antigen checking obtains p value = 0.013 (p < 0.05) which shows that there is significant relationship between leucosite amount and ns1 checking result which is decreasing of leucosite amount and ns1 positive checking result. similar result was claimed by a research irawan10 in 2016 which states that there was a correlation between leucosite and ns1 antigen checking result with p value = 0.000 (p < 0.05) table 5 was illustrated that at the time of admission as many as 13 (43.3%) respondents had platelet counts <100,000 cells / mm3d and 17 (56.7%) respondents still had a platelet count> 100,000 cells / mm3 and of the 17 respondents who had platelet counts> 100,000 cells / mm3 there were 4 (13.3%) having normal platelet counts. these results were indicated that in the initial phase of infection some respondents experienced thrombocytopenia and some did not / had not experienced tombocytopenia. thrombocytopenia usually occurs after the onset of heat on the 3rd 7th day. respondents who 71acivrida mega charisma, et al.: relationship of non structural antigen 1 (ns1) copyright © 2020, ijtid, issn 2085-1103 have not experienced thrombocytopenia may not have entered the platelet decline phase. chi square test result obtains p value = 0.028 (p > 0.05) which shows there is significant result between trombocite amount and ns1 antigen checking result, where the trombocite decreasing is in line with ns1 positive antigen checking result, however, there are some subjects who did not experienced trombocite decreasing. this result is in line with a research muhamad12 in 2017 which claimed that there was a significant relationship between trombocite amount and ns1 antigen checking result with p value = 0.031 (p < 0.05). in table 6 it can be seen that at 3 (10%) the respondents had hematocrit values below normal, 10 (33.3%) respondents had normal hematocrit values and 17 (56.6%) respondents had hematocrit values above normal. this shows that most respondents experienced an increase in hematocrit values during admission. but if it is associated with the criteria for dengue diagnosis applied by who that is an increase in hematocrit value > 20%, then there are only 5 (16.7%) respondents who meet these criteria. this result is in line with the research conducted irawan 10 in 2016 which claimed that there is no significant result between hematocrite and ns1 antigen checking result with p value = 0.810 (p > 0.05). table 7 was described from 30 respondents found 17 (56.7%) had positive ns1 antigen examination results and 13 (43.3%) had ns1 negative antigen examination results. the existence of negative results in this study could be due to misinformation regarding the length of fever experienced by respondents (fever > 4 days) so that the ns1 antigen was undetectable or it could also be because the respondent was really not infected with dengue, therefore further investigation is needed. namely serological examination of dengue ig m and igg which usually begins to be detected on days 5 10 of fever (in the confaleen phase).15 at present, an examination of dengue antigen has been developed, namely non-structural 1 dengue antigen (ns1 antigen) which can detect table 6. distribution of hematocrit value in patients suspected of dengue infection during admission hematocrit value (%) n % adult male <40 1 3,3 40-48 2 6,7 >48 2 6,7 adult female <37 1 3,3 37-43 1 3,3 >43 3 10,0 kids <= 15 years old < 33 1 3,3 33 – 38 7 23,4 >38 12 40,0 total 30 100 adv: n=frequency table 7. distribution of ns1 antigen examination results in patients suspected of dengue infection during admission ns1 antigen n ( % ) positive 17 56,7 negative 13 43,3 total 30 100 adv: n=frequency dengue virus infection earlier even on the first day of onset of fever.16,22 ns1 is a non-structural glycoprotein with a molecular weight of 46-50 kd and is a highly conserved glycoprotein. initially ns1 was described as a soluble complement fixing (scf) antigen in the culture of infected cells. ns1 is needed for the survival of the virus but its biological activity is unknown. existing evidence shows that ns1 is involved in viral replication. ns1 itself is produced in two forms: membrane associated and secreted form. during cell infection, ns1 is found to be associated with intracellular organelles or transferred via secretion pathways to the cell surface (cytoplasmic membrane). ns1 is not part of the structure of the virus, but it is excreted on the surface of infected cells and has group-specific determinants and types. the role 72 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 67–78 copyright © 2020, ijtid, issn 2085-1103 of ns1 in immunopathogenesis has also been submitted based on the findings of anti-scf antibodies in serum patients with secondary infection but not in primary infection.17 ns1 dengue is secreted into the blood system in individuals infected with the dengue virus. ns1 circulates at high concentrations in the serum of patients with primary and secondary infections during the clinical clinic phase (clinical phase of illness) and the first days of the convalescence phase (recovery).18 from the results of the study it was also shown that ns1 detection can provide a specific diagnosis of dengue infection.10 datta and friends in india in 2010 were, compared ns1 in the acute phase was ns1 positive 71.42% in the acute phase, while in the ns1 positive convalescence phase only 6.38%. high sensitivity in the initial phase of fever because ns1 protein circulates in high concentrations in the patient’s blood during the initial acute phase, both in primary infection and in secondary infection. the high level of ns1 until day 5 of fever is related to the time of virenia because it is a period of viral replication and the absence of antibodies against the virus. levels of virenia and ns1 levels also depend on intrinsic characteristics and strains of the virus that infects and immunity status of the patient itself.11 another study was conducted by kumarasamy et al., it was obtained the results that the sensitivity of commercial reagents for ns1 dengue antigen for acute dengue infection was 93.4% and specificity was 100%. positive and negative forecast values are 100% and 97.3%, respectively. lastere et al studied 181 patients with dhf in france polynesia found ns1 sensitivity of 76.5% and specificity of 96.2%.12 table 8 was showed that in this study obtained from 25 respondents who showed no signs of bleeding found 14 (56%) respondents had positive ns1 antigen examination results and 11 (44%) respondents had negative ns1 antigen examination results. while the 5 respondents who showed signs of bleeding found 3 (60%) had a positive ns1 examination results and 2 (40%) had negative ns1 antigen examination results. chi square test results between respondent clinical signs and symptoms during admission of ns1 antigen examination results were obtained p value = 0.310 (p> 0.05) which means there is no relationship between clinical signs and symptoms with ns1 antigen examination results. the results of this study are in line with the research12 in 2007 which states there is no relationship between symptoms and clinical signs with ns1 antigen examination results with p = 0.115 (p> 0.05). signs and clinical symptoms typical of dengue infection are signs of bleeding, the most are skin bleeding such as a torniquet test (positive rl test, weir test), but not all of the signs of bleeding occur in dengue patients.15 torniquet test is positive if there are more than 10 petechiae in a diameter of 2.5 cm at the bottom of the front (volar) including the elbow fold (cubital fossa). a positive tourniquet test shows that capillary fragility is increased, but this condition can also be found in diseases caused by other viral infections such as measles, chikung fever, and abdominal typhus bacterial infection.7 the presence of a variety of early clinical signs and symptoms that are not typical often results in delays in diagnosis. the course of this disease can be very fast in a few days, even in a matter of hours sufferers can enter the critical phase. to avoid delays in diagnosis, physical examination and anamnese alone are not enough, it is necessary to do other examinations, namely laboratory tests as a supporter as well as enforcement of the diagnosis. table 8. relationship between clinical signs and symptoms and results of ns1 antigen examination clinical symptoms and signs dengue (fever, headache, arthritis, nausea) ns1 antigen total value p pr (ik95%)negative positive bleeding signs n (%) 11 14 25 0,310 1,071 (44,0) (56,0) (100) with bleeding signs n (%) 2 3 5 (40,0) 13 (60,0) 17 (100) 30 total (43,3%) (56,7%) (100%) adv: n=frequency 73acivrida mega charisma, et al.: relationship of non structural antigen 1 (ns1) copyright © 2020, ijtid, issn 2085-1103 table 9 was showed that in this study the results obtained when the admission of 30 respondents there were 7 (23.3%) respondents had a normal hb level and 23 (76.7%) respondents had a normal hb level. of the 7 respondents who had <normal 3 hemoglobin levels (42.9%) had a positive ns1 examination result and 4 (57.1%) had a negative ns1 antigen examination result. chi square test results between hemoglobin levels and ns1 antigen examination results obtained values p = 0.235 (p> 0.05) which means there is no relationship between hemoglobin levels and ns1 antigen examination results. these results are in line with research conducted by irawan anasta putra, et al in 2016 which stated that there was no relationship between hemoglobin levels in patients with suspected dengue infection and ns1 antigen examination results with p = 0.483 (p> 0.05). hemoglobin is a molecule consisting of heme (iron) and globin polypeptide chains (alpha, beta, gama and delta), are in the erythrocytes and are responsible for transporting oxygen.8 blood quality is determined by hemoglobin levels. the structure of hb is expressed by mentioning the number and type of globin chains that exist. there are 141 amino acid molecules in the alpha chain and 146 amino acid molecules in the beta chain, gama and delta. hb levels in the first days of the dengue infection are usually normal/slightly tabel 9. relation of hemoglobin levels to ns1 antigen examination results hb levels ns1antigen total value p pr (ik95%)negative positive < normal n (%) 4 3 7 0,235 1,420 (57,1) (42,9) (100) normal n (%) 9 (39,1) 14 (60,9) 23 (100) 4 13 3 17 7 30 total (43,3%) (56,7%) (100%) adv:n=frequency decreased, but then the levels will increase following the increase in hemoconcentration.15 table 10 is showed that in this study the results obtained at the time of admission as many as 22 (73.3%) respondents had leukocyte counts <4,000 cells/mm3 (<normal), and as many as 8 (26.7%) respondents had a leukocyte count of 4,000 10,000 cells/mm3 (within normal limit) and no respondent experienced an increase in leukocyte count. chi square test results regarding the relationship of leukocyte count and ns1 antigen examination results obtained p value = 0.013 (p < 0.05) which indicates that there is a significant relationship between leukocyte count and ns1 antigen examination results in a decrease in the number of leukocytes proportionally/in line with the results of the examination ns1 antigen is positive. similar results were revealed by hottz, et al,19 which in his research found the results of leukocyte counts with ns1 antigen examination results with p = 0.000 (p <0.05). lecocytes are white blood cells that function as the body’s defense against bacteria or viruses in the body. normal levels of leukocytes range from 4,000 10,000 cells/ mm3.lecocytes also have an important role in the body’s immunological function, if there is an increase in leukocytes can be used as a sign that infection occurs in the body. 20 at the beginning of dengue infection (when the fever starts) the leukocyte count is usually normal or decreases with neutrophil cell dominated. the occurrence of lecopenia is mainly caused by mature pmn (polymorphonuclear) leukocytes table 10. the relationship of leukocyte count with ns1 antigen examination results hb value ns1antigen total value p pr (ik95%)negative positive < normal n (%) 6 16 22 0,013 5,816 (27,3) (72,7) (100) normal n(%) 7 1 8 (87,5) 13 (12,5) 17 (100) 30 total (43,3%) (56,7%) (100%) adv: n= frequency 74 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 67–78 copyright © 2020, ijtid, issn 2085-1103 production, while in the last phase pain is found to increase lymphoblastoid cells.11 lekopenia reaches its peak just before the fever drops and is normal again in 2-3 days after defervescence.12 increased lymphoblastoid cells at the end of dengue disease originate from the transformation of t cells in leukocytes. t cells play a role in cellular immuno responses, recognize and destroy virus-infected cells and activate macrophages in phagocytosis due to immunological stimulation of dengue infection. 20 table 11 was showed that in this study results were obtained, when the admission of 13 (43.3%) respondents had platelet counts <100,000 cells/ mm3 and 17 (56.7%) respondents still had a platelet count> 100,000 cells / mm3 and of the 17 respondents who had platelet counts> 100,000 cells / mm3 there were 4 (13.3%) having normal platelet counts. of the 13 responses that had platelet counts <100,000 cells/mm3, 9 (69.2%) responses had a negative ns1 antigen examination result and 4 (30.8%) had positive ns1 antigen examination results. while in respondents with platelet counts> 100,000 cells / mm3 there were 4 (23.5%) had negative ns1 antigen examination results and 11 (76.5%) had positive ns1 antigen examination results. the chi square test results obtained by shine p = 0.028 (p <0.05) which showed a significant relationship between platelet counts table 11. relationship of platelet amounts with ns1 antigen examination results platelet amount (sel/mm3) ns1 antigen total value p pr (ik95%) negative positive <100.000 n (%) 9 4 13 1,400 (69,2) (30,8) (100) >100.000 n (%) 4 13 17 (23,5) 13 (76,5) 17 (100) 30 total (43,3%) (56,7%) (100%) adv: n=frequency and ns1 antigen examination results, where the decrease in platelets was in line with the positive ns1 antigen examination results, but there were some who did not experience a decrease in platelets have positive ns1 antigen examination results. this result is similar to the results of research conducted by muhamad12 which states there is a significant relationship between platelet counts and ns1 antigen examination results with p = 0.031 (p <0.05). thrombocytopenia has an important role in the pathogenesis of dengue infection. thrombocytopenia in dengue infection occurs through the mechanism of bone marrow suppression, platelet destruction and shortening of platelet life. in this study, the lowest platelet count occurred on day 4 since the onset of fever and decreased platelet count (00150000 cells/mm3) generally occurred on the 2-3th day since the onset of fever. decreased platelet count to .000100,000 cells/mm3 or less than 1-2 platelets / large field of view (lpb) with the average inspection carried out at 10 lpb. in general thrombocytopenia occurs before there is an increase in hematocrit and occurs before the temperature drops. platelet count ≤100,000/ mm3 is usually found between days 3 7.14 platelet count can be used as a tool to diagnose dengue because it shows high sensitivity from day 4 of fever at 67.7%, even on day 5 to 7th shows 100%. very high specificity in the use of thrombocytopenia as a parameter is caused by infrequent infectious diseases accompanied by a decrease in platelet count below 150,000 cells/mm3. even if the criteria for thrombocytes below 100,000 cells/mm3 are used, the specificity is almost 100% from the first day, but reduces the sensitivity between 10-20%. 15 thus the daily platelet examination will greatly help the diagnosis of dengue because it increases its sensitivity and specificity. 13 table 12 was showed that the results of the study were obtained during admission as many as 3 (10%) respondents had hematocrit values below normal, 10 (33.3%) respondents had normal hematocrit values and 17 (56.6%) respondents had hematocrit values above normal. this shows that most respondents experienced 75acivrida mega charisma, et al.: relationship of non structural antigen 1 (ns1) copyright © 2020, ijtid, issn 2085-1103 an increase in hematocrit values at the beginning of dengue infection. but if it is associated with dengue diagnosis hematocrit criteria applied by who that is an increase in hematocrit value > 20%, then there are only 5 (16.7%) respondents who meet these criteria. from the results of the chi square test to determine the relationship between hematocrit values with ns1 examination results, hematocrit values in this study were divided into two groups, namely the group with hematocrit value < 39% and the group with hematocrit value> 39%. from the results of the grouping on the chi square test obtained 11 (36.7%) respondents had a hematocrit value <39% where 5 (45.5%) respondents had negative ns1 antigen examination results and 6 (54.5%) respondents had antigen examination results ns1 is positive. and in groups with hematocrit value> 39%, 19 (63.3%) respondents divided into 8 (42.1%) respondents had ns1 negative antigen examination results and 11 (57.9%) respondents had positive ns1 antigen examination results. the results of the chi square test obtained p value = 0.132 (p> 0.05) which means there is no relationship between the hematocrit value of respondents during the admission with ns1 antigen examination results. this result is in line with the results of research irawan10 which states that there is no significant relationship between the matrix value and the ns1 antigen examination results with p value = 0.810 (p> 0.05). table 12. relationship of hematocrit value with ns1 antigen examination results hematocrit value ( % ) ns1 antigen total value p pr (ik95%) negative positive < 39 n (%) 5 (45,5) 6 (54,5) 11 0,132 1,062>=39 n (%) 8 11 19 (42,1) 13 (57,9) 17 30 total (43,3%) (56,7%) (100%) adv: n=frequency in general, a decrease in platelets precedes an increase in hematocrit. in dengue infection, hematocrit values usually begin to increase on day 3 of the course of the disease and increase according to the process of dengue disease.18 increased hematocrit value is a manifestation of hemoconcentration that occurs due to plasma leakage into the extra vascular space with serous fluid effusion through damaged capillaries. as a result of this leakage, plasma volume is reduced, resulting in hypovolemic shock and circulatory failure. in severe cases accompanied by bleeding, generally the hematocrit value does not increase even decreases.14 regular examination of hematocrit is needed in the treatment of dengue infection so as to prevent the possibility of hypovolemic shock that causes blood circulation failure. conclusion in conclusion, there is no significant correlation between signs, clinical symptoms, and haemoglobyn level in dengue suspected patient, moreover, there is significant correlation with the amount of leucosyt, trombocyt, hematocryt toward dengue infection. because of that, we will be able to diagnose dengue patient, as a result, we will be able to clinical symptoms in more detail way. acknowledgements i would like to express my gratitude to stikes anwar medika hospital and the biology laboratory of tikes anwar medika hospital for helping and supporting this research. conflict of interest the authors declare that there is no conflict of interest for this research. 76 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 67–78 copyright © 2020, ijtid, issn 2085-1103 references 1. candra a. 2010. demam berdarah dengue: epidemiologi , patogenesis , dan faktor risiko penularan dengue hemorrhagic fever. aspirator. 2(2):110-9 2. suhendro, nainggolan l, chen k, pohan ht. 2009. demam berdarah dengue. dalam: sudoyo aw, setiyohadi b, alwi i, simadibrata m, setiati s, editor. buku ajar ilmu penyakit dalam jilid iii edisi v. jakarta: pusat penerbitan departemen ilmu penyakit dalam fakultas kedokteran universitas indonesia. hal 2773-9 3. who. 2011. comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. india: who press 4. suwandono a, parwati i, irani p, rudiman f. 2011. perbandingan nilai diagnostik trombosit , leukosit , antigen ns1 dan antibodi igm anti dengue. j indon med assoc. 61(8):326-32 5. da costa vg, marques sac, moreli ml. 2014. a meta-analysis of the diagnostic accuracy of two commercial ns1 antigen elisa tests for early dengue virus detection. plos one. 9(4):1-12 6. libraty dh, paul rt, darren p,timothy pe, siripen k, sharone g, et al. 2002. high circulating levels of the dengue virus nonstructural protein ns1 early in dengue illness correlate with the development of dengue hemorrhagic fever. jid. 186:1165-8 7. idai. 2012. infeksi virus dengue. dalam: editor s.s. poorwo soedarmo, h. garna, s.r. s. hadinegoro & h.i. satari. buku ajar infeksi & pediatri tropis. edisi 2. jakarta: badan penerbit idai. hal155-180 8. kirana pam, agustyas t, risti g. 2018. hubungan nilai mean platelet volume (mpv) dan platelet distribution width (pdw) terhadap jumlah trombosit pada pasien demam berdarah dengue di rs urip sumoharjo. medical journal of lampung university. volume 7 (2). 9. ahmed nh & shobha b. 2014. comparison of ns1 antigen detection elisa, real time rt-pcr and virus isolation for rapid diagnosis of dengue infection in acute phase. j vector borne.dis. 51:194-9 10. irawan ap, ahmad s, armadi d, ave olivia r. korelasi pemeriksaan ns1 ag dan pemeriksaan darah tepi pada anak dengan demam,2016. jmj . vol 4. 2016:106-118 11. rena n, utama s, parwati t. 2009. kelainan hematologi pada demam berdarah dengue. j peny dalam. 10:3santosh st, chincholkar vv, kulkarni dm, nilekar sl, ovhal rs, halgarkar cs. 2013. a study of ns1 antigen and platelet count for early diagnosis of dengue infection. int j curr microbiol app sci. 2(12):40-4 12. muhamad jp. hubungan hasil pemeriksaan antigen non struktural 1 (ns1) terhadap gejala, tanda klinis dan jumlah trombosit pada pasien suspect infeksi dengue , 2017. skripsi fakultas kedokteran universitas lampung 2017. 13. kulkarni rd, et al. 2011. association of platelet count and serological markers of dengue infection-importance of ns1antigen. indian j med microbiol. vol 29:359-62 14. megariani, rinang mariko, amrin alkavar, andani eka putra . uji diagnostik pemeriksaan antigen non struktural 1 untuk deteksi dini infeksi virus dengue pada anak , 2014. sari pediatri.vol 16.2014 15. mayer f.wowor. deteksi dini demam berdarah dengue dengan pemeriksaan antigen ns1, 2011. jurnal biomedik. 2011. vol 3 :1-9 16. pusparini. 2004. kadar hematokrit dan trombosit sebagai indikator diagnosis infeksi dengue primer dan sekunder. jurnal kedokteran trisakti. 23(2):51-6 17. soedarmono sp. infeksi virus dengue dalam : soedarmono sp, garna h, hadinegoro sr,satari hi, penyunting , buku ajar infeksi dan pediatrik tropis. edisi kedua ,jakarta . badan penerbit fkui;2008, hal 155-81 18. halstead b. 2016. pathogenesis of dengue: dawn of a new era [version 1; referees: 3 approved]. f1000 faculty rev.1353:1-8. 19. hottz e, neal dt, guy az, andrew sw, fernando ab. 2011. platelets in dengue infection. drug discovery today: disease mechanisms drug. vol 8:1-2 20. lin ys, yeh tf, lin cf. 2011. molecular mimicry between virus and host and its implications for dengue disease pathogenesis. experiment biol med. 236:515-23 21. juranah, muhadi d, arif m, bahar b. 2011. uji hematologi pasien terduga demam berdarah dengue indikasi rawat inap. kementrian kesehatan republik indonesia. profil data kesehatan indonesia tahun indonesian journal of clinical pathology and medical laboratory. 17(3):139 22. utari fp, efrida, husnil k. 2018. perbandingan nilai hematokrit dan jumlah trombosit antara infeksi dengue primer dan dengue sekunder pada anak di rsup. dr. m. djamil. jurnal kesehatan andalas. vol 7 (1) 23. trung td, le ttt, tran th, nguyen th, nguyen nv, pham tdh, nguyen tc, cameroon s, bridget w. 2010. liver involvement associated with dengue infection in adult in vietnam. the american journal of tropical medicine and hygiene. vol 83 (4):774-780 24. brady oj, peter wg, samir b, jane pm, john sb, anne gh, catherine lm, andrew wf, thomas ws, simon ih. 2012. refining the global spatial limits of dengue virus transmission by evidence based consensus. research article tropical disease. vol 10 (3) library �� vol. 2. no. 1 january–march 2011 basic mechanism of hyperbaric oxygen in infectious disease prihartini widiyanti 1,2 1 science and technology faculty 2 institute of tropical disease airlangga university abstract hyperbaric oxygen therapy (hbot) is the inhalation of 100 percent oxygen inside a hyperbaric chamber that is pressurized to greater than 1 atmosphere (atm). hbot causes both mechanical and physiologic effects by inducing a state of increased pressure and hyperoxia. hbot is typically administered at 1 to 3 atm. while the duration of an hbot session is typically 90 to 120 minutes, the duration, frequency, and cumulative number of sessions have not been standardized. hbo has been use widely in treating gangrene diabetic, stroke, osteomyelitis and accelerating wound healing. the use of hbo in infectious disease is wide, so the mechanism of hyperbaric oxygen in infectious disease should be well-understand. this understanding could bring the proper and wise management of infectious disease and to prevent the side effect of each therapy. key words: hbo, infectious disease, mechanism, proper and wise mechanism introduction this review would discuss the basic mechanism of action of hyperbaric oxygen in infectious disease. it will present the evidence for the bacteriostatic and bactericidal effect of hyperoxia and hyperbaric oxygen on microbial organisms in vitro and in in vivo model of infections. it will also examine the effect of oxygen on the activity of antimicrobial agent and on the function of immune defense mechanisms. regulation of oxygen delivery to tissues tissue oxygen tensions are effected mainly by the concentration on inspired oxygen , cardiac output, local blood flow, cellular metabolism and substrate availability. (kehrer jp et al, 1990; sheffield pj, 1988; silver ia, 1984). different partial pressures of oxygen (po2) are normally found in various body compartment. the po2s may the oven lower. in bacterial osteomyelitis, the po2 range from approximately 100 mm hg within pulmonary alveoli to 15 mm hg in the liver parenchymal cell. in traumatized or septic tissues, po2s may be even lower. in bacterial osteomyelitis, the po2s of bone is lowered by 50%; in experimental abscesses po2s may measure as low as 0 mm hg ( hays rc, mandell gl, 1974) within individual cells, po2s are heterogeneous and are much lower than extracellular po2s. for example, po2s in mitochondria are less than 1 mm hg (wilson df, erecinska m, 1984). normoxia (15%–21%) is defined in this review as the fractional inspired oxygen (fio2) concentration necessary to maintain aerobic metabolism and homeostasis in the body. oxygen tensions outside this normal range will be devined as follows: anaerob (less than 0.01% o2), hypoxia (12% o2 or less), hyperoxia (45%–100% o2), and hyperbaric oxygen (any o2 tension greater than i atmosphere absolute pressure or 760 mm hg). general mechanism of action of oxygen in infections hyperoxia and hyperbaric oxygen (hbo) increase oxygen tensions in tissue to levels which inhibit microbial growth by inhibiting various microbial metabolic reactions. hyperoxia and hbo by themselves also exert direct bacteriostatic and bactericidal effects on selected microorganisms because of increased generation of reactive oxygen species or free radical (jamieson d et al, 1986; raffin ta et al, 1977). free radicals are lethal for microorganisms that ether lack or possess limited antioxidant defenses. hbo is a unique antibacterial agent. �0 indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 49-54 at doses used clinically, hbo is usually bacteriostatic. not all doses of hbo have an antibacterial effect. the use of hbo at pressures of 1.5 ata or less promotes the growth of aerobic bacteria in vitro (olodart rm, 1966). hyperbaric oxygen also raises oxygen tensions in hypoxic tissue to levels necessary for the killing of bacteria by neutrophils (mader jt et. al, 1980). while phagocytosis remains unaffected by low oxygen tensions (karnovsky ml, 1968) killing of microorganisms by the oxidative burst is dependent on oxygen tensions. (babior bm, 1978; beaman l et al, 1984; hasset dj, cohen ms, 1989) polymorphonuclear leukocytes (pmns) from patients with chronic granulomatous disease lack the enzyme nadphoxidase necessary for oxygen –dependent killing of such pathogenic bacteria as pseudomonas aeruginosa and staphylococces aereus (mandell gl, hook ew, 1969). hyperoxia and hbo also influence the activity of selected antimicrobial agens belonging to the following categories antimetabolites, protein synthesis inhibitors and reduction – oxidation cycling agent. oxygen tensions also influence the pharmacokinetics of antimicrobial agent. for example, hypoxemia (pao2-32mm hg) prolongs (2-fold) the serum half –life of aminoglycosides hypoxemia effects both the absorption from muscle as well as the climination of these antimicrobials (miphij mj et al, 1978). hyperbaric oxygen can also effect the outcome of infections indirectly by influencing tissue repair and regeneration responses in infected necrotic tissue.for example, hypoxia (12% o2, 1 ata) retards healing of skins wounds and thus probably favors bacterial growth (knighton dr et al, 1986) hyperbaric oxygen (100% o2, 2 ata, 2t, twice daily)does not effect the healing of vascularized, full-thickness skin wound , but enhances wound closure in ischemic wound (kivisaari j, niinikoski j, 1975). significantly decreased in uninfected bone of rabbits after exposure to hbo (100% o2, 2ata) (stelner b et al, 1984) the hemodynamic changes induced by hyperbaric oxygen may be the results of increased oxygen delivery of tissue. it is also possible that negative inotropic effect on myocardium play a role in these changes. as for as can be judged from work with a model of antibiotic – controled pepsis , the presence of sepsis per se does not cause may hemodynamic changes during exposure to hbo (muhvich kh, 1986). susceptibility of anaerobic and aerobic bacteria to hbo pathogenic bacteria are classified in terms of the partial pressure of oxygen in which they grow. by definition , anaerobic bacteria can not survive in normal oxygen tensions because they lack antioxidant defenses. as such they are very susceptible hbo . for example, hyperbaric oxygen (3 ata for 18 hours) is completely bactericidal for clostridium perfringens in vitro (hill gb, osterhaut s, 1972) however, there are differences in susceptibility to oxygen among clostridium spesies. hyperbaric oxygen (100% o2, 2 ata) block the germination of c. perfringens spores in vitro, but is not bactericidal for the spores (demello fj et al, 1970) facultative anaerobic bacteria are able to grow in normoxia hyperoxia by increasing the synthesis of antioxidant enzymes (gregory em, fridovich i, 1973). the growth of some aerobic bacteria is enhanced by hyperoxia, but is inhibited by hbo. for example, oxygen tentions upo to 1 ata enhance the growth of escherichia coli, whereas oxygen tensions greater than 2 ata inhibit growth in vitro. (olodart rm, 1966) hyperoxia (100% o2, 1 ata) enhances the the growth of p. aerogenesa in vitro (park mk et. al, 1991); hyperoxia (0.2 ata to 0.87 ata) enhances the growth of corynebacterium diphtheriae in vitro (gottlieb sf et al, 1974). prolonged in vitro exposure to oxygen tensions greather than 1.5 ata inhibith the growth of several aerobic and facultative anaerobic bacteria. hyperbaric oxygen (greather than 1.5 ata) is bacteriostatic for e. coli (bochme de et al, 1976; brown or, 1972 ) p. aerugenesa, (bornside gh et al, 1975, c. diphtheriae, lactobacillus casei, (gottlieb sf, 1979) and vibrio anguillarum (keck pe et al, 1980) however, a 1 hour intermittent exposure to hbo (100% o2, 2 ata every 8 hours) has no effect on the growth of pherugenosa or s. aureus (brown gl et al, 1979) prolonged in vitro hyperbaric oxygen exposure (2.9 ata o2, 24 hours) is also bacteriostatic for the following enteric bacteria salmonella thyposa, s. schottmuelleri, s. paratyphi, shigella dysenteriae, s.flexneri, and proteus vulgaris (bornside et al, 1975) the growth of strepstococcus (enterococcus) faeculis is partially in habited by 2.9 ata o2 however, an alpha hemolytic strain of streptococcus is not inhibited by hbo (gottlieb sf, 1979) possibly because of the presebse of a hyaluronic acid-containing capsule ( cleary pp, larkin a, 1979). hyperbaric oxygen is bactericidal for aerobic and facultative anaerobic bacteria usually only at pressures and/ or durations which are greather than can be used clinically. for example, hbo is bactericidal for p.aeroginosa , proteus vulgaris , and s. typhosa. at 3 ata for 24 hours and for e. coli at 20 ata when treated for 6 hours (bornside et al, 1975). mechanisms of bacteriostatic effect of hbo hbo inhibits the growth of aerobic facultative anaerobic bacteria by inducing a variety of metabolic effect involved with the syntesis of proteins. nucleic acids and essential cofactors metabolic reactions: membrane transport function are also effected. these effects where achieved with the use of hyperbaric oxygen in viyo. inhibition of amino acid and protein biosyntesis exposure of e. coli to hyperbaric oxygen (100% o2 at greater than 3 ata) causes a rapid inhibition of growth and respiration (brown or, 1972) the inhibitory effect hbo are most likely caused by free radicals and other reactive oxygen based molecules, because hyperoxia (100% o2, 1 ata) inhibits growth of a superoxide dismutase-deficient double mutant of e.coli (sod a sod b) (carlioz a, touati d, 1986) free radicals probably ��widiyanti: basic mechanism of hyperbaric oxygen in infectious disease inactivate a bacterial enzyme (dihydroxyacid dehyoratase) involved in amino acid biosynthesis (brown or, 1975). dihydroxyacid dehydratase catalizes the formation of alpha-ketoisovalerate, an intermediate in the formation of valine and leucine. hyperbaric oxygen (100% o2. 4.2 ata) deceases the specific activity of dihydroxyacid dehydratase by 78 % (brown or, 1975). the inhibition of amino acid biosynthesis by hbo eventually leads to increase level of trna, which is responsible for inducing stringency response.the stringency response is characterized by increased level of tetraand pentaphosphorylated guanosine which inhibit bacterial carbohydrate, lipid and nucleotide synthesis and enhance proteolysis (cashel m, 1975). the end result is cessation of bacterial growth. the inhibition by hbo of protein synthesis in bacteria may also be caused by free radicalinduced block in the transport of substrates use in rna transcription. hyperoxia or the superoxide anion free radical inhibit the transport of lactose, guanosine and methylglycopyranoside in to e.coli (forman hj et al, 1982). hyperoxia also inhibit the transport of protons and the synthesis of atp in bacterial membranes (wilson dm et al, 1976). however it appears that the growth inhibition caused by hbo begins long before a drop in atp level occurs (mathis rr, 1976). the mechanism of decreased transport cused by hbo is thought to be the oxidation of sulfhydryl-containing protein involved in transport of metabolic substrates. free radicals are able to inactivate other bacterial proteins with key enzymetic function by oxidizing sulfhydryl-containing animo acids such as metionine play a key role in defending against this type of oxidative damage to proteins (brot n et al, 1981). decreased levels of key cofactors of metabolic reactions hyperbaric oxygen also inhibits bacterial growth by decreasing the levels of thiamine and of both the reduced and oxidiced forms of nicotinamide adenine dinnucleootide (nad, nadh) (brown or, 1983) thiamine pyrophosphate is an essentral coenzyme in carbohydrate metabolism and nadph production; nadph is a critical cofactor in a wide range of metabolic reactions. the mechanism of the decrease in nad is in inhibition of the de nooa nad syntesis pathway and possibly also and increase in catabolism of nad (gardner pr, 1990). decreased synthesis in increased degradation of dna and rna hyperbaric oxygen can also inhibit bacterial growth by directly blocking rna transcription and dna syntesis, for example, hbo (4,2 ata) inhibits rna transcription and dna systhensis in both stringent and relaxed strains of e. coli after a 30 minute exposure (brown or, 1983). electron microscopic studies show ultrastructural evidence of degradation of nucleic acids and ribosomal proteins in p.aeruginosa, after bacteriostasis induced by prolonged exposure to hbo (100% o2 ,2.9 ata) for 24 hours (clark jm, 1971). p. aerugioesa undergo market changes in morphologic appearance when exposed to oxygen at pressures that do not induce bacteriostasis (100% o2 ,2 ata). these abnormal shape changes are reversible (kenward ma et al, 1980). another important mechanism of oxygen-induced toxicity to bacteria is via injury to dna. production of to superoxide anion in vitro and in vivo has been linked to mutations in bacteria, hbo is mutagenic induced the reversion of a tryptophan auxotroph (e.coli wp 2 hcr) to prototrophy. paraquat toxicity for e. coli is in large part due to superoxide radical production (hassan and fridovich, 1978). paraquat is highly mutagenic for two strains of s. typhimurium (moody and hassan, 1982). both base-pair substitution and frameshift mutations were noted in dna from the salmonella strains. cell containing high levels of sod are more resistant to toxicity and mutagenecity than cell containing normal levels of this enzyme. from a quantitative standpoint, an important cellular source of superoxide is the nonenzymatic oxidation of cytochrome intermediates of the electron transport chain in mitochondria. superoxide is also generated by the cytochrome-p-450 substrate-oxygen complexes in the endoplasmic reticulum. another cellular organelle producing tonic oxygen spesies is the peroxisome. here h2o2 production occurs by oxidation of substrates such as long chain fatty acids. in all these cellular organelles, the generation of tonic oxygen spesies is dependent on tissue oxygen tensions (turrens jf et al, 1982) xantine oxidase is a major source of o2 in ischemic and hypoxic tissue that undergo reoxygenetation by blood reflow (mccord jm,1985). in summary, the presence of an adequate amound of molecular oxygen is necessary for oxygendependent killing by pmns and macrophages to occur. a variety of enzymatic and nonenzymatic celluler reactions also normally result in the production of o2 and h2o2. the production of these molecules is enhanced by increasing tissue oxygen tensions. free radicals are highly reactive and if not removed by scavengers, may cause extensive cellular injury. bacterial defense mechanism against free radicals for protection against the free radicals generated during normal aerobic metabolism, cells have developed antioxidant defence mechanism. three main antioxidant enzymes are known. superoxide dismutase (sod) is an extremely efficient o2 (gsh peroxidase) catalyzes the reduction of hydrogen peroxide to water and dioxygen, and is capable of converting tonic lipid peroxides into nontonic products. superoxide onion may undergo spontaneous dismutation to form hydrogen peroxygen. the rate of reaction is enhanced markedly by the presence of superoxide dismutase (sod). dismutation of two o2 radicals results in the formation of one hydrogen peroxide molecule. catalase subsequently converts hydrogen peroxide to water and oxygen. the role of catalase is probably more important during hyperoxic conditions than in normoxic conditions. in the presence of trace amount of transition metals , �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 49-54 particularly iron, hydrogen peroxide may participate in the fenton reaction. this reaction serves to produce the highly reactgive oh. radical, removal of h2o2 by catalase is important in order to prevent lipid peroxidation of membranes by oh. free radicals may also be inactivated by reacting with low molecular weight substances located in the cellular membranes or in the cytosol. tocopherol (vitamin e) is in antioxidant located in membranes. ascorbate, beta-carotene and sulfhydryl-containing compound such as cysteine, cysteamine and gluthatione are water soluble antioxidant compound. inder normal metabolic conditions, these free radical cellular injury. however, if host devense mechanism atre overwhelmed, damage to eukaryotic cells as well as procarotic cells will occur. (freeman ba, 1982). it is clear that primary mechanism of toxicity of hbo for eukaryotic cells and for microorganism is through the generation of free radicals, and other tonic oxygen spesies. mammalian cells have various antioxidant defense and utilize free radical reactions for bacterial killing. augmentation of endogenous host antioxidant defenses may permit use of higher doses of hbo than are currently possible in the treatment of infectious disease states. one of the rationales for using hyperbaric in infections is the potential to exploit the enhanced of selected microorganism to tonic oxygen molecules. role of superoxide and hydrogen peroxide in bacterial killing by hyperoxia and hyperbaric oxygen the superoxide anion radical appears to be particularly important in bacterial killing (gregory em, 1974) several in vitro studies have shown that the absence of the enzyme responsible for the detoxification of o2, namely superoxide dismutase (sod), increases the susceptibility of many anaerobic and facultative anaerobic bacteria to oxygen (mccord jm, 1971) on the other hand , by raising bacterial levels of sod , the susceptibility of the bacteria to oxygen can be diminished in vitro. for example, sod levels in b. fragilis can be raised 5-fold by exposure to 2% o2 (privale ct, 1979). the increased sod activity markedly reduces killing of these bacteria by hbo (gregory em, 1973) killing of s. sanguis can also be prevented by increasing sod activity : dimenthylsulfoxide (apermeable oh. scavenger) does not protect againt free radical toxicity (diguiseppi j, fridrovich i, 1982) studies with sod and catalase devicient mutants of e. coli confirm that sod is more important than catalase in protecting against the growth inhibition caused by hyperoxia (schellhorn he, hassan hm, 1988). in some strains of bacteria such as l. plantarum high levels of mn 2+ appear to be an effective substitute for sod in protecting against the toxic effect of o2. other bacteria such as n. gonorrhoeace are particulary susceptible to a different toxic oxygen spesies, namely h2o2. in these bacteria resistance to oxygen induced killing is associated with high levels of catalase, the enzyme responsible for detoxification of h2o2 . aditional antioxidant defence such as peroxidase and high levels glutathione also contribute to survival of these bacterial in aerobic conditions (archibald fs, duong mn, 1986). work done by beaman et al,(1985) has shown that surface associated sod and high levels of catalase in nocardia asteroides act together to resist oxygen dependent microbicidal activity of human pmns. microoganism with adequate antioxidant defenses are resistant to toxic actions of o2 and may use the production of toxic oxygen spesies to injure host cells for example, virulent strains of listeria monocylogenis exhibit maximal production of h2o2 and o2. virulence is correlate with survival of listeria monocytogenesis in macrophage monolayers. the exogeneous h2o2 damage macrophages. an avirulent strain of l. monocytogenes does not release h2o2 or o2 in significant amounts (godfrey rw, wilder ms, 1985). it is not clear if damage to bacterial cytoplasmic membrane caused by hbo is significant enough to be considered an important mechanism of hbo induced killing. in the case of e. coli, very few broken cells and no evidence of membrane lipid peroxidation are seen after the bacterial have been killed by hbo in vivo (harley jb et al, 1981). however the presence of a capsule appears to protect bacteria against oxygen-induced damage, in the case of streptococcus pyogenes the presence of a hyaluronic acid capsule increases resistance to the bacteriostatic effect of oxygen. removal of the capsule from an encapsulated streptococcus strain using hyaluronidase digestion increases susceptibility of this bacterium to the toxic effect of oxygen (cleary pp, larkin a, 1979). genetic mechanism of bacterial resistance to oxygen two regulatory genes responsible for the increased resistance of bacteria to hyperoxia have been indentified and are known as the soxr and oxyr regulons. hyperoxia and superoxide induce the synthesis of 30 proteins; approximately 20 of these proteins are regulated by the soxr or the osyr regulons. (christman mf et al, 1985; greenberg jt et al, 1990; storz g et al, 1990; walkup lkb, kogama t, 1989). many of these bacterial proteins are enzymes involved in detoxification of free radicals and repair of free radicals damage; example are sod , endonuclease iv, and glucose 6-phosphate dehydrogenase (greenberg jt et al, 1990; tsavena jr, weiss b, 1990). example of these proteins include the antioxidant enzymes hydroperoxidase 1 catalase. nad(p)hdependent alkyl hydroperoside reductase, and glutathione reductase, exposure to toxic oxygen species induces the synthesis of several other protective proteins whose specific identify remains to be characterized (christman mf et al, 1985; demple b, halbrook j, 1983; storz g et al, 1990). reference 1. archibald fs, duong m-n, 1986. superoxide dismutase and oxygen toxicity defenses in the genus neisserea” infect innum 51: 631–641. 2. babior bm, 1978. oxygen dependent microbial killing by phagocytes. new eng 1 med : 296: 659–669. ��widiyanti: basic mechanism of hyperbaric oxygen in infectious disease 3. beaman l, beaman bl, 1984. the role of oxygen and its derivate pesin microbial pathogenesis and host defense. ann rev microbial: 38: 27–48. 4. beaman bl, black cm, doughty f,.beaman l”role of superoxide dismutase and catalase as determinant of pathogenecity of norcardia asteroids: importance in resistance to microbicidal activities of human polymorphonuclear neutrophils, 1985. infect immune: 47: 135, 111. 5. bochme de, vincent k. brown or, 1976. oxygen and toxicity inhibition of amino acids biosynthesis, 1976. nature.(lond) 262: 418–420. 6. bornside gh , pakman lm, ordones jr aa.” inhibition of pathogenic enteric bacteria by hyperbaric oxygen : enhanced antibacterial activity in the absence of carbon dioxide, 1975. antimicrop agents chemother. 7: 682–687. 7. brot n, weissbach l, werth j , weissbach h, 1981. enzymatic reduction of protein – board methionine sulfoxide. proc natl acad sei usa 78: 2155–2158. 8. brown or, 1975. reversible inhibition of respiration of escherichia coli by hyperoxia . microbios 5: 7–16. 9. brown or ,song gs, 1980. pyridine nucleotide coenzyme biosyntesis a cellular site of oxygen toxicity. biochem biophys bes commun 93: 172,178. 10. brown gi, thomson pd , madder jt , hilten jg , brown me. wells gh, 1979. effect of hyperbaric oxygen upon s. anoeus, p. aeurigenosa, and c. albicans. aviat space environ med, 50: 717–720. 11. brown or, yein, 1978. dihydroxiacid dehydratase; the site of hyperbaric oxygen p[oisoning in branched-chain amino acid biosynthesis. biochem biophysed res comman 85: 1219–1224. 12. brunker ri, brown or, 1971. effect of hyperoxya on oxidized and reduced nad and nadp concentrations in escherichia coli. microbios 4: 193–203. 13. caldwell p,luk da, weissbach h, bort n, 1978. oxidation of the methionine residues of escherichia coli ribosomal protein 1. 12 decreases the protein’s biological activity. proc natl acid sei usa 75: 53–49–5352. 14. carlios a, touati d, 1986. isolation of superoxide dismutase mutants in eschericheria coli is superoxide dismutase necessary for aerobic life? embo j. 5: 623–630. 15. gashel m, 1975. regulation of bacterial ppgpp .anan rev microbial 29: 301–318. 16. cristman mf ,morgan rw, jacobson fs, ames bn, 1985. positive control of a regulation for devense against oxidative stress and some heat-shock proteins is salmonella typnimurium. cell. 41: 753–762. 17. chark jm, pakman lm, 1971. inhibition of psendomonas aerogionosa by hyperbaric oxygen. ii. ultrastructural changes. infect human 4: 488–491. 18. cleary pp, larkin a, 1978. hyaluronic acid capsules strategy for oxygen resistence in group a streptococa. j bacterial 140: 1090–1097. 19. demello fj, hashimoto t, hitchcock cr, haglin ù, 1970. the effect of hyperbaric oxygen on the generation and toxin production of clostridium perfringens spores, in; wada j, iwa t, eds proceedings of the fourth international conggress on hyperbaric medicine, tokyo: igaku shoin ltd: 276–281. 20. demple b, halbrook j, 1983. inducible repair of oxidative dna damage in escherichia coli.’’ nature (lond) 304: 466–468. 21. digaiseppi j, fridovich i, 1982. oxygen toxitity in streptococcus sanguis. the relative importance of superoxide and hydroxyl radicals. j boil chem. 257: 4046–4051. 22. forman hj ,william jj, nelson j, daniele rp, fisher ab, 1982. hyperoxia inhibits stimulated superoxide release by rat alveolar macrophages. j appl physiol 53: 685–689. 23. freeman ba, grapo j, 1982. biology of disease. free radicals and tissue injury, lab invest 47: 412–426. 24. gardner pr, fihdovich i, 1990. quilonate phosphoribosyl translerase is not the oxygen –sensitive site of nicotinamide adenine dinucleotide biosyntesis. free rad biol med 8: 177–119. 25. godfrey rw, wilder ms, 1985. generation of oxygen species and virulence of listeria monocytogenes. infect immune 47: 837–839. 26. gottlieb se, solosky ja, aubrey r, nedelkolf dd, 1974. synergistic action of increased oxygen tension and paba folic acid antagonists on bacterial growth. aerospace med. 45: 829–833. 27. greenbreg jt, monach p, chon jh, josephy pd, demple b, positive control of a global antioxidant defense regulon activated by superoxide generation against is escherichia coli. proc natl acad sci usa 87: 6181–6185. 28. gregory em, fridovich i, 1973. induction of superoxide dismutase by molecular oygen . j bacteriol 114: 543–541. 29. gregory em. fridovich i, 1973. oxygen toxicity and the superoxide dismutase, j bacteriol 114: 1193–1197. 30. gregory em, goskin sa, fridovich i, 1974. superoxide dismutase and oxygen toxicity in a eukaryote. j bacteriol 117: 456–460. 31. gudewice tm, mader jt, davis gp, 1987. combined effectg of hyperbaric oxygen and antifungal agenst on the growth of candida albicans. aviat space environ mad. 58: 673–678. 32. halliwell b, gutteridge jmg, 1984. oxygen toxicity, oxygen radicals, transition metals and disease. biochem. j 219: 1–14. 33. harle jb, fraks jg, bayer me, goldfine h , rasmusen h, 1981. hyperbaric oxygen toxicity and ribosomal destruction in escherichia coli k12. can j microbiol 27: 44–54. 34. hassan hm, fridovich i, 1978. superoxide radicals and the oxygen enhancement of the toxicity of paraquat in escherichia coli. j boil chem. 253: 8143–8148. 35. hasett dj, gohen ms, 1989. bacterial adaptation to oxidative stress: implication for pathogenesis and interaction with phagocytic cells. faseb j 3: 2574–2582. 36. hays rc, mandell gl, 1974. po2, ph, and redox potential of experimental abscess. proc soc exp boil med: 174: 29–30. 37. hill gb, osterhout s, 1972. experimental effect of hyperbaric oxygen on selected clostridial species h . in vivo studies in mice. j infect dis 125: 17–25. 38. jamieson d, chance b, chedenas e, boveria a, 1986. the relation of free radicals production to hyperoxia. an rev physiol 48: 703–719. 39. karnovsky ml,1968. the metabolism of leukocytes, semin hematol, 5: 156–165. 40. keck pe, gottlieb sf, gonley f, 1980. interaction of of increased pressures of oxygen and sulfonamides the in vitro and in vivo of pathogenic bacteria. undersea biomed res 7: 95–106. 41. kehter jp, jones dp, lemaster jj, jaeschke h, 1990. mechanism of hypoxic cell injury. summary of the symposium presented at the1990 annual meeting of the society of toxicology. toxicol appl pharmacol 106: 165–178. 42. kenward ma, alcock sr, brown mrw, 1980. effect of hyperbaric oxgen on the growth and properties of spedomonas aeruginosa,’’ microbios 28: 47–60. 43. kivisari j, niinikoski j, 1975. effect of hyperbaric oxygenation and prolonged hyperoxia on the healing of open wound. acta chir scan 141: 14–19. 44. kuo cf, mashino t, fridovich i. 1987.α ,ß dihydroxyisovalerate dehydratadse, aa superoxide-sensitive enzyme. biol chem 262: 4724–4727. 45. mcgord jm, 1985. oxygen –derived free radicals in postischemic tissue injury.’’ n eagl j med. 312: 159–163. 46. mcgord jm, keele jr bb , fridovich, 1971. an enzyme-based theory of obligate anaerobiosis the spysiological function of supoeroxide dismutase. proc natl acad sci usa.68: 1024–1027. 47. mader jt, brown gl, guession jg, wells gh. reimars ja, 1980. a mechanism for the amelioranon by hyperbaric oxygen of experimental staphylococcal osteomyelitis an rabbit. j infect dis 142: 915–922. 48. mandell gl, hook ew, 1969. leukocyte bactericidal activity in ehronic granulomatous disease correlation of bacterial hydrogen peroxide production and susceptibility tointracelullar killing. j bacteriol 100: 531–532. 49. mathis rr, brown or, 1976. atp concentration in escherichia coli during oxygen toxicity. biocheva biophys acta 440: 723–732. 50. miphij nj, robert rj, myer mg, 1978. effect of hyperoxia upon aminoglycoside serum parmacocinatoes in animals. antimicrob agent chemoterm 14: 344–347. �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 49-54 51. moody cs, hassan hm,1982. mutagenecity of oxygen free radicals. proc natl acad sci, 79: 2855–2859. 52. muhvich kh, park mk ,myers ram, marzella l, 1989. hyperoxia and the antimicrobial susceptibilily of esacherichia coli and pseudomonas aeruginosa, antimicrob agent chemoterm, 33: 1526–1530. 53. muhvich kh, piano mr, piano g , myers ram, ferguson jl, marzella i, 1989. splanchnic blood flow in a rat model of antibiotic –controled intra abdominal abscess during normoxia and hyperoxia. cire shock 253–262. 54. ollodart rm, 1966. effect of hyperbaric oxygenation and antibiotic on aerobic microorganism. in brown jr iw, gox bg, eds proceding of the third international conference on hyperbaric medicine, washington dc. natl acad sci: 565–671. 55 . pakman lm, 1971. inhibition of pseudomonas aeruginosa by hyperbaric oxygen i, sulmonamide activity enhancement and reversal. infect immune.4: 479–487. 56. park mk, muhvich kh, myer ram, marzella l, 1991. hyperoxia prolongs the aminoglycoside induced postantibioic effect in pseudomonas aeruginosa.’’ antimicrob agent chemotherm 35: 691–695. 57. privalle gt, gregory em. superoxia dismutase and o2 lethality in bacteriodes fragilis. j. bacteriolj. bacteriol 138: 139–145. 58. raffin ta, simon lm. thiodore j, robin ed.1977. effect ofeffect of hyperoxia on the rate of generation of superoxide anions(soa) in free solution and in a cellular alveolar macrophage(am) systein. clin res. 25: 134a. 59. rolfe rd, hengers dj. gamphell bj, barrett jt, 1978. factors related to the oxygen tolerence of anaerobic bacteria. appl environ microbial. 36: 306–313. 60. schellhorn he, hassan hm, 1988. response of hydroperoxidase and superoxide disnutase deticient mutants of escherichia k-12 to oxidative stress. can j microbiol. 34: 1171–1176. 61. seiter rl, brown or, 1982. induction of stringency by hyperoxia in escherichia coli. cell mod boil 28: 285–291. 62. sheffield pja, 1988. tissue oxygen measurement. in. (davic jc, hunt tk,eds.) problems wounds. the role of oxygen . new york: elsevier. 17–51. 63. silver ja, 1984. polarographic techniques of oxygen measurement.in (gottlieb sf, longmutr is, totter jr, eds.) oxygen an in-depth study of its pathophysiology. bethesda, md, undersea medical society, 215–239. 64. steiner b, wong ghw, graves s., 1984. susceptibility of treponema pollideon to the tonic products of oxygen reduction and the nontreponemal nature of its calatase. br j vener dis.60: 14–22.br j vener dis.60: 14–22. 65. sterz g. tartaglia la, arnes bn, 1990. the oxyr regulon. antoniethe oxyr regulon. antonie van leeuwenhoek 58: 157–161. 66. tsavena ir, weiss b, 1990. soxr, a locus governing a superoxide response regulon in escherichia coli k-12. j bacteriol 172: 4197–4205. 67. turrens jf, freeman ba, grapo jd,1982. hyperoxia increaseshyperoxia increases h2o2 release by lung mitochondria and microsomes. arch biochem biophys. 217: 411–419. 68. walkup lkb, kogoma t, 1989. escherichia coli proteins inducible by oxidative stress mediated by superoxide radical. j bacteriol 171: 1476–1484. 69. wilson dm, adlereto jf, maloney pc, wilson th, 1976. proton motive force as the source of energy for adenosine 5’-triphosphate synthesis in escherichia coli. j bacteriol 126: 327–337. 70. wilson df, erecinska m, 1984. the role of cytochrome c oxidase in regulation of cellular oxygen consumption. in: (gottlieb sf, longmuir is, totter jr, eds) oxygen: an in-depth syudy of its pathophysiology, bethesda md. undersea medical society,undersea medical society, 459–492. vol. 9 no. 1 january–april 2021 ijtid, p-issn 2085-1103, e-issn 2356-0991 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ original article correlation between climate factors with dengue hemorrhagic fever cases in surabaya 2007 – 2017 1,3 faculty of medicine of universitas airlangga, surabaya, indonesia 2 departement of public health, faculty of medicine of universitas airlangga, surabaya, indonesia received: 22nd january 2019; revised: 4th february 2019; accepted: 9th february 2021 abstract dengue hemorrhagic fever (dhf) is a disease caused by dengue virus. dhf is mediated by the mosquito vector, the aedes mosquito. the proliferation of dengue vector is influenced by many factors, one of which is climate factors. dhf is one of the main public health problems in indonesia. cases of dengue were first discovered in 1968 in the city of jakarta and surabaya. currently surabaya is one of the dengue endemic areas in indonesia. . the case of dhf in the city of surabaya can be said to be still quite high compared with another city in indonesia, although there is a decrease in the number from year to year. when examined, many factors influence the high number of dengue cases in surabaya, one of which is climate factor. climate factors play a role in the proliferation of dhf vectors. therefore, this study aims to examine for 10 years, namely in 2007 2017 whether there is a correlation between climate factors with dengue cases in the city of surabaya., which in this study the climate factors used are rainfall, average temperature, and average air humidity. this research uses an analytical method namely spearman on the spss software version 20. the results obtained that the case of dhf in the city of surabaya has no relationship with climatic factors such as rainfall and average temperature with a significance value of the relationship p> 0.05. while the climate factor that has a relationship with dhf cases in surabaya city is air humidity with a significance value of p <0.05 and has a positive relationship with the value of r = + 0.190. it can be concluded that not all climate factors have a relationship with the dhf case in surabaya in 2007 2017, which has a relationship with the dhf case is air humidity. abstrak demam berdarah dengue (dbd) merupakan penyakit yang disebabkan oleh virus dengue. dbd diperantarai oleh vektor nyamuk yaitu nyamuk aedes. perkembangbiakan vektor demam berdarah ini dipengaruhi oleh banyak faktor salah satunya adalah perubahan iklim. dbd merupakan salah satu masalah kesehatan utama masyarakat di indonesia. kasus demam berdarah pertama kali ditemukan pada tahun 1968 di kota surabaya. saat ini surabaya merupakan salah satu daerah endemis dbd di indonesia. kasus dbd di kota surabaya sendiri dapat dikatakan masih cukup tinggi apabila dibandingkan dengan kota lain di indonesia walaupun terlihat ada penurunan jumlah dari tahun ke tahun. apabila ditelaah, banyak faktor yang mempengaruhi masih tingginya kasus dbd di kota surabaya, yang salah satunya adalah faktor iklim. faktor iklim berperan dalam perkembangbiakan vektor dbd. maka dari itu, penelitian ini bertujuan untuk meneliti selama 10 tahun, yaitu tahun 2007 – 2017 apakah ada hubungan antara faktor iklim dengan kasus dbd di kota surabaya, yang pada penelitian ini faktor iklim yang digunakan adalah curah hujan, suhu rata-rata, dan rata-rata kelembaban udara. penelitian ini menggunkan metode analitik yaitu spearman pada perangkat spps versi 20. didapatkan hasil bahwa kasus dbd di kota surabaya tidak mempunyai hubungan dengan faktor iklim berupa curah hujan dan suhu rata-rata dengan nilai signifikansi hubungan p>0.05. sedangkan faktor iklim yang memiliki hubungan dengan kasus dbd di kota surabaya merupakan kelembaban udara dengan nilai signifikansi p<0.05 serta memiliki hubungan yang positif dengan nilai r = + 0.190. dapat disimpulkan tidak semua faktor iklim mempunyai hubungan dengan kasus dbd kota surabaya tahun 2007 – 2017, yang memiliki hubungan dengan kasus dbd adalah kelembaban udara. kata kunci: kasus dbd; faktor iklim; kelembaban udara, surabaya, 2007 2017 * corresponding author: nadhilahp@gmail.com open acces under cc-by-nc-sa share alike 4.0 keywords: dhf case; climate factors; humidity; surabaya; 2007 2017 nadhilah putri ghaisani1*, sulistiawati2, maria lucia inge lusida3 nadhilah putri ghaisani, et al.: correlation between climate factors with dengue hemorrhagic 40 ijtid, p-issn 2085-1103, e-issn 2356-0991 how to cite: ghaisani, np., sulistiawati., lusida, mli. correlation between climate factors with dengue hemorrhagic fever cases in surabaya 2007 – 2017.. indonesian journal of tropical and infectious disease, 9(1), 39–44 introduction dengue hemorrhagic fever (dhf) is a disease caused by dengue virus carried by female aedes mosquitoes, especially aedes aegepty and a few aedes albopictus.1 dengue is widespread in the tropics and subtropics, including indonesia. dengue is one of the main health problems in indonesia.2 dhf cases first appeared in indonesia, namely in jakarta and surabaya in 1968.3 dhf incidence rate (ir) in indonesia from 1968 2015 continue to increase.4,5,6,7,8,9 dengue cases found in all provinces in indonesia.10 one of the things that influences this phenomenon is the climate change. climate change causes changes in rainfall, temperature, humidity, and air direction, thus affecting the terrestrial and oceanic ecosystems and also health.11 climate change has a role in dhf vector.12 aedes mosquitoes live in urban habitat and breed specifically in containers. water needs for breeding is very important. it reach its peak during the rainy season.13 this mosquito tend to bite in the morning until noon. dhf has a strong correlation with the climate because the incidence of dhf usually happens on the beginning and the end of the rainy season.14 very high rainfall influences the population of mosquitoes. increased rainfall intensity refers to the increasing place of mosquitoes to breed, resulting in increasing mosquitoes population. increasing the mosquito population increases the risk of female mosquitoes carrying the pathogens which will transmit to the next host.15 aedes mosquito reproduction cycle will be shorter at temperatures higher than 32°c so that the mosquito population will multiply with increasing temperature 16. warm temperatures also accelerate the metabolic process so that the frequency of biting will increase.17 the maximum temperature for mosquito growth is 2527°c. 18 humidity affects the flight behavior and host search, mosquito life span and mosquito reproduction.17 high humidity helps the process of mosquito metabolism which will indirectly increase the frequency of biting. materials and methods in surabaya, the incidence of dhf in the 2007-2017 period as a whole has decreased in numbers although not stable. in below, figure 1 show the number of dhf cases in surabaya from 2007 until 2017. figure 1. number of dhf cases in surabaya in 2007 – 2017 from the data obtained a significant increase occurred in 2010, 2013, and 2016. if it is associated with the time of the el nino occurrence, in those years the el nino events that occur in the moderate and strong category. from previous study, there was an increase in the incidence of dhf when the el nino with the same category occur. this research is an analytical study that uses secondary data in the form of institutional administrative data, namely the report of the meteorology climatology and geophysics agency (bmkg) and the surabaya city health office with a cross-sectional approach. the sampling technique in this study uses a total sampling technique. the data were taken is the bmkg of surabaya city weather report in 2007-2017 and the surabaya city health office report on the incidence of dengue cases in 2007-2017. the collected data is grouped by month in each year and is written using tables and graphs and analyzed descriptively and tested statistically the correlation using spearman method on the spss software version 20. result and discussion dhf cases profile in surabaya open acces under cc-by-nc-sa share alike 4.0 ijtid, p-issn 2085-1103, e-issn 2356-0991 41 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 39–44 judging from the graph above in figure 2, the same pattern was formed in 2007 2009. starting from 2011 to 2017 the number of dengue cases began to decrease so that the pattern formed had changed from before. whereas in 2010, 2013, and 2016 have different patterns from other years. overall, from september to december the number of dengue cases has always been lower than in previous months. figure 2. cases of dhf per month each year based on surabaya city rainfall data for 2007-2017 in figure 3, december to march were months with high rate of rainfall, while july to september were months with low rainfall. however, in 2010, 2013 and 2016 there was a change in the pattern in which high rate of rainfall occurred throughout the year even in the month that was supposed to be the peak of the dry season, making the accumulation of rainfall in those years the highest among the other years. figure 4. average temperature each year figure 3. rainfall per month each year surabaya has monsoonal rain type that is influenced by west and east monsoon winds where the peak of the rainy season occurs in january, and the peak of the dry season occurs in august.19 0 100 200 300 400 500 600 jan feb mar apr mei jun jul agst sept okt nov rainfall 2007 2008 2009 2010 2011 2012 2013 2014 rainfall distribution of surabaya average temperature distribution of surabaya city the average temperature of surabaya city for the last 11 years is within normal range when compared to the 30-year data with an average value of 28,62oc. 28 28.2 28.4 28.6 28.8 29 29.2 29.4 29.6 temperature (oc) from the graph in figure 4, 2011 became a year with the lowest average temperature with a value of 28,6oc, while 2016 was a year with the highest average temperature of 29,4oc. the humidity of the city of surabaya for the last 11 years is within normal range when compared to the 30-year data with a value of 74,33. from the graph in figure 5, 2009 became the year with the lowest humidity with a value of 69,58 and 2017 became the year with the highest average humidity with a value of 76,92. humidity distribution of surabaya city open acces under cc-by-nc-sa share alike 4.0 nadhilah putri ghaisani, et al.: correlation between climate factors with dengue hemorrhagic 42 ijtid, p-issn 2085-1103, e-issn 2356-0991 the effect of rainfall on the incidence of dhf cases is complex, because it is influenced by several other factors.20 rainfall has an influence on the vector growth, which is the density of adult mosquitoes. high rainfall intensity will cause the breeding site of adult mosquitoes to increase, which in turn increase the density of mosquitoes.15 however, in a short period, heavy rain will destroy mosquito larvae and reduce the survival rate of female mosquitoes.16 table 1. spearman correlation test result variable mean ± sd p rainfall 138,265 ± 131,269 0,159 dhf cases 136,71 ± 135,560 from the result of published study in table 1, the correlation test using spearman obtained relationship significance of p >0.05. it can be interpreted that there was no correlation between rainfall and the incidence of dhf cases in surabaya in 2007-2017. however, it must be noted that incidence of dhf cases is influenced by other factors besides than rainfall, such as humidity, evaporation of water, wind speed, and cloudiness.20 these results supported previous study in surabaya which showed no significant fluctuations in certain months of the year regarding the number of dengue cases. these results are also similar with studies in other influ influencing factors.20 in addition, changes in rainfall patterns can also affect human behavior which will later affect lifestyle that further affect the dynamics of aedes mosquito populations, for example, a change in water storing habit.20 however, studies assessing the correlation of rainfall with the incidence of dhf is not suitable to use the spearman method. spearman is suitable for measuring linear and static relationships, while the correlation between weather and dhf events is neither linear nor static.18 this can happened because from the previous study, the correlation between rainfall and the incidence of dhf has several conditions, such as regular rain that may cause an increase in dengue cases, whereas heavy rainfall does not. 18,21 so, the correlation bertween dhf cases and rainfall are not linear nor static. the results of this study supported previous studies in the city of surabaya which showed no significant fluctuations in certain months of the year regarding the number of dengue cases. c figure 5. humidity in surabaya city per year correlation between rainfall with dhf cases correlation between temperature and dhf cases based on previous study, temperature has a role in the transmission cycle of dengue virus.21 research in thailand and singapore showed that there was a correlation between temperature and the incidence of dhf cases. 22,16 table 2. spearman correlation test result variable mean ± sd p temperature 28,909 ± 0,739 0,066 dhf cases 136,71 ± 135,560 correlation test results in table 2 showed the relationship significance of p >0.05 which means there was no correlation between temperature and the incidence of dhf in the city of surabaya in 2007-2017. similar to correlation of rainfall with the incidence of dhf cases, the relationship with temperature is also not a linear relationship or static, thus making this method not suitable for this case.18 it can be seen that the temperature data used is the average temperature, whereas the temperature is not only measured from the average open access under cc-by-nc-sa share alike 4.0 ijtid, p-issn 2085-1103, e-issn 2356-0991 43 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 39–44 average value but there is also a minimum and maximum temperature. this minimum or maximum temperature value may also affect the presence or absence of its relationship with the incidence of dhf. research that uses an epidemiological approach states that in certain months, high temperatures will cause mosquito populations to increase with low virus transmission, which usually causes an increase in virus transmission under conditions of high rainfall, low temperatures, and high humidity. 23 humidity affects the flight behavior of mosquitoes by increasing the metabolism of the mosquito's body which then increase the biting behavior.24 table 3. spearman correlation test result variabel mean ± sd p humidity 73,48 ± 5,614 0,029 dhf cases 136,71 ± 135,560 the correlation test results in table 3 showed the relationship significance of p <0.05 which means there was a significant correlation between the two variables. the strength of the relationship between the two variables is very weak and the direction of the relationship is positive (r =+0.190). it can be interpreted that the higher the humidity, the higher the incidence of dhf. similar to current study which showed that air humidity has a relationship with the incidence of dhf cases through the effect on the density of the dengue virus vector, the aedes aegepty mosquito and the external incubation period of the dengue virus itself, thereby increasing its transmission. 23 accumulation followed by an increase in the incidence of dengue cases. previous studies showed that there was a correlation between the increase in the incidence of dhf cases with the phenomenon of el-nino-southern oscillation (enso) which is a cycle of sea surface temperature in the pacific sea. from the results of studies in venezuela, 2009 2010 were the year with moderate el-nino category, while 2014-2016 were the year with strong el-nino or mega nino. 24 within those 3 years, there was a recorded climate phenomenon that does not usually occur in surabaya. during those years, dengue fever cases in the city of surabaya also showed an increase in number. this result is linear with the previous study, that there is a significant relationship between enso and dengue incidence.25 correlation between humidity with dhf cases correlation between enso and dhf cases based on the available data, year of 2010, 2013 and 2016 were the year with high rainfall d conclusion the climate factor which has an analytical correlation with the dhf case in surabaya in 2007 2017 is humidity, while the climate factor such as rainfall and temperature does not have an analytical correlation with the dhf incidence rate. there is an influence of the el-nino phenomenon on the number of dhf cases in surabaya in a certain year. conflict of interest there is no conflict of interest of this study. references 1. halstead, s. (2008). dengue virus–mosquito interactions. annual review of entomology. 2008; 53(1), pp.273-291 2. karyanti, m. and hadinegoro, s. perubahan epidemiologi demam berdarah dengue di indonesia. sari pediatri. 2009; 10(6) 3. nathan, m. and harun, s. dengue haemorrhagic fever and japanese b encephalitis in indonesia.. [online]. 2019. europepmc.org. available at: https://europepmc.org/abstract/med/2851186 4. ministry of health of the republic of indonesia. infodatin (pusat data dan informasi kementerian kesehatan ri). available at: http://www.depkes.go.id/resources/download/pusdat in/infodatin/infodatin-dbd-2016.pdf open access under cc-by-nc-sa share alike 4.0 nadhilah putri ghaisani, et al.: correlation between climate factors with dengue hemorrhagic 44 ijtid, p-issn 2085-1103, e-issn 2356-0991 5. ministry of health of the republic of indonesia. infodatin (pusat data dan informasi kementerian kesehatan ri). available at: http://www.depkes.go .id/resources/download/profil/profil_kab_koa _2016/3578_jatim_kota_surabaya_2016.pdf. in situasi dbd di indonesia. 2016: 1–12 6. ministry of health of the republic of indonesia. infodatin (pusat data dan informasi kementerian kesehatan ri). available at: http://www.depkes.go. id/resources/download/profil/profil_kab_kot a_2015/3578_jatim_kota_surabaya_2015.pdf 7. ministry of health of the republic of indonesia. infodatin (pusat data dan informasi kementerian kesehatan ri). available at: http://www.depkes.go .id/resources/download/profil/profil_kab_kot a_2014/3578_jatim_kota_surabaya_2014.pdf.201 4: 1–6 8. ministry of health of the republic of indonesia. infodatin (pusat data dan informasi kementerian kesehatanri).availableat:http://www.depkes.go.id/r esources/download/profil/profilkab_kota_20 13/3578_jatim_kota_surabaya_2013.pdf. 2013 9. ministry of health of the republic of indonesia. infodatin (pusat data dan informasi kementerian kesehatan ri). available at: http://www.depkes.go .id/resources/download/profil/profil_kab_kot a_2012/3578_jatim_kota_surabaya_2012.pdf [accessed 9 apr. 2018]. 2012 11. harapan, h., michie, a., mudatsir, m., sasmono, r. and imrie,a. epidemiology of dengue hemorrhagic fever in indonesia: analysis of five decades data from trhe national disease surveillance. bmc research notes. 2019; 12(1) 12. hii, y., rocklöv, j., ng, n., tang, c., pang, f. and sauerborn, r. climate variability and increase in intensity and magnitude of dengue incidence in singapore. global health action. 2009; 2(1), p.2036 13. harapan, h., michie, a., mudatsir, m., sasmono, r. and imrie,a. epidemiology of dengue hemorrhagic fever in indonesia: analysis of five decades data from trhe national disease surveillance. bmc health. [online] 2018. available at: https://www. who.int/newsroom/factsheets/detail/climate-change and-health [accessed 18 march 2018] 15. lowe, r., stewart-ibarra, a., petrova, d., garcíadíez, m., borbor-cordova, m., mejía, r., regato, m. and rodó 15. valdez, l., sibona, g. and condat, c. impact of rainfall on aedes aegepty population. ecological modelling, 2018; 385: 96-105 16. chumpu, r., khamsemanan, n. and nattee, c., the association between dengue incidences and provincial-level weather variables in thailand from 2001 to 2014. plos one. 2019; 14(12): e0226945 17. reinhold,j., lazzari,c. and lahondere, c. (2018). effects of the environmental temperature on aedes aegepty and aedes albopticus mosquitoes: a review. insects, 9(4), p.158 18. ehelepola, n., ariyaratne, k., buddhadasa, w., ratnayake, s. and wickramasinghe, m. (2015). a study of the correlation between dengue and weather in kandy city, sri lanka (2003 -2012) and lessons learned. infectious diseases of poverty. 2015; 4(1) 19. bmkg | badan meteorologi, klimatologi, dan geofisika. artikel : karakteristik rata-rata suhu maksimum dan suhu minimum stasiun meteorologi nabire tahun 2006-2015 | bmkg. 2018. [online] available at: http://www.bmkg.go .id/artikel/?id=xa9q99255011rged5919 20. choi, y., tang, c., mciver, l., hashizume, m., chan, v., abeyasinghe, r., iddings, s. and huy, r. effects of weather factors on dengue fever incidence and implications for interventions in cambodia. bmc public health. 2016; 16(1) 21. lai, y. the climatic factors affecting dengue fever outbreaks in southern taiwan: an application of symbolic data analysis. biomedical engineering online. 2018; 17(s2) 22. campbell, k., lin, c., iamsirithaworn, s. and scott, t. the complex relationship between weather and dengue virus transmission in thailand. the american journal of tropical medicine and hygiene. 2013; 89(6): 1066-1080 23. sintorini, m. (2018). the correlation between temperature and humidity with the population density of aedes aegypti as dengue fever’s vector. iop conference series: earth and environmental science. 2018; 106: 012033 24. ninphanomchai, s., chansang, c., hii, y., rocklöv, j. and kittayapong, p. predictiveness of disease risk in a global outreach tourist setting in thailand using meteorological data and vectorborne disease incidences. international journal of environmental research and public health. 2014; 11(10) : 10694-10709 25. vincenti-gonzalez, m., tami, a., lizarazo, e. and grillet, m. (2018). enso-driven climate variability promotes periodic major outbreaks of dengue in venezuela. scientific reports, 8(1) open access under cc-by-nc-sa share alike 4.0 , x. climate services for health: predicting the evolution of the 2016 dengue season in machala, ecuador. the lancet planetary health. 2017; 1(4), pp.e142-e151 research notes. 2019; 12(1) 14. world health organization. climate change and 10. halstead, s. dengue virus–mosquito interactions. annual review of entomology. 2008; 53(1), pp.273291 http://www.depkes.go/ http://www.depkes.go/ http://www.depkes.go/ http://www.depkes.go/ http://www.bmkg/ ijtid vol 7 no 6 may-august 2019.indd 161 vol. 7 no. 6 september-december 2019 research report mtt formazan replaced wst-8 as a better simple screening method for detection of glucose-6phosphate dehydrogenase deficiency indah s. tantular1,2a, wahyu wulansari1, yasutoshi kido3, daniel k. inaoka4, hilkatul ilmi1, soetjipto1, maria inge lusida1, fumihiko kawamoto1,5 1 institute of tropical disease, universitas airlangga, surabaya, indonesia 2 department of parasitology, faculty of medicine, universitas airlangga, surabaya, indonesia 3 department of parasitology, osaka city university school of medicine, osaka, japan 4 school of tropical medicine and global health, nagasaki university graduate school of medicine, nagasaki, japan 5 department of environmental and preventive medicine, oita university faculty of medicine, yufu, japan a corresponding author: indahst99@yahoo.com abstract we have previously developed a new method using a new formazan substrate wst-8, as a simple and rapid screening test for detection of glucose-6-phosphate dehydrogenase (g6pd) deficiency accomplished by the naked eye. however, it was little difficult to distinguish between faint orange colors developed by heterozygous females and pink colors of normal hemolyzed blood, since both have similar tones, but this was the only simple and rapid screening test can be applied in the field. to solve this problem, we established a newer and simple screening method has been established by replacing a different formazan substrate, mtt (3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2h tetrazolium bromide) in combination with a hydrogen carrier, 1-methoxy phenazine methosulfate to replace wst-8. mtt formazan exhibits a purple color, thus allowing for the ability to easily distinguish the pink colors of hemolyzed blood. however, mtt has been reported to react with hemoglobin non-specifically and to interfere with the interpretation of the color reaction. in our examinations by mixing mtt with hemolyzed blood, we found that the non-specific reaction was very slow, and that the addition of a small amount of blood (5 ~ 10 μl) into a reaction mixture (800 μl) did not interfere the reaction of g6pd activity. in this new mtt method, a strong purple color was generated in normal blood samples at 20~30 min after incubation, which could be distinguished by the naked eye from g6pd-deficient blood samples with less than 50% residual activity and has the same sensitivity and negative predictive value as wst-8 (ca. 85%). in addition, quantitative measurement using a spectrophotometer was also possible despite the fact that mtt formazan is water-insoluble. keywords: g6pd-deficiency, new screening method, formazan substrate, mtt, purple color development abstrak kami sebelumnya telah mengembangkan metode tes skrining sederhana dan cepat untuk mendeteksi defisiensi glukosa-6-fosfat dehidrogenase (g6pd) menggunakan substrat formazan wst-8 yang dapat diamati langsung dengan mata telanjang. namun mengalami sedikit kesulitan dalam membedakan antara warna oranye pudar yang dihasilkan oleh perempuan heterozigot dan warna merah muda yang disebabkan oleh hemolisis pada darah normal karena memiliki warna dasar yang sama, tetapi ini merupakan satu satunya tes skrining cepat yang dapat digunakan di lapangan. untuk mengatasi hal ini, kami mengembangkan metode skrining g6pd baru dan sederhana dengan menggunakan substrat formazan lain, yaitu mtt (3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2h tetrazolium bromide) yang dikombinasikan dengan1-methoxy phenazine methosulfate sebagai pengganti wst-8 . formazan mtt akan menghasilkan warna ungu, sehingga dengan mudah dapat dibedakan dengan warna merah muda yang disebabkan oleh hemolisis pada darah normal. walaupun disebutkan bahwa mtt dapat bereaksi non-spesifik dengan hemoglobin dan mengganggu interpretasi reaksi warna. namun dari hasil penelitian kami dengan mencampurkan mtt dengan darah hemolisis, menunjukkan bahwa reaksi non-spesifik yang terjadi sangat lambat, dengan demikian bila penambahan hanya dengan sejumlah kecil sampel darah (5 ~ 10 μl) ke dalam campuran reaksi 162 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 161–166 (800 μl) tidak akan mengganggu reaksi aktivitas g6pd. dengan metode mtt yang baru ini, akan menampilkan warna ungu yang kuat pada sampel darah normal dalam waktu 20 ~ 30 menit setelah inkubasi sehingga dengan mata telanjang dapat langsung dibedakan dengan sampel darah defisiensi g6pd dengan aktivitas enzim kurang dari 50% dan memiliki sensitivitas dan nilai prediksi negatif yang sama dengan wst-8 (sekitar 85%). selain itu pengukuran kadar g6pd secara kuantitatif dapat dilakukan dengan menggunakan spektrofotometer walaupun disebutkan bahwa formazan mtt tidak larut dalam air. kata kunci: defisiensi g6pd, metode skrining baru, substrat formazan, mtt, perubahan warna ungu. introduction glucose-6-phosphate dehydrogenase (g6pd) deficiency is one of the most frequent hereditary disorders, with an estimated 400 million people affected worldwide, particularly in tropical areas including malaria endemic regions.1 the g6pd gene spans 18 kb on the x chromosome (xq28), containing an open reading frame of 1,545 base pairs encoded in 13 exons and 12 introns. to date, more than 400 g6pd biochemical variants have been described, and 186 mutations among them have been discovered at the molecular level.2 the most frequent clinical manifestation of g6pd deficiency is acute hemolytic anemia, which is usually triggered by taking specific oxidative drugs such as primaquine.1 primaquine has been used for the radical treatment of vivax malaria and for gametocytocidal action against falciparum malaria. primaquine-induced hemolytic crisis is thus a serious problem in chemotherapeutic malaria control efforts. therefore, primaquine should be administered to malaria patients only after normal g6pd activity is confirmed. a number of surveys on malaria and g6pd deficiency of individuals living in malaria endemic areas of southeast asian countries 3-10 have been done using the acridine orange staining method for rapid diagnosis of malaria1112 and the wst-8 method13 for rapid detection of g6pd deficiency. by using these methods, the results of a blood examination could be informed within 30 mins to the malaria patients and prescribed antimalarial drugs, including primaquine, on-site if their g6pd activity was normal. presently, several screening methods for detection of g6pd-deficiency in the field have been reported. the fluorescent spot test 14-15 is the most widely used screening method. however, this method requires an ultraviolet lamp in a dark room, and since it provides only a qualitative result, it is very difficult to identify heterozygous females. other methods, such as the formazan ring method 16 and the sephadex gel method,17 that do not require any equipment or electricity have been used in epidemiological studies.18-22 both of these methods have used a formazan substrate, mtt (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2h tetrazolium bromide) and a hydrogen carrier, phenazine methosulfate (pms). unfortunately, both methods also provide only qualitative results and, thus, it was extremely difficult to diagnose heterozygous females. in addition, pms is strongly photo-sensitive, and special attention is needed to protect against exposure to ordinary light during screening.19 another formazan method, using wst-8 (2-(2methoxy-4-nitrophenyl)-3-(4-nitro phenyl)-5-(2,4disulfophenyl)-2h tetrazolium monosodium salt) and 1-methoxy pms 13 have been reported to overcome the disadvantages in the mtt/pms methods. the 1-methoxy pms is a photo-resistant hydrogen carrier, and wst-8 formazan is highly water-soluble; both are easy to assay qualitatively and quantitatively. however, this method also has a disadvantage: a faint orange color developed by 30~50% g6pd residual activity (i.e, heterozygous female samples) is quite similar in tone to the pink color of hemolyzed blood, which is not so easy to distinguish by the naked eye, making it difficult to confidently identify heterozygous females.9 as mtt formazan exhibits a purple color, the wst-8 in previous method13 was replaced by mtt formazan to be easier distinguish faint orange colors from pink colors (figure 1). a newer rapid screening and detection method of g6pd deficiency by using mtt/1-methoxy pms and naked eye without the interference of non-specific reactions between mtt and hemoglobin is reported herein. figure 1. principle of chemical reactions for detection of g6pd activity by using a formazan substrate, mtt materials and methods chemicals glucose-6-phosphate (g6p) and nicotinamide adenine dinuleotide phosphate (nadp) were obtained from boehringer co. (mannheim, germany). mtt, 1-methoxy pms and the wst-8 diagnostic kit were purchased from dojindo laboratories (kumamoto, japan). 163tantular, et al.: mtt formazan replaced wst-8 as a bettersimple screening method preparation of reaction mixtures for the mtt method the transparent type of microcentrifuge tube should be used in this method. the reaction mixture in a 1.5-ml microcentrifuge tube consisted of: (1) 20 μl of the substrate mixtures containing 50 mm g6p, 4 mm nadp in 400 mm tris-hcl buffer with 100 mm mgcl2 (adjusted ph to 7.2~7.5), (2) 20 μl of 5 mm mtt in h2o, (3) 20 μl of 1 mm 1-methoxy pms in h2o, and (4) 740 μl of h2o. these substrates and dye solutions can be stored at least for 6 months at 4 °c in the dark or for several years at –20 °c. procedures normal blood (g6pd activity, 9.0 iu/g hb), hemizygous male blood (1.0 iu/g hb) and heterozygous female blood (4.1 iu/g hb) were obtained from indonesian donors (the senior author and two volunteers, respectively). written informed consents were obtained from the two volunteers after the explanation of this study to them based on the guidelines ofthe declaration of helsinki. all ius were measured by an ultraviolet spectrophotometric method using a biochemical assay kit (345-b, trinity biotech, ireland). the reaction was commenced after adding 5 μl of whole blood to the reaction tube and mixing by shaking several times. the reaction tube was then left to stand and color photographs were taken at various intervals. development of purple color was observed by measuring the absorbance at 550 nm23 of the reaction tubes at various intervals using an ultraviolet spectrophotometer, hitachi u-2800 (tokyo, japan). examination of the non-specific reaction between mtt and hemoglobin was performed by mixing 5~25 μl normal blood into 800 μl of 0.125 mm mtt in h2o (the same concentration of mtts in the reaction mixture for the screening method) and the color change was observed at different intervals. results non-specific reaction with hemoglobin fairbanks and beutler 23and hirono et al.17 have reported that mtt reacts with hemoglobin non-specifically, and its dark red or brown color strongly interferes with the interpretation of the color reaction. in our examinations of a 1.5-ml tube containing 800 μl of 0.125 mm mtt in h2o, addition of 20~25 μl blood reacted with mtt albeit very slowly and the color of hemoglobin was changed to dark red at 6 hrs after incubation (figure 2). subsequently, small brownish precipitates were formed in the tubes at 8~10 hrs after incubation. however, these non-specific reactions were not observed when a small amount of blood (5~10 μl) was loaded (see tube 1 in figure 3d). these results indicated that the interference by the non-specific reaction was negligible when 5~10 μl of blood were mixed with the 800-μl reaction mixture. indeed, absorbance at 550 nm in the negative control did not change even in the presence of the same concentration of mtt (figure 4). qualitative findings figure 3 shows the development of a purple color that was generated by different g6pd activities in 1.5-ml tubes. appearance of the purple color in normal blood was observed at about 10 min after incubation at room temperature. at 30 min after incubation, a dark purple color in the normal blood sample (tube 4 in figure 3a) was clearly distinguished from only a faint purple color produced by heterozygous female sample (tube 3 in figure 3a). at 1 hr (figure 3b) and 2 hrs (figure 3c) after incubation, purple colors in the normal blood and heterozygous female blood became stronger, respectively, but at 3 hrs after incubation, small aggregates of mtt formazan formed, and the intensity of the purple color gradually decreased as the aggregates precipitated (tube4 in figure 3d). hemizygous male blood showed no change in color (tube 2 in figure 3a-d), nor did the negative control (tube 1 in figure 3a-d). however, the samples of heterozygous female blood (tube 3 in figure 3a-d) showed a slow color development toward purple, and it was possible to visually differentiate them from positive and negative controls. time-course of purple color development figure 4 shows the time-course of the purple color development in normal blood and in the negative controls without substrate. the color reaction of the normal blood reached a maximum after a 1.5~2-hr incubation, while the color development in the negative control did not occur during the 3-hr incubation (figure 4). at 3 hrs after incubation, however, absorbance in the normal blood sample decreased as the formazan aggregates precipitated to the bottom of the tube. these results indicated that a quantitative measurement was possible until 2 hrs after incubation although mtt formazan was water-insoluble. all results taken together suggested that judgment of g6pd activity by the new mtt method should be performed between 30 to 60 min of incubation, particularly for accurate identification of heterozygous female samples. 1 2 3 4 5 6 figure 2. development of dark red color by non-specific reactions between mtt and hemoglobin at 6 hrs after incubation. amount of blood loaded; 15 μl in tubes 1-2; 20 μl in tubes 3-4; 25 μl in tubes 5-6. tubes 1, 3 and 5, controls without mtt; tubes 2, 4 and 6, 0.125 mm mtt in 800 μlh2o. note that hemoglobin colors are changed to dark red in tubes 4 and 6. 164 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 161–166 figure 3. purple color development in reaction tubes with blood samples of different g6pd activities. five μl blood is loaded in each tube. tube 1, normal blood without the substrates (negative control); tube 2, hemizygous male; tube 3, heterozygous female; tube 4, normal blood (positive control). note that at 30 min after incubation, development of a strong purple color is seen in the positive control (tube 4), while a weak color development in tube 3 can be distinguished from the negative control (tube 1). at 3 hrs after incubation, the purple color of the positive control (tube 4) decreased in compared to those at 1~2 hrs since the water-insoluble mtt formazan gradually aggregates and then precipitates at the bottom of the tube. no change in color was observed in the negative control (a-d), even in the presence of mtt. figure 4. time-course of purple color development at 550 nm absorbance as measured by ultraviolet spectrophotometer g6pd activities corresponding to normal blood sample (tube 4 in figure 3). values represent the means of three determinations. at 3 hours after incubation, the absorbance at 550 nm decreased due to the precipitation of formazan aggregates. note that no change in absorbance was seen in the negative control in the presence of mtt. discussion the international standard method for detection of g6pd-deficiency is uv spectrophotometric assay by measurement of absorbance at 340 nm using an uv spectrophotometer with a biochemical assay kit (345-b, trinity biotech, ireland). but this method can be performed only at special hospitals or special institutions. a number of methods for rapid diagnosis of g6pd-deficiency have 165tantular, et al.: mtt formazan replaced wst-8 as a bettersimple screening method been reported. among them, the fluorescent spot test,1415 some mtt formazan methods 16-17, 22 and the wst-8 formazan method 13 have been adopted for application in the field. recently, two rapid chromatographic diagnostic test kits are commercially available, i.e, binaxnow g6pd (alere inc., usa)24 and the carestart g6pd (access bio, usa).25 both are qualitative assays utilizing formazan color development, but quantitative point-of-care tests are currently under development and validation.26 in the mtt/pms methods, many researchers have attempted to resolve the interference problem caused by the non-specific reaction using many techniques for separation of mtt from hemoglobin in reaction mixtures, such as absorption of g6pd enzyme on anion-exchange cellulose paper,23 of hemoglobin on cation-exchange cellulose paper,16and of g6pd enzyme absorbed on deaesephadex gel,17 or dissolving all reagents in agar plates and separating from blood (the formazan ring method16). however, our research on non-specific reactions revealed that many special efforts mentioned above are unnecessary. interestingly, we found that the interference caused by the non-specific reaction can be neglected as a small amount of blood sample is loaded, and that a quantitative measurement is also possible, similar to that of the wst-8 method. therefore, the new mtt method does not require any technique for separation of mtt from hemoglobin, and the only necessary action is to simply mix reagents in reaction tubes. all mtt methods are basically qualitative assays due to the fact that the mtt formazan is water-insoluble. extraction of the produced formazan by organic solvents, such as ether-acetone solution, dimethyl sulfoxide (dmso) or sodium dodecyl sulphate, is possible.23 as shown in figure 4, however, a quantitative assay is possible by the mtt method without the extraction process. nonetheless, the new mtt method may be more practical if it is used for screening for g6pd deficiency in malaria endemic regions by the naked eye without any equipment. mtt is a cheaper dye than wst-8, and it is more widely commercially available worldwide than wst-8. our field trial using the mtt method among the dayak and the melayu peoples in batang lupar district, kalimantan island was successful (unpublished). in this surveillance, 26 deficient individuals among 416 volunteers have been detected. among those, 22 venous blood samples were confirmed mutations by sequencing. these results may indicate that this new screening method using mtt/1methoxy pms is a better method for field detection of g6pd deficiency than the wst-8 method since exhibits a strong purple color which shows production of mtt formazan and describes the high activity of g6pd enzyme which could be easily distinguished by the naked eye. conclusions we found that the interference by non-specific reactions between mtt and hemoglobin can be neglected as a small amount of blood sample is loaded. therefore, mtt could be used as a formazan substrate, instead of wst-8, for better rapid screening of g6pd deficiency. this method is easy, rapid and reliable screening method, especially for field application. conflict of interest we have no conflict of interest to declare. acknowledgements we thank the wcp (world class professor) program supported by the indonesian directorate general of resources science and technology and higher education, ministry of research technology and higher education, indonesia, and prof. dato’ sri tahir, the tahir professorship grant, indonesia for their encouragements during this study. references 1. cappellini md, fiorelli g. glucose-6-phosphate dehydrogenase deficiency. lancet.2008; 371: 64-74. 2. minucci a, moradkhani k, hwang mj, zuppi c, giardina b, capoluongo e. glucose-6-phosphate dehydrogenase (g6pd) mutations database: review of the”old” and update of the new mutations. blood cells mol dis. 2012; 48: 154-65. 3. matsuoka h, arai m, yoshida s, tantular is, pusarawati s, kerong h, et al. five different glucose-6-phophate dehydrogenase (g6pd) variantsfound among 11 g6pd-deficient persons in flores island, indonesia.jhum gen. 2003; 48:541-4. 4. matsuoka h, wang j, hirai m, arai m, yoshida s, kobayasi t. et al. glucose-6-phosphate dehydrogenase (g6pd) mutations in myanmar: g6pd mahidol (487g>a) is the most common variant in the myanmar population. j hum gen. 2004; 49: 544-7. 5. jalloh a, van thien h, ferreira mu, ohashi j, matsuoka h, kanbe t, et al. rapidepidemiologic assessment of glucose-6phosphate dehydrogenase (g6pd) deficiency in malaria-endemic areasinsoutheast asia using a novel diagnostic kit. trop med int health. 2004; 9:615-23. 6. matsuoka h, nguon c, kanbe t, jalloh a, sato h, yoshida s, et al. glucose-6-phosphate dehydrogenase (g6pd) mutations in cambodia: g6pd viangchan (871g>a) is the most commonvariant in the cambodian population. jhumgen. 2005; 50: 468-72. 7. kawamoto f, matsuoka h, kanbe t, tantular is, pusarawati s, kerong h, et al. further investigations of glucose-6-phosphate dehydrogenase variants in floresisland, eastern indonesia. jhum gen. 2006; 51: 952-7. 8. matsuoka h, thuan dtv, van thien h, kanbe t, jalloh a, hirai m, et al. seven different glucose-6-phosphate dehydrogenase variants including a new variant distributed in lam dong province in southern vietnam. acta med okayama. 2007; 61: 181-5. 9. tantular is, matsuoka h, kasahara y, pusarawati s, kanbe t, tuda jsb, et al. incidence and mutation analysis of glucose-6dehydrogenase deficiency in eastern indonesian populations. acta med okayama. 2010; 64: 367-73. 10. kawamoto f, matsuoka h, pham nm, hayashi t, kasahara y, dung nt, et al. further molecular analysis of g6pd deficiency variants in southern vietnam and a novel variant designated as g6pd ho chi minh (173 a>g; 58 asp>gly): frequency distributions of variants compared with those in other southeast asian countries. acta med okayama. 2017; 71: 325-32. 166 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 144–149 11. kawamoto f. rapid diagnosis of malaria by fluorescence microscopy using light microscope and interference filter. lancet. 1991; 337: 200-2. 12. kawamoto f, billingsley pf. rapid diagnosis of malaria by fluorescence microscopy. parasitol today. 1992;8: 69-71 pmid: 15463575 13. tantular is, kawamoto f. an improved, simple screening method for detection ofglucose-6-phosphatedehydrogenase deficiency. trop med int health. 2003; 8: 569-74. 14. beutler e. a series of new screening procedures for pyruvate kinase deficiency, glucose-6-phosphate dehydrogenase deficiency, and glutathione reductase deficiency. blood. 1966; 28: 553-62. 15. beutler e, mitchell m. special modification of the fluorescent screening method for glucose-6-phosphate dehydrogenase deficiency. blood. 1968; 32: 816-8. 16. fujii h, takahashi k, miwa s. a new simple screening method for glucose-6-phosphate dehydrogenase deficiency. acta haematol jap 1984:47: 185-8. 17. hirono a, fujii h, miwa s. an improved single-step screening method for glucose-6-phosphate dehydrogenase deficiency. jap j trop med hyg. 1998; 26:1-4. 18. hirono a, ishii a, kere n, fujii h, hirono k, miwa s. molecular analysis of glucose-6-phosphate dehydrogenase variants in the solomon islands. american j hum gen. 1995; 56:1243-5. 19. tantular is, iwai k, lin k, basuki s, horie t, htay hh, et al. field trials of a rapid test for g6pd deficiency in combination with a rapid diagnosis of malaria. trop med int health. 1999; 4, 245-50. 20. ishii a, nagai n, arai m, kawabata m, matsuo t, bobogare a, et al.chemotherapeuticmalaria control as a selective primary health care activity in the solomon islands. parasitologia. 1999; 41: 383-4. 21. iwai k, hirono a, matsuoka h, kawamoto f, horie t, lin k, et al. distribution of glucose-6-phosphate dehydrogenase mutations in southeast asia. hum gen. 2001; 108: 445-9. 22. suryantoro p. glucose-6-phosphate dehydrogenase (g6pd) deficiency in yogyakarta and its surrounding areas. southeast a j trop med pub health. 2003; 34 suppl. 3: 138-9.fairbanks vf, beutler e. a simple method for detection of erythrocyte glucose-6-phosphate dehydrogenase deficiency (g-6-pd spot test). blood. 1962; 20: 591601. 23. osorio l, carter n, arthur p, bancone g, gopalan s, gupta sk, et al.performance of binaxnow g6pd deficiency point-of-care diagnostic in p. vivax-infected subjects. am j trop med hyg. 2015; 92: 22-7. 24. goo yk, ji sy, shin hi, moon jh, cho sh, lee wj, et al. first evaluation of glucose-6-hosphate dehydrogenase (g6pd) deficiency in vivax malaria endemic regions in the republic of korea. plos one. 2014; 9: e97390. 25. bancone g, gornsawun, chu cs, porn p, pal s, bansil p, et al. validation of the quantitative point-of-care carestart biosensor for assessment of g6pd activity in venous blood. plos one. 2018; 13: e0196716. ijtid vol 6 no 1 jan-april 2016_revisi.indd 5 vol. 6. no. 1 january–april 2016 literature review synthesis of metal-organic (complexes) compounds copper(ii)-imidazole for antiviral hiv candidate teguh hari sucipto1,2, fahimah martak2 1institute of tropical disease, universitas airlangga, surabaya, indonesia. 2natural product compound and synthesis laboratorium, departement of chemistry, mathematics and natural science faculty, sepuluh nopember institute of technology corresponding author : teguhharisucipto@gmail.com abstract the human immunodeficiency virus (hiv) is viruses known as rotaviruses. potential target for therapeutic is reverse transcriptase (rt), possesses an rna-dependent dna polymerase, dna-dependent dna polymerase and ribonuclease h fuctions. imidazoles have high anti-hiv inhibitory activity, some derivates of imidazole reported drugs. 8-chloro-2,3-dihydroimidazole[1,2-b][1,4,2]benzodithiazine-5,5-dioxides and 9-chloro-2,3,4-trihydropyri-mido[1,2-b][1,4,2]benzodithi-azine-6,6-dioxides. this compounds succesfully identified anti-hiv activity. copper is a bio-essential element and copper complexes have been extensively utilized in metal mediated dna cleavage for the generation of activated oxygen species. it has been reported that teraaza macrocyclic copper coordination compounds have anti-hiv activities. studies have shown that these macrocyclic complexes can react with dna in different binding fashions and exhibit effective nuclease activities. complex compounds are compounds in which there is an atom that acts as the central atom and trotter group of molecules that can be either neutral or charged ions. application a metal-organic (complex) compounds, especially copper metal and derivates of imidazole. so, in this study can explore new anti-hiv candidate. key words: complexes compound, copper, imidazole, antiviral, hiv abstrak human immunodeficiency virus (hiv) adalah virus yang termasuk golongan rotavirus. target potensial untuk terapi adalah reverse transcriptase (rt), memiliki sebuah dna-dependent rna polimerase, dna-dependent dna polimerase dan ribonuklease. imidazol memiliki aktivitas penghambatan anti-hiv yang tinggi, beberapa turunan dari imidazol melaporkan obat. 8-kloro-2,3-dihydroimidazole [1,2-b] [1,4,2] benzodithi-azine-5,5-dioksida dan 9-chloro-2,3,4-trihydropyri-mido [1,2-b] [1,4,2] benzodithi-azine-6,6-dioksida. ini senyawa aktivitas anti-hiv berhasil diidentifikasi. tembaga adalah unsur dan tembaga kompleks bio-penting telah banyak digunakan dalam logam dimediasi pembelahan dna untuk generasi spesies oksigen aktif. telah dilaporkan bahwa senyawa koordinasi tembaga teraaza makrosiklik memiliki kegiatan anti-hiv. penelitian telah menunjukkan bahwa kompleks makrosiklik dapat bereaksi dengan dna di mode mengikat yang berbeda dan menunjukkan aktivitas nuklease yang sangat efektif. senyawa kompleks adalah senyawa yang ada atom yang bertindak sebagai atom dan dikelilingi oleh molekul yang dapat berupa ion netral atau ion pengganti. aplikasi logam-organik (kompleks) senyawa, terutama logam tembaga dan turunan dari imidazol. jadi, pada studi ini dapat dipelajari kandidat anti-hiv baru. kata kunci: senyawa kompleks, tembaga, imidazole, antivirus, hiv introduction the human immunodeficiency virus is a number of class of viruses known as rotaviruses, was identifiend as the causative agent in the transmission and development of acquired immune deficiency syndrome (aids). the replicative cycle of hiv provides many potential targets for therapeutic intervation. reverse transcriptase 6 indonesian journal of tropical and infectious disease, vol. 6. no. 1 january–april 2016: 5−11 (rt), possesses an rna-dependent dna polymerase, a dna dependent dna polymerase and ribonuclease h fuctions.1 imidazoles have high anti-hiv inhibitory activity2, some derivates of imidazole reported drugs. imidazole a ring substituted and pirimidine ring for potent inhibitory activity against rt. these cmpund showed minimal cytotoxicity and are therefore suitable for antiviral development. complex compounds are compounds in which there is an atom that acts as the central atom and trotter group of molecules that can be either neutral or charged ions. this trotter group called ligands. complex compounds formed are influenced by the nature of the ligand, which includes the alkalinity, bond, and chelate effects. copper is a bio-essential element and copper complexes have been extensively utilized in metal mediated dna cleavage for the generation of activated oxygen species. it has been reported that tetraaza macrocyclic copper coordination compounds have anti-hiv activities. this papers reviews about imidazole potency and copper for anti-hiv. so, in this study can explore drug from the mixture compound, metal-organic compound, especially cu-imidazole complexes. imidazole compound and derivates brzozowski et al., (2006) prepared new compound with modifications on the imidazole [2]. we present the synthesis 8-chloro-2,3-dihydroimidazole[1,2b][1,4,2] benzodithi-azine-5,5-dioxides and 9-chloro-2,3,4trihydropyrimido[1,2b][1,4,2] benzodithi-azine-6,6-dioxides (figure 1). succesfully identified anti-hiv activity eco50 0.09 μm. this compounds showed minimal cytotoxicity and suitable for antiviral development. in the compounds, methyl group at position 7 showed the highest anti-hiv activity cause electron-donating. compounds showed significant cytotoxicity in cell-based assays even though they were very effective in hiv-1 integrase-based assays.2 figure 1. modification of imidazole.2 figures 2. 5-phenyl-1-phenylamino imidazole.3 anti-hiv of 5-phenyl-1-phenylamino-imidazole have been cytotoxicity data in qsar study. in the qsar study, imidazole derivate presence of hydrogen bond donor groups appears to be an important feature for reducing the cytotoxicity. molecular size can also important for determining the cytotoxicity.3 in 2004, 1-[2-(alkylthio-1-imidazolyl)carbonyl]-4-[3(isopropyl amino)-2-pydridyl] piperazines, the compound were tested for anti-hiv activity and had maximum precent of protection 2x10-5m.1 2-alkylthio-1-[4-(1-benzyl-2-athyl-4-nitro-1himidazole-5-yl)-piperazin-1-yl] ethanones and alkyl-[4(1-benzyl-2-ethyl-4-nitro-1h-imidazol-5-yl)-piperazin-1yl) ketones, the newly synthesized compounds were assayed against hiv-1 and hiv-2 in mt-4 cells. the compounds were showed inhibition of hiv-1 (ec50 0.45 μg ml -1) and hiv-2 (0.50 μg ml-1). the target is non-nucleoside reverse transcriptase inhibitor.4 copper for antiviral hiv activity copper is a bio-essential element and copper complexes have been extensively utilized in metal-mediated dna cleavage for the generation of activated oxygen species. it has been reported that teraaza macrocyclic copper coordination compounds have anti-hiv activities. studies have shown that these macrocyclic complexes can react with dna in different binding fashions and exhibit effective nuclease activities.5 figures 3. macrocyclic copper(ii) complexes.5 at 2010, copper(ii) containing bis-macrocyclic [cu23l2] 2+ has improved anti-hiv potency in vitro (ec50 4.3 nm). the interaction of the metallodrug has been optimized by using ultra rigid chelator units that offer an equatorial site for coordination to the amino acid side chains of the protein.6 cu2-xylyl-bicyclam also exhibits anti-hiv activity. it was used cu2+-cyclam as a paramagnetic probe to investigate interactions of metal-locyclams with the model protein target in solution.7 copper complexes were substrated competitive inhibitors for hiv-1 protease. for example, [bis-(2pyridylcarbonyl)-amido] copper(ii) nitrate dihydrate binds with an inhibiton constant of 480 μm. molecular modeling suggests that the catalytic water between asp25 and asp125 of hiv-1 protease is directly coordinated to the cu(ii) ion.8 7sucipto and martak: synthesis of metal-organic (complexes) compounds copper (ii)-imidazole figures 4. copper(ii) bis-macrocyclic.8 complex compounds of cu(ii)-imidazole complex compounds copper(ii) with monodentate ligand 1 imidazoles ying et al., synthesis complex compounds of cu(ii) as the central atom with ligands that have a monodentate imidazole strutur octahedral geometry. complex compound formed is cu(c3n2h4)2(hl)2 and cu(c3n2h4)2l with c3n2h4 is an imidazole and hl is adipic acid. in the structure of the complex compounds occur hydrogen bonds between the nh---o into a compound supermolecule due to polymerization. in the complex compound cu(c3n2h4)2l, cu atom has five coordination centers of cun2o3 pyramidal, then bind to ligands bridge ligand monodentate adipic acid so as to form a polymerization.9 figures 5. (a) complex compound cu(c3n2h4)2(hl)2 , (b) hydrogen bonding structure.9 figures 6. (a) complex compound cu(c3n2h4)2l, (b) hydrogen bonding structure.9 i n t h e y e a r 2 0 1 1 h a s b e e n s y n t h e s i z e d [cu5(iba)4(n3)2(so4)2] .4h2o with hiba is 4(imidazol1-yl) benzoate -acid by liu. this complex compound has a symmetrical structure with an angle {cu2, cu2a, cu2b, cu2c} is 71.57° and 108.43°. magnetic properties of complex compounds are ferromagnetic.10 figures 7. c o m p l e x c o m p o u n d [ c u 5 ( i b a ) 4 ( n 3 ) 2 (so4)2].4h2o. 10 figures 8. 2d structure [cu5(iba)4(n3)2(so4)2].4h2o of hydrogen bonding effect. 10 8 indonesian journal of tropical and infectious disease, vol. 6. no. 1 january–april 2016: 5−11 by li et al., in 2013 have synthesized a complex compound used for heterogeneous catalysts. this is a complex compound [cu(ima)2]n, synthesized in methanol and ambient temperature. the catalytic properties of complex compounds is very good because it has the results (%) is high and the higher the stability of the compound.11 figures 9. 2d crystal structure of mof with 4 coordination cu2+.11 [cu2l but(imidazole)2br2](clo4)2 have been successfully synthesized by graham et al., in 2005 ago. the molecular structure above has cation [cu2l but(imidazole)2 br2] 2+ and perchlorate anions. lbut ligand is anticonformation so as to cause the complex compounds into centrosymmetry with bridge butane, cu---cu 8446 å. copper (ii) as a coordination center, coordinating with the anions bromide dn monodentate ligand n-imidazole.12 figures 10. [cu2l but(imidazole)2br2] (clo4)2 compound. 12 complex compounds [cu(bhac)(himdz)] has a central atom cu(ii) as a coordination center, acetylacetone benzoilhidrazona tridentat ligand and monodentate ligands imidazole. these compounds can be formed due to metal ion coordination with enolate-o, imine-n and the deprotonated amide-o atom sixth and fifth ring chelate.13 figures 11. complex compound [cu(bhac)(himdz)]. 13 then through intramolecular hydrogen bonds nh---n form a crystalline regularity, this is called polymerization. the effective magnetic moment of these compounds is 1.86 μb. weak antiferromagnetic because their 2-apical equatorial, chloro and bridges asetato.13 figures 12. 2d structure of crystal [cu(bhac)(himdz)]. 13 synthesis of bis (9,10-dihydro-9 oxo-10-acrydinacetato) bis (imidazole) copper (ii)tetrahydrate.14 monomer crystal structure of cu(cma)2(him)2 will react intermolecular hydrogen bond with water molecules. figures 13. complex compound cu(cma)2(him)2. 14 cu complex compounds (cma)2(him)2 can bind hydrogen nh---o form a crystalline order as follows, figures 14. 2d crystal cu(cma)2(him)2. 14 in 2005, song et al., have managed to synthesize [cu4(h3l)(h2l)cl3(h2o)2] cl2.5h2o. this complex compound used as a ligand clorate ligands bridge connecting cu-cu so that a cu-cl-cu. furthermore, with 9sucipto and martak: synthesis of metal-organic (complexes) compounds copper (ii)-imidazole the intramolecular hydrogen bond is formed a polymer complex.15 figures 15. complex compound [cu4(h3l)(h2l)cl3(h2o)2] cl2.5h2o. 15 figures 16. 1d polimer structure of complex compound [cu4(h3l)(h2l)cl3 (h2o)2]cl2.5h2o. 15 carbalo et al., 2004 perform synthesis of [cu(hl)2(im)] with the coordination geometry pyramide structure.16 figures 17. complex compound [cu(hl)2(im)]. 16 the complex compounds will undergo intramolecular hydrogen bonds form a continuous crystal. the crystals were formed as glasses with cu(ii) as the central atom. figures 18. crystal [cu(hl)2(im)] showed 2d after hydrogen donding. 16 complex compounds copper (ii) with a bidentate ligand 1 imidazoles in 2010 pramanik et al., have succeeded in synthesizing complex compounds [cu(ii)(l2)(h2o)(no3)2] with the central atom cu(ii) which has a planar coordination giometry pyramid, nitrate as a monodentate ligand and two atoms n on imidazole freely donate an electron pair to form a common bond of cu(ii) which is referred to as a bidentate ligand. furthermore, there is intramolecular hydrogen bond bond, the n atom of the imidazole with oxygen atoms from nitrate. so as to form a polymerization, namely a crystal.17 figures 19. (a) complex compound [cu(ii)(l2)(h2o)(no3)2], (b) polymeri -zation. 17 complex compounds copper(ii) by ligand tridentat 1 imidazoles sarker et al., 2010 has been to synthesize a complex compound with cu(ii) as the central atom, 1-alkyl-2-(otioalkil) fenilazoimidazol as ligands tridentat, and scn as bridging ligand. molecular formula is [cuii (setaainme) (scn)2]. imidazole donated three pairs of free electrons to bind together with the atom cu(ii). scn as ligands bridge will connect between the molecules form a complex compound supermolecule.18 10 indonesian journal of tropical and infectious disease, vol. 6. no. 1 january–april 2016: 5−11 figures 20. (left) chemistry structure, (right) design of molecule structure [cuii(setaainme)(scn)2]. 18 figures 21. left [cu(biap)br2] and right [cu(biap)(no2)2]. 19 baretta et al., 2000 managed to synthesize two complex compounds with imidazole as a ligand tridentate namely, [cu(biap)br2] and [cu(biap)(no2)2]. complex compounds [cu(biap)br2] has the shape of trigonal geometry bipiramidal with br2 in apical position and 3 nitrogen of ligands and anions bromide in equarorial. hydrogen bonding occurs in bromine and imdazol-nh atom in the molecule itself. complex compounds [cu(biap)(no2)2] asymetric shape. can be seen in the image below ions cu(ii) in a position squer pyramidal with two imidazole and 1 amine, nitroten together with the oxygen on the nitrite on the state of equatorial and equal.19 summary imidazoles have high anti-hiv inhibitory activity, some derivates of imidazole reported drugs. imidazole a ring substituted and pirimidine ring for potent inhibitory activity against rt. copper is a bio-essential element and copper complexes have been extensively utilized in metal mediated dna cleavage for the generation of activated oxygen species. copper can potential for anti-hiv, because copper have inhibitor activity for hiv-proteinase. copper can interact with donor atoms on a biological target via the formation of coordinate bonds rather than a combination of weaker intermolecular force such as h-bonding and chelator. the chelator has high stability complex to retain the copper ion in vivo and exchangeable ligands must be present to allow coordination of amino acid side chains. acknowledgments we thank hotma wardhani harahap for providing valuable comments. this study was supported by the institute of tropical disease (itd) the center of excellence (coe) program by the ministry of research and technology (ristek) indonesia and chemistry departement, sepuluh nopember institute of technology references 1. f. hadizadeh and a. mehrparvar, synthesis of some new 1-[2alkylthio-1-benzyl-5-imidazolyl) carbonyl]-4-[3-(isopropylamino)2-pyridyl]piperazine as anti-hiv, j. sci. 15 (2004) 131-134 2. z. brzozowski, f. saczewski, n. neamati, synthesis and anti-hiv activity of a novel series of 1,4,2-benzodithiazine-dioxides, bioorg. med. chem. lett. 16 (2006) 5298 3. k. roy and j.t., leonard, topological qsar modeling of cytotoxicity data of anti-hiv 5-phenyl-1-phenylamino-inidazole derivatives using gfa, g/pls, fa and pcra techniques, ind. j. chem. 45a (2006) 126-137 4. y.a. al-soud, n.a. al-masoudi, h.g. hassan, e.d. clercq, c. pannecouque, nitroimidazoles. v. synthesis and anti-hiv evaluation of new 5-substituted piperazinyl-4-nitroimidazole derivatives, acta pharm. 57 (2007) 379-393 11sucipto and martak: synthesis of metal-organic (complexes) compounds copper (ii)-imidazole 5. j. liu, h. zhang, c. chen, h. deng, t. lu, l. ji, interaction of macrocyclic copper (ii) complexes with calf thymus dna: effects of the side chains of the ligands on the dna-binding behaviors, dalton trans., (2003) 114-119 6. a. khan, g. nicholson, j. greenman, l. madden, g. mcrobbie, c. pannecouque, e.d. clercq, r. ullom, d.l. maples, r.l. maples, j.d. silversides, t.j. hubin, s.j. archibald, binding optimization through coordination chemistry: cxcr4 chemokine receptor antagonists from ultra rigid metal complexes, j. am. chem. soc. 131 (2009) 3416-3417 7. t.m. hunter, l.w. mcnae, x. liang, j. bella, s. parsons, m.d. walkinshaw, p.j. sadler, protein recognition of macrocycles: binding of anti-hiv metallocyclams to lysozyme, pnas 107 (2005) 22882292 8. e. maggers, exploring biologically relevant chemical space with metal complexes, current opinion in chem. bio. 11 (2007) 287292 9. e. ying, y. zheng, h. zhang, syntheses, crystal structures and properties of two cu(ii) coordination polymers: cu(c3n2h4)2(hl)2 and cu(c3n2h4)2l with c3n2h4=imidazole, h2l=adipic acid, j. mol. struc. 93 (2004) 73-80. 10. g. liu, x.wang, h. zhou, s. nishihara, synthesis, structure and magnetic properties of pentanuclear cu(ii) coordination polymer with 4-(imidazole-1-yl)-benzoic acid, inorg. chem. comm. 14 (2011) 1444-1447. 11. z. li, l. xue, l. wang, s. zhang, b. zhao, two-dimensional copperbased metal-organic framework as a robust heterogeneous catalyst for the n-arylation of imidazole with arylboronic acids, inorg. chem. comm. 27 (2013) 119-121. 12. b. graham, l. spicca, b.w. skelton, a.h. white, d.c.r. hockless, imidazole derivatives of binuclear copper (ii) and nickel (ii) complexes incorporating bis(1,4,7-triazacyclononan-1-yl) ligands, inorg. chim. act. 358 (2005) 3974-3982 13. z. gu, g. li, p. yin, y. chen, h. peng, m. wang, f. cheng, f. gu, w. li, y. cai, temperature-induced two copper (ii) supermolecular isomers constructed from 2-ethyl-1h-imidazole-4,5-dicarboxlylate, inorg. chem. comm. 14 (2011) 1479-1484. 14. s. das, s. pal, self-assembly of copper(ii) complexes with a dibasic tridentate ligands and monodentate n-hetrocycles: structural, magnetic and epr studies, j. mol. struc. 741 (2005) 183-192 15. y. song, c. massera, o. roubeau, a.m.m. lanfredi, j. reedijk, chloro-bridged cu(ii) pairs linked into a 1d coordination polymer through a dinucleating imidazole-based ligand: 3d structure and magnetism, polyhedron. 24 (2005) 1599-1605 16. r. carballo, a. castineiras, b. covelo, e. martines, j. niclos, e.m. lopes, solid state coordination chemistry of monoclear mixedligand complexes of ni(ii) and zn(ii) with α-hydroxycarboxylic acids and imidazole, polyhedron. 23 (2004) 1505-1518 17. a. pramanik, a. basu, g. das, coordination assembly of p-substituted aryl azo imidazole complexes: influences of electron donating substitution and couter ions, polyhedron. 29 (2010) 1980-1989 18. k.k. sarker, s.s. halder, d. banerjee, t.k. mondal, a.r. paital, p.k. nanda, p. raghvaiah, c. sinha, copper-thioarylazoimidazole complexes: structures, photochromism and redox interconversion between cu(ii) <-> cu(i) and correlation with dft calculation, inorg. chim. act. 363 (2010) 2955-2964 19. m. beretta, e. bouwman, l. casella, b. douziech, w.l. driessen, l. gutierres-soto, e. monzani, j. reedijk, copper complexes of a new tridentate imidazole-containing ligand: spectroscopy, structures and nitrite reductase reactivity – the molecular stuctures of [cu(biap) (no2)2] and [cu(biap)br -2], inorg. chim. act. 310 (2000) 41-50 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 3 september–december 2022 original article antibiotic sensitivity against klebsiella spp. in the post debridement culture an open fracture in emergency department of dr. soebandi hospital jember dini agustina1* , endiningtyas cahyaningrum2, cicih komariah3, i nyoman semita4 , yudha ananta khaerul putra5 1laboratory of microbiology, faculty of medicine, universitas jember, jember, indonesia 2 faculty of medicine, universitas jember, jember, indonesia 3laboratory of pharmacology, faculty of medicine, universitas jember, jember, indonesia 4department of surgery, dr. soebandi general hospital, jember, indonesia 5emergency department unit, dr. soebandi general hospital, jember, indonesia received: july 13th, 2022; revised: october 31st, 2022; accepted: december 2nd, 2022 abstract surgical site infection (ssi) in open fracture is often caused by bacterial contamination in the management of open fracture. because of that, one of the most important thing in handling open fracture is debridement. prophylactic antibiotics given are cephalosporin and aminoglycosides. post-debridement culture is important in predicting the incidence of infection. one of the bacteria that is often found in post-debridement culture is klebsiella spp. which can produce esbl to fight β-lactam class of antibiotics. the purpose of this study was to determine antibiotic sensitivity against klebsiella spp. in the post-debridement culture of cases of open fractures in the emergency department of dr. soebandi hospital jember. this study uses a laboratory exploratory research design. the sample of this study was the isolate of klebsiella spp. which amounts to 5 from post debridement culture of open fracture patients in the emergency department of dr. soebandi hospital jember from march to may 2019.the method used is diffusion (kirby baurer) by matching using the clsi standard table to determine sensitive, intermediate, or resistant. the results of this study showed that most antibiotics had resistance to klebsiella spp., including βlactam antibiotics, such as amoxicillin, ceftriaxone, cefixime, penicilin, meropenem, and cefadroxil. vancomycin antibiotics are still sensitive to klebsiella spp. in all patients. gentamicin, ciprofloxacin, tetracycline, and chloramphenicol antibiotics were sensitive in 1 patient. erythromycin intermediates antibiotics against klebsiella spp.. the conclusion of this study is that all β-lactam group antibiotics are resistant to klebsiella spp while the most sensitive antibiotic is vancomycin. keywords: antibiotic sensitivity; esbl; klebsiella spp.; open fracture; post debridement abstrak infeksi luka operasi pada patah tulang terbuka seringkali disebabkan oleh kontaminasi bakteri pada manajemen patah tulang terbuka. oleh karena itu, salah satu hal yang penting pada penanganan patah tulang terbuka adalah debridemen. kultur setelah debridemen penting dalam memprediksi kejadian infeksi. salah satu bakteri yang sering ditemukan pada kultur setelah debridemen adalah klebsiella spp. yang dapat menghasilkan extended-spectrum β-lactamase (esbl) untuk melawan antibiotik golongan β-lactam. tujuan penelitian ini untuk mengetahui sensitivitas antibiotik terhadap klebsiella spp. pada kultur post debridement kasus patah tulang terbuka di emergency department dr. soebandi hospital jember. penelitian ini menggunakan desain penelitian eksploratif laboratorik. sampel penelitian ini adalah isolat bakteri klebsiella spp. hasil kultur post debridement 5 pasien patah tulang terbuka di * corresponding author: dini_agustina@unej.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-1861-0056 https://orcid.org/0000-0003-0363-0254 https://orcid.org/0000-0002-6008-5988 190 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 189–197 emergency department dr. soebandi hospital jember periode maret-mei 2019. metode yang digunakan adalah difusi (kirby baurer) dengan hasil pengukuran yang dikonversikan dengan tabel standar clinical laboratory standards institue (clsi) untuk menentukan sensitif, intermediet, atau resisten. hasil: hasil penelitian ini menunjukan sebagian besar antibiotik mengalami resistensi terhadap klebsiella spp., termasuk antibiotik golongan β-lactam, seperti amoxicillin, ceftriaxone, cefixime, penicilin, meropenem, dan cefadroxil. antibiotik vancomycin masih sensitif terhadap klebsiella spp. pada seluruh pasien. antibiotik gentamicin, ciprofloxacin, tetracycline, dan chloramphenicol sensitif pada 1 pasien. antibiotik erythromycin intermediet terhadap klebsiella spp. kesimpulan dari penelitian ini adalah semua antibiotik golongan β-lactam resisten terhadap klebsiella spp. sedangkan antibiotik yang paling sensitif adalah vancomycin. kata kunci: esbl; klebsiella spp.; patah tulang terbuka; sensitivitas antibiotik; setelah debrimen how to cite: agustina, d., cahyaningrum, e., komariah, c., samita, i. n., putra, y. a. k. antibiotic sensitivity against klebsiella spp. in the post debridement culture an open fracture in emergency department of dr. soebandi hospital jember. indonesian journal of tropical and infectious disease. 10(3). 189–197. dec. 2022. introduction surgical site infection (ssi) in the open fracture is often caused by bacterial contamination in open fracture management. because of that, debridement is one of the most important things in handling open fractures. an open fracture is a break in the continuity of the bone with injury to the skin above the site of fractures, with traffic accidents the most cause.1–3 in open fractures, contact with the outside environment is susceptible to infection. in the treatment of open fractures, one of the important things to do is debridement.4–7 in a study at third hospital of hebei medical university, most ssis (81.8 %,18/22) were found during subsequent hospitalizations. the total incidence of ssis was 6.0 % (22/364). the superficial ssis accounted for 2.4 % (9/364) and deep ssis for 3.6 % (13/364).8, whereas in the second hospital of tangshan, the overall incidence of ssi was 18.6%, with 17.0% and 1.6% for superficial and deep infection, respectively. there were 2027 males and 665 females of the study sample.9 post-debridement culture is more important in predicting infection incidence than pre-debridement culture. in postdebridement cultures of open fractures, infections are often caused by gram-negative bacteria such as klebsiella spp., e. coli, pseudomonas spp., acinetobacter spp., and enterobacter spp..10,11 research by sitati et al. (2017) mentioned that bacteria found in post-debridement culture of open fractures are klebsiella spp., s. aureus, pseudomonas spp., con (negative coagulase) staphylococci, and e. coli. this is in line with the preliminary study conducted from march to may 2019 at the emergency department dr. soebandi hospital jember, in 30 patients with open fractures, it was found that in the culture of post-debridement, the most common bacteria were klebsiella spp.12 klebsiella spp. is a gram-negative, rodshaped bacterium and lactose-positive colonies cultivated on macconkey agar.13 klebsiella spp. is the main bacterium of the family enterobacteriaceae, which can produce extended-spectrum β-lactamase (esbl) to fight the β-lactam class of antibiotics, such as the penicillin, cephalosporin, carbapenem, and monobactam groups. this enzyme hydrolyzes the β-lactam ring from antibiotics so that antibiotic resistance can occur.14 prophylactic antibiotics such as the aminoglycosides and cephalosporin first generation are often given in cases of open fractures to prevent infection. this is what allows antibiotic resistance.15 antibiotic resistance could occur in some ways, such as destroying antibiotics with the enzymes produced, improving antibiotic capture point receptors, improving the physicochemical targets of antibiotic targets in bacterial cells, and antibiotics could not penetrate bacterial cell walls due to changes in bacterial cell wall properties. if someone is infected with 191 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license dini agustina, et al. antibiotic sensitivity against klebsiella spp. resistant bacteria, the effort to deal with infection with antibiotics is more complicated.16 research conducted at voi county hospital, kenya, by sitati et al. in 2017, stated that gram-negative bacteria klebsiella spp. and pseudomonas spp. pre and postdebridement experienced high resistance to tetracycline and amoxicillin-clavulanic acid by 27% and 23% and experienced resistance of 87.5%, 91%, and 47.6% in gentamicin, amikacin, and cefuroxime.12 it was giving ceftriaxone therapy as a prophylactic antibiotic and cefixime when outpatient to patients with open fractures in the emergency room of emergency department dr. soebandi hospital jember is not based on culture results and antibiotic sensitivity testing. this is what underlies the author's research on the sensitivity of 12 types of antibiotics, namely amoxicillin, tetracycline, ceftriaxone, gentamicin, cefixime, ciprofloxacin, penicillin, meropenem, erythromycin, vancomycin, cefadroxil, and chloramphenicol against klebsiella spp. in the post-debridement culture of cases of open fractures in emergency department dr. soebandi hospital jember. materials and methods materials the population of this study was 12 bacterial isolates from the post-debridement culture of open fracture patients in the emergency room of emergency department dr. soebandi hospital jember from march to may 2019 consisted of klebsiella spp. (5 patients), pseudomonas spp. (3 patients), shigella spp. (2 patients), salmonella spp. (1 patient), and proteus spp. (1 patient). the sample of this study was klebsiella spp. amounting to 5. the 12 types of antibiotics, namely amoxicillin, tetracycline, ceftriaxone, gentamicin, cefixime, ciprofloxacin, penicillin, meropenem, erythromycin, vancomycin, cefadroxil, and chloramphenicol. the research used mcfarland standards, mueller-hinton agar, plates, sterile cotton swabs, aluminum foil, tweezers, syringes, caliper, and ruler. methods this study uses a laboratory explorative research design that is research that does not aim to look for relationships between variables, is only descriptive and is carried out at the laboratory of microbiology, faculty of medicine, university of jember. the method used is diffusion (kirby baurer) by matching the inhibition zone diameters using the standard clinical laboratory standards institution (clsi) table to determine sensitive, intermediate, or resistant. the steps of the research procedure was to prepare 0.5 mcfarland standards made from 1% bacl2 and 1% h2so4 and shake before use to adjust the turbidity of the bacterial suspension and mueller-hinton agar from 15.2 grams of mueller-hinton and 400 ml aquadest. antibiotic sensitivity testing in this study used the method disc diffusion (kirby-baurer test) with mueller-hinton agar. the steps taken were to make inoculums from klebsiella spp. from each plate using a loop into 2 ml of nacl 0,9%. the inoculum turbidity was adjusted to ensure an even or nearly even growth yield using mcfarland standard. after turbidity obtained the same results as mcfarland standard, the plate was inoculated using a sterile cotton swab dipped in the inoculum in laminar flow biobase. before being swabbed on mueller-hinton agar, excess inoculum was removed by pressing and rotating the cotton swab firmly against the side of the tube. the swab was evenly distributed over the entire surface of mueller-hinton agar by rotating the plate at an angle of 60° and allowed to dry for several minutes at room temperature with the cup closed. then given 4 discs of antibiotics in each medium. 192 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 189–197 discs are aseptically placed on the surface of mueller-hinton agar using sterile tweezers to avoid contamination with other bacteria. the media that had been given an antibiotic disc was incubated for 24 hours at 370c. repetition was carried out three times on different media. measurements were made the next day after 24 hours of media incubation at 370c. measuring the diameter of the bacterial growth inhibition zone using a caliper or ruler is done on the back of the mueller-hinton agar media so that you don't have to open the lid. the measurement results are adjusted to the clinically and laboratory standards institute (clsi) in the classification of sensitive, intermediate, or resistant. ethical approval this research received ethical approval from the health research ethics comitte of faculty of medicine, university of jember with the letter number 1.408/h25.1.11/ke/2020. results and discussion based on preliminary studies carried out from march to may 2019 at the igd rsd, dr. soebandi jember, in 30 patients with open fractures, there were data that there were 12 patients in the culture of positive postdebridement growing bacteria, 42% klebsiella spp., 25% pseudomonas spp., 17% shigella spp., 8% salmonella spp., and 8% proteus spp.. klebsiella spp. is the most common bacteria in post-debridement culture, 5 isolates. patients positive for klebsiella spp. in the post-debridement culture there were 5 people with 4 male and 1 female. in contrast, the age distribution of patients was 1 person in range 17-25 years, 2 people in range 36–45 years, 1 person in range 56–65 years, and 1 person in range ≥ 66 years. all patients were diagnosed with varying degrees of open fracture according to the gustilo-anderson classification. most patients experience open fractures due to traffic accidents. the clinical characteristics data of 5 patients with open fractures, such as diagnosis and mode of injury (moi) (shown in table 1). table 1. diagnosis, grade, and moi of the open fractures patients the results of medical record data on the type of antibiotic prophylaxis, antibiotics during hospitalization, and antibiotics consumed at home used by patients with open fractures as a sample of this study can be seen in table 2. the antibiotics tested were adjusted to the clsi standard for klebsiella spp. as shown in table 3. patients diagnosis grade mode of injury (moi) p1 traumatic amputation digiti 2, open fracture head metacarpal 2, and open fracture digiti 3 phalanx distal manus sinistra iiib exposed to a wood-cutting knife p2 open fracture fibula dextra and fracture iliac wing dextra iiia traffic accidents between 2 motorcycle riders p3 open fracture tibia-fibula 1/3 medial sinistra iiib traffic accidents between 2 motorcycle riders p4 open fracture digiti 1,2,3 phalanx proximal pedis dextra iiia traffic accidents between 2 motorcycle riders p5 open fracture kominutif tibia-fibula sinistra ii traffic accidents between 2 motorcycle riders 193 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license dini agustina, et al. antibiotic sensitivity against klebsiella spp. table 2. antibiotics are given to patients with open fractures at dr. soebandi hospital patients antibiotic prophylaxis inpatient outpatient p1 ceftriaxone ceftriaxone cefixime p2 ceftriaxone ceftriaxone cefixime p3 ceftriaxone ceftriaxone cefixime p4 ceftriaxone ceftriaxone cefixime p5 ceftriaxone cefazoline and gentamicin cefixime table 2. the result of the antibiotic sensitivity test for klebsiella spp. sample aml cro cfm p mem cfr cn cip te e c va p1 r r r r r r r i r i i s p2 r r r r r r s i i r s s p3 r r r r r r r i r i i s p4 r r r r r r r s i i i s p5 r r r r r r r i s i i s r= resistent, i= intermediate, s= sensitive, p1= patient 1, p2= patient 2, p3= patient 3, p4= patient 4, p5= patient 5, aml= amoxicillin, cro= ceftriaxone, cfm= cefixime, p= penicilin, mem= meropenem, cfr= cefadroxil, cn= gentamicin, cip= ciprofloxacin, te= tetracycline, e= erythromycin, c= chloramphenicol, va= vancomycin. this study found positive patients klebsiella spp. in the post-debridement culture. there were 5 people, 4 male, and 1 female, with an age range of 22-70 years. the results of this study are consistent with research by agarwal et al2, which states that of the 70 open fracture patients studied, 63 patients (90%) were men with ages ranging from 3-75 years.2 research by gupta et al10 states that most open fracture patients have a 13 times higher incidence of males than females. this is because men are more often outdoors activities.10 traffic accidents are the most common cause of open fractures, followed by work-related injuries and falls from heights. high-energy trauma is the most common mode of injury (moi) causing open fractures. diagnosis of patients with open fractures dominated by phalanx, tibia, and fibula according to research by sop and sop4, which states that phalanx fractures are the most common fractures, followed by tibia and fibula fractures.4 degree iii open fractures, according to the gustilo-anderson classification, have a significantly higher infection rate than grades i and ii. this is related to the severity of the wound and the degree iii treatment’s length of time.17–19 management of open fracture cases requires the administration of antibiotic prophylaxis to prevent surgical site infection (ssi). prophylactic antibiotics used in treating open fractures in rsd dr. soebandi jember uses the ceftriaxone antibiotic, while the antibiotic given while outpatient is cefixime. ceftriaxone and cefixime are included in the antibiotic. a cephalosporin is an antibiotic option in line with guidelines for treating open fractures in a journal by zalavras20. the journal also describes the administration of cephalosporin and aminoglycosides antibiotics to prevent infection. cephalosporin is given to prevent gram-positive bacteria in first and seconddegree open fractures. in contrast, thirddegree open fractures require antibiotics to protect against gram-positive and negative, and aminoglycosides (gentamicin) are given.20 according to sop and sop4, when antibiotics are given 66 minutes after injury, the infection rate is 0% and increases to 17% if it exceeds this time.4 the british orthopedic association/british association of plastic reconstruction and aesthetic surgeons (boa/bapras) supports the opinion of experts that antibiotics are given 194 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 189–197 24 to 48 hours for the degree i and a maximum of 72 hours in degrees ii and iii.21 in this study, a sensitivity test for 12 antibiotics was carried out on the klebsiella spp. these bacteria can cause many of disease, cause problem to people with immunocompromised and the most common cause of hospital acquired pneumonia. in this study klebsiella spp. that found in culture most likely caused of open fracture, even the management has been carried out according to the procedure.22,23 open fracture treatment depends on location of fracture but generally need irrigation, debridement, and antibiotic. initial debridement in should be performed within 24 hours. the goals of open fracture management include decreasing risk of infection and promoting fracture union.24,25 klebsiella spp. cultured after debridement in patients with open fractures developed resistance to β-lactam antibiotics. these antibiotics include amoxicillin, ceftriaxone, cefixime, penicillin, meropenem, and cefadroxil. however, antibiotics such as gentamicin, ciprofloxacin, tetracycline, chloramphenicol, and vancomycin, these bacteria are sensitive. associated with the resistance of these bacteria to β-lactam class antibiotics, it proves that these bacteria are extended-spectrum β-lactamase (esbl) producing bacteria. the results of this study different from studies conducted at voi county hospital, kenya by sitati et al12 which states that patients with open fractures after culture in pre and post debridement, obtained high resistance data against tetracycline, erythromycin, and amoxicillin-clavulanic acid in gram positive and negative bacteria. gram negative bacteria such as klebsiella spp. and pseudomonas spp. found in pre and post debridement experienced high resistance to tetracycline and amoxicillin-clavulanic acid by 27% and 23% and experienced resistance of 87.5%, 91%, and 47.6% in gentamicin, amikacin and cefuroxime. this is likely due to differences in study locations and differences in the selection of antibiotics used in the treatment of open fracture patients.12,27 the results of research that have been done have shown that antibiotics are still sensitive to klebsiella spp. namely gentamicin in 1 patient, ciprofloxacin in 1 patient, tetracycline in 1 patient, chloramphenicol in 1 patient, and vancomycin in all patients. esbl-producing bacteria can be given vancomycin antibiotics which can kill bacteria by breaking peptide bonds between amino acids in the peptidoglycan wall. although using vancomycin for open fractures is safe, it is still controversial, except for patients who are allergic to penicillin. this is because vancomycin added to cefazoline has no benefit in patients with open fractures. however, a recent 2016 publication by tennent et al shows the benefits of using vancomycin powder in local wounds of rats to prevent biofilm formation.28 gentamicin antibiotics were still sensitive according to the study of ashwin and thomas29 , which stated that bacterial culture in third-degree open fractures was 83.3% klebsiella spp. sensitive to gentamicin.29 chloramphenicol antibiotics are sensitive to klebsiella spp. this is in accordance with research by nitzan et al30 which states that members of enterobactericeae, such as the bacteria klebsiella spp., e. coli, and enterobacter spp. achieve statistical significance of a lower level of resistance to chloramphenicol of 18.4%.16 ciprofloxacin antibiotics are sensitive to klebsiella spp. by the 2018 mangala study, which states that ciprofloxacin has a 69% sensitivity to the enterobacteriaceae family.31 research on carbapenem-resistant klebsiella pneumoniae (crkp) states that in data analysis from 1998–2010, resistance to tetracycline increased only slightly. nextgeneration tetracycline may be helpful in the treatment of crkp because of increased tissue penetration, antibiotic activity, and the decreased tendency for antibiotic resistance.32 this is in line with research that has been done because the tetracycline 195 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license dini agustina, et al. antibiotic sensitivity against klebsiella spp. antibiotic was found to be sensitive in 1 patient. erythromycin antibiotics were concluded intermediates against the bacteria klebsiella spp. because intermediates were obtained in 4 patients and resistant in 1 patient. research by khan et al33 states that the bacteria klebsiella spp. found to be more resistant to macrolide antibiotics, equal to 41.67% against erythromycin.33 resistant antibiotics and intermediates cause the treatment of open fractures to be suboptimal, resulting in surgical site infection (ssi). the management of open fractures is focused on effective debridement measures, appropriate antibiotic therapy, and initial wound closure to prevent infection.21 the older age (71.5%) due to experiencing immune deficiency (decreased immune system) resulting in a longer recovery time. degree iii open fractures according to the gustilo-anderson classification have a significantly higher infection rate than grades i and ii. this is related to the severity of the wound and the length of time of the third degree treatment.13 the occurrence of ssi in these patients is likely due to the above ssi risk factors, such as age 70 years (> 60 years), male, experiencing high energy injuries due to accidents, and the degree of open fracture iiib. conclusions the conclusion of this study after an antibiotic sensitivity test was conducted on 5 samples of klebsiella spp. in postdebridement culture in emergency department soebandi general hospital jember is resistant to klebsiella spp. the βlactam class of antibiotics used in this study are amoxicillin, ceftriaxone, cefixime, penicillin, meropenem, and cefadroxil. klebsiella spp. was still sensitive to other antibiotics such as chloramphenicol gentamicin, ciprofloxacin, tetracycline, and vancomycin. erythromycin antibiotics are stated intermediates to the bacterium klebsiella spp. for the dr. soebandi hospital jember. the institution needs to have periodic culture tests and antibiotic sensitivity tests for inpatients so that an antibiogram can be made and used as a basis for the definitive treatment of diseases, especially in the field of orthopedic. the antibiotic sensitivity test towards klebsiella spp. which contaminated the postdebridement procedure in patients with open fracture, showed an evidence that a comprehensive evaluation of the empirical antibiotic prophylactic strategies preand post-operative procedures so far need to be considered. this statement is based on the total resistance of ceftriaxone and cefixime in all klebsiella spp samples. based on this research, we also humbly recommend other antibiotics that can be alternative options as prophylactic agents to prevent perioperative contamination and postoperative surgical site infection in patients with open fractures, such as gentamicin, ciprofloxacin, tetracycline, chloramphenicol, and vancomycin. acknowledgement the authors would like to thank dr. soebandi hospital jember and medical faculty, universitas jember. conflict of interest the authors declare that they have no conflict of interest. references 1. calandruccio jh. fractures, dislocations, and ligamentous injuries of the hand and wrist clinicalkey. in: campbell’s operative orthopaedics. 2021. p. 3497–559. 2. agarwal d, maheshwari r, agrawal a, chauhan vd, juyal a. to study the pattern of bacterial isolates in open fractures. j orthop traumatol rehabil. 2022;8(1):1. 196 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 189–197 3. halawi mj, morwood mp. acute management of open fractures: an evidence-based review. orthopedics. 2015;38(11):e1025–33. 4. sop jl, sop a. open fracture management statpearls ncbi bookshelf [internet]. statpearls publishing. 2022. p. 1–23. 5. perry kl. management of open fractures: part 1. http://dx.doi.org/1012968/coan2016213165. 2016;21(3):165–70. 6. ali am, noyes d, cogswell lk. management of open fractures of the lower limb. http://dx.doi.org/1012968/hmed20137410577. 2013;74(10):577–80. 7. griffin m, malahias m, khan w, hindocha s. update on the management of open lower limb fractures. open orthop j. 2012;6(1):571–7. 8. zhu c, zhang j, li j, zhao k, meng h, zhu y, et al. incidence and predictors of surgical site infection after distal femur fractures treated by open reduction and internal fixation: a prospective single‐center study. bmc musculoskelet disord. 2021;22(1):1–10. 9. hu q, zhao y, sun b, qi w, shi p. surgical site infection following operative treatment of open fracture: incidence and prognostic risk factors. int wound j. 2020;17(3):708. 10. gupta s, saini n, sharma r, kehal j, saini y. a comparative study of efficacy of pre and post debridement cultures in open fractures. internet j orthop surg [internet]. 2012;19(3). 11. cherian jj, lobo jo, ramesh lj. a comparative study of bacteriological culture results using swab and tissue in open fractures: a pilot study. j orthop case reports. 2019;9(1):33–6. 12. sitati fc, mosi po, mwangi jc. early bacterial cultures from open fractures differences before and after debridement. ann african surg [internet]. 2018;14(2). 13. finka r, agustina d, rachmawati da, suswati e, mufida dc, shodikin a. the role of pili protein 38,6 kda klebsiella pneumoniae as a hemagglutinin and adhesin protein which serves as a virulence factor. j agromedicine med sci. 2019;5(2):9. 14. ghafourian s, sadeghifard n, soheili s, sekawi z. extended spectrum beta-lactamases: definition, classification and epidemiology. curr issues mol biol 2015, vol 17, pages 11-22. 2014;17(1):11–22. 15. tandirogang y, esa t, sennang n. kuman dan kepekaan antimikroba di kasus patah tulang terbuka. indones j clin pathol med lab. 2018;19(2):88. 16. syahputra rri, agustina d, wahyudi ss. the sensitivity pattern of bacteria against antibiotics in urinary tract infection patients at rsd dr. soebandi jember. j agromedicine med sci. 2018;4(3):171–7. 17. matos ma, lima lg, de oliveira laa. predisposing factors for early infection in patients with open fractures and proposal for a risk score. j orthop traumatol. 2015;16(3):195– 201. 18. elniel ar, giannoudis p v. open fractures of the lower extremity: current management and clinical outcomes. efort open rev. 2018;3(5):316–25. 19. agel j, rockwood t, barber r, marsh jl. potential predictive ability of the orthopaedic trauma association open fracture classification. j orthop trauma. 2014;28(5):300–6. 20. zalavras cg. prevention of infection in open fractures. infect dis clin north am. 2017;31(2):339–52. 21. o’brien c, menon m, jomha n. controversies in the management of open fractures. open orthop j [internet]. 2014;8(1):178–84. 22. ashurst j v., dawson a. klebsiella pneumonia. statpearls. 2022; 23. bengoechea ja, sa pessoa j. klebsiella pneumoniae infection biology: living to counteract host defences. fems microbiol rev. 2019;43(2):123. 24. hull pd, johnson sc, stephen djg, kreder hj, debridement of severe open fractures is associated with a higher rate of deep infection. https://doi.org/101302/0301-620x96b332380. 2014;96-b(3):379–84. 25. you dz, schneider ps. surgical timing for open fractures. ota int open access j orthop trauma. 2020;3(1):e067. 26. agustina d, nadyatara k, mufida dc, elfiah u, shodikin ma, suswati e. faktor virulensi outer membrane protein 20 kda klebsiella pneumoniae sebagai protein hemaglutinin dan adhesin. ejournal kedokt indones. 2020;7(3):200–4. 27. tri nugroho supranoto y, habibi a, zulaikha s, mutia r, nyoman semita i, agustina d, et al. a case report: surgical site infection of open fracture grade iiic caused by methicillinresistant staphylococcus aureus (mrsa). j asian med students’ assoc. 2021;9(1). 28. tennent dj, shiels sm, sanchez cj, niece kl, akers ks, stinner dj, et al. time-dependent effectiveness of locally applied vancomycin powder in a contaminated traumatic orthopaedic wound model. j orthop trauma. 2016;30(10):531–7. 29. ashwin h, thomas g. a prospective study on results of bacterial culture from wound in type iii compound fractures. int j res orthop. 2018;4(6):935–9. 30. nitzan o, suponitzky u, kennes y, chazan b, raul r, colodner r. is chloramphenicol making 197 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license dini agustina, et al. antibiotic sensitivity against klebsiella spp. a comeback? pubmed. isr med assoc j. 2010;12(6):371–4. 31. mangala a, arthi k, deepa r. comparison of predebridement and debridement cultures in predicting postoperative infections in compound fractures. j clin diagnostic res [internet]. 2018;12(7):dc06–9. 32. sanchez g v., master rn, clark rb, fyyaz m, duvvuri p, ekta g, et al. klebsiella pneumoniae antimicrobial drug resistance, united states, 1998–2010 volume 19, number 1—january 2013 emerging infectious diseases journal cdc. emerg infect dis. 2013;19(1):133–6. 33. khan n, hassan f, naqvi b, hasan s. antimicrobial activity of erythromycin and clarithromycin against clinical isolates of escherichia coli, staphylococcus aureus, klebsiella and proteus by disc diffusion method pubmed. pak j pharm sci. 2011;24(1):25–9. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid vol 3 no 1 jan-maret 2012.indd 23 vol. 3. no. 1 january–march 2012 pain relieved using extra anatomy pathway in acute infection abdurachman anatomy-histology department, medical faculty, airlangga university abstract acute infection is characterized especially by pain as major complaint of patients. in this following case report, it will be shown that pain cause of acute infection can be relieved using acupuncture technique. acupuncture use meridian as extra anatomy pathway. key words: pain, meridian, extra anatomy pathway introduction during the long history of traditional chinese acupuncture, the results of needling acupoints have been described both clinically and theoretically. the concepts of chi, blood, meridians, and acupoints are integral to the understanding and application of traditional chinese medicine (tcm). since its introduction into western culture, there were many experiments and writings to attempt to explain these concepts in western scientific terms. many early explanations were shallow and simplistic, for example, that acupuncture is simply a primitive way of describing stimulation of the nervous system, or that it is only placebo treatment (starwynn, 2001). recently a growing number of insightful researchers have penetrated further into the common truth between tcm and western science, and their paths have led into the realms of electromagnetics and quantum physics (starwynn, 2001). acupuncture involves the insertion and manipulation of needles into specific points on the body to relieve pain or for therapeutic purposes. in acupuncture medicine technique, a communication path exists aside from nervous, blood vessels and lymph vessels communication path. meridian is not a nerve path, not a lymph vessels path, is also not a path of blood vessels. this particular communication path is known as energy communication path (chi) or is specially named as meridian. in acupuncture theory, it is mentioned that chi flows through the body's meridians. if this chi flows is disrupted, complains or symptoms according to the degree of disruption and the meridian where disrupted will appear (yanfu,1 2002). as early as in the ancient age, people began to use stone needle for medical treatments. acupuncture therapy uses acupuncture points as the stimulating points and the relationship of meridian as basis of the treatment. meridian consists of major channel and branches of channels, which refer to the network that runs chi, contact the viscera, communicate the internal and external and run through up and down inside the body (yanfu,1 2002) according to gellman (2002), the body's bio energy flows through specific channels called meridian and regulates the whole body function of the body's organ. meridian is channels which connect all the body's components. aside from connecting all of the body's energy internally, meridian also connects the body's internal energy with external energy (natural energy) through "doors" called acupuncture points or acupuncture points. stimulation on acupuncture points will be transmitted meridian communication path. then stimulation will affect circulation of the existing energy system, creating a healing effect, especially to meridian connected directly to the stimulated acupuncture point (gellman, 2002). diameters of the acupuncture points are approximately between on to three millimeters. the depths from the surface of the skin are according to the place and different in each individual (wensel, 1980). it has long been known that acupuncture points have some specific characteristics, at superficial acupuncture points, there are high electric potential (can reach as high as 300mv), high electric capacitance (0.1-lmf), low electric resistance, increased skin respiration, high local temperature, radiating light which spontaneously visible from jing and yuan points, and sound signals (frequency case report 24 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 23−25 2–15 hz, amplitude: 0.5–l mv). at profound acupuncture points, there are low perception threshold to electric stimulation, high capacitance, electric resonance with the other acupuncture points, high conductivity to isotopic tracers (starwynn, 2001). darras (1992) investigated the pathways of acupuncture meridians in the human body through the injection of radioactive tracers at acupuncture points. technetium 99 m (99 mtc) as sodium pertechnetate, the most common radioactive tracer in nuclear medicine, has been used. the migration patterns were recorded with a scintillation camera associated with computer imaging capabilities. his findings show that the preferential pathways taken by the radiotracer coincide with acupuncture meridians as described in chinese traditional medicine. more, it has been established that these pathways are distinguishable from either lymphatic or vascular routes. case report a 68-year-old mother came up with right foot pain. right foot pain caused by an iron rod pierced. iron was piercing of the plantar toward back foot about 3 cm long (see fig. 1). diameter of the iron in about 6 mm. puncture occurred 20 hours ago. figure 1. wound location (private document, camera in black berry bold 9500) injury caused pain. pain was felt increasing. injuries also generate signs of inflammation do to infection. the foot swelled especially in the area around the wound, the plantar foot and the dorsum of the foot (fig. 1). the color of the area around the wound flushed skin, increased body temperature. the temperature increase is felt throughout the body, the patient feels cold. related to the pain increases, the patient could not stand upright, because the inversion of the foot should be positioned. after examination, anamnesis and physical examination, the physician who examined propose to do therapy using acupuncture techniques. before performing the acupuncture therapy, the physician did conventional therapy in the form: first, the doctor did conventional therapy in the form: 1. clean the wound (debridement) 2. closing the wound using gauze soaked in liquid antiseptic solution (betadine) and then closes the wound using hypafix (fig. 2a). figure 2. a. closes the wound using hypafix (private document) b. punctured in ki-3 acupoint (private document) furthermore, inspectors perform acupuncture therapy by acupuncture needle (stainless steel) sterile size 0.25 x 40 mm. puncture perpendicularly 0.3–0.5 inch at the kidney point-3 (ki-3, taixi), on the medial border of the foot. posterior to the medial malleolus, in the depression between the tip of the medial malleolus and achilles tendon (yanfu,2 2002), (fig. 2b). needle direction and rotated counter-clockwise. in this case report, point was chosen as an effective point for the body's energy flow in kidney meridian energy lines, especially for unblocked of body energy flow in plantar of foot (yanfu,2 2002). pain was relieved in about 45 seconds. patients feel the pain decreased to about 80%. after that, acupuncture needle revoked, the person can stand in the direction normal standing foot (not invertion), a little pain. conclusion 1. pain in acute infection can be relieved by puncturing the point of acupuncture. 2. the way of communication used in acupuncture is extra anatomy pathway. 3. this case report impressed the existence of meridian pathway. another pathway common use in anatomy terminology (abdurachman, 2005, 2009). 4. this case report impressed the existence of acupuncture points. references 1. abdurachman, 2010. acupuncture therapy to relieve pain in dextral hypochondrial area. case report, folia medica indonesiana, vol. 45 no. 3. 2. abdurachman, 2005. laser puncture effect at pishu (bl-20) acupoint on stz induced diabetic rats. the european journal, vol. 3 august 2005 pp. 32–33. 25abdurachman: pain relieved using extra anatomy pathway 3. gellman h, 2002. acupuncture treatment for musculoskeletal pin. a textbook for orthopedics, anesthesia and rehabilitation. taylor and francis, new york. 4. darras j-c, albarède p, de vernejoul p, 1993. nuclear medicine investigation of transmission of acupuncture information. acupunct med; 11: 22–28. 5. starwynn d, 2001. electrophysiology and the acupuncture systems. medical acupuncture vol. 13, number 1. 6. wensel md, 1980. acupuncture for americans. virginia: reston publ. comp. inc. a prentice hall company. 7. yanfu(1) z, jingsheng z, zhaoguo l, renying c, 2002. basic theory of traditional chinese medicine. publishing house of shanghai university of traditional chinese medicine. shanghai. china. 8. yanfu(2) z, jingsheng z, zhaoguo l, renying c, 2002. chinese acupuncture and moxibution. publishing house of shanghai university of traditional chinese medicine. shanghai. china. 9. li zhaoguo, published by publishing house of shanghai university of traditional chinese medicine. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 �� vol. 2. no. 1 january–march 2011 expression of b-xylosidase encoding gene in phis����/ bacillus megaterium ms��� system sri sumarsih, ni nyoman tri puspaningsih, ami soewandi,js. departement of chemistry, faculty of science and technology airlangga university abstract b-xylosidase encoding gene from g. thermoleovorans it-08 had been expressed in the phis1525/ b. megaterium ms941 system. the b-xylosidase gene (xyl) was inserted into plasmid phis1525 and propagated in e. coli dh10b. the recombinant plasmid was transformed into b. megaterium ms941 by protoplast transformation. transformants were selected by growing the recombinant cells on solid lb medium containing tetracycline (10 µg/ ml). the expression of the b-xylosidase gene was assayed by overlaid the recombinant b. megaterium ms941 cell with agar medium containing 0.2% methylumbelliferyl-b-d-xyloside (mux). this research showed that the b-xylosidase gene was succesfully sub-cloned in phis1525 system and expressed by the recombinant b. megaterium ms941. thethe addition of 0.5% xylose into the culture medium could increase the activity of recombinantactivity of recombinant of recombinantb-xylosidase by 2.74 fold. the recombinant b. megaterium ms941 secreted 75.56% of the expressed b-xylosidase into culture medium. the crude extract b-xylosidase showed the optimum activity at 50° c and ph 6. the recombinant b-xylosidase was purified from culture supernatant by affinity chromatographic method using agarose containing ni-nta (nickel-nitrilotriacetic acid). the pure b-xylosidase showed a specific activity of 10.06 unit/ mg protein and relative molecular weight ± 58 kda. key words: expression, b-xylosidase, geobacillus thermoleovorans it-08, phis1525, bacillus megaterium ms941geobacillus thermoleovorans it-08, phis1525, bacillus megaterium ms941, bacillus megaterium ms941 introduction β-d-xylosidases (1,4-β-d-xylan-xylo-hydrolase, e c 3 . 2 . 1 . 3 7 ) a r e h e m i c e l l u l a s e s t h a t h y d r o l y z e xylooligosaccharides to xylose and are essential for the complete utilization of xylan. the branching and variability of the xylan structure requires the concerted action of several hemicellulolytic enzymes including endo-1, 4-β-xylanases that hydrolyze the xylan backbone, and β-xylosidases that cleave the resulting xylooligomers into xylose monomers.1 plasmid ptp510 is a recombinant plasmid containing xylanolytic enzyme encoding gene from g. thermoleovorans it-08. the xylanolytic enzymes were expressed intracellularly by the recombinant ptp510/ e. coli dh5α. the recombinant plasmid containing three genes: exo-exoxylanase (exo-xyl), α-l-arabinofuranosidase (abfa), and β-xylosidase (xyl) (�en�ank accession �os. d�38704�,(�en�ank accession �os. d�38704�, d�387047, and d�345777, respectively). escherichia coli plays a prominent role as a heterologous protein production host due to extensive research efforts over the last several decades. albeit well known and used for protein production, several intrinsic problems hamper the system’s unrestrained usage. e. coli lacks protein export mechanisms therefore the protein produced accumulates intracellularly, mostly in the form of inactive inclusion bodies due to the high concentrations present. the presence of endotoxins and the inability to attach glycosidic residues constrict its application for pharmaceutical proteins. the �ram-positive bacterium bacillus megaterium offers several advantages over e. coli as a protein production system. it does not possess endotoxins and has a high secretion capacity. compared to bacillus subtilis it is distinguished by higher plasmid stability and a lower intrinsic protease activity which is a significant advantage for a secretory protein production system. important prerequisites like an efficient transformation system, stable and freely replicating plasmids and the ability to integrate heterologous genes into the genome are met.2 �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 25-29 the use of a gram-positive bacterium could facilitate protein production due to the lack of an outer membrane allowing direct secretion of proteins into the growth medium. in contrast to b. subtilis, b. megaterium does not produce alkaline proteases. another advantage of this bacterium is the high stability of plasmids during growth, which allows a stable gene expression in long term cultivations and bioreactors. b. megaterium has been used for the production of several recombinant proteins, e.g. dextransucrase,3 and lysozyme specific single chain fv (scfv) fragment d1.3.4 recently, a set of free replication vectors and genetically optimized b. megaterium strains for the intraand extracellular production of affinity tagged recombinant proteins were developed. they were successfully employed for the production and purification of dextransucrase,5 levansucrase,3 and penicillin amidase.6 plasmid phis1525 is a shuttle vector for e. coli/ b. megaterium, containing a signal peptide for secretion of heterologous protein of interest into the culture medium. the phis1525 system is also equipped with a 6x his-tag sequence for purification and detection of the expressed his-tagged target proteins.2, 3 in this work, we reported the expression of β-xylosidase encoding gene from g. thermoleovorans it-08 in the phis1525/ b. megaterium ms941 system. materials and methods cultures escherichia coli dh10β and bacillus megaterium ms941 were grown in lb (luria bertani) medium (composition : 1% nacl, 1% tripton, 0.5%1% nacl, 1% tripton, 0.5% yeast extract). recombinant e. coli dh5α, containing xylanolytic enzyme encoding gene (ptp510) from g. thermoleovorans it-08, was grown in lb medium containing ampicillin (100μg/ ml). chemicals all analytical chemicals and media component used were pure grade and available commercially. amplification and sub-cloning β-xylosidase encoding gene (xyl) was amplified from ptp510 using a pair of primers which designed according to the sequence of β-xylosidase of g. thermoleovorans it-08 (genebank no. dq345777) : fxyl 5’-ttattgagc tctcgaatattctaacccag-3’ and rxyl : 5’-caa gagcatgcaatatttcaggaatatatttaaacc3’. the xyl gene was inserted into plasmid phis1525 and propagated in e. coli dh10β before transforming into the b. megaterium. the recombinant plasmids were analyzed by restriction analysis and sequencing. the recombinant plasmid was transformed into b. megaterium ms941 by protoplast transformation method in the isotonic medium containing polietilen glikol.7 transformants were selected by growing the recombinant b. megaterium ms941 on the solid lb medium containing tetracycline (10 μg/ ml). assay of β-xylosidase expression the expression of the β-xylosidase gene was assayed using 0.5% agar containing 0.2% methylumbelliferyl-βd-xyloside (mux). recombinant b. megaterium ms941 cells on lb medium (+tet 10 μg/ml) were overlaid with suspension of phosphate buffer ph 6.0 containing 0.5% agar and 0.2% methylumbelliferyl-β-d-xyloside (mux). the assayed petri plates were incubated at 600 c overnight. the β-xylosidase expression was monitored on the uv-trasluminator. enzyme assay the enzyme activity was determined by measuring the release of p-nitrophenol from p-nitrophenyl-β-dxylopyranoside (p�px). the mixture of 100 µl enzyme sample and 900 µl of l mm p�px, was incubated at 50° c for 30 min. the reaction was stopped by adding 0,1 ml of 0,4 m �a2co3 solution. the release of p-nitrophenol was measured using spectrophotometer uv-vis at λ 405 nm. 1 (one) unit of the β-xylosidase activity was defined as the amount of the enzymes releasing 1 �mol p-nitrophenol�mol p-nitrophenolmol p-nitrophenol equivalent per minute under the assay condition. effect of xylose on β-xylosidase activity the effect of xylose on β-xylosidase activity was studied by producing the enzyme in various concentration by producing the enzyme in various concentration of xylose as an inducer and time of addition into the culture medium. the enzyme activities were determined toward pnpx as a subtrate. the enzyme production was carried out in 100 ml cotton plugged erlenmeyers which contained 20 ml fresh lb medium and 1% over-night pre-culture of recombinant b. megaterium ms941, and cultivated 37° c 250 rpm. the concentration of xylose was varied: 0, 0.25 and 0.50% (w/v), and the time of addition was varied: 0, 1, 2, 3, and 4 hours after cultivation (od578 of 0–0.2. the enzyme was harvested after 10 h cultivation. the supernatant was collected by centrifugation at 10,000 rpm, 4° c for 20 min. the° c for 20 min. the c for 20 min. the for 20 min. the β-xylosidase activity was determined toward pnpx as a substrate. production of recombinant β-xylosidase the enzyme production was carried out in 500 ml cotton-plugged erlenmeyers containing 100 ml fresh lb medium, and inoculated by 1% of pre-culture of recombinant b. megaterium ms941 and cultivated 37° c, 250 rpm. expression of xyl was induced by adding 0.5% sterile xylose at an od578 of 0.1. the enzyme was harvested after 10 h cultivation. the supernatant i (called secreted enzyme) was collected by centrifugation at 10,000 rpm, 4° c for 10 min. the pellet cell was resuspended in4° c for 10 min. the pellet cell was resuspended in° c for 10 min. the pellet cell was resuspended in c for 10 min. the pellet cell was resuspended in for 10 min. the pellet cell was resuspended in citrate-phosphate buffer ph 6.0 and lysed by two passages through a ultrasonicator at 20 khz for 60 sec. debris cells was removed by centrifugation at 10,000 rpm, 4° c for4° c for° c for c for for 10 min. the supernatant ii called non-secreted enzyme. the β-xylosidase activities of enzymes, both secreted and ��sumarsih, et al.: expression of β-xyloside encoding gene non-secreted enzyme were determined toward pnpx as a substrate. effect of ph and temperature on β-xylosidase activity the optimum ph of the crude β-xylosidase activity was determined by measuring the enzymes activity toward pnpx as a substrat in ph range of 5–8 at 50° c for 30 min. the optimum temperature of the crude β-xylosidase activity was determined by measuring the enzymes activity toward substrat pnpx in defferent temperature range 50–70° c. purification of recombinant β-xylosidase the recombinant β-xylosidase (xyl) was purified by affinity chromatography method using agarose containing �i-�ta ((nickel-nitrilotriacetic acid). protein was precipitated from supernatant by adding 40% saturated (�h4)2so4. the precipitant was collected by centrifugation at 13,000 rpm, 4° c for 10 min. the desalted protein was resuspended with buffer a (50 mm bufer fosfat ph(50 mm bufer fosfat ph50 mm bufer fosfat ph = 8,0 + 250 mm �acl + 5 mm imidazole + 5 mm βmerkaptoetanol), then loaded onto a column containingloaded onto a column containing 1 ml �i–�ta agarose (�iagen) and pre-equilibrated with buffer a and incubated 2 h at cool room. after discarding the flow through, the column was washed with five column volumes of buffer a, and eluted with five column volumes, and eluted with five column volumesand eluted with five column volumes of buffer � (50 mm phosphate ph 8.0, 250 mm �acl, 100 mmimidazole and 1 mm β-mercaptoethanol). all of the fractions were analysis by sds-pa�e method.8 results and discussion amplification and sub-cloning �ased on the structure analysis, β-xylosidase gene was successfully amplified from ptp510 and sub-cloned in plasmid phis1525/ e. coli dh10β. the recombinantβ. the recombinant the recombinant plasmid named psmx (fig.1). figure 1. the psmx map β-xylosidase expression test the expression of β-xylosidase in b. megaterium ms941 was assayed using methylumbelliferyl-β-dxyloside (mux) reagent. this work showed that the recombinant β-xylosidase was successfully expressed by recombinant b. megaterium ms941. among 12 colonies of assayed recombinant b. megaterium ms941, there were two colonies (bm1 and bm3) flourescented on the uv transluminator. it was been concluded that the recombinants b. megaterium ms941 bm1 and bm3 expressed the β-xylosidase extracellularly. the β-xylosidases catalyse the hydrolysis of glycosidic linkage of mux, and release xylose and umbelliferon (fig 2). figure 2. hydrolysis of mux by β-xylosidase the effect of xylose addition recombinant plasmid psmx contains xyla promoter (pxyla) and xylr repressor. the expression recombinant β-xylosidase in b. megaterium ms941 system was xyloseinducible. the addition of 0.5% xylose into ke culture medium at od578 = 0.332 could improve the growing of the recombinant b. megaterium ms941. the influence of xylose concentration and the time of addition into the culture medium toward the activity of β-xylosidase, were studied. the addition of 0.25–0.50% xylose into the culture medium at 2 h cultivation (od578 = + 0,1) increased the enzyme activity by 2.0–2.74 fold. the enzyme showed an activity of 0.0343 + 0.0022 unit/ ml at the absence of xylose. the optimum condition to induce the β-xylosidase activity was the addition of 0.50% xylose at 2 h cultivation (fig 3.). figure 3. the effect of xylose concentration and time of addition into culture medium toward β-xylosidase activity �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 25-29 effect of ph and temperature to enzyme activity the optimum activity of the recombinant enzyme was reached at 50° c and ph 6 (fig 4.). the crude extracts of β-xylosidase from recombinant b. megaterium ms941 the optimum activity at 50° c and ph 6. figure 4. the enzyme activity toward pnpx at different ph and temperature this condition was also reported by puspaningsih (2005) for recombinant β-xylosidase expressed in petxyl/ e. coli �l21 de3 [10]. however, the optimum temperature and ph optimum was different for the recombinant β-xylosidase expressed in ptp510/ e. coli dh5α. the enzyme had optimum activity at 70° c dan ph 5. on other hand, the β-xylosidase expressed in the origin strain, geobacillus thermoleovorans it-08, showed the optimum activity at 70° c and ph � this different optimum temperature and ph may be caused by the differential host and surrounding protein. the recombinant ptp510/ e. coli dh5α and its origin strain (geobacillus thermoleovorans it-08) expressed a xylanolytic enzyme, exo-xylanase,exo-xylanase, α-l-arabinofuranosidase, and β-xylosidase.9 wagschal et al. (2008) has synthezed and cloned the g. thermoleovorans it-08 β-xylosidase into pet29b/ e. coli bl21 (de3). the recombinant β-xylosidase (gbtxyl43a) showed the optimum activity at ph 5.0 thermal atability (t1/2) 970 min at 51,2° c.11 enzyme secretion the analysis of enzyme secretion showed that the recombinant b. megaterium ms941 was capable to secrete 75.56% of the expressed β-xylosidase enzyme, into the culture medium. the using of b. megaterium ms941 with a detectable extracellular protease deleted showed the improvement the secretion of dextrasucrase [2]. one of the factors in protein secretion is the protein folding. in the production of recombinant penicillin g amidase (pga) in b. megaterium, calcium ion was an important factor in protein folding and maturation. the presence of calcium ion impacted the secretion of pga protein in b. megaterium ms941. the addition of 2.5 mm cacl2 into the lb medium improved the pga secretion by 2.6 fold compared to the absence of cacl2 [6]. the preliminary study, showed that metal ions such as mg++, mn++, zn++, ca++ and fe++ impacted the activity of β-xylosidase from g. thermoleovorans it-08. the addition of 0.5–2.5 mm metal ions improved the β-xylosidase toward substrate pnpx. purification of recombinant enzyme the sds-page analysis showed that the protein purification revealed a pure β-xylosidase protein in fraction b2. the pure enzyme was showed by a single protein band + 58 kda on the electrophoregram (fig 5.) figure 5. sds-page analyzed of pure β-xylosidase b2 = fraction 2 of eluted enzyme by buffer b m= proteinm= protein penanda (16-213 kda, intron biotechnology) t h e p u r i f i c a t i o n o f β x y l o s i d a s e b y a f f i n i t y chromatography using agarose coloum containing ninta revealed a pure enzyme with specific activity of 10.06 units/mg protein, or 24.19 fold compared to the activity of supernatant. conclusion β-xylosidase encoding gene from g. thermoleovorans it-08 was succesfully expressed in phis1525/ b. megaterium ms941. acknowledgements this research was supported by directorat general of higher education departement of national education indonesia, throught “hibah bersaing project” 2006-2007. the authors would like to thank prof. dr. dieter jahn and laboratorium microbiology, technical university braunschweig, germany for the giving of plasmid phis1525 and strains (bacillus megaterium ms941 and e. coli dh10β). ��sumarsih, et al.: expression of β-xyloside encoding gene referrences 1. �ravman t, zolotnisky �, shulami s, �elakhov v, solomon d, �aasov t, shoham �, and shoham y. 2001.stereochemistry of family 52 glycosyl hydrolase β-xylosidase from bacilus stearothermophilus t-� is a retaining enzyme. febs letter, 495: 39–43. 2. hollmann r, malten m, �ien r, yang y, wang w, jahn d and deckwer w d. 200�. bacillus megaterium as a host for recombinant protein production. eng. life sci., �: 470–474. 3. malten m, biedendieck r, gamer m, drews a, stammen s, buchholz k, dijkhuizen l, and jahn d. 2006. a bacillus megaterium plasmid system for the production, export and one-step purification of affinity-tagged heterologous levansucrase from growth medium. applied and environmental microbiology, 72: 1677–1679. 4. jordan e, hust m, roth a, biedendieck r, schirrmann t, jahn d, and dübel s. 2007. production of recombinant antibody fragments in bacillus megaterium, microbial cell factories, 6: 2. 5. malten m, hollmann r, deckwer wd, jahn d. 2005. production and secretion of recombinant leuconostoc mesenteroides dextransucrase dsrs in bacillus megaterium. biotechnol bioeng, 89: 206–218. 6. yang y, biedendieck r, wang w, gamer m, malten m, jahn d, deckwer wd. 206. high yield recombinant penicillin g amidase production and export into the growth medium using bacillus megaterium, microbial cell factories, 5: 36. 7. barg h, malten m, jahn m, jahn d. 2005. protein and vitamin production in bacillus megaterium. microbial processes and products, 18: 205–224. 8. rohman a, oosterwijk n, kralj s, dijkhuizen l, dijkstra bw, and puspaningsih nnt. 2007. purification, crystallization and preliminary x-ray abalysis of a thermostable glycoside hydrolase family 43 β-xylosidase geobacillus thermoleovorans it-08. acta crystallographica, f63: 932–935. 9. puspaningsih,nnt. 2003. cloning of xylolytic gene in e.coli dh5α, jsps-short course program, september-november, mie university, japan. 10. puspaningsih,nnt.2005. cloning, over-expression, and application of β-xilosidase from recombination of local strain for degradation palm waste, research report of rut the ministry of reseach and technology indonesia. 11. wagschal k, heng c, lee cc, robertson gh, orts wj and wong dws. 2008. purification and characterization of a glycosidic hydrolase family 43 β-xilosidse from geobacillus thermoleovorans it-08, appl. biochem biotechnol; doi 10.1007/s12010-008 8362-5. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid vol 8 no 2 may-agustus 2020_newfromsarah.indd vol. 8 no. 2 may–august 2020 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ original article association between sepsis risk calculator and infection parameters for neonates with risk of early-onset sepsis trias kusuma sari1, irwanto1, risa etika1, mahendra tri arif sampurna1, ni made mertaniasih2 1department of pediatrics, dr. soetomo academic-teaching hospital, faculty of medicine, universitas airlangga, surabaya, east java, indonesia 2department of clinical microbiology, dr. soetomo academic-teaching hospital, faculty of medicine, universitas airlangga, surabaya, east java, indonesia received: 11th december 2018; revised: 24th december 2019; accepted: 23rd april 2020 abstract c-reactive protein (crp) is an acute-phase reactant protein that is primarily induced by the il-6 action during the acute phase of an infl ammatory or infectious process. the bacterial infection is a potent stimulus, leading to a rapid elevation of crp levels within hours while the cbc and symptom are often misleading and/or absent. american academy of pediatrics (aap) is recommended routine blood examination test complete blood count (cbc), c-reactive protein (crp), and blood culture along with empirical antibiotic in neonates with early onset sepsis risk (eos) risk even asymptomatic. the previous study is showed there were no correlation of crp and eos risk. this study aims to evaluate the crp and cbc profi le in neonate with risk of eos. methods of this study are using the sepsis risk calculator (src) to calculate the probability of neonatal early ons5et sepsis (eos) based on maternal risk and infant’s clinical presentation. neonates with ≥34 weeks of gestation who were started on antibiotic treatment after laboratory examination and blood culture were taken. eos risk estimation were compared including crp, leukocyte, and thrombocyte count. anova applied to distinguished laboratory examination between stratifi ed risk groups. the result is showed using 82 subjects who met the inclusion and exclusion criterias, the eos risk level was stratifi ed into green, yellow, and red group. the p-value of crp level, platelets, white blood cells were 0.35,0.54 and 0.48 where p-value was considered as signifi cant if < 0.05. the conclusion of this study is there were no correlation of crp level and eos risk keywords: sepsis risk calculator, infection parameter, risk of early onset sepsis, c-reactive protein, complete blood count abstrak c-reactive protein (crp) adalah suatu reaksi fase akut protein yang diinduksi oleh aktivasi dari il-6 selama fase akut dari infl amasi atau proses infeksi. crp adalah sebuah indikator yang penting pada pasien dengan risiko sepsis. infeksi bakterial adalah suatu stimulus yang berpotensi meningkatkan kadar crp dalam beberapa jam dimana darah lengkap dan klinis pasien seringkali tidak berubah secara signifi kan. american academy of paediatrics (aap) merekomendasikan pemeriksaan darah rutin antara lain darah lengkap, crp dan kultur darah bersamaan dengan pemberian antibiotik namun penelitian sebelumnya menemukan bahwa tidak didapatkan hubungan antara kadar crp dengan risiko sepsis. tujuan dari penelitian ini adalah untuk mengevaluasi kadar crp dan darah lengkap pada bayi baru lahir dengan risiko sepsis awitan dini. metode yang digunakan pada penelitian ini dengan menggunakan sepsis risk calculator (src) untuk menghitung probabilitas risiko sepsis awitan dini berdasarkan risiko ibu dan klinis pasien. bayi baru lahir dengan risiko sepsis awitan dini dengan usia gestasi ≥34 minggu dilakukan pengambilan darah lengkap, kultur darah dan crp sebelum pemberian antibiotic. laboratorium yang dibandingkan diantara ketiga kelompok risiko sepsis termasuk crp, leukosit, dan jumlah trombosit. anova diterapkan untuk menilai perbedaan antara kelompok risiko. hasil dari penelitian ini yang melibatkan 82 subjek yang memenuhi kriteria inklusi dan eksklusi, kelompok berdasarkan rekomendasi src dikelompokkan menjadi kelompok hijau, kuning, dan merah. nilai p dari crp, trombosit, sel darah putih adalah * corresponding author: triaskusumasari07@gmail.com 109trias kusuma sari, et al.: association between sepsis risk calculator and infection parameters copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 0,35,0,54 dan 0,48 di mana nilai p dianggap signifi kan jika <0,05. kesimpulan dari penelitian ini adalah tidak didapatkan hubungan antara risiko sepsis awitan dini dan crp. kata kunci: sepsis risk calculator, parameter infeksi, risiko sepsis awitan dini, c-reactive protein, darah lengkap how to cite: sari, trias kusuma., irwanto, irwanto., etika, risa., sampurna, mahendra tri arif., ni made mertaniasih. (2020). association between sepsis risk calculator and infection parameters for neonates with risk of early-onset sepsis. indonesian journal of tropical and infectious disease, 8(2), 1–8. introduction early onset sepsis (eos) can be related to microorganisms obtained from the mother where pathogenic colonization occurs in the perinatal period. with rupture of the amniotic membrane, microorganisms in the vaginal flora or other pathogenic bacteria can reach the amniotic fl uid and fetus.1 increasing risk of early onset of sepsis is in line with increasing of maternal temperature (≥ 37.5°c), rupture duration of the membranes (≥ 18 hours) along with gestational age (less than 34 weeks and more than 40 weeks of gestation) and also low birth weight.2 american academy of pediatrics (aap) recommends neonates from chorioamnionitis mother, to take laboratory examination and received antibiotic treatment even if the baby is asymptomatic.3 this cbc counts and c-reactive proteins (crps) recommendation can be used as guidance of antibiotic treatment decisions in well-appearing infants, and the potential utility of clinical examination to identify eos in at-risk infants.4 the use of antibiotics may cause several complications, longer length of stay on nicu, several pain procedures, lower rate of breastfeeding, changes of intestinal microbes, necrotizing enterocolitis and antibiotic resistance.5 sepsis risk calculator (src) is the interactive calculator produces the probability of early onset sepsis per 1000 babies by entering values for the specifi ed maternal risk factors along with the infant’s clinical presentation.6 src can be calculated in an infant born ≥ 34 weeks gestation. after entering the clinical presentation (wellappearing, equivocal, and clinical illness), src recommendation were assessed and considered in each group (green, yellow and red). the red group is the most vulnerable to suff er or have higher probability of eos. sepsis risk calculator (src) is originally introduced by kaiser permanente, and a validated tool which has been used and studied in many countries in predicting eos.7-8 kerste et al on 2016 study the implementation of src, there were reduced of antibiotics used 50%.9 even the src was promising tools, the comparison between each group has not been evaluated yet. the aim of this study is to evaluate the result of src on complete blood count and crp level in neonates with early onset of sepsis. materials and methods the study was approved by the ethical committee in health research of dr. soetomo academic-teaching hospital surabaya (625/ panke.kke/x/2017). this observational study with the cross-sectional design was conducted in nicu dr. soetomo academic-teaching hospital from november 2017 until april 2018, on newborns with gestational age ≥ 34 weeks who had eos risks and were born in this hospital within the study period. the subject was selected using a consecutive total sampling method and sample size was determined using a prospective-cohort calculation. routine laboratory examination comprising of cbc and crp was performed in all subjects. blood culture was only obtained in 42 subjects. the inclusion criteria of this study were newborns who had gestational age≥ 34 weeks, eos risks, appropriate gestational age (aga). subject are excluded if any of major congenital abnormality. 110 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 108–115 neonatal sepsis risk calculator: src can be accessed through https://neonatalsepsiscalculator. kaiserpermanente.org/ website or smartphone. the required information in src application are the incidence of eos was set as at 0.5/1000 live births according to cdc national incidence. group b streptococcus (gbs) status was set as unknown because gbs status was not routinely assessed in dr. soetomo academic-teaching hospital surabaya. the score will be shown as personal risk stratifi cation of eos for each newborn according to the clinical presentation (well-appearing, equivocal, and clinical illness) and eos risk level (green, yellow, dan red). with the src method, the baby will be grouped based on three groups, namely the green, yellow and red groups. where the green group is the group that does not need blood tests or antibiotics. in the yellow group, patients are recommended to do a blood culture examination without empirical antibiotics and it is recommended to monitor vital signs in the nicu. in patients who enter the red group, empirical antibiotics are recommended to be given immediately blood culture: as blood culture is a gold standard of bacteremia we also observed the characteristic of the patient and the result of cbc and crp between src group. the blood will be obtained through a peripheral vein (equal to 1 cc) as the gold standard diagnosis of eos. bact system was used as the microbial culture method and transferred into the mullerhinton agar to check antimicrobial susceptibility (ast) in vitex 2 compact. abnormal leukocytes: leukocyte abnormality values are less or more than normal values. less if <5,000 / mm3 and more if> 34,000 / mm3 in infants aged 0 days 1 week. blood counts measurement is using cellpack dcl from sysmex. blood count were taken before antibiotic admission, in the fi rst 12 hours of life. c-reactive protein (crp) is expressed in units of mg / l. normal crp value is <10 mg / l and abnormal if more than 10 mg/l. measurement of crp using flex® cartridge from sysmex. crp were taken before antibiotic admission, in the fi rst 12 hours of life. statistics data were analyzed using spss (statistical package for the social sciences). the value was presented as the mean + standard deviation (sd). normality test was tested using kolmogorovsmirnov test. if the data distribution was normal, t-paired test would be used and wilcoxon test would be performed if the distribution was not normal. chi-square test was utilized to assess the homogenity of the subjects according to the demographic characteristic and laboratory examination. results and discussion the population of this study is infants who had the risk of early onset sepsis (born to mothers who had a history of premature rupture of membranes for more than 18 hours, mothers with chorioamnionitis and had indications for intrapartum prophylactic antibiotics but inadequate). there were 82 patients were included in this study but only 42 patients that have blood culture results. characteristics of the subject that have blood culture were described in table 1. an inadequate intrapartum antibiotic is the most cause of risk of early onset of sepsis. inadequate intrapartum antibiotics are the higher percentage of eos risk in this study population. gestational age, maternal highest temperature, and prom have a nonlinear correlation with eos risk. 10previous study is table 1. characteristics of the subject characteristics (n=42) maternal chorioamnionitis n (%) rupture of the membrane ≥ 18 hours n (%) inadequate intrapartum antibiotic n (%) infant mean gestational age, (week) mean body weight, gram mean heart rate (time/minute) mean respiratory rate (time/minute) oxygen support (mechanical ventilation) 4 (9.5%) 22 (53%) 26 (62%) 36.7 ± 2.2 2523 ± 566.3 150 ± 155 46 ± 47.6 4 * data are in number and percentage. this is the characteristic of 42 patients, the most eos risk in this study was an inadequate intrapartum antibiotic. four patients with needed oxygen support more than room oxygen. 111trias kusuma sari, et al.: association between sepsis risk calculator and infection parameters copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 showed that an adequate antibiotic which used by mothers with premature rupture of membranes will reduce the risk of infection in neonates with relative risk [rr] = 0.67, 95% ci 0.52–0.85) 11 an inadequate antibiotic in patient with prom will increase the risk of eos with or 37.0 (95% ci 9.7–140.9). the mean of gestational age on the population are below than 37 weeks, this event increases the incident of early onset of sepsis with incidence 3.0 cases per 1000 birth life.12 sepsis risk calculator recommends the management of the patient with eos risk according to clinical presentation such as vital sign (tachycardia, tachypnea, and abnormality of body temperature), usage of mechanical ventilation used and vasoactive drugs. in this study, vital signs on the red group had abnormal mean heart rate (166.4(6.2)) and respiratory rate 64.3(4.38). cbc and crp analysis between src groups were described in table 2. the laboratories were complete blood count (cbc) values and crp in 82 patients, where all blood samples were taken 8 hours after birth 1 time and repeated if the clinical deterioration has occurred. in this study, there were no signifi cant diff erences as a statistic between the three groups of both cbc and crp values with mean values still in the normal range. similar to the previous study, acthen et al 2017 found eos risk was not correlated with changes in infection parameters. they found negative correlations between both eos risk, crp level and leukocyte count within 6 h of the start of antibiotics, as well as crp level between 6 and 24 h after start of treatment.13 crp production is a non-specifi c response to disease and cannot be used alone as a diagnostic test for septicaemia. the sensitivity and specifi city of crp (at 72 hours of admission) in diagnosis of acute neonatal sepsis were 76.92% and 53.49% respectively while it had a positive predictive value of 80% and negative predictive value of 48.94%. over all the diagnostic accuracy of crp in diagnosis of neonatal sepsis was 70.07%.14 p a t i e n t w i t h p o s i t i v e b l o o d c u l t u r e ’s characteristics, and laboratory results were described in table 3. this study is found that two patients with positive blood culture have a normal level of crp and one patient on the green group have abnormal crp level. contradiction with this result, a study in india (2016) have found the abnormality of crp in 92.95% of positive culture cases. there is also a statistically signifi cant relationship between positive blood cultures and crp. the crp test is positive at 64.34% of early onset sepsis and 35.66% of late onset sepsis.15 in the study by carola et al 2017, the management recommendations based on the eos calculator after clinical evaluation are presented including the 5 neonates with culture-proven sepsis and 142 neonates with culture-negative sepsis who were treated with antibiotics for ≥7 days. empiric antibiotics would have been recommended in 23.5% of the neonates in the cohort. blood culture only was recommended for 8.9% of the neonates. no empiric antibiotics or laboratory evaluations were recommended for the remaining 66.7%. in that cohort, 142 neonates were treated with prolonged antibiotics (7 days or more) for suspected culture-negative sepsis. table 2. cbc and crp analysis between src groups laboratory results groups green yellow red p complete blood count (mean ± sd) haemoglobin 16.7 (2.28) 15.8 (2.37) 15.49 (1.85) 0.19 white blood cell 18747 (6472) 15646 (4712) 14817 (7331) 0.54 platelet 242043 (59622.7) 252875 (70656) 250909 (87464) 0.48 c –reactive protein (mg/l) 2.73(8.6) 0.45(0.56) 8.36 (31.75) 0.35 * data are in number and p values are the results of anova. patients on the red group had higher crp level than green and yellow group. 112 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 108–115 all 5 neonates with positive blood cultures had abnormal cbc and crp values.8 the sensitivity and the specifi city of each crp was 92.96% and 50.39%.16 c-reactive protein has the best predictive value when measured within 24 to 48 hours after infection. in healthy individuals, the crp level is generally below 2 mg/l but can be up to 10 mg/l. there may be slight variations with age, sex, and race. it has a half-life of approximately 19 hours, begins to rise after 12–24 hours, and peaks within 2–3 days. normal crp values at two examinations (8 to 24 hours after birth and 24 hours later) were shown to have 99.7% negative predictive values and negative likelihood ratios of 0.15 which were proven to be sepsis.17 in the diagnosis of early-onset sepsis, previous studies are reported on widely diff ering sensitivities and specifi cities of crp ranging from 29 to 100% and from 6 to 100%, respectively. the delayed induction of the hepatic synthesis of crp during the infl ammatory response to infection lowers its sensitivity during the early phases of sepsis.18 from the results of complete blood count results, there were no significant differences between the three groups and in patients with positive blood cultures only one in three patients had a positive crp score. total white blood cells have a low positive predictive value (ppv) for sepsis while platelet counts are insensitive or specifi c for the diagnosis of sepsis and are not very helpful for monitoring response to therapy.19 the blood culture results of patients belong to green group, positive culture was found in 2 patients (micrococcus luteus and acinetobacter lwofi i), while in red group, 1 patient had positive blood culture (multistrain resistant aerococcus viridans). all patients with positive blood culture, table 3. patient with positive blood culture characteristics and laboratory results initial/culture result src groups bw/ga hb wbc plt crp n.s/micrococcus luteus green 2600/37 21.5 24370 360000 0.66 m/acinetobacterlwofi i green 2600/38 16 11680 190000 13.68 n.f/aerococcusviridans red 3600/41 16.2 23030 296000 2.56 * bw = body weight, ga = gestational age, hb = haemoglobin, wbc = white blood cell, plt = platelet, crp = c-reactive protein. had risk factor of meconial amniotic fl uid with inadequate antibiotics treatment. meconial amniotic fl uid could be sign of chorioamnionitis, which may enhanced the growth of bacteria in amniotic fl uid and caused both maternal and neonatal infections.20 among 42 patients there were 3 patients with positive blood cultures (7.5%). the results of blood cultures obtained were micrococcus luteus (1), acinetobacter lwofi i (1), aerococcus viridans (1) which had more than one class of antibiotic resistance. blood culture is the gold standard for the diagnosis of sepsis, and when the adequate volume is obtained, culture has excellent sensitivity even when the baby has a very low level of bacteremia. however, many culture results were found to be negative especially when the baby appeared ill or antibiotics were received before culture was obtained. based on the recommendation at least 1 ml of blood, either in 1 or divided into 2 0.5 ml cultures, obtained from infants with suspected sepsis before initiation of antibiotic therapy. however, sampling is limited by blood volume, especially in very low birth weight babies, who are at the highest risk for sepsis but have the lowest total blood volume. however, the sensitivity of blood culture decreased by 10% to 40% when 0.5 ml was inoculated compared to 1 ml. therefore, adequate volume for culture must be ensured.21 the sensitivity of blood culture is almost 100% when 1 ml is inoculated and the baby has bacteremia concentration of at least 4 colonyforming units (cfu) per milliliter. the optimal time for culture taking in bacteremic conditions is as soon as possible in fever episodes based on heat followed by bacteremia or endotoxaemia in one or two hours. in newborns often have a shorter threshold for the commencement of antibiotics, 113trias kusuma sari, et al.: association between sepsis risk calculator and infection parameters copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 which results in low opportunities for isolated organisms in blood culture. this coincides with the low specifi city of signs of sepsis in newborns compared to children and adults that contribute to a low positive rate in blood culture.22-23 two patients had gram-positive blood cultures and one patient with gram-negative on this study has normal blood count and have an inadequate antibiotic as a risk factor. newborns with mothers who received intrapartum antibiotic prophylaxis (iap) due to colonization of group b streptococci or chorioamnionitis, had a lower risk for early onset sepsis than infants with mothers who did not receive an adequate iap.24 the classic study focusing on escerchia coli infection, newborns were found to have bacteriemia with high colonies. however, more recent studies include pathogens other than eserchia coli in infants. a newborn with a risk of sepsis found that 68% of septic infants had bacteremia with a low colonization rate (≤ 10 colony-forming units (cfu) / ml) and 42% had a 1 cfu / ml colony count. calculation of low bacterial colonies will cause as much as 60% of culture to be false negative with a sample volume of 0.5 ml. many blood cultures can help improve these test results, but studies in the newborn period have shown confl icting results.25 on this study patient with red group, there were only 1 patient who had positive culture, these results diff er from those of other researchers where a clinical evaluation of sepsis compared with blood cultures in patients diagnosed with sepsis which is showed sensitivity (62.5% [95% confidence interval (ci): 35.4384.80%]}, specificity [63% (95% ci: 47.55-76.79%)], positive predictive value [37% (95% ci: 19.4057.63%)] and value negative predictive [82.8% (95% ci: 66.35 -93.44%)]. there were statistically signifi cant diff erences between blood culture results and clinical sepsis (p 0.014). 26 one patient with clinically ill appearance had the results of an aerococcus viridans culture that had multi-resistance to antibiotics also have a normal range of blood count and crp. patients with aerococcus viridans culture results in this study had risk factors for meconeal amniotic fl uid and inadequate antibiotic administration. the organisms most commonly involved in early-onset sepsis in term infants and fewer term infants are gbs and escherichia coli, which account for around 70%. additional pathogens are other streptococci (viridans group streptococci, streptococcus pneumoniae).27 aerococcus, abiotrophia which is a gram-positive-coccus bacteria catalase negative is a group of rarely isolated bacteria as opportunistic agents of infection, although this organism can become a pathogen in immunocompromised patients. aerococcus is an environmental isolate that can also be found in human skin. these bacteria have low virulence and only become opportunistic pathogens in immunocompromised hosts. infection that occurs is often in the form of tissue damage (for example a heart valve) or may be nosocomial and is associated with prolonged hospitalization, antibiotic therapy, invasive procedures and the presence of foreign objects. the association of infection with aerococcus viridans in humans found an almost signifi cant value in those with rupture of membranes during childbirth (p 0.073), prolonged rupture of membranes (p 0.058), those receiving intrapartum antibiotic prophylaxis (iap) (p 0.059) and women who smoked during pregnancy (p 0.062)28there were several limitations of this study. first, the number of samples was relatively small. second, the lack of gbs status data of the subject’s mother, since this test is not a routine in indonesia. hird, due to fi nancial restraints, blood culture test were only performed in half of the subjects. in this study, two neonates with green recommendation had positive blood culture. the src tools incidence input in this study follows cdc recommendation (0.5 ‰). the result is similar to retrospective cohort study of carola et al, in which, from 1159 infants born to mothers with clinical chorioamnionitis, the calculator would have missed 2 of 5 infants with cultureproven, early-onset sepsis. the src tools has been updated to enable the possibility of eos incidence, as high as 4‰. this update would enable to capture the two missed sepsis infants into the right risk and management category. 114 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 108–115 conclusions the results of complete blood count and crp levels between each group of src recommendation shown no signifi cant diff erences. the analysis indicate that crp level is uncorrelated with eos risk, thus clinical judgement is necessary to accompany laboratory examination. acknowledgement the author wishes to thank the other member arrend f bos as professor in visiting professor from beatrix children hospital, the netherland for giving a solution and advices to this study. we are appreciate the help of many doctors especially our head of pediatrics department, muhammad faizi, md conflict of interest there is no confl ict of interest of this paper. references 1. tita, a. t. and andrew w. diagnosis and management of clinical chorioamnionitis. clin perinatol. 2010; 37: 339–54. 2. puopolo km, draper d, wi s, newman tb, zupancic j, lieberman e, et al. estimating the probability of neonatal early-onset infection on the basis of maternal risk factors. j pediatr. 2011; 128: 1155–63. 3. polin ra, papile l-a, baley je, et al. management of neonates with suspected or proven early-onset bacterial sepsis. j pediatr. 2012; 129 4. joshi n, gupta a, allan j, et al. clinical monitoring of well-appearing infants born to mothers with chorioamnionitis; j pediatr. 2018; 141: 1–10 5. brecht, m., clerihew, l. and mcguire, w. prevention and treatment of invasive fungal infection in very low birthweight infants. arch dis child fetal neonatal. 2009; 94: 65–9. 6. escobar, g. j., puopolo, k. m. and wi, s. stratifi cation of risk of early-onset sepsis in newborns ≥ 34 weeks’ gestation. j pediatr. 2014; 133: 30–6. 7. warren s, garcia m, hankins c. impact of neonatal early-onset sepsis calculator on antibiotic use within two tertiary healthcare centers. nat publ gr [internet]. 2016; (october): 1–4. available from: http://dx.doi. org/10.1038/jp.2016.236 8. carola d, vasconcellos m, sloane a, mcelwee d, edwards c, greenspan j, et al. utility of early-onset sepsis risk calculator for neonates born to mothers with chorioamnionitis. j pediatr. 2017; 11: 1–6 9. kerste, m., corver, j., sonnevelt, m. c., et al. application of sepsis calculator in refer to author guideline newborns with suspected infection. j matern fetal neonatal med. 2016; 29: 3860–5. 10. puopolo k, draper d, wi, s, dan newman, et al. estimating the probability of neonatal early-onset infection on the basis of maternal risk factors. j pediatr. 2011; 128: 1155–63. 11. kenyon s, boulvain m, neilson j. antibiotics for preterm rupture of membranes. cochrane database syst rev. (abstrak). 2010 12. weston j, pondo t dan lewis m, et al. the burden of invasive early-onset neonatal sepsis in the united states, 2005-2008. pediatr infect dis j. 2011; 30: 937–41. 13. achten n, zonneveld r, tromp e, plötz f. association between sepsis calculator and infection parameters for newborns with suspected early onset sepsis, j clin neonatol. 2017; 6: 159–162 14. hisamuddin e, hisam a, wahid s, raza g. validity of c-reactive protein (crp) for diagnosis of neonatal sepsis. pak j med sci. 2015; 31(3): 527–531. 15. bhatia s, verma c, tomar b, et al. correlation of crp and blood culture in evaluation of neonatal sepsis. ijbamr. 2016; 6: 663–70 16. kamble r and rajesh ovhal r. bacteriological profi le of neonatal septicemia. int.j.curr.microbiol app.sci. 2015; 2: 172–182 17. meem, m., modak, j. k., mortuza, r., morshed, m., islam, m. s. dan saha, s. k. biomarkers for diagnosis of neonatal infections: a systematic analysis of their potential as a point-of-care diagnostics. j glob health. 2011; 1: 201–9. 18. hofer n, zacharias e, müller w, resch b. an update on the use of c-reactive protein in early-onset neonatal sepsis: current insights and new tasks. j clin neonatol. 2012; 102: 25–36 19. manzoni p, m. m., galletto p, gastaldo l, gallo e, agriesti g, dan farina d. is thrombocytopenia suggestive of organism-specifi c response in neonatal sepsis? j pediatr. 2011; 51: 206–10. 20. siriwachirachai t, sangkomkamhang us, lumbiganon p, laopaiboon m. antibiotics for meconium-stained amniotic fl uid in labour for preventing maternal and neonatal infections. cochrane database syst rev. (abstrak). 2014 21. nora h, eva z, wilhelm m, and bernhard r. an update on the use of c-reactive protein in early-onset neonatal sepsis: current insights and new tasks. j clin neonatol. 2012; vol 102: 25–36. 22. derek s. wheeler, m.d., hector r. wong, m.d., and basilia zingarelli. pediatric sepsis – part i: “children are not small adults. open infl amm j. 2011; 4: 4–15 115trias kusuma sari, et al.: association between sepsis risk calculator and infection parameters copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 23. james l. wyn. defi ning neonatal sepsis. curr opin pediatr. 2016; 28(2): 135–140. 24. jonathan m. wortham, nellie i, stephanie j, schrag, et al. chorioamnionitis and culture-confi rmed, earlyonset neonatal infections. j pediatr. 2016; 137(1): 1–11 25. alonso t dan theresa o. challenges in the diagnosis and management of neonatal sepsis. j trop pediatr. 2015; 61: 1–13 26. somaia e, mervat e, reem h, doaa a, qassem, dan gameel. the role of 16s rrna gene sequencing in confi rmation of suspected neonatal sepsis. j trop pediatr. 2016; 62: 75–80 27. simonsen, k. a., anderson-berry, a. l., delair, s. f. dan davies, h. d, 2014. early-onset neonatal sepsis. clin microbiol rev, 27: 21–47. 28. rasmussen. aerococcus: an increasingly acknowledged human pathogen. clin microbiol infect. 2015; 22: 22–7 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid vol 3 no 1 jan-maret 2012.indd 5 vol. 3. no. 1 january–march 2012 the changing clinical performance of dengue virus infection in the year 2009 soegeng soegijanto1,2,3, helen susilowati3, kris cahyo mulyanto3, eryk hendrianto3 and atsushi yamanaka4 1 department of child health dr. soetomo hospital surabaya 2 medical faculty of airlangga university surabaya 3 institute of tropical disease center – airlangga university 4 kobe university graduate school of medicine abstract background: dengue (den) virus, the most important arthropod-borne human pathogen, represents a serious public health threat. den virus is transmitted to humans by the bite of the domestic mosquito, aedes aegypti, and circulates in nature as four distinct serological types den-1 to 4). the aim of study: to identify dengue virus serotype i which showed mild clinical performance in five years before and afterward showed severe clinical performance. material and method: prospective and analytic observational study had been done in dr. soetomo hospital and the ethical clearance was conduct on january 01, 2009. the population of this research is all cases of dengue virus infection. diagnosis were done based on who 1997. all of these cases were examined for igm & igg anti dengue virus and then were followed by pcr examination to identify dengue virus serotype. result and discussion: den 2 was predominant virus serotype with produced a spectrum clinical illness from asymptomatic, mild illness to classic dengue fever (df) to the most severe form of illness (dhf). but den 1 usually showed mild illness. helen at al (2009–2010) epidemiologic study of dengue virus infection in health centre surabaya and mother and child health soerya sidoarjo found many cases of dengue hemorrhagic fever were caused by den 1 genotype iv. amor (2009) study in dr. soetomo hospital found den 1 showed severe clinical performance of primary dengue virus infection as dengue shock syndrome two cases and one unusual case. conclusion: the epidemiologic study of dengue virus infection in surabaya and sidoarjo; in the year 2009 found changing predominant dengue virus serotype from dengue virus ii to dengue virus 1 genotype iv which showed a severe clinical performance coincident with primary infection. key words: changing clinical performance, dengue infection. introduction dengue (den) virus, the most important arthropodborne human pathogen, represents a serious public health threat. den virus is transmitted to humans by the bite of the domestic mosquito, aedes aegypti, and circulates in nature as four distinct serological types den-1 to 4. den virus has been recognized in over 100 countries, and 2.5 billion people live in areas where den virus is endemic.16 dengue, an emerging arboviral and arthropod borne disease, is a major cause of morbidity throughout the tropical and sub-tropical regions of the world.1 dengue virus (dv) infection with any 1 of 4 serotypes produces a spectrum of clinical illness, ranging from an asymptomatic or mild febrile illness to classic dengue fever (df) to the most severe form of illness, dengue hemorrhagic fever (dhf). dhf is characterized by plasma leakage and a hemorrhagic diathesis near the time of differences, typically after 5 days of fever.2 in severe dhf, morbidity and mortality are the result of hypotension and shock, at times accompanied by severe coagulation abnormalities and bleeding. since early hospitalization and careful supportive care can reduce the case-fatality rate of dhf, the rapid identification of patients at risk for developing dhf is desirable in regions where dv is endemic. dengue hemorrhagic fever is one of the important health problem in indonesia, although the mortality rate has been decreased but many dengue shock syndrome cases is very difficult to be solving handled. natural course of dengue virus infection is very difficult to predict of the earlier time research report 6 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 5−9 of severity occur; it is may be due to the new variant of dengue virus that infect a child could be severe and can not be identified earlier. previous study show that some of den 2 and den 3 virus cases could show a clinical performance of severe dengue virus infection such as dengue shock syndrome. based on halstead hypothesis, the severe dengue virus infection could be correlated with secondary infection. the infant cases show a severe clinical manifestation. in thailand and cuba, many cases of dengue virus infection were identified as secondary infection and some of them showed dengue shock syndrome, but this case did not found in other countries. moren (1980) found that the differences of growing dengue virus in monocyte could be a predictor of severity or mild cases for dengue virus infection. the first outbreak of dhf in indonesia was reported in java island in 1968, all types (den vi-4) were isolated from patient in jakarta in 1973–1974. indonesia has approximately 100.000 annual dengue cases. since then some outbreak in other cities and island were reported and the type of circulating den virus varies in each province and island. based on setiati te et al (2006), recently predominant type as follow: jakarta den v3; palembang den v3; bandung den v2; manado den v1; merauke den v3; yogyakarta den v3. in the year 2009, dengue virus team of institute tropical disease had done epidemiologic study in surabaya. material & method prospective and analytic observational study had been done in dr. soetomo hospital and the ethical clearance was conduct on january 01, 2009. the population of this research is all cases of dengue virus infection that in tropical ward of children, diagnosis were done based on who 1997. cases of dengue virus infection were collected & involving in research based on inform concern. all of these cases were examined for igm & igg anti dengue virus and then followed by pcr examination to identify dengue virus serotype. blood examination should be done everyday. x-ray examination were also done base on clinical performance of pleural effusion & ascites. data of all cases dengue virus infection should be analyze using method of kruskal walles & mann whitney and regression logistic multivariet. result & analysis 150 cases of primary and secondary of dengue virus infection were studied. dengue virus was isolated from vero cell and 120 samples have positive cpe. 70 samples were found as serotype by doing rt-pcr examination. serotype den 1: there ware only 3 cases (see table 3) consisted of 2 cases had age 1-4 years and 1 had age 5–14 years. they showed a severe clinical performance as dss 2 cases and 1 case as unusual case (see table 1). table 1. distribution of serotype and clinical performance of dengue virus infection clinical performance & diagnostic serotype df dhf dss unusual total den 1 0 0 2 1 3 den 2 30 26 7 2 65 den 3 1 0 1 0 2 den 4 0 0 0 0 0 total 31 26 10 3 70 kruskal-wallis: p = 0,03* * = significant (p < 0,05) table 2. distribution of clinical performance of dengue virus infection clinical performance & diagnostic type of infection df dhf dss unusual total primary 16 7 1* 2 26 secondary 15 19 9 1 44 total 31 26 10 3 70 mann-whitney; p = 0,035* * = significant (p < 0,05) serotype den 1 was usually mild case but in this study 1 case showed a severe clinical performance as dss and identified as primary infection (see table2). table 3. distribution of primary and secondary infection and serotype that were correlated with clinical performance of dengue virus infection clinical performance & diagnostic type of infection df dhf dss unusual total primary den 1 0 0 1* 0 1 den 2 16 7 0 2 25 den 3 0 0 0 0 0 den 4 0 0 0 0 0 total 16 7 1 2 26 secondary den 1 0 0 1 1 2 den 2 14 19 7 0 40 den 3 1 0 1 0 2 den 4 0 0 0 0 0 total 15 13 9 1 44 7soegijanto et al.: the changing clinical performance of dengue virus the second case of den 1 was identified as secondary dengue virus infection and the third case was an unusual case which showed secondary of dengue virus infection (see table 3). based on yamanaka this serotype den 1 might be have genotype iv or mention as den 1 genotype iv. discussion aryati (2005), fedik (2007), had done an epidemiologic study of dengue hemorrhagic fever cases this in surabaya, found that den virus 2 was a predominant types. the study in health center of surabaya den v2 was predominant in surabaya (see table 4). all of them showed clinical manifestation of dengue virus infection with produces a spectrum of clinical illness, ranging from an asymptomatic or mild febrile illness to classic dengue fever (df) to the most severe form of illness as dengue hemorrhagic fever (dhf). dhf is characterized by plasma leakage and a hemorrhagic diathesis near the time of differences, typically after 5 days of fever (2). most of them showed severe dengue hemorrhagic fever as the result of hypotension and shock, at the times accompanied by severe coagulation abnormalities and bleeding. since early hospitalization and careful supportive care can reduce the case-fatality rate of dhf, the rapid identification of patients at risk for developing dhf is desirable in regions where dv is endemic. on the year 2007 13% (7 cases) showed very severe clinical performance of dengue virus infection due to combining virus of den 2 and den 3 infected in one host of dengue hemorrhagic fever case that could induce viremia. but based on epidemiologic study in surabaya & sidoarjo on 2009 and 201027 found many cases of dengue hemorrhagic fever were caused by virus den v1 (see table 5). the clinical performance of cases dengue virus infection who came in health center of surabaya in year 2008 with 2169 cases showed clinical performance of dengue fever 87% and 10% dengue hemorrhagic fever and dengue shock syndrome and 3% unusual manifestation. in the year 2009 with 2268 cases dengue virus infection showed clinical performance of dengue fever 71.5% and dengue hemorrhagic fever and dengue shock syndrome 28% and unusual cases of dengue virus infection 0.5% (see table 6). this finding supported study of mosquito bites to some peoples live surrounding dengue hemorrhagic cases who had been admitted in hospital (see table 7). table 4. prevelance dengue virus infection based on serotype virus that was found in surabaya on the year 2003–2005, 2007, 2008. year den v1 den v2 den v3 den v4 d2+d3 total 2003–2005 0 20 (80%) 4 (16%) 1 (4%) 25 2007 0 46 (87%) 0 0 13% 53 2008 0 20 (100%) 0 0 20 table 5. prevalence dengue virus infection in surabaya & sidoarjo in 2009–2010. year den v1 den v2 den v3 den v4 total 2009 79 (87%) 6 (6.5%) 0 6 (6.5%) 91 2010 (jan–feb) 27 (100%) 0 0 0 27 table 6. clinical performance of dengue virus infection in health centre of surabaya year total patients dengue fever dhf + dss unusual 2008 2169 1890 (87%) 216 (10%) 63 (3%) 2009 2268 1601 (71,5%) 656 (28%) 11 (0,5%) table 7. virus isolation from mosquito mosquito 2008 total pool cpe immune staining pcr sequencing ae.aegypti 271 12 2 dengue d2 d2 cx.quinquefasciatus 336 10 4 dengue d2 d2 cx.tritaeniorhynchus 131 3 – – – – cx.vishnui 71 1 – – – – cx.pseudovishnui 42 1 1 dengue d2 d2 8 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 5−9 table 7 supported previous epidemiologic study that found den v2 as predominant types in the year 2008 but table 8 supported epidemiologic study in the year 2009 found den v1 as predominant types. the study in dr. soetomo hospital since january 1, 2009 as followed den 1 showed clinical performance of dengue shock syndrome 2 cases and unusual case with total 3 cases, den 2 were found clinical performance of 30 cases dengue fever, 26 dengue hemorrhagic fever 7 dengue shock syndrome and 2 unusual cases, with total 65 cases. den 3 were found clinical performance of dengue fever 1 case dengue shock syndrome 1 case, with total 2 cases. den 4 virus was not found. the differences of result were found due to the differences of population of study. but den v1 were always found in this study.27 virus isolation from mosquito bites showed den v1 has been isolated and identified on den 1 genotype iv, it was new variant virus that correlated with phylogenetic dengue virus came from beijing which had severe clinical performance of dengue virus infection. figure 1. phylogenetic dengue virus in the world in the year 2009 we have many experience to care severe performance of dengue virus infection with unusual manifestation that could not followed who criteria 1997. more cases showed criteria for severe dengue virus infection, as followed: severe plasma leakage (leading to: shock/dss, fluid accumulation with respiratory distress), severe bleeding (as evaluated by clinician), severe organ involvement (liver: ast or alt > = 1000, cns: impaired consciousness, heart and other organ). therefore for managing the unusual dengue virus infection we should followed new who criteria diagnosis and classification of cases as followed. during three decades, the world health organization (who) has recognized and recommended the classification of dengue in: dengue fever (df) and dengue hemorrhagic fever (dhf) with or without dengue shock syndrome (dss).6 however in some severe cases the clinical manifestations sometimes doesn't fit to these definition and classification. in this who recommendation clinical manifestation in df are mild form than dhf/dss, but in this case df with severe hemorrhagic manifestation and that may be life threatening. dengue can also express itself by means of the so-called "atypical" forms or unusual manifestation.1,5 these unusual clinical manifestations may delay recognition of potentially severe disease. lately, several publications that appeared worldwide emphasize the need to revise the classification of severe dengue.1 one of the revised dengue classification proposed by denco (dengue control) has been applied and studied in several countries in asia and latin america with good result.1,7 the denco study concluded that 18 to 40% of the cases could not be classified by means of the current who classification, and over 15% of unusual cases with shock could not be classified as severe cases of dengue either, since they did comply with some of the criteria to be regarded as a case of dhf/dss.1,7 the pathogenesis of bleeding in df is poorly understood. thrombocytopenia may enhance the risk, but the primary cause of bleeding is unknown. limited data suggest that activation of coagulation and fibrinolysis play role in the pathogenesis (srichaikul). an imbalance in the regulation of coagulation and fibrinolysis, as in disseminated intravascular coagulation syndrome (dic), in conjunction with the characteristic thrombocytopenia may contribute to the bleeding tendency in df. in the year 2009, the study found that den v1 genotype iv showed a severe clinical performance. of a primary dengue virus infection. this study supported to gubler table 8. virus isolation from mosquito mosquito 2009–2010 total pool cpe immune staining pcr sequencing ae.aegypti 1784 45 13 dengue d1 d1 cx.quinquefasciatus 74 4 1 dengue d1 figure 2. suggested dengue case classification and levels of severity. dengue guidelines for diagnosis, treatment, prevention, and control. world health organization, unicef, undp. new edition 2009 9soegijanto et al.: the changing clinical performance of dengue virus hypothesis which gave information that a new virulent variant den v1 can cause a severe clinical performance of dengue virus infection. summary the epidemiologic study of dengue virus infection in surabaya. in the year 2009 found a changing predominant dengue virus from dengue virus 2 to dengue virus 1 genotype 4 which showed a severe clinical performance coincident with primary infection. referrences 1. torres em. dengue. estudos avancados 2008; 22(64): 22–52. 2. guzman mg, kouri g. dengue diagnosis, advances and challenges. int j infect dis 2004; 8: 69–80. 3. world health organization. dengue: guidelines of diagnosis, treatment, prevention and control-new edition. geneva: who, 2009. 4. malavige gn, fernando s, fernando dj, et al. dengue viral infections. postgrad med j 2004; 80: 588–601. 5. martinez e. dengue. in: gonzalez-saldana n. et al. (ed.) infectologia clinica pediatrica. mexico, df: editorial trillas, 1997: p. 589–95. 6. world health organization. dengue haemorrhagic fever: diagnosis, treatment and control. 2nd edition. geneva: who, 1997: p. 17–27. 7. wills b. janisch t. 2007. denco-current state of play. available from http://conganat.sld.cu/instituciones/ipk/memorias/dengue2007/ conf/wills-b2.pdf. [cited on november 28th 2008.] 8. seneviratnea sl, malavige gn, de silva hj. pathogenesis of liver involvement during dengue viral infections. transactions of the royal society of tropical medicine and hygiene 2006; 100: 608–14. 9. abdul wahid sfs, sanusi s, zawawi mm, azman ali r. comparison of the pattern of liver involvement in dengue hemorrhagic fever with classic dengue fever. southeast asian j trop med public health 2000; 31: 259–63. 10. de souza l, carneiro h, filho j, de sauza t, cortez v, neto c, et al. hepatitis in dengue shock syndrome. braz j infect dis 2002; 6: 322–7. 11. subramanian v, shenoy s, joseph a. dengue hemorrhagic fever and fulminant hepatic failure. digest dis sci 2005; 50: 1146–7. 12. lawn s, tilley r, lloyd g, tolley h, newman p, rice p. dengue hemorrhagic fever with fulminant hepatic failure in an immigrant returning to bangladesh. clin infect dis 2003; 37: 1–4. 13. lam sk. dengue infections with central nervous system manifestations. neurol j southeast asia 1996; 1: 3–6. 14. wali jp, biswas a, chandra s, et al. cardiac involvement in dengue hemorrhagic fever. int j cardiol 1998; 64: 31–6. 15. promphan w, sopontammarak s, preukprasert p, kajornwattanakul w, kongpattanayothin a. dengue myocarditis. southeast asia j trop med public health 2004; 35(3): 611–3. 16. lateef a, fisher da, tambyah pa. dengue and relative bradycardia. emerging infectious diseases 2007; 13(4): 650–1. 17. obeysekara i, yvette h. arbovirus heart disease. myocarditis and cardiomyopathy following gangue fever and chickengunya fever. a follow up study. am heart j 1973; 85: 186–94. 18. arif sm, ahmed h, khokon kz, azad ak, faiz ma. dengue haemorrhagic fever with bradycardia. j medicine 2009; 10: 36–7. 19. nair vr, unnikrishnan d, satish b, sahadulla mi. acute renal failure in dengue fever the absence of bleeding manifestations or shock. infect dis clin pract 2005; 13: 142–143. 20. chaivisuth a. renal involvement in dengue infection. thai pediatric journal 2005; 12(3): 261. 21. vasanwala ff, puvanendran r, ng jm, suhail sm. two cases of self-limiting nephropathies secondary to dengue haemorrhagic fever. singapore med j 2009; 50(7): e253–5. 22. batra p, saha a, vilhekar k, chaturvedi p, thampi s. dengue fever in children. j mgmis 2006; 11: 13–8. 23. gubler d. dengue and dengue hemorrhagic fever. clin microbiol rev 1998; 11: 480–96. 24. shu p, huang j. current advances in dengue diagnosis. clin diag lab immunol 2004; 11: 642–50. 25. malavige g, fernando s, fernando d, seneviratne s. dengue viral infection. postgrad med j 2004; 80: 588–601. 26. lei h, yeh t, liu h, lin y, chen s, liu c. imunopathogenesis of dengue virus infection. j biomed sci 2001; 8: 377–88. 27. atsushi yamanaka, kris c mulyatno, helen susilowati, eryk hendrianto, amor p ginting, dian d sary, fedik a rantam, soegeng soegijanto, eiji konishi. displacement of the predominant dengue virus from type 2 to type 1 with a sub seguence genotype shift from iv to i in surabaya, indonesia 2008–2010. plos one 2011: 6: 1–8. vol. 8 ● no. 1 january-april 2020 e issn 2356–0991 p issn 2085–1103 e-journal.unair.ac.id/index.php/ijtid indexed by: clinical and hemoglobin profile of malaria patients in karitas hospital, southwest sumba district, indonesia during 2017 effect of patient's personal character on prevention of transmission of pulmonary tb polymerase chain reaction and serology test to detect rubella virus in congenital rubella syndrome patients with hearing loss sensitivity of erythromycin against toxigenic strain of corynebacterium diphtheriae cp-1 levels and atypical lymphocytes in early fever of dengue virus infection with non-structural protein 1 (ns-1) antigen test in dr. darsono hospital, pacitan prognostic factors of severe dengue infections in children a survey for zoonotic and other gastrointestinal parasites in pig in bali province, indonesia relationship of non structural antigen 1 (ns1) to clinical signs, symptoms and routine blood examination dengue suspected e issn 2356 0991 p issn 2085 1103volume 8 number 1 january–april 2020 editorial team of indonesian journal of tropical and infectious disease editor in chief prihartini widiyanti, indonesia editorial board mark alan graber, united states kazufumi shimizu, japan masanori kameoka, japan hak hotta, japan fumihiko kawamoto, japan nasronudin nasronudin, indonesia maria inge lusida, indonesia puruhito puruhito, indonesia retno handajani, indonesia kuntaman kuntaman, indonesia soegeng soegijanto, indonesia bambang prajogo, indonesia ni nyoman sri budayanti, indonesia achmad fuad hafid, indonesia tri wibawa, indonesia irwanto irwanto, indonesia marcellino rudyanto, indonesia yulis setiya dewi, indonesia laura navika yamani, indonesia secretariat zakaria pamoengkas nur diana fajriyah secretariat office publishing unit of indonesian journal of tropical and infectious disease, institute of tropical disease universitas airlangga kampus c, jalan mulyorejo surabaya 60115, jawa timur – indonesia. phone 62-31-5992445-46 faximile 62-31-5992445 e-mail: ijtid@itd.unair.ac.id homepage: e-journal.unair.ac.id/index.php/ijtid e issn 2356 0991 p issn 2085 1103 contents page printed by: universitas airlangga press. (rk 023/01.20/aup). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, fax. (031) 5992248. e-mail: adm@aup.unair.ac.id volume 8 number 1 january–april 2020 1. clinical and hemoglobin profile of malaria patients in karitas hospital, southwest sumba district, indonesia during 2017 alvin johan, audrey natalia, william djauhari, rambu farah effendi ................................... 1–8 2. effect of patient's personal character on prevention of transmission of pulmonary tb herdianti, entianopa, sugiarto ...................................................................................................... 9–15 3. polymerase chain reaction and serology test to detect rubella virus in congenital rubella syndrome patients with hearing loss sabrina izzattisselim, nyilo purnami ............................................................................................. 16–23 4. sensitivity of erythromycin against toxigenic strain of corynebacterium diphtheriae alif mutahhar, dwiyanti puspitasari, dominicus husada, leny kartina, parwati setiono basuki, ismoedijanto moedjito ........................................................................... 24–28 5. mcp-1 levels and atypical lymphocytes in early fever of dengue virus infection with nonstructural protein 1 (ns-1) antigen test in dr. darsono hospital, pacitan indah agustinaningrum, jusak nugraha, hartono kahar .......................................................... 29–42 6. prognostic factors of severe dengue infections in children senja baiduri,dominicus husada, dwiyanti puspitasari, leny kartina, parwati setiono basuki, ismoedijanto ............................................................................................ 43–53 7. a survey for zoonotic and other gastrointestinal parasites in pig in bali province, indonesia ni komang aprilina widisuputri, lucia tri suwanti, hani plumeriastuti ............................... 54–65 8. relationship of non structural antigen 1 (ns1) to clinical signs, symptoms and routine blood examination dengue suspected acivrida mega charisma ................................................................................................................. 66–76 indonesian journal of tropical and infectious disease this journal is a peer-reviewed journal established to promote the recognition of emerging and reemerging diseases spesifically in indonesia, south east asia, other tropical countries and around the world, and to improve the understanding of factors involved in disease emergence, prevention, and elimination. the journal is intended for scientists, clinicians and professionals in infectious diseases and related sciences. we welcome contributions from infectious disease specialists in academia, industry, clinical practice, public health and pharmacy, as well as from specialists in economics, social sciences and other disciplines. for information on manuscript categories and suitability of proposed articles see below and visit e-journal. unair.ac.id/index.php/ijtid/ index. i. instructions to authors article structure 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avoiding a detailed literature survey or a summary of the results. material and method provide sufficient detail to allow the work to be reproduced. methods already published should be indicated by a reference: only relevant modifications should be described. result and discussion results should be clear and concise results presented in the tables and figures should not be repeated unnecessarily in the text. result should be presented without interpretation and followed by the discussion section which should provide an interpretation of the results in relation to findings of other investigators. discussion should explore the significance of the results of the work, not repeat them. a combined results and discussion section is often appropriate. avoid extensive citations and discussion of published literature. conclusion conclusions should be stated clearly.the main conclusions/summary of the study may be presented in a short conclusions section, which may stand alone or form a subsection of results and discussion section. conclusion section is for research report/original article and summary is for literature review/review article. acknowledgement all acknowledgements including financial support should be mentioned under this heading. list here those individuals who provided help during the research (e.g., providing language help, writing assistance or proof reading the article, etc.). references please ensure that every reference cited in the text is also present in the reference list (and vice versa). minimum 15 references for research report/original article and 30 references for review. references wrote on vancouver (superscript) style. in the vancouver style, citations within the text of the essay/paper are identified by arabic numbers in superscript. this applies to references in text, tables and figures. the vancouver (superscript) system assigns a number to each reference as it is cited. a number must be used even if the author(s) is named in the sentence/text. e.g. smith10 has argued that... the original number assigned to the reference is reused each time the reference is cited in the text, regardless of its previous position in the text. when multiple guide for author references are cited at a given place in the text, use a hyphen to join the first and last numbers that are inclusive. use commas (without spaces) to separate non-inclusive numbers in a multiple citation e.g. 2,3,4,5,7,10 is abbreviated to… the placement of citation numbers within text should be carefully considered e.g. a particular reference may be relevant to only part of a sentence. as a general rule, reference numbers should be placed outside full stops and commas and inside colons and semicolons, however, this may vary according to the requirements of a particular journal. examples – there have been efforts to replace mouse inoculation testing with in vitro tests, such as enzyme linked immunosorbent assays57, 60 or polymerase chain reaction20-22 but these remain experimental. moir and jessel maintain “that the sexes are interchangeable”.1 tables tables should be embedded in page. provide footnotes and other information (e.g., source/copyright data, explanation of boldface). tables should be no wider than 17 cm. condence or divide larger tables. please submit tables as editable text and not as images. number tables consecutively in accordance with their appearance in the text and place any table notes below the table body. be sparing in the use of tables and ensure that the data presented in them do not duplicate results described elsewhere in the article. please avoid using vertical rules and shading in table cells. figures provide figures embedded in page. figures should be drawn professionally. photographs should be sharp (contrast). provide footnotes and other information (e.g., source/copyright data, explanation of boldface) in figure legend. submit image files (e.g., electromicrograph) without text content as high-resolution (300 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ijtid@itd.unair.ac.id ijtid vol 6 no 2 mei-agustus 2016_edit.indd 46 vol. 6. no. 2 mei–agustus 2016 a nosocomial infection manifested as erysipelas in pemphigus foliaceus patient under intravenous dexamethasone treatment a nosocomial infection manifested as erysipelas achmad yudha pranata1a, hendra gunawan1, endang sutedja1, oki suwarsa1, hartati purbo dharmaji1 1 departemen ilmu kesehatan kulit da kelamin universitas padjadjaran/rs dr. hasan sadikin bandung a corresponding author: achmadyudhapranata@gmail.com abstract introduction: puncture wound in diagnostic interventions permits the entry of bacteria into the skin or soft tissue, thus precipitating nosocomial infection, such as erysipelas. there are other risk factors of nosocomial infections including old age, immunosuppressive drugs, and underlying diseases. pemphigus foliaceus (pf) is an autoimmune disease with corticosteroid treatment as the mainstay therapy, which could cause immunosuppression and predispose patients to infection. the objective of this paper was to report erysipelas as one of the manifestations of nosocomial infection in patients under immunosuppressive therapy. case: a case of erysipelas acquired on the 9th day of hospitalization in a pf patient underwent intravenous dexamethasone injection, with history of puncture wounds on the previous day on the site of erysipelas was reported. the clinical findings of erysipelas were well defined, painful erythema and edema that felt firm and warm on palpation, with blisters and pustules on top. gram staining from the pustules and blisters fluid revealed gram (+) cocci. patient was given 2 grams intravenous ceftriaxone for 7 days and saline wet compress. improvement on the erysipelas was seen the day after ceftriaxone injection. the patient was discharged after 12 days of hospitalization with improvement both on the pf and the erysipelas. on the next visit 7 days later, the erysipelas lesion disappeared. conclusion: puncture wound and immunosuppresive treatment are the factors that could cause erysipelas as a nosocomial infection, and an appropriate treatment of the infection would decrease the functional disability of the patient. key words: erysipelas, nosocomial infection, immunosuppresive, pemphigus foliaceous, ceftriaxone abstrak pendahuluan: luka suntikan pada intervensi diagnostik memungkinkan masuknya bakteri ke dalam kulit atau jaringan lunak, sehingga dapat mencetuskan infeksi nosokomial, seperti erisipelas. faktor risiko lain untuk terjadinya infeksi nosokomial antara lain usia tua, penggunaan obat imunosupresif, dan penyakit yang mendasari. pemfigus foliaseus (pf) adalah penyakit autoimun dengan pengobatan utama kortikosteroid, yang dapat menyebabkan imunosupresi dan membuat pasien rentan terhadap infeksi. tujuan tulisan ini adalah untuk melaporkan erisipelas sebagai salah satu manifestasi infeksi nosokomial pada pasien yang mendapatkan terapi imunosupresi. kasus: dilaporkan satu kasus erisipelas pada pasien pf yang terjadi pada perawatan di rumah sakit hari ke-9, yang mendapatkan terapi injeksi deksametason intravena, dengan riwayat tusukan jarum sehari sebelumnya pada lokasi erisipelas. manifestasi klinis erisipelas berupa makula eritema dan edema berbatas tegas, nyeri, teraba keras dan hangat, dengan vesikel serta pustula pada permukaan lesi. pada pewarnaan gram yang diambil dari isi vesikel dan pustula didapatkan bakteri kokus gram (+). pasien diterapi dengan injeksi seftriakson intravena selama 7 hari dan kompres terbuka dengan larutan salin. perbaikan pada erisipelas didapatkan satu hari setelah pemberian seftriakson. pasien dipulangkan pada hari ke 12 perawatan dengan perbaikan baik pada pf dan erisipelasnya. pada saat kontrol 7 hari kemudian, sudah tidak terdapat lesi erisipelas. kesimpulan: luka tusukan dan terapi imunosupresi dapat menjadi faktor penyebab infeksi nosokomial berupa erisipelas. pengobatan yang tepat pada infeksi tersebut akan mengurangi gangguan fungsional pasien. kata kunci: erisipelas, infeksi nosokomial, imunosupresi, pemfigus foliaseus, seftriakson case report 47pranata, et al.: a nosocomial infection manifested as erysipelas introduction nosocomial infections are infections acquired during hospital care, which are not present or incubating at admission, occurring more than 48 hours after admission. important factors influencing nosocomial infection include old age, immune status, immunosuppressive drugs, underlying disease, injuries to skin, diagnostic and therapeutic interventions.1 skin and soft tissue infections (sstis) such as impetigo and erysipelas,2 are one of the most frequent nosocomial infections found.1 erysipelas is usually caused by staphylococcus aureus (s. aureus) or beta-hemolytic streptococci.2 the etiology of sstis may be normal host flora transferred through a break in the barrier, such as instrumentation (eg, needles), that could permit the entry of normal skin flora and indigenous flora from the instrument of penetration.3 esmaili et al.4 reported that s. aureus responsible for 93,7% of skin infections in pemphigus patients. pemphigus foliaceus is an autoimmune disorder, with generalized crusts and erosion as clinical findings, and immunosuppressive drugs as the mainstay of treatment.5 hospitalization in addition to corticosteroid therapy (with or without adjuvant immunosuppressive agents) would predispose the pemphigus patients to infection, with skin infection as one of the most frequently acquired.4 this is a case report of nosocomial infection manifested as erysipelas in a patient under immunosuppressive therapy. case a 57 year-old man was hospitalized at department of dermatology and venereology, dr. hasan sadikin hospital, bandung, indonesia with pemphigus foliaceous (pf). the clinical findings were generalized erythema, superficial loose blisters, erosions, and crusts, that affect 70% of body surface area. histopathology examination showed subcorneal acantholysis, and direct immunofluorescence revealed intra epidermal igg deposition, thus the diagnosis was established. the patient was treated with 15 mg intravenous dexamethasone injection daily, along with intravenous ranitidine injection, and antihistamine. on the 9th day of hospitalization, the patient complained about a painful, erythematous macules and edema on the upper right arm. there was history of repeated needle puncture, due to multiple failed trials of blood aspiration the day before, just below the site of the edema. from the physical examination, besides the generalized erythema and crusts, on the right arm there were irregular, well defined, painful erythema and edema with blisters and pustules on top, measuring 10 x 15 cm, that felt firm and warm on palpation (figure 1). the blisters and pustules were aspirated for gram staining, that revealed gram (+) cocci and polymorphonuclear cells. blood examination showed leukocytosis (28,400/mm3). thus the erysipelas diagnosis was added, and considered as a nosocomial infection. the patient then given 2 grams intravenous ceftriaxone injection daily for 7 days, and wet compress with saline solution was applied to the erysipelas lesion. improvement on the erythema and edema was seen the day after. the patient was discharged from the hospital on the 12th day, with improvement on the pf lesions, all the erosions have dried, and most of the crusts dissapeared. the erysipelas lesion was also improved, marked as decreased erythema and edema, and the pain diminished. after discharged from the hospital, the pf treatment was changed into 80 mg methylprednisolone tablet, to be tappered every 7 days. the erysipelas treatment was saline wet compress. seven days later on the next visit, the pf lesions already became hyperpigmented macules, and the painful erysipelas lesion had already dissapeared. figure 2. the erysipelas lesion on the final observation, leaving behind hypo pigmented and erythematous macules discussion skin and soft tissue infection is one of the most frequent nosocomial infections found. injuries to skin or mucous membranes due to diagnostic or therapeutic interventions could bypass natural defense mechanism of the skin. other factors including old age that are associated with decreased resistance to infection, and immunosuppressive drugs that may lower the resistance to infection.1 erysipelas is an sstis caused by s. aureus or beta-hemolytic streptococci,2 with bimodal incidence figure 1. erysipelas lesion 48 indonesian journal of tropical and infectious disease, vol. 6. no.2 mei–agustus 2016: 46−48 distribution, peaking among young children and the elderly. there is also an increased risk in the immunocompromised, including patients underwent corticosteroid therapy. erysipelas diagnosis is largely based on clinical findings, and might demonstrate leukocytosis.6 counted 20,000/ mm3 or more.7 the unique signs of erysipelas are welldemarcated erythema and edema, of which a diagnosis can be made confidently, and bullae or vesicles may complicate about 5% cases. the most commonly involved site was the leg, followed by the arm, and face.8 the clinical findings of erysipelas in this case were tender, well-defined erythema and edema that felt warm on palpation, with blisters and pustules on top. there was also leukocytosis (28,400/mm3). in this case, erysipelas was considered as nosocomial infection, as the patient acquired it on the 9th day of hospitalization, and there were no signs of infection on admission. the predisposing factors were old age and corticosteroid consumption for pf treatment. conditions that lead to disruption in the skin barrier predisposed patients to erysipelas.8 the etiology of skin and soft tissue infection may be normal host flora, with several means in penetrating the skin barrier. the most common route is through a break in the barrier, such as instrumentation (eg, needles), that could permit the entry of bacteria from the instrument of penetration.3 in this case, multiple puncture wound from repeated trials of blood aspiration with needle the day before, on the site of erysipelas precipitated the disease. antibiotics are the mainstay of treatment for erysipelas and most patients experience a complete recovery after antibiotics and few experience recurrences.6 a series of antibiotics have been suggested with highly successful clinical response, above 88%.9 ceftriaxone is a third generation cephalosporin, with sstis as one of the indications.10 based on the bacterial susceptibility data to antibiotics in dr. hasan sadikin hospital, ceftriaxone is still sensitive in 100% cases.11 pavlov and slavova12 reported that 3rd generation cephalosporin given in a parenteral route for 5–7 days showed good clinical resolution in severe erysipelas. clinical improvement will be seen in 24-48 hours after treatment initiation, and several days usually needed for disease resolution.7 in this case, the blisters are aspirated while keeping the roof intact. intravenous ceftriaxone as the mainstay treatment. the skin lesions improved within 24 hours and complete resolution was seen on the 14th day. hospital-acquired infections add to functional disability and emotional stress of the patient and may, in some cases, lead to disabling conditions that reduce the quality of life. nosocomial infections are also one of the leading causes of death. the economic costs are considerable. the increased length of stay for infected patients is the greatest contributor to cost.1 prevention of nosocomial infections requires integrated, monitored programs, which includes the following key components; 1) limiting transmission of organisms between patients in direct patient care through adequate hand washing and glove use, and appropriate aseptic practice, isolation strategies, sterilization and disinfection practices, and laundry, 2) controlling environmental risks for infection, 3) protecting patients with appropriate use of prophylactic antimicrobials, nutrition, and vaccinations, 4) limiting the risk of endogenous infections by minimizing invasive procedures, and promoting optimal antimicrobial use, 5) surveillance of infections, identifying and controlling outbreaks, 6) prevention of infection in staff members, and 7) enhancing staff patient care practices, and continuing staff education.13 as a conclusion, puncture wound and immunosuppresive treatment could cause erysipelas as a nosocomial infection, and an appropriate treatment of the infection would decrease the functional disability of the patient. references 1. epidemiology of nosocomial infections. dalam: prevention of hospital-acquired infections a practical guide. 2nd edition. world health organization. who/cds/eph/2002.12. page 4–8. 2. saavedra a, weinberg a, swartz mn, johnson ra. soft-tissue infections: erysipelas, cellulitis, gangrenous cellulitis, and myonecrosis. dalam: wolff k, goldsmithe la, katz si, dkk., editor. fitzpatrick’s dermatology in general medicine. 8th edition. new york: mcgraw-hill; 2012. page 1720–31. 3. ki v, rotstein c. bacterial skin and soft tissue infections in adults: a review of their epidemiology, pathogenesis, diagnosis, treatment and site of care. can j infect dis med microbiol. 2008 mar; 19(2): 173–84. 4. esmaili n, mortazavi h, normohammadpour p, dkk. pemphigus vulgaris and infections: a retrospective study on 155 patients. hindawi autoimun dis. 2013: 1–5. 5. payne as, stanley jr. pemphigus. dalam: wolff k, goldsmithe la, katz si, et al., editor. fitzpatrick’s dermatology in general medicine. 8th edition. new york: mcgraw-hill; 2012. page 586–99. 6. celestin r, brown j, kihiczak g, schwartz ra. erysipelas: a common potentially dangerous infection. acta dermatoven apa. 2007; 16(3): 123–7. 7. james wd, berger tg, elston dm. editor. bacterial infections. dalam: andrew’s diseases of the skin. 10th edition. philadelphia. wb saunders company; 2006. page 258–63. 8. chong fy, thirumoorthy t. blistering erysipelas: not a rare entity. singapore med j. 2008; 49(10): 809–13. 9. ribeiro a, oliveira al, batigalia f. the in-hospital treatment of erysipelas using cephalosporin, ciprofloxacin or oxacillin. j phleb lymph. 2012; 5: 6–8. 10. sadick ns. systemic antibacterial agents. dalam: wolverton se, editor. comprehensive dermatologic drug therapy. indianapolis: wb saunders;2001. page 28–54. 11. kepekaan bakteri terbanyak di instalasi rawat inap dari berbagai spesimen terhadap antibiotika periode januari-juni 2013. dalam: peta bakteri dan kepekaannya terhadap berbagai antibiotika di rumah sakit dr. hasan sadikin bandung semester i tahun 2013. tim program pengendalian resistensi antimikroba. smf/departemen patologi klinik rs. dr. hasan sadikin bandung. 12. pavlov s, slavova m. antibiotic therapy and prophylaxy of patients with erysipelas. j of imab. 2004; 10(1): 31–3. 13. prevention of nasocomial infection. dalam: prevention of hospitalacquired infections a practical guide. 2nd edition. world health organization. who/cds/eph/2002.12. page 30–7. indonesian journal of tropical and infectious disease this journal is a peer-reviewed journal established to promote the recognition of emerging and reemerging diseases spesifically in indonesia, south east asia, other tropical countries and around the world, and to improve the understanding of factors involved in disease emergence, prevention, and elimination. the journal is intended for scientists, clinicians and professionals in infectious diseases and related sciences. we welcome contributions from infectious disease specialists in academia, industry, clinical practice, public health and pharmacy, as well as from specialists in economics, social sciences and other disciplines. for information on manuscript categories and suitability of proposed articles see below and visit e-journal. unair.ac.id/index.php/ijtid/ index. i. instructions to authors article structure please kindly check our template. language all manuscripts must be in english, also the table and figure texts. title page give complete information about each author (i.e., full name, graduete degree(s), affiliation and the name of the institution in which the work was done). clearly identify the corresponding author and provide that author’s mailing address (including phone number, fax number, and email address). use uppercaser for title except the name of species, only the first letter using uppercase. abstract a concise and factual abstract is required. the abstract should state briefly the purpose of the research, the principal results and major conclusions. abstract should be 250-300 words. it should include objectives and rationale of the study, method used, main findings and significance of findings. it should be accompanied by up to 5 keywords. abstract should be available in english and bahasa. introduction state the objectives of the work and provide an adequate background, avoiding a detailed literature survey or a summary of the results. material and method provide sufficient detail to allow the work to be reproduced. methods already published should be indicated by a reference: only relevant modifications should be described. result and discussion results should be clear and concise results presented in the tables and figures should not be repeated unnecessarily in the text. result should be presented without interpretation and followed by the discussion section which should provide an interpretation of the results in relation to findings of other investigators. discussion should explore the significance of the results of the work, not repeat them. a combined results and discussion section is often appropriate. avoid extensive citations and discussion of published literature. conclusion conclusions should be stated clearly.the main conclusions/summary of the study may be presented in a short conclusions section, which may stand alone or form a subsection of results and discussion section. conclusion section is for research report/original article and summary is for literature review/review article. acknowledgement all acknowledgements including financial support should be mentioned under this heading. list here those individuals who provided help during the research (e.g., providing language help, writing assistance or proof reading the article, etc.). references please ensure that every reference cited in the text is also present in the reference list (and vice versa). minimum 15 references for research report/original article and 30 references for review. references wrote on vancouver (superscript) style. in the vancouver style, citations within the text of the essay/paper are identified by arabic numbers in superscript. this applies to references in text, tables and figures. the vancouver (superscript) system assigns a number to each reference as it is cited. a number must be used even if the author(s) is named in the sentence/text. e.g. smith10 has argued that... the original number assigned to the reference is reused each time the reference is cited in the text, regardless of its previous position in the text. when multiple guide for author references are cited at a given place in the text, use a hyphen to join the first and last numbers that are inclusive. use commas (without spaces) to separate non-inclusive numbers in a multiple citation e.g. 2,3,4,5,7,10 is abbreviated to… the placement of citation numbers within text should be carefully considered e.g. a particular reference may be relevant to only part of a sentence. as a general rule, reference numbers should be placed outside full stops and commas and inside colons and semicolons, however, this may vary according to the requirements of a particular journal. examples – there have been efforts to replace mouse inoculation testing with in vitro tests, such as enzyme linked immunosorbent assays57, 60 or polymerase chain reaction20-22 but these remain experimental. moir and jessel maintain “that the sexes are interchangeable”.1 tables tables should be embedded in page. provide footnotes and other information (e.g., source/copyright data, explanation of boldface). tables should be no wider than 17 cm. condence or divide larger tables. please submit tables as editable text and not as images. number tables consecutively in accordance with their appearance in the text and place any table notes below the table body. be sparing in the use of tables and ensure that the data presented in them do not duplicate results described elsewhere in the article. please avoid using vertical rules and shading in table cells. figures provide figures embedded in page. figures should be drawn professionally. photographs should be sharp (contrast). provide footnotes and other information (e.g., source/copyright data, explanation of boldface) in figure legend. submit image files (e.g., electromicrograph) without text content as high-resolution (300 dpi/ ppi minimum) tiff or jpg files. ensure that each illustration has a caption. supply captions separately, not attached to the figure. a caption should comprise a brief title (not on the figure itself) and a description of the illustration. keep text in the illustrations themselves to a minimum but explain all symbols and abbreviations used. equations equations (refer with: eq. 1, eq. 2,..) should be indented 5 mm (0.2”). there should be one line of space above the equation and one line of space below it before the text continues. the equations have to be numbered sequentially, and the number put in parentheses at the right-hand edge of the text. equations should be punctuated as if they were an ordinary part of the text. punctuation appears after the equation but before the equation number. the use of microsoft equation is allowed. c2 = a2 + b2. (1) plagiarism check the manuscript has not been published previously (partly or in full), unless the new work concerns an expansion of previous work (please provide transparency on the re-use of material to avoid the hint of text-recycling (“self-plagiarism”). please kindly tell us if you already use plagiarism check (turnitin, etc.). the writing process of article is suggested to use reference manager program (mendeley, etc.) article processing charge due to the necessity of increasing the ijtid quality, starting from january 1st 2018, there would be ratified article processing charge for publication in indonesian journal of tropical infectious disease, idr 1 million or usd 75. payment applies after the manuscript is accepted in the editing process. ii. types of articles research report/original article articles should be under 3000 to 4000 words and should include references. use of subheadings in the main body of the text in recommended. photographs and illustrations are 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ijtid@itd.unair.ac.id 96 vol. 5. no. 4 january–april 2015 research report lymphocyte response to mycobacterium leprae antigens in reversal reaction state of leprosy an in vitro study of lymphocyte stimulation index using mtt method fanny iskandar,1 irawanyusuf,1 mansur arif,1 indropo agusni1,2 1hasanuddin medical faculty, makassar 2leprosy study group, institute of tropical disease, universitas airlangga, surabaya, indonesia abstract reversal reaction (rr) in leprosy is a sudden inflammatory episode in the chronic course of the disease due to rapid change of cellular immunological status. the aim of the study is to measure the in vitro results of lymphocyte stimulation index (lsi) rr leprosy derived lymphocytes after challenged with m.leprae antigens. twenty three borderline leprosy with rr and 11 borderline leprosy patients without rr were included in the study. peripheral blood mononuclear cells (pbmc) were separated from peripheral blood of these patients using ficol-hypaque column and cultured in laboratory. using the colorimetric tetrazole (mtt) method these lymphocytes were challenged with pha, dharmendra antigen (1/100 and 1/10 dilutions), lam (50 and 100 nanograms). stimulation index were calculated and superanatans were collected for measuring the ifn-γ and il-10 production (elisa). all of lymphocytes from rr patients showed higher stimulation index after challenged with the five m.leprae antigens compared to lymphocytes from non rr patients (p <0.05) . ifn-γ and il-10 also increased but not significant (p>0.05). it is concluded that lymphocytes of leprosy patients during rr state are more sensitive to antigenic stimuli compared to non-rr leprosy patients. further extended studies are needed to determine the “cut off” value of lymphocyte stimulation index that is useful for clinicians in the field in the prediction of rr before starting anti leprotic treatment. key words: leprosy, reversal reaction, lymphocyte stimulation index, mtt, borderline type abstrak reversal reaction (rr) dalam kusta adalah periode inflamasi mendadak pada jalur kronis penyakit akibat perubahan status imunologi selular yang cepat. tujuan penelitian ini untuk mengukur secara in vitro hasil indeks stimulasi limfosit (isl) rr kusta berasal dari limfosit setelah menantang dengan m.leprae antigen. dua puluh tiga batas kusta dengan rr dan sebelas batas penderita kusta tanpa rr dimasukkan dalam studi. sel mononuklear darah perifer (sldp) dipisahkan dari darah perifer dari para pasien menggunakan kolom ficol-hypaque dan dibiakkan di laboratorium. menggunakan metode colorimetric tetrazole (mtt), limfosit ini ditantang dengan pha, antigen dharmendra (1/100 dan 1/10 pengenceran), lam (50 dan 100 nanograms). indeks stimulasi dihitung dan supernatan dikumpulkan untuk mengukur ifn-γ dan produksi il-10 (elisa). semua limfosit dari rr pasien menunjukkan indeks stimulasi lebih tinggi setelah ditantang dengan lima antigen m.leprae dibandingkan dengan limfosit yang berasal dari pasien non rr (p < 0.05). ifn-γ dan il-10 juga meningkat tapi tidak signifikan (p > 0.05). hal ini disimpulkan bahwa limfosit pada pasien lepra selama masa rr lebih sensitive terhadap rangsangan antigen dibandingkan dengan pasien lepra non rr. penelitian selanjutnya diperlukan untuk menentukan nilai cut off dari indeks stimulasi limfosityang berguna untuk klinisi dalam memprediksi rr sebelum memulai perawatan anti lepra. kata kunci: lepra, reversal reaction, indeks stimulasi limfosit, mtt, batas kusta 97iskandar, et al.: lymphocyte response to mycobacterium leprae antigens introduction leprosy is still a public health problem in indonesia, especially in the eastern part of the country.1 one of the problems in the field is the reversal reaction (rr), which often occurred during the multi-drugs therapy (mdt) course for leprosy. it is an acute inflammatory episode that occurred during the chronic course of the disease and sometimes cause disability to the patient. clinically it is manifested by acute inflammatory skin lesions that previously relative “silent” and acute neuritis can occurred that led to disability.2 by histopathological examination of the skin lesions in rr, inflammatory process could be found in the granuloma with more lymphocytic cells due to influx of lymphocytes from surrounding tissue came to the granuloma with its inflammatory mediators.3 activation of these lymphocytes could be a result of lymphocyte stimulation by many substances, including several antigens originated from mycobacterium leprae, the cause of the disease. the aim of this study is to conduct an in vitro study on the response of lymphocytes from leprosy patients during the rr episode. figure 1. type 1 leprosy reaction (reversal reaction) material and methods thirty four blood samples obtained from 23 borderline leprosy patients with rr and 11 blood samples from borderline leprosy patients without rr were included for in vitro study. the peripheral blood mono nuclear cells (pbmc) were separated using ficoll hypaque column and lymphocyte culture were performed.4 phytohaemagglutinine (pha) were used as nonspecific stimulans, dharmendra 1/10 and dharmendra 1/100 as specific protein stimuli from m.leprae , lipoarabinomannan (lam) 50 nanogram and 100 nanogram as specific carbohydrates stimuli were also used to the lymphocyte cultures. lymphocyte proliferation test (ltt) were performed using the the colorimetric tetrazole (mtt) procedures as recommended by mosmann in 1983.5 stimulation index (si) is ratio between stimulation index result and threshold. si was read by computer and regarded as positive results if the value >1. the level of ifn-gamma and il-10 from supernatant were measured by elisa procedure using appropriate kits. statistical significant differences between the rr and non rr group were analyzed using mann whitney u test and fisher’s exact test. spearmans rho test was also used for calculating correlation coefficient. results & discussion stimulation with non-specific mitogen (pha) resulted si positive in 20/23 rr patients compared to 7/11 in non rr patients (p<0.05). all of specific m.leprae antigens using for lymphocytes stimulation showed significant statistical differences between the the rr and non rr group. figure 2. (a) lymphocyte culture, (b) formazan coated lymphocytes a b 98 indonesian journal of tropical and infectious disease, vol. 5. no. 4 january–april 2015: 96–99 acute inflammatory process in reaction state in leprosy is a result of sudden changes in immunological response stability during the chronic course of the disease. treatment with mdt drugs will kill a big amount of lepra bacilli inside the host body and many new antigens including protein and carbohydrates antigens from dead m.leprae spread to the surrounding tissues and circulation. these antigens will stimulate t-lymphocytes, especially in table 1. results of stimulation index with non-specific and specific antigens of m.leprae antigen positive si in rr patients positive si in non-rr patients p pha 20 / 23 7 / 11 0.013 dharmendra 1/100 22 / 23 6 / 11 0.002 dharmendra 1/10 22 / 23 3 / 11 0.000 lam 50 22 / 23 5 / 11 0.000 lam 100 21 / 23 5 / 11 0.000 table 2. mean of ifn-gamma level in supernatans after stimulation with nonspecific and specific antigens of m.leprae antigen mean level of ifn-π in rr patients mean level of ifn-π in non-rr patients p pha 22.98* 24.27 0.699 dharmendra 1/100 23.97 21.18 0.299 dharmendra 1/10 25.86 23.40 0.522 lam 50 20.26 20.46 0.839 lam 100 28.05 24.06 0.368 *unit/ml (elisa) table 3. mean of il-10 level in the supernatan after stimulation with nonspecific and specific antigens of m.leprae antigen mean level of il-10 in rr patients mean level of il-10 in non-rr patients p pha 29.41 29.70 0.593 dharmendra 1/100 30.28 30.26 0.974 dharmendra 1/10 29.59 29.55 0.934 lam 50 29.44 29.47 0.942 lam 100 29.36 29.91 0.259 *unit/ml (elisa) areas surrounding the location of lepra bacilli. some of these lymphocytes are already sensitized previously by the same antigen (t-memory cells).6 the result of lymphocyte re-activation is the proliferation, differentiation and production of interleukin as il-2, ifn-gamma, il10 and other interleukins. in this study the proliferation of lymphocytes was performed in-vitro using the mtt method that requiring a fresh peripheral blood from patients. previously lymphocyte transformation test (ltt) with radioactive labeled thymidine were often used for measuring lymphocyte activation. recently this procedure has been changed to mtt technique that relatively save and accurate. this colorimetric technique is based on the enzyme utilities that needed during proliferation of lymphocytes, which can be labeled by certain dyes.7 the amount of labeled enzyme used by cells indicated the amount of cell’s proliferation. during the reaction state in reversal reaction of leprosy, many t-memory cells are activated and proliferation occurred. as a result of proliferation, the number lymphocytes increase including the t-memory cells. subsequent stimulations by antigens from lepra bacilli will stimulate memory t-lymphocytes that already accumulate during granuloma formation in the pathogenesis of leprosy.8 stimulation index in this study showed that lymphocytes from rr patients gave a higher proliferation process compared to non-rr patients. not only stimulation by protein antigens, carbohydrate antigens were also showed higher results. proliferation of lymphocytes is always followed by release of many inflammatory mediators and resulted an acute inflammatory reaction of the skin lesions.9,10 in this study, the production of interleukins were not significant difference between rr and non-rr patients. this results need to be studied further to find the reason, it might be due to technical or time of harvesting the lymphocytes after stimulation. the results of this study showed that not only a single antigen involved in rr process, but many antigens can stimulate lymphocytes of leprosy patients. how the lymphocytes can be stimulated by carbohydrate antigens from m.leprae need to be investigated and which antigen is predominated the lymphocyte stimulation in rr still a question. conclusion lymphocytes from leprosy with rr patients showed significant higher stimulation index compared with non rr patients. both protein and carbohydrate antigens from m.leprae can stimulate lymphocytes in leprosy patients. more investigations are needed to clarify the true mechanism of rr. refferences 1. depkes r.i. buku pedoman pemberantasan kusta (2009). subdit p2kusta ri. 99iskandar, et al.: lymphocyte response to mycobacterium leprae antigens 2. agusni i (2011). clinical manifestation of leprosy. in (makino m, matsuoka m, goto m eds) leprosy. from 3. ridley ds (1988). pathogenesis of leprosy and related diseases. 1st edition. butterworth & co. publ. 4. abbas ak, lichtman ah, pillai s (2011). chapter 4. cell-mediated immune responses. basic immunology. saunders, philadelphia. pp. 89–112., 5. mosmann d (1983). mtt assay, a colorimetric technique for the study of lymphocyte stimulation . j immunol 92, 247–252. 6. hussain rf, nouri ame, olver rtd (1993). a new approach for measurement of cytotoxicity using colorimetric assay, j of immunol method; 160(1993) 89–96. 7. britton wj (1998). the management of leprosy reversal reaction. lepr rev, 74 8. bjune gb, ridley ds, kronvall g (1976). lymphocyte transformation test in leprosy; correlation of the response with clinical inflammation of lesions. clin exp immunol 25, 85–94. 9. kawahita ip, walker sl, lockwood dnj. (2008). leprosy type1 reactions and erythema nodosum leprosum. an bras dermatol, 83(1),75–82. 10. walker sl & lockwood dnj (2008). leprosy type 1 (reversal) reactions and their management. lep rev 78, 372–386. ijtid vol 8 no 2 may-agustus 2020_newfromsarah.indd vol. 8 no. 2 may–august 2020 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ research article diff erences of interleukin-18 and interleukin-10 levels in pulmonary rifampicin resistant dan rifampicin sensitive tuberculosis patients in dr. soetomo hospital surabaya audrey gracelia riwu1a, jusak nugaraha2, yoes prijatna dachlan3 1department of immunology postgraduate school, universitas airlangga, surabaya, east java, indonesia 2department of clinical pathology, faculty of medicine universitas airlangga, dr. soeotomo hospital surabaya,east java, indonesia 3department of parasitology, faculty of medicine universitas airlangga, surabaya, east java, indonesia received: 1st january 2019; revised: 14th march 2019; accepted: 19th december 2019 abstract rifampicin is an anti-tuberculosis drug used in short-term treatment regimen for tuberculosis (tb) patients. resistance to rifampicin causes the prolonged duration of tuberculosis treatment. interleukin-18 (il-18) is a pro-infl ammatory cytokine which acts in controlling the growth of m. tuberculosis, while interleukin-10 (il-10) is an anti-infl ammatory cytokine which acts in limiting tissue damage and maintain tissue homeostasis. il-18 and il-10 is important in explaining the diff erent degrees of infl ammation (mild, moderate and severe) in rifampicin-resistant (rr) and rifampicin-sensitive (rs) pulmonary tb patients. the purpose of this study is to determine diff erent levels of il-18 and il-10 in new tb patients with rr and rs. a retrospective cohort study with a cross-sectional design. 50 subjects were examined and grouped into two groups, namely pulmonary tb with rr (n = 25) and pulmonary tb with rs (n = 25). il-18 and il-10 were measured using the elisa method. diff erences in il-18 and il-10 levels between groups were analyzed using the mann-whitney test. the mean level of il-18 (pg/ml) in rr and rs pulmonary tb patients were 1273.53±749.86 and 787.96 ±589.28 respectively. the mean level of il-10 (pg/ml) in rr and rs pulmonary tb patients were 125.25±118.32 and 128.81±135.77 respectively. the mean level of il-18 in rr and rs pulmonary tb patients were found to have a signifi cant diff erence, while the mean level of il-10 did not have a signifi cant diff erence. this circulating level of il-18 and il-10 can be used as a marker of infl ammation degrees in pulmonary rr-tb and rs-tb patient. keywords: interleukin-18, interleukin-10, tuberculosis, rifampicin resistant, rifampicin sensitive abstrak rifampisin adalah rejimen dasar pengobatan jangka pendek untuk penderita tuberculosis (tb). resistensi terhadap rifampisin menyebabkan durasi pengobatan tuberculosis menjadi lebih lama. interleukin-18 (il-18) adalah sitokin proinfl amsi yang berperan dalam mengontrol pertumbuhan m. tuberculosis, sedangkan interleukin 10 (il-10) adalah sitokin anti-infl amasi yang berperan membatasi kerusakan jaringan dan mempertahankan homeostatis jaringan. il-18 dan il-10 berperan penting untuk menjelaskan derajat infl amasi (ringan, sedang dan berat) yang berbeda pada penderita tb paru dengan rifampicin resistant (rr-tb) dan rifampcin sensitive (rs-tb). tujuan penelitian ini adalah mengetahui perbedaan kadar il-18 dan il-10 pada penderita rr-tb dan rs-tb. penelitian ini merupakan penelitian cohort retrospektif dengan rancangan cross-sectional. sebanyak 50 subjek penelitian diperiksa dan dikelompokkan menjadi dua kelompok yaitu kelompok rr-tb (n=25) dan kelompok rs-tb (n=25). pemeriksaan il-18 dan il-10 dilakukan dengan metode elisa. perbedaan kadar il-18 dan il-10 antara kelompok dianalisis menggunakan mann-whitney. rerata kadar il-18 (pg/ml) pada penderita rr-tb dan rs-tb adalah 1273.53±749.86 dan 787.96±589.28. rerata kadar il-10 (pg/ml) pada penderita rr-tb dan rs-tb adalah 125.25±118.32 dan 128.81±135.77. rerata kadar il-18 pada penderita rr-tb dan rs-tb ditemukan memiliki perbedaan signifi kan, sedangkan rerata kadar il-10 pada penderita rr-tb dan rs-tb tidak * corresponding author: riwuaudrey@gmail.com 117audrey gracelia riwu, et al.: differences of interleukin-18 and interleukin-10 levels copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 memiliki perbedaan yang signifi kan. nilai kadar il-18 dan il-10 ini dapat digunakan sebagai penanda derajat infl amasi pada penderita rr-tb dan rs-tb. kata kunci: interleukin-18, interleukin-10, tuberculosis, rifampicin resistant, rifampicin sensitive how to cite: riwu, audrey gracelia., nugaraha, jusak., dachlan, yoes prijatna. diff erences of interleukin-18 and interleukin-10 levels in pulmonary rifampicin resistant dan rifampicin sensitive tuberculosis patients in dr. soetomo hospital surabaya. indonesian journal of tropical and infectious disease, 8(2), 1–8 introduction in 2018, the world health organization (who) was stated that tuberculosis (tb) is one of the top ten causes of death worldwide. about 10.4 million people suffer from tb and 1.7 million people die from this disease. more than 95% of deaths from tb occur in low and middleincome countries. india, indonesia, china, the philippines, pakistan, nigeria, and south africa are countries that accounted the most cases of tb.1 according to the basic health research of indonesia the prevalence of patients diagnosed with tb in 2013 was 0.4% with the fi ve highest provinces which are west java, papua, dki jakarta, gorontalo, banten and west papua. of the entire population diagnosed with tb, only 44.4% were treated with a program medicines.2 rifampicin resistant is defi ned as a tb case that is declared resistant to rifampicin. tb strains resistant to rifampicin may be either sensitive or also resistant to isoniazid, which for the latter is considered as multidrug resistant-tuberculois (mdr-tb) based on the genexpert test results. this is due to the lower mutation rate of isoniazid (2.56 x 108 cfu / ml m. tuberculosis colonies) compared to the mutation rate of rifampicin (6 x 1010 cfu / ml m. tuberculosis colonies), so that it can be said that tb patients that are resistant to the rifampicin drug are also resistant to isoniazid, but this comparison varies greatly between countries and patient groups.3,4 rifampicin is an antibiotic that has efficient antimicrobial action which combined with isoniazid which considered to be the basis of a short-term treatment regimen for tb. rifampicin in m. tuberculosis targets the rna polymerase β-subunits by binding and inhibiting the extension of rna messenger. the role of rifampicin is to inhibit active growth and slow metabolism (slow-growing) of bacilli.3 interleukin-18 (il-18) was fi rst described and used in rat serum which was intraperitoneally inoculated with endotoxin and was referred to as “interferon-gamma (ifn) inducing factor”.5 inside the human body, il-18 is constitutively expressed by several cells, namely macrophages, kupff er cells, keratinocytes, osteoblasts, adrenal cortex cells, intestinal epithelial cells, microglial cells, and synovial fi broblasts.6 il-18 is a proinfl ammatory cytokine that works synergistically with interleukin-12 (il-12) to induce ifn production. the expression of il-18 is regulated in chronic infl ammatory diseases mediated by th1. il-18 can also contribute to the protection against mycobacteria. it is found that rats with il-18 defi ciency also have a decrease in ifn levels.7 interleukin-10 (il-10) is an anti-infl ammatory cytokine which has a crucial role in preventing inflammatory, pathological autoimmune8 and allergies.9 defi ciency or decreased expression of il-10 can increase the infl ammatory response to microbes but on the other hands, it can also cause the development of infectious diseases such as tb and several of autoimmune diseases.8 il-10 can also increase the continuity of m. tuberculosis and its growth in macrophages by suppressing the partial maturation of phagosomes which depend on the activity of the signal transducer and activator of transcription 3 (stat3)10. currently, il-10 increases survival and intracellular growth mycobacteria by suppressing innate and adaptive immune responses.11 this study will describe how diff erent levels of il-18 and il-10 in pulmonary tb patients with rifampicin resistant and rifampicin sensitive, 118 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 116–123 where il-18 and il-10 can play an important role in explaining the diff erent degrees of infl ammation between these two groups. materials and methods study population this study was conducted in the department of clinical pathology, dr. soetomo hospital from august to november 2018. this study included 50 patients who were selected from the tbdots/mdr clinic of dr. soetomo hospital. the study protocol has been approved by the ethical review committee of dr. soetomo hospital (0488/kepk/viii/2018). the data of all patients were collected after taking informed consent from patients. the age of patients ranged from 17 to 75 years old. the patients were assigned into two groups. the fi rst group consisted of 25 patients with rifampicin-resistant pulmonary tb and the second group also consisted of 25 patients with rifampicin-sensitive pulmonary tb. patients with hiv-aids, hepatitis, autoimmune diseases, diabetes mellitus, liver and kidney disease were excluded from this study. also, patients treated with corticosteroid or immunosuppressive drugs were excluded, along with patients who had received anti-tuberculosis for more than one month because it can cause bias in the results of the examination sample preparation four milliliters of blood were drawn aseptically from the basilic vein of each patient. blood specimens were collected by using vacutainer venipuncture then stored in the serum separator tube. the tube contains a separation gel in the base of the tube which separates the serum from the whole blood. the blood sample was collected then was centrifuged at 3000 rpm for 10 minutes, the serums were then stored and freeze at -80°c for further use. enzyme-linked immunosorbent assay (elisa) analysis the frozen serums were thawed at room temperature and cytokine il-18 and il-10 levels were then measured using the human sandwich-elisa kit from elabscience® done as the manufactures instructions. the cytokine concentrations in samples were calculated using the standard curve generated from recombinant cytokines, and the results are expressed in picograms per milliliter (pg/ml). statistical methods the result is presented as the mean ± s.d. statistical signifi cance was calculated by the mann-whitney test to see diff erences between il18 and il-10 in patients with pulmonary rr-tb and pulmonary rs-tb. the p values< 0.05 were considered statistically signifi cant. results and discussion clinical characteristics of subjects the clinical characteristics of the 25 patients with pulmonary rr-tb and 25 patients with pulmonary rs-tb are summarized in table 1. the clinical type of all tb patients were all pulmonary tb. il-18 level the highest level of il-18 found in pulmonary rr-tb patients was 2486 pg/ml, and the lowest 58.39 pg/ml, while the highest level of il-18 in pulmonary rs-tb patients was 1990 pg/ml and the lowest was 106.06 pg/ml. the mean, standard deviation, and p-values of il-18 levels in these two groups are shown in table 2. the mean level of il-18 between pulmonary rr-tb and rs-tb patients were showed signifi cant diff erences (p <0.05). the diff erences of il-18 in pulmonary rr-tb and pulmonary rs-tb patients are shown in the boxplot in figure 1. table 1. clinical characteristics of the population studied. pulmonary rr-tb pulmonary rs-tb gender, male/female 18/7 11/14 median age (range) 37.00 (23-67) 43.00 (18-63) 119audrey gracelia riwu, et al.: differences of interleukin-18 and interleukin-10 levels copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 the il-18 level between pulmonary rr-tb and rs-tb patients found in this study has a mean of 1273.53 ± 749.86 pg/ml and 787.96 ± 589.28 pg/ml respectively. this shows that the increasing level of il-18 in the blood was found to be signifi cantly higher in pulmonary rr-tb than in pulmonary rs-tb. this results in this study also in accordance with the result of wang et al12 study. wang et al12 also stated that the il-18 serum was found to be higher in patients with pulmonary rr-tb (131.03 ± 94.92) compared to drug sensitive tb (94.28 ± 57.10) and healthy controls (61.66 ± 24.78). the resistance to rifampicin in tb is caused by mutations in the bacterial chromosome (rpo gene). mutations in this gene will cause changes in the structure and activity of drug targets that results in generating bacterium m. tuberculosis that cannot be eliminated using rifampicin which has an impact on increasing the number of said bacteria in the host body.13 this increase in the number of bacteria causes macrophages as a fi rstline defense against the invasion of these bacteria and mediates the innate immune response through the introduction of pathogens and an increase in infl ammatory reactions. increased macrophage activation in rr pulmonary tb infection will increase the production of proinflammatory cytokines that play a role for the mechanism of killing m. tuberculosis.14 rifampicin plays an important role in tb treatment because of its bactericidal eff ect that can eliminate m. tuberculosis.15 when pulmonary tb patients are resistant to rifampicin, the table 2. the mean and standard deviation of il-18 in pulmonary rr-tb and pulmonary rs-tb group n mean standard deviation p-value pulmonary rr-tb 25 1273.53 749.86 0.017 pulmonary rs-tb 25 787.96 589.28 n = number of samples p < 0,05 = signifi cant groups il -1 8 l ev el s figure 1. il-18 levels in pulmonary rr-tb and pulmonary rs-tb. this result shows that an increase in il-18 levels in the blood was found to be signifi cantly higher in pulmonary rr-tb patients compared to pulmonary rs-tb, meaning a higher increase in the infl ammatory process for pulmonary rr-tb patients compared to pulmonary rs-tb patients. this results is also accordance with the result of wang et al12 study. 120 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 116–123 growth of m. tuberculosis will increase and cannot be controlled. macrophages as the fi rstline defense will fi ght the bacterial invasion and mediate innate immune responses through the introduction of pathogens and the activation of infl ammatory reactions. macrophages will polarize to various functional conditions such as m1 which is classically activated and m2 which is alternatively activated. macrophage polarization into m1 is important for the elimination of intracellular m. tuberculosis. activation of m1 macrophages through the tlr2 signal pathway can be benefi cial for the host to inhibit growth and the survival of m. tuberculosis.16,17 increased activation of m1 macrophages in newly infected rr pulmonary tb will produce pro-infl ammatory cytokines which play a role in the mechanism of eliminating m. tuberculosis. this causes the level of pro-infl ammatory cytokines to be higher in rr pulmonary tb serum compared to rs pulmonary tb. the level of pro-infl ammatory cytokines in both rr and rs pulmonary tb is found to be higher compared to the level of anti-infl ammatory cytokines to suppress growth and the survival of m.tuberculosis.12 increased level of il-18 in the patients’ serum is also suspected to indicate that there has been a leak of cytokines from the tissues to the circulation. this is supported by various studies which stated that a high concentration of il-18 are found in tb patients with advanced disease, high fever, and extensive radiographic infi ltrates.7, 18 increased levels of il-18 as a proinflammatory cytokine in rr pulmonary tb patients are associated with various pathological conditions in the patients themselves. patients with pulmonary rr-tb with high levels of il18 were also found to have higher esr and crp levels compared to pulmonary rs-tb patients and healthy people. esr and crp have been used as markers for the diagnosis of pulmonary tb that reflect pathological processes in the patient’s body. increased crp and esr indicate that an acute infl ammatory process has occurred in pulmonary tb patients.12 higher il-18 levels found in pulmonary rr-tb patients compared to pulmonary rs-tb patients in this study confi rmed various previous studies which stated that il-18 levels were signifi cantly increased in patients with severe pulmonary tb. il-10 level the highest level of il-10 in pulmonary rrtb patients was 465.77 pg/ml, and the lowest was 1.57 pg/ml, while the highest level of il-10 in pulmonary rs-tb patients was 552.11pg/ml and the lowest level was 1.36 pg/ml. the mean, standard deviation, and p-values of il-10 level in these two groups are shown in table 3. the mean of il-10 level between patients showed no signifi cant diff erences (p>0.05). the diff erences of il-10 in pulmonary rr-tb and pulmonary rs-tb patients are shown in the boxplot in figure 2. il-10 is an anti-infl ammatory cytokine that works by inhibiting the ability of myeloid cells such as macrophages and dendritic cells to activate th1 cells. initially, il-10 is known to be secreted by antigen-stimulated th2, but it is now known that il-10 is not only secreted by th2, but also secreted by a subset of cd4 + t cells, including th1 and th17, b cells, neutrophil cells, and macrophages.17 il-10 is generally thought to modulate the ability of the immune response and allow bacterial elimination without damaging the host tissue, but in some cases the absence of il-10 makes the immune response more eff ective in eliminating pathogens, but resulting in more damage to the tissue and aff ects the survival of the host.20, 21 the mean level of il-10 in pulmonary tb patients with rs and rr in this study were 128.81 ± 135.77 pg/ml and 125.15 ± 118.32 pg/ ml respectively. this shows that il-10 levels in rs were found to be higher than in rr pulmonary table 3. the mean and standard deviation of il-10 in pulmonary rr-tb and pulmonary rs-tb group n mean standard deviation p-value pulmonary rr-tb 25 125.15 118.32 0.961 pulmonary rs-tb 25 128.81 135.77 n = number of samples p > 0,05 = not signifi cant 121audrey gracelia riwu, et al.: differences of interleukin-18 and interleukin-10 levels copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 tb, although statistically did not have a signifi cant diff erence (p> 0.05). the results of this study are following a study conducted by butov et al22 which stated that the mean level of il-10 in mdr-tb patients’serum before and after 2 months of treatment were found to be lower when compared to non-mdr tb patients and healthy people. this result is in accordance with the result of lihawa23 and peñaloza24 study. lihawa and yudhawati23 in dr. soetomo hospital showed that descriptively il-10 levels in mdr-tb patients were found to be lower than non-mdr tb, but statistically no signifi cant diff erences were found. peñaloza24 was stated that during non-mdr m. tuberculosis infection, il-10 production is important for host survival, but the role of il-10 in the immune response of patients with mdr pulmonary tb molecularly has not been found with certainty. this insignifi cant diff erence in il-10 may indicate a tendency of static state occuring during the acute phase of tb levels il10 due to the role of macrophages which secrete more proinfl ammatory cytokines to protect the host from m. tuberculosis. it is evidenced in this study by the discovery of il-18 levels that were higher than the il-10 levels in each group. high levels of il-10 can only be found in chronic tb infections.25 conclusions the level of il-18 is higher in patients with pulmonary rr-tb compared to pulmonary rstb. this circulating level of il-18 and il-10 can be used as a marker of infl ammation degrees in pulmonary rr-tb and rs-tb patients. acknowledgment the author would like to thank the postgraduate school of universitas airlangga and dr. soetomo hospital specifi cally for the department of research i l 1 0 l ev el s groups figure 2. il-10 levels in pulmonary rr-tb and pulmonary rs-tb patients. the results shows showed no signifi cant diff erences between these two groups. 122 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 116–123 and development which has permitted them to conduct this research in the tb-dots/mdr polyclinic. the author would also like to thank dr. soedarsono, dr., sp.p(k) who has been willing to become a clinical supervisor, to the research and development department of the clinical pathology installation who has helped to carry out the examination using the elisa method and all of the patients who donated the samples. conflict of interest there is no confl ict of interest that has to be declared in this study. references 1. who. global tuberculosis report 2018. geneva, switzerland: world health organization; 2018. 2. kemenkes ri. pedoman nasional pengendalian tuberculosis. jakarta: kementrian kesehatan republik indonesia; 2014. 3. dasilva p, palomino j. molecular basis and mechanisms of drug resistance in mycobacterium tuberculosis: classical and new drugs. j antimicrob chemother. 2011; 66(7): 1417–30. doi: 10.1093/jac/dkr173 4. kurbativa ev, cavanaugh js, shah sn, wrisht a, kim hj, metchock b. rifampicin-resistant mycobacterium tuberculosis susceptibility to isoniazid and other antituberculosis drugs. int j tuberc lung dis. 2012; 16(3): 355–7. doi: 10.5588/ijtld.11.0542. 5. wawrocki s, druszczynska m, kowalewics m.k, rudnicka w. interleukin 18 (il-18) as a target for immune intervention. acta biochim pol. 2016; 63(1): 59–63. doi: 10.18388/abp.2015_1153. 6. dinarello c, novick d, kim s, kalplanski g. interleukin18 and il-18 binding protein front immunol. 2013; 4: 289. doi: 10.3389/fi mmu.2013.00289. 7. han m, yue j, lian y, zhao y, wang h, liu l. relationship between single nucleotide polymorphism of interleukin-18 and susceptibility to pulmonary tuberculosis in the chines han population. microbiology and immunology. 2011: 55: 388–93. doi:10.1111/ j.1348-0421.2011.00332.x 8. iyer ss, cheng g. role of interleukin 10 transcriptional regulation in infl ammation and autoimmune disease. crit rev immunol. 2012; 32(1): 23–63. 9. ng th, britton gj, hili ev, verhagen j, burton br, wrauth dc. regulation of adaptive immunity; the role of interleukin-10. front immunol. 2013; 4; 129. doi:10.3389/fi mmu.2013.00129 10. o’leary s, o’sullivan mp, keane j. il-10 blocks phagosome maturation in mycobacterium tuberculosisinfected human macrophages. am j respir cell mol biol. 2011; 45: 172–80. 11. abdalla ae, lambert n, duan x, xie j. interleukin10 family and tuberculosis: an old story renewed. int j biol sci 2016; 12(6): 710–717. doi:10.7150/ ijbs.13881 12. wang y, chunmei h, zailang w, hui k, weiping x, hong w. serum il-1 and il-18 correlate with esr and crp in multi-drug resistant tuberculosis patients. j biomed res. 2015; 29(5): 426–28. doi: 10.7555/ jbr.29.20150077 13. amalia e, nindatama m.r, hayati l, handayani d. (2015). identifi kasi mutasi gen rpob ser531leu mycobacterium tuberculosis yang berhubungan dengan resistensi rifampsin. biomed j of indo, vol. 1 no.1. 14. domingo-gomzales r, prince o, cooper a, khader s. cytokines and chemokines in mycobacterium tuberculosis infection. microbiol spectr. 2016; 4(5). doi: 10.1128/microbiolspec.tbtb2-0018-2016. 15. zhang, x., & guo, j. advances in the treatment of pulmonary tuberculosis. j thoracic dis. 2012; 4(6): 617–623. 16. lim yj, yi mh, choi ja, lee j, han jy, jo sh, et al. roles of endoplasmic reticulum stress-mediated apoptosis in m1-polarized macrophages during mycobacterial infections. sci rep. 2016; 6:37211doi: 10.1038/srep37211 17. wang s, zhang j, sui l, xu h, piao q, qu x, et al. antibiotics induce polarization of pleural macrophages to m2-like phenotype in patients with tuberculous pleuritis. sci rep. 2017; 7(1): 14982. doi: 10.1038/ s41598-017-14808-9. 18. elarab ae, garrad h. serum level of interferon gamma (inf), il-12, and il-18 in active pulmonary. aamj. 2012; 10(3). 19. redford p, murray j, o’garra a. the role of il-10 in immune regulation during m. tuberculosis infection. mucosal immunol. 2011; 4(3): 261–70. doi: 10.1038/ mi.2011.7. 20. peñaloza hf, schultz bm, nieto pa, salazar ga, suazo i, gonzalez pa, et al. opposing roles of il-10 in acute bacterial infection. cytokine growth factor rev. 2016; 32: 17–30. doi: 10.1016/j.cytogfr.2016.07.003. 21. ng th, britton gj, hill ev, verhagen j, burton br, wraith dc. regulation of adaptive immunity; the role of interleukin-10. front immunol. 2013; 4: 129. doi: 10.3389/fi mmu.2013.00129 22. butov do, mykhalio k, kuzhko bt. interleukin-10 gene polymorphisms is associated with multi-drug resistance tuberculosis in ukranian population. intl j of mycobac. 2016; 5: 152–3. 123audrey gracelia riwu, et al.: differences of interleukin-18 and interleukin-10 levels copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 23. lihawa n, yudhawati r. hubungan kadar il-10 dan tuberculosis multi-drug resistant. jurnal respirasi. 2015; 1(2): 41–47 24. penaloza h, noguera l, riedel c, bueno s. expanding the current knowledge about the role of interleukin10 to major concerning bacteria. front microbiol. 2018; 9: 2047. doi: 10.3389/fmicb.2018.02047 25. o’garra, redford p.s, mcnab f.w, bloom c.i, wilkinson r.j, berry m. the immune response in tuberculosis. annurev immunol. 2013; 31: 475–527. ijtid vol 3 no 1 jan-maret 2012.indd 19 vol. 3. no. 1 january–march 2012 reccurent laryngeal papilloma nyilo purnami, rizka fathoni department of otorhinolaryngology head and neck surgery medical faculty airlangga university abstract a case of respiratory papillomatosis was reported. the patient suffered from the disease since eight months old with chief complaint progressive hoarseness and dyspnea. it was diagnosed with respiratory papillomatosis and scheduled for performing tracheotomy and continued with the first microlaryngeal surgery (mls). decanulation was taken after 2nd surgery of removing papillomas. finally was reported she got serial of surgery for 22 times during 18 years of age. it was costly and deteriorating quality of life. the problem remains persisted because of frequent recurrences and need for repetitive surgeries. specimen biopsy for histologic examination was shown the signs of hpv infection, papilomatic coated squamous epithel with mild dysplasia and coilocytosis. the threatening of upper airway obstruction is the main important reason for patient's coming. the patency of airway assessed by direct laryngoscopy then the next treatment was decided with schedule of micro laryngeal surgery (mls). finally the mls treatment is just only for temporarily recovery. a further research to define the proper treatment in the future is required, especially for prevention of the diseases related to the viral causes of infection. key words: recurrent, respiratory papilloma, hpv, microlaryngeal surgery introduction respiratory papilloma has been known since the 17th century ago. this disease was first discovered by marcellus donalus as "warts in the throat" that grows on the throat area. papillomas may grow on the mucosa throughout the respiratory tract. vocal cords are a common predilection obtained. the growth of tumors usually occurs in multiple and tends to grow recurrent.1,2 papilloma of the nose is rarely obtained. moreover, it can also be found type of sinonasal papilloma (inverted papilloma) and carcinoma can mimic the form of papilloma in the nose area.3 patients were commonly found in difficulty to breath, dyspnea state, because of airway obstruction and indicated for performing tracheotomy. the disease leads to the expansion of papilloma growth and tends to increase morbidity. considering from this point, early diagnosis is very important and need some efforts to avoid tracheotomy in patients.4 since now papilloma is remain a problem because of its frequent relapses and potential to threat airway obstruction that endangers the lives of patients. this problem more over complicated, cause there is no right treatment to overcome this problem so far. even though various theories have been published but the results are not satisfactory yet.5 the purpose of this paper is to report a case of laryngeal papilloma in our departmen, departement of otorhinolaryngology head and neck, airlangga university, dr. soetomo general hospital. case report march 17, 1994, in ent outpatient, a young woman (raf) 8 months old, was referred from a doctor, orlhns specialist at the dr. soedono hospital, madiun city. she complaint with hoarsness since 3 months before. she looked cahectic. examination on the ear and nose and thorat, showed no abnormalities. on direct laryngoscopy examination with the following results; anamnesis hoarseness, sometimes dyspnea, coughing was not found and ate and drank well. physical examination found mild stridor and intercostals case report 20 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 19−22 retractions. direct laryoscope showed bump of mass which colour white pellucid, uneven, lookslike papilloma pharynx and larynx, in the glottis and supraglottis. it planned for tracheotomy and extirpation with micro laryngeal surgery (mls). picture 1. a scheme of direct laryngoscopy showed mass in the larynx, glottis and supraglottis one day later the patient was performed tracheotomy and followed with mls one month later with the following result, was seen mass bumps, which color is translucent white alike papilloma, located in the pharynx, posterior middle aritenoid, cricopharynx at 3, 6 and 9 hours. then the tumor is extracted until it was clean and performed histopathologic examination. picture 2. specimen from papilloma in the vocal cord biopsy when performing mls, showing tissue sections shaped papilomatic coated with squamous epithel with mild dysplasia and coilocytosis, epithel in the surface and shown stroma with fibrous. patient came at may 11, 1994 (13 days later), without any complaints. on examination found trakeocanul installed and functioning properly. the pathologic anatomy result (no. l. 1598/94) with the conclusion: papilloma with coilocytosis (signs of hpv infection). on september, 1994, four months later papillomas were still in the pharynx and larynx. second mls was planned next one month. tha patient returned 1 month after the mls, there was no complaint and the examination didn't find growth of papilloma again. decanulation was planned. on december, 1994 (one month later), still found little papillomas in the oropharynx and one month later the situation remain similar. decanulation was performed. on february 15, 1995, papilloma became the less prominent. likewise the following months, the situation remains the same until the month of november 1995 (20 months since the patient first came). recently status coming, the patient was 18 years old. she had been performed mls for 22 times surgeries. we recorded the endoscopic examination at august, 2011. the picture wass shown below and after that examination, she performed the 22nd mls for removed the papillomas. picture 4. endoscopic examination on larynx. left picture: papillomas growth on the pharynx, right: papillomas in the glottis. picture 5. endoscopic examination after performed the 22nd, showed minimal papillomas in the larynx. discussion it was found a patient with papillomas which age were 8 months old when in the first arrived. most patients with papilloma have age under 5 years.6 in adults, men are tends common occured, but the incidence in children is almost the same.8 in this case, the patient is a women. picture 3. endoscopic examination (illustration) on larynx, minimal growth of papilloma, and airway is wide enough 21purnami, fathoni: reccurent laryngeal papilloma tumors can grow along the respiratory tract and mouth (aero-digestive tract) and predilection the most common is in the larynx (97.9%–100%).3,4 the growth of papilloma of the nose, are often in the histopathologic form of inverted papilloma (47%) and fungiformis papilloma (50%) than the cylindrical papilloma (3%).15 one of the factors causing papillomas is due to a viral infection. any signs of hpv infection are found in both patients in the form of coilositosis cells, so that convince suspicion the virus as the etiological factor of disease.1,9 this can cause by transmission from mother during delivery (60%).4 but, the gynecological examination form the mother of the patient didn't found signs of condyloma. this possibility can occur because the patient's mother may have recovered from her illness at the time when examination performed (some time later after giving birth). at first, papilloma is often confused with suspicion of allergic disease, asthma or croup.5 similar with the 2nd case, the complaint of runny nose and frequent epistaxis has suffered since 2 years before. papilloma was diagnosed after one year later after the appearance growing mass in the left nose. three months later, there were complaints of sound breathing and short of breath. patients referred with the airway inflammation. but, the thoracic x-ray showed no abnormalities. finally, the direct laryngoscope showed multiple masses in the pharynx and larynx, suggest papillomas. papilloma can show remission with increasing age.6 in this case, a minimal tumor growth after 20 months later and the mls has done frequently. following the papilloma growth getting fewer and steady, therefore, the tracheo-canule could be pulled out. based on studies about papilloma that grows outside the larynx, it gives a better response to treatment (mls).5 serial of microlaryngeal surgery (mls) were performed repeatedly to they that need to excise the tumor, because of that, airway is free and sounds normal again. decreasing of papilloma is expected to facilitate the body's defense system to eradicate the residual lesion, and then would accelerate healing. as is well known, larger size of the tumor, there is a lot of virus and more difficult to control.17 all patients performed emergency tracheotomy at the first time came at the emergency room (second case) and tracheotomy preparation for mls a day after the examination of direct laryngoscope (first case). actually, tracheotomy could be avoided if the patient came and diagnosed earlier. this procedure will cause a wound that may facilitate the implantation of new lesions in lower respiratory tract. expansion to the tracheobronchial founded approximately 83% after tracheotomy.2 this is a concern, especially in the second case where there is growth of laryngeal papilloma, with using tracheocanule can cause new lesions caused by friction of the canule. therefore, it's needed to evaluate the subglottic and trachea due to the expansion of laryngeal papillomas. the first case, where the larynx is clear from papilloma, there is no papillomas growth in tracheobronchial region although it has been performed tracheotomy. this is corresponding with the state that the papillomas growth in the trachea is always preceded by a laryngeal papilloma after tracheotomy.8 we only have two papilloma patients without tracheotomy in our hospital. examination on 11 and 13 months after first mls didn't found any papillomas extension to the tracheobronchial. in 14 patients who performed tracheotomy, several of them were found down expansion to the tracheobronchial after 2nd or 3rd mls (approximately 6–12 months). after that, interval time between mls more short (1–2 months), even in one case the papillomas expansion has reached the left bronchus after the 23rd mls (34 months later). tracheotomy is necessary when there is upper airway obstruction with grade iii jackson or show signs of respiratory failure. meanwhile, when in grade i-ii, could performed mls with insufflations anesthesia techniques. however, this technique has never been applied so far, so tracheotomy performed for procedures such as in the case of the first mls. decanulation done as early as possible when conditions are stable and papilloma growth stopped for at least 6 months. likewise in the first case, growing of the papilloma was slight then pulled out the canule performed 10 months later and next 10 months showed minimal lesion.5 in addition, there are also two patients who have been decanulation after 6th and 10th mls (2 yr and 3yr 5mo). until tracheal, papilloma growth has stopped. the existence of a large papilloma growth (diffuse, multiple) possibly because patient with low immune state (since the age of 8 months has reccurence of cough) thereby increasing aggressiveness of the disease. one factor in accelerating the remission of disease is to increase the immunity of patients, namely how to immunotherapy such as vaccination and administration of interferon.10,16 this treatment is not yet a standard treatment at our institution. inverted papilloma of the nose, which its epithelial growth folding in to the stroma. hpv virus is a one of suspected etiology factor, these tumors are potentially associated with multiple papillomas along the respiratory tract and mouth. this is consistent with the results of studies using pcr techniques (polymerase chain reaction) which have found hpv virus types 6, 11, 16 and 18 in the genital tract and respiratory tract. in the genital tract hpv types 6 and 11 found in the exophytic condyloma, but types 16 and 18 are found on flat condyloma with a high degree of dysplasia and invasive carcinoma. similarly in the respiratory tract, hpv types 6 and 11 associated squamous papilloma and inverted papilloma, while hpv types 16 and 18 are found in squamous carcinomas.18 an important thing to differentiated from squamous papilloma is the nature of the invasive and the tendency to malignancy in inverted papilloma. therefore, patients with inverted papilloma need to be having long-term follow-up of recurrence and risk factors of transformation towards malignancy. interval changes of malignancy ranging from 22 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 19−22 5 to 20 years, with the incidence of 1.5 to 2%.19 there was a report the occurrence of malignancies at the age of 20 years from one patient papillomas since childhood and has performed tracheotomy, ie bronchogenik carcinoma. eventually the patient died after occured metastasis. some experts associate inadequate incision and exposure to carcinogens such as radiation with materials, cigarette smoke with risk factor of reccurence and malignancy. histologic examination found a representation of atypical epithelium and dysplasia.17 aggressiveness of papilloma growth may be explained by histopathology examination, among others associated with the type of papilloma, the degree of cell atipia, mitotic index, the ratio of neoplastic epithelium with the stroma, and the presence of inflammatory cells.13,15 it required a clear description of histopathology analysis results by including the factors mentioned above. likewise, signs of viral infection should be included, for example coilocytosis, nuclear inclusion bodies or multinucleated epithelial cells.15 where possible to do on a regularly, such information will be able to add the epidemiological data that may be useful in overcoming this disease. conclusion it has been reported a case of recurrent laryngeal papilloma, threatened the airway and lead to obstruction in the larynx. tracheotomy should be avoided if patients can come earlier and early diagnosis is established. the problem was still persisted with the high recurrence in children and treated temporary by micro laryngeal surgery. inverted papillomas might have a greater risk for the occurrence of malignant transformation, then long-term follow-up is required. following study is necessary to explore further about pathogenesis of suspected viral infection in pregnant patients as resources to find a strategy in the epidemiological approach to disease prevention. references 1. wallenborn pa jr, roanoke. papillomas of the larynx and pharynx: two case reports. laryngoscope 1976; 11: 1663–8. 2. doyle dj, gianoli gj, espinola t, miller rh. recurrent respiratory papillomatosis: juvenile versus adult forms. laryngoscope 1994; 104: 523–7. 3. buchwald c, franzmann mb, jacobsen gk, lidenberg h. human papillomavirus and normal nasal mucosa: detection of human papillomavirus dna in normal nasal mucosa biopsies by polymerase chain reaction and in situ hybridization. laryngoscope 1994; 104: 755–7. 4. sri herawati. pengobatan papilloma laryngx dengan isoprenosin. dalam kumpulan referat dokter. book iii. lab.upf tht rsud dr. soetomo. surabaya. 5. cohen sr, seltzer s, geller ka, thomson jw. papilloma of the laryngx and tracheobronchial tree in children. 6. strong ms, vaughan cw, cooperband sr, haly gb, clemente macp. recurrent respiratory papillomatosis. management with the co2 laser. ann otol rhinol laryngol 1978: 508–16. 7. dedo hh, jeckler rk. laryngeal papilloma: result of treatment with the co2 laser and podophyllum. ann otol rhinol laryngol 1982; 91: 425–30. 8. kashima hk, shah f, lyles a et al. a comparison of risk factors in juvenile-onset and adult-onset recurrent respiratory papillomatosis. laryngoscope 1978; 89: 1689–95. 9. helinger ph, schild ja, maurizi dg. laryngeal papilloma, review of etiology and therapy. laryngoscope 1968; 78: 1462–74. 10. stephens cb, arnold ge, butchko gm, hardy c. autogenous vaccine treatment of juvenile laryngeal papillomatosis. laryngoscope 1978; 89: 1689–95. 11. arends mj, wylie ah, bird cc. papillomavirus and human cancer. hum pathol 1990; 21: 686–7. 12. jocklin wk. virology. 3th ed. london: apleton & lange, 1998: pp 8–10. 13. shapshay sm, rebeiz ee. benign lesions of the larynx. in: bailey bj, ed. head and neck surgery otolaryngology. vol i. philadelphia: jb lippincott co, 1993: 636–7. 14. lawson w, ho bt, shaari cm, biller hf. inverted papilloma: a report of 112 cases. laryngoscope 1995; 105: 282–8. 15. nielsen pl, buchwald c, nielsen lh, tos m. inverted papilloma of the nasal cavity: pathological aspects in a follow-up study. laryngoscope 1991: 101: 1094–101. 16. lusk rp, mc cabe bf, mixon jh. three year experience of treating recurrent respiratory papilloma with interferon. ann otol rhinol laryngol 1987; 96: 158–61. 17. singleton gt, adkins wy. cryosurgical treatment of juvenile laryngeal papillomatosis. ann otol 1972; 81: 784–9. 18. kashima hk, kessis t, hruban rh, et al. human papilloma virus in sinonasal papillomas and squamous cell carcinoma. laryngoscope 1992; 102: 973–6. 19. friedman i, osborn da. pathology of granulomas and neoplasmas of the nose and paranasal sinuses. new york: churchill livingstone, 1982: 103–13. ijtid vol 7 no 6 may-august 2019.indd 131 vol. 7 no. 6 september-december 2019 research report diversity, dominancy, and periodicity of mosquitoes in filariasis endemic areas in samborejo village tirto district pekalongan regency raden roro upiek ngesti wibawaning astuti1,2, budi mulyaningsih3, raden wisnu nurcahyo4, rc. hidayat soesilohadi5, and suwarno hadisusanto6 1 postgraduate student of biological science faculty of biology, gadjah mada university 2 parasitology division laboratory of animal systematic faculty of biology, gadjah mada university 3 parasitology department faculty of medicine, gadjah mada university 4 parasitology department faculty of veterinary medicine, gadjah mada university 5 laboratory of entomology faculty of biology, gadjah mada university 6 laboratory of ecology faculty of biology, gadjah mada university a coresponding author: upiekastuti@ugm.ac.id abstract vector-borne mosquito diseases are still as a public health problem in the world, including in indonesia. many of mosquitoes species are significantly as vectors of patogen, such as virus, bacteria, protozoan, and helminths due to human health. samborejo village is one of filariasis endemic areas and it is still in a high microfilaria rate each of mosquito species has a differential of distribution, bioactivities pattern, and type of habitat of their breeding sites with others. the objective of this study was to determine the diversity, dominancy, and periodicity pattern of mosquitoes during night time in samborejo village tirto district pekalongan regency. mosquitoes collections were done by landing method, from 6 pm 6 am of in an hour period of collection, for biting and resting activities and also for indoor and outdoor collection respectively. mosquitoes were then identified and the diversity was analized by shannon-wienner index. the total number of each species was served in percent. totally there were 339 collected mosquitoes, consisting of 165 (48.67%) females and 174 (51.33%) males. of all, there were 4 species identified which were culex quinquefasciatus (92.1%), culex tritaeniorhynchus (0.6%), culex vishnui (1.8%), and aedes aegypti (5.5%). samborejo village showed in low diversity with the index of 0.338, and cx. quinquefasciatus to be the dominant species in this area. culex quinquefasciatus also became the frequent species in each period of collection for indoor and outdoor, and it showed the indoor active biting at 9 pm, 01 am, and 03 am; furthermore, the outdoor active biting was at midnight (00) and at 03 am. however, aedes mosquito was showed active biting in earlier, it was at 6 pm, 7 pm, and at 02 am. keywords: diversity, dominancy, mosquito, periodicity, samborejo village. abstrak penyakit tular vektor oleh nyamuk masih menjadi masalah kesehatan masyarakat di dunia termasuk di indonesia. berbagai jenis nyamuk memiliki peran sebagai inang perantara (vektor) dalam penularan agen penyakit baik dari jenis virus, bakteri, protozoa, hingga cacing, dan kalurahan samborejo adalah salah satu daerah endemik filariasis di kabupaten pekalongan dengan tingkat mikrofilaria yang masih cukup tinggi. pola distribusi, keragaman, perilaku setiap nyamuk beserta lingkungannya merupakan kajian yang masih sangat terbatas informasinya. tujuan penelitian ini adalah menjelaskan keragaman, dominansi, dan pola aktivitas nyamuk pada malam hari di kalurahan samborejo kecamatan tirto kabupaten pekalongan. koleksi nyamuk dilakukan dari pukul 18.00 06.00wib pada periode setiap satu jam, dengan metoda landing biting, dan nyamuk dikoleksi pada saat biting dan resting baik indoor maupun outdoor. nyamuk hasil koleksi diidentifikasi dan data keragaman dianalisis dengan shannon-wienner index, data jenis dan jumlah nyamuk ditampilkan dalam persen. sejumlah 339 ekor nyamuk dikoleksi terdiri atas 165 ekor (48.67%) adalah nyamuk betina dan 174 ekor (51.33%) adalah nyamuk jantan, dengan jenis culex quinquefasciatus (92.1%), culex tritaeniorhynchus (0.6%), culex vishnui (1.8%), dan aedes aegypti (5,5%). kelurahan samborejo menunjukkan keragaman nyamuk yang rendah, dengan nilai index 0,338, dan nyamuk 132 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 131–136 cx. quinquefasciatus menunjukkan dominansi dan dapat ditemukan pada setiap periode koleksi baik indoor maupun outdoor. pada periode waktu pukul 21.00, 01.00 dan 03.00 wib nyamuk cx. quinquefasciatus menunjukkan puncak periode aktif indoor, sedangkan puncak aktivitas outdoor adalah pada pukul 24.00 dan 03.00 wib. untuk nyamuk ae. aegypti dapat ditemukan pada periode lebih awal yaitu pukul 18.00, 19.00, dan pukul 02.00 wib. kata kunci: diversitas, dominansi, periodisitas, nyamuk, kalurahan samborejo. introduction mosquitoes have been known as one of haematophagus insects and some of them can serve as vector for transmitting the protozoan, worms, or virus, agent of human diseases in many parts of the world, including in indonesia.1,2 lymphatic filariasis (lf) is one of public health problems, caused by one of 3 species of filarial nematodes, namely, wuchereria bancrofti, brugia malayi, and brugia timori. the microfilariae are found in the human blood only around midnight.3-4 it is transmitted by mosquitoes and globally about 120 million people in 73 countries have been affected.1,2,4,5 in indonesia, there is an increasing number of lymphatic filariasis endemic areas in which filariasis cases are increased significantly. it was about 8,000 cases in the year 2004 and to more than 14,900 cases in 2016.6-8 study by febrianto9 found that there was 7.6% from 76 of respondents showed the positive microfilaremia in samborejo village. some mosquitoes are cosmopolitan and live nearby human habitations.10 in indonesia, 20 mosquitoes species have been known as filariasis vectors, 8 species of anopheles spp., 3 species of culex spp., 6 species of mansonia spp., one species of aedes, armigeres, and coquillettidea,1 and some of them have biting behaviour in the night time.11 the nocturnal active biting of vector could be considered as an intermediary for filariasis transmission. studies on the distribution, behavior, and ecology of mosquitoes in filariasis endemic areas are significant studies in studying parasitic-vector interactions and are studies of novelty that are still very limited in information.12 samborejo village is located in pekalongan regency central java and the ecotype is a semiurban areas. this village was an endemic area for urban filariasis caused by wuchereria bancrofti.9 however, there is unwell known in the distribution, species variation, dominancy and periodicity of mosquito population in samborejo village pekalongan regency. the purpose of this study was to examine the diversity, dominancy, and active period of mosquitoes during night time in samborejo village. material and method materials mosquitoes were the subjects of this study. the materials for mosquitoes collection were aspirator, hand net, paper cup/coffee cup with gauze covered, and cotton with sugar solution. microscope, cover glass petridish, and small brush were provided for mosquitoes identification. methods the study was under consideration with the ethical commission of faculty medicine gadjah mada university, number ref: ke/fk/0612/ec/2017. study area. samborejo village in tirto district pekalongan regency was selected as the study area. it was semiurban types for the settlement. mosquitoes collection. mosquitoes collections were done every hour from 6 pm 6 am by landing bite method and mosquito’s net in the selected house of the area. mosquitoes were put in paper cup covered by gauze. then, mosquitoes were identified by using identification key book, mosquito pictorial key of indonesia.13 data were tabulated and analyzed. the environmental parameter, air temperature (ºc) and humidity (%), of selected location of settlement were measured. data analysis. the number of mosquitoes from the study area was performed in percent. the diversity of mosquito species was analyzed by shannon weinner diversity index.14 correlation analysis of ecological parameters with the active period of the mosquitoes was done by correlation test (spss 20. version). result and discussion diversity from samborejo village, totally there have been 340 collected mosquitoes that consisted of 175 males and 165 females (table 1). based on the shannon-wienner index samborejo village was low in diversity (0,338). it was understood that there were only 2 genera, culex and aedes has collected. this finding was similar to our previous study in other the endemic areas, in pasirsari villages (pekalongan city) and simbangkulon villages (buaran distric pekalongan regency). in the both areas the total number of 323 adult female mosquitoes had been collected and consisted of 8 species of the genus culex and 1 species of aedes aegypti.15 however, in jenggot village (pekalongan city) it is showed moderate diversity and more various the mosquitoes genus, which were 5 of mosquito genera, which were genera of culex, aedes, armigeres, anopheles, and the genus of lutzia. this finding is different from the results of the research conducted by arimurti (2008) which concluded that the cx. quiqueefas ciatus mosquito from pekalongan 133astuti, et al.: diversity, dominancy, and periodicity of mosquitoes table 1. diversity and total number of female mosquitoes in samborejo village tirto district pekalongan regency. species number of mosquitoes total % indoor biting outdoor biting indoor resting outdoor resting culex quinquefasciatus 61 49 7 35 152 92.12 % culex tritaeniorhynchus 0 1 0 0 1 0,60 % culex vishnui 1 1 0 1 3 1,83 % aedes aegypti 8 0 0 1 9 5,45 % total number of mosquito 165 100 % city and regency showed a high level of diversity with polymorphism reaching 100% based on the rapd-pcr method.16 the culex and aedes are culicinae mosquito, they were belong to culicidae family and the order of diptera. morphologically the adult female mosquito in the genera of culex and aedes may distinguished mostly by the head and thorax character. in both genera, at the head the palpus were shorter than the proboscis, however in the scutum of thorax’s culex mosquito covered pale brown scales and in aedes the scales usually dark and sometimes with contrast white scales.13 the adult female cx. quinquefasciatus may be recognized easily with other culex species (cx. tritaeniorhynchus or cx. vishnui) by the dark brown without pale band of the proboscis and the terga of abdomen was dark with broad white basal bands.13 geographically, mosquito is cosmopolite insect, and it widespreads in the tropic and subtropic region,10 and the changing of the environment will affect the insect activity in which will have an impact on diversity and distribution.1,4,10 dominancy and periodicity culex quinquefasciatus to be the dominant in the area of study (92.12%). the dominancy this mosquito also is showed in the different endemic areas in pekalongan, such as 66.67% in pekalongan selatan district17, 97.3% in buaran district18, and in samborejo the density was 5.25 mosquito/human/ hour.9 as in table 1, cx. quinquefasciatus was abundant and it was the most frequent active in each of period of collection for indoor and outdoor collection. every mosquito species has its own distribution, behaviour pattern and character of its habitat different from others. the daily behaviour pattern of mosquito activities will occur at day or night time depending on the species.19 there was limited information in the periodicity of biting and resting behaviour of cx. quinquefasciatus, especially in filariasis endemic areas. this study will improved the collecting data of its bionomic. in this study, the period of collection from 6 pm-6 am showed that only cx. quinquefasciatus was the most frequent active, and the indoor activities was rather higher than the outdoor ones (figure 1). resting activities was showed the earlier period of collection, it was around 9 pm for indoor and 7 pm for outdoor (figure 2). this phenomenon may be due to the moving of this mosquitoes from the breeding sites to the houses before they get for the source of blood. this mosquito was mostly active in outdoor biting and the period of active was at 01 and 4 am18, while in pasir sari village pekalongan selatan district the peak of active biting period was at midnight and around 02 pm.15 this peak timing of the mosquito periodicity may synchronous with the abundant of mf in the peripheral blood. as sack20 has said that the secretion of overnight host melatonin may introduce the release of w. bancrofti mf in the blood circulation. the host’s melatonin profile study is revealed that there was significantly increased and the peak of the concentration was at 0 4 am. besides that, the total mf also significantly increased from about 82% at 22 pm and reached out 100 % at 1 am. at the next period the total number of mf was still high, it was around 98% and 80% at 2 am and 3 am respectively, then it was drastically reduced, from 62% at 4 am to 2% at 10 am.20 the study in the some filariasis endemic areas in pekalongan is showed that cx. quinquefasciatus caughted more frequently during the time of collection rather than other species. as in pasirsari village, in pekalongan selatan district pekalongan city and in paweden village pekalongan regency is showed that 76%, 57.7% and 98.9% respectively were cx. quinquefasciatus.15,18,19 the mosquito existence was important for transmitting the parasite. some studies was reported that the environment around settlement and human activities in the night time had significant effect for increasing the risk factor of people to get the infection.9,19 windiastuti et al.17 has said that the existence of breeding places, resting places around houses will increased the risk of infection 8.7 times and 2.17 times respectively, even it would be 9.03 times to get infection for people who has active in the night time. this study is revealed that on the period of midnight to 4 am was the active peak of cx. quinquefasciatus (figure 1b). there was new finding that ae. aegypti mosquito was active indoor in the early night, it was about at 6 pm and at 2 am in early morning (figure 1a). other species, cx. vishnui and cx. tritaeniorhyncus were known as japanese encephalitis (je) virus.2,4 in this area the occurrence proportion of the two species was low. this may because of they more like the animal rather than human as their source of blood, such as chicken and domestic birds. 134 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 131–136 a b 18.00 19.00 20.00 21.00 22.00 23.00 00.00 01.00 02.00 03.00 04.00 05.00 06.00 18.00 19.00 20.00 21.00 22.00 23.00 00.00 01.00 02.00 03.00 04.00 05.00 06.00 figure 1. mosquitoes diversity and periodicity of indoor (a) and outdoor (b) biting activities in samborejo village. a 18.00 19.00 20.00 21.00 22.00 23.00 00.00 01.00 02.00 03.00 04.00 05.00 06.00 b 18.00 19.00 20.00 21.00 22.00 23.00 00.00 01.00 02.00 03.00 04.00 05.00 06.00 figure 2. mosquitoes diversity and periodicity of indoor (a) and outdoor (b) resting activities in samborejo village. 135astuti, et al.: diversity, dominancy, and periodicity of mosquitoes ecological parameter in this study the ecological parameter affected in various indoor and outdoor activities of mosquitoes, especially for cx. quinquefasciatus both for the biting and for the resting activities (table 2). table 2. correlation of ecological parameter to the activities of mosquito in samborejo village tirto district pekalongan regency. species level of coefisien correlation indoor biting outdoor biting indoor resting outdoor resting temperature (°c) 0,0663 -0,484 0,414 0,384 humidity (%) -0,513 0,398 0,150 -0,437 wind velocity -0,977* -0,107 0,017 0,801 each mosquito species needs specific ecological requirement for their survival, distribution, and abundance.4,21 there was limited information of the effects of temperature and humidity to the mosquito activities in such filariasis endemic areas. moise et al.21 has stated that cx. quinquefasciatus abundance increased significantly in may and june annually as their observed in 2006 to 2010, and temperature was positively affected for the mosquito abundance. as shown in table 2, there was no correlation among the temperature to the indoor biting, humidity to the indoor resting, and wind velocity to outdoor biting and indoor resting. low correlation in positive and negative was showed by the temperature, humidity to the indoor or outdoor, and to biting and resting activities. however, there was a strongly negative correlation of wind velocity to the indoor biting activities and strong positive correlation to the resting activities. it is said that by increasing the wind velocity will significantly reduce the indoor biting activities. however, it significantly will increase the number of mosquitoes to rest. for comparison, the study in paweden village pekalongan regency is showed that there was moderate negative correlation (-0.50 and -0.64) between temperature and the cx. quinquefasciatus mosquito for indoor and outdoor biting activities. it was mean that as increasing temperature will reduce the indoor and outdoor biting activities, however the humidity has strong positive correlation (0.75) to the activities.18 based on the results, the abundance and periodicity were likely match with the microfilaria in the blood of patient. this condition will maintain the high risk of filariasis transmission. for that purpose, vector surveillance should be considered to gain the filariasis elimination program together with mass drug administration (mda). conclusion this study is revealed that samborejo village showed low diversity (0,338) of mosquitoes fauna, and culex quinquefasciatus to be the dominant species in this area. the indoor biting activities of cx. quinquefasciatus were at 9 pm, 01 am, and 03 am; furthermore, the outdoor biting activities were at midnight (00) and at 03 am. however, aedes mosquito was showed active biting in earlier periods, which were at 6 pm, 7 pm, and at 02 am. conflict of interest there was no conflict of interest for this paper. acknowledgement our sincere thanks, goes to indonesia ministry of research and technology-dghe, that funded this research through “hibah disertasi doktor”, no. 2535/un1-p.iii/ ditlit/lit/2017; local government and medical community services of samborejo village, and our field assistant atikah fitria s.si; ika nurcahyani s.si; rudi nirwantoro s.si; and muhammad yuski,s.si. references 1. sutanto i, ismid is, sjarifuddin pk, and sungkar s. parasitologi kedokteran. edisi ke 4. badan penerbit fk ui. p: 245-279. 2013 2. vythilingam i. mosquitoes of public health important: mosquitoes and public health. labert academic publisher. pp: 80. 2016. 3. manguin s, mouchet j, and carnevale p. main topics in entomology: insects and disease vector, green trends in insect control, chapter 1. ed: lopez, o and fernandez-balanos j. rsc green chemistry no. 11. royal society of chemistry. www.rsc.org. p: 1-16. 2011 4. fenwick a. accelerating progress towards elimination of some neglected topical diseases (ntds). international congress on parasitology (icopa). 2018. 5. who. elimination of lymphatic filariasis in the south-east asia region. reports of the 8th meeting of the regional programme review group, srilanka, 28-29 april, 2011. 6. aditama ty. control policy ntd’s in indonesia. asean seminar on neglected tropical diseases committee report. jakarta 28-29 september. 2012. 7. kemenkes ri. filariasis di indonesia. buletin jendela epidemiologi. pusat data dan surveilans epidemiologi kemenkes ri., vol 1. issn 2087-1546. juli 2010. 8. kemenkes ri. peraturan menteri kesehatan ri no. 29 tahun 2014 tentang penanggulangan filariasis. 190 hal. 2015. 9. febrianto b, maharani aip, dan widiarti. faktor resiko filariasis di desa samborejo kecamatan tirto kabupaten pekalongan jawa tengah. bul. penel kesehatan, vol.2036, no. 2, pp: 48-58. 2008. 10. rahman gms. manholes as an important breeding sites for culex mosquitoes in gazipur city corporation, bangladesh. bangladesh j. zool. 45(2): 139-148. 2017. 136 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 131–136 11. astuti unw, hadisusanto s, dan sari iy. periodisitas nyamuk culex quinquefasciatus say, 1823 dalam hubungannya dengan potensi transmisi filariasis di kelurahan ngampilan dan notoprajan kecamatan ngampilan yogyakarta. seminar nasional biologi. yogyakarta. pp: 1108-1116. 2010. 12. takken w and koenraadt cjm. ecology of parasite-vector interaction, ecology and control of vector-borne diseases 3, doi: 10.3920/978-90-8686-744-8_1. wageningen acad.publ. 2013. 13. depkes ri. buku identifikasi nyamuk di indonesia. 2015. 14. ismaini lm, lailati, rustandi d, sunandar. analisis komposisi da keanekaragaman tumbuhan di gunung dempo, sumatera selatan. pros sem nas masy biodiv indon (1):1397-1402. 2015. 15. astuti unw, poerwanto sh, handayani ns, and hadisusanto s. abundance and periodicity of culex quinquefasciatus say 1823 (diptera: culicidae) as early indicator for filariasis transmision in pekalongan central java indonesia. aip conference proceedings.1744.020045.doi:10.1063/1.4953519. 2016. 16. arimurti arr. keanekaragaman genetik nyamuk vektor filariasis culex quinquefasciatus say. 1823 (diptera: culicidae) di kota dan kabupaten pekalongan dengan metode pcr-rapd. the journal of muhammadiyah medical laboratory technologist. 2008. 1(2): 42-61. 17. windiastuti ia, suhartono, and nurjazuli. hubungan kondisi lingkungan rumah, sosial ekonomi, dan perilaku masyarakat dengan kejadian filariasis di kecamatan pekalongan selatan kota pekalongan. jurnal kesehatan lingkungan indonesia. vol 12 (1): 51-57. 2013. 18. astuti unw and jasmi ra. periodisitas nyamuk nokturnal di daerah endemik filariasis di pekalongan jawa tengah. laporan penelitian, hibah penelitian biodiversitas tropika dosen untuk pengembangan materi pembelajaran. fakultas biologi ugm. 2015. 19. price pw, denno rf, eubanks nd, finke di, and kaplan i. insect ecology: behaviour, populations and communities. cambridge university press. 2011. 20. sack rl. host melatonin secretion is a timing signal for the release of w. bancrofti microfilaria into the circulation. medical hypotheses, elsevier, 73: 147-149. 2009. 21. moise ik, riegel c, and muturi ej. environmental and socialdemographic predictors of the southern house mosquito culex quinquefasciatus in new orleans, louisiana. parasites & vectors, 11:249.2018. 79 vol. 6. no. 4 january–april 2017 research report proportion of hbsag and hbeag positive in maternal patients and their hbsag positives babies with immunoprophylaxis of hbv immunization in dr. soetomo general hospital, surabaya melina rosita tanadi1a, maria inge lusida2, hermanto tri joewono3 1 faculty of medicine universitas airlangga surabaya 2 departement of microbiology, tropical disease center, universitas airlangga, surabaya, indonesia 3 departement of obstetrics and gynecology, soetomo general hospital, surabaya, indonesia a corresponding author: melinarosita@gmail.com abstract hepatitis b virus (hbv) can be transmitted vertically from mother to her baby. mothers with hbsag and hbeag positives have more risk of transmitting hbv to her baby rather than hbsag positives only. the aim of this study is to determine the proportion of maternal patient with hbsag and hbeag positives and their hbsag positives babies with immunoprophylaxis of hbv immunization. this study was performed by analytical observation using medical records in 2013-2014 at obstetric and gyn ecology department, dr. soetomo hospital. the samples were all maternal patients (3796) during that period and also their babies from hbsag positives mothers. unfortunately, several original medical records were not available. thirty two (0,85%) out of 3781 maternal patients were found to be hbsag positives, and three (9,37%) of 32 patients with hbsag positives were hbeag positives. from 32 mothers who were positive hbsag, 22 complete medical records of their babies were found and all of them (100%) had been given hepatitis b immunoglobulin (hbig) and hepatitis b vaccine less than twelve hours after birth. in three cases of the babies from hbeag positives mothers which had been given prophylaxis properly, two cases each of which was with caesarean and spontaneous delivery were hbsag negatives. interestingly, the other one which born with spontaneous delivery was found to be hbsag positives. further study in this hbsag positives baby, especially in analyzing its hbv dna is needed. the epidemiology of hepatitis b in maternal patients, especially that with complete and neat data needs further research. keywords: hbsag, hbeag, hepatitis b, maternal patient, vertical transmission abstrak virus hepatitis b (vhb) dapat ditularkan secara vertikal dari ibu ke anak. pada ibu dengan hbsag dan hbeag positif lebih beresiko menularkan vhb pada anaknya daripada hanya positif hbsag. penelitian ini bertujuan untuk mengetahui proporsi ibu bersalin dengan hbsag dan hbeag positif dan bayi dengan hbsag positif yang telah diberi imunoprofilaksis terhadap hbv. penelitian ini dilakukan dengan teknik observasional analitik menggunakan rekam medis pasien ibu bersalin periode tahun 2013-2014 yang dirawat di departemen obstetri dan ginekologi rsu dr. soetomo. sampel penelitian ini adalah semua ibu bersalin (3796) pada periode tersebut serta bayi dari ibu bersalin yang positif hbsag. sayangnya, ada beberapa data asli yang tidak tersedia. dari 3781 pasien ibu sebanyak 32 (0,85%) pasien ibu positif hbsag. dari ibu positif hbsag ditemukan tiga (9,37%) pasien hbeag positif. dari 32 pasien ibu positif hbsag, dapat dikumpulkan 22 rekam medis bayi yang lengkap dan semuanya (100%) sudah diberi imunoglobulin hepatitis b dan vaksin hbv kurang dari 12 jam sejak lahir. pada tiga kasus anak dari ibu positif hbeag yang telah diberi profilaksis, ditemukan negatif 80 indonesian journal of tropical and infectious disease, vol. 6. no. 4 january–april 2017: 79–83 introduction hepatitis b virus (hbv) infection was one of major global health problems because about two billion people in the world have been infected with hbv and more than 350 million people are chronic carriers.1 in 2013, indonesian basic health research (riskesdas) also found the highest cause of the prevalence of hepatitis in indonesia is by hbv (21,8%).1 hepatitis b virus can be transmitted parenterally, in contact with blood or other body fluids. in endemic area of chronic hbv infection, transmission mainly through vertical transmission, especially during perinatal and early childhood.2 approximately 3,9% of pregnant women in indonesia in 2007 as a carrier of hbv infection.3 risk factors of hbv infection can be divided based on agent, host, and environment. people who are susceptible to the infection of hbv are people who live in asia, africa, and other regions with high prevalence of hbv infection, has not been vaccinated, had sexual intercourse with someone who had hbv infection, living with a person infected with hbv, homosexuals, using parenteral drugs, undergoing hemodialysis, and someone who is undergoing chemotherapy or other immunosuppressive treatment.4 in hemodialysis patients study, the most common hbv agent in east java, especially in surabaya had genotype b,5 whereas the most common subtype in java was adw.6 hepatitis b virus infection can be diagnosed with a laboratory test. screening of hbv infection commonly by hepatitis b surface antigen (hbsag) tests. hepatitis b surface antigen appears in blood serum after 1-10 weeks of exposure to hbv. hepatitis b surface antigen test has a specificity of 99.7% and a sensitivity of 100%.7 after discovering someone has positive hbsag, further examination with hbeag test is also recommended. hepatitis b envelope antigen is a test to determine whether there has been virus replication.8 centers for disease control and prevention (cdc) recommends that all pregnant women should be screened with hbsag marker. if it is found only hbsag positive, then the risk of perinatal transmission is 10%.9 while newborns of an hbv carrier woman with hbsag and hbeag positive, have 90% risk of becoming infected and carrier.10 it is because of the baby tolerance to virus antigen. if these babies are not treated properly, it can develop into hepatocellular carcinoma (hcc) and leads to death after decades. however, hbv infection can be prevented by providing effective vaccination. hepatitis b virus vaccine is effective to 90% adults and children if given in three doses (three injections with a period of 6 months). injections with hepatitis b immunoglobulin (hbig) and hbv vaccine may also be granted because hbig can give immediate but temporary protection against the virus until hbv vaccine is effective.11 infection of hbv is also very likely to transmit in medical personnel who assist the delivery. prevalence in this group varies between 10%-20%.9 therefore, right education and techniques are needed for helping the delivery process in pregnant women and to care the infants who are at risk to be vertically infected by hbv. this study will analyze the proportion of maternal hbsag and hbeag positive and hbsag positives babies with hbv immunoprophylaxis (hbig and hbv vaccine) which was administrated to the babies of hbsag positive maternal patients in dr. soetomo general hospital during the period of 2013-2014. this information is expected to be useful as information to the public and government in order to develop the prevention to vertical transmission of hbv infection. this study was conducted in dr. soetomo general hospital because: firstly, it has a lot of patients so that we can get a large number of samples. secondly, it is a type a hospital in indonesia which means it is supposed to have the best health care service in indonesia. therefore from knowing how is the result of prevention of vertical transmission of hbv in dr. soetomo general hospital, this study will show how far indonesia has been handling the prevention of hbv transmission. material and method this study used a cross-sectional design. materials used in this research were secondary data: medical records documents of maternal patients who were checked using hbsag and hbeag test, and her babies were born during the period of 2013-2014 in the departement of obstetric and gynecology dr. soetomo general hospital surabaya. the samples of this study were chosen by total sampling technique. variables examined in this study were hbsag and hbeag positives status of maternal patients and immunization status (passive and active) of her babies from hbsag positive mothers. maternal patients that hbsag positive, will be tested with hbeag marker. at that time, the examination of hbsag and hbeag were done in laboratory of clinical pathology at dr. soetomo general hospital using elisa technique. data from the observation that had been collected, then it was processed and analyzed descriptively using simple statistics (percentages). hbsag pada dua kasus dengan tindakan persalinan caesar dan spontan. menariknya, satu kasus lainnya dengan tindakan persalinan spontan ditemukan positif hbsag. diperlukan penelitian lebih lanjut mengenai analisis dna vhb pada bayi yang positif hbsag ini. perlu pula penelitian lebih lanjut mengenai epidemologi hepatitis b pada ibu bersalin dengan data yang jelas dan lengkap. kata kunci: hbsag, hbeag, hepatitis b, pasien bersalin, penularan vertikal 81tanadi, et al.: proportion of hbsag and hbeag positive result and discussion from the data in the period of january 2013-december 2014 that had been collected, 47 out of 3796 (1,24%) maternal patients were valid and found to be hbsag positives, but unfortunately only 32 patients out of 3781 (0,85%) that were valid and met hbsag and hbeag data. this was because medical records are used for another research by another medical personnel. this proportion of maternal patients who were hbsag positives (0,85%) is smaller compared with the results in 2007 which prevalence of maternal patients with hepatitis b in indonesia was 3,9%.3 the result of this study does not represent the population because this study took samples from a refer center hospital. therefore, only rare and complicated cases are handled here. besides that, many cases of maternal patients with hbsag positives can be handled in the local hospital. the proportion of hbsag negative was 99,15%. out of 32 hbsag positive maternal patients, there were 3 (9,37%) hbsag and hbeag positive maternal patients. from 32 positive hbsag maternal patients, there were 22 medical records of the babies that successfully collected. in table 1, if the frequency of hbsag positive maternal patients compared between 2013 and 2014, the higher proportion was in 2014 (0,97%) although there were the fewer number of maternal patients than in 2013. in table 2, from the maternal patients with complete hbsag and hbeag data, the highest frequency of hbsag positive maternal patients in 2013 was in may, while in year the 2014 were in february, march, may, and october which had a same number of patients (two patients). bhatt (2000) study showed that no seasonal distribution for hbv infection.12 there was no incidence of hbv infection that had drastic increase in a particular season, only hepatitis a virus (hav) which has a certain seasonal cycle. its peaks in march and september.13 hepatitis a virus infection is an acute disease and can be cured and all of the transmission of hav through the fecal-oral route. hepatitis b virus and hepatitis c virus (hcv) infection can develop to be a chronic disease so that the carrier of hbv and hcv infection always transmit the virus every year and month. according to soemoharjo,6 the transmission of hbv can pass through contact with the not intact body with blood, mucus of infected persons and also through sexual intercourse to infected persons. therefore, this allows hbv infect anyone who had not infected and contact with blood on body fluid of hbv patients. based on delivery techniques in hbsag positive maternal patients (table 3), the most frequent technique used was caesarean (62%). based on chang et al (2014) research, were found a decrease of hbv transmission in caesarean delivery technique,14 because the doctors who helped childbirth process knew that there were higher risks for spontaneous delivery for increasing the transmission of hbv. hepatitis b virus from amniotic fluid can enter the body through the wound in the baby’s skin that happened because of entering the birth canal or accidentally swallowed if there was a contraction of the uterus (materno fetal micro infusion).6 if there is no contraction during table 1. the frequency of positive hbsag and hbeag maternal patients in 2013 and 2014 year n hbsag positives %incomplete data complete data 2013 7 2541 20/2541 0.79% 2014 8 1240 12/1240 0.97% total 15 3781 32/3781 0.85% table 2. frequency of maternal patients with hbsag positive, and hbsag, hbeag positive month 2013 2014 hbsag positives hbsag positives hbeag positives hbsag positives hbsag positives hbeag positives jan 3 1 1 0 feb 0 0 2 0 mar 1 0 2 0 apr 1 0 0 0 may 5 0 2 0 jun 1 1 0 0 jul 0 1 1 0 agt 0 0 1 0 sept 0 0 1 0 oct 1 0 2 0 nov 1 0 0 0 dec 4 0 0 0 total 17 3 12 0 table 3. delivery techniques in hbsag positives maternal patients delivery techniques hbsag positives (%) hbsag and hbeag positives (%) spontan 11/29 (38%) 2/3 (67%) caesar 18/29 (62%) 1/3 (33%) total 29 (100%) 3 (100%) 82 indonesian journal of tropical and infectious disease, vol. 6. no. 4 january–april 2017: 79–83 caesarean delivery then it will decrease the risk of hbv transmission. in this study out of three maternal patients who had hbsag and hbeag positive, there were two patients who had spontaneous birth. it cannot be a reference because a small number of patients cannot represent the overall picture of delivery techniques that often used in hbeag positive maternal patients. in table 4, both hbsag positive and hbsag, hbeag positive maternal patients had the significant difference in the gestation age distribution, which term gestation had the higher frequency. incidence of low birth weight and prematurity increased in women with acute hepatitis b.15 another study found that carrier hbsag women had higher risk to become preterm labor.16 however, wong et al research didn’t find any association between hbv infection in maternal patients with preterm labor, same as this research results in dr. soetomo general hospital.17 based on the occupation as seen in figure 1, the most frequent occupation of maternal patients was housewife (13 patients), followed by private employees (10 patients). in 2014, a study in nigeria at a university of medicine, the highest number of jobs in the antenatal women who were hbsag positive was unemployment (13 among 42 women).18 the result study in nigeria is similar to the result study in dr. soetomo. general hospital which is the most frequent occupation was housewife or unemployment. the previous result’s study showed that low socioeconomic status that initiates multi-sexual partners, unprotected sexual intercourse was more susceptible to get sexually transmitted disease.18 table 4. gestation age distribution in hbsag positives and hbsag, hbeag positives patients gestation age hbsag positives (%) hbsag and hbeag positives (%) preterm 3/29 (10,34%) 0/3 (0%) aterm 26/29 (89,65%) 3/3 (100%) total 29/29 (100%) 3/3 (100%) figure 1. bar diagram of hbsag positive maternal patiens based on occupation distribution based on their education level, highest frequency in hbsag positive maternal patients were senior high school graduate (50%). hbsag positive maternal patients prophylaxis action quantity (n=22) percentage hepatitis b vaccine 22 100% hbig 22 100% the remaining 10 files were not found in the medical records storage. all of 22 babies were given hbig and hbv vaccine less than 12 hours after birth (100%). this result suggests that the prevention of transmission of hbv infection in dr. soetomo general hospital had been done properly. as seen in table 6, all children from hbeag positive maternal patients had been given immunoprophylaxis (hbv vaccine and hbig) less than 12 hours after birth and had undergone complete vaccination three times. table 6. data of babies from hbeag positive maternal patients delivery technique gravida administration of hepatitis b vaccine and hbig complete vaccination (3 x) hbsag status of the child at this time mother a spontaneous multigravida yes complete positive mother b spontaneous multigravida yes complete negative mother c caesarean multigravida yes complete negative two children from two maternal patients (one mother who delivered with caesarean and another mother with spontaneous delivery) were found to be negative hbsag. but interestingly, the other child of the mother who had spontaneous delivery was hbsag positive even though her child had been given phrophylaxis properly. this positive hbsag of the child may be caused by the presence of escape mutant hbsag in infants who had been given hbig and hbv vaccination. hepatitis b surface antigen (hbsag) with arginine replacement for glycine at amino acid 145 is the most common escape mutant hbsag found in several clinical samples.19 all infants who fail to respond to immunophrophylaxis were born from hbeag positive mothers who had hbv dna levels ≥6 log10 copy/ml. the existence of hbv dna in cord blood also reflects the failure to respond passive and active immunization.20 from the result in this study, there was no tendency of transmission of hbv through certain delivery technique, same as in hu et al jhs e shs b uk figure 2. circles diagram of hbsag positive maternal patients based on the education based on their education level, highest frequency in hbsag positive maternal patients were senior high school graduate (50%) (figure 2). the raw education data showed that the population with lower education status (elementary, junior high school, and senior high school) were more prone to be infected with hepatitis b. it was because of lack of education in promotive prevention of the disease given in early grade school (elementary, junior high school, and senior high school) (figure 2). this study found only 22 complete medical record documents of the babies from 32 hbsag positive maternal patients (table 5). the remaining 10 files were not found in the medical records storage. all of 22 babies were given hbig and hbv vaccine less than 12 hours after birth (100%). this result suggests that the prevention of transmission of hbv infection in dr. soetomo general hospital had been done properly. table 5. frequency of prophylactic administration of the baby from hbsag positive maternal patients prophylaxis action quantity (n=22) percentage hepatitis b vaccine 22 100% hbig 22 100% table 6. data of babies from hbeag positive maternal patients delivery technique gravida administration of hepatitis b vaccine and hbig complete vaccination (3 x) hbsag status of the child at this time mother a spontaneous multigravida yes complete positive mother b spontaneous multigravida yes complete negative mother c caesarean multigravida yes complete negative 83tanadi, et al.: proportion of hbsag and hbeag positive as seen in table 6, all children from hbeag positive maternal patients had been given immunoprophylaxis (hbv vaccine and hbig) less than 12 hours after birth and had undergone complete vaccination three times. two children from two maternal patients (one mother who delivered with caesarean and another mother with spontaneous delivery) were found to be negative hbsag. but interestingly, the other child of the mother who had spontaneous delivery was hbsag positive even though her child had been given phrophylaxis properly. this positive hbsag of the child may be caused by the presence of escape mutant hbsag in infants who had been given hbig and hbv vaccination. hepatitis b surface antigen (hbsag) with arginine replacement for glycine at amino acid 145 is the most common escape mutant hbsag found in several clinical samples.19 all infants who fail to respond to immunophrophylaxis were born from hbeag positive mothers who had hbv dna levels ≥6 log10 copy/ml. the existence of hbv dna in cord blood also reflects the failure to respond passive and active immunization.20 from the result in this study, there was no tendency of transmission of hbv through certain delivery technique, same as in hu et al (2013) study which stated that if hepatitis b vaccine and hbig were given immediately after birth, the choice of labor procedure didn’t determine the tendency of hbv transmission. conclusion the proportion of maternal patients who was hbsag positive in dr. soetomo general hospital in period of 2013-2014 was 0,85%. some missing data should take into consideration. out of 32 hbsag positive maternal patients, there were three maternal patients who were positive hbeag (9,37%). all of the children with complete medical record documents in this study (22 children) had been given hbig and hbv vaccine properly (100%). a good management of the medical records is needed in every hospital so that the practitioner who used data for research will get correct and complete data, therefore the results will be more accurate. acknowledgement the author would like to thank to all reviewers for their excellent review of the manuscript. references 1. riset kesehatan dasar [internet]. badan penelitian dan pengembangan kesehatan kementerian kesehatan ri. 2013. available from: http:// www.depkes.go.id/resources/download/general/hasil riskesdas 2013.pdf 2. df de pm, t m, dj t, ra ves, jl n-s, f st, et al. prevalence and factors associated with hepatitis b virus infection among senior citizens in a southern brazilian city. hepat mon. 2013;13(5). 3. kusumawati l, mulyani ns, pramono d. faktor-faktor yang berhubungan dengan pemberian imunisasi hepatitis b 0-7 hari. ber kedokt masy. 2007;23(1):21–7. 4. hepatitis b are you at risk? vol. 21, department of health & human services centers for disease control and prevention. 2010. 5. lusida mi, sakugawa h, nagano-fujii m, handajani r, setiawan pb, nidom ca, et al. genotype and subtype analyses of hepatitis b virus ( hbv ) and possible co-infection of hbv and hepatitis c virus ( hcv ) or hepatitis d virus ( hdv ) in blood donors , patients with chronic liver disease and patients on hemodialysis in surabaya , indones. microbiol immunol. 2003;47(12):969–75. 6. soemoharjo s. hepatitis virus b edisi 2. ed. 2 cet. jakarta: egc; 2008. 7. ashraf h, alam nh, rothermundt c, brooks a, bardhan p, hossain l, et al. prevalence and risk factors of hepatitis b and c virus infections in an impoverished urban community in dhaka, bangladesh. bmc infect dis. 2010;10(208):1–8. 8. indonesia dkr. pedoman pengendalian hepatitis virus. jakarta: kementrian kesehatan ri; 2012. 9. hepatitis b epidemiology and prevention of vaccine-preventable diseases [internet]. centers for disease control and prevention. 2012. available from: https://www.cdc.gov/vaccines/pubs/pinkbook/hepb. html 10. hepatitis b [internet]. world health organization. 2002. available from: http://www.who.int/csr/disease/hepatitis/hepatitisb_ whocdscsrlyo2002_2.pdf 11. horn t, learned j. hepatitis virus dan hiv. aids community research initiative of america (acria); 2005. 12. bhatt cp. prevalence of viral hepatitis b in bpkihs dharan, nepal. j nepal med assoc. 2000;39:281–3. 13. memish za, knawy b al, el-saed a. incidence trends of viral hepatitis a, b, and c seropositivity over eight years of surveillance in saudi arabia. int j infect dis. 2010;14(2):115–20. 14. ms c, s g, pc a, j m-b. caesarean section to prevent transmission of hepatitis b: a meta-analysis. can j gastroenterol hepatol. 2014;8(8):439–44. 15. mm j. hepatitis b and pregnancy: an underestimated issue. liver int. 2009;29(s1):133–9. 16. aghamohammadi a, nooritajer m. maternal hbsag carrier and pregnancy outcome. aust j basic appl sci. 2011;5(3):607–10. 17. s w, ly c, v y, ho l. hepatitis b carrier and perinatal outcome in singleton pregnancy. am j perinatol. 1999;16(9):485–8. 18. ikeako l, ezegwui h, ajah l, dim c, okeke t. seroprevalence of human immunodeficiency virus, hepatitis b, hepatitis c, syphilis and co-infections among antenatal women in a tertiary institution in south-east nigeria. ann med health sci res. 2014;4(6):954–8. 19. cooreman mp, leroux-roels g, paulij wp. vaccineand hepatitis b immune globulin-induced escape mutations of hepatitis b virus surface antigen. j biomed sci. 2001;8(3):237–47. 20. zou h, chen y, duan z, zhang h, pan c. virologic factors associated with failure to passive–active immunoprophylaxis in infants born to hbsag-positive mothers. j viral hepat. 2011;19(2):e18–25. ijtid vol 3 no 1 jan-maret 2012.indd 35 vol. 3. no. 1 january–march 2012 research report digital detection system design of mycobacterium tuberculosis through extraction of sputum image using neural network method franky chandra satria arisgraha,1 prihartini widiyanti,1,2 retna apsari1 1 department of physics, science & technology, faculty airlangga university, surabaya, indonesia 2 institute of tropical disease airlangga university, surabaya, indonesia abstract tuberculosis (tbc) is an dangerous disease and many people has been infected. one of many important steps to control tbc effectively and efficiently is by increasing case finding using right method and accurate diagnostic. one of them is to detect mycobacterium tuberculosis inside sputum. conventional detection of mycobacterium tuberculosis inside sputum can need a lot of time, so digitally detection method of mycobacterium tuberculosis was designed as an effort to get better result of detection. this method was designed by using combination between digital image processing method and neural network method. from testing report that was done, mycobacterium can be detected with successful value reach 77.5% and training error less than 5%. key words: mycobacterium tuberculosis, digital image processing, neural network introduction tuberculosis (tbc) is an dangerous disease and many people has been infected. accurate and fast detection of mycobacterium tuberculosis is needed and very important to prevent tb before getting worse. one of detection method that has been developed to detect mycobacterium tuberculosis is by sputum examination. conventional detection method of mycobacterium tuberculosis through sputum examination using microscope can need a lot of time, so digitally detection method of mycobacterium tuberculosis was designed as an effort to get better result of detection. the advantage of computation technology is in digital imaging and image detection. computation technologies that can be used in medical research are digital image processing and neural network (nn). theory tuberculosis tuberculosis (tbc) is an dangerous disease, most of mycobacterium infect lungs but it can infect another part of body. tuberculosis can infect by cough, patient bring mycobacterium to the air in sputum drop (droplet nuclei). once cough can result about 3000 sputum drops. usually, tuberculosis can infect in a long time. ventilation can reduce number of drop. quantity of mycobacterium in lungs can increase infection quality of tuberculosis. digital image processing digital image processing is used in many kind of aim. one of them is to convert 24 bit true color image to binary format through some steps character extraction, noise filtering, grayscale, and threshold. 36 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 35−38 grayscale by using representation of rgb (red, green, blue) value, a true color image is converted to white color and gradation of black color that is usually called by grayscale image. threshold threshold is data conversion of image in refer to the image just has two value 0 and 1. this step is done to get an information of pixel "high" or "low". neural network neural network (nn) is a method than can useful in many goals, for detection, identification, and control. nn was designed to solve some problem through learning technique. nn algorithm is easy to learned and can be used in medical research is to identify medical image. basic configuration of multilayer neural network is like figure 1. figure 1. basic configuration of multilayer neural network back propagation is one of some architectures of neural network. this architecture consist input layer, hidden layer and output layer, and every layer consists one or more artificial neuron. the name of this architecture is multilayer neural network. methodology this research was done by preparing digital image of sputum. analog image of sputum was converted to digital using computer. 24 bit digital image of sputum was extracted to binary through digital image processing method. pixel value at digital image was used as input of detection program. program to detect digital image of mycobacterium tuberculosis was designed using neural network method. neural network method was tested by using sputum image and was compared to database that was recognized. digital image of mycobacterium tuberculosis or not was used as input data of nn learning. if results of error compared to database, the value less than 5 percent and the conclucion should be done, but if error more than 5 percent then the design of program need to be repaired. designed of research that was done was like at figure 2. figure 2. algorithm of detection using neural network detection method of mycobacterium tuberculosis through image extraction using neural network method was designed by using back propagation type. neural network was build using 3 layer objects (one input layer, one hidden layer and one output layer). result input data of neural network in bitmap format with dimension is 70 pixels x 70 pixels. training data of neural network is digital image of sputum which consist mycobacterium and not that was already detected. 20 images data was used as inputs in neural network training process with specifications 10 images consists mycobacterium and 10 images are not. example of neural network image data inputs is like in figure 3. (a) (b) figure 3. input image of neural network (a)mycobacterium (b) not mycobacterium 37arisgraha, et al.: digital detection system desig'n of mycobacterium 40 images data was used as inputs in testing of neural network with specifications 10 images consists mycobacterium and 20 images are not. input image that was used in neural network testing process was not input image that was used in neural network training process. neural network training was used to classify sputum image according to type of object that had been researched, sputum images which consists mycobacterium and sputum images which not consists mycobacterium. training of neural network was done by using delphi 6.0 software. program that was designed is shown in figure 4. figure 4. training process of neural network in training process of neural network, some parameters value was set according to the aim. parameters value that was set are dimension of input image, hidden number, output number, learning rate value, momentum value, limit of epoch, and limit of maximum error. parameters value of neural network was shown in table 1. table 1. parameters value specification of neural network parameters value number of input (pixel) 4900 number of hidden 3 number of output 1 learning rate 0,1 momentum 1 number of epoch 269 output value 0,1399 target: – mycobacterium – not mycobacterium 0,1 0,9 error 0,0399 parameters value in table 1 was used in neural network testing process. from the result of neural network testing that had been done using training parameter from table 1, neural network training can be done well and error value of training reach criteria less than 5 percent. table 2. successful level of neural network testing no. image type number of input successful detection successful level 1. mycobacterium 20 16 80% 2. not mycobacterium 20 15 75% according to data in table 2, mean of successful to detect mycobacterium and not mycobacterium image is about 77.5 percent. result of neural network testing by using 20 input images of mycobacterium and 20 input images of not mycobacterium, 16 input images of mycobacterium and 15 input images of not mycobacterium was successfully detected according to the target. some results does not match because the dimension of mycobacterium is not suitable with the dimension of mycobacterium in program setting. that is caused by mycobacterium which were grow up a become longer than limit of dimension. this condition as difficulty in identification. there were noise of image so it can be affected performance of mycobacterium. the resolution of camera was low so the contrast of mycobacterium image was low too. it can caused by position of mycobacterium and another image and it can influenced value, and result of detection. coloring at sputum image can affected different illumination so comparison of image value can be different. cropping position was not suitable, it can affected pixel value because maybe some part of image was out of cropping area. threshold value was not suitable and it can be affected input of neural network. successful level of mycobacterium detection is like table 2 that is 80 percent of mycobacterium images and 75 percent of not mycobacterium images or mean of successful level was reach 77,5 percent by using 40 inputs of data. this result is still need to be repaired by choosing better method so detection result will be more accurate, some efforts to get better result are by adding inputs of training data with many variations, repairing characteristic detection or characteristic extraction of mycobacterium tuberculosis and not mycobacterium tuberculosis images, modification value of neural network parameters so success level of mycobacterium will higher than before, repairing digital image processing method, then performance of mycobacterium image could be contrast, or need modification of detection method by adding good method. 38 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 35−38 conclusions the result of digital detection system of mycobacterium tuberculosis has been reached 80 percents succesful level of identification and less than 5 percents error value of neural network training. references 1. bharath r, drosen j. 1994. neural network computing. windcrest. 2. crane c. 1997. a simplified approach to image processing: classical and modern techniques in c, prentice hall ptr. 3. desiani a. 2007. kajian pengenalan wajah dengan menggunakan metode face-arg dan jaringan syaraf tiruan backpropagation. media informatika, vol. 5, no. 2. 4. fausett l. 1994. fundamental of neural network. prentice hall inc. 5. gonzales r, wintz p. 1987. digital image processing, second edition. addison-wesley publishing company. 6. kabra sk., lodha r, seth v. 2004. some current concepts on childhood tuberculose. review article. indian j. med. res 120, october: 387–397. 7. korn ag. 1992. neural network experiments on personal computers and workstations. massachusetts institute of technology. 8. novrianto ada, andayana i. 2009. pengembangan sistem pemeriksaan dahak secara mikroskopis untuk diagnosis penyakit tbc dengan menggunakan teknik pengolahan citra dijital, biomedik elektro itb. 9. pedoman nasional penanggulangan tuberkulosis. 2002. departemen kesehatan republik indonesia, jakarta. 10. pedoman nasional penanggulangan tuberkulosis. 2007. departemen kesehatan republik indonesia, jakarta. 11. fajarianto o. 2008. penyakit tbc. http://ofajar88.wordpress.com/ page/10/ 12. perkumpulan pemberantasan tuberkulosis indonesia. 2006. jurnal tuberkulosis indonesia. vol. 3 no. 2. issn 1829–5118. 13. rao vb, rao hv. 1993. c++ neural networks and fuzzy logic, mis: press. 14. rich e, knight k. 1991. artificial intelligence, 2nd ed., mcgraw-hill, inc. 15. rodrigues jr, thome ac. 2003. cursive character recognition – a character segmentation method using projection profile-based technique, http://www.nce.ufrj.br/labic/downloads/. 16. subagyo a. aditama yt. et. al. 2006. pemeriksaan interferongamma dalam darah untuk deteksi infeksi tuberkulosis. bagian pulmonologi dan ilmu kedokteran respirasi fkui – rs persahabatan, laboratorium patologi kinik rs persahabatan, jakarta. jurnal tuberkulosis indonesia vol. 3 no. 2. 17. subekti rm, achmad b, and suyitno g. …. analisis kondisi ginjal pasien menggunakan metode jaringan syaraf tiruan. majalah batan. 18. supatman, 2009. deteksi pembesatran kelenjar getah bening pada paru dengan pengolahan citra digital untuk mendiagnosa penyakit primer kompleks tuberkulosis (pktb). seminar nasional aplikasi teknologi informasi 2009 (snati 2009). yogyakarta. 19. survey kesehatan rumah tangga, 2004. departemen kesehatan indonesia. 20. susmikanti m. 2010. pengenalan pola berbasis jaringan syaraf tiruan dalam analisa ct scan tumor otak beligna. seminar nasional aplikasi teknologi informasi (snati 2010). yogyakarta. 21. wirawan s, guritno s, and harjoko a. 2006. perancangan format penulisan informasi diagnosa hasil x-ray pada citra medis dengan format svg. 1st seminar on application and research in industrial technology. yogyakarta. 22. zain my, and nasution tma. 2009. identifikasi bakteri tuberculosis berdasar ciri morfologi dan warna. its-undergraduate 10581paper.pdf. �� vol. 2. no. 1 january–march 2011 hepatitis virus infection in repeatedly transfused thalassemia patients mia ratwita andarsini, ari setyawati, dwi putri, i dewa gede ugrasena, sjamsul arief department of child health medical faculty, airlangga university-dr. soetomo hospital abstract patients of thalassemia who are conventionally treated by a regular transfusion regimen, are at a risk of developing transfusion transmitted infections, including hepatitis. the present study was conducted to evaluate the prevalence of hepatitis virus infection in repeated transfused thalassemia patients. a total of 83 patients of thalassemia who had received at least 10 transfusions were tested for hbs ag, anti hbs and anti-hcv using elisa. amongst these patients, hbs ag, anti hbs and anti hbc were detected in 1.2%, 26.5% and 12% patients respectively. the prevalence of hbv and hcv infection were in agreement with the findings in other study. key words: thalassemia, repeated transfusion, hepatitis viral infection introduction thalassemias are inherited disorders of hemoglobin (hb) synthesis. their clinical severity widely varies, ranging from asymptomatic forms to severe or even fatal entities. worldwide, 15 million people have clinically apparent thalassemic disorders. reportedly, disorders worldwide, and people who carry thalassemia in india alone number approximately 30 million. these facts confirm that thalassemias are among the most common genetic disorders in humans; they are encountered among all ethnic groups and in almost every country around the world. thalassemia major (cooley anemia) is characterized by transfusiondependent anemia.1 the management of thalassemia major essentially comprises of regular blood transfusion and a life long ironchelation therapy. thalassema patients are prone to develop complication such as transfusion transmitted infection particularly hepatitis virus infection.2,3 in developed country, prevalence of hepatitis b infection in blood dependent patients are varies from 0.53% in shiraz iran to 22,5% in palestine.4,5 meanwhile hepatitis c infection prevalence varies from 15.7% to 37.9%.3–5 in case of hepatitis b, since an effective vaccine is available, immunization against this virus before transfusion management is started would effectively protect against transfusion transmitted hepatitis b. however, since no such vaccine is so far available against hepatitis c, the only effective protective measure against this virus is provision of hcv negative blood for transfusion. therefore, screening of transfused blood for hcv in not only mandatory, but also it is essential to use the most sensitive screening methods with least possible false-negative results. the aim of this study was to look into the prevalence of hepatitis virus infection in repeated transfused thalassemia major patients in our setup. patients and methods this study was conducted at hematology oncology outpatient clinic, department of pediatric, dr soetomo hospital surabaya from june to november 2009. a total of 83 cases of thalassemia that have been followed up routinely and had been transfused, as a part of their management, irrespective of their age, sex, and history of jaundice were included in this study. a detailed clinical data was noted included age, interval of transfusion, hemoglobin level and hepatitis b immunization status. all the patients who met the inclusion criteria tested for hbs ag, anti-hbs and anti-hbc using elisa. informed consent was taken for each patient involved. about five ml of patient’s blood sample was collected by a clean venepuncture. positive result of hbs ag was ��andarsini, et al.,: hepatitis virus infection in repeatedly transfused thalassemia patients considered as hepatitis b infection and anti-hcv positive was considered as hepatitis c infection. the result was reported descriptively and expressed as mean + standart deviation (sd). result in a total of 83 patients of thalassemia enrolled the study, 49 were males and 34 were females. the age at the time of this study ranged between 2 yrs and 18 yrs with a mean age of 10.6 yrs. the interval between transfusions varied between 2 to 6 weeks in different patients with hemoglobin level ranged between 4,4 – 12 g/dl with mean 7,62 g/dl. hbs ag were detected in 1 (1,2%) patient and anti hbs antibodies were detected also in 22 (26,5%) patients. eleven (13%) of patients have no history of hepatitis b immunization. anti-hcv antibodies were detected in 10 (12%) of patients. all of these patients have no history of jaundice and clinical evidence of hepatitis viral infection before entered the study. the characteristic of patients were summarized in tabel 1. tabel 1. characteristic of patients with non hepatitis infection, hbv infection and hcv infection non hepatitis infection (n= 72) hbv infection (n=1) hcv infection (n=10) sex m f age (years) interval of transfusion (weeks) history of hbv vaksinasion yes no hemoglobine level (g/dl) 44 (61.1%) 28 (38.9%) 10.6 + 3.6 3–6 41 (56.9%) 31 (43.1%) 7.6 + 2.4 1 8 4 1 8.9 6 (60%) 4 (40%) 10.8 + 4.3 2–6 1 (100%) 8.1 + 2.6 discussion patients with severe thalassemia require medical treatment, and a blood transfusion regimen was the first measure effective in prolonging life. in the process of experimenting with blood transfusion, it was found to provide patients with many benefits, including reversal of the complications of anemia, elimination of ineffective erythropoiesis and its complications, allowance of normal or near-normal growth and development, and extension of patients’ life spans. blood transfusion should be initiated at an early age when the child is symptomatic and after an initial period of observation to assess whether the child can maintain an acceptable level of hb without transfusion.1 the major complications of blood transfusions are those related to transmission of infectious agents, especially hcv, hbv and hiv infections.1,2 in this study prevalence of hbv infection was 1.2%. among developed country, study in iranian patients showed the prevalence varied from 0.53–6% [4,6], but it is lower than in palestine patients which revealed 22.5% of the blood transfusion dependent patients. [5] report from england in 1991–1997 showed that the prevalence of hepatitis b infection associated with transfusion was 0.57%.7 hbv infection can be prevented by a immunization. although 11 (13%) of our patients have no history of hbv infection, only 1 or 83 thalassemia patients in this study has hbs ag positive. means, screening of hbs ag done by indonesian red croos was effective to prevent hepatitis infection in the transfusion dependent patients. hcv infection has gained importance particularly as one of the major complications in multiply transfused patients during the last decade. this is especially true for counties where hcv is more prevalent in general population and therefore also amongst blood donors. the prevalence of hcv seropositivity in multiply transfused β-thalassemia patients has been observed to vary greatly, varies from 15.7% to 37.9%.3–5 but study by younus resulted a high prevalence of hcv seropositivity (42%).2 in our study, anti-hcv antibodies were detected in 10 (12%) of patients, which was lower than previous study in developed country. although indonesian red croos’ screening of hbv and hcv infection was effective and the prevalence of hbv and hcv infection were in agreement with the findings in other study, serious attempts have to be made to ensure a safe blood transfusion, so as to cut down the prevalence of hcv hepatitis in multiply transfused thalassemic patients. education regarding transfusion transmitted infections, including hcv, hbv & hiv infections, is of prime importance. references 1. hm. yaish, “thalassemia”, available at www.emedicine.com, accessed on july 2010. 2. m. younus, k. hassan, n. ikram, l. naseem, h.a. zaheer, mf. khan, “hepatitis c virus seropositivity in repeatdly transfused thalassemia major patients”, international journal of pathology, vol.2, 2004, pp. 20–3. 3. mg. borou, maa. zadegan, km. zandian, mh. rodan, “prevalence of hepatitis c virus (hcv) among thalassemia patients in khuzestan province, southwest iran”, park j med sci, vol. 25, 2009, pp.113–7. 4. m. karimi, aa. ghavanini, “seroprevalence of hepatitis b, hepatitis c and human immunodeficiency virus antibodies among multitransfused thalassaemic children in shiraz,iran”, j paediatr child health, vol. 37, 2001, pp. 564–6. �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 46-48 5. ri. jadallah, gm. adwan, ma. abu hasan, km. adwan, “prevalence of hepatitis b virus markers among high risk groups in palestine”, medical journal of islamic world academy of sciences, vol. 15, 2005, pp.157–60. 6. s. mirmomen, sm. alavian, b. hajarizadeh, j. kafaee, b. yektaparaz, mj. zahedi, et al, “epidemiologi of hepatitis b, hepatitis c, and human immundeficiency virus infection in patients with betathalassemia in iran: a multicentre study”, arch iranian med, vol. 9, 2006, pp. 319–23. 7. k. soldan, m. ramsay, m. collins, “acute hepatitis b infection associated with blood transfusion in england and wales, 1991-7: review of database”, bmj, vol. 318, 1999, pp. 95. ijtid vol 6 no 1 jan-april 2016_revisi.indd 19 vol. 6. no. 1 january–april 2016 case report mycobacterium leprae bacillemia in both twins, but only manifest as leprosy in one sibling netty sukmawati,1 indropo agusni,1 m. y ulianto listiawan,1 cita rosita s prakoeswa,1 dinar adriaty,2 ratna wahyuni,2 iswahyudi2 1 dept. of dermatology, school of medicine, universitas airlangga, surabaya, indonesia. 2 institute of tropical disease, universitas airlangga, surabaya, indonesia. abstract leprosy in twins is rarely reported. a 19 years-old male student, from lamongan district, was diagnosed as multibacillary (mb) leprosy in the skin and std clinic of dr. soetomo general hospital surabaya. multiple anesthetic skin lesions were found, but the bacteriologic examination was negative for acid fast bacilli (afb). histopathology examination support the diagnosis of bl type of leprosy. his twin brother that has been lived together since born until present seems healthy without any complaints of skin lesions and have no signs of leprosy. when a serologic examination for leprosy was performed, a high anti pgl-1 antibody level was found in patient (igm anti pgl-1 2937 and igg anti pgl-1 3080 unit/ml) while his healthy twin brother showed only low level (igm 745 and igg 0 unit/ml). interestingly when a pcr study was performed to detect m.leprae in the blood, both of them showed positive results. using the ttc method, a genomic study of for m.leprae, it is revealed that both samples were identic ( 27x ttc repeats). according to patient’s history, he had a traffic accident and got a wound in the knee seven years ago, while the skin lesions seems started from this area around three years ago before it spread to other parts of the body. the patient was treated with multi-drug therapy (mdt) while his sibling got a prophylactic treatment for leprosy. after 6 months of treatment, the leprosy skin lesions were diminished and the serologic anti pgl-1 has been decreased. his healthy brother also showed a decrease in anti pgl-1 level and no skin signs of leprosy. key words: leprosy, twin, bacillemia, pcr, prophylactic treatment abstrak. penyakit kusta pada pasien bersudara kembar merupakan peristiwa yang jarang terjadi. dilaporkan seorang pemuda berumur 19 berstatus mahasiswa yang datang berobat ke rsud dr soetomo surabaya dengan keluhan bercak di kulit kaki, badan dan muka. pasien berasal dari daerah lamongan dan bersaudara laki-laki kembar, tetapi dalam keadaan sehat. diagnosa penyakit kusta ditegakkan berdasarkan lesi kulit yang anestesi, meskipun tidak ditemukan basil tahan asam (bta) dari lesi kulit. pemeriksaan histopatologis menunjang diagnosa yang sesuai dengan kusta tipe bl. saudara kembarnya yang telah tinggal bersama sejak kecil tidak menunjukkan adanya lesi kulit ataupun bta. pada pemeriksaan serologi anti phenolic glycolipid-1 (pgl-1) pada pasien didapatkan kadar yang tinggi (igm 2937 u/ml dan igg 3080 u/ml) sedangkan saudara kembarnya menunjukkan igm anti pgl-1 745 u/ml, sedangkan iggnya 0. yang menarik adalah saat dilakukan pemeriksaan pcr untuk mendeteksi adanya m.leprae dalam darah, ternyata keduanya samasama menunjukkan hasil pcr yang positif. selanjutnya dengan metode ttc dilakukan studi genomic dari m.leprae yang ditemukan. hasil sekuensing pengulangan ttc menunjukkan bahwa ke 2 sampel tersebut identik (27x pengulangan ttc). pasien diobati dengan obat multi-drug therapy (mdt) sedangkan untuk saudara kembarnya diberikan obat pencegahan kusta. evaluasi setelah 6 bulan menunjukkan perbaikan klinis pada pasien dan penurunan titer antibodi anti pgl-1, sedangkan saudara kembarnya tetap tidak menunjukkan adanya gejala kusta serta semakin rendahnya titer antibodi. kata kunci: kusta, saudara kembar, basilemia, pcr, terapi pencegahan 20 indonesian journal of tropical and infectious disease, vol. 6. no. 1 january–april 2016: 19−23 background leprosy is a chronic disease caused by mycobacterium leprae that primarily effects the peripheral nerves and secondary affects the skin and other organs.1 transmission leprosy dependent on immunological status and susceptibility, household contact, the environment and social conditions such as economic status, lacking of ventilation at home or poor hygiene.2 genetic factors are also to be a important factor in transmission of leprosy disease. studies suggest that, among monozygotic (identical) twins if one had leprosy, the other almost always had leprosy, while this was not the case with dizygotic twins.3 it is also influenced by human leukocyte antigen (hla) that affect susceptibility.4 the main transmission route of m.leprae is droplet infection, but transmission such as skin contact, through the placenta during pregnancy, breast-feeding and trauma should not be ruled out even though there is no conclusive evidence.5 who recommends the multi-drug therapy (whomdt) regiment for leprosy and the program have began since 1980 in indonesia.6 although most of leprosy cases have been treated, there are still new leprosy cases were detected every year, indicating that transmission of leprosy still occurred in the community.7 one of the reasons for explaining the continuing of new detected leprosy cases is the non-human resource of m.leprae. since the human source (leprosy patients) are already treated by mdt and become non-infectious anymore, the role of non-human resources should be kept in mind.. these non-human resources including water, soil or other contaminated agents.7 several studies reports existence of viable m.leprae outside the human body. detection of viable mycobacterium leprae (rna m.leprae) found in soil samples in ghatampur india.8 dna m.leprae also found in water sources (wells) in along coast of east java9 m.lepra in soil and wells water were reported in leprosy endemic areas of east java, including lamongan regency.10 cases twins ( y and d) , 21 years-old students, unmaried, from lamongan, visited the skin and vd clinic of dr soetomo general hospital surabaya. one sibling, y, complained anasthetic red patch, which firstly appeared in front of right knee since 3 years ago. y and d was born on 1994 in payaman, solokuro, part of lamongan distric. both of them were normaly born in one placenta (monozygotic). they spent time together in one house and sharing one bedroom since childhood. when they were 13 years old, they had junior school in sendang agung village, paciran, part of lamongan district. in 2012 they became students in malang and still lived in one the dorm rooms. in 2007, y was 14 years old, he got accident falling to the ground in lamongan. he got trauma behind the right knee. at that time the wound just treated with antiseptic and healed. six years later, in 2013, y complained red patch which first appeared in front of the right knee. he went to a doctor and got some medications but the skin lesions still persist. then the patient and his sibling visited the out patient clinic of dr. soetomo hospital surabaya. figure 1. twins a. y (leprosy patient) b. d (healthy twin). figure 2. a. first anesthetic lesion on the knee multiple anaesthetic skin lesions were found over the right extremity and face. negative result of skin slit smear for acid fast bacilli (afb) were noted from bacteriological examination using ziehl neelsen staining. skin biopsy from the skin lesion at the right extremity revealed a bl type leprosy. serological examination (elisa anti pgl-1 antibody) serology to both of twins, elisa results of igm anti pgl-1 in y patient was 2937 unit/ml and igg anti pgl-1 was 3080 unit/ml. in the other health twin, serology results showed levels of igm anti pgl-1 was 745 unit/ml and igg anti pgl-1 was 0. skin biopsy from the skin lesion at the right extremity revealed a bl type leprosy. (figure 3 & 4) 21sukmawati, et al.: mycobacterium leprae bacillemia in both twins figure 3. epidermal atrophy, flattened rete ridges and grenz zone were observed (h/e 400x) figure 4. dilated capillary blood with lymphocyte infiltration. . (h/e 400x) polymerase chain reaction (pcr) study was performed to the bloods of the twin, using the lpf and lpr nested primers to the bloods of the twin (figure 5) 1 2 3 4 5 6 note : 1. pbmc from leprosy patient (y) 2. pbmc from healthy sibling (d) 3. skin lesion of patient (y) 4. neg control 5. pos control – m.leprae thai53 6. 100bp dna ladder figure 5. pcr results from blood and skin lesion (lpf –lpr nested primers) . further study was conducted to compare the genomic pattern between the two m.leprae dna from the amplicon leprosy patient (y) healthy sibling (y) ttc 27x ttc 27 x figure 6. direct sequencing of ttc area from both samples and number of ttc repeats 22 indonesian journal of tropical and infectious disease, vol. 6. no. 1 january–april 2016: 19−23 products of pcrresults . (figure 6 ). using the ttc method, the number of ttc repeats from both of samples were similar, 27x repeats, which indicates the two samples were identic or similar strain. the examination results of these twins can be summarized as follows : data mr. y (leprosy patient) mr. d (healthy siblings) skin lesions multiple anesthetic macules no skin lesions bacterial examination (afb) negative negative histopathology from skin lesion bl type of leprosy not done serology (anti pgl-1 ) igm 2937 igg 3080 u/ml igm 745 igg 0 u/ ml pcr from skin lesion positive not done pcr from blood (pbmc) positive positive direct sequencing ttc area 27 x repeats 27 x repeats the leprosy patient (mr. y) was treated with rifampicine, dapsone and lamprene (who-mdt regiment) for 12 months while his healthy sibling was treated with a prophylactic dose of rifampicine and ofloxacine for two weeks. after six months later, the skin lesions disappear and the titer of anti pgl-1 antibodies were decreased. his healthy sibling does not develop any sign of leprosy and the anti pgl-1 titer became normal. discussion leprosy in twin is relatively rare and seldom reported in the literature. chakravartti & vogel (1973) conducted an epidemiologic study leprosy in twins. among 62 pairs of monozygotic twins and 40 pairs of dizygotic, they found that the monozygotic twins have a greater risk to get leprosy if the sibling affected the disease.3 several studies reported several genes and substance may have a role in the susceptibility to leprosy, including hla, tap2, vdr, ptpn22 in adaptive immunity and nramp1, tlr2,mica etc. in innate immunity.4 in our case, they are monozygotic twin which is theoretically will have a similar pattern. they live together since birth until adolescent in leprosy endemic area of lamongan. this area has been known as leprosy endemic area in east java since a long time ago.11 if the source of infection is the same, usually via droplet infection, they will got the same exposures and same long time duration. then the incubation period will be the same and both of them will manifest leprosy on the same time. but in fact, leprosy manifest only in one sibling and the different life experience between them is the traffic accident seven years previously. the site of the first skin lesion of leprosy was very close with the scar of the wound during the accident three years ago. it might be possible that m.leprae entered the body via the wound and then spread to other organ. nonhuman resource of m.leprae have been reported from some leprosy endemic areas and also some of them found the viable m.leprae from the soil and water.12. in our case, mr. d who got traffic accident probably infected by the bacilli from environment, which become manifest leprosy after 4 years. the diagnosis of leprosy in this case is confirmed by the typical anesthetic skin lesions and histopathological examination. although the other cardinal signs of leprosy (peripheral nerves enlargement and the present of acid fast bacilli / afb) was negative, the pcr results showed that the specific dna of m.leprae was present in the skin lesion and peripheral blood. the serological test result of mr.y supported the diagnosis of manifest leprosy (high titer of igm and igg anti pgl-1) while the antibody titer of mr.d showed a low sero-positive result (igm anti pgl-1 745 u/ml with cut off 605 u/ml) that indicated a subclinical leprosy. one can assume that the process of leprosy in mr.d is still in the initial stage, which will progress to manifest leprosy within certain years ahead.13 the use of the ttc technique, one procedure of variable number tandem repeat (vntr) method for genetic study of m.leprae. this technique was chosen because it is relatively easy, simple and relatively low cost.14 the results showed 27x ttc repeats in both samples indicated similar pattern of the strain, which means they were originated from one similar source. after got the disease, mr. d became a source of infection for his twin brother. positive pcr test from the blood indicates that the healthy brother was in subclinical stage of leprosy. this stage will develop toward the manifest leprosy after certain years, if no prophylactic treatment was given.15 up to present time. there is still no guidance yet about chemoprophylactic treatment in leprosy, therefore the use of rifampicine and ofloxacine for the subclinical leprosy in this case was based on the author’s experience.16 referrences 1. jopling wh, mcdougall ac. handbook of leprosy. 5th ed. . india cbs publ & distr. 1996. 2. brycesson a, pfalzgraff re. leprosy 3rd ed. churchill livingstone. 1990 3. chakravartti mr and vogel f (1973). a twin study on leprosy. top hum genet 1 : 1-123. 4. rajni rani (2010). genetic susceptibility and immunogenetics. in (kar hk & kumar b, eds) ial textbook of leprosy. jaypee brothers medical publ. 5. agusni i. (2003). leprosy. an ancient disease with a lot of mysteries. inaugural speech. airlangga university press. 6. world health organization study group (1982). chemotherapy of leprosy for control programmes. who geneve, switzerland. 7. world health organization (2009). global leprosy situation. weekly epidemiological record.no.33. 14 august 2009.. 23sukmawati, et al.: mycobacterium leprae bacillemia in both twins 8. lavania m, katoch k, katoch vm et al. (2008). detection of viable mycobacterium leprae in soil samples: insight into possible source of transmission of leprosy.(2008). infection genetic snd evolution. elseiver. 2008;8:627-31. 9. wahyuni r. (2009). the existence of mycobacterium leprae in the water and soils of leprosy endemic area in east java province. thesis. postgraduate program. airlangga university surabaya. 10. agusni i, izumi s, adriaty d, iswahyudi. (2004) m.leprae study in the environment of leprosy endemic area. indonesian med j. 58(8) : 319-324. 11. health municipality of east java province. (2008). leprosy report. dinkes jatim; 2008. 12. wahyuni r, adriaty d, iswahyudi et al. (2010). mycobacterium leprae in daily water resources of inhabitants who live in leprosy endemic area of east java. indonesian j tropic infect dis 1 (2) : 65-68. 13. godal t, nagassi k (1973). subclinical infection in leprosy. br med j 3:557-9. 14. matsuoka m, shang i, budiawan t et al. (2004). genotyping of mycobacterium leprae on the basis of the polymorphisme of ttc repeats for analysis of transmission. j clin microbiol. 42(2): 741745. 15. agusni i. kardjito t, soedewo fh et al. (2001) .subclinical leprosy in mandangin island, madura. (part ii). a preliminary study of serial surveys in leprosy endemic area. indonesian med j 51 (12): 393400. 16. one year evaluation of preventive treatment in subclinical stage of leprosy. indropo agusni , cita rosita s prakoeswa, m yulianto listiawan et al. 18th international leprosy congress, brussel, belgium, november 2013. ijtid vol 6 no 1 jan-april 2016_revisi.indd 1 vol. 6. no. 1 january–april 2016 research report effect of free alkaloid and non-free alkaloid ethanol 70% extract of justicia gendarussa burm f. leaves against reverse transcriptase hiv enzyme in vitro and chemical compound analysis bambang prajogo1, prihartini widiyanti2,3, hafrizal riza4 1 department of pharmacognosy, faculty of pharmacy, universitas airlangga, surabaya, east java, indonesia 2 faculty of science and technology, universitas airlangga, surabaya, east java, indonesia 3 institute of tropical disease (itd), universitas airlangga, surabaya, east java, indonesia 4 department of pharmaceutical biology, faculty of pharmacy, universitas tanjungpura, pontianak, west kalimantan, indonesia corresponding author: prajogo_ew@yahoo.com abstract hiv-aids is a global problem and the deadliest disease in the world. one of hiv and aids prevention strategy can be done with traditional medicine research program from natural resource that has anti-hiv aids activity. it has been found that 70% ethanol extract of justicia gendarussa burm.f leaves, alkaloid free and alkaloid non-free, has a strong inhibitory activity against hiv reverse transcriptase enzyme, as an effort to find a solution in the face of hiv aids prevalence that is still high with problem of hiv-aids treatment such as side effects and resistances. justicia gendarussa had already known for having an effect anti-hiv and therefore we were looking at the mechanism of inhibition of hiv reverse transcriptase enzyme. both types of extracts were tested in vitro using elisa technique and analysed chemical content of gendarusin a as anti-hiv using high performance liquid chromatography. elisa test results obtained percent inhibition, respectively for 254.2, 254.2, 235.6, and 279.7 for the concentration of 5 ppm, 10 ppm, 15 ppm and 20 ppm of free alkaloid extract and 169.0, 164.0, 130.5 and 369.5 for the concentration of 5 ppm, 10 ppm, 15 ppm and 20 ppm of non-free-alkaloid extract. the results of high performance liquid chromatography obtained gendarusin a in the free-alkaloid extract at retention time 8.402 minutes and non-free alkaloid extract at retention time 8.381. therefore, these results concluded that the justicia gendarussa burm.f can be a useful resource for the isolation and development of new anti-hiv. key words: justicia gendarussa; 70% ethanol extract; free and non-free alkaloid; reverse transcriptase; anti-hiv abstrak hiv-aids merupakan permasalahan global dan penyakit yang mematikan di dunia. salah satu strategi pencegahan hiv-aids dapat dilakukan dengan program penelitian pengobatan tradisional dari sumber daya alam yang memiliki aktivitas anti-hiv aids. telah ditemukan bahwa ekstrak ethanol 70% daun justicia burm.f gendarussa, bebas alkaloid dan mengandung alkaloid, memiliki aktivitas inhibitor yang kuat terhadap enzim hiv reverse transcriptase, sebagai upaya dalam rangka mencari solusi menghadapi prevalensi hiv aids yang masih tinggi dengan masalah pengobatan hiv-aids seperti efek samping dan resistansi. justicia gendarussa telah diketahui memiliki efek anti-hiv dan perlu diketahui mekanisme penghambatan enzim hiv reverse transcriptase. kedua jenis ekstrak diuji in vitro menggunakan teknik elisa dan dianalisis kandungan kimia gendarusin a sebagai anti-hiv menggunakan high performance liquid chromatography (hplc). hasil tes elisa diperoleh persen inhibisi, masing-masing untuk 254.2, 254.2, dan 279.7, 235.6 untuk konsentrasi 5 ppm, 10 ppm, 15 ppm, dan 20 ppm ekstrak bebas alkaloid dan 169.0 bebas, 164.0, 369.5, 130.5 untuk konsentrasi 5 ppm, 10 ppm, 15 ppm, dan 20 ppm dari ekstrak yang mengandung alkaloid. hasil high performance liquid chromatography (hplc) menunjukkan gendarusin a pada ekstrak bebas alkaloid pada waktu retensi 8.402 menit dan ekstrak yang 2 indonesian journal of tropical and infectious disease, vol. 6. no.1 january–april 2016: 1−4 mengandung alkaloid pada waktu retensi 8.381. hasil tersebut mengarah pada kesimpulan bahwa justicia gendarussa burm.f dapat berpotensi bagi isolasi dan pengembangan anti-hiv baru. kata kunci: justicia gendarussa; ekstrak ehanol 70%; bebas dan tidak bebas alkaloid; anti-hiv figure 1. gendarusin a structure. (prajogo bew, 2010) introduction hiv-aids is a global problem and the deadliest disease in the world. according to who global report, the number of aids deaths in the world in 2009 reached 1.8 million people1. whereas based on the health ministry data, although the total hiv and aids cases nationwide declined from 2011 as many as 21.031 and 4162 into 9.883 and 2.224 in 2012, but it is still relatively high.2 seen from a treatment, medical efforts of the hiv-aids treatment service still face some problem. for example, the antiretroviral utilization which is the dose and side effect are very limited. toxicity and side effects affecting patient obedience to antiretroviral. furthermore, antiretroviral utilization this time are resistant that cause the failure of therapy.3 one of hiv and aids prevention strategy can be done with traditional medicine research program from natural resource that has anti-hiv aids activity. traditional medicine research is directed to find a scientific evidence on it.4 natural resources still have an important role as an initial material invention of new drugs.5 based on a published report in 2007, there were 974 molecular compound that 63% of them come from natural or semisynthetic derivatives from natural materials.6 the reverse transcriptase enzyme has an important role in the life cycle of hiv because reverse transcription is an early phase of viral replication in the cell host. all the proteins and enzymes that play an important role in the new virus formation are not carried by the virus but using enzymes and proteins in host cells.7 furthermore, the reverse transcriptase enzyme together with an integrated enzyme is derived from a virus that enters the host cell in fusion phase.8 therefore, the drugs development that act on the reverse transcriptase enzyme will inhibit the next cycle process directly, starting from reverse transcription of the virus rna into dna, the integration of dna virus on host cell dna, and core replication to the virus proteins formation. inhibition on phase after transcription is still possible to set an infection in the host cell because the virus dna can settle along with the host cell dna. therefore, inhibition of the enzyme reverse transcriptase can reduce a hiv infection.7 currently, it is being developed an anti-hiv drugs derived from natural medicine, namely justicia gendarussa burm.f. research has been done on them to test the effect of hexane, methanol and ethanol of justicia gendarussa burm.f drug against hiv virus in vitro methanol and ethanol extracts obtained 70% alkaloid-free give a decrease in the amount of virus results.9 the part of justicia gendarussa burm.f herb showed inhibitory activity analog reverse transcriptase enzyme substrates in vitro.10 isolate of the pure compound flavonoid apigenin showed inhibition of hiv reverse transcriptase enzyme activity as a substrate analog and hiv protease enzyme in vitro.11 the main content of 70% ethanol extract justicia gendarussa burm.f is apigenin. a few compounds either major or minor component can give a synergistic effect as an anti-hiv through the same or different mechanism.12 therefore, this study aimed to test the inhibitory activity of the reverse transcriptase hiv enzyme using 70% ethanol extract free and non-free alkaloid. materials and methods materials justiciagendarussaburm.f leaf obtained from cultivated plants in trawas, mojokerto, east java. roche rt activity kit obtained from pt. roche, germany. materials to extract, alkaloid test and chemical content are 70% ethanol, dichloromethane, methanol, hexane, distilled water, citric acid, filter paper, dragendorf reagent, silica gel gf 254.extraction tool set such as macerator extraction, memmert oven, evaporator buchi, julabo usr 3 ultrasonic, imark microplate absorbance reader, hplc tool set: agilent1100, reversephase column c18 nova-pak® sized 3,9 × 150 mm. research place research had been done in two laboratories, laboratory pharmacognosy faculty of pharmacy, airlangga university and institute of tropical disease laboratory, airlangga university. 3prajogo, et al.: effect of free alkaloid and non-free alkaloid ethanol 70% extract of justicia gendarussa methods simplicia of justicia gendarussa burm.f leaves were divided into two sample groups. first group acidified to release the alkaloids and the second group did not to acidified. both two sample groups were macerated with 70% ethanol and then concentrated. to ensure of free alkaloid, the first group tested the alkaloid free using thin layer chromatography with stationary phase silica gel gf 254 and the mobile phase dichloromethane: methanol, 9:1 with the spray dragendorf reagent. both of samples tested its activity in an enzyme activity inhibition reverse transcriptase hiv using elisa and determined the type of inhibitor. both samples were also analysed to determine levels of chemical content gendarusin a based on previous research that isolates apigenin has reverse transcriptase hiv inhibitory activity. the conditions that used in hplc was methanol eluent: water (30:70), flow 1 ml/min, stop time 25 minutes, a wavelength of 254 nm. results and discussion in this study, it had been tested to know the inhibitory activity of justicia gendarussa burm.f 70% ethanol extract against reverse transcriptase hiv enzyme and acidification differences treatment to know the effect of alkaloid on the inhibition of the enzyme reverse transcriptase hiv. extraction was done by ethanol 70% maceration because the previous studies showed the extract can decrease the amount of hiv virus in vitro culture. the first group was tested to ensure that it was free from alkaloid as shown in figure 2. the stain a that was free from alkaloid extract was not showed red-orange stain like stain b and c. inhibition testing of reverse transcriptase hiv enzyme was obtained by using the formula (1). %inhibition = (1) explanation: a0: blank absorbance a1: sample absorbance table 1 shows the both extracts had inhibitory activity of the reverse transcriptase hiv enzyme that was indicated by the percent inhibition. table 1. inhibition percentage of 70% ethanol extract alkaloidfree and non-free of justicia gendarussa burm.f leaves to the activity of the reverse transcriptase hiv enzyme sample concentration % inhibition a 20 ppm 15 ppm 10 ppm 5 ppm 279,7 235,6 254,2 254,2 b 20 ppm 15 ppm 10 ppm 5 ppm 369,5 130,5 164,0 169,0 k+ 100 μg/ml 83 kexplanation: a : non-free alkaloid extract b : free alkaloid extract k+ : positive control (doksorubisin 100 μg/ml) k: negative control (tested solution without enzyme and sample) for the chemical substitute analysis of gendarusin a obtained at retention time 8.402 minutes for the alkaloidfree extract and retention time 8, 381 minutes in fig 3 compared with standard gendarusin a 9.6 ppm with a retention time of 8.590 minutes as shown in fig 2. figure 2. gendarusin a 9,6 ppm standard chromatogram 4 indonesian journal of tropical and infectious disease, vol. 6. no.1 january–april 2016: 1−4 a b figure 3. ethanol 70% extract of justicia gendarussa burm.f, leaves chromatogram (a) alkaloid-free and (b) non alkaloid-free. conclusions from these results, it can be concluded that the 70% ethanol extract of justicia gendarussa burm.f leaves 60th alkaloidfree and non-alkaloid-free have inhibitory activity of the reverse transcriptase hiv enzyme. references 1. casiday r and frey r, drug strategies to target hiv: enzyme kinetics and enzyme inhibitors, department of chemistry, washington university, 2001. 2. feher m and schmidt j.m, property distributions: differences between drugs, natural products, and molecules from combinatorial chemistry, journal of chemical information and computer science, vol. 43, pp. 218–227, 2003. 3. flexner c, hiv drug development: the next 25 years, natural review drug discovery, vol. 6, pp. 959–966, 2007. 4. gilbert b and alves lf, synergy in plant medicine, current medical chemistry, vol. 10, pp. 13–20, 2003. 5. kementerian kesehatan republik indonesia, laporan situasi perkembangan hiv&aids di indonesia sampai dengan juni 2011, (online), (http://www.aidsindonesia.or.id/download/lt2menkes2011. pdf, accessed on 13 november 2011), 2011. 6. komisi penanggulangan aids, strategi nasional penanggulangan hiv dan aids 2007-2010, (online), (http://www.undp.or.id/ programme/pro-poor/the%20national%20hiv%20&%20aids%20 strategy%2020072010%20%28indonesia%29.pdf, accessed on 3 november 2011), 2007. 7. newman dj and cragg gm, natural products as sources of new drugs over the last 25 years, journal of natural product,.vol. 70, pp. 461–477, 2007. 8. pommier y, johnson aa, marchand c, integrase inhibitors to treat hiv/aids, nature review: drug discovery, vol. 4, pp. 236–248, 2005. 9. prajogo bew, prihartini w, nasronudin, bimo a. the effect of gendarussin a isolates of justicia gendarussa burm.f. leaf in reverse transcriptase inhibition of hiv type i in vivo, indonesian j. of tropical and infectious disease vol. 5 no. 5, pp. 136-141, 2015 10. woradulayapinij w, soonthornchareonnon n, and wiwat c, in vitro hiv type 1 reverse transcriptase inhibitory activity of thai medicinal plants and canna indica l. rizhomes, journal of ethnopharmacology, vol. 101, pp.84–89, 2005. 11. world health organization, golbal summary of the hiv aids epidemic, on december2009,(online),(http://www.who.int/hiv/ data/2009_global_summary.png, accessed 10 november 2011), 2009. 12. yeon-ju k, hyun-jeong o, hyo-min a, ho-jung kang, jung-hyun k, and young-hwan k, flavonoids as potential inhibitors of retroviral enzymes, journal of the korean society for applied biological chemistry, vol. 52, pp. 321–326, 2009. 13. yuliangkara b, prajogo bew, dan widiyanti p, pengaruh ekstrak heksan, metanol, dan etanol tanaman obat justicia gendarussa burm.f terhadap virus hiv in vitro, skripsi, fakultas farmasi, universitasairlangga, surabaya. ijtid vol 8 no 2 may-agustus 2020_newfromsarah.indd vol. 8 no. 2 may–august 2020 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ research article lower perceived-stigmatization by health workers among hiv-aids patients of key population backgrounds jihan qonitatillah1, samsriyaningsih handayani2a, ernawati3, musofa rusli4 1faculty of medicine, universitas airlangga, surabaya, east java, indonesia 2department of public health and preventive medicine, faculty of medicine, universitas airlangga, surabaya, east java, indonesia 3department obstetrics and gynecology, faculty of medicine, dr. soetomo hospital, surabaya, east java, indonesia 4department of internal medicine, division of infectious and tropical disease, dr. soetomo hospital, surabaya, east java, indonesia received: 22nd january 2019; revised: 29th october 2019; accepted: 25th february 2020 abstract the stigma of people living with hiv-aids (plwha) by health workers may have a broad impact, so it is necessary to identify the factors that infl uence the occurrence of stigma. identifi cation of factors that cause a decrease in stigmatization by health workers will have an impact on improving the quality of life of people with hiv, increasing compliance with medication, and ultimately reducing the incidence of hiv infection itself. the purpose of this study was to analyze factors related to plwha’s perception of stigma among health workers in the community health center. this research applied a cross-sectional design using interviews. ninety-four patients from the infectious disease intermediate care of dr. soetomo hospital surabaya, a tertiary level hospital, were interviewed. the stigma perception was assessed using a questionnaire modifi ed from the standardized brief questionnaire by health policy project with cronbach’s alpha of 0.786. the data were simultaneously analyzed with binary multiple regressions on ibm spss statistics 22.0 for windows software. there were 30 out of 94 patients with key population backgrounds, and most population was injecting drug users (idus) and female sex workers (fsws). plwha perceived most stigmatized community health workers when they drew blood, provided care, and considered they were involved in irresponsible behavior. there were relationships between age (p=0.008), marital status (p=0.013), and the history of key population (p=0.006)to people living with hiv-aids (plwha)’s perception of stigma among health workers in east java community health center. future research on factors infl uencing hiv-related stigma is needed to improve patients’ quality of life. keywords: health workers, hiv-aids, key population, stigma abstrak stigma terhadap orang dengan hiv-aids (odha) oleh tenaga kesehatan dapat berdampak luas, maka perlu dilakukan identifi kasi faktor-faktor yang memengaruhi terjadinya stigma. identifi kasi faktor-faktor yang menyebabkan penurunan stigmatisasi oleh tenaga kesehatan akan berdampak terhadap peningkatan quality of life orang dengan hiv, meningkatnya kepatuhan minum obat, dan akhirnya akan mengurangi angka kejadian infeksi hiv itu sendiri. tujuan dari penelitian ini yaitu untuk menganalisis faktor-faktor yang berhubungan terhadap persepsi orang dengan hiv-aids (odha) atas stigma oleh tenaga kesehatan puskesmas. penelitian ini menggunakan rancangan penelitian cross-sectional dengan metode wawancara. sembilan puluh empat pasien dari poli rawat jalan instalasi pipi rsud dr. soetomo, yang merupakan rumah sakit tersier diwawancarai. persepsi stigma pasien dinilai menggunakan kuesioner standar oleh health policy project dengan nilai cronbachs alpha 0,786. data dianalisis dengan uji regresi logistic berganda dengan perangkat lunak ibm spss statistics 22.0 for windows. didapatkan 30 dari 94 pasien yang memiliki riwayat kelompok risiko, dengan kelompok risiko terbanyak adalah penasun dan wps. gambaran stigmatisasi oleh tenaga kesehatan terhadap odha yaitu khawatir ketika mengambil darah, a corresponding author: samsri.handayani@gmail.com 91jihan qonitatillah, et al.: lower perceived-stigmatization by health workers copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 memberikan perawatan berkualitas rendah, dan menganggap seseorang terinfeksi hiv karena mereka terlibat perilaku yang tidak bertanggung jawab. terdapat hubungan antara usia (p=0,008), status perkawinan (p=0,013), dan odha beriwayat kelompok risiko (p=0,006) dengan persepsi odha atas stigma oleh tenaga kesehatan puskesmas. usia yang muda, menikah, dan memiliki riwayat kelopok risiko merupakan faktor-faktor yang signifi kan terhadap rendahnya persepsi odha atas stigma oleh tenaga kesehatan puskesmas jawa timur. penelitian terkait faktor-faktor yang berhubungan dengan stigma hiv dibutuhkan untuk meningkatkan kualitas hidup odha. kata kunci: tenaga kesehatan, hiv-aids, kelompok risiko, stigma how to cite: qonitatillah, jihan. handayani, samsriyaningsih. ernawati, ernawati. rusli, musofa. lower perceivedstigmatization by health workers among hiv-aids patients of key population backgrounds. indonesian journal of tropical and infectious disease, 8(2), 1–8 introduction the stigma against plwha, which arises from the mind of an individual or society who believes that aids is a result of immoral behavior that cannot be accepted by society, is refl ected in cynical attitudes, feelings of excessive fear, and negative experiences to plwha1. stigma and discrimination are not only carried out by commoners who do not have enough knowledge about hiv and aids but can also be carried out by health workers2. the opinion that states aids is a curse because of immoral behavior also greatly aff ects how people comport themselves and behave towards plwha3. in 2014, unaids established a program in accordance with millennial developmental goals (mdgs) namely 3 zeros, which includes zero new infections, zero aids-related deaths, and zero stigma and discrimination4. this program is a humancentered hiv prevention and treatment service to end the aids epidemic by 20305. however, this has not been in contrary to the reality in the fi eld. research by stringer involving 651 health workers found that almost 90% of health workers gave at least one stigma to plwha. 18.9% of health workers agreed that plwha had a large number of sexual partners, 33.3% agreed that plwha could avoid hiv infection if they wanted to, and 35.3% thought that suff erers could become infected with hiv due to irresponsible sexual behavior6. research in indonesia in 2014 also found stigma by health workers, including landfi lls that are diff erentiated and labeled hiv, feeding under the door, not changing patient’s bedsheets, excessive use of protective equipment, isolation, and taking action without informed consent7. stigma by health workers towards people with hiv certainly still has a strong impact. eventually, this will impact how others perceive a person, social rejection, decreased acceptance of social interaction, increased discrimination, and adding family burden8. the impact of this stigma is not good and can be fatal for hiv patients, as mentioned in the study conducted by ardani9. drug-addict-plwha who feel stigmatized will reduce the possibility of seeking treatment, for those who have undergone treatment may choose to end the treatment. furthermore, stigma aff ects the lives of plwha by causing depression and anxiety, sadness, guilt, and feelings of worthlessness. besides, stigma can reduce the quality of life and limit access and use of health services9. labeling and discrimination against people living with hiv-aids are the foremost eff ective barriers in preventing hiv and also in providing drugs, care, and support10. because of the stigma of people with hiv can have a wide-ranging impact, it is necessary to identify the factors that infl uence stigma to plwha by health workers. identification of factors that cause a decrease in stigmatization by primary health center workers will have an impact on improving the quality of life of people with hiv, improving medication adherence, so the incidence rate of hiv itself will be reduced. therefore, this study was aimed to identify the correlating factors between plwha and stigmatization by community health center’s workers using subjects of people with hiv 92 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 90–100 in the outpatient care clinic of intermediate and infectious disease care unit (perawatan intermediet penyakit infeksi pipi) dr. soetomo hospital surabaya. it is hoped that the results of this study can provide input to policymakers to initiate a stigma reduction program for people with hiv that can be started from plwha who has the highest stigma, to make it easier for plwha to disclose their status and treatment. also, it is hoped that the prevention of hiv transmission to the community will be more controlled and help improve the quality of life with hiv-aids (plwha). materials and methods this study used an observational analytic study with cross-sectional study design. the sample of this study was 94 hiv positive patients in the outpatient care clinic of intermediate and infectious disease care unit dr. soetomo hospital surabaya from october to december 2018 who were referral patients from a community health center or had received health services at a community health center in east java after being diagnosed with hiv. the sampling technique used was consecutive. respondents were interviewed using a modifi ed questionnaire by the health policy project available at www.stigmaindex. com, which has been tested for reliability and validity with a cronbach’s alpha coeffi cient of 0.786. the standardized brief questionnaire by the health policy project was developed and verifi ed through a calculated collaborative process that involved experts from various countries. there are four areas which are pertinent to stigma and discrimination in health care environment that the experts are complied to focus on: 1) fear of hiv infection among health facility staff ; 2) stereotypes and prejudice related to people living with or thought to be living with hiv; 3) observed and secondary stigma and discrimination; and 4) policy and work environment11. in the questionnaire by the health policy project, the health workers’ point of view is used as the object. what is new in this study is using the perspective of people living with hiv-aids. the questionnaire was about socio-demographic data and hiv-related questions that illustrate the understanding, awareness, and experience of attitudes by health center workers towards plwha. this questionnaire was divided into four sections. the fi rst section was background information containing questions about sex, age, marital status, duration of hiv diagnosis, the origin of residence, occupation, and history of key population. the second section, infection control, contained questions about the stigma that has been experienced related to hiv infection control at the time of examination. the third section, health facilities’ environment, contained questions related to stigma in the health facility environment. the fourth section, opinion about people living with hiv, contained statements related to the opinion of health workers towards people living with hiv-aids. the choice of answers to each question was how often the stigma occurred so that it would describe which stigma is most often obtained. results and discussion sociodemographic characteristics the sample in this study was varies based on the gender, age, marital status, occupation, duration of patient diagnosed with hiv, hiv control/check-up, residence, and history of key population as described in table 1. patients from surabaya were grouped according to the sub-district of residence. the distribution of patients from surabaya is shown in table 2. the number of females infected with hiv-aids was higher than males, in contrast to data released by the ministry of health in 2017. the higher number of infected females is because females are vulnerable to hiv due to biological factors, reduced sexual autonomy, and it is explained that women want to prevent hiv but do not have enough strength to against12. prospective studies of serodiscordant couples and male contact with fsw show that women are twice as likely to be infected if exposed to hiv13. the age classifi cation in table 1 is based on the indonesian ministry of health in the annual hiv-aids disease progress 93jihan qonitatillah, et al.: lower perceived-stigmatization by health workers copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 report, which used the same age classifi cation so that the comparison of results is appropriate. the age of most plwha obtained from this study was 25-49 years because it is the age of sexually active. the same data is issued by the indonesian ministry of health in the report on the development of hiv-aids & sexually transmitted infectious diseases for the first quarter 2017, that is 69.6% is the 25-49 years age group, 17.6% is the 20-24 years age group and 6.7% is the age group of >50 years14. most marital status was marriage, which could be a clue that sexual contact was the most cause. the longest hiv diagnosis was one year or less, which could be understood because dr. soetomo hospital surabaya is a third-level health facility that accepts referral cases and cannot be resolved at a fi rst or second level health facility. arvs were taken at the dr. soetomo so that many new patients immediately went to the dr. soetomo hospital surabaya to get treatment. the most times of having hiv control to health services was once in a month at dr. soetomo hospital surabaya due to the rules of taking antiretroviral drugs. table 1. sociodemographic characteristics sociodemographic characteristics frequency percentage (%) gender male 45 47.9 female 49 52.1 age 20-24 years old 2 2.1 25-49 years old 84 89.3 >50 years old 8 8.6 marital status married 58 61.7 single 23 24.5 widowed 13 13.8 occupation housewife 25 26.6 female sex worker 45 47.9 health worker 1 1.1 others 23 24.6 duration of patient diagnosed with hiv 1 year 26 27.7 2 years 7 7.4 3 years 17 18.1 4 years 9 9.6 5 years 8 8.5 6 years 8 8.5 7 years 4 4.3 8 years 2 2.1 9 years 3 3.2 >10 years 10 10.7 hiv control/check-up twice or more in a month 11 11.7 once in a month 79 84 once in three months 2 2.1 once in 4-6 months 2 2.1 residence blitar 2 2.1 bondowoso 1 1.1 gresik 3 3.2 jombang 1 1.1 mojokerto 1 1.1 ngawi 1 1.1 pasuruan 3 3.2 sidoarjo 9 9.6 sumenep 2 2.1 surabaya 71 74.3 trenggalek 1 1.1 history of key population yes 30 33.9 no 64 68.1 table 2. distributions of patients from surabaya sub-districts frequency percentage (%) benowo 2 2.9 bubutan 1 1.4 genteng 1 1.4 gubeng 6 8.6 karang pilang 1 1.4 kenjeran 1 1.4 krembangan 7 10 mulyorejo 3 4.3 pabean cantian 2 2.9 rungkut 2 2.9 sawahan 10 14.3 semampir 2 2.9 sukolilo 3 4.3 sukomanunggal 1 1.4 simokerto 1 1.4 tambaksari 12 17.1 tegalsari 7 10 wiyung 3 4.3 wonocolo 1 1.4 wonokromo 4 5.7 94 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 90–100 most patients lived in surabaya, precisely in tambaksari district. this can be understood because it is located near to dr. soetomo hospital surabaya, which is about 2 km measured using the google maps application. there are four community health centers in this district, namely pacarkeling health center, tambakrejo health center, rangkah health center, and gading health center. the second most was from sawahan district. this is consistent with data from the ministry of health of the republic of indonesia, which is as many as 139 patients tested positive for hiv in the fi rst quarter of 2017, the most after health center of putat jaya surabaya14. the number of patients who did not have a history of key population was greater than those who had a history of key population, which is as much as 68.1%. the distribution of key population background of people living with hiv-aids (plwha) history of key population was obtained through interviewing the patients using questionnaires. the data obtained is displayed in table 3. the results have been obtained that patients with the most history of key population are injected-type drug users (idus) and prostitute (fsw) as many as nine people. the same data issued by the ministry of health of the republic of indonesia shows the data of idu has the highest prevalence of 41% compared to other key populations15. hiv prevalence in the idu group is high because they inject drugs more than once a day and more than 60% of them using needles that are not sterilized. while risky sexual behavior that causes hiv prevalence among fsws remains high, because of unprotected sex. msm groups of 7 people followed this. it was reported that condom use in msm consistently lower than fsw, despite the higher level of hiv prevention knowledge16. d e s c r i p t i o n o f p lw h a’ s p e r c e i v e d stigmatization by health center workers the description of stigmatization by health workers at the community health center perceived by plwha was obtained from interviewing the patients using questionnaires. the data obtained is displayed in table 4, 5, 6, and 7. in section 2: infection control, was divided into two parts. part 1 was health center workers’ concern when examining people living with hivaids since part 2 was exclusive protection in treating people living with hiv-aids. from 13 questions on the questionnaire that describe stigmatization by health workers at the health center, the stigmatization of health workers was taken which was often obtained from the number of subjects who have been stigmatized, the answers to that are least worried, worried, very worried in section infection control. also, the answer once or twice, several times, and almost every time in section health facilities’ environment and health workers opinion about people living with hiv-aids. in section infection control, the most stigmatization was obtained when health workers were worried when they did blood sampling. a study by sismulyanto17 conducted at a hospital in banyuwangi shows that from 96 nurses, as many as 7.5% of the nurses were afraid to take laboratory samples, such as blood and urine. according to sismulyanto17, this is because they were afraid of contracting hiv when in direct contact with the patient’s blood. in section health facility’s environment, the most stigmatization was obtained when health care workers provide low-quality care to hiv table 3. distribution of key population background of plwha category frequency percentage (%) patient with history of key population female sex workers (fsw) 9 9.6 injecting drug user 9 9.6 fsw sex partner 4 4.3 men who have sex with men (msm) 7 7.4 transvestite homosexual 1 1.1 patient without history of key population housewife 28 29.8 private sector worker 20 21.3 others 16 17.0 95jihan qonitatillah, et al.: lower perceived-stigmatization by health workers copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 patients compared to other patients, including rejecting patients with hiv-aids because they consider hiv-aids patients are people who have a great risk if direct contact with patients7. a study in aceh, indonesia, shows that some doctors treat plwha with disrespect, push other patients away from them, and keep them away from care services18. it was also found that most stigmatization was obtained when health workers talk badly about hiv patients. this was due to the high stigma in the community and health workers which causes health workers to stay away from them, so they tended to provide low-quality care. in section health workers’ opinions of people living with hiv-aids, the most stigmatization was obtained when health care workers assume that someone who is infected with hiv because of irresponsible behavior. this was because the community thinks that “bad” behavior is seen from free sex and blames plwha as a source of aids transmission7. table 4. description of plwha’s perceived stigmatization on infection control: part 1 form of stigma not worried a little worried worried very worried never experienced n % n % n % n % n % worried when touching the clothes 82 87.2 3 3.2 1 1.1 0 0 8 8.5 worried when dressing wounds 47 50.0 21 22.3 3 3.2 1 1.1 22 23.4 worried when drawing blood 66 70.2 19 20.2 7 7.4 0 0 2 2.1 worried when taking the temperature 81 86.2 7 7.4 1 1.1 0 0 5 5.3 table 6. description of plwha’s perceived stigmatizationon-health facilities’ environment form of stigma never once or twice several times almost every time n % n % n % n % health workers unwilling to care for you 91 96.8 2 2.1 1 1.1 0 0 health workers providing poorer quality of care to relative to other patients 87 92.6 4 43 2 2.1 1 1.1 health workers talking badly about you 87 92.6 6 6.4 1 1.1 0 0 health workers do not want to do blood sampling 92 97.9 1 1.1 1 1.1 0 0 health workers treat in a place that is not closed 91 96.8 3 3.2 0 0 0 0 disclose the status of hiv patients to others without consent 93 98.9 0 0 1 1.1 0 0 using an hiv-related name when calling you when waiting in sequence number 93 98.9 0 0 1 1.1 0 0 during the examination, health workers call improperly 93 98.9 0 0 0 0 1 1.1 during examinations or other activities at the health center, health workers say that you are hiv patient with a loud tone 93 98.9 0 0 1 1.1 0 0 table 5. description of plwha’s perceived stigmatization on infection control: part 2 form of stigma never rarely often always n % n % n % n % avoid physical contact 83 88.3 9 9.6 2 2.1 0 0 wear double gloves 87 92.6 3 3.2 2 2.1 2 2.1 wear gloves during all treatments 78 83.0 4 4.3 4 4.3 8 8.5 use any special infection-control that are not used while examining other patients 78 83.0 4 4.3 4 4.3 8 8.5 96 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 90–100 relationship analysis relationships between variables were tested using ibm spss statistics 22.0. all data about age, sex, marital status, occupation, place of residence, history of risk groups, and duration of hiv diagnosis were transformed into binomial table 7. description of plwha’s perception of health workers’ opinions of people living with hiv-aids form of stigma never once or twice several times almost every time not know n % n % n % n % n % hearing health workers say most of plwha do not care if they infect other people 88 93.6 2 2.1 1 1.1 1 1.1 2 2.1 hearing health workers say hiv patients should feel ashamed of themselves 88 93.6 4 4.3 0 0 0 0 2 2.1 hearing health workers say most hiv patients have multiple sexual partners 81 86.2 6 6.4 2 2.1 0 0 5 5.3 hearing health workers say someone infected with hiv because they engage in irresponsible behavior 78 83.0 12 12.8 1 1.1 0 0 3 3.2 hearing health workers say hiv is punishment for bad behavior 85 90.4 6 6.4 2 21 0 0 1 1.1 forms for analysis. the statistical test used is the binary logistic multiple regression test. relationship of stigmatization data by health center’s workers with age, sex, marital status, occupation, residence, history of risk groups, and duration of hiv diagnosis are shown in table 8 table 8. bivariate analysis of stigmatization variables on independent variables dependent variables stigma signifi cance (chi-square test)low stigma greater stigma n % n % age <37 25 52,1 23 47,9 p = 0.019 >37 13 28,3 33 71,7 gender male 14 31,1 13 68,9 p = 0.078 female 24 49 25 51 marital status married 29 50 29 50 p = 0.016 single 9 25 27 75 occupation low risk 36 40 54 60 p = 0.690 high risk 2 50 2 50 duration of hiv diagnosis >5 years 15 42,9 20 57,1 p = 0.711 < 5 years 23 39 36 61 residence surabaya 8 34,8 15 65,2 p = 0.526 outside of surabaya 30 42,3 41 57,7 history of key population do not have any history 32 50 32 50 p = 0.006 have history 6 20 24 80 97jihan qonitatillah, et al.: lower perceived-stigmatization by health workers copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 table 9. multivariate logistic regression analysis of stigmatization variables against independent variables dependent variables independent variables p exp (b) signifi cance stigma perception age 0.008 0.249 signifi cant gender 0.950 1.033 not signifi cant marital status 0.013 0.251 signifi cant occupation 0.339 3.174 not signifi cant duration of hiv diagnosis 0.140 0.444 not signifi cant residence 0.092 2.713 not signifi cant history of key population 0.006 0.180 signifi cant using the chi-square test and again tested using the binary logistic multiple regressions test in table 9. the binary logistic multiple regressions test was carried out to eliminate confounding variables, fi nd out which groups received greater stigma, and get an exponential rate of plwha perceptions of stigma by health center workers. the history of key population was divided into two groups. having a history of key population was one of the fsws, fsw’s sex partners, msms, transvestites, and injecting drug users (idus). choices other than fsws, fsw’s sex partners, msms, transvestites, and idus were included as do not have a history of key population. the chosen cut-off for the stigma was 24. it was a high stigma if greater or equal to 24, while smaller than 24 was a low stigma. the score of 24 indicates that the respondent answered never or not worried, which is score 1, in all of the 24 questions, which means that the respondent never got any form of stigma from the health center workers. once or twice, got 2 on the score. score 3 for worried, often, and several times. if the answer was very worried, always, and almost every time got score 4. the score of each respondent was obtained from the sum of each question. the cut-off for age was the mean of them, which was 37.46 rounded to 37. if greater or equal to 37 years old, it was said to be old age. while it was said to be young if smaller than 37 years old. jobs were categorized into 2, high and low-risk jobs. highrisk jobs were health workers, doctors, nurses, security, ward attendants, sex workers, and fl ight attendants. meanwhile, choices other than those mentioned were low-risk jobs. the cut-off chosen residence was surabaya, where patients from the city of surabaya were said to live near and outside surabaya said to be distant. the cut-off time for hiv diagnosis was its mean, which was 4.29. if greater or equal to 4.29 years, it was old patients. while it is new patients if smaller than 4.29 years. analysis of the relationship between age, sex, marital status, occupation, residence, history of key population, and duration of hiv diagnosis with stigmatization by health workers in east java community health centers on patients in outpatient care clinic of intermediate and infectious disease care unit (perawatan intermediet penyakit infeksi pipi) provided signifi cant results on the variables of age, marital status, and key population history. whereas sex, occupation, residence, and duration of hiv diagnosis variables provided insignifi cant results. the history of key population had exp (b) of 0.18, which means plwha who have the history of key population get a stigma 0.18 times compared to those without a history of key population. so, it showed a protective factor of stigmatization by health workers. plwha who have the history of key population got a lower stigma than plwha who did not have. this was because plwha who have the history of key population have a psychological mentality that is accustomed to being stigmatized in the community. pala, villano, and clinton19 explained that hiv stigma is not because someone is hivpositive but also because of other conditions of social stigmatization, such as having same-sex partners with other people, female sex workers, and her partner/s, and injecting drug users 98 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 90–100 (idus). both female sex workers (prostitute) and plwha face the same type of stigma, which is seen as “unclean”, a danger to public health, and making decisions that are detrimental to their families and communities. for fsw living with hiv, they get these two stigmas. sex workers living with hiv are regularly exposed to negative stereotypes about themselves and consider them ‘worthy’ to become hiv positive20. due to the frequent exposure to negative stereotypes from the community, plwha’s psychological state who have a history of key population is more vulnerable to stigma. plwha who do not have a history of key population, have a different mentality than plwha who have a history of key population because they are not accustomed to experiencing stigma from the community. hiv-aids brings an unprecedented problem for that person, regardless of background. a person suff ering from hivaids experiences severe psychological distress and feels hopeless about the future, including work, family life, health, and self-esteem21. old age, above 37 years old, gets a higher stigma compared to the age below 37 years old. this is because older adults are at a signifi cant risk of experiencing hiv stigma22. research has shown that older plwha may experience greater stigma due to the double stigma of being hiv positive plus age discrimination, which is usually referred to as layering23. emlet has stated that layering or co-occurring stigmas of ageism and hiv stigma had been experienced by about 68% of older hiv positive adults in washington dc. internalized stigma has a negative impact on the self-esteem and psychological well-being of older adults living with hiv24. plwha who were married got lower stigma compared to plwha who were not currently married, which was 0.251 times. in this case, the factor of being married is associated with social support. plwha who are married has higher social support compared to plwha who are single. research conducted by emlet explains that social support is associated with lower levels of hiv stigma25. a signifi cant relationship had been proven found between the participation of peer groups and the improvement of the quality of life of plwha26,27. reducing the impact of stigma and perceived behavior of plwha can be done by changing individual and community perceptions about hiv-aids by using peer support and counseling approaches28. it was also explained that social support aff ects lower levels of depression and anger29. sex, occupation, residence, and duration of hiv diagnosis variables gave insignifi cant results related to stigmatization by health workers. some factors that are thought to cause this result include the research method in the form of interviews so that there could be biased information. the cut-off values that do not have standard rules yet in categorizing continuous variables can affect the relationship and interpretations of the research results. also, it will randomize the research fi ndings30,31. categorizing variable will make some information loss, so the statistical power to know the relation between variables will be lower32. this is well understood because if the threshold for the defi nition of “exposure” changes, the magnitude of the estimated eff ect such as the odd ratio (or), will vary too30. conclusions stigma against people living with hiv-aids (plwha) by health workers is still often found in the community health center in east java. the stigma could have a wide impact, so it is necessary to identify the factors that infl uence the occurrence of stigma, which is expected to reduce stigmatization by health workers. factors related to plwha’s perception of stigma among health workers found in this research were the history of key population, age, and marital status. plwha who have a history of key population, got a lower stigma than plwha who do not have because plwha who have a history of key population have a psychological mentality that the score to being stigmatized in the community. old age got higher stigma compared to the young age, because of having the double stigma of being hiv positive and age discrimination. plwha who were married, got lower stigma compared to plwha who were not currently married because they have higher social support compared to plwha 99jihan qonitatillah, et al.: lower perceived-stigmatization by health workers copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 who are single. it is hoped that the results of this study can provide input to policymakers to initiate a stigma reduction program for people with hiv that can be started from plwha who has the highest stigma, to make it easier for plwha to disclose their status and treatment. besides, it is hoped that the prevention of hiv transmission to the community will be more controlled and to help improve the quality of life people living with hiv-aids (plwha). acknowledgment the authors would like to express gratitude to those who had helped in the implementation of this study, including staff in the intermediate and infectious disease care unit (perawatan intermediet penyakit infeksi pipi) dr. soetomo hospital surabaya, all patients that had been willing to take part in this study. conflict of interest there is no confl ict of interest of this study. references 1. maman s, dkk. a comparison of hiv stigma and discrimination in five international sites: the infl uence of care and treatment resources in high prevalence settings. soc sci med. 2009; 2271–8. 2. paryati t, dkk. faktor-faktor yang mempengaruhi stigma dan diskriminasi kepada odha (orang dengan hiv/aids) oleh tenaga kesehatan: kajian literatur. pustaka unpad. 2013; 3. musthofa sb, shaluhiyah z, widjarnoko b. stigma masyarakat terhadap orang dengan hiv/aids. j kesehat masy nas. 2015; 9(4): 333–9. 4. komisi penanggulangan aids. strategi dan rancana aksi nasional 2015-2019 penanggulangan hiv dan aids di indonesia. 2015. 5. unaids. fast track: ending the aids epidemic by 2030. 2014. 6. stringer kl, turan b, mccormick l, durojaiye m, nyblade l, kempf mc, et al. hiv-related stigma among health care providers in the deep south. aids behav. 2016; 115–25. 7. maharani r. stigma dan diskriminasi orang dengan hiv/aids (odha) pada pelayanan kesehatan di kota pekanbaru tahun 2014. j kesehat komunitas. 2014; 2(5): 225–32. 8. khairiyah r. peningkatan self regard untuk menyikapi stigma masyarakat terhadap orang dengan hiv/aids di yayasan abdi asih surabaya. diligib uin surabaya. 2018; 9. ardani i, handani s. stigma terhadap orang dengan hiv/aids (odha) sebagai hambatan pencarian pengobatan: studi kasus pada pecandu narkoba suntik di jakarta tahun 2017. bul penelit kesehat. 2017; 45(2): 81–8. 10. shisana o, rehle t, simbayi lc, zuma k, jooste s, zungu n, et al. south african national hiv prevalence, incidence, and behaviour survey, 2012. hsrc press. 2014; 11. health policy project. measuring hiv stigma and discrimination among health facility staff: monitoring tool for global indicators. 2015. 12. sern tj. the knowledge, perceptions, attitudes, and perceived risk in hiv/aids among woman in malaysia: a cross-sectional study. int j soc sci. 2018; 8(9): 725–34. 13. higgins ja, hoff man s, dworkin sl. rethinking gender, heterosexual men, and women’s vulnerability to hiv/aids. am j public. 2011; 435–45. 14. kementerian kesehatan republik indonesia. laporan perkembangan hiv/aids 7 penyakit menular seksual (pims) triwulan i tahun 2017. faktor-faktor risiko penularan hiv/aids pada laki-laki dengan orientasi seks heterose. 2017. 15. kementerian kesehatan republik indonesia. surveilans terpadu biologis dan perilaku 2011. 2011. 16. aw o f a l a a a , o g u n d e l e o e . r e v i e w h i v: epidemiology in nigeria. saudi j biol sci king saudi univ. 2018; 967–703. 17. sismulyanto, supriyanto s, nursalam. model to reduce hiv-related stigma among indonesian nurses. int j public heal sci. 2015; 4(3): 184–91. 18. harapan h. sciverse sciencedirect discriminatory attitudes toward people living with hiv among health care workers in aceh, indonesia: a visa from a very low hiv caseload region. cegh clin epidemiol glob heal. 2013; 29–36. 19. pala an, villano p, clinton l. attitudes of heterosexual men and women toward hiv negative and positive gay men. j homosex. 2017; 64(13): 1778–1792. 20. nswp. stigma and discrimination experienced by sex workers living with hiv. 2015. 21. sharma p, kirmani mn. psychotherapy in hiv/aids. int j indian psychol. 2015; (3): 115. 22. leblanc a. aging with hiv/aids. in r. settersten jr & j. angel (eds.). handb social aging. 2011; 495–512. 23. emlet ca. the impact of hiv-related stigma on older and younger adults of aids/hiv care. psychol sociomedical asp aids/hiv. 2014; 24. emlet ca. understanding the impact of stigma on older adults with hiv. psychology and aids exchange newsletter. 2014; 25. emlet c, brennan d, brennenstuhl s, rueda s, hart t, rourke s, et al. protective and risk factors associated with stigma in a population of older adults living with hiv in ontario canada. aids care. 2013; 1330–9. 100 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 90–100 26. fajriyah yl, demartoto a, murti b. the eff ect of depression, stigma, and peer support group, on the quality of life of people living with hiv/aids in solo plus peer support group, surakarta, central java. j heal promot behav. 2018; 3(1): 27–36. 27. kurniasari ma, murti b, demartoto a. association between participation in hiv/aids peer group, stigma, discrimination, and quality of life of people living with hiv/aids. j epidemiol public heal. 2016; 1(2): 127–34. 28. vyavaharkar m, moneyham l, murdaugh c, tavakoli a. factors associated with quality of life among rural women with hiv disease. aids behav. 2012; 16(2): 295–303. 29. whitehead n, hearn l, burrel l. the association between depressive symptoms, anger, and perceived support resources among underserved older hiv positive black/african american adults. aids patient care std’s. 2014; 507–12. 30. heavner k, burstyn i. a simulation study of categorizing continous exposure variables measured with error in autism research: small changes with large eff ects. int j environ res public health. 2015; 12: 10198–234. 31. decoster j, gallucci m, iselin a. best practices for using median splits, artifi cial categorization, and their continous alternatives. j exp psychopathol. 2011; 2(2): 197–209. 32. gyimesi ml, vilsmeier jk, voracek m, tran us. no evidence that lateral preferences predict individual differences in the tendency to update mental representations: a replication-extension study. collabra psychol. 2019; 5(1): 38. vol. 8 no. 3 september–december 2020 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ research report the epidemiological pattern and risk factor of esbl (extended spectrum β-lactamase) producing enterobacteriaceae in gut bacterial flora of dairy cows and people surrounding in rural area, indonesia agusta reny soekoyo1, sulistiawati2, wahyu setyorini3, k. kuntaman3,4,5 1 master program of basic medical sciences, faculty of medicine, universitas airlangga 2 department of public health, faculty of medicine, universitas airlangga 3 institute of tropical disease, universitas airlangga 4 department of medical microbiology, faculty of medicine, universitas airlangga 5 dr soetomo academic hospital, surabaya, indonesia received: 23rd january 2020; revised: 28th january 2020; accepted: 6th october 2020 abstract livestock would be a risk factor of resistant bacteria that impact on human health. rural area with farms as major economic source has become a risk of the spread of the esbl producing enterobacteriaceae the aim of the study was to explore the distribution and risk factor of esbl (extended-spectrum β-lactamase) producing enterobacteriaceae in the gut bacterial fl ora of dairy cows and people surrounding farming area. total of 204 fecal swab samples were collected, 102 from dairy cows and 102 from farmers. samples were sub-cultured by streaking on macconkey agar supplemented with 2 mg/l cefotaxime. the growing colonies were confi rmed of the esbl producer by modifi ed double disk test (m-ddst) and identifi cation of enterobacteriaceae by biochemical test. esbl genes were identifi ed by pcr. esbl producing bacteria were found 13.7% in dairy cows and 34.3% in farmers. esbl producing enterobacteriaceae in dairy cows were 6.9% and in farmers of 33.3%. statistical analysis showed: distribution of esbl producing enterobacteriaceae strain were insignifi cant among dairy cows and farmers while blatem distribution was signifi cantly diff erent (p= 0,035) and use of antibiotic was identifi ed as a risk factor of colonization of esbl producing enterobacteriaceae in farmers (p= 0,007). farmers had suspected as the source of esbl producing enterobacteriaceae based on higher prevalence. further education of appropriate use of antibiotic need to enhance to control risk factor and prevent the colonization of esbl producing enterobacteriaceae. keywords: enterobacteriaceae, esbl, gut fl ora, dairy cow, farmer, rural abstrak hewan ternak diduga sebagai faktor risiko kejadian bakteri resisten yang berdampak terhadap kesehatan manusia. area rural dengan potensi ekonomi di sektor peternakan merupakan area yang berisiko terhadap penyebaran enterobacteriaceae penghasil esbl. penelitian bertujuan mengeksplorasi pola distribusi dan faktor risiko enterobacteriaceae penghasil esbl pada bakteri fl ora usus sapi perah dan penduduk sekitarnya. total 204 sampel swab feses, terdiri dari 102 swab feses sapi perah dan 102 swab feses peternak. swab feses ditanam pada media macconkey yang ditambahkan 2 mg/l cefotaxime. koloni yang tumbuh dikonfi rmasi sebagai penghasil esbl dengan metode modifi ed double disk test (m-ddst) and diidentifi kasi dengan uji biokimia. identifi kasi gen esbl menggunakan metode pcr. prevalensi bakteri penghasil esbl di sapi perah sebesar 13.7% dan di peternak sebesar 34.3%. distribusi enterobacteriaceae penghasil esbl pada sapi perah 6.9% dan pada peternak 33.3%. analisis statistik menunjukkan: tidak ada perbedaan signifi kan antara distribusi bakteri enterobacteriaceae penghasil esbl pada sapi perah dan peternak, distribusi blatem pada sapi perah dan peternak berbeda signifi kan (p = 0,035), dan penggunaan antibiotik sebagai faktor risiko kolonisasi enterobacteriaceae penghasil esbl pada peternak (p= 0,007). peternak diduga sebagai sumber enterobacteriaceae penghasil esbl. penyuluhan * corresponding author: kuntaman@fk.unair.ac.id 145agusta reny soekoyo, et al.: the epidemiological pattern and risk factor of esbl copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 tentang penggunaan antibiotik secara tepat perlu ditingkatkan untuk mengendalikan faktor risiko dan mencegah kolonisasi enterobacteriaceae penghasil esbl. kata kunci: enterobacteriaceae, esbl, fl ora usus, sapi perah, peternak, rural how to cite: the epidemiological pattern and risk factor of esbl (extended spectrum β-lactamase) producing enterobacteriaceae in gut bacterial flora of dairy cows and people surrounding in rural area, indonesia. soekoyo, ar. sulistiawati, setyorini, w. kuntaman, k. indonesian journal of tropical and infectious disease, 8(3), 144–151. introduction the inappropriate use of antibiotics in human and animal health is a major cause of pathogenic bacterial resistance.1 resistant p a t h o g e n i c b a c t e r i a t h a t h a v e i n c r e a s e d significantly over the past few decades are esbl-producing bacteria (extended-spectrum β-lactamase).2 esbl mainly distributed among gram-negative bacilli of the enterobacteriaceae group.3 the use of antibiotics as growth promotion and the prevention of disease in veterinary 4 was correlated with the increase of esbl producing gram-negative bacteria.5 it thus, the livestock are identifi ed as a risk factor for esbl producing enterobacteriaceae (esbl-e).6 in 2018, east java province was identifi ed has the highest population of dairy cows in indonesia, about 283,311 cows.7 most of the dairy farms in east java province are located in rural areas to empowering the community's economy. kalipucang village in district of pasuruan, east java was established as the fi rst center of public dairy farming in indonesia at 2016.8 e s b l p r o d u c i n g e n t e ro b a c t e r i a c e a e (esbl-e) bacteria cause various infections in humans, such as: bacteremia, gastroenteritis, respiratory infections, urinary tract infections, and infections of the central nervous system.9 in dairy cows, escherichia coli and klebsiella spp are identifi ed as an agent that causing infl ammation of mammary gland and udder tissue (mastitis) which impact on decreasing quantity and quality of milk production, increasing the rejected prematurely, and death.10 esbl-e becomes a serious challenge in therapy for infection includes prolong of diagnosis and expensive, a longer duration of treatment, limited antibiotic choices that impact on higher cost of therapy for an infection, as well as increased morbidity and mortality.11 multiple resistance to fl uoroquinolones, aminoglycosides, and trimethoprim are commonly found in esble.3 it also causes carrier in both humans12 and livestock.6 since esbl-e has been identifi ed as one of the causes of mastitis in dairy cows in 2000,6 dairy farming was suspected to be at risk as a source of esbl-e transmission. it thus the epidemiological profi le of esbl-e in farm needs to be explored. this study is the fi rst study to analyze the epidemiological patterns of esbl producing enterobacteriaceae in livestock and humans in rural areas in indonesia. the aim of the study was to identify and analyzed the distribution and risk factor of esbl producing enterobacteriaceae in gut bacterial fl ora of dairy cows and people/farmer who have close contact with dairy cows. materials and methods design this study was conducted a cross-sectional design. this study was approved by research ethics committee in faculty of medicine of universitas airlangga, no: 82/ec/kepk/ fkua/2019. samples collection fecal samples were collected from april until july 2019 from dairy cows and farmers. samples collected using amies transport medium (deltalab, spanyol). the total dairy farming in the district of pasuruan, east java, are 648 clusters, of which as many as 102 were randomly included as the samples in this study, consisting 146 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 3 september–december 2020: 144–151 of 102 samples from dairy cows and 102 from humans that living around and have close contact with dairy cows. these clusters were located in kalipucang village. the samples transportation were using a cool box and ice pack (4-8˚c). samples were processed within 24 hours after taken from the sample source. bacterial and esbl identifi cation the isolation, confi rmation, and identifi cation of esbl-e were conducted in the clinical microbiology laboratory of dr. soetomo hospital, surabaya. amies swab was streaked on macconkey selective medium supplemented by cefotaxime 2 mg/l and incubated for 18-24 h at 37˚c. the growing colonies were esbl confi rmed by modifi ed double disk synergy test (m-ddst). colonies which grow were inoculated in mueller hinton medium (0,5 macfarland) with five (5) antibiotics disk : amoxicillin/ clavulanic (amc) 30 ug, ceftazidime (caz) 30 ug, cefotaxime (ctx) 30 ug, ceftriaxone (cro) 30 ug, and aztreonam (atm) 30 ug which placed within 15 mm of distance from edge to edge of amc disk.13 incubated for 18-24 hours at 37˚c. the inhibition zone which show synergy zone between one of cephalosporin disk or aztreonam disk with amoxicillin/clavulanic disk was confi rmed as esbl producer. esbl positive strain were bacteriologically identifi ed using the biochemical test: triple sugar iron (tsi) test, indol test, methyl red (mr) test, voges proskauer (vp) test, urease test, and motility test. all bacterial isolates were then stored in deep freeze minus 80˚c. genotypic examination genotypic examination was held in institute of tropical diseases, universitas airlangga, surabaya. dna extraction dna extraction was conducted by boiling method. the identifi ed esbl producing bacteria were re-cultured on mueller hinton medium, incubated at 37˚c for 18 – 24 h. four to fi ve colonies were taken and suspended in sterile distilled water in 1,5 ml eppendorf tube. the suspension was homogenized with vortex for 15 seconds and immersed in a thermostat at 95˚c for 10 minutes, then centrifuged at 14.000 rpm for 1 minutes. the supernatant was used as dna template in pcr and stored in -20˚c.14 dna amplifi cation three esbl gene primers are used to amplify and identify the esbl gene, as follow (table 1) : [15] pcr reaction was run in volume of 20 μl: 10 ul of gotaq green master mix 2x (promega),1 ul for each of forward and reverse primers, 3 μl nuclease free water, and 5 μl dna template. pcr was run as follow: for blactx-m: denaturation on 94ºc for 7 minutes and the following 35 cycles on 94ºc for 50 seconds, annealing on 50ºc for 40 seconds, extension on 72ºc for 1 minute, and fi nal extension on 72ºc for 5 minutes; for blashv: denaturation on 96ºc for 5 minutes and the following 35 cycles on 96ºc for 1 minute, annealing on 60ºc for 1 minute, extension on 72ºc for 1 minute, and fi nal extension on 72ºc table 1. primers of esbl genes gen sekuens primer (5’-3’) amplicon size (bp/base pair) blactx-m f : 5’ atgtgcagyaccagtaargt 3’ r : 5’ tgggtraartarctsaccaga 3’ 593 blashv f : 5’ ggttatgcgttatattcgcc 3’ r : 5’ ttaggttgccagtgctc 3’ 867 blatem f : 5’ atgagtattcaacatttccg 3’ r : 5’ ctgacagttaccaatgctta 3’ 867 147agusta reny soekoyo, et al.: the epidemiological pattern and risk factor of esbl copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 for 10 minutes; and for blatem: denaturation on 96ºc for 5 minutes and the following 35 cycles on 96ºc for 1 minute, annealing on 58ºc for1 minute, extension on 72ºc for 1 minute, and fi nal extension on 72ºc for 10 minutes. pcr amplicon were visualized in 2% gel electrophoresis. questionnaire to find risk factors information about risk factor of esbl producing enterobacteriaceae in dairy cows and farmers were obtained through interview and questionnaires. enterobacteriaceae strain and genotype distribution among dairy cows and farmers and risk factors were analyzed by chi square/fisher exact test on spss 22 version program. results d i s t r i b u t i o n o f e s b l p r o d u c i n g enterobacteriaceae in dairy cows and farmers prevalence of esbl producing bacteria in dairy cows was 13.7% (14/102) and in farmers 34.3% (35/102). esbl producing enterobacteriaceae in dairy cows were 6.9% (7/102) and in farmers 33.3% (34/102) (table 2). the esbl producing enterobacteriaceae in dairy cows were mostly: escherichia coli 85.7% (6/7) and enterobacter spp 14.3% (1/7), whereas among 34 esbl-e in human were escherichia coli 82.4% (28/34), enterobacter spp 14.3% (3/34), klebsiella pneumoniae 5.9% (2/34), and klebsiella oxytoca 2.9% (1/34). there were no signifi cant diff erences in distribution of esbl producing enterobacteriaceae strain in dairy cows and in farmers (table 2) and fig.1. escherichia coli were identifi ed as dominant strain of esbl producing enterobacteriaceae in dairy cows and farmers (85.7% vs. 82.4%). distribution of esbl gene among dairy cows and human (farmers) a m o n g s e v e n e s b l p r o d u c i n g enterobacteriaceae in dairy cows, six isolates were harbored blactx-m (85.7%) and one an unidentifi ed gene (14.3%). among 34 isolates of esbl producer in farmers, 26 isolates harbored blactx-m (76.5%), 15 isolates blatem, and three table 2. distribution of esbl producing enterobacteriaceae strain in dairy cows and farmers esbl producer dairy cows (n=102) farmers (n=102) p value esbl producing bacteria 14 (13.7%) 35 (34.3%) nonenterobacteriaceae 7 (6.9%) 1 (0.9%) enterobacteriaceae 7 (6.9%) 34 (33.3%) escherichia coli 6 (85.7) 28 (82.4) p = 1,000 enterobacter spp 1 (14.3) 3 (8.8) p = 0,542 klebsiella pneumoniae 0 / 0 2 (5.9) p = 1,000 klebsiella oxytoca 0 / 0 1 (2.9) p = 1,000 note: esbl-e: esbl producing enterobacteriaceae figure 1. the double disk synergy test (ddst) for identifying esbl producer bacteria. note: the increasing of inhibition zone in area between cephalosporin disk and clavulanic acid disk was marked as positive esbl producer. table 3. esbl genes distribution of esbl producing enterobacteriaceae in dairy cows and farmers esbl gene dairy cows (n=7) farmers (n=34) p value blactx-m 6 (85.7) 26 (76.5) p = 1,000 blashv 0 (0) 3 (8.8) p = 1,000 blatem 0 (0) 15 (44.1) p = 0,035 unidentifi ed gene 1(14.3) 0 (0) p = 0,171 blactx-m, blatem 0 (0) 8 (23.5) blactx-m, blashv, blatem 0 (0) 1 (2.9) note: unidentifi ed gene: gene of esbl producing enterobacteriaceae which couldn’t detected with specifi c primer used in this study 148 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 3 september–december 2020: 144–151 isolates blashv (8.8%), respectively. there was a signifi cant diff erence of blatem distribution in dairy cows and in farmers (p = 0, 035) (table 3). combination of two and three of esbl genes were found in enterobacteriaceae producing esbl isolates in farmers. eight isolates harbored blactx-m dan blatem (23.5%) and one isolate harbored blactx-m, blashv, blatem (2.9%) (table 3). risk factor for colonization of esbl producing enterobacteriaceae age, origin of dairy cows, history of illness during the last 3 months, history of drug use, type of drug given last 3 month, and type of feed were not risk factors for colonization of esbl-producing enterobacteriaceae in dairy cows. risk factor for esbl producing enterobacteriaceae colonization in farmers was the use of antibiotics (p = 0.007). gender, age, education level, household, hygiene sanitation of environment (location of dairy cow shed and type of toilet), personal hygiene sanitation (frequency and how to wash hands), and frequency of going out of the city during the last 3 months were not a risk factor. discussion c o l o n i z a t i o n o f e s b l p r o d u c i n g enterobacteriaceae in dairy cows by 6,9% in this study is similar with study in healthy ruminant (cows and buff aloes) in rural areas in cambodia by 7%16 and lower than in cattle farm in german by 54.5%.6 colonization of esbl producing enterobacteriaceae in farmers by 33,33% lower than the colonization of esbl-producing bacteria in healthy individuals in rural areas in thailand by 65.7%,17 in china by 73.9%,18 and workers in cattle farms in germany: 12.5%. 6 human and animal gut were the natural habitat of many bacterial especially enterobacteriaceae and become a reservoir of various infections.12 non-appropriate and overuse of antibiotic caused selective pressure that supports the growth of resistance bacteria.9 colonization of resistance bacteria in human and animal gut causing transmission of resistance genes in gut flora bacterial through horizontal gene transfer by conjugative plasmid.12 esbl mostly encoded by genes in plasmids.19 enterobacteriaceae was identifi ed as having plasmid carrying resistant genes. incfii plasmid group known as a plasmid group that encoded esbl genes and it widely distributed in enterobacteriaceae. it called epidemic resistant plasmid group.20 this study identifi ed escherichia coli as the dominant esbl producing bacteria in dairy cows (85.7%) and farmers (82.4%). distribution of escherichia coli as an esbl producer in dairy cows in this study was 85.7%, higher than in cattle farms in mecklenburg-western pomerania, germany by 54.5%.6 at farmers, distribution of esbl producing escherichia coli by 82.4% is lower than the distribution of escherichia coli in healthy individuals in rural areas in thailand by 85.4% 17 and in china 88%,18 but higher than workers in cattle farms in germany: 12.5%. 6 escherichia coli is the main organism that produces esbl in communities21 and associated with urinary tract infections (uti). it is related to their role as gut bacterial fl ora and are pathogenic to humans and animals.12 the resistance of commensal escherichia coli to antimicrobial agents has been found in healthy individuals.22 this bacterium also acts as an indicator of 'acquired antibiotic resistance genes’ in the community.23 distribution of blactx-m in the esbl producing enterobacteriaceae in dairy cows by 85.7% is higher than the distribution of blactx-m in cattle farms in germany 80%.6 at farmers, figure 2. electrophoresis of amplifi ed gene of blactx-m (593 bp) 149agusta reny soekoyo, et al.: the epidemiological pattern and risk factor of esbl copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 distribution of blactx-m in the esbl-producing enterobacteriaceae by 76.5% higher than in healthy individuals in rural areas in china by 68.1%18 and in thailand by 65.7%.17 blactx-m that mostly integrated with conjugative plasmid, and conjoint with the other resistant gene, has a higher transferability among bacteria, and impact on higher prevalence epidemiologically.24 blactx-m, blashv, and blatem are dominant esbl genes in various regions worldwide and found from isolates of humans, animals and the environment. blatem and blashv mainly found in escherichia coli and klebsiella pneumoniae.25 the study identifi ed blatem in escherichia coli and enterobacter spp isolates and blashv in klebsiella pneumoniae and escherichia coli isolates. blactx-m is the dominant esbl gene worldwide, especially in community and has increased in incidence since 2000.12 it were identifi ed in livestock and pets with escherichia coli as the main producing bacteria.26 our study showed that blactx-m was identifi ed in escherichia coli, enterobacter spp, and klebsiella oxytoca. dissemination of blactx-m occurs rapidly, extensive, and significantly. plasmids are known to carry genes which encode resistance for antibiotic.27 conjugative plasmids play an important role in facilitating the horizontal dissemination of blactx-m among bacteria.28 blactx-m was identified in various epidemic resistant plasmid groups, including: groups incf, incn, inci1, incl / m, and inchi2.24 these plasmid group are able to capture and transfer resistant genes among bacteria.29 the incfii group is the largest plasmid group encoding blactx-m and widely found in enterobacteriaceae24 and isolates from human and animal.20 isecp1 is the genetic element which associated with all variants of blactx-m, play a role incoding transposase and inducing blactx-m expression. transposase is an enzyme that mobilizes blactx-m in certain plasmids.28 other types of is include: iscr1 plays a role in blactx-m group 2 and 9 expression, is10 in blactx-m group 8 expression,24 and is26 in blactx-m group 1 and 9 expression. isecp1 and iscr1 play a role in the mobilization of class 1 integron that encodes various types of resistant genes (mdr cassettes).28 clones of escherichia coli were identifi ed having a signifi cant role in the dissemination of blactx-m, among others, such as st131, st38, st393, st405. st131 serotype 025: h4 phylogenetic group b2 is an extra-intestinal pathogenic e. coli strain and was mainly involved in blactx-m dissemination especially blactx-m15 in worldwide.24 it identifi ed having incfii plasmid group and found in isolates originated from the animal, environment, and especially human.28 combination of two or three esbl genes in one bacterial isolate is due to integron and plasmid that carry several resistant genes. enterobacteriaceae would be harboring of 5 to 6 plasmids in one isolate.30 class 1 integron which related to blactx-m were identified encoding several types of resistant genes (mdr cassettes).28 the unidentifi ed gene is thought to be an esbl gene in addition to blactx-m (group 1), blashv, and blatem. the fi nding of antibiotic use as risk factor of esbl producing enterobacteriaceae in farmers in this study (p= 0,007) related according to the study of luvsansharav et al,17 which identifi ed the use of antibiotics in the last 3 months (or 1,883; 95% ci 1,221-2,903) as a risk factor for colonization of esbl producing bacteria in healthy individuals in rural area in thailand and zang et al18 that identifi ed antibotic use in the previous 6 months (or 1,892; 95% ci 1,242–2,903; p = 0.034) as a risk factor for colonization of esbl-producing bacteria in healthy individuals in rural area in china. in dairy cows in this study, there was not any antibiotic use detected based on data on questionnaires. the total of 52% of dairy cows were given anthelmintic every three months. risk factors for colonization of esbl producing enterobacteriaceae in dairy cows was not identifi ed. dissemination of esbl producing bacteria occurs from animals to humans or vice versa.6 esbl producing gram negative in dairy cows have the potential as a zoonotic risk,31 especially through close contact during daily care.6 150 copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 indonesian journal of tropical and infectious disease, vol. 8 no. 3 september–december 2020: 144–151 the results of the study showed that the rural community could act as a reservoir of esblproducing enterobacteriaceae. the fi nding of escherichia coli and blactx-m as the dominant strain and esbl gene epidemiologically indicated alarming sign. e. coli st131 consider as virulent strain,24 multiple resistance, easily colonize and spread between humans, animals and environment isolates.27 it contributes to the spread of blactx-m globally through horizontal gene transfer.24 esbl-producing e. coli and blactx-m have driven the spread of esbl gene in the community. colonization of esbl-producing enterobacteriaceae is a risk factor for infection of esbl-e.3 the colonization of esbl-producing enterobacteriaceae in community were predicted increasingby about 5% annually.24 this certainly becomes a challenge in therapy of infectious disease. conclusion farmers had suspected as the source of esbl producing enterobacteriaceae based on higher prevalence. the use of antibiotic in human, was identifi ed as risk factor for colonization of esbl producing enterobacteriaceae while not identifi ed in dairy cows. conflict of interest there is no confl ict of interest of this study. acknowledgement we thank staff and cadre of sumberpitu public health centre for assistance of collecting samples, health offi ce of district of pasuruan for the study support. microbiology laboratory of dr. soetomo and institute tropical diseases, surabaya for processing samples. thank you to tahir professorship grant universitas airlangga no. 1149/un3/2018, that supported this study. references 1. health ministry of the republic of indonesia. antimicrobial resistance control becomes world attention. news release. 2018. [cited 30 january 2019]. available from:<http://www.depkes.go.id/ article/view/18112900002/pengendalian-resistensiantimikroba-jadi-perhatian-dunia.html>. 2. shaikh s, fatima j, shakil s, rizvi smd, kamal ma. antibiotic resistance and extended spectrum β-lactamase : types, epidemiology and treatment, saudi j bio sci. 2015 ;22(1) ;90-101. doi: http://dx.doi. org/10.1016/j.sjbs.2014.08.002 3. b r o l u n d a . o v e r v i e w o f e s b l p r o d u c i n g enterobacteriaceae from a nordic perspective. review, infect ecol epidemiol, 2014;4: 1-9. doi:https:// doi.org/10.3402/iee.v4.24555 4. madigan mt, martinko jm, bender ks, buckley dh, stahl da. brock biology of microorganism, 14th edn, pearson education inc.s; 2015: 820-821. 5. dandachi i, chabou s, daoud z, and rolain j m . p r e v a l e n c e a n d e m e rg e n c e o f e x t e n d e d spectrum cephalosporin,carbapenem and colistinresistantgram negative bacteria of animalorigin in the mediterranean basin. front microbiol. review. 2018;9(2299):1 – 26. doi: https://doi.org/10.3389/ fmicb.2018.00550 6. dahms c, hubner no, kossow a, mellmann a, dittmann k, kramer a. occurance of esbl-producing escherichia coli in livestock and farm workers in mecklenburg-western pomerania, germany, plos one. 2015; 10(11): 1-13 https://doi.org/10.1371/journal. pone.0143326 7. central bureau of statistics of republic indonesia. dairy cows population by province, 2009-2018. animal husbandry. dynamic table. [cited 29 december 2019]. available from: (https://www.bps.go.id/ linktabledinamis/view/id/1018). 8. goverment of district of pasuruan. offi cial website of pasuruan regency goverment : pasuruan regency has the first county of dairy farm centre in indonesia, [cited 4 nopember 2019]. available from:<https:// www.pasuruankab.go.id/>. 9. carroll kc, morse sa, mretzner t, miller s. jawetz, melnick & adelberg’s medical microbiology, 27th edn, mcgraw hill education: lange medical books; 2016, pp.351-360. 10. nurhayati, is dan martindah, e. pengendalian mastitis subklinis melalui pemberian antibiotik saat periode kering pada sapi perah. wartazoa. 2015; 25(2): 065 – 074. doi:http://dx.doi.org/10.14334/wartazoa. v25i2.1143 11. amelia a, nugroho a, and harijanto pn. diagnosis a n d m a n a g e m e n t o f i n f e c t i o n s c a u s e d b y enterobacteriaceae producing extended-spectrum β-lactamase. acta med indones-indones j intern med. 2016; 48(2):156-166. 151agusta reny soekoyo, et al.: the epidemiological pattern and risk factor of esbl copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 12. woerther pl, burdet c, chachaty e, and andremont a. trends in human fecal carriage of extended spectrum β-lactamases in the community : toward the globalization of ctx-m. clin microbiol rev. 2013; 26(4): 744-758. doi: http://dx.doi.org/10.1128/ cmr.00023-13 13. schreckenberger p and rekasius v, 2007. procedure double disk diff usion confi rmation of esbl. layola university medical center. pp 1 – 7. 14. tawfi ck mm, el-moghazy an, hassan ma. pcrbased moleculer detection of esbls encoding genes blatem, blactx-m, and blashv among mdr escherichia coli isolates from diarrhoea stool cultures in cairo, egypt. international journal of research studies in microbiology and biotechnology (ijrsmb). 2016; 2(3): 7-14. doi : http://dx.doi.org/10.20431/24549428.0203002 15. ferreira cm, william af, almeida ncods, naveca fg, barbosa mdgv. extended spectrum beta lactamase producing bacteri isolated from hematologic patients in manaus, state of amazonas, brazil. braz j microbiol. 2011 jul-sep; 42(3): 1076-1084. doi: 10.1590/s1517-838220110003000028. 16. atterby c, osbjer k, tepper v, rajala e, hernandez j, seng s, holl d, bonnedahl j, borjesson s, magnusson u, and jarhult jd. carriage of carbapenemase‐ and extended‐spectrum cephalosporinase‐producing escherichia coli and klebsiella pneumoniae in humans and livestock in rural cambodia; genderand age diff erences and detection of blaoxa‐48 in humans. wiley. zoonoses public health. 2019 sep;66(6):603– 617. doi: 10.1111/zph.12612 17. luvsansharav uo, hirai i, nakata a, imura k, yamauci k, niki m, komalamisra c, kusolsuk t, and yamamoto y. prevalence of and risk factors associated with faecal carriage of ctx-m β-lactamase-producing enterobacteriaceae in rural thai communities, journal antimicrobial chemotherapy 2012 jul; 67(7): 1769 – 1774. doi : https://doi.org/10.1093/jac/dks118 18. zhang h, zhouy, guo s,and chan w. high prevalence and risk factors off ecal carriage of ctx-m typeextendedspectrumbeta-lactamase-producingenterobacteriaceae from healthy. front microbiol. 2015 march; 6(239): 1 – 5. doi: https://doi.org/10.3389/fmicb.2015.00239 19. paterson dl and bonomo ra. extended spectrum β-lactamases : a clinical update. clin microbiol rev. 2005 oct;18(4): 657–686. doi: 10.1128/ cmr.18.4.657-686.2005. 20. rozwandowicz m, brouwer msm, fisher j, wagenaar ja, gonzalez-zorn b, guerra b, mevius dj, and hordijk j. plasmids carrying antimicrobial resistance genes in enterobacteriaceae.journal of antimicrobial chemotherapy. 2018 may;73(5): 1121–1137. doi: https://doi.org/10.1093/jac/dkx488 21. de a. extended spectrum beta-lactamase infections, in : parthasarathy a, kundu r, agrawal r, choudhury j, shastri dd, yewale vn, shah ak, uttam kg, textbook of pediatric infectious diseases, iap, national publication house, jaypee brothers medical publishers (p) ltd, new delhi; 2013. 22. rolain jm. food and human gut as reservoirs of transferable antibiotic resistance encoding genes. front microbiol. review. 2013 june; 4(173): 1 – 10. doi: https://doi.org/10.3389/fmicb.2013.00173 23. araque m, and labrador i. prevalence of fecal carriage of ctx-m-15 beta-lactamase-producing escherichia coli in healthy children from a rural andean village in venezuela. osong public health res perspect. 2018;9(1):9 –15. doi: https://doi. org/10.24171/j.phrp.2018.9.1.03 24. canton r, gonzalez-alba jm, and galan jc. ctx-m enzymes: origin and diffusion.front microbiol. review. 2012 april; 3(110): 1-19. doi: https://doi. org/10.3389/fmicb.2012.00110 25. doi y, lovleva a, and bonomo ra. the ecology of extended-spectrum-β-lactamase (esbls) in the developed world. journal of travel medicine. review. 2017 ; 24(suppl. 1) : s44-s51. doi: 10.1093/ jtm/taw102. 26. rossolini gm, d’andrea md, and mugnaioli c. the spread of ctx-m-type extended-spectrum b-lactamases. journal compilation 2008 european society of clinical microbiology and infectious diseases, cmi. review. 2008; 14 (suppl. 1): 33–41. doi: https://doi.org/10.1111/j.1469-0691.2007.01867.x 27. dubey rc. a textbook of biotechnology with biotechnology practicals for class xii. revised edition. s. chand & company pvt. ltd. ram nagar. new delhi; 2014. 28. bevan er, jones am, and hawkey pm. global epidemiology of ctx-m β-lactamases: temporal and geographical shifts in genotype. journal of antimicrobial chemotherapy, editor’s choice. 2017 august; 72(8): 2145–2155. doi: https://doi.org/10.1093/ jac/dkx146 29. carattoli a. plasmids in gram negatives : molecular typing of resistance plasmids. international journal of medical microbiology. mini review. abstract. elsevier. 2011 december; 301(8): 654-658. doi: https:// doi.org/10.1016/j.ijmm.2011.09.003 30. livermore dm. bacterial resistance: origins, epidemiology, and impact. clinical infectious diseases. 2003 jan; 36(issue supplement_1): s11–s23. doi: https://doi.org/10.1086/344654 31. olsen rh, bisgaard m, lohren u, robineau b, and christensen h. extended – spectrum beta-lactamaseproducing escherichia coli isolated from poultry : a review of current problems, iilustrated with some laboratory findings. avian pathology. 2014 apr; 43(3): 199–208. doi: https://doi.org/10.1080/0307945 7.2014.907866 �� vol. 2. no. 1 january–march 2011 effect of cynammyldehyde from cinnamon extract as a natural preservative alternative to the growth of staphylococcus aureus bacteria saka winias1, ariyati retno1, raudhatul magfiroh1, nasrulloh1, ryan m1, dr.retno pudji rahayu,2 1 faculty of dentistry airlangga university surabaya 2 oral biology departement of dentistry faculty airlangga university surabaya abstract food is one of the best media for the microorganism to live and grow. therefore, food is often broken because it has been contaminated by the microorganism. in industry country, approximately 30% of population infected by food borne disease. food borne disease is caused of phatogen bacteria food borne. staphylococus aureus is a kind of bacteria that can make food rotten and also it is a phatogen bacteria cause food born disease, no forming spora, positive gram bacteria and the food substance which is contaminated by staphylococus aureus will cause poisoned becaused of enterotoxin which is heat resisting. essential oil is antimicrobial and anti bacterial that the most effective, it can inhibit the growing of microba and bacteria. one of the example of essential oil is cinnamon.sp oil. cinnamon oil is antimcroba agent for bacteri and fungi because it contain cynammyldehyde and cynammyl alcohol and also eugenol. the aim of this study is to understand the antimcrobacterial potential of cynammyldehyde from cinnamon extract to staphylococus aureus. this study is laboratory experimantal research. essential oil from cinnamon by destilation, then redistilation was done to get cynammyldehyde from cinnamon. cynammyldehyde was tested to staphylococus aureus. test method was done as dilution in the form. from this result, it show that cynammyldehide from cinnamon extract has ability in inhibit the staphylococus aureus growth. we can conclude that cynammaldehyde from cinnamon extract has antibacterial effect especially for positive gram bacteria that is staphylococcus aureus. the optimum inhibiting effort is 0.09%. key words: cinnamon, cynammyldehyde, antibacterial, staphylococcus aureus introduction food is one of the medium for bacteria growth so it can break due to microorganism contamination. microorganism can breaks components in the food into simpler compounds. it will changes, decomposition both nutrition and organoleptic.1 more than two million people dead because of food borne disease. food borne disease caused by pathogenic bacteria of food borne. so, it need aan alternative method which eliminate pathogenic bacteria of food borne disease.1 staphylococcus aureus is the kind of decaying food bacteria which pathogenic bacteria of food borne disease, not producing spore, gram positive bacteria and contaminant from it can be toxic because of enterotoksin.2 preservation food is one of the ways to prevent food which contaminated. one of the kind of preservation food is using synthetic materials likes boraks.3 boraks is used by people but it has toxicity which danger if consume for along day. recently, formalin and boraks are agent which have high reactivity so they can reacts with macromolekul on body system. consuming formalin continuously can effet cancer. preservation substances which can use is antimicroba and antibacterial substances.4,5 essential oil is the effective antimicroba and antibacterial which can inhibit bacteri and microba growth. one of the kind of essential oil is cinnamon oil. cinnamon oil is antimicroba to bacteria and fungi,6 because they have cynammyldehyde, cynammyl alcohol and eugenol,7 so cinnamon oil can inhibit pathogenic food borne bacteria growth.8 in industrial country find about 30% population suspect food borne disease. so it need a new method to decrease and eliminate pathogenic bacteria cause of food borne disease.1 ��winias, et al.: effect of cynammyldehyde from cinnamon extract laboratory experiment needed to determine the concentration of cynammyldehyde can optimally inhibit staphylococcus aureus bacteria growth. the researches want cynammyldehyde of cinnamon extract can use as antibacterial to keep food quality and it can realize to society. natural preservation of cynammyldehyde is safe to consume if in appropriate dose. method and materials materials this experiment is laboratory experimental to prove the ability antibacterial cynammyldehyde of cinnamon extract to standard laboratory bacteria such as staphylococcus aureus. using laboratory tools such as micropipette, petridisc, test tube, test tube rack, spectrophotometer, incubator, brender, and standard oase. the materials are brain hearth infusion, muller hinton, aquades steril, sinamat aldehid, and dmso. bacterial test bacterial test is standard bacteri which sensitive to standard therapy. bacteria found in microbiologi laboratory medicine faculty airlangga university. producing extract the material is cynammyldehyde of cinnamon extract. firstly, determine cinnamon which has thickness about 1,5 mm, long about 1 m and good smell if it broken. after that, wash and dry to produce extract. producing extract in research institute for industrial research and standards surabaya. do steam destilation process to get esential oil from cinamon. the cinnamon size is reduced about ± 2 cm by 5 kg, and cinnamon was processed with a tool distiller so it can result essential oil about 5 ml. next essential oil is the next process is redestilation oil bath process to separate the content of eugenol and cynammyldehyde contained in the essential oil. bath oil redestilation process is performed to obtain names of cynammyldehyde of 3 ml. the oil lab tested to know the size of the content of cynammyldehyde. in the oil we found water content of 0.03%, cinnamic names of aldehydes 72.86% 18.78% and eugenol. this will be the basis of dilution of cynammyldehyde, which will be tested to staphylococcus aureus. preparation of bacteria test bacteria prepared by creating suspense in accordance with the methods of microbiology laboratory. bacteria grown in bhi liquid medium, then turbidity adjusted to mc farland turbidity standard 0.5 (1 ×× 108 cfu / ml) and then diluted to concentrations of bacteria 1 × 106 cfu/ml. dilution test materials then performed a serial dilution: 0.18%, 0:14%, 0.10%, 0.06%, 0.02% and then added bacterial suspension with an equal volume of 1 ml so that the concentration is half that of the original, which is 0.09%, 0.07%, 0.05%, 0.03 %, and 0.01%. determining the activity test solution concentrations that have been given the suspense of bacteria were incubated for 24 hours at 37°c. furthermore, all media were incubated for 24 hours grown on muller hinton for 24 hours at a 37° c to determine the number of colonies that are still growing. and media that have been incubated the absorbance values read using a spectrophotometer at a wavelength of 600nm to determine the percentage of inhibition, respectively each concentration. using the formula:9 % inhibition = [(abs control–abs sample)/abs control] × 100% then count of the colony. each bacterial test was done 5 times. the independent variables in this study were from the names of cynammyldehyde from cinnamon extract that had been serially diluted in several concentrations. meanwhile, as the dependent variable is the presence of bacterial growth. data analysis was performed by descriptive statistical one way anova after the data obtained from 5 times repetition of the staphylococcus aureus bacteria (gram positive). results and discussion results and characterization of materials experiments with 5 times the repetition in the concentration of 0.01%, 0.03%, 0.05%, 0.07%, 0.09% obtained by the addition of 0.18%, 0.14%. 0.10% 0.06% 0.02% with each of the levels provided and 1 ml of 1 × 106 of staphylococus aureus bacteria cfu/ml. after incubated for 24 hours and counting the number of bacteria with the spectrophotometer giving the following results. figure 1. graph of percentage inhibition then, to know the number of bacteria that live at each concentration in every experiment performed with bacterial cultures growing on muller hinton solid medium. one plate media in each experiment were divided into 4 sections, and each part drops 50 μl droplets of liquid medium with concentration of 0.01%, 0.03%, 0.05%, 0.07%, and 0.09%. after planting, each plate were incubated in an incubator for �0 indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 38-41 24 hours to determine colony growth on each plate section. these calculations show the following results. table 1. colony count results concentrarion of cynammyldehyde number of research 1 2 3 4 5 control + ∞ ∞ ∞ ∞ ∞ 0.01 % ∞ ∞ ∞ ∞ ∞ 0.03 % ∞ ∞ ∞ ∞ ∞ 0.05 % ∞ ∞ ∞ ∞ ∞ 0.07 % � 15 8 18 27 0.09 % 4 8 5 10 0 statistical analysis using one-way annova produces data that has been attached. in descriptive tests 0.01% concentration, the average value is 17.04%, the minimum value is 5.2%, and 27% for the maximum value. for concentration of 0.03%, the average value is 28.12%, 1.7% is the minimum value and the maximum value is 58.8%. for concentration of 0.05%, the average value is 36.56%, 29% is the minimum value, and the maximum value is 59%. for concentration of 0.07%, the average value is 78.8%, 41% is the minimum value, and the maximum value is 99.4%. for concentrations of 0.09% has an average rating of 98.0%, 95% is the minimum value, and the maximum value is 100%. it can be concluded that the highest inhibition at a concentration of 0 : 09% and the lowest at 0.01% concentration. in the test for homogenity of variances obtained value of significance of the 0.00 (0.00 < 0.05 (α)). this results indicate that there are differences of the variance of the inhibition for each concentration. in annova test showed that the value of f test is 18.945 and p value is 0.00 < 0.05 (α), it shows that h1 is accepted which means that there is an average difference of inhibition for each concentration. in the post hoc test, it prove that there is a difference between the concentration of 0.01% with concentration of 0.07%, and 0.09%. for the concentration of 0.03%, there is a difference with the concentration of 0.07%, and 0.09%. at a concentration of 0.05%, there is a difference with the concentration of 0.09%. the activity of cynammyldehyde against staphylococcus aureus the results showed that cynammyldehyde from extracts of cinnamon can inhibit the growth of staphylococcus aureus. this would have been due to a chemical compound as cynammyldehyde, eugenol, and alcohol in the extract of cynnamon, especially the compound of cynammyldehyde. that compounds as the active ingredient, which can inhibit growth of staphylococcus aureus. it inhibited the growth of bacteria or bacterial death by an antibacterial agent can be caused by inhibition of the synthesis of cell walls, the inhibition of the cell membrane function, inhibition of protein synthesis, or inhibition of the synthesis of nucleic acids.10 cynammyldehyde from cinnamon extract has the potential to inhibit cell wall synthesis. this is based on the content of cynammyldehyde that is aldehyde compounds.11 potential cynammyldehyde from cinnamon extract inhibits staphylococcus aureus by cell wall protein agglomerate, so that the cell wall can not functionate anymore. staphylococcus aureus is a gram-positive bacteria.the cell wall of gram-positive bacteria consist of a very thick peptidoglycan that provides rigidity to maintain the integrity of the cell. bacterial cell wall assembly process begins with the formation of peptide chains that will form the cross bridge peptide chains that incorporate glican chains from peptidoglycan to the another chain leading to complete cell wall assembly. if there is damage to the cell walls or any obstacles in its formation can occur in bacterial cell lytic which makes the bacteria lost the ability to form colonies, and it will cause bacterial cell death. in staphylococcus aureus, the delivery of antimicrobial can inhibit cell wall assembly and cause generate merger glican chain is not connected to cross the cell wall peptidoglycan, being weak structures and cause death of bacteria. any compound that blocks any step in the synthesis of peptidoglycan will cause bacterial cell wall is weakened and cell lysis.10 bacterial cell lysis does not work anymore because the cell wall that maintains shape and protects the bacteria that have a high osmotic pressure. staphylococcus aureus is a gram-positive bacteria that have an osmotic pressure in 3–5 times larger than gram-negative bacteria, making them more susceptible to lysis.10 without a cell wall, bacteria can not survive against outside influence and soon die.12 therefore, the lysis of bacteria suspected of interference or inhibition of cell wall assembly and lysis of the cell wall can explain the bacteriostatic effect of cynammyldehyde of extract of cinnamon. the use of the concentration of cynammyldehyde of different extracts of cinnamon to give different levels of influence in the growth of staphylococcus aureus. at a concentration of 0.07% and 0.09% there are colonies of bacteria which grow, but less in number in comparison with the cultivated in a concentration of 0.01%, 0.03%, 0.05% and the positive control group. bacterial growth was really inhibited at the concentrations of extract of 0.07% and 0.09%. all indicated that higher concentrations of extract of cinnamon the growth of the bacteria staphylococcus aureus increasingly hampered because the active ingredient in the test solution. therefore, this study found that treatment with the potential to inhibit the growth of the bacteria staphylococcus aureus is the initial concentration of 0.07%. in other words, the lowest concentration to inhibit the total growth of staphylococcus aureus is a 0.07%, and the optimal concentrations have the potential to inhibit the growth of the bacteria staphylococcus aureus is 0.09%. ��winias, et al.: effect of cynammyldehyde from cinnamon extract acknowledgements thanks to dr. retno pudji rahayu, drg., m. kes as mentors who has provided us a lot of valuable direction and guidance. sudarmawan , drg.,m.kes, who has shared his research experience, the institute tropical disease c enter, microbiology laboratory in dentistry faculty of airlangga university, and institute for research and stan dardization surabaya industry, that given us the opportuni ty to conduct research and thanks to friends and also those who have helped us both morally and materially to the completion of this research. references 1. iraj rasooli, 2007. food preservation – a biopreservative approach. food global science book. pp. 111–13�. 2. palmer, sa., stewart j., fyfe, l. 1998. antimikrobial properties of plant essential oils and essences againts five important food-borne pathogen. letters appl. microbiol. 26: 118–122. 3. eyyup rencuzogullari. 2000. the cytogenic effects of sodium metabissulfite, a food preservative in root tip cells of allium cepa l. j. biol. 25. pp. 361–370. 4. martina, restuati. 2008. perbandingan chitosan kulit udang dan kulit kepiting dalam menghambat pertumbuhan kapang aspergillus flavus. universitas lampung. hlm. 582–289. 5. �orlina, s. et al. 2009. the health risk of formaldehyde to human beings. american journal of pharmacology and toxicology. 4(3): 98–10�. 6. bulleran, i,a.,f.y. lien and seir. 1997. inhibition of crowd andaflatoxin protection cinnamon and clove oil. cinnamomy and eugeno y. fd sei . 42. pp. 1107–1109. 7. herwita, idris. 2007. the impact cinnamon bio-insecticide to the insect biologic aspect epilachum varivestis, mulsant. jurnal akta agrosia. 1. pp. 99–105. 8. puanpronpitag, et al. 2009. antimicrobial properties of cinnamomum verum aqueous extract. assian journal of biological science. 2(2) pp. 49–53. 9. chang, tzen sang and tsair bon yen. 2006. synergisitic effects of cinnamaldehyde in combination with eugenol against wood decay fungi. bioresource technology xxx (2006) xxx–xxx. 10. jawetz e, melnick �e, and adelberg ca. 2001. mikrobiologi kedokteran. edisi i. diterjemahkan oleh penerjemah �agian mikrobiologi fakultas kedokteran universitas airlangga. salemba medika, surabaya. 11. harborne j�. 1987. metode fitokimia. penuntun cara modern menganalisis tumbuhan. diterjemahkan oleh padmawinata k & soediro. penerbit it�, �andung. 12. morin r� & �orman m. 1995. kimia dan biologi antibiotik b-lactam (chemistry and biology of β-lactam antibiotics). edisi iii. diterjemahkan oleh mulyani s. ikip semarang press, semarang. copyright © 2020, ijtid, issn 2085-1103 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 8 no. 1 january-april 2020 research article a survey for zoonotic and other gastrointestinal parasites in pig in bali province, indonesia ni komang aprilina widisuputri1, lucia tri suwanti2,3,a, hani plumeriastuti4 1postgraduate student, faculty of veterinary medicine, universitas airlangga, surabaya, east java, indonesia. 2department of veterinary parasitology, faculty of veterinary medicine, universitas airlangga, surabaya, east java, indonesia. 3institute of tropical diseases, universitas airlangga, surabaya, east java, indonesia. 4department of veterinary pathology, faculty of veterinary medicine, universitas airlangga, surabaya, east java, indonesia. acorresponding author: tswant@gmail.com; phone number: +6281226094872 received: 8th november 2018; revised: 21st december 2018; accepted: 25th february 2019 abstract pigs have potentially to transmit zoonotic gastrointestinal parasite disease both caused by protozoa and worm. the aim of this study was to identify gastrointestinal parasites that were potentially zoonotic in pigs in the province of bali. a total of 100 fresh feces samples was collected from several pig farms in bali, from badung and tabanan districts, each consisted of 50 samples. pig feces samples were examined for the presence of eggs worms, cysts and oocysts for protozoa based on the morphology and size. identification for protozoa and worms used native, sedimentation and sucrose flotation methods. parameters measured were sex, feed and cage management. the result showed that the characteristic parameters for pigs in both district were generally female. cage management for raising pigs mostly used group cage. feed that provided in both district mostly used bran and concentrate. all of 100 pig feces samples that examined positive for parasites. there were 8 types of gastrointestinal parasites that have been identified. four types of protozoa found were entamoeba sp. (99%), balantidium sp. (79%), eimeria sp. (78%), blastocystis sp. (69%) and four types of worms were ascaris sp. (20%), trichuris sp. (20%), strongyloides sp. (19%), and oesophagostomum sp. (8%). all pigs were infected with two or more parasites. the prevalence of parasitic gastrointestinal infections was different for each district, six genera (entamoeba sp., balantidium sp., blastocystis sp., eimeria sp., oesophagostomum sp. and trichuris sp.) were higher found in tabanan district and the two genera (ascaris sp. and strongyloides sp.) were higher in badung district. oesophagostomum sp. was only found to infect pigs in tabanan district. the conclusion is gastrointestinal parasites that found in pigs at badung and tabanan district bali province mostly have zoonotic potential. keywords: zoonotic parasite, gastrointestinal parasite, pig, bali indonesia abstrak babi memiliki potensi untuk menularkan penyakit parasit gastrointestinal zoonotik yang disebabkan oleh protozoa dan cacing. tujuan dari penelitian ini adalah untuk mengidentifikasi parasit gastrointestinal yang berpotensi zoonosis pada babi di provinsi bali. sebanyak 100 sampel feses segar dikumpulkan dari beberapa peternakan babi di bali, dari kabupaten badung dan tabanan masing-masing terdiri dari 50 sampel. sampel feses babi diperiksa terhadap keberadaan telur cacing, kista dan ookista protozoa berdasarkan morfologi dan ukuran. identifikasi protozoa dan cacing menggunakan metode natif, sedimentasi dan flotasi sukrosa. parameter yang diukur adalah jenis kelamin, pakan dan manajemen kandang. hasil penelitian menunjukkan bahwa karakteristik parameter pada babi di kedua kabupaten umumnya betina. manajemen kandang untuk beternak babi kebanyakan menggunakan kandang kelompok. pakan yang disediakan di kedua kabupaten sebagian besar menggunakan dedak dan konsentrat. dari total 100 sampel feses babi yang diperiksa positif terhadap parasit. terdapat 8 jenis parasit gastrointestinal yang telah diidentifikasi. empat jenis protozoa yang ditemukan adalah entamoeba sp. (99%), balantidium sp. (79%), eimeria corresponding author. e-mail: tswant@gmail.com; telp: +6281226094872 sp. (78%), blastocystis sp. (69%) dan empat genus cacing yaitu: ascaris sp. (20%), trichuris sp. (20%), strongyloides copyright © 2020, ijtid, issn 2085-1103 ni komang aprilina , et al.: a survey for zoonotic and other gastrointestinal parasites 55 sp. (19%), and oesophagostomum sp. (8%). setiap babi terinfeksi oleh dua atau lebih parasit. prevalensi infeksi parasit gastrointestinal berbeda untuk tiap kabupaten, enam genus (entamoeba sp., balantidium sp., blastocystis sp., eimeria sp., oesophagostomum sp. dan trichuris sp.) lebih tinggi ditemukan di kabupaten tabanan dan dua genus (ascaris sp. dan strongyloides sp.) lebih tinggi di kabupaten badung. oesophagostomum sp. hanya ditemukan menginfeksi babi di kabupaten tabanan. kesimpulannya adalah parasit gastrointestinal yang ditemukan pada babi di kabupaten badung dan tabanan provinsi bali sebagian besar memiliki potensi zoonosis. kata kunci: parasit zoonotik, parasit gastrointestinal, babi, bali indonesia how to cite: widisuputri, ni komang aprilina; suwanti, lucia tri; plumeriastuti, hani. a survey for zoonotic and other gastrointestinal parasites in pig in bali province, indonesia. indonesian journal of tropical and infectious disease, [s.l.], v. 8, n. 1, p. 55-66, mar. 2020. issn 2356-0991. available at: <https://ejournal.unair.ac.id/ijtid/article/view/10393>. date accessed: 04 apr. 2020.doi:http://dx.doi.org/10.20473/ijtid.v8i1.10393. introduction pigs are one of the commodities in the livestock sector, which has great potential to be developed in the recent decades. the pig population in indonesia continues to increase along with the increasing number of large-scale pig farms and individual pig farmers. one of the regions in indonesia where most people raise pigs is in bali province. bali provincial livestock service1 reports that the total pig population in 2016 reached 803,517. in bali province, pigs are an important commodity and most people in bali maintain pigs as their primary and secondary business. in addition, pigs also play an important role in fulfilling daily food needs and as a complement to religious ceremonies.2 generally, pigs in bali are traditionally raised with low nutritional value and poor hygiene. this condition make pigs are more vulnerable to various diseases and has potential to spread the diseases.3 the existence of the diseases can cause considerable economic losses for pig farmers. losses include a decrease in production due to inhibition of livestock growth and increase medical costs.4 one of the diseases that can infect pig is gastrointestinal parasites. economic losses caused by gastrointestinal parasites were significant, but farmers may not realize it because the symptoms tend to be subclinical and pigs may still look healthy.5 gastrointestinal parasites in pigs are protozoa and worms. the types of protozoa that can infect gastrointestinal tract of pigs include entamoeba sp.; balantidium sp.; eimeria sp.; and isospora sp.6 recent study by yoshikawa et al.,7 in east nusa tenggara found the presence of protozoa blastocystis sp. as much as 87.1%. research about blastocystis sp. in pigs in bali province, previously have not been reported. according to suryastini et al.,8 several types of gastrointestinal worms that can infect pigs were gnathostoma hispidum, hyostrongylus rubidus, macracanthorhyncus hirudinaceus, globocephalus urosubulatus, strongyloides ransomi, ascaris suum, oesophagostomum dentatum and trichuris suis. some gastrointestinal parasites in pigs have potentially to transmit zoonotic diseases to human. according to schar et al.,9 there are five gastrointestinal parasites that can be detected in pigs with zoonotic potential, were ascaris sp., trichuris sp., capillaria spp., balantidium coli and entamoeba sp. in addition, wang et al.,10 stated that blastocystis sp. in pigs also had zoonotic potentially. it will certainly have an impact on the animal welfare as well as pig farmers and surrounding communities close to the farm area. therefore, the aims of this study was to determine zoonotic and other gastrointestinal parasites in pig at bali province, indonesia based on fecal examination and discuss their zoonotic potential. materials and methods study area this study was conducted in two district in bali: badung and tabanan districts. in badung district https://ejournal.unair.ac.id/ijtid/article/view/10393 http://dx.doi.org/10.20473/ijtid.v8i1.10393 copyright © 2020, ijtid, issn 2085-1103 56 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 55–66 figure 1. map of sampling location. dark blue colour is badung district and pink colour is tabanan district. samples were taken in north kuta sub-district, and in tabanan district samples were taken in baturiti sub-district. geographically, badung district located between 08˚14’20” 08˚50’48’’ south latitude and 115˚05’00” 115˚26’16” east longitude. north kuta sub-district has an area of 33.86 km2 with an altitude of 0-65 meters above sea level. north kuta sub-district was located in the lowlands close to urban areas. geographically, tabanan district located between 08˚14’30” 08˚30’07” south latitude and 114˚54’52” east longitude. baturiti sub-district has an area of 99.17 km2 with an altitude of 465-2082 meters above sea level. baturiti sub-district was located in the highlands of rural areas. dark blue colour is badung district and pink colour is tabanan district (figure 1). a total of 100 pig fecal samples were taken randomly, from badung and tabanan districts consisted each 50 samples. samples collection were conducted from 15 22 january 2018. feces samples collection feces samples from several pig farmers are taken directly using gloves from the ground and after defecation and accompanied by a veterinarian from the local livestock department. all feces samples were collected in urine steril container and were preserved in 2.0% potassium dichromate for protozoa examinations and 10% formalin for helminths examination, then stored in cool box for transportation. for each animal was recorded with different code. parameters included sex, feed and cage management. examination of feces samples samples were observed at vet erinar y parasitology laboratory, faculty of veterinary medicine, universitas airlangga, surabaya indonesia. samples were examined for eggs worm, cyst and oocyst for protozoa. identification for protozoa and egg worm using native, sedimentation and sucrose flotation methods. feces were diluted with aquadest and then filtered. for native examination, the feces sample is stirred first using a stirring rod and then a small portion of feces sample is taken and placed on the object glass and the lid uses a cover glass after that check under the microscope 400x magnification. for sediment examination, filtrate were centrifugation at 1.500 rpm for 5 minutes (by centrifuge hc 1180t 8 hole with timer, china), then removed supernatant. this step repeated until 3 times. take the sediment slowly and place it on the object glass then cover with a cover glass. the remaining sediment was added with sucrose solution until complete 12 ml to be centrifuged at 1.500 rpm for 10 min in a 15 ml plastic tube. floated was added sucrose solution until mouth of tube and was covered by a cover glass. after 5 min, cover glass was transferred to object glass, and the eggs of worm were observed at 100x magnification and the cysts and oocysts of protozoa were observed at 400x magnification for identification by light microscopy. identification for both protozoa and worm were based on the morphology and size of the eggs, cysts or oocysts.11,12,13 baturiti, tabanan north kuta, badung copyright © 2020, ijtid, issn 2085-1103 ni komang aprilina , et al.: a survey for zoonotic and other gastrointestinal parasites 57 table 1. characteristic parameters pigs for sampling characterictics places total badung district (n=50) tabanan district (n=50) sex male 18 16 100 female 32 34 feed bran + concentrate 35 28 bran + concentrate + banana trunk 7 9 bran + concentrate + banana trunk + leftlovers house 5 0 bran + leftlovers house 1 0 100 bran + consentrate + banana trunk + taro stems 0 9 bran + consentrate + taro stems 0 3 bran + chicken innards+ leftlovers house 2 0 bran + banana trunk 0 1 management individual cage 11 8 100 group cage 39 42 results and discussion a total of 100 pig feces samples from badung and tabanan districts bali province, indonesia were identified. information from each pig characteristics were provided in (table 1). table 1 shows that the majority of the pig population in tabanan and badung districts are female and feed given to almost pigs is bran and concentrate. in badung district some pigs were fed by leftovers from the kitchen while in tabanan district some pigs were fed using plant origin ingredients, banana stems and taro leaves. the cage management in both districts mostly pig farmers are using group cages. the results of identification indicate that the pigs in bali are infected by 8 genera of parasites: entamoeba sp., balantidium sp., eimeria sp., blastocystis sp., strongyloides sp., trichuris sp., ascaris sp. and oesophagostomum sp. the morphological of the gastrointestinal parasites found in pigs in bali province are described in figure 2. all of the feces samples that have been examined, overall positive for gastrointestinal parasites (table 2). it means all of pigs were infected with gastrointestinal parasites. the highest prevalence was entamoeba sp. (99%) respectively, was followed by balantidium sp. (79%), eimeria sp. (78%), blastocystis sp. (69%), ascaris sp. (20%), trichuris sp. (20%), strongyloides sp. (19%), and oesophagostomum sp. (8%). the prevalence of parasitic gastrointestinal infections was different for each district, six genera (entamoeba sp. balantidium sp., blastocystis sp., eimeria sp., oesophagostomum sp. and trichuris sp.) were higher found in tabanan district and the two genera (ascaris sp. and strongyloides sp.) were higher in badung district. oesophagostomum sp. was only found to infect pigs in tabanan. one pig could infected with two or more parasites, even, the pigs were infected with seven species of parasites. in detail, the mix infection was presented in table 3. almost all of mix infections involve entamoeba sp. there is no single infection. in indonesia, especially in bali province, studies about gastrointestinal parasites have been widely reported. however, most of these studies focus on one type of parasite. there have not been many studies that discuss about mixed infection between protozoa and worms in each pig. from the results of this study showed that gastrointestinal parasites in pigs in badung and tabanan districts found several parasites that have zoonotic potential. copyright © 2020, ijtid, issn 2085-1103 58 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 55–66 figure 2. morphology of gastrointestinal parasites in pig in bali province under light microscope. a). entamoeba sp. (bar: 10μm); b) and c). balantidium sp. cyst and tropozoite (bar: 50μm); d). eimeria sp. (bar: 10μm); e). blastocystis sp. (bar: 10μm); f). strongyloides sp. (bar: 50μm); g). trichuris sp. (bar: 50μm); h). ascaris sp. (bar: 50μm); and i). oesophagostomum sp. (bar: 50μm). table 2. prevalence of gastrointestinal parasites infections in pig in bali province based on each genus of parasite places samples number of positive (%) en ba bl ei as oe st tr badung district 50 49(100) 34(76) 35(70) 29(48) 12(24) 0(0) 13(26) 5(10) tabanan district 50 50(100) 45(90) 34(68) 49(98) 8(16) 8(16) 5(10) 15(30) total 100 99(99) 79(79) 69(69) 78(78) 20(20) 8(8) 18(18) 20(20) en, entamoeba sp.; ba, balantidium sp.; bl, blastocystis sp.; ei, eimeria sp.; as, ascaris sp.; oe, oesophagostomum sp.; st, strongyloides sp.; tr, trichuris sp. h g i e f d c b a copyright © 2020, ijtid, issn 2085-1103 ni komang aprilina , et al.: a survey for zoonotic and other gastrointestinal parasites 59 table 3. prevalence gastrointestinal parasites in pig in bali province based on mix infection parasites number of positive (%) badung district (n=50) tabanan district (n=50) total (n=100) en+ba 2 (4) 0 (0) 2 en+bl 4 (8) 0 (0) 4 en+ba+bl 7 (14) 1 (2) 8 en+ba+ei 7 (14) 5 (10) 12 en+bl+ei 4 (8) 0 (0) 4 en+bl+as 2 (4) 0 (0) 2 en+ei+oe 0 (0) 1 (2) 1 en+ei+st 0 (0) 1(2) 1 en+ei+tr 0 (0) 1 (2) 1 en+ba+bl+ei 3 (6) 13 (26) 16 en+ba+bl+as 3 (6 0 (0) 3 en+ba+bl+st 2 (4) 0 (0) 2 en+ei+as+st 2 (4) 0 (0) 2 ba+bl+ei+st 1 (2) 0 (0) 1 en+bl+ei+st 3 (6) 0 (0) 3 en+bl+ei+tr 0 (0) 1 (2) 1 en+ba+ei+as 1 (2) 2 (4) 3 en+ba+ei+oe 0 (0) 3 (6) 3 en+ba+ei+tr 2 (4) 2 (2) 4 en+ba+ei+st+tr 1 (2) 0 (0) 1 en+ba+bl+as+st 1 (2) 0 (0) 1 en+ba+bl+ei+as 1 (2) 4 (8) 5 en+ba+bl+ei+oe 0 (0) 3 (6) 3 en+ba+bl+ei+st 1 (2) 1 (2) 2 en+ba+bl+ei+tr 1 (2) 9 (18) 10 en+ba+ei+as+st+tr 0 (0) 1 (2) 1 en+bl+ei+as+st+tr 0 (0) 1 (2) 1 en+ba+bl+ei+oe+st 0 (0) 1 (2) 1 en+ba+bl+ei+as+st 1 (2) 0 (2) 1 en+ba+bl+ei+as+st+tr 1 (2) 0 (0) 1 en, entamoeba sp.; ba, balantidium sp.; bl, blastocystis sp.; ei, eimeria sp.; as, ascaris sp.; oe, oesophagostomum sp.; st, strongyloides sp.; tr, trichuris sp. protozoa are the most common parasites that infect pigs in both districts. the higher prevalence of protozoa is dominated by entamoeba sp. (100%). this result was higher than the study by suryawan et al.,14, which stated that out of 102 faecal samples of pigs in papua, 34.2% were infected with entamoeba sp. research by agustina et al.,.15 in bali province found that the prevalence of amoeba sp. in pig fecal samples as much as 82.4%. this is certainly a concern, because all pig samples examined in this study were 100% positive for entamoeba sp. entamoeba sp. is a protozoa that can infect human and animals. according to matsubayashi et al.,16 states that there are 6 species from genus entamoeba that have been identified to infect human and animals, namely e. histolytica, e. polecki, e. coli, e. dispar, e. moshkovskii and copyright © 2020, ijtid, issn 2085-1103 60 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 55–66 e. hartmanni. research by gomez et al.,17 from samples of pigs and human on four pig farms in colombia showed that pig faecal samples were positive for e. coli, human faecal samples were also positive for e. coli and e. hystolitica / dispar. the presence of e. coli species in pigs and humans in colombia shows the possibility of zoonotic potential of these parasites, so further molecular identification needs to be done. however, study by agustina et al.,18 about the incidence of entamoebiasis in pigs in bali province showed negative pcr results on e. polecki, so the zoonotic potential needs to be studied further. in this study, another protozoa found was balantidium sp. the prevalence in this study was 83%. in indonesia, the incidence of balantidium sp. in pigs had been widely studied by agustina et al. and yuliari et al.,.18,19 with a prevalence of 61.2%, and 36.4%, respectively. in korea6 was recorded the prevalence of balantidium sp. in pigs was 64.7%, in china20 was 22.79%, and in cambodia9 was 15.8%. balantidium sp. is a protozoa that can cause balantidiosis. balantidiosis is a zoonotic disease that can infect human and animals through the world. pigs are natural reservoir for balantidium sp. transmission of the disease by faecal-oral route. in pigs it is usually asymptomatic and these protozoa live in the lumen of the cecum and colon. transmission between human and animals can occur as well as humans to humans. in human, the incidence of balantidiosis can be asymptomatic. severe infection can cause diarrhea and abdominal discomfort. balantidiosis can occur due to several factors, such as sanitation, climate conditions, and community culture. an important factor in the spread of disease to humans is the presence of infected pigs and careless disposal of animal waste. this often occurs in poor rural areas where people tend to live near their livestock, so the disease is easily spread. some sectors that have a high risk of being infected by balantidium sp. are veterinarians, animal handlers and butchers.21,22 eimeria sp. is a protozoa that can cause coccidiosis. the prevalence of eimeria sp. in this study was 83%, higher than the study yuliari et al.,19 in pigs in papua, indonesia, with an average prevalence was 68.2%. in bali province, the prevalence of eimeria sp. in pigs was reported by agustina et al.,15 as much as 54.8%. the incidence of coccidiosis in several countries has also been reported13,20,23,24,25 with a prevalence 16.53%, 16.7%, 47%, 89.2% and 3%, respectively. coccidiosis in young pigs can cause diarrhea and can be predispose to secondary infections by viruses or bacteria. in severe cases, pigs can become dehydrated with a 10-59% chance of death. animals that have been repeatedly infected have no clinical symptoms, and can transmit to other animals and pollute the surrounding environment.15 research about blastocystis sp. in pigs in bali province, previously have not been reported. in this study, the prevalence of blastocystis sp. in pigs was 60%. in indonesia, research on blastocystis sp. was reported for the first time7 and blastocystis sp. was found in humans, pigs, chickens and rodents in the winyapu area, southwest sumba district, east nusa tenggara province, and evidenced by pcr methods. so far, there are 17 blastocystis sp. subtypes that have been identified based on gen analysis of small subunits ribosomal rna (ssu rrna).26 humans can be infected by 9 subtypes (st1 st9).27 in china28 was reported that there were 3 zoonotic subtypes in pigs, namely st1, st3 and st5, which showed that blastocystis sp. in pigs could be zoonotic. several factors that related to the emergence of blastocystis sp. infection are lack of the environmental hygiene, poor communit y sanitation, socio-economic status and lifestyle. blastocystis sp. can infect humans and some animals including pigs, cows, monkeys and chickens. some zoonotic subtypes of these animals have been isolated, therefore, they can act as reservoir hosts. transmission can occur from human to human, from human to animal and from animal to human by faecal-oral route.29,30 in this study found various types of nematode worms namely strongyloides sp., trichuris sp., ascaris sp. and oesophagostomum sp. this result is also evidenced by the existence of investigations in indonesia found various types of worms that often infect pigs. study by agustina31 copyright © 2020, ijtid, issn 2085-1103 ni komang aprilina , et al.: a survey for zoonotic and other gastrointestinal parasites 61 in pigs in bali found oesophagostomum sp. with the prevalence of 47.5%. in addition, research by fendryanto et al.,3 on piglets in bali found the prevalence of ascaris sp., trichuris sp. and strongyloides sp. with the prevalence of 33.2%, 14.0% and 57.6%, respectively. in poland, study by wictor and jarosz32 noted the prevalence of worms in pigs was found ascaris sp. (22.2%), trichuris sp. (5.6%), strongyloides sp. (36.1%) and oesophagostomum sp. (36.1%). in malaysia25 noted the prevalence of strongyloides sp. (45.6%) and trichuris sp. (8.7%). in cambodia, inpankaew et al.,33 noted the prevalence of oesophagostomum sp. (76.6%), strongyloides ransomi (23.3%), ascaris suum (13.3%) and trichuris suis (6.6%). research by nonga and paulo34 in tanzania showed that differences in the prevalence of gastrointestinal worms in some areas may arise due to differences in environmental conditions that are conducive to the parasite survival, the number of definitive hosts infected, type of feed and animal diet and the hosts immune system. strongyloides sp. is an important parasite that can be infected most of the suckling piglets. the worms predilection is in the small intestine. common clinical symptoms that may occur are diarrhea followed by progressive dehydration. in severe infections, death usually occurs before piglets are between 10 and 14 days old, but if piglets can survive, dwarfism can occur. recent research by giang et al.,35 states that the type of strongyloides sp. in pigs in vietnam based on molecular identification is s. ransomi. s. ransomi has a similar morphology to s. papillosus, but in molecular analysis based on 18s rdna, s. ransomi is close to s. venezuelensis. the zoonotic aspect and importance of strongyloides sp. in veterinary medicine are discussed more detail in thamsborg et al.,36 which states that until now s. ransomi in pigs has not been zoonotic, but there are other species such as s. stercoralis in dogs have zoonotic potential to humans. trichuris sp. is a type of worm that commonly infect pigs and live in the large intestine. pigs are considered as the natural host of trichuris sp, although primates and humans may be infected. trichuris sp. infection can cause ulceration in the lining of the intestinal mucosa, damage to blood capillaries and secondary infections can occur by bacteria. clinical symptoms in pigs include anorexia, slimy and bloody diarrhea, dehydration and death occur in severe cases. trichuris sp. can survive for several years outside the hosts. so far, it is still a question of whether or not trichuris sp. is zoonotic. according to nejsum et al.,37 stated that the species trichuris trichiura in humans can be found in pigs, but until now most worms did not survive. this shows that human cross infection can occur with t. suis in pigs under experimental conditions. ascaris sp. is disease that can cause ascariasis and commonly found in pigs. this typical worm species also found in wild pigs. if pig infected with a severe infection, intestinal obstruction can occur, loss of appetite, vomiting, jaundice and death. in the case of moderate infection can occur low appetite, low food efficiency and slow growth. ascaris sp. is zoonotic and can infect humans and other mammals by consuming food or water contaminated by infective eggs. ascaris sp. eggs in a dry environment can last 2 to 4 weeks, while in a humid and cold environment they can survive eight weeks and become an infective stage in the environment. after ingestion, eggs hatch into larvae through the intestinal wall, pass through the liver and migrate to the lungs, and adult worms have a predilection in the small intestine.38 the occurrence of zoonosis ascaris sp. has been reported39 which identified 14 cases of ascariasis in humans in contact with pigs in maine, usa. in addition, research conducted by nejsum et al.,40 stated that ascariasis is a case of zoonosis in denmark, where humans are in direct contact with pigs and pig feces. in t h i s s t u d y , t h e p r e v a l e n c e of oesophagostomum sp. only found in tabanan district. oesophagostomum sp. is known as a worm nodule that has predilection in the large intestine in cecum and colon. oesophagostomum sp. worm infection occurs when pigs eat plants or foods that contaminated by infective larvae31. oesophagostomum sp. infection in pigs can cause lack of appetite, poor growth rates, easy secondary infection and can cause death41. so far there have been no studies that discuss the copyright © 2020, ijtid, issn 2085-1103 62 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 55–66 possibility of zoonosis of oesophagostomum sp. in pigs. in this study, all pig were infected with mixed parasite protozoa and worms. according to tolistiawaty et al.,42 parasitic infections generally occur due to the weakness resistance of the animal to parasites. mixed infections often occur, and making it difficult to know the specific symptoms that seen. infection that occurs is usually caused by several types of worms in the intestine and other organs. the way of animal treatment also very influential on the incidence of gastrointestinal parasitic infections. this is supported by research from supriadi et al.,43 which was stated that gastrointestinal parasitic infections in pigs can be caused by poor management. poor cage sanitation is also a factor that increases the risk of parasitic infection and does not rule out the possibility of transmission to humans, especially for pig owners (zoonosis). in addition, according to roesel et al.,44 stated that the most important factors associated with gastrointestinal parasitic infections in pigs are related to sanitation, especially cleaning of pig stool regularly from the cage and the use of disinfectants. in badung and tabanan districts, most people use group cages to raising pigs. this type of maintenance system includes intensive maintenance where the pig is caged in a cage. according to lai et al.,20 raised pigs traditionally have a higher prevalence of the disease, this is because intensive pig farming has better maintenance management. although intensive maintenance implements better management, it seems that it cannot help reduce the incidence of disease infection effectively. the possibility of a parasitic infection occurs due to a lack of public awareness about the good sanitation, besides that habit from pigs by eat in soil contaminated with faeces can be predispose to infection. research by mutua et al.,45 stated that pig needs energy, amino acids, minerals, vitamins and water. these elements are needed for the process of growth, reproduction and lactation. conclusion gastrointestinal parasites that found in pigs in badung and tabanan districts bali province mostly have zoonotic potential. these parasites included entamoeba sp., balantidium sp., eimeria sp., blastocystis sp., strongyloides sp., trichuris sp., ascaris sp. and oesophagostomum sp. this study is expected to provide information to improve the hygiene and sanitation in terms of raising pigs, to provide a basis for further control and treatment in pigs that infected with gastrointestinal parasites as well as providing information about zoonotic potential that can arise. acknowledgements the author would like to thank the department of parasitology, faculty of veterinary medicine, universitas airlangga for supporting this research. thank you to the regional development planning agency of badung and tabanan bali district for giving permission to take samples and i would like to thank the agency of animal husbandry and fisheries and field officers who have helped me so that this research can be done in accordance with the expected time. conflict of interest the authors declare that there is no conflict of interest for this research. references 1. bali provincial animal husbandry. information on livestock data in bali province in 2016. bali: bali province animal husbandry; 2016. 2. oka ibm, dwinata im. strongyloidiasis in pre-weaned piglets. bul vet udayana [internet]. 2011 [cited 2018 sep 12];3(2). available from: http://download. portalgaruda.org/article.php?article=13828&val=941 &title=strongyloidosis pada anak babi pra-sapih 3. fendriyanto a, dwinata im, oka ibm, agustina kk. identification and prevalence of gastrointestinal nematode worms in pigs in bali. indones med veterinus. 2015;4(5). http://download/ copyright © 2020, ijtid, issn 2085-1103 ni komang aprilina , et al.: a survey for zoonotic and other gastrointestinal parasites 63 4. fao. swine health management. food and agriculture organization of the united nations; 2012. 5. kumsa b, kifle e. internal parasites and health management of pigs in burayu district, oromia regional state, ethiopia. j s afr vet assoc [internet]. 2014 feb 26 [cited 2018 sep 12];85(1):913. available from: http://www.ncbi.nlm.nih.gov/pubmed/24831203 6. ismail haha, jeon h-k, yu y-m, do c, lee y-h. intestinal parasite infections in pigs and beef cattle in rural areas of chungcheongnam-do, korea. korean j parasitol [internet]. korean society for parasitology; 2010 dec [cited 2018 sep 12];48(4):347–9. available from: http:// www.ncbi.nlm.nih.gov/pubmed/21234241 7. yoshikawa h, tokoro m, nagamoto t, arayama s, asih pbs, rozi ie, syafruddin d. molecular survey of blastocystis sp. from humans and associated animals in an indonesian community with poor hygiene. parasitol int [internet]. 2016 dec [cited 2018 sep 12];65(6):780–4. available from: http://www.ncbi.nlm. nih.gov/pubmed/27080248 8. suryastini kad, dwinata im, damriyasa im. accuracy of the ritchie method in detecting gastrointestinal worm infection in pigs. indones med veterinus. 2012;1(5). 9. schär f, inpankaew t, traub rj, khieu v, dalsgaard a, chimnoi w, chhoun c, sok d, marti h, muth s, odermatt p. the prevalence and diversity of intestinal parasitic infections in humans and domestic animals in a rural cambodian village. parasitol int [internet]. 2014 aug [cited 2018 sep 12];63(4):597–603. available from: http://www.ncbi.nlm.nih.gov/pubmed/24704609 10. wang w, owen h, traub rj, cuttell l, inpankaew t, bielefeldt-ohmann h. molecular epidemiology of blastocystis in pigs and their in-contact humans in southeast queensland, australia, and cambodia. vet parasitol. elsevier b.v.; 2014;203(3–4):264–9. 11. meutchieye f, kouam mk, miegoué e, nguafack tt, tchoumboué j, téguia a, theodorooulos g. a survey for potentially zoonotic gastrointestinal parasites in domestic cavies in cameroon (central africa). bmc vet res [internet]. biomed central; 2017 dec 26 [cited 2018 sep 12];13(1):196. available from: http:// bmcvetres.biomedcentral.com/articles/10.1186/s12917 017-1096-2 12. krishna murthy cm, ananda kj, adeppa j , satheesha mg. studies on gastrointestinal parasites of pigs in shimoga region of karnataka. j parasit dis. 2016;40(3):885–9. 13. nathaniel ao, anyika kc, frank mc, jatau jd. prevalence of gastro-intestinal parasites in pigs in jos south local government area of plateau state, nigeria. haya saudi j life sci [internet]. 2017 [cited 2018 sep 13];2(5). available from: http://scholarsmepub.com/ 14. suryawan gy, suratma na, damriyasa im. potential of pigs as a source of transmission of zoonosis entamoeba spp. bul vet udayana. 2014;6(2). 15. agustina kk, sudewi nmaa, d har ma yu d ha aago, oka ibm. identification and prevalence of gastrointestinal protozoa in piglets that sold in a traditional market in bali. bul vet udayana [internet]. 2016 [cited 2018 sep 12];8(1). available from: https://ojs.unud.ac.id/index.php/buletinvet/article/ view/19667 16. matsubayashi m, murakoshi n, komatsu t, tokoro m, haritani m, shibahara t. genetic identification of entamoeba polecki subtype 3 from pigs in japan and characterisation of its pathogenic role in ulcerative colitis. infect genet evol [internet]. 2015 dec [cited 2018 sep 12];36:8–14. available from: http://www. ncbi.nlm.nih.gov/pubmed/26318541 17. mendoza-gómez mf, bact, pulido-villamarín a, barbosa-buitrago a, aranda-silva m. presence of gastrointestinal parasites in swine and human of four swine production farms in cundinamarcacolombia. rev mvz córdoba [internet]. universidad de córdoba; 2015 [cited 2018 sep 12];20:5014–27. available from: http://www.scielo.org.co/scielo.php?script=sci_ arttext&pid=s0122-02682015000400015 18. agustina kk, dharmayudha aago, oka ibm, dwinata im, kardena im, dharmawan ns, damriyasa im. case of entamoebiasis in pigs raised with a free range systems in bali, indonesia (kasus entamoebiasis pada babi yang dipelihara dengan cara diumbar di bali, indonesia). j vet [internet]. 2016 [cited 2018 sep 12];17(4):570–5. available from: https://ojs.unud.ac.id/ index.php/jvet/article/view/26559 19. yuliari pk, damriyasa im, dwinata im. prevalence of protozoa digestive tract in pigs in the baliem valley and the arfak mountains of papua. indones med veterinus. 2013;2(2). 20. lai m, zhou rq, huang hc, hu sj. prevalence and risk factors associated with intestinal parasites in pigs in chongqing, china. res vet sci [internet]. 2011 dec [cited 2018 sep 13];91(3):e121–4. available from: http://www.ncbi.nlm.nih.gov/pubmed/21349561 21. sangioni la, botton s de a, ramos f, cadore gc, monteiro sg, brayer pereira di, vogel fsf. balantidium coli in pigs of distinct animal husbandry categories and different hygienic-sanitary standards in the central region of rio grande do sul state, brazil. acta sci vet. 2017;45(june):1–6. 22. schuster fl, ramirez-avila l. current world status of balantidium coli. clin microbiol rev [internet]. 2008 oct 1 [cited 2018 sep 13];21(4):626–38. available from: http://www.ncbi.nlm.nih.gov/pubmed/18854484 23. zhang wj, xu lh, liu yy, xiong bq, zhang ql, li fc, song qq, khan mk, zhou yq, hu m, zhao j. prevalence of coccidian infection in suckling piglets in china. vet parasitol [internet]. 2012 nov 23 [cited 2018 sep 13];190(1–2):51–5. available from: http:// www.ncbi.nlm.nih.gov/pubmed/22694832 24. tsunda, g. ybw a. porcine coccidiosis: prevalence study in ganye southeasthern admawa state, http://www.ncbi.nlm.nih.gov/pubmed/24831203 http://www.ncbi.nlm.nih.gov/pubmed/21234241 http://www.ncbi.nlm/ http://www.ncbi.nlm.nih.gov/pubmed/24704609 http://scholarsmepub.com/ http://www/ http://www.scielo.org.co/scielo.php?script=sci_ http://www.ncbi.nlm.nih.gov/pubmed/21349561 http://www.ncbi.nlm.nih.gov/pubmed/18854484 http://www.ncbi.nlm.nih.gov/pubmed/22694832 copyright © 2020, ijtid, issn 2085-1103 64 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 55–66 nigeria [internet]. 2013 [cited 2018 sep 13]. available from: https://www.semanticscholar. org/paper/porcine-coccidiosis-%3a-prevalence study-in-ganye-%2c-tsunda-y.b.wanga./ a2414e6d691de7496a63394682a651565ca99946 25. mat yusof a, md isa ml. prevalence of gastrointestinal nematodiasis and coccidiosis in goats from three selected farms in terengganu, malaysia. asian pac j trop biomed [internet]. no longer published by elsevier; 2016 sep 1 [cited 2018 sep 13];6(9):735–9. available from: https://www.sciencedirect.com/ science/article/pii/s2221169116301873 26. alfellani ma, taner-mulla d, jacob as, imeede ca, yoshikawa h, stensvold cr, clark cg. genetic diversity of blastocystis in livestock and zoo animals. protist [internet]. 2013 jul [cited 2018 sep 13];164(4):497–509. available from: http://www.ncbi. nlm.nih.gov/pubmed/23770574 27. wawrzyniak i, poirier p, viscogliosi e, dionigia m, texier c, delbac f, alaoui he. blastocystis , an unrecognized parasite: an overview of pathogenesis and diagnosis. ther adv infect dis [internet]. 2013 oct 12 [cited 2018 sep 13];1(5):167–78. available from: http://www.ncbi.nlm.nih.gov/pubmed/25165551 28. song j-k, hu r-s, fan x-c, wang s-s, zhang h-j, zhao g-h. molecular characterization of blastocystis from pigs in shaanxi province of china. acta trop [internet]. 2017 sep [cited 2018 sep 13];173:130–5. available from: http ://www. ncb i.nlm.nih.go v/ pubmed/28619673 29. tan ksw. new insights on classification, identification, and clinical relevance of blastocystis spp. clin microbiol rev [internet]. 2008 oct 1 [cited 2018 sep 13];21(4):639–65. available from: http://www.ncbi. nlm.nih.gov/pubmed/18854485 30. palasuwan a, palasuwan d, mahittikorn a, chiabchalard r, combes v, popruk s. subtype distribution of blastocystis in communities along the chao phraya river, thailand. korean j parasitol [internet]. korean society for parasitology; 2016 aug [cited 2018 sep 13];54(4):455–60. available from:http://www.ncbi. nlm.nih.gov/pubmed/27658597 31. agustina kk. identification and prevalence of strongyle type worms in pigs in bali. bul vet udayana. 2013;5(2). 32. mizgajska-wiktor h, jarosz w. potential risk of zoonotic infections in recreational areas visited by sus scrofa and vulpes vulpes. case study--wolin island, poland. wiad parazytol [internet]. 2010 [cited 2018 sep 13];56(3):243–51. available from: http://www. ncbi.nlm.nih.gov/pubmed/21174955 33. inpankaew t, murrell kd, pinyopanuwat n, chhoun c, khov k, sem t, sorn s, muth s, dalsgaard a. a survey for potentially zoonotic gastrointestinal parasites of dogs and pigs in cambodia. acta parasitol [internet]. 2015 jan 1 [cited 2018 sep 13];60(4):601–4. available from: http://www.ncbi.nlm.nih.gov/pubmed/26408577 34. nonga he, paulo n. prevalence and intensity of gastrointestinal parasites in slaughter pigs at sanawari slaughter slab in arusha, tanzania. livest res rural dev. 2015;27. 35. giang nth, hoan td, huyen ntt, lan ntk, doanh pn. morphological and molecular characterisation of strongyloides ransomi (nematoda: strongyloididae) collected from domestic pigs in bac giang province, vietnam. tap chi sinh hoc. 2017;39(3). 36. thamsborg sm, ketzis j, horii y, matthews jb. strongyloides spp. infections of veterinary importance. parasitology [internet]. 2017 mar 4 [cited 2018 sep 13];144(3):274–84. available from: http://www.ncbi. nlm.nih.gov/pubmed/27374886 37. nejsum p, betson m, bendall rp, thamsborg sm, stothard jr. assessing the zoonotic potential of ascaris suum and trichuris suis: looking to the future from an analysis of the past. j helminthol [internet]. 2012 jun 19 [cited 2018 sep 13];86(2):148–55. available from:http://www.ncbi.nlm.nih.gov/pubmed/22423595 38. midha a, janek k, niewienda a, henklein p, guenther s, serra do, schlosser j, hengge r, hartmann s. the intestinal roundworm ascaris suum releases antimicrobial factors which interfere with bacterial growth and biofilm formation. front cell infect microbiol [internet]. 2018 aug 7 [cited 2019 apr 7];8:271. available from:https://www.frontiersin.org/ article/10.3389/fcimb.2018.00271/full 39. miller la, colby k, manning se, hoenig d, mcevoy e, montgomery s, mathison b, de almeida m, bishop h, dasilva a, sears s. ascariasis in humans and pigs on small-scale farms, maine, usa, 2010–2013. emerg infect dis [internet]. 2015 feb [cited 2018 sep 13];21(2):332–4. available from: http://www.ncbi.nlm. nih.gov/pubmed/25626125 40. nejsum p, parker ed, frydenberg j, roepstorff a, boes j, haque r, astrup i, prag j, skov sorensen ub. ascariasis is a zoonosis in denmark. j clin microbiol [internet]. 2005 mar 1 [cited 2018 sep 13];43(3):1142–8. available from: http://www.ncbi. nlm.nih.gov/pubmed/15750075 41. jufare a, awol n, tadesse f, tsegaye y, hadush b. parasites of pigs in two farms with poor husbandry practices in bishoftu, ethiopia. onderstepoort j vet res [internet]. 2015 apr 30 [cited 2018 sep 13];82(1):839. available from: http://www.ncbi.nlm. nih.gov/pubmed/26017194 42. tolistiawaty i, wdjaja j, lobo lt, isnawati r. gastrointestinal parasites in livestock at the sigi district slaughterhouse, central sulawesi. balaba. 2016;12(2). 43. supriadi, muslihin a, roesmanto b. pre-elimination of gastrointestinal parasites in pigs from suranadi village, narmada district, west lombok. media bina ilm. 2014;8(5). 44. roesel k, dohoo i, baumann m, dione m, grace d, clausen p-h. prevalence and risk factors for http://www.sciencedirect.com/ http://www.ncbi.nlm.nih.gov/pubmed/25165551 http://www.ncbi.nlm.nih.gov/ http://www/ http://www.ncbi.nlm.nih.gov/pubmed/26408577 http://www.ncbi.nlm.nih.gov/pubmed/22423595 http://www.frontiersin.org/ http://www.ncbi.nlm/ http://www.ncbi.nlm/ copyright © 2020, ijtid, issn 2085-1103 ni komang aprilina , et al.: a survey for zoonotic and other gastrointestinal parasites 65 gastrointestinal parasites in small-scale pig enterprises in central and eastern uganda. parasitol res [internet]. springer berlin heidelberg; 2017 jan 26 [cited 2018 sep 13];116(1):335–45. available from: http://link.springer. com/10.1007/s00436-016-5296-7 45. mutua fk, dewey c, arimi s, ogara w, levy m, schelling e. a description of local pig feeding systems in village smallholder farms of western kenya. trop anim health prod [internet]. 2012 aug 5 [cited 2018 sep 13];44(6):1157–62. available from: http://www. ncbi.nlm.nih.gov/pubmed/22219174 http://link.springer/ http://www/ vol. 9 no. 1 january–april 2021 ijtid, p-issn 2085-1103, e-issn 2356-0991 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ original article validity of method for mtbc and ntm detection in fnab specimens from tuberculous lymphadenitis using microscopy, xpert mtb / rif and culture method herisa nataliana junus 1*, ni made mertaniasih 2, soedarsono 3 1 clinical microbiology study program, faculty of medicine, universitas airlangga, surabaya, indonesia 2 department of clinical microbiology , faculty of medicine, universitas airlangga, surabaya, indonesia 3 department of pulmonology and respiratory medicine, faculty of medicine, universitas airlangga, surabaya, indonesia received:7th november 2019; revised: 23th july 2020; accepted: 5th january 2021 abstract mycobacterium tuberculosis and nontuberculous mycobacteria usually cause infection in tuberculous lymphadenitis. to improve accuracy of the detection mtb and ntm bacteria it is necessary to select valid methods. this study aims to compare validity of diagnostic methods from fnab specimens for determining tuberculous lymphadenitis patients. a descriptive observational laboratory study involved 35 samples were obtained from tuberculous lymphadenitis patients in dr. soetomo hospital surabaya east java. all specimens examined ziehl-neelsen staining microscopy, xpert mtb/rif , culture method middlebrook7h10 solid media and mgit as gold standard. identification of mtb dan ntm with sd bioline tb ag mpt64 and niacin paper strip bd . used diagnostic test 2x2 to analyze sensitivity, specificity, negative predictive value and positive predictive value. ziehl-neelsen staining microscopy sensitivity 83,33 % and specificity 95,65% of , ppv 90,91%and npv 91,67%, diagnostic accuracy 91,43 % . xpert mtb/rif sensitivity 75% and specificity 95,65% , ppv 90 % and npv 88 %, diagnostic accuracy 88,57 % with 95% ci (confidence interval ) . characteristics female dominated 23/35 (65.7%) while male numbered 12/35 (34.3%), age range distribution of tb lymphadenitis patients is highest in young adults 17 years to 25 years as many as 15/35 (42.9%) the second highest is the age group of 36 years to 45 years by 8/35 (22.9%), clinicial presentation are mostly lymph node enlargement in cervical 37% patients other locations supraclavicular ,mamae. clinical symptoms mostly lymphadenopathy 31,5% and other lymphadenopathy with fever. microscopy method still have the good validity shoul be conjunction with the molecular keywords: candida albicans; fluconazole; gastric perforation; histopathological; nsaids; peritonitis abstrak mycobacterium tuberculosis dan non tuberculous mycobacteria merupakan penyebab infeksi pada limfadenitis tuberkulosis. untuk meningkatkan akurasi isolasi bakteri mtb dan ntm diperlukan pemilihan metode yang valid. penelitian ini bertujuan membandingkan validitas metode diagnostik deteksi mtb dan ntm serta karakteristik pasien limfadenitis tb dari spesimen fnab . penelitian dilakukan di rumah sakit dr. soetomo surabaya jawa timur bersifat deskriptif observasional diperoleh sebanyak 35 sampel fnab pasien limfadenitis tb . pemeriksaan yang dilakukan adalah pemeriksaan mikroskopis menggunakan pewarnaan ziehl-neelsen, tes cepat molekuler xpert mtb/rif, kultur media padat middlebrook7h10 . standar emas pada penelitian ini menggunakan metode kultur media cair mgit. identifikasi mtb dan ntm dilakukan dengan sd bioline tb ag mpt64 dan niasin paper strip bd. analisis sensitivitas, spesifisitas,nilai duga negatif dan nilai duga positif menggunakan uji diagnostik tabel 2x2 . . pemeriksaan mikroskopis pewarnaan ziehl-neelsen dengan ci ( confidence interval ) memiliki 95% memiliki nilai sensitivitas 83,33 % dan spesifisitas 95,65%, nilai duga positif 90,91%, nilai duga negatif 91,67%, diagnostik akurasi 91,43 %. gj metode diagnostik tes cepat molecular xpert mtb/rif sensitivitas 75 % , spesifisitas 95,65 %, nilai duga positif 90 %, nilai duga negatif (88 %, dan diagnostik akurasi 88,57 %. karakteristik perempuan mendonina * corresponding author: herisa22junus@gmail.com open access under cc-by-nc-sa share alike 4.0 rapid tests and culture as gold standard in determining the diagnosis of tb lymphadenitis. herisa nataliana junus, et al.: validity of method for mtbc and ntm detection in fnab specimens 34 ijtid, p-issn 2085-1103, e-issn 2356-0991 si sebanyak 23/35 (65.7%) laki-laki berjumlah12/35 (34.3%), 17 tahun sampai 25 tahun sebanyak 15 orang 15/35 (42.9%) terbanyak kedua adalah kelompok usia 36 tahun sampai 45 tahun sebanyak8/35 (22.9%). pasien dengan gejala benjolan pada leher sebanyak 37%, lokasi lain supraklavikula, mamme. gejala klinis paling banyak mengalami gejala klinis pembesaran kelenjar getah bening saja sebanyak 31,5%,dan gejala klinis lainnya berupa pembesaran kelenjar getah bening dan febris pemeriksaan metode mikroskopik masih memiliki validitas yang baik , pemeriksaan yang dilakukan dengan dukungan tes cepat molekular dan kultur sebagai gold standar dapat membantu menegakkan diagnosis limfadenitis tb. kata kunci: candida albicans; fluconazole; gastric perforasi; histopatological; nsaids, peritonitis how to cite: junus, hn., mertaniasih, nm., soedarsono. validity of method for mtbc and ntm detection in fnab specimens from tuberculous lymphadenitis using microscopy, xpert mtb / rif and culture method. indonesian journal of tropical and infectious disease, 9(1), 33–38. introduction tuberculosis is an infectious disease that is still a health problem in the world because of the high burden of mortality and morbidity. world health organization (who ) reported in 2017 number of tb cases are currently 254 per 100,000 or 25.40 per 1 million population.1 indonesia has the number of new cases 420,994 cases in 2017,2 in 2018 estimated 10.0 million range (9.0 – 11,1 million ) people ill with tb incidence of extra pulmonary tb cases in the world estimated increase 14% from total of 6.4 million tb cases in 2017. detection of tuberculous lymphadenitis is quite difficult because the symptoms are often not typical, course of infection depends on patient risk factors and definitive diagnostics can be established based on the discovery of microbes causing infection.3 this study aims to compare validity of diagnostic methods specimens examined ziehlneelsen staining microscopy, xpert mtb/rif, culture method middlebrook7h10 solid media and mgit as gold standard from fnab specimens for determining tuberculous lymphadenitis patients. microscopic examination with ziehlneelsen staining method is first examination to identify acid fast bacilli (afb) using a binocular microscope but rarely positive results because of paucibacillary nature of mtb bacteria in tissues.5 ziehl-neelsen staining microscopy sensitivity range 78,3%83,33 % 20. molecular test xpert mtb/rif assay (cepheid, usa) is a diagnostic tool recommended by who and available in various various health care facilities in indonesia, detect dna mtb as well as mutations in the rpob gene that cause resistance to rifampicin.6 the relevant literature. culture method from specimen is still a gold standard in growing bacilli with sensitivity range 70% to 80%. advantage culture examination to avoid risk of false negative results and obtain mtb isolates for identification. positive results do not always indicate the presence of living or viable microorganisms. a quick and accurate diagnosis is very important so that there is no over diagnose or under diagnose. moreover patient can get tb treatment immediately and provide other benefits, such as reducing disability and death rates and preventing tb transmission to others.7 open access under cc-by-nc-sa share alike 4.0 materials and methods this research is a descriptive observational laboratory testing the validity. fnab spesimens were taken from april 2019 until june 2019 at the anatomical pathology laboratory. material culture method using microbiology systems-.bd bactec™ mgit 960 system . identification of mtb dan ntm with sd bioline tb ag mpt64 and niacin paper strip from becton dickson company. method ziehl-neelsen staining microscopy procedure using fuchsine staining 3 minutes, alcohol acid solution staining for 30 seconds and methylene blue staining for 30 seconds, xpert mtb /rif using aspirate samples ijtid, p-issn 2085-1103, e-issn 2356-0991 35 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 33–38 results and discussion because some people have been exposed to people who are suspected of having tuberculosis. other studies also get the same distribution in the age range of 22 years to 44 years due to the productive age affects the high risk of tb.11 tb lymphadenitis cases can occur 60 to 80% in people with hiv because tb is an opportunistic infection in people with hivaids and this disease is found so that in patients with suspected tb lymphadenitis should be screened for hiv co-screening. clinical presentation lymph node enlargement mostly in cervical 37% patients other locations supraclavicular mamae. clinical symptoms mostly lymphadenopathy 31,5% and other lymphadenopathy with fever. clinical symptoms are one of the conditions for establishing a diagnosis of tb lymphadenitis, but in other studies it was found that clinical syndromes cannot be used as a single basis in determining the diagnosis of tb lymphadenitis because there are many variations in results because it is difficult to determine when the patient first experiences a complaint, subjectivity patients various.12 mostly lymph node enlargement sites in collie area, other studies also have the same data which is located in the cervical area.13 totally 35 specimens were examined 11/35 (31.4%) showed positive smear acid fast bacilli (afb) and 24/35 (68.6%) negative similar to the results of other studies a total of 120 patients affected by tb lymphadenitis as many as 26 samples (21.7%) found positive smear and 94 samples (78.3%) did not find smear,14 mtb bacilli were rare found in lymph node tissue because of its paucibacillary nature, low positivity depends on the average number of aspirate aspirates fnab 1000 to 10,000 / ml sample, liquid culture method mgit and solid culture method showed positive results 12/35 (34,3 %) and negative 23/35 (65,7%) (figure 1) (figure 2), to confirm presence of mycobacteria from positive culture using rapid identification tests sd bioline tb ag mpt64 (figure 3) and niacin paper test showed positive mtbc species 10 /12 (84%) and 2/12 (16%) reported as ntm species. ssamples according manufacture ‘s protocol sample reagent was added in a 2:1 ratio to unprocessed falcon tube, incubation 15 minute at room temperature. and add 2 ml material to cartridge and loaded to genexpert machine. culture was put up after decontamination samples on media slopes following the standard protocolmicrobiology systems-.bd bactec™ mgit 960 system. used diagnostic test 2x2 to analyze sensitivity, specificity, negative predictive value and positive predictive value. ethical clearance this study received approval from the health research ethics committee of dr. soetomo hospital surabaya with the statement of ethical clearance of 1232 / kepk / v / 2019. open access under cc-by-nc-sa share alike 4.0 demographic data obtained from medical records april to june 2019 most lymphadenitis patients came from surabaya with 21/35 (60%) and 14/35 outside surabaya (40%). no data available incidence of tuberculous lymphadenitis disease at the hospital dr. soetomo therefore needs further epidemiological research. characteristics of gender female dominated as many as 23/35 (65.7%) while male numbered 12/35 (34.3%) in line with other studies women dominated as many as 120 people (58.8%).8,9, 10 female factors are more dominant because of several factors such as differences in biological, hormonal, social, environmental and different behavior from men. male and female immune systems can be indicative of underlying causes for various patterns of disease in women. socially in developing countries, women are more vulnerable because they have low economic status, which makes it late for someone to come to a health care center.10 the age range distribution of tb lymphadenitis patients is highest in young adults 17 years to 25 years as many as 15/35 (42.9%) the second highest is the age group of 36 years to 45 years by 8/35 (22.9%). based on the background of the work status there were most 16 /35 employees (46%), the second highest was the non working group. herisa nataliana junus, et al.: validity of method for mtbc and ntm detection in fnab specimens 36 ijtid, p-issn 2085-1103, e-issn 2356-0991 middlebrook faster than the growth in loweinsten jensen media.17 the sensitivity of the mpt 64 tbag test kit in differentiating mtb and mott 100%.18 the results of the examination molecular test xpert mtb / rif assay (cepheid, usa) 35 patients with suspected tb lymphadenitis showed that 10/35 (28,69%) patients were positively detected by the m. tuberculosis bacterial gene and negative results in 25/35 (71,4%) . results of the reading of the detected mtb genexpert can be known quantitatively the level of mtb detection using xpert mtb/rif tool and categorized as follows: mtb detected low rif resistance not detected 8 samples (80%) and 2 sampels (20%) mtb detected very low rif resistance not detected. the validity test results in this study used the analysis of the sensitivity and specificity of the fnab specimens by using the wilson diagnostic analysis table with 95% confidence interval microscopic method of ziehl-neelsen staining (zn), comparison with the gold standard culture method mgit obtained a sensitivity value of 83.33% the ability of ziehl neelsen microscopic staining methods to identify with smear positive results in tb lymphadenitis patients is quite high. specificity value of 95.65% with 95% ci means that the ability to find out negative and true results of no afb in tuberculous lymphadenitis patients is 95.65%. a positive predictive value of 90.91% means that the probability of afb being present on microscopic examination if the results of a positive diagnostic test is 90.91%. a negative estimate value of 91.67% means that the probability of not having afb if the diagnostic test is negative is 91.67%. diagnostic accuracy of 91.43%. in line with other studies namely sensitivity of 83% and specificity of 98%.19 table 1. the result of diagnostic test figure 1. mtb colonies on middlebrook7h10 medium figure 2. mott colonies on middlebrook7h10 medium the culture method is a gold standard examination aimed at isolating mtb bacteria from samples of patients suspected of having tb lymphadenitis. the sensitivity value is quite high because the number of bacteria from 10 to 100 bacilli / ml from concentrated specimens can be detected.15 examination of culture methods at the clinical microbiology laboratory dr. soetomo uses the microbiology systems -.bd bactec ™ mgit 960 system diagnostic tool. process of decontamination can potentially cause the death of mtb bacilli, too acidic and too alkali conditions can also cause mtb bacilli death and failure to thrive.16 other studies comparing the loweinsten jensen and middlebrook 7h10 solid growth media found differences in the growth period of middlebrook open access under cc-by-nc-sa share alike 4.0 ijtid, p-issn 2085-1103, e-issn 2356-0991 37 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 34–38 sensitivity value: 83,33% specificity value: 95,65% with 95% ci positive predictive value of 90.91%: 90,91% negative estimate value of 91.67%: 91,67% diagnostic accuracy of 91.43%.: 91,43 % the validity test results in this study use the wilson wilson diagnostic analysis table 1 with 95% ci method of molecular xpert mtb / rif rapid test comparison with the mgit culture method as the gold standard. the results of this study obtained a sensitivity value of 75% which means that the ability of the examination of the rapid molecular xpert mtb / rif method in identifying mtb bacterial dna in tb lymphadenitis patients is 75%.specificity value of 95.65% with 95% ci means that the ability to find negative and true results of absence of mtb bacterial dna in tb lymphadenitis patients is 95.65%, positive predictive value of 90% means that the probability of mtb bacterial dna if positive diagnostic test results is 90%. an estimated negative value of 88% means that the probability of the absence of mtb bacterial dna if the diagnostic test is negative is equal to 91.67%. diagnostic accuracy of 88.57%. specificity (95.65%) in this study is in line with other studies where the specificity of xpert mtb / rif is 91%.20,21,22 regarding the negative results of the molecular nuclei acid amplification test need procedure of specimen collection . although the sensitivity value of this study is lower than the specificity value, a high enough specificity value of 95.65% can help in establishing the diagnosis that the patient did not have tb lymphadenitis. conclusion conflict interest there is no conflict interest of this paper. acknowledgement author would like to thank the chief of dr. soetomo hospital, surabaya, indonesia. head of clinical microbiology study program, faculty of medicine airlangga university. this report would not have been possible without contribution and collaboration dr. willy sandhika, dr. m.si, sp.pa (k) head of clinical research from department of anatomical pathology, faculty of medicine universitas airlangga, chairman of institute of tropical disease, universitas airlangga, surabaya, indonesia and dean of faculty of medicine, universitas airlangga. and also for contribution from agnes dwi sis perwitasari, s.si, a staff of tuberculosis laboratory, institute of tropical diseases universitas airlangga and sugeng harijono, a.md.a.k, medical staff of department of clinical microbiology dr. soetomo academic hospital. references 1. world health organization, et al. mycobacteriology laboratory manual. global laboratory initiative advancing tb diagnosis. geneva switzerland: world health organization, 2014 2. marlina, i. infodatin tuberkulosis. jakarta selatan. 2018 available from: https://pusdatin. kemkes.go.id 3. purohit m, & mustafa t. laboratory diagnosis of extra-pulmonary tuberculosis (eptb) in resourceconstrained setting: state of the art, challenges and the need. journal of clinical and diagnostic research: jcdr. 2015; 9(4): ee01 4. gandhare, avinash, et al. tuberculosis of the lymph nodes: many facets, many hues. astrocyte, 2017; 4(2): 80 5. tadesse m g, abebe k, abdissa d, aragaw k, abdella a, bekele l. rigouts.genexpert mtb/rif assay for the diagnosis of tuberculous ly lymphadenitis on concentrated fine needle aspirates in high tuberculosis burden settings. plos one. 2015; 10(9): e0137471 6. kementerian kesehatan ri. petunjuk teknis pemeriksaan tb menggunakan tes cepat molekuler. 2017 available from : https://www.who.int/tb/ laboratory/mycobacteriology-laboratory-manual.pdf 7. mertaniasih, ni made, et al. nontuberculous mycobacterial species and mycobacterium tuberculosis complex coinfection in patients with pulmonary tuberculosis in dr. soetomo hospital, sut tb lymphadenitis diagnostic examination can penot be done only rely on one method in establishing the diagnosis of tb lymphadenitis the distribution of mtb and ntm is very helpful in providing therapy based on the causative agent for tb lymphadenitis infection. open access under cc-by-nc-sa share alike 4.0 https://www.who.int/tb/%20laboratory/mycobacteriology-laboratory-manual.pdf https://www.who.int/tb/%20laboratory/mycobacteriology-laboratory-manual.pdf herisa nataliana junus, et al.: validity of method for mtbc and ntm detection in fnab specimens 38 ijtid, p-issn 2085-1103, e-issn 2356-0991 surabaya, indonesia. international journal of mycobacteriology. 2017; 6(1): 9-13 8. singh, saurabh kumar; tiwari, kamlesh kumar. tuberculous lymphadenopathy: experience from the referral center of northern india. nigerian medical journal: journal of the nigeria medical association. 2016; 57(2): 134 9. kamal, mohammad shah, et al. cervical tuberculous lymphadenitis: clinico-demographic profiles of patients in a secondary level hospital of bangladesh. pakistan journal of medical sciences. 2016; 32(3): 608 10. pang, yu, et al. epidemiology of extrapulmonary tuberculosis among inpatients, china, 2008– 2017. emerging infectious diseases, 2019; 25(3): 457 11. khandkar, chinmay, et al. epidemiology of peripheral lymph node tuberculosis and genotyping of m. tuberculosis strains: a case-control study. plos one. 2015; 10(7): e0132400 12. agatha, i. gst ngr pt mandela, et al. uji klinis sindroma klinis limfadenitis tuberkulosis dengan fine needle aspiration biopsy (fnab) sebagai baku emas. e-jurnal medika udayana 13. yashveer, j. k.; kirti, y. k. presentations and challenges in tuberculosis of head and neck region. indian journal of otolaryngology and head & neck surgery. 2016; 68(3): 270-274 14. mitra, shaila k.; misra, rajiv k.; rai, priyanka. cytomorphological patterns of tubercular lymphadenitis and its comparison with ziehlneelsen staining and culture in eastern up.(gorakhpur region): cytological study of 400 cases. journal of cytology, 2017; 34(3): 139 15. kant, kamla, et al. microbiological evaluation of clinically suspected cases of tubercular lymphadenopathy by cytology, culture, and smear hh microscopy–a hospital-based study from northern india. journal of family medicine and primary care. 2019; 8(3): 828 16. chatterjee, mitali, et al. effects of different methods of decontamination for successful cultivation of mycobacterium tuberculosis. the indian journal of medical research. 2013; 138(4): 541 17. naveen, g.; peerapur, basavaraj v. comparison of the lowenstein-jensen medium, the middlebrook 7h10 medium and mb/bact for the isolation of mycobacterium tuberculosis (mtb) from clinical specimens. journal of clinical and diagnostic research: jcdr. 2012; 6(10): 1704 18. arora, jyoti, et al. utility of mpt64 antigen detection for rapid confirmation of mycobacterium tuberculosis complex. journal of global infectious diseases. 2015; 7(2): 66 19. maynard-smith, laura, et al. diagnostic accuracy of the xpert mtb/rif assay for extrapulmonary and pulmonary tuberculosis when testing nonrespiratory samples: a systematic review. bmc infectious diseases. 2014; 14(1): 1-15 20. akhter, hasina, et al. diagnosis of tuberculous lymphadenitis from fine needle aspirate by pcr. bangladesh journal of medical microbiology. 2014; 8(1): 2-6 21. bunger, r., et al. evaluation of bactec micro mgit with lowenstein jensen media for detection of mycobacteria in clinically suspected patients of extra pulmonary tuberculosis in a tertiary care hospital at mullana (ambala). j med microb diagn. 2013; 2(123): 2161-0703.1000123 22. mertaniasih, ni made. buku ajar tuberkulosis diagnostik mikrobiologis. airlangga university press, 2019 open access under cc-by-nc-sa share alike 4.0 copyright © 2020, ijtid, issn 2085-1103 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 8 no. 1 january–april 2020 research article effect of patient's personal character on prevention of transmission of pulmonary tb herdianti1*, entianopa2, sugiarto3 1environmental health study program, stikes ibnu sina, batam 2,3public health study program, stikes harapan ibu, jambi a corresponding author: herdianti@stikesibnusinabatam.ac.id received: 3rd march 2019; revised: 22nd march 2019; accepted 9th january 2020 abstract tuberculosis (tb) is an infectious disease that is still a problem for health in the world, caused by mycobacterium tuberculosis. muaro jambi showed second ranks of tb patients number in jambi province. based on the available data, the biggest positive tb patients were in the muara kumpeh health center working area which was 54 people (2016) and 68 people (2017). the purpose of this study was to determine the relationship and risk of self efficacy and interpersonal relations to the prevention of pulmonary tb transmission in the muara kumpeh health center in muaro jambi district in 2018. this research is a quantitative research with cross sectional study research design. the sample of this study was 68 people interviewed using questionnaire with the total sampling. the research was carried out in the work area of muara kumpeh health center in april-august 2018. the data was collected then analyzed in univariate and bivariate. a total of 37% of respondents were adults (26-45 years), 53% of male patients and 52% of respondents were treated by their spouses during illness. there was a significant relationship (p-value = 0.011) between self efficacy and tb transmission prevention behavior and low self efficacy at 5.14 times the risk of transmitting pulmonary tb compared to high self efficacy. with high self confidence from respondents can provide good motivation for their recovery. there was no significant relationship (p-value = 0.104) between interpersonal relationships with tb transmission prevention behaviors and interpersonal relationships not risk factors for preventing tb transmission behavior. this could be due to not too much percentage difference between respondents who have low and high interpersonal relationships. besides that, the knowledge factor of the patient's family can also be confounding on this variable. low self efficacy has a risk of 5.14 times transmitting tb and this is statistically significant. it is better to do more routine care of the patient's family so that it can improve self efficacy and interpersonal relationships of the patient. keywords: self efficacy, interpersonal relationship, prevention of pulmonary tb transmission abstrak tuberkulosis (tb) adalah penyakit menular yang masih menjadi masalah di dunia kesehatan sejauh ini merupakan penyakit menular yang disebabkan oleh mycobacterium tuberculosis. wilayah muaro jambi menempati urutan kedua terbanyak di tb di provinsi jambi. berdasarkan data yang tersedia, pasien tb positif terbesar adalah di wilayah kerja pusat kesehatan muara kumpeh yang terdiri dari 54 orang (2016) dan 68 orang (2017). tujuan dari penelitian ini adalah untuk mengetahui hubungan dan risiko efektivitas diri dan hubungan interpersonal untuk pencegahan infeksi tb paru di pusat kesehatan muara kumpeh di kabupaten muaro jambi pada tahun 2018. penelitian ini adalah penelitian kuantitatif dengan penelitian cross sectional study. sampel penelitian ini adalah 68 orang yang diwawancarai menggunakan kuesioner dengan number of sampling. penelitian dilakukan di wilayah kerja pusat kesehatan muara kumpeh pada bulan april-agustus 2018. data yang dikumpulkan kemudian dianalisis secara univariat dan bivariat. sekitar 37% responden adalah orang dewasa (26-45 tahun), 53% pasien pria dan 52% responden dirawat oleh pasangan mereka selama sakit. ada hubungan yang signifikan (p-value = 0,011) antara corresponding author. e-mail: herdianti@stikesibnusinabatam.ac.id efikasi diri dan perilaku pencegahan penularan perilaku mailto:herdianti@stikesibnusinabatam.ac.id mailto:herdianti@stikesibnusinabatam.ac.id copyright © 2020, ijtid, issn 2085-1103 10 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 9–15 dan efikasi diri yang rendah pada 5,14 kali risiko transplantasi tb paru dibandingkan dengan efikasi diri yang tinggi. dengan harga diri yang tinggi dari responden dapat memberikan motivasi yang baik untuk pemulihan mereka. tidak ada hubungan yang signifikan (p-value = 0,104) antara hubungan interpersonal dengan perilaku pencegahan tb dan hubungan interpersonal alih-alih faktor risiko untuk mencegah penularan perilaku tb. ini mungkin disebabkan oleh perbedaan persentase yang rendah di antara responden dengan hubungan interpersonal yang rendah dan tinggi. selain itu, faktor pengetahuan keluarga pasien juga dapat membingungkan tentang variabel ini. efikasi diri yang rendah memiliki risiko 5,14 kali radiasi tb dan ini signifikan secara statistik. cara terbaik adalah melakukan perawatan keluarga yang lebih rutin untuk meningkatkan efikasi diri dan hubungan interpersonal pasien. kata kunci: efikasi diri, hubungan interpersonal, pencegahan penularan tb paru how to cite: herdianti, herdianti; entianopa, entianopa; sugiarto, sugiarto. effect of patient's personal character on prevention of transmission of pulmonary tb. indonesian journal of tropical and infectious disease, [s.l.], v. 8, n. 1, p. 9-15, mar. 2020. issn 2356-0991. available at: <https://e-journal.unair.ac.id/ijtid/article/view/12318/9918>. date accessed: 04 apr. 2020. doi:http://dx.doi.org/10.20473/ijtid.v8i1.12318. introduction pulmonary tb is a chronic and contagious infection that is closely related to environmental conditions and community behavior. pulmonary tb is an in fect io u s d isease cau sed by mycobacterium tuberculosis. this disease is transmitted through the air which is spit, sneezing and coughing. mycobacterium tuberculosis enters the lung tissue through the airways (droplet infection) to the alveoli, primary infection occurs. then spread to the local lymph nodes and formed a complex primer. primary and primary complex infections are called primary tb, most of whom will experience healing on a further journey. pulmonary tb disease usually attacks the lungs but can also attack other body organs.1,2 pulmonary tb is still a public health problem in the world. pulmonary tb disease attacks many productive age groups. most come from low socioeconomic groups and low levels of education.3 the world health organization (who) is stated that the situation of the world tuberculosis (tb) is getting worse, where the number of tb cases has increased and many have not been cured. who declare tb as a global emergency, especially due to the epidemic of cases of multi drug resistance (mdr), a suspect with pulmonary tuberculosis can easily transmit it through sputum by spreading the germs into the air4. tuberculosis (tb) is an infectious disease that is still a problem in the world of health to date. global tb incidence is reported to have decrease at a rate of 2.2% in 2016-2017.1,2 the who report in 2017 concludes that there are estimated 8.6 million tb cases in 2016 of which 1.1 million people (13%) are hiv positive of tb patients and around 75% of these patients are in the african region. in 2012, there were an estimated 450,000 people suffering from mdr tb and 170,000 of them died.1,5 indonesia is the 4th country with the highest number of tuberculosis patients in the world. treatment of tuberculosis is one way to control infection and reduce transmission of tuberculosis. the nat io nal tu bercu lo sis pro gram has successfully achieved the target of the sustainable development goals in the form of increasing the discovery of new cases of positive smear as much as 70% and cure rates of 85%, but some hospitals and private practices still have not implemented the directly observed treatment short course (dots) and international standards for tuberculosis strategies care (istc). efforts to expand the application of the dots strategy are still a major challenge for indonesia in controlling tuberculosis.6,7 indonesia have achievements in reducing tuberculosis mortality. in 2007, indonesia ranked third among countries with the most tb cases. in 2015 it was ranked 5th under india, china, kenya and south africa with a decrease in mortality which was 168,000 / year (in 2010) to 64,000/ year (in 2015).7 https://e-journal.unair.ac.id/ijtid/article/view/12318/9918 http://dx.doi.org/10.20473/ijtid.v8i1.12318 copyright © 2020, ijtid, issn 2085-1103 herdianti, et al.: effect of patient's personal character 11 based on data obtained from the jambi provincial health office, it can be seen that there has been an increase in tuberculosis cases in muaro jambi district in 2015 the number of pulmonary tb patients was 368 people while in 2016 the number of pulmonary tb patients was 377 people, with a prevalence of 0, 10%. from these data there is an increase in tuberculosis case finding each year, the second largest number of pulmonary tb patients is muaro jambi regency while the lowest is sungai penuh city. every year data shows an increase in tb sufferers in muara jambi district. based on the available data, the biggest positive tb patients are in the muara kumpeh health center working area which is 54 people in 2016 and 68 people in 2017.8 given that pulmonary tuberculosis can have fatal consequences and death, the family or community should know and understand the various problems and impacts of the disease, especially pulmonary tb. with the existence of a self efficacy and a good interpersonal relat ionship, it plays an important role in efforts to prevent transmission of pulmonary tb. the purpose of this study was to determine the relationship and risk of self efficacy and interpersonal relations to the prevention of pulmo nary tb transmissio n in the muara kumpeh health center in muaro jambi district in 2018. materials and methods this research is a quantitative study with a cross sectional study design to see the relationship between dependent and independent variables at one time.9 the sample of this study amounted to 68 people using the total sampling questionnaire. the study was conducted door to door to interview respondents. and was held in april august 2018. the data used in this study consisted of secondary data and primary data. secondary data is obtained from the data from pkm muara kumpeh medical recording and address data from the village office. while the primary data is data collected by researchers with interview and observation techniques. the research instrument used was a questionnaire with 10 questions for each variable. the questionnaire was first tested for validity and reliability. the data collected was then analyzed in univariate and bivariate ways. univariate analysis is to an obtained the description of the characteristics of respondents related to age, sex and who treated during illness with chi-square and odd ratio to determine the relationship and the magnitude of the risk of the independent variables on the dependent variable. results and discussion this chapter describes the results of research both univariately and bivariately between the relationship of self-efficacy and interpersonal relationships to the prevention of transmission of pulmonary tb. the results of this study are presented in percentage form and are presented in the form of graphs and tables. characteristic of respondents based on univariate analysis of the data obtained consisting of age groups, genders and caring families can be seen clearly in figure 1, 2 and 3 below. age of respondents is grouped according to age classification according to the ministry of health of the republic of indonesia consisting of children under five, children, figure 1. distribution of frequency of respondents by age of patients with pulmonary tb in the work area of muara kumpeh health center in 2018 copyright © 2020, ijtid, issn 2085-1103 12 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 9–15 table 1. analysis of relations self efficacy on prevention of tb transmission no. self efficacy prevention total poor good p value or (ll ul) figure 2. distribution of frequency of respondents based on gender of pulmonary tb patients in the work area of muara kumpeh health center in 2018 source: primary data, 2018 table 2. analysis of interpersonal relationship to prevention of tb transmission no. interpersonal relationship prevention total poor good p value or (ll ul) figure 3. distribution of frequency of respondents by family who care for pantients with pulmonary tb in the work area of muara kumpeh health center in 2018 adolescents, adults, elderly and the elderly. in full, the percentage of each age group can be seen in figure 1. based on figure 1 it can be seen that the most respondents were the adult group (37%) and the least were the older group (4%). based on figure 2, it is known that out of 68 respondents, 53% of respondents were male and 47% of respondents were female. as many as 52% of respondents during illness were treated by their wifes and husbands and no respondents were treated other than the main family as shown in figure 3 above. bivariate analysis this analysis is aims to prove the existence of a meaningful relationship between the independent variables and the dependent variable, the analysis using chi-square test and the significance level of p <0.05. if the calculation results show the value of p <alpha (0.05), then statistically the two source: primary data, 2018 variables studied have a meaningful relationship. in addition, an odd ratio (or) analysis was also conducted to see how much risk the variable was examined. the level of self-efficacy and interpersonal relationships is divided into two categories, namely low and high, while the prevention of tb transmission are also two categories namely poor and good. the relationship and the amount of risk between the independent and dependent variables can be seen in the table 1. based on table 1, 88.9% of respondents had a low self efficacy which prevented the poor and there were also 11.1% of respondents who, despite their high self efficacy, still did poor prevention of transmission. from the results of chi-square analysis seen in the p-value, it was found that there was a significant relationship between self efficacy and tb prevention behavior. whereas from or analysis, it was found that self efficacy was a risk factor for the prevention of tb transmission behavior of 5.14. based on table 2 as many as 88.6% of respondents have low interpersonal relationships that do poor prevention and there are also 11.4% n % n % n % 1. low 40 88.9 5 11.1 45 100 5.14 2. high 14 60.9 9 39.1 23 100 0.011 (1.47 17.97) total 54 79.4 14 20.6 68 100 n % n % n % 1. low 31 88.6 4 11.4 35 100 3.37 2. high 23 69.7 10 30.3 33 100 0.104 (0.94 12.11) total 54 79.4 14 20.6 68 100 copyright © 2020, ijtid, issn 2085-1103 herdianti, et al.: effect of patient's personal character 13 of respondents have taken good preventive despite low interpersonal relationships. from the results of chi-square analysis, it can be seen that there is no significant relationship between interpersonal relationships with tb transmission prevention behavior. from or analysis it was found that interpersonal relations were not risk factors for preventing tb transmission behavior. characteristics of respondents from the results of the analysis of figure 1 it can be seen that the most sufferers are the adult age group (26-45 years). this is likely to be attributed to occupational factors that are at risk for developing pulmonary tb. there are 9% of children under five who experience tb. based on the results of in-depth interviews that researchers obtained that most likely this was caused by the transmission of the behavior of mothers who did not know the causes and dangers of tb because on average they were confused and did not realize where their children could get tb. based on sex, also most men. according to researchers this is almost the same as age, which is likely to come from occupational risks. the average male tb patient works as a worker/ employee who does not pay much attention to hygiene around. they also include laymen on pulmonary tb disease. the average patient is treated by their husband/ wife. this should be good for the patient's motivation to recover because the caring family cares about the patient's health. this relates to interpersonal relationships. from the respondents' answers, most received positive support coming from their families for their recovery. bivariate analysis based on the results in 68 respondents are showed that 45 respondents who had low self efficacy. this was because the patient was still unfamiliar with tb disease so they did not have the confidence to recover even though they came for treatment. then other results were found that 23 respondents had high self-efficacy. high self efficacy group in this study consisted of patients to the adhering treatment program for six to nine months, maintaining environmental hygiene, obeying the supervisor taking medication by taking medication on time, being able to adjust to the side effects of tb drugs, taking the correct dose and correct medication time, perform sputum examination to find out the progression of the disease, take adequate rest, and check into health services if there are complaints that aggravate the disease. bivariate analysis shows that there is a relationship between self efficacy and prevention of pulmonary tb transmission and patients who have low self efficacy are at risk of transmitting pulmonary tb 5 times compared to patients with high self efficacy. according to researchers, this is only natural because most sufferers are indifferent to tb so they don't even know the causes and dangers. this of course affects the self efficacy to recover. this patient's ignorance can be seen from most respondents who do not know the cause of tb. they also did not separate equipment used by sufferers in the house. so that what is used by the patient such as towels, toiletries, food and other possibilities is also likely to be used by other family members who live at home. in addition, patients also do not understand that tb can be transmitted through the air. this increases transmission in the patient's home. the results in this study are in line with the research conducted by hendiani, which shows that the average tb patient has a high self-efficacy of 56.8%. this is because respondents want to recover from tb disease so that they comply with all health worker advice, various efforts are made to recover, such as taking vitamins, avoiding cigarette smoke and maintaining a clean house.10 respondents have low confidence in the prevention of transmission of tuberculosis due to previous failure treatment.11 self-efficacy also influences the choices they make, helps determine how much effort a person spends in a behavior, how long they will survive in the face of obstacles and how strong they are in dealing with adverse situations. self-efficacy plays an important role in predicting behavior and results. self-efficacy has been linked to various clinical problems.3,12 according to bandura13 the process of self efficacy is one of cognitive or knowledge. in this copyright © 2020, ijtid, issn 2085-1103 14 indonesian journal of tropical and infectious disease, vol. 8 no. 1 january-april 2020: 9–15 case the action taken by someone who comes from his mind. then the thought gives direction for the actions taken. if the higher knowledge, level of education, and work that is owned will contribute to the formation of high self-efficacy and high self-efficacy can not be separated from the presence of influencing factors such as previous individual experiences, experiences of others who are the same, social persuasion and physiological and emotional state.11 according to the study tambunan14 individuals who have good self-efficacy have a 5.850 times chance of showing a better quality of life compared to individuals who have poor self-efficacy. health workers should emphasize again the importance of awareness of knowing pulmonary tuberculosis ranging from causes, the process of transmission and prevention. therefore, the role of health workers contributes to the self efficacy of tb patients.15 an interpersonal relationship analysis on prevent ing transmissio n of pulmo nary tb shows no relationship and is not a risk factor. unrelated to these two variables is likely due to the respondent's good answers from 10 questions related to interpersonal relationships. the average respondent has received positive support from both family and health workers. from the results of the analysis, it was found that 7.4% said that families and officers rarely reminded them to take precautions. according to researchers there are still respondents who have a family role and the role of health workers who are not good is due to various things such as the busyness of family members so that they do not have effective time between family members. besides that, there is an assumption that the problem can still be handled independently by respondent, so that the family's attention with the respondent decreases as well as the role of the officer. there is still other opinion of respondent that the officer is not really in pro vid ing understanding or complete informat ion on prevention of transmission of pulmonary tb. in the treatment of tuberculosis, the role of the family is very important. the role of families in the treatment of tuberculosis is supervision of swallowing drugs. the family must encourage the recovery of the patient well. families as supervisor for drugs are views and assessments of tb patients for interaction with family in the form of information, attention, encouragement and assistance from drugs supervisor so that the quality of relationships can affect the recovery of tb patients.10,16 this research differs from yurida's17 research which is stated that the main social support comes from family support, because family support plays an important role in the lives of tuberculosis patients struggling to recover, think ahead, and make their lives more meaningful due to lack of social support from their families and the environment surrounding causes psychological disorders in tuberculosis sufferers include: depression, adjustment disorders, anxiety, loss of life goals, weakening productivity, phobias and others18. given that pulmonary tuberculosis can have fatal consequences and death, the family or community should know and understand various problems and impacts of the disease, especially pulmonary tuberculosis.19,20 it is expected that the role of the family in preventing transmission of pulmonary tb will be higher, so that respondents take precautions on themselves and their environment. in addition, the role of officers in the muara kumpeh health centre area involves posyandu cadres and certain organizations in counseling, especially in preventing transmission of pulmonary tb. conclusions there is a relationship between self efficacy on prevention of pulmonary tb transmission and patients who have low self efficacy are at risk of transmitting pulmonary tb 5 times compared to patients with high self efficacy. there is no relationship between interpersonal relationships with the prevention of pulmonary tb transmission and interpersonal relationships are not a risk factor for preventing transmission of pulmonary tb in the work area of the muara kumpeh health center. copyright © 2020, ijtid, issn 2085-1103 herdianti, et al.: effect of patient's personal character 15 acknowledgements the researcher expressed their hig hest gratitude to the directorate of research and community service of the directorate general of strengthening and development of the ministry of research, technology and higher education which has provided 2018 budget grants for this research. not forgetting also the gratitude to the foundation and manager of the stikes harapan ibu who have provided full support for this research. conflict of interest there was no conflict of interest for this research. references 1. kemenkes. tuberkulosis (tb). 2017 [internet]. 2018;1(april):2018. available from: www.kemenkes. go.id 2. pdpi. pedoman penatalaksanaan tb (konsensus tb). in: perhimpunan dokter paru indonesia [internet]. 2011. p. 1–55. available from: http://klikpdpi.com/ konsensus/xsip/tb.pdf 3. herawati e. hubungan antara pengetahuan dengan efikasi diri penderita tuberkulosis paru di balai kesehatan paru masyarakat surakarta. 2015; 4. who. mdr-tb. burden, glob treat enroll o n mdr-tb outcomes, treat. 2016;2015–6. 5. who. who | tb/hiv facts 2015. who. 2010;4. 6. kementrian kesehatan republik indonesia. profil kesehatan indonesia 2015 [internet]. vol. 70, kesehatan. 2016. 1780-1790 p. available from: www. kemkes.go.id. 7. menteri kesehatan ri. toss tb: temukan tb obati sampai sembuh [internet]. www.depkes.go.id. 2016. p. 1–2. available from: http://www.depkes.go.id/pdf. php?id=16040400008 8. dinas kesehatan provinsi jambi. profil kesehatan privinsi jambi. 2017. 9. notoatmodjo s. metodologi penelitian kesehatan. jakarta:pt rineka cipta. 2012; 10. hendiani n, sakti h, widayanti cg. hubungan antara persepsi dukungan keluarga sebagai pengawas minum obat dan efikasi diri penderita tuberkolosis di bkpm semarang. j psikol undip. 2014;13(1):82–91. 11. arias ms. determinants of self efficacy to seek care for tuberculosis and complete tuberculosis treatment among hiv-positive individuals attending hiv/aids clinics in honduras. diss abstr int sect b sci eng [internet]. 2010;71(6–b):3624. available from: http://ovidsp.ovid. com/ovidweb.cgi?t=js&page=reference&d=psyc7 &news=n&an=2010-99240-239 12. mason p, singh p, ho j, anh n, fox g, marks g. coping self-efficacy for tb treatment in ca mau. in: 5th conference of the union asia pacific region. 2015. 13. bandura a. bandura self-efficacy defined [internet]. encyclopedia of human behavior. 1994. p. 71–81. available from: http://www.uky.edu/~eushe2/bandura/ banency.html 14. pratomo ip, burhan e, tambunan v. malnutrisi dan tuberkulosis. j indon med assoc. 2012;62((6) juni 2012):230–6. 15. naghib salam, jila abed s, ezzati zhaleh, zayeri farid. self-efficacy and compliance with treatment regimen in tuberculosis patient in kurdistan [farsi]. j nurs midwifery [internet]. 2012;22(77):1p. available from: http://search.ebscohost.com/login.aspx?direct=true&d b=cin20&an=2012122488&site=ehost-live 16. silvani h, sureskiarti e, kunci k, aktif keluarga p, paru kt, program sarjana ilmu keperawatan stikes muhammadiyahsamarinda m, et al. hubungan peran aktif keluarga sebagai pengawas minum obat (pmo) dengan angka kekambuhan tb paru di ruang seruni rsud abdul wahab sjahranie samarinda. j ilmu kesehat. 2016;44(2):66–74. 17. olviani y. hubungan dukungan pasangan penderita tb dengan kepatuhan minum obat pada penderita tb paru di wilayah kerja puskesmas pekauman banjarmasin tahun 2016. 2016; 18. leung cc, lange c, zhang y. tuberculosis: current state of knowledge: an epilogue. vol. 18, respirology. 2013. p. 1047–55. 19. verdier je, de vlas sj, kidgell-koppelaar id, richardus jh. risk factors for tuberculosis in contact investigations in rotterdam, the netherlands. infect dis rep. 2012;4(2):101–5. 20. shanmugam ts, subramaniam p, parthiban n, sivapalan n. are we preventing tuberculosis transmission? respirology. 2015;20:76. http://klikpdpi.com/ http://www/ http://www.depkes.go.id/pdf http://ovidsp.ovid/ http://www.uky.edu/~eushe2/bandura/ http://search.ebscohost.com/login.aspx?direct=true&amp;d introduction materials and methods results and discussion characteristic of respondents bivariate analysis characteristics of respondents bivariate analysis (1) conclusions acknowledgements conflict of interest references available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 8 no. 2 may–august 2020 research report identification of scc mec methicillin-resistant staphylococcus aureus (mrsa) from hospitals’ clinical samples in jambi using polymerase chain reaction (pcr) humaryanto 1* , hanina 1 , lipinwati 1 , charles apul simanjuntak 1 1 faculty of medicine and health science, university of jambi, jambi indonesia received: 8th april 2019; revised: 29th january 2020; accepted: 23rd april 2020 abstract staphylococcal cassette chromosome mec (sccmec) is one of the mobile genetic elements of methicillin-resistant staphylococcus aureus (mrsa) that carries many resistance genes and allows sccmec to move from one bacterium to another. twelve types of sccmec have been identified throughout the world. identification of sccmec type is needed to determine the pattern of mrsa resistance in a particular region. this study aimed to identify the type of sccmec mrsa from clinical samples. specifically, this study was conducted at the biomolecular laboratory of the faculty of medicine and health sciences of jambi university in june 2018-february 2019. culture was carried out on 100 clinical specimens of festering wound swabs from inpatients at hopitals in jambi city. a total of 32 samples of staphytect plus test positive were tested using cefoxitin disc diff usion method and meca polymerase chain reaction (pcr). there were 14 samples identified as mrsa isolates, namely twelve samples (85.72%) of sccmec type iii, one sample (7.14%) of sccmec type ii, and one sample (7.14%) of sccmec type ivb. the results were diff erent from previous studies where all mrsa isolates (100%) in indonesia were sccmec type iii, although most sccmec types were still dominated by sccmec type iii. this study concludes that there has been a shift in the content of sccmec in mrsa isolate originating from hospitals in jambi city. keywords: mrsa, meca, sccmec, genetic, resistance abstrak staphylococcal cassette chromosome mec (sccmec) merupakan salah satu elemen genetik yang mobile pada methicillin resistant staphylococcus aureus (mrsa) yang membawa beberapa gen resistensi dan memungkinkan sccmec berpindah dari satu bakteri ke bakteri lainnya. terdapat dua belas tipe sccmec yang telah teridentifi kasi di seluruh dunia. identifi kasi tipe sccmec sangat diperlukan untuk mengetahui pola resistensi mrsa di suatu wilayah tertentu. penelitian ini bertujuan untuk mengidentifi kasi tipe sccmec mrsa dari sampel klinik. penelitian ini dilakukan di laboratorium biomolekuler fakultas kedokteran dan ilmu kesehatan universitas jambi pada bulan juni 2018-februari 2019. kultur dilakukan terhadap 100 spesimen klinik berupa swab luka yang bernanah pada pasien yang dirawat inap di rumah sakit di kota jambi. sebanyak 32 sampel yang positif pada uji staphytect plus diuji dengan cefoxitin disk difusion metode dan polymerase chain reaction (pcr) meca. terdapat 14 sampel yang teridentifi kasi sebagai isolat mrsa. sebanyak 12 sampel (85,72%) merupakan sccmec tipe iii, satu sampel (7,14%) sccmec tipe ii dan satu sampel (7,14%) sccmec tipe ivb. hasil penelitian ini berbeda dengan penelitian sebelumnya dimana seluruh (100%) isolat mrsa di indonesia merupakan sccmec tipe iii, meskipun tipe sccmec terbanyak masih didominasi oleh sccmec tipe iii. kesimpulan dari penelitian ini adalah mulai ditemukannya perubahan kandungan sccmec pada isolat mrsa yang berasal dari rumah sakit di kota jambi. kata kunci: mrsa, meca, sccmec, genetic, resistensi * corresponding author: humaryanto_fkik@unja.ac.id copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 78 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 77–82 how to cite: humaryanto., hanina., lipinwati., chaeles apul simanjuntak. identification of scc mec methicillinresistant staphylococcus aureus (mrsa) from hospitals’ clinical samples in jambi using polymerase chain reaction (pcr). indonesian journal of tropical and infectious disease, 8(2), 1–8 introduction s. aureus is a common bacterial pathogen that causes minor to serious disease in human. s. aureus can be treated with methicillin (mssa) and resistant to methicillin (mrsa). infection of mrsa becomes an important concern throughout the world and associated with infection in both hospital-acquired methicillin-resistant staphylococcus aureus (ha-mrsa) and community-acquired methicillin-resistant staphylococcus aureus (ca-mrsa). 1,2,3 infection caused by mrsa keeps increasing year to year. according to research in indonesia, the prevalence of mrsa is approximately 30–40%. the prevalence of mrsa in cipto mangunkusumo hospital on 2010 and abdul moeloek hospital lampung on 2013 were 32% and 38%, respectively. 4,5 the resistance of mrsa against beta-lactam antibiotic is encoded by the meca gene. meca gene is a part of the conserved mrsa genetic elements of the staphylococcal cassette chromosome mec (sccmec), encoding pbp2a or pbp2 mutants. 6,7 meca gene is located in a genetic element called the staphylococcal cassette chromosome (sccmec). sccmec is integrated into the chromosome of s. aureus at a unique site located near the s. aureus origin of replication. sccmec is a mobile genetic element that carries many resistance genes and allows sccmec to move from one bacterium to another. 8 thirteen types of sccmec have been identified throughout the world. 9 the components of sccmec are recombinase genes (ccr complexes), mec complex genes, additional resistant genes, and insertion sequences (is). 8,10 differences between sccmec are determined by variations in the ccr complex and the mec complex. sccmec type i about 39 kb, in the 1960s era, has a composition of type 1 ccr complex and class b mec complex. sccmec type ii about 52 kb, dominant in the 1980s era, has a composition of type 2 ccr complex and the class a mec complex. sccmec type iii about 67 kb, dominant in the 1980s, has the composition of the type 3 ccr complex and the class a mec complex. sccmec type iv (a and b) about 20.9–24,3 kb, found in 2002, has a composition of type 2 ccr complex and class b mec complex. 4,5,6 various findings of mrsa patterns in the last decade have shown the changes in distribution, sensitivity to various antibiotics, and possible changes in the sccmec type. 11,12 identification of sccmec type is needed to determine the pattern of mrsa resistance in a particular region. based on the previous description, it is important to identify the type of sccmec mrsa from clinical samples. materials and methods this study was a cross-sectional study. this study was conducted in the biomolecular laboratory of the faculty of medicine and health sciences in jambi university from june 2018 to february 2019. a hundred samples of swabs from festering wound were collected from three secondary referral hospitals in jambi (raden mattaher hospital, dr. bratanata hospital, and kambang hospital). the swabs were incubated at 30 ºc on mannitol salt agar (msa) for 18-24 hours, the yellowish colony would be confirmed by gram staining. gram-positive coccus bacteria were tested using staphytect plus test dr 850 m (oxoid) to detect clumping factor, protein a and type 5 and 8 capsules of polysaccharide. positive samples were tested for resistance to cefoxitin antibiotics by using the disc diff usion method in mueller hinton (mh) agar. the susceptibility testing was conducted as a standard of clsi 2011. 13 identification of meca gene and the type of sccmec were using polymerase chain reaction (pcr). primers used are shown in table 1. copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 humaryanto, et al.: identification of scc mec methicillin-resistant staphylococcus aureus (mrsa) 79 preparation of bacterial dna samples, pcr mec a and pcr sccmec dna samples 5 μl of bacterial suspension (0.5 mc farland) from yellowish colonies were incubated at 30°c 18-24 hours on msa. pcr was performed in a final volume of 25 μl consisting of 5 μl of dna samples, 10 μl of 2x gotaq green master mix (promega), 2 μl 1mm forward primer (mec a1), 2 μl 1mm reverse primer (mec a2) and 6 μl of nuclease-free water. positive control and negative control were s. aureus atcc 43300 and s. aureus atcc 25923. the mixture was denatured at 94°c for 5 minutes followed by 30 cycles, 94°c for 45 seconds, 72°c for 90 seconds, and 72°c for 10 minutes. dna was amplified with a thermocycler (thermo scientific, usa). multiplex pcr sccmec was carried out on positive samples of meca gene to detect sccmec chromosomes. primers used are shown in table 1. pcr was performed in a final volume of 25 μl consisting of 5 μl of dna samples, 12.5 μl of 2x gotaq green master mix (promega), 0.5 μl 1 mm of forward primer, 0.5 μl 1 mm of reverse primer (scc mec primers type i, ii, iii, iva, and ivb) and 2.5 μl nuclease-free water. pcr to identify the type of sccmec began with an initial denaturation at 94°c for 5 minutes followed by 10 cycles of denaturation at 94°c for 45 seconds, annealing at 55°c for 45 seconds, extension at 72°c for 90 seconds, then continued with 25 cycles of denaturation at 94°c for 45 seconds, annealing at 50°c for 45 seconds, extension at 72°c for 90 seconds, and final extension 72°c 10 minutes. the amplicons were visualized in 0.8% agarose stained using sybr safe dna (invitrogen), and images were obtained using a gel documentation system. results and discussion a total of 100 festering wound swab samples were obtained from hospitalized patients in raden mattaher hospital, dr. bratanata hospital, and kambang hospital. thirty-two samples were positive s. aureus through staphytect plus test. there were 14 isolates of mrsa based on cefoxitin resistance in disc diff usion method and pcr meca positive (figure 1). multiplex pcr was performed on 14 mrsa isolates to identify the type of sccmec in the samples. there were 12 samples (85.72%) of sccmec type iii, 1 sample (7.14%) of sccmec type ii, and 1 sample (7.14%) of sccmec type ivb (figure 2). the sccmec types distribution were depended on geographical manner. most mrsa isolates from eastern and middle eastern countries hospitals contain sccmec type iii. 15 this sccmec type is common in some south east asia countries hospitals such as thailand, singapore, indonesia and malaysia. 16 diff erent with some south east asian countries, mrsa isolates from table 1. sequence of oligonucleotide primers.14 target gene primer nucleotide sequence (5’-3’) amplicon (bp) meca gene meca1 gta gaa atg act gaa cgt ccg ata a 310 meca2 cca att cca cat tgt ttc ggt cta a sccmec i i-f gct tta aag agt gtc gtt aca gg 613 i-r gttctctcatagtatgacgtcc sccmec ii ii-f cgttgaagatgatgaagcg 398 ii-r cgaaatcaatggttaatggacc sccmec iii iii-f ccatattgtgtacgatgcg 280 iii-r ccttagttgtcgtaacagatcg sccmec iva iva-f gccttattcgaagaaaccg 776 iva-r ctactcttctgaaaagcgtcg sccmec ivb ivb-f tctggaattacttcagctgc 493 ivb-r aaacaatattgctctccctc copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 80 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 77–82 figure 1. agarose gel electrophoresis of pcr product amplified from meca gene (310 bp). m is dna marker; k(+) is positive control, lane 1-14 are meca fragments. figure 2. agarose gel electrophoresis of pcr product amplified from sccmec type. m is dna marker; lane 1-3,5,7-14 are sccmec type iii fragments (280 bp). lane 4 is sccmec type ii fragment (398 bp). lane 6 is sccmec type ivb fragment (493 bp). korea and japan predominantly contain sccmec type ii. 16 while some european countries mrsa isolates contain sccmec type iv. 17 in this study, the majority of sccmec types was type iii (85.72%). these results were consistent with studies conducted in seven countries in asia including indonesia and studies conducted in iran where sccmec type iii was the most common in mrsa isolates. 16,18,19 in addition to sccmec type iii, this study also found a small proportion of mrsa isolates contained sccmec type ii and type ivb. sccmec type i, ii, and iii were the commonly found types in hospitals (ha-mrsa), while sccmec type iv and v were the commonly found types in communities (ca-mrsa). 20,21,22 sccmec type ii also found in jakarta, a study mentioned that the majority of mrsa isolates in hospitals were sccmec type ii. 23 while sccmec type iv also found in denpasar (12.5%) and malaysia (3.18%) among mrsa isolates in hospitals. 24,25 this means that there has been a shift in the content of sccmec in mrsa isolates in indonesia. the discovery of sccmec type iv in the hospital raises concerns because this type is more mobile, generally causes more severe clinical symptoms, and is more difficult in the selection of suitable antibiotics. 21,24 in comparison to other sccmec elements, sccmec iv is small in size and more variable, which has possibly enabled it to spread easily within s. aureus. conclusions based on the results revealed in this study, there has been a change in the type of sccmec in mrsa isolates from hospitals. therefore, it is copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 humaryanto, et al.: identification of scc mec methicillin-resistant staphylococcus aureus (mrsa) 81 recommended to conduct further research with a larger sample size, both from hospitals and communities to identify the sccmec type and its relationship to patterns of sensitivity to antibiotics. keeping in view, the finding of sccmec type iv in jambi should be investigated, whether it is a circulator or a persisting invader. further molecular analysis of these mrsa isolates by pulsed-field gel electrophoresis or mlst (multi locus sequence typing) may provide much useful information regarding the origin and the epidemiology of local isolates. acknowledgement this project was funded by a grant from the faculty of medicine and health sciences, jambi university. conflict of interest the authors declare that they have no conflict of interest. references 1. alrabiah k, al alola s, al banyan e, al shaalan m, al johani s. characteristics and risk factors of hospital acquired e methicillin-resistant staphylococcus aureus (ha-mrsa) infection of pediatric patients in a tertiary care hospital in riyadh, saudi arabia. int j pediatr adolesc med. 2016; 3(2): 71–7. 2. thomas r, ferguson j, coombs g, gibson pg. community-acquired methicillin-resistant staphylococcus aureus pneumonia: a clinical audit. asian pacific soc respirol. 2011; 16: 926–31. 3. bukharie ha. a review of community-acquired methicillin-resistant staphylococcus aureus for primary care physicians. j fam community med. 2010; 17(3): 117–20. 4. mahmudah r, soleha tu, ekowati c. identifikasi methicillin resistant staphylococcus aureus (mrsa) pada tenaga medis dan paramedis di ruang intesive care unit (icu) dan ruang perawatan bedah rumah sakit umum daerah abdoel moeloek. med j lampung univ. 2013; 2(4): 70–8. 5. liana p. gambaran kuman methicillin-resistant staphylococcus aureus (mrsa) di laboratorium mikrobiologi departemen patologi klinik rumah sakit dr. cipto mangunkusumo (rscm) periode januaridesember 2010. mks. 2014; 46(3): 171–5. 6. hill-cawthorne ga, hudson lo, fouad m, el a, piepenburg o, et al. recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in staphylococcus aureus. plos one. 2014; 9(6). 7. paterson gk, harrison em, holmes ma. the emergence of mecc methicillin-resistant staphylococcus aureus. trends microbiol. 2014; 22(1): 42–7. 8. ito t, hiramatsu k, oliveira dc, de lencastre h, zhang k, westh h, et al. classification of staphylococcal cassette chromosome mec (sccmec): guidelines for reporting novel sccmec elements. antimicrob agents chemother. 2009; 53(12): 4961–7. 9. kaya h, hasman h, larsen j, stegger m, johannesen b. sccmecfinder, a web-based tool for typing of staphylococcal cassette chromosome mec in staphylococcus aureus using whole-genome sequence data. am soc microbiol. 2018; 3(1): 1–9. 10. nitschke h, pfohl k, monecke s, jatzwauk l, mu e, et al. diversity of scc mec elements in staphylococcus aureus as observed in south eastern germany. plos one. 2016; 11(9): 1–24. 11. yuwono, sunarjati s, masria s, supardi i. staphylococcus aureus dengan polymerase chain reaction identification of staphylococcal cassette chromosome mec methicillin resistant staphylococcus aureus using polymerase chain reaction. maj kedokt bandung. 2009; 43(2): 60–5. 12. sudigdoadi s. analisis tipe staphylococcal cassette chromosome mec (sccmec) isolat methicillin resistant staphylococcus aureus (mrsa). maj kedokt bandung. 2014; 42(4): 149–54. 13. cockerill fr, wikler ma, bush k, craig wa, dudley mn, eliopoulos gm, et al. performance standards for antimicrobial susceptibility testing ; twenty-first informational supplement. vol. 31, clsi document. 2011. m100-s21 p. 14. mcclure-warnier j-a, conly jm, zhang k. multiplex pcr assay for typing of staphylococcal cassette chromosome mec types i to v in methicillin-resistant staphylococcus aureus. j vis exp. 2013; (79). 15. holden mtg, hsu l, kurt k, weinert la, mather ae. a genomic portrait of the emergence, evolution, and global spread of a methicillin-resistant staphylococcus aureus pandemic. cold spring harb lab press. 2013; 23: 653–64. 16. asghar ah. molecular characterization of methicillinresistant staphylococcus aureus isolated from tertiary care hospitals. pak j med sci. 2014; 30(4): 698–702. 17. kinnevey pm, shore ac, brennan gi, sullivan dj. extensive genetic diversity identified among sporadic methicillin-resistant staphylococcus aureus isolates recovered in irish hospitals between 2000 and 2012. antimicrob agents chemother. 2014; 58(4): 1907– 17. copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 82 indonesian journal of tropical and infectious disease, vol. 8 no. 2 may–august 2020: 77–82 18. ghanbari f, saberianpour s, ghanbari n. staphylococcal cassette chromosome mec (scc mec) typing of methicillin-resistant staphylococcus aureus strains isolated from community-and hospital-acquired infections. avicenna j clin microb infec. 2017; 4(2). 19. peters b, liu j, chen d, peters bm, li l, et al. staphylococcal chromosomal cassettes mec (sccmec): a mobile genetic element in methicillin-resistant staphylococcus aureus microbial pathogenesis staphylococcal chromosomal cassettes mec (sccmec): a mobile genetic element in methicillin-resistant sta. microb pathog. 2016; 101 (july 2018): 56–67. 20. ahmad n, ruzan in, kamel m, ghani a, hussin a, nawi s, et al. characteristics of communityand hospitalacquired meticillin-resistant staphylococcus aureus strains carrying scc mec type iv isolated in malaysia. j med microbiol. 2009; 58: 1213–8. 21. ouchenane z, smati f, rolain j, raoult d. molecular characterization of methicillin-resistant staphylococcus aureus isolates in algeria. pathol biol. 2011; 59: e129–32. 22. monecke s, schwarz s, hotzel h, ehricht r. rapid microarray-based identification of diff erent meca alleles in. antimicrob agents chemother. 2012; 56(11): 5547–54. 23. sabir m, dwiyanti r, hatta m, buntaran l, sultan ar. sccmec type ii gene is common among clinical isolates of methicillin-resistant staphylococcus aureus in jakarta, indonesia. bmc res notes. 2013; 6(1): 110. 24. santosaningsih d, santoso s, setijowati n, rasyid ha, budayanti ns, et al. prevalence and characterisation of staphylococcus aureus causing community-acquired skin and soft tissue infections on java and bali, indonesia. tropical medicine and international health. 2018; 23(1): 34–44. 25. hannan a, javed f, saleem s, tahira k, jahan s. frequency of staphylococcal cassette chromosome mec type iv and type v in clinical isolates of methicillin resistant staphylococcus aureus. open j med microbiol. 2015; 5(june): 69–75. copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid vol 3 no 1 jan-maret 2012.indd 53 vol. 3. no. 1 january–march 2012 modern wound dressing for wound infection: an overview novida rizani department of physics science & technology faculty airlangga university, surabaya, indonesia abstract when the tissue of skin is break means a wound is happens. to seal it, many choices of wound healing are available. moist wound dressing can be better optional than the conservative ones. a bioactive agent that being added at the dressing in fact can increase healing rate of wound, moreover can subjugate wound infection caused by the pathogens, and also capable to prevent it. in this review, there are summary of modern moist wound healing, the wound pathogens, and some of sturdy bioactive agent for wound dressing. the treatment of wound infections using impregnated wound dressing by bioactive agent can also by antimicrobial agent that will striven against bacteria colonial. key words: moist wound healing, wound infection, wound pathogen, antimicrobial introduction skin and wound the skin serves as the body's interactive surface with the environment. as such, it is vulnerable to a variety of wounds, which differ by the type and severity of skin injury.10 a wound is a break in the continuity of a tissue of the body, either internal or external. wounds are classified as open or closed. an open wound is a break in the skin or in a mucous membrane. a closed wound involves underlying tissues without a break in the skin or a mucous membrane. any injury, unless it is very minor, may be harmful not only to the tissues directly involved but also to the functions of the entire body. wounds that threaten life include those that produce cassation of breathing, severe bleeding shock, or damage to the brain, heart, or other vital organ. the local effects of an open or closed wound may include loss of blood, interference with blood supply, destruction of tissues, nerve injury, functional disturbances, and contamination with foreign material. these effects often involve nearby uninjured tissues. even superficial wounds sometimes take a week or more to heal. the healing process includes absorption of blood and serum that have seeped into the area, repair of injured cells, replacement of dead cells with scar tissue, and recovery of the body from functional disturbances, if there were any.11 open wounds are injuries to the body that range from very minor, like a scratch that bleeds very little, to severe, such as complete removal of a body part. open wounds can also range from those that bleed severely but are relatively free from the danger of infection to those that bleed little but have a greater potential for becoming infected. treatment options vary according to the nature of the lesion. the two most serious first aid problems caused by open wounds are a large, rapid loss of blood, which may result in shock, and contamination and infection of exposed body tissue. promotion of wound healing with minimal disfigurement and prevention of infection remain the goals of treatment.10,11 modern moist wound dressing alginates alginates are biodegradable dressings derived from seaweed. these highly absorptive dressings are soft, nonwoven, and nonadhesive, and conform to the shape of the wound. when in contact with drainage, they form a gel. alginates (calcium or calcium/sodium) have an active ion literature review 54 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 53−59 exchange of calcium ions for sodium ions at the wound surface forms soluble sodium alginate gel that provides a moist wound environment. calcium dressings need moisture/exudate from the wound to function, therefore they are not suitable for dry wounds or wounds with hardened eschar. alginates are most useful for wounds with heavy exudate. don't use them for dry or eschar-covered wounds, because they won't form a gel and may stick to the wound, causing tissue trauma when they are removed. the fibrous nature of most alginates can leave residual fibres in the wound if there is insufficient wound exudate to gel the fibres. this may precipitate an inflammatory reaction as it stimulates a foreign body response. caution is also needed when using alginate rope dressings in very deep or narrow sinuses, as complete removal can be difficult. studies have shown that some calcium alginate dressings promote haemostasis in bleeding wounds due to the active release of calcium ions that aid the clotting mechanism. some alginates assist with debridement of nonviable tissue, and some contain silver, whose broad-spectrum antimicrobial activity minimizes bio-burden—the number of bacteria present on the product. if a patient has a silver dressing, it may need to be removed before magnetic resonance imaging (mri). alginates come in sheets that can be cut to size. they also come in rope or ribbon form, which is especially good for areas of undermining or tunneling. for large wounds, though, alginates can be an expensive choice. when using an alginate, it is necessary to cover the wound with a secondary dressing to hold the product in place and to protect the wound from outside contaminants. leave the alginate in place for one to three days, until it begins to gel and shows evidence of breakthrough drainage. the way alginates absorb exudate is dependant on the make up of the alginate, for example some retain their integrity and can be removed in one piece; others disintegrate and need to be irrigated away from the wound bed. before reapplying the alginate, the wound must be irrigate thoroughly with sterile normal saline solution. foams foam dressings are made from polyurethane, which may in some cases have been heat-treated on one side to create a semi-permeable membrane. this allows the passage of exudate through the non-adherent, semi-permeable surface into the insulating foam. this type of dressing is nonadhesive and comes in various sizes, shapes, and degrees of thickness. foams are available in sheets or cavity filling shapes. most foam dressings are available in bordered or non-bordered formats, which latter to be used if the patient has a skin sensitivity to adhesives. several advantages of foams are highly absorbent, cushioning and protective, insulate and conform well to body surfaces, provide thermal insulation. they facilitate a moist wound environment and absorb excess exudate to decrease the risk of maceration. some lock fluid within the core of the dressing, others transform into a gelling foam. foam dressings are also available with charcoal impregnation for malodorous wounds. foams may be used as a primary or secondary dressing, to promote autolytic debridement, and to inhibit hypergranulation. when using a foam dressing, make sure it's one to two inches larger than the wound. depending on the level of exudate, foams can be left in place for up to seven days. they indicate when they need to be changed through the spreading discolouration that appears on the dressing. foam wound cavity dressings reduce dead space in the wound, conform to wound shape and absorb large amounts of exudate, therefore reducing the need for frequent dressing changes. cavity foam dressings require secondary dressings or tape/bandage to keep in place. care is needed when adhesives are used to fix dressings in the elderly, as their skin is often fragile and prone to breakdown. tubular retention bandages to fix dressing in place are a safer option in the elderly. hydrocolloids hydrocolloids are moisture-retentive dressings, which contain gel-forming agents such as sodium carboxymethylcellulose and gelatin. although the name begins with the word hydro, these dressings do not contain moisture, but instead form a 'seal' at the wound surface. this prevents the normal daily evaporation of moisture from the skin. many dressings combine the gel-forming properties with elastomers and adhesives which are applied to a carrier such as foam or film to form an absorbent, selfadhesive, waterproof wafer. hydrocolloid dressings contain hydrophilic colloidal particles in an adhesive compound laminated to a flexible wafer. in the presence of wound exudate, hydrocolloids absorb liquid and form a gel, the properties of which are determined by the nature of the formulation. in sheet form the polymer outer layer can be either semi-occlusive or occlusive. like foams, they come in numerous sizes, shapes, and levels of thickness. some have tapered edges that are less likely to curl up. hydrocolloids have minimal absorptive capabilities. they help keep the wound moist and promote autolysis of necrotic areas. don't use them on wounds that are infected or have heavy exudate. to avoid damaging fragile skin by removing the dressings too frequently, keep them in place for as long as possible, but no longer than seven days. hydrocolloid interaction debrides by autolysis and can reduce dressing frequency to up to seven days wear time depending on the amount of exudate and the type of hydrocolloid dressing. hydrocolloids are also available in paste and powders for increased absorption and to decrease dead space in the wound cavity. warming the hydrocolloid dressing prior to application (while still in the packet) will make application easier and the dressing more conformable. hydrocolloid dressings may also be cut to size to help conformability. the dressing should exceed the size of the wound by at least 2 cm. the wound itself will indicate when a dressing change is required by the accumulation of moisture within the dressing. when 55rizani: modern wound dressing for wound infection removing a hydrocolloid dressing, support the surrounding skin. if the dressing is stubborn then submerge the limb in warm water in a bath or shower. generally, hydrocolloids with a waterproof backing are not recommended on clinically infected wounds due to the semi-occlusive nature of the dressing. there have been reports of hypergranulation with prolonged use of hydrocolloids in moderate to highly exudating wounds so wound tissue assessment is paramount when applying hydrocolloids for long periods. hydrocolloids should be discontinued before hypergranulation occurs. hydrogels hydrogels are insoluble hydrophilic polymers with few absorptive properties, that expand in water. they are available in sheet, amorphous gel, sheet hydrogelimpregnated dressings, or gauze impregnated with various percentages of water. however, dressings generally contain between 60–70% water, which, with other constituents, is held in a viscous form known as the hydrogel. alternatively, there are also hydrogel sheet dressings available, which contain less water. hidrogels designed to hydrate wounds, rehydrate eschar and aid in autolytic debridement. they add moisture to the wound bed and are nonadherent, provide a moist environment for cell migration and absorb some exudate. they're used mainly for dry and minimally exudative wounds, such as wounds containing necrotic or dead tissue. dead tissue becomes hard and desiccated due to the loss of a blood supply and the application of a hydrogel dressing donates water to the dead tissue, softening it and aiding the body's process of autolytic debridement. hydrogels have marked cooling and soothing effect on the skin, which is valuable in burns and painful wounds. if stored in the refrigerator, they can provide cool relief to painful wound sites. autolytic debridement without harm to granulation or epithelial cells is another advantage of hydrogel dressings. any hydrogel dressing should be large enough to cover the wound and at least 3cm of surrounding skin. it is important not to apply excessive amounts of hydrogel as this may cause skin maceration. the viscosity varies between dressings. the thick gels available which helps them stay in the cavity of the wound, while the thin gels allowing easy spread over a large area. some amorphous gels contain propylene glycol that can cause allergic reactions in elderly skin. amorphous hydrogels are applied liberally onto or into a wound and covered with a secondary dressing such as foam or film. mostly hydrogel dressing require a secondary dressing to hold it close against the wound bed, either a film dressing or a hydrocolloid dressing can be used for this purpose. if the patient is known to have skin sensitivities, then a hydrogel sheet should be used in place of a hydrogel dressing and this should be covered with padding and a bandage. hydrogel sheet dressings can also be used in preference to hydrogel dressings if the patient has localised pain and cannot tolerate an adherent dressing. in this case, the hydrogel sheet can be held in situ with padding and bandaging or a film dressing. hydrogel dressings may require changing every 2–3 days and care must be taken not to macerate the surrounding skin with excessive amounts of hydrogel. but some of them can remain in situ for up to three days. the patient/carers should be cautioned that on removal the dressing may have changed in colour/consistency as a result of it removing debris from the wound bed. for easy removal of hydrogels the wound is irrigated. in addition to their use in wounds the thin hydrogels are helpful in the management of lesions such as chicken pox and shingles.2,4,8 hydrofibre dressings h y d r o f i b r e d r e s s i n g s a r e n o n w o v e n s o d i u m carboxymethyl cellulose spun into white fibres and manufactured into sheet or ribbon packing dressings, which is applied dry. aquacel, a hydrofibre dressing, maintains a moist wound healing environment as fibres convert to form a gel on contact with exudate. the vertical wicking of exudate reduces maceration of surrounding skin. patients may occasionally mention a 'drawing' sensation as the dressing absorbs the exudate. it is used on moderate to heavily exuding wounds and must be changed when fully saturated with exudate. the dressings are claimed to be more absorbent than alginates and to promote nontraumatic dressing removal. however, these dressings may occasionally stick to the edges of a wound so it is advisable not to overlap onto the surrounding skin.2,8 hydroactive dressings these multilayered highly absorbent polymer dressings, some with a surface adhesive and a waterproof outer layer, are similar to hydrocolloids. however, instead of forming a gel in contact with exudate, the fluid is trapped within the dressing, to maintain a moist environment. semi-permeable film dressings film dressings are adhesive, thin transparent polyurethane, which are permeable to gas but impermeable to liquid and bacteria. films are elastic, conformable and transparent allowing inspection of the wound. as films are non-absorbent they are not suitable for exudating wounds although island dressings with a central nonstick pad are available and can absorb slightly more exudate that the simple films. films can also be used as secondary dressings to waterproof a primary dressing such as foam. incorrect removal of film dressings may cause trauma to surrounding skin.2 wound infection a wound infection happens when germs enter break the skin. this germs, called bacteria, attach to tissue causing wound to stop healing, and other signs and symptoms. wounds can be punctures (holes), lacerations (tears), 56 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 53−59 incisions (cuts), abrasions (scrape), or burn. deep uclers (open sores), large burns, or bite wounds are more likely than other wounds to get infected. wound infection can also happen in small wounds that were not treated.12 terminology it is important to have a clear understanding of the terms used for wound infection. since 1985 the most commonly used terms have included wound contamination, wound colonisation, wound infection and, more recently, critical colonisation. these terms can be defined as: wound contamination the presence of bacteria within a wound without any host reaction. wound colonisation the presence of bacteria within the wound which do multiply or initiate a host reaction. critical colonisation multiplication of bacteria causing a delay in wound healing, usually associated with an exacerbation of pain not previously reported but still with no overt host reaction. wound infection the deposition and multiplication of bacteria in tissue with an associated host reaction. in practice it seems that some experienced tissue viability nurses and medical practitioners use the term 'critical colonisation' to describe wounds that are considered to be moving from colonisation to local infection. the challenge within the clinical setting, however, is to ensure that the majority of practitioners recognise this situation with confidence and for the bacterial bioburden to be reduced as soon as possible, perhaps through the use of topical antimicrobials.7 signs and symptoms the signs and symptoms of a wound infection are any of the following: (12) high or low body temperature, low body prssure, or a fast heart beat. increased discharge (blood or other fluid) or pus coming out of the wound. the discharge or pus may have an odd color or a bad smell. increased swelling that goes past the wound area and does not go away after five days. swollen areas usually look red, feel painful, and feel warm when it is touched. wounds that do not heal or get better with treatment. an old wound that bleeds easily. a wound that is painful, even though it does not look like it should be. wound pathogens when a large number of bacteria get into wound, it can get infected. there are different types of bacteria. more than one type may infect wound at the same time. normal bacteria that lives on skin often enter a wound first. a break in the skin gives them a chance to enter it and cause infection. bacteria may also come from the invironment, such as soil, air, or water. if an object such as nail cause the wound, bacteria may come from that. a bit wound by an animal or person can cause infection from their saliva (spit). the majority of micro-organisms are less than 0.1mm in diameter and can therefore only be seen under a microscope. they can be categorised into different groups, such as bacteria, fungi, protozoa and viruses, depending on their structure and metabolic capabilities.7,12 bacteria these are relatively simple cells that can be further categorised according to differences in their shape and cell wall. cocci (spherical shaped cells), bacilli (rods) and sprirochaetes (spirals) can be arranged singly; however cocci and bacilli can also be found in pairs, chains and irregular clusters. they can be visualised using a bacteriological staining process called gram staining; after gram staining, gram-positive bacteria are purple and gram-negative bacteria are red. species that fail to stain with the gram reaction, such as clostridia, require specialised stains. the growth and survival of all bacteria is dependent upon environmental factors, for example strict aerobes require oxygen whereas anaerobes are rapidly killed by oxygen. it is important to note, however, that both aerobes and anaerobes can survive in close proximity to each other and that some can survive in both conditions by growing aerobically and then switching to anaerobic metabolism in the absence of oxygen; these are known as facultative anaerobes. fungi these are composed of larger more complex cells than bacteria. they are either single-celled yeasts or multi-cellular organisms with a nuclei contained within a cell membrane. fungi can be responsible for superficial infections of the skin, nails and hair and, although they have been isolated from wounds, they are rarely pathogenic in this setting. protozoa these are single celled organisms within a fragile membrane and without a cell wall. they are most significantly associated with infected skin ulcers. viruses these are composed of genetic material (nucleic acid) enclosed within a protein coat or a membranous envelope. although viruses do not generally cause wound infections, bacteria can infect skin lesions formed during the course of certain viral diseases. it is important to remember that different microorganisms can exist in polymicrobial communities and this is often the case within the margins of a wound.7 57rizani: modern wound dressing for wound infection treatment once a diagnosis of wound infection has been confirmed and antibiotic sensitivities identified, appropriate management regimens should be considered, with a high priority given to reducing the risk of cross infection. it is important to treat the patient as a whole and not the infection alone, so management strategies must be based on data derived from an holistic assessment of the needs of the individual. the main treatment objective will be to reduce rather than eradicate the bacterial burden within the wound margins. in addition to antibiotic therapy, there are two main generic groups of wound management products that have the potential to reduce the bacterial burden in the wound, these are compounds containing silver or iodine.7 iodine iodine is an element that has antiseptic properties. it is active against a number of pathogens, so that iodine dressings can control bacteria on the surface of the wound. iodine dressings have a maximum dosage that may be used at any one time and a maximum length of time over which they can be used. the dressing changes colour from deep yellow to white as the iodine is used, clearly showing when the antimicrobial activity is exhausted. in wound management iodine is used in two forms, they are cadexomer iodine a polysaccharide starch lattice containing 0.9% elemental iodine that is released on exposure to wound exudate, and pvp-1 (povidone iodine) an iodophor composed of elemental iodine and a synthetic polymer. both have different physical characteristics that relate to the component parts and the iodine concentration of available iodine that is released when in use. clinically iodine is indicated for wound cleansing, wound bed preparation (the stimulation and influence of specific cells involved with the immune system) and the prevention and management of wound infection. in the past, iodine use has been limited by the fact that elemental it can be absorbed systemically, is almost insoluble and can be an irritant to the skin. contraindications include patients with thyroid disease, patients with large or deep wounds, and patients with a known allergy to iodine. iodine products can cause thyroid disruption. if patients have a history of a thyroid disorder, their thyroid function should be checked before and while using the product. silver recently a number of dressings containing silver have become available, although silver and silver compounds have been routinely used in clinical practice as bactericidals for over a century. silver have a broad spectrum antimicrobial, effective against a range of aerobic, anaerobic, gram positive and gram negative bacteria as well as filamentous fungi and viruses; pseudomonas aeruginosa and staphylococcus aureus are some of microorganisms that silver effectively can handle. silver interferes with the bacterial electron transport system and inhibits the multiplication of the bacteria. however, to achieve this, silver ions have to be able to enter a cell. the chemical bonding of silver with a sulphonamide antimicrobial sulphadiazine has resulted in the development of a safe broad-spectrum agent for topical use (eg flamazine). in this formulation silver is released slowly from the transport medium in concentrations that are selectively toxic to micro-organisms such as bacteria and fungi. this type of silver product has been used successfully in the management of acute and chronic wounds. products that can sustain the interaction of silver with micro-organisms in the exuding wound are likely to be more effective in preventing/controlling local infection as potentially more silver ions will be available to enter bacterial cells. this assumes that the concentration of silver in the solution is both correct and maintained. another advatage of silver is that no resistant strains have been discovered. despite the absence of resistance in clinical practice, it has been possible to produce resistance in the laboratory setting by using sub-therapeutic levels of silver. there is controversy about the optimal amount of silver required to achieve a balance between efficacy, toxicity and the potential for resistance. when a silver dressing is selected, the amount of available silver should be considered as well as whether the silver is released from the dressing or the bacteria is drawn into the dressing. the importance of choosing a product with a clinically relevant dose of available silver and recommends that treatment stops once the objective for selecting that dressing has been achieved. silver products may cause localised discolouration if they come into contact with the skin surrounding a wound; this should wash/wear off within a few days. a rare but more serious complication of using silver products is argyria, which occurs when cells absorb silver salts; this results in a permanent discolouration of the skin (called argyria). if the silver is absorbed by internal organs it may impair their function.1,3,7 honey most research carried out on honey has focused on the role of manuka honey, which, like most honeys, releases hydrogen peroxide but is also believed to have an additional antimicrobial agent known as the unique manuka factor (umf). it is widely claimed that honey is able to deodorise and debride wounds and these additional properties may be particularly beneficial in infected wounds. there are no standardised protocols for the frequency with which honey should be applied and the type of secondary dressing that should be used.3 antimicrobial therapy antimicrobial are chemical substances that have the capacity, in dilute solutions, to selectively inhibit the growth of or to kill other micro-organisms. antimicrobial agents can be as synthetic compunds or natural compounds. many research has proved that plants can provide strongly 58 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 53−59 recomended agents as antimicrobial which also have high activity to bear micro-organism. antibiotics are a kind of antimicrobial that produced by a micro-organism. whereas it is now generally accepted that systemic antibiotics are essential for the management of clinically infected wounds, the choice of antibiotic to be used is not always apparent. only after a comprehensive assessment process including consideration of patient characteristics, the results of microbiological investigations and the identification of both the nature and location of the wound, can the most appropriate antibiotic be identified. the routine use of topical antibiotics is not justified for colonised or infected wounds. unfortunately, the resistance to antibiotics has become a serious problem in recent years particularly with the rise of epidemic strains of mrsa (methicillin-resistant staphylococcus aureus). the overuse of broad-spectrum antibiotics will only serve to exacerbate the situation. it could therefore be argued that all antibiotic use should be based on known sensitivities. that is why, many other antimicrobial agents are still been hunted until this seconds, whether they are from synthetic precursors or from isolated substances of natural extract. in addition, a recent systematic review of antimicrobial agents has concluded that systemic or topical antimicrobials are not generally indicated for the management of chronic wound infections. however, there may be some value in the prophylactic use of topical antimicrobials for the initial management of acute cellulitus, whilst awaiting clarification of antibiotic sensitivity and the establishment of a therapeutic regimen.7 antimicrobial agents from plants a wide source of bioactivity compounds is plants. finding healing powers in plants is an ancient idea. clinical microbiologists have two reasons to be interested in the topic of antimicrobial plant extracts. first, it is very likely that these phytochemicals will find their way into the arsenal of antimicrobial drugs prescribed by physicians. second, the public is becoming increasingly aware of problems with the overprescription and misuse of traditional antibiotics.6 garcinia mangostana (mangosteen) is one of plant that have a precious substances, called mangostin, since they are known for having a very high activity as antioxidant and antimicrobial. mangosteen it self is a small purple fruit about the size of a tangerine, which has traditionally been used to treat inflammation, dysentery, and skin disorders. it was also used to fight infection by boiling up the fleshy part of the rind, also know as the pericarp, into a tea. as early as 1855, a german scientist identified mangostin, a powerful antioxidant, known as a xanthone, which appeared to fight infection, fungus, bacteria, histamines and possibly cancer. xanthones can be extracted from the root bark, stem bark and sap as well as rind [exocarp]of the mangosteen fruit.5 among more than 40 mangosteen xanthones found in the whole mangosteen fruit, alpha-mangosteen and gamma-mangostin have been the main subject of many studies by mangosteen researchers around the world. scientists demonstrated that gamma-mangostin proved to be a stronger antioxidant than other compounds long known for their antioxidant properties. medical researchers in thailand and taiwan discovered that gamma-mangostin was shown to have even more potent antioxidant activity than vitamin e, one of the most powerful antioxidants known to science. a l p h a m a n g o s t i n w a s p r o v e n t o h a v e s t r o n g antibacterial activity against staphylococcus aureus. scientists demonstrated alpha-mangostin's activity against vancomycin-resistant enterococci (vre) and methicillinresistant staphylococcus aureus. they found that alphamangostin, alone or in combination with gentamicin, against vancomycin-resistant enterococci (vre) and used in combination with vancomycin hydrochloride against methicillin-resistant staphylococcus aureus (mrsa) might be useful in controlling vre and mrsa infections. further studies shown that alpha-mangostin works well with and enhances the effects of other commercially available antibiotics such as ampicillin and minocycline. in another laboratory experiment, scientists studied the antimicrobial activity of alpha-mangostin and found that it possesses strong inhibitory effects against mycobacterium tuberculosis and pseudomonas aeruginosa. researchers also found that alpha-mangostin is a histaminergic receptorblocking agent: alpha-mangostin is effective in preventing or stopping allergies.9 i m p r e g n a t e d w o u n d d r e s s i n g b y antimicrobial antimicrobial dressings can be used on acute or chronic wounds which are critically colonised, or when local and/or systemic infection is already compromising the wound or could compromise wound healing. when choosing an appropriate wound dressing it is vital to assess whether the wound is colonised, critically colonised or infected. the presence of microorganisms in a wound does not indicate that wound infection is inevitable; indeed, they may have a protective effect. the presence of multiplying bacteria in a wound with no host reaction is termed colonisation. critical colonisation has been defined as the multiplication of bacteria causing a delay in wound healing, usually associated with an exacerbation of pain not previously reported, but still with no overt host reaction. signs of critical colonisation include: a continued delay in healing despite appropriate treatment, thick slough that does not respond to standard debridement techniques, fast returning slough after sharp or larval debridement, and malodour. levels of bacteria in critically colonised wounds need to be reduced to allow the wound to heal. the topical 59rizani: modern wound dressing for wound infection application of an antimicrobial is probably the most effective way to do this. the development of an infection will be influenced largely by the virulence of the organism and the immune status of the patient. patients considered most at risk are those being treated with long-term steroids and those receiving chemotherapy. if a wound appears to be infected, it is important to confirm this and identify the causative organism(s) and possible sensitivities to antimicrobial, as antibiotic. a wound swab should be sent for microbiology, culture and sensitivity. the use of systemic antimicrobial is supported where there are clear signs of infection. this is best achieved by starting a topical antiseptic dressing as soon as the signs of critical colonisation are detected.1 conclusions many of kind modern moist wound dressings can facilitate wound treatment to be more effectively and comfortable. healing rate also can be faster than using an conservatism dressings. however, threat of wound infection can exacerbate wound conditions and delaying healing process. wound infection happens because of the presence of pathogens, such as bacterial, fungi, protozoa, and viruses. some eforts that's been taken to heal and prevent these are by using additional stuff in dressing, like kind of iodine, silver, honey, or many other antimicrobial agents. unfortunately, mrsa case shows that antimicrobial from kind of antibiotics has decending activity against some micro-organism which already having high resistance toward those agents. luckly, nature has always provide wide of plenty antimicrobial agent source that can be taken just by extracting and isolating from plants. mangosteen is one of plants that have a high potential as antimicrobial agent source since they have mangostin which have a high level of antimicrobial activity, as well as antioxidant. alpha-mangostin in fact has been reported that can wipe off apprehensive about mrsa infections. impregnation antimicrobial agent in moist mound dressing can increase healing rate and prevent wound infection, by battling microbial colonisation. references 1. ashley flores, (2007), "topical antimicrobal derrsings: an overview", wound essentials • volume 2. 2. carolina weller, geoff sussman, (2006), "wound dressing update", journal of pharmacy practice and research, volume 36, no. 4. 3. jacqui fletcher, (2006), "best practice choosing an appropriate antibacterial dressing", vol: 102, issue: 44, page no: 46. 4. linda snyder, rn., (2008), "ce: wound basics: types, treatment, and care", http://www.modernmedicine.com/modernmedicine/ ce+library/ce-wound-basics-types-treatment-and-care/ articlestandard/article/detail/535258 5. "mangosteen xanthones: alpha-mangostin and gamma-mangostin", http://naturalhomecures.net/mangosteen/science/m-to-z/alphamangostin-and-gamma-mangostin 6. marjorie murphy cowan, (1999), "plant products as antimicrobial agents", clin. microbiol. rev., 12(4): 564 7. mark collier, (2004), "recognition and management of wound infections", http://www.worldwidewounds.com/2004/january/collier/ management-of-wound-infections.html 8. pauline beldon, (2010), "how to choose the appropiate dressing for each wound type", wound essentials, volume 5. 9. "the benefits of mangosteen", http://www.natural-plus-healthy. com/benefits-of-mangosteen.html 10. tina andrews, 2011, http://www.livestrong.com/article/130677different-kinds-open-wounds/ 11. "types & causes of open wounds", http://nursingcrib.com/nursingnotes-reviewer/types-causes-of-open-wounds/ 12. "wound infection", http://www.drugs.com/cg/wound-infection. html 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid vol 3 no 2 april juni 2012.indd 65 vol. 3. no. 2 april–juni 2012 ethnic and atopic dermatitis: what have we learned in asian population? cita rosita sigit prakoeswa dermato-venereology department medical faculty universitas airlangga / dr soetomo hospital surabaya indonesia abstract atopic dermatitis (ad) is a chronic inflammatory skin disease, with relapsing remitting course. management of ad is challenging due to the complexities of this disease. two hypotheses concerning the mechanism of ad have been proposed. one holds that the primary defect resides in an immunologic disturbance that causes ig e-mediated sensitization, with epithelial barrier dysfunction regarded as a consequence of the local inflammation. the others propose that an intrinsic defect in the epithelial cells leads to the barrier dysfunction; the immunologic aspects is considered to be an epiphenomenon. many studies support that ad is a complex trait which has interactions between genes and environmental factors contributing to disease manifestation, but the result of replicate association between genetic markers and ad is inconsistence. an important factor contributing to this inconsistency is related to population diversity. it is possible that certain genetic markers might contribute to increase the risk in certain ethnic population but not in others, either because of the differences in frequencies of the risk alleles and the specific genes interaction. there is limited information about the role of ethnicity in asian population. the overall purpose of this review is to present an update on ethnicity approach of ad in asian population. research on prevalence, risk factor, innate and adaptive immune response genes, skin barrier dysfunction genes and geneenvironment interaction such as epigenetic, is discussed. it is generally approved that the ethnicity of study subject is a key factor in interpreting genetic polymorphism studies. therefore, discussion of some current areas of research about polymorphism are presented, including filaggrin (flg) gene and cd14 c-159tsnp. addressing the issues described above may improve our understanding of ad pathogenesis that has implications for the clinical management of ad. keywords: atopic dermatitis, ethnic, asian, immune system, skin barrier, gene-environment interaction abstrak latar belakang: dermatitis atopik (da) merupakan penyakit keradangan kulit yang bersifat kronis dengan periode remisi dan eksaserbasi. kompleksitas dari penyakit tersebut menjadikan tantangan bagi dokter spesialis kulit dan kelamin dalam melakukan penatalaksanaan. patogenesis da didasari oleh dua hipotesis. pertama, gangguan imunologi sebagai penyebab primer, menyebabkan sensitisasi yang diperantarai oleh ig e, selanjutnya terjadi kerusakan barrier kulit sebagai konsekuensi dari keradangan local yang terjadi. kedua, gangguan primer pada sel-sel epitel kulit menimbulkan disfungsi kulit sebagai barrier, sehingga memudahkan terjadi keradangan dan diikuti dengan proses imunologi. berbagai penelitian mendukung kontribusi interaksi antar gen serta lingkungan sebagai factor penting dalam manifestasi klinis da, tetapi asosiasi antara petanda genetic dan da masih inkonsisten. faktor penting yang berkontribusi pada hal tersebut adalah diversitas populasi. petanda genetic tertentu dapat berperan untuk meningkatkan resiko terjadinya da pada populasi etnik tertentu karena adanya perbedaan frekuensi alel dan interaksi spesifik berbagai gen. informasi tentang peran etnisitas pada populasi asia sangat terbatas. tujuan: dari penulisan ini adalah untuk menggambarkan pendekatan terkini tentang etnis pada da di populasi asia. diskusi meliputi penelitian yang telah dilakukan tentang prevalensi, factor resiko, gen yang bertanggung jawab pada respon imun alami maupun adaptif, gen yang bertanggung jawab pada disfungsi barrier kulit serta interaksi antara gen dengan lingkungan seperti epigenetic. secara umum disepakati bahwa etnisitas subyek penelitian merupakan faktor kunci dalam interpretasi penelitian polimorfisme. oleh karena itu, penelitian polimorfisme termasuk gen penyandi filaggrin research report 66 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 65−75 introduction a topic dermatitis (ad) is a common inflammatory skin disease that may affect individuals in any age, race, or ethnicity. it is commonly affected during childhood or infancy with chronic and relapsing course. the etiology of ad is partially understood, genetics and environmental factors are thought to play important roles in the pathogenesis.1 asia is the largest continent in the world and has many different geographic areas. it extends from countries in the sub tropic zones, the far east (including japan, korea, and china) to countries in the tropics, the south and southeast asian countries (including singapore, malaysia, the philippines, indonesia, thailand, vietnam, cambodia, laos, and myanmar) and to countries in the subtropics (including the indian subcontinent and countries in the middle east). most of the asian population has fitzpatrick skin photo type iii, iv, and v, whereas a small proportion has skin photo type ii and vi. wide variety of different ethnicity, culture, hygiene, nutrition, and socioeconomic status also present. the manifestation of dermatologic conditions in one part of asia is commonly different from those seen in another part.2 racial differences in both the expression of ad and genetics are important areas of research for several reasons. first, there might be different phenotypic expression of the disease in a given racial and or ethnicity group (that might or might not be confounded by environmental differences). secondly, the frequency of genetic variation varies between races.3 ethnic (racial) differences in ad have been minimally investigated. the current experimental human model for skin is commonly based upon physical and biochemical properties known about caucasian skin. thus, anatomical or physiological properties in skin of different races may influence a disease process or a treatment of that disease it self. there is limited information about the role of ethnicity in asian population.4 the overall purpose of this review is to present an update on several approaches to understanding the susceptibility and expression (severity) of ad in asian population. four key questions are addressed in this review: (1) what is the difference between race and ethnic? (2) do the biophysical properties of asian skin differ from others? (3) are there any differences between prevalence and risk factor of ad in asians and others? (4) what about the research on innate and adaptive immune response genes, skin barrier dysfunction genes and gene-environment interaction such as epigenetic of ad based on ethnicity in asians? dan cd 14 c-159t juga dibahas pada tulisan ini. pemahaman tentang pathogenesis da diharapkan dapat meningkatkan kualitas penatalaksanaan klinis da. kata kunci: dermatitis atopik, etnik, asia, sistemimun, barrier kulit, interaksi gen lingkungan. question 1: what is the difference between race and ethnicity? definitions of ethnicity and race based on new oxford american dictionary are:5 ethnicity is the population subgroup (within a larger dominant national or cultural group) with a common national or cultural tradition. race is each of the major divisions of human-kind, having distinct physical characteristic. race also means a group of people with common features. question 2: do the biophysical properties of asian skin differ from others? racial variability should be considered in terms of different skin responses to topical and environmental agents. race provides a useful implement to investigate and compare the effects of lifetime sun exposure and ambient relative humidity. evolution provided over 100,000 years of genetic advantage to survive for those races living in a specific area with specific climatic conditions. to survive in harmful environment requires an optimal adaptation of outermost layers of our body, the skin relates to structural, biochemical, and molecular level.4 stratum corneum (sc) is equally thick in different races. however, weigand demonstrated that the sc in blacks contained more cell layers and required more cellophane tape strips to be removed than the sc of caucasians, while kampaore and tsuruta showed that asian skin was significantly more sensitive tostrip than black skin. no correlation was found between the degree of pigmentation and the number of cell layers. these data clarified that the greater intercellular cohesion in blacks, consequences in an increased number of cell layers and an increased resistance to stripping. this mechanism may involve lipids, because the lipid content of the sc ranges from 8.5% to 14%, with higher values in blacks.4 corcuff investigated the corneocyte surface area and the spontaneous desquamation and found no differences between black, white, and oriental skin. however, an increased desquamation (up to 2.5 fold) was found in blacks. they concluded that the differences might be related to a different composition of the intercellular lipids of the sc.6 sugino found significant differences in the amount of ceramides in the sc, with the lowest levels in blacks followed by caucasians, hispanics, and asians. in this experiment, ceramide levels were inversely correlated with transepidermal water loss (tewl) and directly correlated with water content.7 meguro confirmed these correlations.8 these data may partially explain the controversial findings in the literature on the mechanisms of skin sensitivity. changes in skin permeability and barrier 67prakoeswa: ethnic and atopic dermatitis function have been reported. kompaore evaluated tewl and lag time after application of a vasoactive compound (methyl nicotinate) before and after removal of the sc by tape stripping. before tape stripping, tewl was 1.3 times greater in blacks and asians compared to caucasians. no difference was found between blacks and asians, whereas after stripping they found a significantly higher tewl in blacks and asians than in whites. in particular, after stripping asians showed the highest tewl (asians 1.7 times greater than caucasians).9,10 they concluded a resemblance with previous studies,11,12 which concluded that skin permeability measured by tewl, was higher in blacks than in caucasians. they also concluded that asian skin had the highest permeability among the study groups. however, these findings have not yet been confirmed by other groups. infact, sugino, that also included asians in their study found that the baseline tewl decreased, blacks were greater than caucasians greater than or equal to hispanics and greater than or equal to asians.7 another study about asian skin, has compared the tewl between asians and caucasians and found no statistically significant difference at baseline or after tape stripping without vasoactive substance applied.13 reed found differences in the recovery of the barrier between subjects with skin type ii/iii compared to skin type v/vi, and no differences between general caucasians and asians.14 darker skin recovered faster after the barrier damage was induced by tape stripping.4 racial differences in skin conductance are difficult to interpret in terms of sc water content, because other physical factors, such as the skin surface or the presence of hair, can modify the quality of the skin electrode contact. volar and dorsal forearms present a significant differences between all races.15 these results are apparently contrast with tewl recordings. indeed, increased sc water content, correlates with a higher tewl.16 these data may be explained on the basis of the differences of intercellular cohesion or lipid composition. the increasing of skin water content could possibly occur due to the greater cell cohesion with a normal tewl.4 there are reasonable evidences that exist to support that black skin has a higher tewl compared to white skin by means of objective measurements. although some deductions have been made about asian skin, the results are contradictive, and further evaluation of asian skin needs to be performed. perhaps, more specificity about the origin of their heritage should also be included because “asian” encompasses a broad spectrum of people.4 question 3: are there any differences between prevalences and risk factors of ad in asian and others? there are still limited studies available pertaining the epidemiologic data of ad in asian populations. however, several population studies have demonstrated considerable geographic and racial/ethnic variations in the prevalence of ad.18,19,20 based on partially understood environmental factors, ad appears to be more common in industrialized nations and urban settings than in developing countries and rural communities.21 survey study about population in northern europe, the united states, and japan has reported prevalence rates of 15.6%, 17.2%, and 21%, respectively, whereas a prevalence of 8.5% was reported in a recent study from south eastern nigeria.22,23,24,25 however, with expanded urbanization and adoption of western lifestyle, the prevalence of ad appears to be rising in developing countries, as in more industrialized nations.25 the international study of asthma and allergies in childhood (isaac) steering committee evaluated the frequency of ad among 463,801 children aged 13–14 years old from 56 countries.18 in 1999, high prevalence of ad was reported in several regions where non-caucasian individuals predominated, the centers of africa (nigeria, kenya), asia (japan, pakistan), and of south america (paraguay, chile). several epidemiologic studies have shown increasing prevalence of ad among blacks and asian/pacific islanders when compared with caucasians. recently, a 12-month observational prospective study of 182 babies (62 caucasian, 61 chinese, and 59 vietnamese infants) born in melbourne, australia, indicated that the incidence rate of ad was varied by ethnicity.20 in those populations, ad prevalences of the caucasian, chinese, and vietnamese infants were 21%, 44%, and 17% respectively. because the caucasian and the chinese infants were in similar socioeconomical backgrounds, genetic differences likely played a role in the different incidence rates. in contrast, since the vietnamese infants had a lower socioeconomic background, but were skill in the same racial group as the chinese infants, environmental factors likely contributed more than genetic factors to these incidence differences.20 ad was found to be more prevalent among chinese infants born in san francisco and honolulu than among the local caucasian population.26 similar findings were presented in london-born black caribbean children compared with their white counterparts. among londonborn black caribbean children, the prevalence of ad was 16.3% compared with 8.7% in white children.19 however, a study of indian and caucasian preschool children in leicester, united kingdom, failed to show any ethnic differences in the prevalence of ad.27 the reasons for the observed differences in prevalence may be based at least in part, on variations of genetic and environmental factors. however, differences of research methodology between epidemiologic studies must be considered when comparing prevalence rates among populations. further research on the epidemiology of ad among asians is warranted.17 a study about prevalence of ad by ethnic group was conducted by baker in suburban area of san diego. ethnic the data were available for 5912 patients in the study population. ethnicity was designated as white (not of hispanic origin), hispanic, black, filipino, other asian, or mixed race. the prevalence of ad was determined to be 3.2% in the overall study population, 3.7% of which were black, 8.5% were filipino, 2.0% were hispanic, 2.8% were 68 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 65−75 white (not of hispanic origin), 3.2% were mixedrace, and 5.6% were other asian origins.28 because this study was conducted by only one physician in a limited geographic area, it couldn't be assumed that the population study typically represents the overall filipino population in the united states or in the philippines. the actual prevalence of ad probably varies considerably by geographic area. moreover, the population is not ethnically representative of the overall us population. in particular, although he observed a higher prevalence of ad among patients of “other asian” origin, this group included too many subsets for separate analysis (ie, japanese, korean, chinese, laotian, vietnamese, cambodian, and other ethnic groups were included).28 a prospective study was undertaken to investigate several perinatal predictors of ad occurring in the first 6 months of life which were reported from 1005 mothers and their infants who participate in a us cohort study of pregnant women and their offspring. the main outcome measurement was maternal report of an ad diagnosis in the first 6 months of life and analyzed using multiple logistic regression models to assess the association between several simultaneous predictors and incidence of ad. the cumulative incidence of ad in the first 6 months of life was 17.1%. the adjusted odds ratio (or) for risk of ad among infants born from black mothers was 2.41 (95% confidence interval [ci]: 1.47, 3.94), compared with infants born from white mothers, and was 2.58 (95% ci: 1.27, 5.24), compared with infants born from asian mothers. male infants had an or of 1.76 (95% ci: 1.24, 2.51). increased gestational age at birth was a predictor (or: 1.14; 95% ci: 1.02, 1.27, for each 1-week increment), but birth weight for gestational age was not. infants born by mothers with a history of ad had an or of 2.67 (95% ci: 1.74, 4.10); paternal history of ad was also predicted, although maternal atopic history was more predictable than paternal history. several other figure 1. summary of genome-wide linkage studies of ad: representation of the 23 human chromosomes, highlighting those loci for which genome screens have identified linkage to ad. loci are mapped to short orlong chromosomal arms and colorcoded according to the studies listed in the legend.30 69prakoeswa: ethnic and atopic dermatitis perinatal, social, feeding, and environmental variables were not related to the risks of ad. in conclusion, black and asian race/ethnicity, male gender, higher gestational age at birth, and family history of atopy, particularly maternal history of ad, were associated with increased risk of ad in the first 6 months of life. these findings suggest that genetic and pre and perinatal influences the essential in the early manifestation of this condition.29 question 4: what about the research on innate and adaptive immune response genes, skin barrier dysfunction genes and gene-environment interaction such as epigenetic of ad based on ethnicity in asians? strategies for investigating the genetic basis of atopic dermatitis a large amount of research have been undertaken worldwide in the exploration of genetic factors as the etiology of ad. three main approaches have been used: candidate gene association, selecting genes for study based on a hypothesis of known biological function; genome-wide linkage screens, which are hypothesis free and compare the transmission of genetic information between cases and controls in family pedigrees; and dna microarray studies, which look at gene expression in selected regions of interest, or across the whole genome. each of these strategies has been added to the understanding of genetics in ad.29 up to present, there have been 5 genome-wide linkage studies performed on ad, plus a genome-wide linkage screen originally designed for asthma with analyses repeated for the ad outcome (fig 1). all but one of these screens were performed on families of european ancestry: (1) 199 german and scandinavian (2) 148 british (3) 109 swedish (4) 100 danishand (5) 295 frenchfamilies, of which 62 affected sib pairs for ad were available for reanalysis. the non-european study was performed on 77 japanese families selected through 111 sib pairs with ad figure 2. genes associated with ad in at least 1 published study. genes are grouped according to how many positive association studies have been reported. the y-axis indicates the number of genes (corresponding to the yellow boxes) for each time that a positive association was reported.30 70 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 65−75 (287 individuals) and relied on a linkage mapping panel of 5,861 single nucleotide polymorphisms (snps) rather than the microsatellite panel traditionally used for linkage screens. in the japanese families, demonstrated linkage at two locus on chromosome 1 and 15 didn’t show replication with other studies.30 in a search of the public database in june 2009, 111 studies were identified for which results of the tests associated to ad were reported on a candidate gene. the major outcome was limited to ad as a qualitative trait or ad severity. from these 111 published studies, only 42 studies were performed in asian populations. there are reports on 81 genes, of which more than half (46 genes) had at least 1 positive association study reported (fig 2). of these 46 genes, 15 studies failed to replicate associations, and 13 were positively associated in at least 1 other independent study. one of these genes, flg, has been associated with ad in 20 different reports. there are 35 additional genes studied for which there has been no evidence for a positive association to date.30 adaptive and innate immune response genes one approach toward distinguishing potential genes interactions and systematically evaluating the role of candidate genes/polymorphisms in ad susceptibility, for which there is compelling evidence for association, is to implement the program ingenuity pathways analysis (ingenuity systems, inc, redwood city, calif; www. analysis.ingenuity.com). as a proof of the concept, barnes (2010) evaluated the 81 genes summarized in fig 2 using the ingenuity pathway analysis. there were slightly more than half (n = 48) of the 81 genes studied clustered in 2 major networks, both of which were associated with immune dysregulation, specifically the pathway which is associated with antigen presentation and cell-mediated and humoral immune response, the pathway associated with cell signaling and interaction cellular movement. six genes (monocyte differentiation antigen cd14 [cd14], gata-binding protein 3 [gata3], interleukin 4 [il4], il18, nucleotide-binding oligomerization domain 1 [nod1], and toll-like receptor 2 [tlr2]), which had previously been significantly associated with ad, were clustered into the antigen presentation and immune response pathway. meanwhile, 9 previously associated genes (bcl2related protein a1 [bcl2a1], brain-derived neutrophilic factor [bdnf], regulated upon activation,normally texpressed, and presumably secreted [rantes], colony stimulating factor 2 [csf2], glutathione s-transferase p 1 [gstp1], il5, interleukin 12 beta [il12b], interleukin 12 receptor beta 1 [il12rb1], and suppressor of cytokine signaling 3 [socs3]) were clustered into the cell-signaling/ movement pathway. although the studies for which those candidates genes were evaluated did not specifically tested for genes interaction. this investigation potency serves as an example of the power of this approach in selecting optimal candidates for genetic association studies.30 in asian population, there were three genes explored from the antigen presentation and immune response pathway (cd14, il4 and il18) and six genes from the cellsignaling/movement pathway (bdnf, rantes, csf2, il5, il12b, il12rb1), from total of 42 studies. barnes presented significant correlation between il4 and il18 and ad in asian population. and also discovered significant correlation between bdnf, rantes, il5, il12b and il12rb1 in asian population (japanese, chinese and korean, table 1). due to the minimal amount of studies study, these results have a limitation to make a conclusion about the role of snp in there genes of ad from asian population.30 on account of the historical isolation and environmental positive selections, major racial/ethnic groups differ in allele frequencies.31 consequently, ethnic differences may contribute to the variations in allele frequencies of phenotype-associated genes in humans. the cd14 protein, which is the receptor for lipopolysaccharides (lps) and other bacterial wall-derived components, has been suggested to play a critical role in th1 differentiation of naive t cells, with the likelihood of developing a t helper 1 (th1)-type response by up-regulating the expression of il-12, which in turn decreases ige-mediated immunity.32zhang reviewed the cd14c-159t snp in the promoter region of cd14 in asthma population. there are significant variations in allele frequencies in different ethnic groups, summarizing that c allele frequencies on the highest in those of african descent, followed by caucasians and asians. this indicates that there are significant genetic variations between these ethnic groups in terms of predominant alleles and linkage disequilibrium structure of the snp cd14c-159t. it is generally approved that the ethnicity of study subjects is a key factor in interpreting genetic polymorphism studies. in ad, there is an inconsistent result of studies of cd14c159t snp. there is significant association in american and german population, but not in german and chinese population. therefore, we still could not make a conclusion about the role of snp cd14c-159t in ad from asian population.33 skin barrier dysfunction genes it is increasingly appreciated that both genetic and environmental factors that affect skin barrier function contribute to ad susceptibility, as well as barrier dysfunction which is an essential feature of ad. a disrupted barrier would allow penetration of microbes and allergens and other environmental insults, such as toxins, irritants, and pollutants, with consequences such as inflammation, allergen sensitization, and bacterial colonization. although the epidermis functions as the primary defense to the external environment, considerably barrier function is regulated by the sc and by the tight junctions (tj), which reside at the level of the stratum granulosum. when the sc is compromised, either by the reduced levels of sc lipids, mechanical trauma resulting from extensive scratching that 71prakoeswa: ethnic and atopic dermatitis is precipitated by intensive itch (the hallmark of ad), or as a result of genetic defects in sc proteins (ie, flg), tjs are the next line of defense. linkage screens performed on ad to date have not elucidated specific candidate genes per sc, but they have implicated loci harboring clusters of genes associated with skin barrier dysfunction. specifically, one of the earliest screens indicated linkage at the epidermal differentiation complex (edc) locus on chromosome 1q21, which contains a very large and diverse family of genes associated with skin barrier dysfunction, including loricrin, involucrin, members of the s100 gene family, and most notably, flg.34association studies on genes related to the edc cluster and other barrier dysfunction candidates have been restricted to flg, also known as filament-aggregating protein, and within flg, most associations have been limited to 2 null mutations (r501x and 2282del4). in fact, flg is the most consistently associated gene with risk of ad, as shown in fig 2, by mid-2009, there were 20 positive reports on genetic associations between flg mutations and ad.35 the gene encoding human flg was firstly cloned in 1989, when it was found to contain numerous tandem flg repeats localized in chromosome 1q21, and because of its tight regulation at the transcriptional level in terminally differentiating epidermis, it was postulated to be an important candidate for disorders of keratinization.36it was subsequently evaluated for its function in the formation of the sc and found to be a critical protein involved in epidermal differentiation and in maintaining barrier function.37 full sequencing of the flg gene has revealed multiple additional polymorphisms with varying frequency across the ethnic groups. however, with a combined allele frequency among patients with ad of 18% and 48% for the r501x and 2282del4 mutations respectively, the 2 null mutations represent the strongest and most compelling genetic risk factors for ad.38in the largest meta-analysis performed, that on the r501x and 2282del4 mutations, rodriguez analyzed data from 24 independent studies, which included 6,448 cases, 26,787 control subjects, and 1,993 families (all selected forad) and determined that the effect size’s risk effect of eczema caused by the 2 flg null mutations was not dissimilar to previous reports at an or of just over 3.39 other candidate genes showing somewhat comparable or include il-4, or 1.88, p = 0.0140; il-13, or 1.7, p = 0.0341 and relative risk (rr) 2.5, p = 0.01442; and mast cell chymaseor 2.17, p = 0.009.43 brown reviewed about genotype-phenotype correlation between flg and ad, showed the phenotype of early onset (before the age of 2 years), persistent, and severe ad has shown the strongest and the most highly significant statistical association with the combined flg null genotype, having an or of up to 7.7 (95% ci 5.3–10.9).44,45,46 in asian population three studies have been conducted to explore the snp of flgin ad from japanese, all of these studies showed positive association (table 1). further research are required to establish this finding.30 epigenetic epigenetics is the study of mitotically heritable changes in phenotype (alterations in gene expression) that occur without direct alterations of the dna sequence. these epigenetic changes include methylation of dna by the covalent addition of a methyl group to a cytosine residue in a cpg site; posttranslational modification of the amino acid tails of histones by means of acetylation, phosphorylation, methylation and aberrant expression of micrornas (mirnas), each of which is capable of posttranscriptionally regulating the expression of a cohort of cognate target genes.47 dna methylation requires the activity of dna methyltransferases (dnmts).the mechanism of dna demethylation is less clear. loss of binding to methylated dna-binding proteins might allow the promoter to enter a transcriptional state.47 ad is a complex disease for which the risk is convinced to be determined by a complicated interplay of genetics and environmental exposures that have been discussed and debated for many years. the recent understanding of epigenetics as a mechanism mediating gene-environment interaction offers new opportunities to advance novel concepts and re-examine established ones about this disease.29 ho reviewed regarding environmental epigenetics of asthma, including the epigenetic effects of tobacco smoke, microbial allergens, oxidants, airborne particulate matter, diesel exhaust particles, polycyclic aromatic hydrocarbons, dietary methyl donors and other nutritional factors, and dust mites. the discovery and validation of epigenetic biomarkers linked to exposure asthma, or both might lead to better epigenotyping of risk, prognosis, treatment prediction, and development of novel therapies.47 in ad, environmental factors, possibly react through epigenetic mechanism, and it may contribute to disease pathogenesis. in support of this, dna methyltransferase (dnmt1) transcripts in peripheral blood mononuclear cells of ad patients with high ige levels are significantly lower than the control.48 the effect of reduced dnmt1 level on ige may be indirect, with dna hypomethylation of t cells resulting in their increase production of il4, which then stimulates ige production by b cells.49,50 enhanced immune response at epidermal surfaces, caused by dna hypomethylation of methylation-sensitive immune genes, along with the activation of the genes to critical to t/bcells interactions and inflammation, could initiate a sustained immune response.51 ho also reviewed the trigger factos that influence epigenetics of asthma. that finding in agreement with previous studies reporting that benzopyrene(bap)is able to decrease global dna methylation, inhibit dnmin vitro, and interfere with recruitment of the methylation machinery. bap is frequently used as a prototype polycyclic aromatic hydrocarbon (pah) for many experimental studies. in 72 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 65−75 table 1. genes associated with atopic dermatitis in at least one study based on ethnicity in asian population30 gene chromosomal location variant association population no. of subjects* adam33 20p13 rs2853209 yes japanese 140/258 bdnf 11p13 c270t yes chinese 160/169 ccr4 3p24 c1014 t no japanese 198/183 cd14 5q31.1 c-159t/c-260t no chinese 171/160 no chinese 113/67 cma1 (mcc) bstxi yes japanese 100/100 yes japanese 145/706 no japanese 100/101 yes japanese 169! csf2 5q31.1 t3606c no japanese 181/100 defa4 8p23.1 g-6298c no korean 631/458 defa5 8p23.1 g-2819a no korean 631/458 defa6 8p23.1 g-4844a no korean 631/458 defb1 8p23.1 t-2266c yes korean 631/458 eotaxin(ccl11) 17q21.1-q21.2 c-426 t no japanese 140/140 no japanese 140/140 a-384 g no japanese 140/140 no japanese 140/140 g67a no japanese 140/140 no japanese 140/140 flg 1q21.3 r501x, 3321dela, s1695x, q1701x, s2554x, s2889x, s3296x yes japanese 118/134 s2554x yes japanese 105 families, 376/923 s2554x, s2889x, s3296x, and 3321dela yes japanese 125/133 ifng 12q14 str at first intron no chinese 94/186 il4 5q31.1 c-589t (c-590 t) yes japanese 88 families no japanese 302/122 no chinese 94/186 asthma, (pahs) are one of the most widespread classes of pollutants of the environment and in food. they are present in crude oil, coal, and tar deposits and are derived from incomplete combustion of fossil fuel, oil, garbage, and cigarettes. they are major components of airborne particulate matter (pm) of urban aerosols and widely exist in food products, including grains, vegetables, oils, and fats. pahs are emitted to the air during the production of coke and aluminum. cooked meats are contaminated when they are charcoal grilled, roasted, or smoked.47 the trigger factor that could reduce dnmt1 in ad is still unknown, it is possible that bap has a role in ad as stated above, further studies are required. summary an important factor contributes to the failure of reproducing associations between genetic markers and a complex trait, such as ad, in independent populations is also related to population diversity. it is possible that certain genetic markers might contribute to disease risk in a particular (ie, ethnic or racial) population but not in others, either because of differences in frequencies of the risk alleles or because of specific genes interactions. it is still difficult to evaluate the effect of ethnicity on genetic associations of ad in asian population, because however, there is relatively little diversity in the populations that have been studied. flg is probably the best example of a candidate gene for which ethnicity likely influences the extent to which a polymorphism confers risk. in relevance to clinical practice, individuals with flg insufficiency are at increased risk of severe and persistent ad, which future therapy may place greater emphasis on barrier repair for this subgroup of patients. ad is a disease in which pharmacogenetics may facilitate the development of primary prevention and treatment regiments tailored according to the individual genetic predisposition in asian population. 73prakoeswa: ethnic and atopic dermatitis gene chromosomal location variant association population no. of subjects* c-3112 t yes japanese 202/150 t33c no chinese 94/186 il4ra 5p13 c-703 t yes japanese 451/116 ile50val no japanese 27/29 no japanese 302:122 a184g no japanese 202/150 g186a yes japanese a326c yes japanese c327a yes japanese glu375ala no japanese 27/29 no japanese 302/122 e375a no chinese 94/186 l389l no chinese 94/186 cys406arg no japanese 27/29 c406r no chinese 94/186 s503p no chinese 94/186 glu 551arg yes japanese 27/29 no japanese 302/122 q576r no chinese 94/186 t1803c yes japanese 202/150 c3112 t yes japanese 202/150 il5 5q31.1 24597t/a yes korean 646/474 il5r 3p26-p24 28380c/a no korean 646/474 il8 4q12-q13 2352a/t no korean 646/474 il8ra (cxcr1) 2q35 3047c/t no korean 646/474 il8r (cxcr2) 2q35 l262l no korean 646/474 il10 1q31-q32 a-1082 g no korean 276/140 no chinese 94/186 t-819c yes korean 276/140 no chinese 94/186 a-592c yes korean 276/140 no chinese 94/186 il12b 5q31.1-q33.1 a1188c yes japanese 164/100 g4237a no chinese 94/186 il12rb1 19p31.1 a-111 t yes japanese 382/658 il13 5q31 c-1112t no chinese 94/186 a704c no japanese 185/102 no japanese 185/102 c1103t no japanese 185/102 no japanese 185/102 g4257a yes japanese 185/102 yes japanese 185/102 g4464a no chinese 94/186 t7488c no japanese 160/103 il18 (igif) 11q22.2-q22.3 t113g yes korean 646/474 g2137c no japanese 160/104 irf2 4q35.1 c-829t (c-830 t) no japanese 49 families lmp2 6p21.3 lmp2*r no korean 53/184 lmp7 6p21.3 lmp7*a no korean 53/184 mip1a (ccl3) 17q12 c954t no japanese 39/65 rantes (ccl5) 17q11.2-q12 g-401a yes japanese 62/14 yes japanese 389/177 yes japanese 389/177 smpd2 6q21 haplotype yes korean 284/248 spink5 (lekti) 5q32 g-206a yes chinese 669/711 asp106asn 41 no japanese families table 1 continuation. 74 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 65−75 refferences 1. desai n, alexis af. atopic dermatitis and other eczema. in: kelly ap, taylor sc. editors. dermatology for skin of color. new york: mcgraw-hill companies; 2009. p. 163–6. 2. joyce tel, yuin cc. common skin diseases and treatment in asia. in: kelly ap, taylor sc. editors. dermatology for skin of color. new york: mcgraw-hill companies; 2009. p. 611–26. 3. meyers da. genetics of asthma and allergy: what have we learned? j allergy clin immunol 2010; 126(3): 439–46. 4. primavera g, berardezka e. biophysical properties of ethnic skin. in: berardesca e, leveque jl, maibach hi. editors. ethnic skin and hair. new york: informa health care; 2007. p. 13–8. 5. stevenson a, lindberg ca. new oxford american dictionary. 3rd ed. new york: publisher oxford univ press; 2010. 6. corcuff p, lotte c, rougier a, maibach h. racial differences in corneocytes. acta derm venereol (stockh) 1991; 71: 146–8. 7. sugino k, imokawa g, maibach h. ethnic difference of stratum corneumlipid in relation to stratum corneum function. j invest dermatol 1993; 100: 597. 8. meguro s, arai y, masukawa y, uie k, tokimitsu i. relationship betweencovalently bound ceramides and transepidermal water loss (tewl). arch dermatol res 2000; 292(9): 463–8. 9. kompaore f, tsuruta h. in vivo differences between asian, black and white inthe stratum corneum barrier function. int arch occup environ health 1993; 65(suppl 1): s223–5. 10. kompaore f, marty jp, dupont ch. in vivo evaluation of the stratum corneumbarrier function in blacks, caucasians and asians with two noninvasivemethods. skin pharmacol 1993; 6: 200–7. 11. wilson d, berardesca e, maibach hi. in vitro transepidermal water loss: differencesbetween black and white human skin. brit j dermatol 1988; 199: 647–52. 12. berardesca e, maibach hi. racial differences in sodium lauryl sulphate inducedcutaneous irritation: black and white. contact dermatitis 1988; 18: 136–40. 13. yosipovitch g, theng cts. asian skin: its architecture, function, and differencesfrom caucasian skin. cosmet toiletr 2002; 117(9): 57–62. 14. reed jt, ghadially r, elias pm. effect of race, gender and skin type on epidermalpermeability barrier function. j invest dermatol 1994; 102: 537. 15. berardesca e, de rigal j, leveque jl, maibach hi. in vivo biophysical characterizationof skin physiological differences in races. dermatologica 1991; 182: 89–93. 16. rietschel rl. a method to evaluate skin moisturizers in vivo. j invest dermatol1978; 70: 152–5. 17. the international study of asthma andallergies in childhood (isaac) steeringcommittee. worldwide variation inprevalence of symptoms of asthma, allergicrhinoconjunctivitis, and atopiceczema. lancet 1998; 351: 1225–32. 18. williams hc, pembroke ac, forsdyke h, et al. london-born black caribbean childrenare at increased risk of atopic dermatitis. j am acad dermatol 1995; 32: 212–7. 19. mar a, tam m, jolley d, marks r. thecumulative incidence of atopic dermatitisin the first 12 months among chinese,vietnamese, and caucasian infants bornin melbourne, australia. j am acad dermatol 1999; 40: 597–602. 20. diepgen tl. atopic dermatitis: the roleof environmental and social factors, theeuropean experience. j am acaddermatol 2001; 45: s44–8. gene chromosomal location variant association population no. of subjects* yes japanese 124/110 yes japanese 41 families no japanese 124/110 no japanese 41 families his396his yes japanese 124/110 glu420lys yes japanese 124/110 yes japanese 118 yes japanese 41 families glu420lys no chinese 669/711 glu825asp no japanese 41 families a1103g no chinese 669/711 g1156a no chinese 669/711 g2475 t no chinese 669/711 ivs12-26c/t yes japanese 124/110 ivs12-10a/g yes japanese 124/110 ivs13-50g/a yes japanese 124/110 ivs14119g/a yes japanese 124/110 st2 11p14.3-p12 a-27639g no japanese 452/636 stat6 12q13 str at exon 1 no chinese 94/186 tap1 6p21.3 tap1*a no korean 53/184 tap2 6p21.3 tap2*a haplotype korean 53/184 tarc (ccl17) 16q13 c-431t no japanese 193/158 c2134t no japanese 148/158 tim1 12q12-q13 5383_5397del yes korean 112/201 tnfa 6p21.3 g-238a no chinese 94/186 g-308a no chinese 94/186 c-857 t no chinese 94/186 c-863a no chinese 94/186 t-1031c no chinese 94/186 table 1 continuation 75prakoeswa: ethnic and atopic dermatitis 21. schultz larsen f, diepgen t, svensson a.the occurrence of atopic dermatitis innorth europe: an international questionnaire study. j am acad dermatol1996; 34: 760–4. 22. laughter d, istvan ja, tofte sj, hanifin jm. the prevalence of atopic dermatitisin oregon schoolchildren. j am acad dermatol 2000; 43: 649–55. 23. sugiura h, umemoto n, deguchi h, etal. prevalence of childhood and adolescentatopic dermatitis in a japanese population:comparison with the diseasefrequency examined 20 years ago. acta derm venereol 1998; 78: 293–4. 24. nnoruka en. current epidemiology ofatopic dermatitis in southeastern nigeria. int j dermatol 2004; 43: 739–44. 25. worth r. atopic dermatitis amongchinese infants in honolulu and sanfrancisco. hawaii med j 1962; 22: 31–4. 26. neame rl, kurinczuk jj, graham-brownrac. prevalence of atopic dermatitis inleicester: a study of methodology andexamination of possible ethnic variation. br j dermatol 1995; 132: 772–7. 27. baker rb. incidence of atopic dermatitis and eczema by ethnic group seen within a general pediatric practice. the permanente journal/ winter 1999; 3(1): 31–2. 28. moore mm, shiman slr, edwards jwr, kleinman kp, camargo ca, gold dr, et al. perinatal predictors of atopic dermatitis occuring in the first six months of life. pediatrics 2004; 113(3): 468–74. 29. brown sj, mclean whi. eczema genetics: current stateof knowledge and future goals. j invest dermatol 2009; 129: 543–52. 30. barnes kc. an update of the genetics of atopic dermatitis: scratching the surface in 2009. j allergy clinimmunol 2010; 125: 16–29. 31. akey jm, eberle ma, rieder mj,carlson cs, shriver md, nickerson daet al. population history and naturalselection shape patterns of genetic variationin 132 genes. plosbiol 2004; 2: e286. 32. baldini m, lohman ic, halonen m,erickson rp, holt pg, martinez fd. apolymorphism in the 5_ flanking regionof the cd14 gene is associated withcirculating soluble cd14 levels and withtotal serum immunoglobulin e. am jrespir cell mol biol 1999; 20: 976–83. 33. zhang g, goldblatt j, lesouef pn. does the relationship between ige and the cd14 gene depend on ethnicity? allergy 2008; 63: 1411–7. 34. hoffjan s, stemmler s. on the role of the epidermal differentiation complex in ichthyosisvulgaris, atopic dermatitis and psoriasis. br j dermatol 2007; 157: 441–9. 35. o’regan gm, sandilands a, mclean wh, irvine ad. filaggrin in atopic dermatitis. j allergy clin immunol 2008; 122: 689–93. 36. mckinley-grant lj, idler ww, bernstein ia, parry da, cannizzaro l, croce cm,et al. characterization of a cdna clone encoding human filaggrin and localizationof the gene to chromosome region 1q21. proc natlacadsci usa1989; 86: 4848–52. 37. gan sq, mcbride ow, idlerww, markova n, steinert pm. organization, structure, and polymorphisms of the human profilaggrin gene. biochemistry 1990; 29: 9432–40. 38. sandilands a, terron-kwiatkowski a, hull pr, o’regan gm, clayton th, watson rm, et al. comprehensive analysis of the gene encoding filaggrin uncoversprevalent and rare mutations in ichthyosis vulgaris and atopic eczema. nat genet 2007; 39: 650–4. 39. rodriguez e, baurecht h, herberich e, wagenpfeil s, brown sj, cordell hj, et al. meta-analysis of filaggrin polymorphisms in eczema and asthma: robustrisk factors in atopic disease. j allergy clinimmunol 2009; 123: 1361–70, e7. 40. kawashima t, noguchi e, arinami t, yamakawa-kobayashi k, nakagawa h, otsuka f et al. linkage and association of an interleukin4 gene polymorphism with atopic dermatitis injapanese families. j med genet 1998; 35: 502–4. 41. liu x, nickel r, beyer k, wahn u, ehrlich e, freidhoff lr et al. an il13 codingregion variant is associated with a high total serum ige level and atopic dermatitis in the german multicenter atopy study (mas-90). j allergy clin immunol 2000; 106: 167–70. 42. he jq, chan-yeung m, becker ab, dimich-ward h, ferguson ac, manfreda j et al. genetic variants of the il13 and il4 genesand atopic diseases in at-risk children. genes immun 2000; 34: 385–9. 43. mao xq, shirakawa t, yoshikawa t, yoshikawa k, kawai m, sasaki s et al. associationbetween genetic variants of mastcellchymase and eczema. lancet 1996; 348: 581–3. 44. barker jn, palmer cn, zhao y, liao h, hull pr, lee sp et al. null mutations in the filaggrin gene (flg) determine major susceptibility to early-onset atopic dermatitis that persists into adulthood. j invest dermatol 2007; 127: 564–7. 45. weidinger s, rodriguez e, stahl c, wagenpfeil s, klopp n, illig t et al. filaggrinmutations strongly predispose to early-onsetand extrinsic atopic dermatitis. j invest dermatol 2007; 127: 724–6. 46. brown sj, sandilands a, zhao y, liao h, relton cl, meggitt sj et al. prevalent andlow-frequency null mutations in the filaggringene are associated with early-onset andpersistent atopic eczema. j invest dermatol 2008c; 128: 1591–4. 47. shuk mh. environmental epigenetics of asthma: an update. j allergy clin immunol 2010; 126: 453–65. 48. nakamura t, sekigawa i, ogasawara h, mitshuishi k, hira k, ikeda s, ogawa h. expression of dnmt-1 in patients with atopic dermatitis. arch dermatol res 2006; 298: 253–6. 49. kuwubara n, kondo n, fukutomi o, fujii h, orii t. methylation pattern of i epsilon region in b cells stimulated with interleukin 4 and epstein barr virus inpatients with a high level of serum ige. eur j immunogonet 1995; 22: 265–75. 50. lorenzo pr, kuwabara n, kondo n, orii t. ige production by b cells stimulated with inteleukin 4 and epstein barr virus in patients with elevated serum ige level. j invest allergol clin immunol 1995; 5: 78–81. 51. strickland fm, richardson bc. epigenetics in human autoimmunity. autoimmunity 2008; 41(4): 278 ijtid vol 3 no 2 april juni 2012.indd 86 vol. 3. no. 2 april–juni 2012 bradycardia and tachycardia detection system with artificial neural network method delima ayu s1, franky chandra s.a1, retna apsari1 1 biomedical engineering faculty of science and technology, universitas airlangga email: delima_namaku@yahoo.com abstract heart disease is one disease with high mortality rate in the world. based on who records from 112 countries at 2004, the rate is 29% of all deaths each year. medical devices are necessary to diagnose one's health as an indication of a disease. nowadays, indonesia still imports medical devices, for the diagnosis of heart failure, from abroad. this research aims to assist the monitoring of cardiac patients with bradycardia and tachycardia appearances of message condition patient’s heart rate at the same time. the results were displayed with the output of bradycardia condition of the heart rate (heart rate less than 60 beats per minute) or tachycardia (heart rate over 100 beats per minute). the system displayed the data read from the heart to the pc embedded system to monitor the condition of the patients under decisions based on backpropagation neural network. classification system could be performed quite well, training data and by testing the 10 pieces, the optimal weight gain was 1727 iteration, the learning rate was 0.1122, and the error was below 0.001 (0.0009997). keywords: heart rate, heart, tachycardia, bradycardia, backpropagation abstrak latar belakang: penyakit jantung adalah salah satu penyakit yang memiliki angka kematian tinggi di dunia sebesar 29% kematian global setiap tahun, perhitungan ini didasarkan pada catatan kematian dari 112 negara pada 2004 dari data who (world health organization) (rusciano, 2004). penggunaan alat medis sangat diperlukan untuk diagnosa kesehatan seseorang sebagai indikasi adanya penyakit. saat ini indonesia masih mengimpor alat-alat medis tersebut, termasuk untuk diagnosis gagal jantung. tujuan: untuk membantu pasien penyakit jantung dalam pemantauan bradikardi dan takikardi dengan tampilan berupa pesan kondisi denyut jantung pasien saat itu. metode: hasil yang didapat akan ditampilkan dengan keluaran berupa kondisi denyut jantung yaitu bradikardi (denyut jantung kurang dari 60 kali per menit) atau takikardi (denyut jantung lebih dari 100 kali per menit). sistem melakukan pembacaan data jantung dari sistem embedded ke pc untuk memonitoring kondisi penderita dengan keputusan berbasis jaringan saraf tiruan backpropagation. hasil: sistem dapat melakukan klasifikasi dengan cukup baik, dengan data pelatihan dan pengujian masing-masing 10 buah, memperoleh bobot yang optimal pada iterasi ke 1727, learning rate sebesar 0.1122 dan error di bawah 0.001 (0.0009997) kata kunci: heart rate, jantung, takikardi, bradikardi, backpropagation case report introduction heart is one of the most vital organs. the impaired cardiac function greatly influences other organs, especially kidneys and brains. the main function of heart as a single pump is to pump blood foward the entire body to provide nutrition for the metabolism of the survival cells. internally, heart is separated into two parts, the right side of the heart functions as a blood pump to the lungs and the left side of the heart pumps blood foward the entire body. at each there are two halves of the heart chambers of the heart. blood from each atrium is sent to the ventricles. the blood from the right ventricle flous to the lungs at the pump lung and blood from the left ventricle flous through the body in the pump. it is still considered normal that heart atrium contracts for approximately six seconds 87ayu, et al.: bradycardia and tachycardia detection system trillionth preceeding the ventricular contraction, allowing ventricular filling before the ventricles pump blood to the lungs and the entire body. the contraction of the heart works automatically and is generated by electrical currents in the form of action potentials or cardiac conduction and controllable cardiac rhythm. heart has a special system generated in cardiac conduction to the rhythmic electrical impulses which causes a rhythmic contraction of the heart muscle called the heart rhythm. it sendsaction potentials through the heart muscles toword the heart1. as cardiac impulse flows the heart, the electrical current will spread into the tissues surrounding the heart and a small portion of the flow will be spreaded to the body surface. heart is depicted in figure 1. figure 1. heart14 the direction of the conduction of heart is sinotrial (sa) node towards atriventricular (av) node, then to the bundle of his and branched into the left bundle and the right bundle branches. left bundle branch's impulses are sent to the left the ventricle, and those of the right bundle branch are sent to the right ventricle. impulses proceed to the purkinje fibers and a network of fibers they spread rapidly to the ventricular wall3. cardiac conduction is associated with the amount of heart rate (heart rate) per minute. heart rate is used as an indication of any abnormalities in the heart. normal heart rate ranges from 60 to 100 times/min. figure 2. cardiac conduction direction3 heart abnormalities tachycardia in normal circumstances, the electrical impulses are generated by a pacemaker called sa node. this electrical impulse is passed into the ventricle through an av node, in which the node will be slowing down the flow of the impulses. the next impulse will spread throughout the ventricles.2 fast heart rate, called tachycardia, means that the heart rate exceeds 100 beats per minute. tachycardia is divided into two main types: supraventricular and ventricular. the emergence of tachycardia is usually indicated by a shortness of breath or wheezing, rapid pulse, chest pain, cold sweat, and uncons ciousness. but in some people, tachycardia does not imply any symptoms. bradycardia bradycardia or bradyarrhythmias are terms used to indicate the presence of heart rhythm disorders and conduction that causes heart rate to be less than 60 beats/min. the onset of bradycardia is associated with a decrease or failure of impulse formation and of obstacles / interference electrical conductivity. several causes of bradycardia are as follows: a. barriers sa node this condition is relatively common in the elderly due to the failure of the sinus node impulse to spread to the atrium. b. barriers av node in this state atrial impulse is blocked in several places on its way into the ventricle. c. bundle branch block the disorder is more common nowadays with increasing age in both branches of the left and the right. when encountered individually, these disorders are not dangerous, join what? it could be bad. designing the hardware and the software designing the hardware hardware design utilizes arduiono. arduino is an open-source single-board micro-controller, derived from the wiring platform and designed to facilitate the use of electronics in a variety of fields. the hardware arduino has on atmel avr processor while the software arduino has its own programming language figure 3. arduino board 88 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 86−91 this study made the hardware detect cardiac abnormalities “tachycardia” and “bradycardia”. these procedures were performed in several stages, namely, preparation of design diagramming tools, hardware design, and software design. the diagram tool is described in figure 4. figure 4. hardware diagrams the explanation to figure 4: in this study, the design of the sensor used is the plethysmograph reflection mode as depicted in figure 5 which depicts the installation of led and ldr on the finger used as the heart rate detection sensors. figure 5. sensor plethsymograph. in this study there are 2 microcontrollers as that functioned for the transmitter and the receiver. the transmitter circuit consisted of a sensor, a power supply, a wireless module and an arduino duemilanove. the first work of the transmitter was to detect the heart rate sensors on the fingers and to transmit the data to the wireless module which was enabled as the transmitter controlled by the microcontroller. receiver circuit consisted of wireless modules that functioned as a receiver that received data from the transmitter and the data of which were processed by a microcontroller to be then displayed on the lcd with an output in the form of heart condition. the software design can be seen in figure 6. figure 6. software diagrams design of intelligent network backpropagation artificial neural networks is a system in which the computing architecture and its operation can be inspired from the knowledge of biological nerve cells in the human brain. neural network is one of the artificial representation of the human brain. to find out more about the origin and how the structure of the neural network is created and can be used as a counter, these will be reviewed briefly by the terms that have generally been used. the structure in figure 7 is the standard form of the basic units of the human brain tissue that has been simplified. the structure of this standard will change in the future if scientists can find a better standard form or improve the standard form used today. human brain tissue is composed of 1013 pieces, each neuron is connected by about 1015 pieces of dendrites. the function of dendrites is to transmit signals from neuron to other neurons connected to it. as the output channel, each neuron has axons, while the signal receiver is called synapses. figure 7. simple structure a neuron. 89ayu, et al.: bradycardia and tachycardia detection system figure 10. neural network flowchart figure 9. software flowchartfigure 8. diagram system in general, neural network is composed of one trillion (even more) neurons that are interconnected and integrated with each other by a trillion synapses so that they can carry out activities to store (memorize) knowledge regularly and continuously as needed.6 backpropagation is one of the unsupervised training methods (supervised learning) and is usually designed for operations on multi-layer neural network. according to rumelhart’s backpropagation method that has been applied widely, approximately 90% of backpropagation has been successfully applied in various fields, such as finance, pattern recognition handwriting, 90 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 86−91 voice pattern recognition and medical image processing. this algorithm has a training process that is based on a simple interconnection, for example, if the output gives the wrong result, it will be corrected so that the weight can be reduced and subsequent neural network response could be expected to detect the correct value better. backpropagation is also capable of transforming and improving the weight of the hidden layer. the system was created as a diagram figure 11. data training and testing source: intelligent system theory13 figure 12. results classification of heart disease source: intelligent system theory13 figure 13. weight training results source: intelligent system theory13 (figure 8), the sensor started reading to determine the decision to backpropagation. software design cardiac abnormality detection system (bradycardia and tachycardia with a method of backpropagation neural networks). neural networks were prepared using 3 pieces of object layers (one input layer, one hidden layer and one output layer). input layer in a dynamic array was arranged according to the image 91ayu, et al.: bradycardia and tachycardia detection system size of the object which roled as an input to the first layer. a number of output nodes were determined based on the number of characters that were supposed to be recognized. the number of nodes in the hidden layer was determined based on the experimental results. the diagram design procedure of artificial neural network method is shown in figure 9. software testing was performed using the type of feedforward artificial neural networks. artificial neural network weights that was going to be used was the value of the weight at the time of learning. data from the heart to the pc embedded system were compared with the data of the patients who had identified cardiac abnormalities. when the error detection results compared with the data value was less than five percent, the detection result would succeed. the diagram learning procedure and the test methods of artificial neural network are depicted in figure 10. results and discussion testing of backpropagation neural network tests were performed on intelligent systems by observing the system’s ability to perform the data clustering. training data and test data were observed as depicted in figure 11. classification system could perform quite well as depicted in figure 12, with data such as the training the table 1. training data heart disease no condition data clasification disease 1 0.05 0.02 1 0 tachycardia 2 0.09 0.11 1 0 tachycardia 3 0.12 0.2 1 0 tachycardia 4 0.15 0.22 1 0 tachycardia 5 0.4 0.7 1 1 normal 6 0.5 0.4 1 1 normal 7 0.8 0.83 0 1 bradycardia 8 0.82 0.8 0 1 bradycardia 9 0.9 0.89 0 1 bradycardia 10 0.95 0.8 0 1 bradycardia table 2. test data no condition data clasification disease 1 0.09 0.04 1 0 tachycardia 2 0.1 0.1 1 0 tachycardia 3 0.14 0.21 1 0 tachycardia 4 0.18 0.24 1 0 tachycardia 5 0.22 0.28 1 0 tachycardia 6 0.38 0.72 1 1 normal 7 0.6 0.4 1 1 normal 8 0.84 0.82 0 1 bradycardia 9 0.94 0.93 0 1 bradycardia 10 0.98 0.99 0 1 bradycardia testing of table 1 and table 2. the system obtained optimal weight by the number of iterations of 1727 patterns 10 pieces of data, learning rate of 0.1122 and the error below 0.001 (0.0009997) weight training results were stored in the memory and then were used during testing with different data. table 1 shows the training data and table 2 shows the test data. discussion the testing of the data above indicated that the system could work as expected that it could classify patients into normal or diseased conditions by the type of tachycardia or bradycardia. conclusion classification system could be performed quite well as depicted in figure 13, with data such as the training and the testing of table 1 and table 2. the system obtained optimal weight by the number of iterations of 1727 patterns 10 pieces of data, learning rate of 0.1122 and the error below 0.001 (0.0009997). references 1. kurachi, yoshihisa., 2001, heart physiology and pathophysiology, boston, massachusetts: 9–10. 2. guyton and hall. 2006. textbook of medical physiology eleventh edition. pennsylvania: elsevier saunders. 3. jones, shirley a., 2005, ecg notes interpretation and management guide, f.a davis company, philadelphia : 16. 4. ganong, william f. 1985. fisiologi kedokteran. jakarta. egc penerbit buku kedokteran harris, tom. 2008. how light emitting diodes work. [online]. tersedia:http//electronics.howstuffworks. com/led.htm [15 desember 2011] 5 purnomo, mauridhi hery 2006. supervised neural networks dan aplikasinya. 6. setiawardhana, eepis 2010 intelligent system theory. 7. mascaro, stephen a dan h. harry asada. 2001. photoplethysmograph fingernall sensor for measuring forces without haptic obstruction. ieee transactions on robotics and automation, vol 17, no. 5. 8. pallister, c. 1994. blood physiology and pathophysioloogy. oxford: butterworth-heinemann. 9. peacock, todd, chongmeng teh, k’lvin sui dan craig williamson. (2001). design of a heart monitor. department of electrical and computer enginnering mississipi state university mississpi. 10. ramli, ni . 2011. design and fabrication of a low cost heart monitor using reflectance photoplethysmogram. united kingdom: world academy of science, engineering and technology. 11. rs khandpur.1997. handbook of biomedical instrumentation. mcgraw-hill. 12. rusciano, florence. 2004. global burden of disease. switzerland: who (world health organization). 13. santoso, p. 2010. rancang bangun alat pendeteksi frekuensi detak jantung berbasis mikrokontroler. bandung: jurusan fisika universitas pendidikan indonesia. 14. tortora, g. j. & n. p. anagnostakos. 1984. principles of anatomy & physiology, 4 edition. new york: harper & row publishers. 15. wang, j paul. mark and estes. 2002. supraventricular tachycardia. american heart association. 7272 greenville avenue, dallas, tx 72514. �2 vol. 5. no. 1 january–april 2014 research report the utilization of achatina fulica mucus in alginate membrane as wound healing accelerator and antiinfection material fatkhunisa rahmawati1, dita ayu mayasari1, satrio adhitioso1, alfian pramudita putra1, eko budi kuntjoro2, prihartini widiyanti3,4 1 bachelor of biomedical engineering study program, faculty of science & technology, universitas airlangga, surabaya 2 environmental technique study program, department of biology, faculty of science & technology, universitas airlangga, surabaya 3 institute of tropical disease, universitas airlangga, surabaya, indonesia 4 biomedical engineering study program, faculty of science & technology, universitas airlangga, surabaya abstract wound should be covered with bandage that is called wound dressing. most people use synthetic materials such as gauze dressing. gauze has high absorption of nacl, which is often used to cleanse the wound. however, discomfort and pain arise since the gauze becomes sticky on the wound. therefore, we need other alternatives instead of gauze to cover wound. one such alternative is the alginate membrane. this study used alginate membrane with mixture of mucous of the snail achatina fulica, which contain proteins such as proline, serine asparagine, glycosaminoglycan, hydroxylysine, trionin and so forth, to activate the growth factor. alginate powder and carboxymethl cellulose (cmc) was dissolved in distilled water mixed with mucus of the snail achatina fulica in four variations (4:0; 4:1, 4:2, 4:3) through a magnetic stirrer, and casted on a baking sheet covered with sterile gauze. high performance liquid chromatography (hplc) test showed that the glycosaminoglycan content was found on the mucous of achatina fulica. this was indicated by the appearance of peak at 325–350 second. the most optimum alginate and mucus composition was in ratio of 4:2. this ratio resulted in a wound dressing that was still able to absorb the exudate and optimally accelerated wound healing. key words: alginate, achatina fulica, wound healing accelerator abstrak luka seharusnya ditutup dengan perban yang dinamakan dengan penutup luka. banyak orang menggunakan material sintetik seperti penutup kasa. kasa memiliki daya serap nacl, yang biasa digunakan untuk membersihkan luka. bagaimanapun, ketidaknyamanan dan rasa sakit timbul ketika kasa menjadi lengket pada luka. oleh karena itu, kita membutuhkan alternative kasa untuk menutup luka. salah satunya ialah membran alginat. pada penelitian ini menggunakan membran alginat yang dicampur dengan lender bekicot achatina fulica,yang mengandung protein seperti proline, serine asparagines, glycosaminoglycan, hydroxylysine, trionin, dan sebagainya untuk mengaktifkan growth factor. bubuk alginat dan carboxymethl cellulose (cmc) dilararutkan pada air suling dan dicampur dengan lender bekicot achatina fulica dengan empat variasi komposisi (4:0; 4:1, 4:2, 4:3) menggunakan magnetic stirrer dan diletakkan pada tempat oven dengan dilapisi kasa steril. uji high performance liquid chromatography (hplc) menunjukkan bahwa kandungan glycosaminoglycan ditemukan pada lendir bekicot achatina fulica. hal diindikasikan dengan munculnya puncak pada detik 325-350. alginat yang paling optimum dan dan lendir pada komposisi 4:2. komposisi ini menghasilkan penutup luka yang tetap bisa menyerap nanah dan optimal dalam mempercepat penyembuhan luka. kata kunci: alginate, achatina fulica, percepatan penyembuhan luka, , glycosaminoglycan, carboxymethl cellulose �3rahmawati f, et al.: the utilization of achatina fulica mucus in alginate membrane introduction traffic accident is one of the causes of high mortality in indonesia. based on data from the jakarta police department, the number of accidents during 2010 reached 8059 cases, from which the number of people killed was as many as 1,032, seriously injured 3,429 people, and minor injuries 5,679 people. data toward the end of 2011 stated, 8,468 victims of accidents in and around jakarta, as many as 11.04% died. a total of 2,241 people were seriously injured and those with slight injury were as many as 5,292 people (62.49%).1 each accident victim requires treatment to his injuries. by default of wound management, the wound should be covered with a membrane or a bandage called the wound dressing. usually people use a synthetic form of gauze dressing as wound dressings. the gauze is keeping the wound from the surrounding trauma. however, the gauze quickly absorbs nacl which is previously used to wash wounds. this raises the patient’s discomfort and worry if infection may occur due to sticky gauze on the wound.2 there are some research that conducted an experimental study comparing the use of conserved amnion with synthetic wound dressing material coated with gauze to cover circumcision wound in 16 children. from the results, it can be concluded that the use of conserved amnion as a circumcision wound closure is more effective in reducing pain when removing dressings circumcision and can reduce the risk of infection in treating circumcision wound, compared to the use of gauze coated synthetic materials on the outside. besides gauze, wound dressings as amniotic membrane and membrane alginate have been widely used today. amnion has great benefits as a wound cover because it contains growth factors that help natural process of cell proliferation. however, not all pregnant women and their families allow to donate the placental membranes to take the amnion, so that the source of the amnion becomes very limited.3 therefore, we need another alternative as a substitute for the amnion to function as wound closure. one alternative is the alginate membrane. currently, the widely used wound closure is pad or wick-shaped alginate. imported foam products are typically that of hydrogel material. based on studies, it is known that the foam or sponge can be made as alginate material that have a high absorption of wound containing liquid such as exudate. to accelerate wound healing, the membranes are usually given with medication or substance that could cause more active growth factor in human skin. the main element that can activate the growth factors are proteins such as proline, serine asparagine, hydroxylysine, trionin and so forth.4 the material contained in one species achatina fulica snail mucus. during this time, snails are only be used as a food ingredient in the form of chips or sate. moreover, villagers apparently use the mucus from these molluscs as toothache medicine. it is less hygienic. therefore, we need the development of research on snail mucus to be used as a wound healer.5 the combination of alginate, carboxymethyl cellulose (cmc) as a thickener, and snail mucus, is a solution to the needs of eco-friendly wound closure membrane and accelerate wound healing that is expected to reduce the incidence of wound infection and treatment costs. materials materials used in this study were achatina fulica snail mucus, sodium alginate with brand sigma aldrich 71238, and carboxymethyl cellulose (cmc) of brataco technical types. methods early preparation the first stage was the process of snail mucus snail mucus removal by preparing some snails from which the mucus would to be removed. then, part of the shell was washed with water until clean. after that, the tip of its taper-shaped shell was cut about 0.5 cm. samples preparation alginate membranes – snail mucus – carboxymethyl cellulose (cmc) was made from mixing powdered sodium alginate and cmc that had been diluted with a solvent, such as distilled water, and snail mucus. each solution was mixed and stirred with a magnetic stirrer in order to be homogeneous. this solution was then casted on a round baking pan that has been coated with sterile gauze, and then stored in a deep freezer with a temperature range of –80oc to –100oc for ± 24 hours. after 24 hours in the freezer, the samples were removed and immediately lyophilized for 72 hours at a temperature of about –105oc and pressures in militorr. there were four variations of composition in this study using a total weight of 1% (table 1). table 1. composition variations sampel alginate: achatina fulica mucus alginate (gr) distilled water solvent (gr) achatina fulica mucus (gr) a 4 : 0 1.4 140 0 b 4 : 1 1.1 110 2.8 c 4 : 2 0.92 92 4.6 d 4 : 3 0.80 80 6.0 �� indonesian journal of tropical and infectious disease, vol. 5. no. 1 january–april 2014: 12–15 test taxonomy biological characterization test taxonomy is the earliest process prior to the study as it aimed to get achatina fulica snail species, according to the characteristics of the animal molluscs, based on accountable literature references. characterization of fourier transform infra red (ftir) fourier transform infra red (ftir) test was used to determine the peak characteristics of functional groups described as transmittance curve (%) against wave number (cm-1) on the material that has been made. characterization of high performance liquid chromatography (hplc) to find out how much glycosaminoglican levels, one of the important growth factors activating proteins contained in the snail mucus wound closure as the main ingredient in accelerating wound healing. characterization of anatomic histopathology (ahp) wound healing and histology test were in vivo tests by observing the development of wound healing in mice (mus musculus) macroscopically by the intensity of wound color, wound fluid, and wound type.10 characterization of antibacterial test t h i s t e s t u s e d a d i s k d i f f u s i o n m e t h o d w i t h staphylococcus aureus and e. coli bacterial culture, whose inhibitory zone were analyzed. the qualitative disk diffusion test here used the colony units forming of 105 e. coli cultured on nutrient agar agent. results and discussion snails used in this research belonged to the phylum mollusca, classis gastropoda, order stylommatophora, familia achatinidae, genus achatina, and species achatina fulica. snail shell length was 5.3 cm, it has smooth surface without ornament and has a color pattern of longitudinal stripes, alternating dark brown and yellowish white, the color lines are uneven edge, the dark brown part is wider than the white part. the shell is circular cone-shaped, the bottom coil is much larger than the other coil, and the concave coil has very clear boundaries. the shell appears strong and bold, but has very thin edge of the aperture, and the aperture is without cover.6,7 the result of fourier transform infra red (ftir) test revealed a peak in the wave number 3750–3000 (showing o-h bond in alginate), 1900–1650 (showing c=o bond on the alginate), 1250–1050 (showing c-o bonds on the alginate) in accordance with existing references.8 high performance liquid chromatography (hplc) test showed that glycosaminoglycan content was found on the mucous of achatina fulica. this finding is consistent with the literature, where the appearance of peak was at 165,477 in minutes.9 the final characteristic was the result of anatomic histopathology test conducted in mice (mus musculus). the mice were given cut wound, and the healing process was observed through macroscopic test until the proliferative phase (around 13 days). each sample group consisted of four mice. parameters observed were the intensity of wound color, wound fluid, and wound type. the formed wounds contained liquid with a reddish color in each animal. the type of wound was open wound. on day 14, it could be seen that the composition of 4:2 was the most optimal composition in healing wounds since the reddish color in this group as a whole has faded, wound fluid in mice 1,3, 4 from a total of 4 mice had been absorbed by the wound healing accelerator membrane, whereas wounds in mice 1,2, and 4 had been completely closed. the next best composition was 4:3, then 4:1, and the last was a 4:0 or a group of negative samples. through these observations, we observed that when the sample does not contain snail (achatina fulica) mucus, the benefits of wound closing does not work et al. the antibacterial test results showed that from various concentrations used in tube dilution method, the concentration of 8% was the minimum dose of bacterial growth inhibition (mic) and the concentration of 15% was the minimum concentration to kill the bacteria (mbc). antibacterial test was only performed in a solution of 4:2 ratio since in macroscopic test the ratio had been proved as having most optimum wound healing. d:\sampel\teknobiomedik unair\fatkhunisa\92 ml.0 92 ml solid 28/06/2013 38 49 .4 6 38 32 .3 8 34 32 .4 6 29 25 .1 4 23 54 .6 8 20 91 .8 3 16 31 .2 5 15 04 .8 8 14 13 .3 1 13 38 .7 4 12 37 .7 8 12 00 .2 2 11 12 .3 3 89 7. 00 87 2. 96 72 2. 82 62 0. 64 50 2. 39 5001000150020002500300035004000 wavenumber cm-1 0 20 40 60 80 10 0 12 0 14 0 tr an sm itt an ce [% ] page 1/1 figure 1. results of ftir test on wound healing accelerator sample figure 1. results of ftir test on wound healing accelerator sample. respon detektor retention time figure 2. spectrum of hplc test results on achatina fulica snail mucus. ��rahmawati f, et al.: the utilization of achatina fulica mucus in alginate membrane since the healing is known to be affected by tissue bacteria concentrations higher than 105 microorganisms per gram,11 the use of dressing materials were able to reduce content of these microorganisms in surgical dermal wounds, as performed in this study, might be useful to avoid wound infection and, therefore, favor wound healing.12 in addition, the release of angiogenesisassociated growth factors, such vegf (vascular-endothelial growth factor) and pdgf (platelet-derived growth factor), after inflammatory chronification, is a key-step to the development of the granulation tissue during wound healing,13 which could support our findings regarding to enhanced vascularization. the composite of alginate, carboxymethyl cellulose (cmc) and achatina fulica mucus can be produced in the early stages as an alternative wound dressings that have the potential of accelerating wound healing, absorbs excess exudate and prevent infection. from the above data it can be concluded that the most optimum composition of alginate and mucus are in 4:2 ratio. this comparison produces wound dressing that is still able to absorb exudate and optimally accelerate wound healing. in conclusion, we have demonstrated that the mucous secretion of achatina fulica presents antibacterial properties. in addition, the use of dressing films based on this mucous secretion improved wound healing model. conclusion the physical characteristics of accelerator wound healing membrane pore size are approximately 1,457-2,687 μm. the effect of achatina fulica mucus as accelerator wound healing has been proved by the fact of faster healing process of wound compared with the group without the mucus. references 1. ali gp and findrawaty. perbedaan kecepatan penyembuhan luka bersih antara penggunaan lendir bekicot (achatina fulica) dengan povidone iodine dalam perawatan luka bersih pada marmut (cavia porcellus). digilib.unimus.ac.id. accessed on 20th of september 2014; 2002. 2. ismail ddsl, modern dressing improve the healing process in diabetic wound. malang: universitas brawijaya; 2002. 3. kartawijaya h. pengaruh pemberian topikal low molecular weight hyaluronate pada epitelialisasi luka superfisial tikus putih yang dirawat dengan membran amnion freeze-dried, departemen/smf ilmu bedah plastik fakultas kedokteran universitas airlanggarsud dr. soetomo surabaya. surabaya; 2013. 4. lee ky, david jm. alginate: properties and biomedical applications. elsevier; 2011. 5. grahacendikia. perbedaan kecepatan penyembuhan luka bersih antara penggunaan lendir bekicot (achatia fullica) dengan povidone iodine 10% dalam perawatan luka bersih pada marmut (cavia porcellus). malang: universitas brawijaya; 2009. 6. sabelli b. guide to shells and schuster. new york: 1979. p. 40–46. 7. bamers rd. invertebrate zoology. holth-sauders international editions; 1980. 8. erizal, abidin, z. jurnal ilmiah aplikasi isotop dan radiasi sintesis hidrogel campuran poli (vinil alkohol) (pva)-natrium alginat dengan kombinasi beku-leleh dan radiasi gamma untuk bahan pembalut luka. jakarta selatan: pusat aplikasi teknologi isotop dan radiasi batan; 2011. 9. jeong ja, toida t, muneta y, kosiishi l, imanari t, linhardt rj, choi hs, wu sj, kim ys. localization and characterization of acharan sulfate in the body of the giant african snail achatina fulica. comp. biochem; physiol. 130; 2001. p. 513–519. 10. manjas m et al. the use of amnion cream in wound healing of wistar rats wound incision, department of pathologic anatomy faculty of medicine andalas university; padang; 2010. 11. chong hc, tan mj, philippe v, tan sh, tan ck, ku cw, goh yy, wahli w, michalik l, tan ns. regulation of epithelial-mesenchymal il-1 signaling by ppar beta/delta is essential for skin homeostasis and wound healing. j. cell biol., 184(6): 817–31: 2002. 12. ojiegbe gc, njoku-obi an, ojukwu jo. incidence and parametric determinants of post-operative wound infections in a university teaching hospital. cent. afr. j. med., 36(3): 637: 1990. 13. diegelman ev, evans cs. official analytical chemists. 13th ed. washington dc., official methods of analysis of the association of official analytical chemists; 2004. ijtid vol 3 no 2 april juni 2012.indd 100 vol. 3. no. 2 april–juni 2012 the exon 5, 6, 7, 8 of p53 mutations in oral squamous cells carcinoma retno p. rahayu 1 department of oral pathology, faculty of dentistry, universitas airlangga 1 institute of tropical disease, universitas airlangga abstract genetic instability may underlie the etiology of multistep carcinogenesis. the altered p53 gene observed in tumors may represent the expression of such instability and may allow the accumulation of other gene alterations caused by multiple mechanism. p53 gene is the guardian of the genome, that is why we pay more attention to this gene. in this study, we evaluated the significance of p53 mutation in 55 patient with oral squamous carcinoma. thirty among them underwent well-differentiated carcinoma, while the remaining 25 patients underwent poorly differentiated carcinoma. the mutations were detected by pcr-sscp (single strand conformational polymorphism) analysis in the region between exon 5 and exon 8. the results indicated that the p53 mutation in exon 5 (40%), exon 6 (28%), exon 7 (24%) and exon 8 (8%) were associated with poorly differentiated carcinoma, whereas mutation in exon 5 (10%), exon 6 (30%), exon 7 (40%) and exon 8 (20%) were associated with well-differentiated carcinoma. these observations suggest that p53 mutation in exon 5, 6, and 7 have strong correlation with poorly differentiated in oral squamous carcinoma while well-differentiated level was related with mutation in exon 6,7 and 8. keywords: p53, oral squamous carcinoma, mutation, pcr-sscp abstrak latar belakang: ketidakstabilan genetik mungkin mendasari etiologi multistep karsinogenesis. perubahan gen p53 yang diamati pada tumor dapat mewakili ekspresi ketidakstabilan tersebut dan memungkinkan akumulasi perubahan gen lain yang disebabkan oleh mekanisme multiple. gen p53 adalah perwakilan dari genom, sehingga gen ini yang menjadi perhatian. tujuan: evaluasi mutasi p53 pada 55 pasien dengan karsinoma skuamosa oral. metode: tiga puluh di antaranya mengalami karsinoma diferensiasi baik, sedangkan 25 pasien menjalani karsinoma diferensiasi buruk. mutasi yang terdeteksi oleh pcr-sscp (untai tunggal polimorfisme konformasi) analisis di daerah antara ekson 5 dan ekson 8. hasil: hasil menunjukkan bahwa mutasi p53 pada ekson 5 (40%), ekson 6 (28%), ekson 7 (24%) dan ekson 8 (8%) dikaitkan dengan karsinoma diferensiasi buruk sedangkan mutasi pada ekson 5 (10%) , ekson 6 (30%), ekson 7 (40%) dan ekson 8 (20%) dikaitkan dengan karsinoma diferensiasi baik. pengamatan ini menunjukkan bahwa mutasi p53 pada ekson 5, 6, dan 7 memiliki korelasi yang kuat dengan karsinoma diferensiasi buruk pada skuamosa oral sementara mutasi pada ekson 6,7 dan 8 memiliki korelasi kuat dengan karsinoma diferensiasi baik. kata kunci: p53, karsinoma skuamosa oral, mutasi, pcr-sscp literature review introduction squamous cell carcinoma is the most common intraoral malignancy and is the most frequently occurring malignant tumor of the oral structures. in the malignant oral lesions, the squamous cell carcinoma is about 82–90%, and mainly affects male patients with ages varying between 40–80 years, although recent studies have reported the occurrence of this malignancy in younger patients.1 tobacco and betel use, alcohol consumption, viruses, and other occupational and environmental factors have been implicated in the etiology of the disease. epidemiological studies have shown that the incidence of oral squamous cell carcinoma varies significantly among the continents and within developed and developing countries.2 101rahayu: the exon 5, 6, 7, 8 of p53 mutations alterations in tumour suppressor genes like p53 gene are the most commonly implicated genetic events in a number of human tumors and are frequently found in various types of cancer and have been considered as molecular markers of cancer.3 mutation or increased expression of the p53 protein has also been reported in the head and neck squamous cell carcinoma. however, only few data are available on the frequencies of p53 mutation in oral squamous cell carcinoma correlated with poorly and well differentiated tumor grades. the different exon on p53 may be mutated in certain stages because it may serve as a regulatory marker in the process of tumorigenesis. polymorphism in p53 codon 72 may contribute to oral cancer susceptibility.4 one possibility is that p53 alteration can enhance genomic instability and thus augment the accumulation of subsequent genetic event necessary for tumor development.3,5 alteration of the specific gene, such as p53, p27, p16 and cyclin d-1, will induce oral cancer development through mutation, amplification, or deactivation mechanism.4 therefore, hsieh and his research team suggest that the pattern of p53 gene mutation in oral squamouse cell carcinoma conduet further study.5 single-strand conformation polymorphism (sscp) is a screening method for detection of unknowm point mutations. sscp analysis was orginally described by orita et al. (1989). the general idea is to take a small pcr product, denature it, and electrophorese it through a non-denaturing polyacrylamide gel. thus, as the pcr product moves into and through the gel (and away from the denaturant), it will regain secondary structure that is sequence dependent.6 the mobility of the single-stranded pcr products will be different at different sequences. therefore, pcr-sscp can be used to detect mutational products. the major advantage of sscp is that many individual pcr products may be screened for variation simultaneously. many of pcr products may be analyzed on each full-size sequencing gel (depending upon the comb size used).7 most researchers use sscp to reduce the amount of sequencing necessary to detect new alleles at loci of interest or to estimate allele frequencies of populations better. the main purpose of the study is to evaluate the significance of p53 mutation in various stages of patients with oral squamous cell carcinoma. to determine whether mutation of p53 gene was associated with degree of tumorigenesis, we used pcr-sscp method to detect the mutation in exons 5–8 of p53 gene. materials and methods tissues mucosal biopsies of 55 patients with oral squamous cell carcinomas (oscc) from the floor of the mouth were obtained from the surgical pathology files of the department of oral pathology, faculty of dentistry, universitas airlangga, surabaya. the 55 cases of squamous cell carcinoma comprised of poorly and well differentiated tumor grades. the a verage of the patient ages was 58.7 (ranging from 21–74) years. specimens from these patients, from 29 men and 26 women, were screened for adequacy of lesional tissue by staining the tissue sections with hematoxylin and eosin. the histological diagnosis of each specimen was independently reviewed based on the established histological criteria. sixteen biopsies of normal control epithelium from the floor of the mouth were obtained from non-smoker 6 men and 10 women with the average age of 52.2 years (ranging from 35 to 73). pcr-sscp fragments of p53 gene exon 5 to 8 were amplified using the following oligonucleotides. exon 5, ex 5/s 5’-tgttcacttgtgccctgact-3’ ex 5/as 5’-agtaatcagtgacgaatcag-3’ exon 6, ex 6/s 5’-tggttgcccagggtccccag-3’ ex 6/as 5’-ggagggccactgacaacca-3’ exon 7, ex 7/s 5’-cttgccacaggtctccccaa-3’ ex 7/as 5’-aggggtcagcggcaagcaaga-3’ exon 8, ex 8/s 5’-atttccttactgcctcttgc-3’ ex 8/as 5’-tccaccgcctcttgtcttgc-3’ the sequences of all primers were obtained from laboratoires eurobio, paris for the amplification of oral squamous cell carcinoma dna. the pcr conditions were denaturated at 94° c for 1 min, annealed at 59° c for 1 min and extended at 72° c for 1.5 min, with the total number of cycles of 35. the amplified fragments were analyzed by pcrsscp. single strand of the pcr product was made by snap-freeze technique and loading on 6% polyacrylamide containing 5% glycerol. the gel was running on 0.5√tbe (tris-borate-edta) buffer at 20 w constant power at room temperature for 12–16 h, until the xylene cyanol dye reached 3 cm from the top of the gel. the gel was stained with silver nitrate staining. results in this study, we evaluated the significance of p53 mutation in 55 patients with oral squamous carcinoma. the male to female distribution in this study was ignored. thirty among them underwent well-differentiated carcinoma, while the remaining 25 patients underwent poorly differentiated carcinoma. the mutations were detected by pcr-sscp analysis in the region between exon 5 and exon 8. the results indicated that the p53 mutation in exon 5 (40%), exon 6 (28%), exon 7 (24%) and exon 8 (8%) were associated with poorly differentiated carcinoma whereas mutation in exon 5 (10%), exon 6 (30%), exon 7 (40%) and exon 8 (20%) were associated with well-differentiated carcinoma. 102 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 100−103 table 1. prevalence of mutation in p53 gene associated with tumor grade in oral squamous cell carcinoma. position of mutation number of patients with mutation poorly differentiated (25 patients) well differentiated (30 patients) exon 5 10 (40%) 3 (10%) exon 6 7 (28%) 9 (30%) exon 7 6 (24%) 12 (40%) exon 8 2 (8%) 6 (20%) these observations suggest that p53 mutation in exon 5, 6, and 7 have strong correlation with poorly differentiated in oral squamous carcinoma while the well-differentiated level was related with mutation in exon 6, 7 and 8. pcr-sscp analysis showed that mutation on p53 gene could be detected by extra band eruption (fig.1). the mobility of the single-stranded pcr products depended upon their secondary structure. therefore, pcr products that contained change sequence differences as well as insertions, deletions or other mutation, had different mobilities in poly-acrylamide gel electrophoreses. figure 1. pcr-sscp analysis of p53 gene was drawn in lane 1, 2, 3, 4, respectively. mutations were detected and signed by extra bands. discussion oral squmous cell carcinoma (oscc) is the most common cancer found approximately 80% of all cancer in the oral cavity.8 in the developed country like us, which has managed to eradicate infectious disease, cancer is the second cause of death after cardiovascular diseases. in indonesia, cancer was recorded as the cause of death besides heart attack, diabetes and hepatitis. oral squamous cell carcinoma is one of the most common cancers worldwide, with incidences of more than 30 per 100000 population in india (oral cancer) and in france and hong kong (nasopharyngeal cancer). epidemiological studies have shown that the sites of oral cancer differ widely. tongue, lip and floor of the mouth are the most frequent sites of lesions of oral squamous cell carcinoma.2 oral cancer constitutes about 4% of all cancers in the united states and 5% in the united kingdom. a total of 2940 new cases of lip, mouth, and pharyngeal cancer in men were reported in the united kingdom in 1996: an incidence of 10.2 per 100000 population.9 early detection should be a priority, given the excellent prognosis of early stage disease compared with the poor results in advanced stages. in indian screening programmes, community health workers have been trained in primary prevention and early detection of oral cancer and premalignant lesions, but no evidence indicates that this reduces mortality. screening is the most cost effective procedure when targeted at high risk groups, for example, heavy drinkers and smokers.9,10 oral squamous carcinogenesis is a multistep process in which multiple genetic events occuring, alter the normal functions of oncogenes and tumour suppressor genes. this can result in increased production of growth factors or numbers of cell surface receptors, enhanced intracellular messenger signalling, and/or increased production of transcription factors.10 genetic alterations are known to occur during carcinogenesis including point mutations, amplifications, rearrangements, and deletions. point mutations (single base changes) can lead to overactivity or inactivity of gene products. these are common in genes such as k-ras and p53. alterations of these genes have frequently been reported in malignant neoplasms.11 oncogenes themselues are not sufficient to cause oral cancer and appear to be initiators of the process. the crucial event in the transformation of a premalignant cell to a malignant cell is the inactivation of cellular negative regulators tumour suppressor genes, and is regarded to be a major event leading to the development of malignancy. tumour suppressor genes are mostly inactivated by point mutations, deletions, and rearrangements in both gene copies. the p53 gen mutation will not cause cells apoptotic therefore the cancer cells will proliferate.12 if p53 gene mutated, there was no repair into the apoptosis cell which underwent dna damage, that mutant cell could enter the cell circulation and it will happen with uncontrolled cell development.13 one of the important tumor suppressor genes is p53, mainly roles as the guardian of the genome in apoptotic gene. this protein blocks cell division at the g1 to s boundary, stimulates dna repair after dna damage, and also induces apoptosis. these functions are achieved by the ability of p53 to modulate the expression of several genes.14 the p53 protein transcriptionally activates the production of the p21 protein, encoded by the waf1/cip gene, and p21 become an inhibitor of cyclin and cyclin dependant kinase complexes. p21 transcription is activated by wild-type p53, not mutant p53.15 however, waf1/cip expression is also induced by p53 independent pathways such as growth factors, including platelet derived growth factor, fibroblast growth factor, and transforming growth factor β. wild-type p53 has a very short half life (4–5 minutes) whereas mutant forms of protein are more stable, with a six hour half life.14,15 mutation of p53 occurs either as a point mutation, which results in a structurally altered protein that sequesters 103rahayu: the exon 5, 6, 7, 8 of p53 mutations the wild-type protein, thereby inactivating its suppressor activity, or by deletion, which leads to a reduction or loss of p53 expression and protein function. the tumour suppressor gene p53 is known to be mutated in approximately 70% of adult solid tumors.15 mutations of exons 4 to 9 of the p53 gene were found in 72 of 106 patients with head and neck squamous cell carcinoma (hnscc).15 p53 mutations were associated with loss of heterozygosity at chromosome 17p. the prevalence of p53-mutated tumors was higher in the group of patients with nonresponse to neoadjuvant chemotherapy than that in the group of responders (81% v 61%, respectively).4 according to these research the p53 mutation on poorly differentiated level mostly occurs in exon 5 (40%), whereas the least mutation occurs in exon 8 (8%), followed by exon 6 (28%) and exon 7 (24%). however the least number of p53 mutation in well differentiated mutation occurs in exon 5 (10%), and most of the mutation occur in exon 7 (40%), followed by exon 6 (30%) and exon 8 (20%). a high prevalence of p53 mutations on poorly differentiated level was found in exons 5 to 7. but high prevalence of p53 mutations on well differentiated level was found in exons 6 to 8. it is relevant to some studies which state that mutation in oral squamous cell carcinoma occurs beetwen exon 5 and 8. these observation suggest that p53 mutation in exon 5, 6, and 7 have strong correlation with poorly differentiated level in oral squamous carcinoma while well-differentiated level was related with mutation in exon 6,7 and 8. references 1. regezi ja and jordan rc, 2001. oral cancer in the molecular age. journal of california dentistry association, 29: 578–84. 2. gervasio, olas, dutra, r.a., tartaglia, s.m.a., vasconcellos, w.a., barbosa, a.a., and aguiar, m.c.f., 2001. oral squamous carcinoma: aretrospective study of 740 cases in a brazilian population. brazilian dental journal, 12: 57–61. 3. nagler, r. m. molecular aspects of oral cancer. anticancer res., 22: 2977–80, 2002. 4. cabelguenne, a., blons, h., de waziers, i., carnot, f., houllier, a., soussi, t., brasnu, d., beaune, p., laccourreye, o., and laurent-puig, p., 2000. p53 alterations predict tumor response to neoadjuvant chemotherapy in head and neck squamous cell carcinoma: a prospective series. journal of clinical oncology, 18(7): 1465–73. 5. scully, c.; porter, s. 2000. oral cancer. bmj, 321: 97–100. 6. shen h, zheng y, sturgis em, spitz mr, and wei q, 2002. p53 codon 72 polymorphism and risk of squamous cell carcinoma of the head and neck: a case control study. cancer letter, 183: 123–30. 7. mendelsohn j, holley pm, israel ma, gray jw and thomson cb, 2008. the molecular basis of cancer. 3rd ed elsevier saunders, philadelphia: 205–20. 8. samer a bsoul bds ms, michael a huber dds, geza terezalmy, dds, ma, 2005. squamous cell carcinoma of the oral tissue : a comprehensive review for oral healthcare providers. the journal of contemporary dental practice, vol. 6 no 4. november 15. 9. sanderson, r.j., and ironside, j.a. d., 2002. squamous cell carcinomas of the head and neck bmj, 325(7368): 822–7. 10. williams, h.k., 2000. molecular pathogenesis of oral squamous carcinoma. journal of clinical pathology, 53(4): 165–72. 11. mehrota r and syadav, 2006. oral squamous cell carcinoma, etiology, phatogenesis and prognostic of genomic aleration. indian journal of cancer departement of pathology. motilai nehru medical college university of alhabad: 162. 12. kleinsmith lj, 2006. tumor suprresor genes and cancer overview. pearson international edition, inc. publishing as benjamin cummings, 1301 sansome st. 179–86. 13. claudia t et al, 2006. p53 immunoexpression in oral squamous carcinomas from different anatomical sites: a comparative study, int. j. morphol, 24(2): 231-8, 2006. 14. pelengaris s,and khan m, 2006. dna replication and cell cycle in the molecular biology of cancer, toronto: blackwell publishing: 88–119. 15. hsieh, l.l., wang, p.f., chen, i.h., liao, c.t., wang, h.m., chen, m.c., chang, j.t., and cheng, a.j., 2001. characteristic of mutations in the p53 gene in oral squamouse cell carcinoma associated with betel quid chewing and cigarette smoking in taiwanese. carcinogenesis, 22: 1497–503. 23 vol. 5. no. 1 january–march 2014 clinical description and diagnosis of hiv/aids suryono1, nasronudin1,2 1 infectious and tropical disease division department of internal medicine, dr. soetomo hospital airlangga university school of medicine 2 institue of tropical disease airlangga university abstract infections of hiv/aids currently has become very serious problems for the world health. in the country the first case of hiv/aids was discovered in bali in 1987, in its progress has not the meaning but after 1985 hiv transmission increased considerably. the complex problem that the living and the increasing number of cases should indeed, medical practitioners understand more the clinical and how to diagnose infections of hiv/aids. a snapshot of the clinical hiv infection/aids can be seen from grievances and a disease that often accompanies it, a complaint which is found at hiv/aids sufferers in the form of suds retroviral acute: fever, weight loss, diarrhea chronic, disphagi, limpadenopati, infections in the skin respiratory disorders and nervous breakdown center. while a disease that often been gained by those with hiv / aids as candidiasis, tuberculosis, pneumonia bakterialis, toksoplasmosis and pneumonia pneumocystic carinii. diagnose hiv infection created based on clinical symptoms which includes major symptoms and symptoms of minor, and the result of the examination of the laboratory. key words: hiv, aids, transmission, epidemiology, infectious abstrak infeksi hiv/aids saat ini telah menjadi masalah yang serius bagi dunia kesehatan. di indonesia kasus hiv/aids pertama kali ditemukan di bali tahun 1987, dalam perkembangannya tidak mengalami perkembangan yang berarti akan tetapi setelah tahun 1985 penyebaran hiv meningkat dengan tajam. kompleknya permasalahan yang dihadapi odha dan semakin meningkatnya jumlah kasus ini perlu kiranya praktisi kesehatan lebih memahami gambaran klinis dan cara mendiagnosis infeksi hiv/aids. gambaran klinis infeksi hiv/aids dapat dilihat dari keluhan dan penyakit yang sering menyertainya, keluhan yang sering ditemukan pada penderita hiv/aids berupa sindroma retroviral akut: demam, penurunan berat badan, diare kronis, disphagi, limpadenopati, infeksi pada kulit, gangguan pernapasan dan gangguan saraf pusat. sedangkan penyakit yang sering didapatkan pada penderita hiv/aids seperti kandidiasis, tuberkulosis, pneumonia bakterialis, toksoplasmosis dan pneumonia pneumocystic carinii. diagnose infeksi hiv dibuat berdasarkan gejala klinis yang meliputi gejala mayor dan gejala minor, serta hasil pemeriksaan laboratorium. kata kunci: hiv, aids, transmisi, epidemiologi, infeksi introduction hiv/aids infection currently has become very serious problems for the world health. aids first found in 1981, but identification of the virus that causes aids new found about 1983–1984.1,2 this virus were given the name of the human immunodeficiency virus (hiv) that can be found the body fluid especially blood, a liquid sperm, vaginal discharge, and water milk mother. aids has been scattered more than 150 countries, until december 2000 58 million people estimated to be infected with hiv, 22 million have died, 3 million died the year 2000.two thirds of the number of hiv found in the countries of africa, part of sahara africa about 70% and in asia pacific more than 20%.16.000 world‘s people estimated to be infected with hiv in every day.1,3,4 in the country the first case of hiv/aids was discovered in bali in 1987, in its progress has not the meaning but after 1985 hiv transmission increased considerably. since 1999, there are new phenomenon of the spreading of hiv/aids cases included hiv infection started looking for a drug injection users or infecting drug users (idu). in both groups literature review 2� indonesian journal of tropical and infectious disease, vol. 5. no. 1 january–march 2014: 23–27 idu happening quickly because of the use of hypodermic needles together. the 2000 increase the pandemics are explicitly through sex workers (dept. of health, 2003). in 2002, they are prone to get hiv in the country among the 13 million to 20 million, the people living with hiv/ aids (odha) an estimated 90,000 people until we got 130,0005. the problems facing opdha very complex, includes physic health problem because decrease cd4, because their physical health psychological problems as shock, depression, denial, angry and sad and sorry and also psychological problem as isolated, expelled from the lives and so on.6 the complex problem that the living and the increasing number of cases should indeed, medical practitioners understand more the clinical and how to diagnose infections of hiv/aids. this will be the following clinical and diagnoses of hiv/aids infections. hiv/aids clinical descripton the clinical description hiv/aids can be seen from the disease, a disease that is often found and often accompanying.1,7,8 the route of hiv/aids beginnings arise after hiv infection retroviral acute, called suds, this decline in suds is showed cd4 increasing rna-hiv levels (and viral load). cd4 count tend to decline gradually within a few years with cd4 faster rate of decrease in 1.5–2,5 years before patients fall in a state of aids. viral load going up fast at the start of an infection and then went down to a point. with continued infectious viral load gradually rising. in the late phase diseases will be found cd4 count & it; 200/mm3, onset, followed an opportunistic infection the emergence of certain cancers weight down quickly and complication of neurological.5,9,10 symptoms from hiv divided into 4 steps: acute infection stage: no symptoms typical, arising after 6 first week be either fever, taste tired muscle pain and joints, pain ingest, and enlargement of lymph nodes. may also accompanied inflammation of the membranes of the brain (meningitis aseptic she fever, headache, spasms and nerve paralysis the brain. asimtomatic stage: at this stage usually without symptoms and complaint, this stage can last six weeks to months even years after infection. simtomatic stage light to severe: at this stage weight declining not until 10%, thrush that recurs at the mouth, inflammation of the angles of the mouth, can also found bacterial infection of the breath the top but sufferers can doing activities normal. on the stage that further decline weight more 10%, diarrhea that more than 1 months, heat unknown s why over a month, candidiasis oral, oral hairy leukoplakia, pulmonary tuberculosis, and pneumonia bacteria. at this stage of lying in bed more than 12 hours a day during last month. aids stage: at this stage sufferers was attacked by one or several kinds an opportunistic infection, e.g. pneumocystis carinii, toxoplasmosis the brain, diarrhea due to kriptosporidiosis, viral disease sitomegalo, a viral infection herpes, candidiasis esophagus, trachea, the bronchi or lungs and fungal infection other histoplasmosis, e.g. koksidiodomikosis. can also found some cancers; e.g. cancer lymph nodes and sarcoma.5,9,10 symptomps related on hiv/aids 1. fever: rushes often found in people with hiv, cd4 the number could help in evaluating and distinguish a likely cause its fever. on early disease (cd4 > 500) cause fever can occur because of tuberculosis, pneumonia or an acute infection of hiv her. midstage disease (cd4 200–500) can occur because the spread tubrkulosis or pneumonia. in adults with sexual activity active could by sexual because transmitted disease or infection anorektal. late disease (cd4 75–200) fever can occur because infection opurtunistic like pneumocystis carinii or malignansi. another causes can because the spread of tuberculosis, nonthypoid bacteriemia, salmonella bartonellosis, and fungal infection histoplasmosis, and cryptococcosis. advanced disease (cd4 < 75) all diseases in late disease can occur in this stage and can be found mycobacterium avium complex and infection cytomegalo virus1,10,12 2. diarrhea chronicle: diarrhea can be caused for infection, fierceness or hiv his own. infection can be caused by clostridium: defficile, salmonella, campylobacter, shigella, entamoeba histolytica; giardia lambia, isosporabelli, enterovirus, and strangyloides stecoralis. in late disease besides cause above may also caused by cryptosporidium parvum microsporidium and directorate. on advanced disease can occur because mycobacterium avium complex and citomegalovirus1,10,12 3. d y s p h a g i a : d y s p h a g i o f t e n a c c o m p a n i e d b y odynophagia and can be developed into a esophagitis. in midstage disease can occur sprue and esophageal disconfort. on late disease can occur infection mucous esophagus kandida accompanied with a lesion in the mouth. on advanced disease equal to late disease but often found infection citomegalo viruses and ulcer aphthous.1,12 4. respiratory disorder: can happen because bacterial infection of pyogenic, mikobacterium, fungi, parasitic, virus and ferocity of lymphoma or sarcoma sarcoma. this complaint be either shortness or cough12. in late disease can occur because pneumocystis carinii, fungal infection coccidiodes immitis, cryptococus neoformis 2�suryono and nasronudin: clinical description and diagnosis of hiv/aids or histoplasma capsulatum, can also occurred sarcoma sarcoma. on advanced disease besides cause above can be found again pseudomonas aeuruginosa and aspergilus species especially on the circumstances of netropenia or in hospital.1 5. skin infection: infection in the skin can be varied to suit imunosupresinya degrees. on early disease be either rash, lesions because sexual transmited disease, folliculitis, impetigo, ecthyma and sellulitis. on midstage disease can occur mucocutaneous candidiasis, oral hairy leukoplekia, shingles, psoriasis, dermatitis seborreic, and dermatitis atopy. in late disease skin infection that occurred previously become more chronic and refractory to therapy, can also occurred infection opurtunistic (cryptococcolis or histoplasmosis) and can occur lesion on skin. on advanced disease lesion on the skin not typical so it takes biopsy to its diagnoses, can occur bacillary angiomatosis and moluscum contangiosum.1 6. central nerve disorder: central nervous breakdown can include the status change, mental and pain the changing status the kognitive mental disorder; impairment of consciousness, delirium and it is psychosis. in early may occur aseptic meningitis disease happens because of its own. on disease midstage aseptic meningitis may become more frequent and chronic meningitis. in late disease can occur cryptococcal meningitis, toxoplasma encepalitis and aids dementia complex. on advanced disease the disease may occur in late on this phase and often accompanied prymary cns lympoma.1 7. limphadenopathy: caused by bacterial infection, syphilis; mikobakterium, a virus or fungus, may also caused skin disorder wide as dermatitis seborroik, and pioderma, when swollen lymph nodes happened two locations excess of 1 centimeter, and lasted more than three months called persistent generaled limphadenopathy (pgl). pgl arising during over 50 percent living, is symmetrical, no pain, often in glands behind ears and epitrochlear.12 8. weight loss: weight loss is a complaint often obtained in people with hiv, weight loss in line with the progesifitas disease spread. when weight loss more than 10% accompanied diarrhœa chronic over a month or fever over a month not caused another disease called hiv wasting syndrome.5,13 hiv/aids clinical manifestation 1. candidiasis: fungal infection kandida this could be this infection at the folding moist, paronychia, angles of the mouth, balanitis, and onychomikosis. symptoms clinical usually more weight if there infection of the oral mucous, pharinx, and genital. an opportunistic infection by kandida usually more easy to when there is infection bacterium or virus other staphylococcal, like streptococcus, mikobakterium avium complex, cytomegalo herpes viruses and simplek or abrasion the skin and mucosa which are port’ entry for kandida to get in circulation and next an undesired effect pathological on an organ local for example in eyes occurring retinitis and endopthalmitis. candidiasis can cause malnutrition on living due to the lurch swallow (disfagi) and pain ingest (odinofagi).6 2. tuberculosis: infection by mycobacterium tuberkolose occurs more frequently in hiv/aids sufferers compared with the general population, infection this could happen on all stadium hiv infection and usually occurs in cd4 about 400/ml3. in an advanced state of the risk of infection mikobakterium tuberkolose by those with hiv 8–10% per year. marriott, (smith, 1997; merati, 2004). of this number is far higher in a developing country like indonesia where tuberkolosis is still in endemi (merati, 2004). tuberkolosis can be a manifestation of the beginning of hiv so that patients who terdiagnose tuberkolosis should be thought to do with hiv infection especially to a group of high risk are infected with hiv, manifestasinya can include infection of the pulmonary (pulmonary tuberculosis) or infection outside/extra pulmonary stenosis (smith, 1997; merati, 2004). tb extra stenosis occurs more frequently in hiv to 70% in the general population, tb extra stenosis this may include: limpadenitis tb, the genital tract infections, urinary, the nerve center and spinal cord. the diagnoses built upon: disease history, the risk of hiv. photographs thorax hilum, which looks gland enlargement lung, infiltrate at the apex effusion of the pleura, cavity pulmonary or tuberculosis a billion.6 3. pneumonia bacterialis: at the hiv pneumonia with bacteria pyogenic occur more often than the general population, but germs the cause same as: streptococci pneumonia, hemopilus influenza, and brahamella catarrhalis. can also occurred infection with staphylokokus aureus, and gram-negative bacteria. symptoms may include high heat a sudden, asphyxiate, chest pain, and coughing productive with sputum being purulent. can also occurred lung infection chronicle suppurative and sinusitis (smith, 1997). pneumonia bacterialis often occurs in cd4 & it; 250/ml3, while suppurative infections happens when cd4 & it; 100/ml3.12,14 4. toxoplasmosis: infection by toxoplasma gondii is an opportunistic infection that often occurs in hiv infection. common symptom infection toksoplasmosis be either high fever headache and vomiting vomiting, may also form of symptoms ensepalitis neurological or focal plane, as headache, spasm, impaired function cognitive and impairment of consciousness. in disorders more difuse can occur symptoms sudden accompanied fever and spasms or existing bleeding intra cerebral, disorientation, mental disturbance and comma. in the eye can happen retino choroiditis while in mielopathia can occur weakness ektremitas with impaired sensory 2� indonesian journal of tropical and infectious disease, vol. 5. no. 1 january–march 2014: 23–27 and disorders spinter. diagnose can be made by complaint above accompanied ct a brain scan shown any lesions multiple ring-shaped, the picture is that clearer by contrast or with mri, lesions lie in corticomedullary junction or in basal ganglia. serology tests can help where obtained immunoglobulin g (igg) a specific for toksoplasma.6 5. pneumonia pneumocystic carinii: pneumonia pneumocystic carinii (ppc) was opportunistic infection frequently found on the hiv (smith ai, morris a 2002), in america rate occurrence 70–80% on all the hiv who do not get propilaksis (zimmerman, 1994). ppc often arise if cd4 < 200/ml3, symptoms can light to severe form of: dry cough, asphyxiate progressive start from tightness while working until shortness at rest, fever and sweating.11,15 photograph roentgen thorax ppc on light, maybe normal, or a little hormonal perihilar, on ppc are occurring abnormality difuse interstitial bilateral shadowing, and at ppc heavy no abnormality that extensive form of bilateral interstitial alveolar and marking. to examination gas blood, ppc light show pao2 normal, and saturation oxygen declining while working, ppc being pao2 between 60–80 mmhg, ppc and heavy pao2 & it; 60 mmhg.11 the diagnosis made based on to be above accompanied microscopic examination to identify pneumocystis of sputum, preparation broncoalveolar fluid or lung tissue and pcr.11,15 hiv/aids diagnosis the diagnosis of hiv infection/aids can be made based on clinical classification organization or cdc (see appendix 1 and 2) (levy, 1993). in indonesia diagnose aids for the purposes of epidemiology surveillance made if showing hiv testing positive and lack of symptoms was obtained 2 major and one minor symptoms.7 symptoms major 1. the weigh decrease more than 10% in 1 month 2. chronic diarrhea for a month 3. fever more than a month 4. impairment of consciousness and neurological disorders 5. dementia/hiv encefalopati symptoms minor 1. cough more than a month 2. dermatitis generalisata 3. herpes zoster multisegmental and or repeated 4. kandidiasis oro-faringial 5. simplek chronic progressive herpes 6. limfadenopati generalisata 7. fungal infection of recurring at female genitals 8. retinitis cytomegalovirus when acquired one mark/symptoms down here, reported as aids cases, without examination laboratory: 1. sarkoma kaposi 2. repeated pneumonia and life-threatening hiv examination to detect a person suffering from hiv, the test can be done directly on the hiv virus or indirectly by way of finding an antibody. if someone found antibodies against hiv infected with hiv (fauci, 2003; dept. of health, 2004). inspection strategy for diagnostic lab can be seen in annex.3 hiv serology examination examination first antibodies against hiv can be used rapid a test to tapis test, when acquired positive results done reexamination by using test that having the basic principle different and or using preparasi antigens different of the tests first, usually used enzym-linked immunosorbent assay (elisa). when available means could pretty conducted trial confirmation to western blot (wb), indirect immunofluorescence assays (ifa), or with radioimmunoprecipitation assay (ripa) (depkes, 2003; crowe, 2004) other checks that can be used to detect antibodies tyerhadap hiv can be used material of saliva (orasure) and urine (calypte hiv-1 urine elisa).1 hiv virus hiv virus in the body could be detected by a polymerase chain reaction (pcr) technology. this technique was done if the serology test several times not conclusive; in order to make sure there is someone at phase of a window (a window period) to be knowing hiv infection on the baby, and to the interest in certain research. pcr this method includes dna-pcr can, pcr, rna (b) dna assay and p24 antigen joined the.2 hiv risk factors hiv epidemiological risk factors infection covering (depkes, 2001): 1. behavior risky (now or past) • sexual intercourse gogglesexual partners high risk without use condoms • narcotics addict syringe • sexual intercourse unsecured − having many sexual partners − sexual partners known patient hiv/aids − sexual partners from villages in prevalence hiv aids that a high − homosexual 2�suryono and nasronudin: clinical description and diagnosis of hiv/aids 2. workers and customers entertainment as: massage parlor, discotheque, karaoke or prostitution veiled 3. have the acts of sexually transmitted infection (ims) 4. the acts of received transfusions blood recurring 5. the acts of wound leather, tattoo, piercing, sirkumsisi or with an instrument not sterile. summary hiv infection has become a serious breakdown in health, the disease have an impact of crimes against victims, their families and surroundings so we needed getting the prompt, therefore understanding to picture clinical and manner diagnoses should be perceptible by practition health. a snapshot of the clinical hiv infection/aids can be seen from grievances and a disease that often accompanies it, a complaint which is found at hiv/aids sufferers in the form of suds retroviral acute: fever, weight loss, diarrhea chronic, disphagi, limpadenopati, infections in the skin respiratory disorders and nervous breakdown center. while a disease that often been gained by those with hiv/aids as candidiasis, tuberculosis, pneumonia bakterialis, toxoplasmosis and pneumonia pneumocystic carinii. diagnose hiv infection created based on clinical symptoms which includes major symptoms and symptoms of minor, and the result of the examination of the laboratory. reference 1. zavasky dm et al. (2001). special patient populations patients with aids. in: a lange medical book current diagnosis & treatment in infectious disesase. editors wilson wr et al. mcgraw-hill, new york, p. 315–327. 2. fauci as and lane ac. (2003). epidemiologi hiv/aids, in: harrison principle of internal medicine. editor braunwald et al. 15th ed, new york, p. 1852–1861. 3. french rf et al. (1997). how hiv produces immune deficiency. in: managing hiv. editor stewart gj. australasian medical publishing co. limited, sydney, p. 22–28. 4. unaids/who (2002). aids epidemic update, geneva. available from: http/www.unaids,org /en/resources. accessed 2/11/2004 5. departemen kesehatan republik indonesia (2003). pedoman nasional perawatan, dukungan dan pengobatan bagi odha. depkes, jakarta. 6. merati tp (2004). gambaran klinis dan diagnosis mutahir hiv/ aids. dalam naskah lengkap workshop hiv/aids, editor akmal sya’roni, lembaga penerbit bagian ipd fk unsri. hal. 7–26. 7. departemen kesehatan republik indonesia (2001). pedoman tatalaksana klinis infeksi hiv di sarana pelayanan kesehatan. depkes, jakarta. 8. gerberding jl. (2003). occupational exposure to hiv in health care settings. new england journal of medicine. vol. 348; p. 826–833. 9. levy ja. (1993). pathogenesa of hiv infection. microbiol rev 57: p. 183–189. 10. carr a, boyle mj. (1997). primary hiv infection. in: managing hiv. editor stewart dj. australasian medical publishing co. limited, sydney, p. 9–10. 11. smith ai and pigot pc. (1997). hiv and respiratory disease. in: managing hiv. editor stewart gj. australasian medical publishing co. limited, sydney, p. 87–90. 12. who (1998). clinical management of hiv and aids at district level. world health organization regional office for south asia, new delhi. 13. kelly dm et al. hiv. (1997). weight loss and wasting syndrome. in: managing hiv. editor stewart gj. australasian medical publishing co. limited, sydney, p. 113–114. 14. departemen kesehatan republik indonesia (1998). surveilans aids. katalog dalam terbitan departemen kesehatan 616.979.2, jakarta. 15. thomas cf and limper ah. (2004). medical progress pneumocyctis pneumonia, new england journal of medicine, vol. 350; p. 2487– 2498. 16. crowe s and mills j. (2003). aids & other virus infections of the immune system, in: a lange medical book medical immunology. editor parslow tg. 10 th edition, mcgraw-hill, new york, p. 636–654. 77 vol. 1. no. 2 may–august 2010 the role of hyperbaric oxygen to platelet aggregation in diabetic patients type ii (niddm) prihartini widiyanti institute of tropical disease science & technology faculty airlangga university abstract prevalency of diabetes mellitus in indonesia has tendency to be increased from year to year. hyperbaric oxygenation (hbo) has been used as treatment of diabetes mellitus's complication especially diabetic gangrene. but the effect of hbo to the rheology's disfunction especially platelet's aggregation in the patients of niddm was investigated. the randomized pretest-posttest design was used in this study. an experimental laboratory study was performed at naval health institution in surabaya. 32 patients of niddm,women, 40–75 years old, normal physic's diagnosa, normal thorak's photo, normal ekg, normal ear nose and throat, diabetes mellitus's family's record, normal weight (bmi), blood glucose level didn't exceed 400 mg/dl (including controlled dm fbg < 120 mg/dl dan 2 h bg < 160 mg/dl), niddm, normal level of hba1c (4–5,9), as long as this research they couldn’t take their oral hipoglikemic agent, oral anti trombotic, vitamin c and vitamin e. they are divided into 2 group: group of hbo 100% o2 2,4 ata for 3x30 minutes with interval 5 minutes to inhalate air once a day daily for 5 days subsequently and the extraction of data (pat) had been held before oxygenation hyperbaric therapy at the first day and the end of fifth days, in control group only giving 20% o2 with the pressure 1 ata for 90 minutes once a day daily for 5 days subsequently and the extraction of data (pat) had been held before normoxia normobaric therapy at the first day and the end of fifth days. the results were significant decrease of the platelet’s aggregation level especially percent of aggregation after 5 days from 76,56 ± 8,06 become 69,13 ± 6,03. latent periode has also decrease from 28,75 ± 3,87 become 25,75 ± 2,82. speed of aggregation has also decrease from 66,25 ± 3,17 become 62,50 ± 3,44. index of aggregation has also decrease from 0,763 ± 0,071 become 0,581 ± 0,083. using paired t-test, it could be seen the decrease of latent periode (p= 0,001) and index of aggregation (p= 0,000) significantly after exposure of oxygenation hyperbaric hbo 2,4 ata 100% o2 3×30 minutes with interval 5 minutes inhalate air once a day for 5 days,subsequently. speed of aggregation (p = 0,022) and percent of aggregation (p= 0,013) are nonsignificantly. the conclusion of this research is that oxygenation hyperbaric 2,4 ata 100% o2 3×30 minutes with interval 5 minutes inhalate air once a day for 5 days,subsequently could decrease latent periode, speed of aggregation, index of aggregation and percent of aggregation in niddm. key words: hbo, niddm, hba1c, platelet’s aggregation (latent periode, speed of aggregation, index of aggregation and percent of aggregation) research report introduction the prevalence of diabetes mellitus patient in indonesia has a tendency to be increased from year to year (suyono, 1996). the latest report of mccarthy on 1994 the sum of diabetic patient in the world is 110,4 million and it would be reached 1,5 fold on 2000 (175,4 million) and 2 fold on 2010 (239,3 million). diabetes mellitus (dm) is endocrine disease with abnormal metabolic that has long term complication in several part of body such eye, kidney, neuron and vessel. diagnosis of diagnosis dm based on symptoms, diuretic osmotic and hyperglikemia (foster, 1998). long term hyperglikemia would induce rheology disfunction such as platelet agregation (pa), eritrocyte, leucocyte and blood viscosity (tjokroprawiro, 1997). hyperbaric oxygenation 78 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 77-81 (hbo) has been used for therapy of dm complication especially gangrene diabetik in diabetic center of niguarda hospital, milan, italy on 1980 (oriani, 1995). diabetic patient has been experienced hemorheologic disorder (pathorheology) and hyperactive platelet factor which play main role in the pathogenesis of hyperviscosity and microthrombus. hemoreologic disorder would disturb the blood flow in micro-sirculation region such as arteriolecapillary-venule. microsirculation region is the area of oxygen and nutrient exchange in the tissue and taking process of some waste product (muller, 1986). blood fluidity disorder could induce the inclination of platelet aggregation, abnormal hb o2 binding, the inclination of hba1c, erythrocyte aggregation, the decline of erythrocyte deformability, the inclination of plasma viscosity and hypercoagulability. all of the disorder above could yield ischemic and necrosis condition (mcmillan, 1987; colwell, 1980; soeharjono, 1985; muller, 1986). ditzel (1967) is showed that static vena and the destruction of capillaryvena flow in diabetic patient is caused by 3 factor, such as functional alteration (pathofisiology) vessel wall which related with redistribution flow and the leak of plasma component through venule, erythrocyte aggregation and the inclination of blood viscosity. bridges (1965) has been found the increase of platelet viscocity and the alteration of plasma composition in diabetic patient. in the small vessel, it has been found the increase of speed among 2 liquid divide with the distance between 2 layer. shear rate could stimulate the platelet disability. this condition would release some of mediators which activated tromboxane-a2. the interaction of txa2 and adenosine diphosphate (adp) could stimulate platelet aggregation. (hendromartono, 1997). hbo is therapy using 100% o2 in the hyperbaric chamber (> 1 atmospher absolute (ata). it could handle cellular hypoxia and increase o2 supply to the destruct tissue. hbo could decrease adp and collagen with its function as agonist of platelet aggregation (ersoz et al, 1998). hbo 100% o2 3 ata for 2 hours could support the no• regeneration (ito, 1996). this no• could activate guanylate cyclase to stimulate c-gmp production (schmidt hh, 1993). c-gmp could inhibit platelet aggregation (radomsky, 1993). hbo therapy is performed by inhaling the oxygen through mask, and endotrachea channel which has valsava technique previously in 10–14 session on 2,4 ata for 3×30 minutes with 5 minutes interval inhale the air (epsthein, 1998). material and method this research has been done in diabetes departement navy hospital dr. ramelan surabaya. the patient was giving the lottere. before they become the research subject, they should fulfilled letter of agreement (inform concent) and quiz. the selected patient (who fulfilled the inclution criteria, who agree to become research subject, who has been follow the medical examination which needed for this research), has been randomized using random number. sample has been taken from the population randomly and divided into 2 group such as control group (nonb): patient in normoxia normobaric (20% o2 1 ata) for 90 minutes once a day for 5 days subsequently and treatment group (hbo): patient in hyperoxia hyperbaric (100%o2 2,4 ata) for 3×30 minute with 5 minute interval inhale air once a day for 5 days subsequently. by using random sampling, then the sample was determined. the determination of platelet aggregation has been done twice: 1. in the first day before the hbo treatment. 2. in the end of 5th day after the hbo treatment. in the beginning of the research, we have did the randomization based on inclution criteria, (hba1c = 4,5– 5,9). blood sample which has been taken from the patients in diabetic clinic of navy hospital dr.ramelan surabaya then has been delivered to prodia clinical laboratory. the blood’s patient from control group (normoxia normobaric therapy (20% o2 1 ata) 90 minutes once a day for 5 days) and treatment group hyperoxia hyperbaric (100% o2 2,4 ata) 3×30 minutes with interval 5 minutes inhale air once per day for 5 days subsequently. blood sample then have been delivered to clinical laboratory catholic hospital st.vincentius paulo surabaya. result there are several variable in this research such as age, weight, height, fasting blood glucose (mg/dl), 2 hours post prandial blood glucose (mg/dl), hb a1c (%), hbo (oxygenation hyperbaric) 2,4 ata 100%o2 3×30 minutes with interval 2×5 minutes inhale air and aggregation parameter: latent period (second), aggregation speed (°), aggregation index (dh/dt), aggregation procentage (%) with collagen aggregator in niddm patient. the data has been processed by descriptive statistic and inferential such as normality, homogenity, anava same subject, correlation test, anakova. the result of normality test of all variable such as age, weight, height, fasting blood glucose (mg/dl), 2 hours post prandial blood glucose (mg/dl), hb a1c (%), in both group (nonb and hbo) is in normal distribution.using the uji univariate (tests of between-subjects effects) and multivariate test, the data in both group is homogen. the result of correlation test between moderator variable and dependent variable in both group (hbo and nonb) is non significant.he normality test of aggregation parameter such as latent period, speed of aggregation, aggregation of index, aggregation procentage in hbo and nonb has normal distribution. based on u nivariate test (tests of between-subjects effects) and multivariate is showed that all the aggregation parameter (laten phase, aggregation speed, aggregation index, aggregation procentage in hbo group and nonb group are homogen. 79widiyanti: the role of hyperbaric oxygen the result showed the differences of latent phase between hbo and nonb group (p = 0.001; p < 0.05) with the significant role of hba1c (p = 0.039, p < 0.05). there is no significancy of aggregation speed between 2 groups (hbo and nonb) (p = 0.439; p > 0.05) with the influence of 2 hours post prandial blood glucose test. there is significant difference of aggregation index between 2 groups (hbo and nonb) (p = 0.043; p < 0.05). the result did not showed difference of aggregation procentage between hbo and nonb group (p = 0.545; p > 0.05). there are 2 significant variable such as latent period alteration from the beginning to the end (p = 0.009; p < 0.05) and the alteration of aggregation index (p = 0.006; p < 0.05). discussion using correlation test among moderator variable such as age, weight, height, fasting blood glucose, 2 hours post prandrial blood glucose to aggregation parameter such as latent period, aggregation speed, aggregation index, and aggregation procentage, there is no significant correlation (p > 0.05). this result means that all the moderator variables above do not influence the aggregation parameter such as latent period, aggregation speed, aggregation index, and aggregation procentage before and after the treatment. aggregation parameter (latent period, aggregation speed, aggregation index, and aggregation procentage) in hyperbaric oxygenation (hbo) group the normal value of platelet aggregation using collagen aggregator is 35–50. there is the decrease of latent period of platelet aggregation in this group after the treatment compared with the value before the treatment (28,75 ± 3,87 become 25,75 ± 2,82). latent period (p = 0.001) has been showed the significant differences (p < 0.05). this result mean that hbo treatment 2,4 ata 100% o2 3×30 minutes with interval 5 minutes inhale air once a day for 5 days subsequently could decrease latent period of platelet aggregation in niddm patient. normal value of platelet’s aggregation speed of platetet with collagen aggregator is 52–80. there is a declination of platelet’s aggregation speed in hbo group after the treatment compared with before the treatment (66,25 ± 3,17 become 62,50 ± 3,44). platelet aggregation speed ( p = 0 . 0 2 2 ) w a s s h o w e d s i g n i f i c a n t d i f f e r e n c e (p < 0.05), this means hbo 2,4 ata 100% o2 3×30 minutes with interval 5 minutes inhale air once a day for 5 days subsequently could decrease platelet aggregation speed in niddm patients. normal value of platelet aggregation index with collagen aggregator is 0.3–0.7. it seems the declination of platelet aggregation index in hbo group after the fifth day compared with the value from the first day before hbo treatment (0.763 ± 0.072 become 0,581 ± 0,083). this means hbo 2.4 ata 100%o2 3×30 minutes with interval 5 minutes inhale air once a day for 5 days subsequently could decrease platelet aggregation index in niddm patients. normal value of platelet aggregation procentage with collagen aggregator is 50–75. there was the declination of platelet aggregation procentage in hbo group in the fifth day compared with the value in the first day before the hbo treatment (76,56 ± 8,06 become 69,13 ± 6,03). there was significant difference (p = 0,013), it means that hbo 2,4 ata 100% o2 3×30 minutes with interval 5 minutes inhale air once a day for 5 days subsequently could decrease platelet aggregation procentage in niddm patient. from this research, we could conclude that hbo 100% o2 3×30 minutes with interval 5 minutes inhale air once a day for 5 days subsequently could decrease platelet latent period, aggregation speed, aggregation index and aggregation procentage. this result was linear with the research of ito et al (1996) which stated that hbo 100% o2 3 ata 2 hours could support no• regeneration and oury et al (1992) which stated that no• could stimulate cguanosine mono phosphate (c-gmp) production. radomsky et al. (1993) was stated that c-guanosine monophosphate production could inhibited platelet activation. schmidt hh (1993) also support the theory above that no• could activate guanylate cyclase to arrange cyclic guanosine monophosphate (c-gmp). no• could inhibite the adhesion and platelet aggregation through c-gmp mechanism (moncada s, 1993). la croix (1990) has been stated that the hbo exposure 100% o2 3×30 minutes with interval 5 minutes inhale air once a day for 5 days subsequently could decrease adp and collagen as aggregator (platelet aggregation’s trigger). according to nadler jl dan natarajan r, (2000) diabetes mellitus could decrease production and action of no• that is the interaction with glycosylation end products. dm could impair the production and action of no•. dm mechanism could alter the action of no• by decreasing the production and action of no• such as the interaction with glycosylation end products, no• reaction with super anion to yield peroxyinitrite lipid, increase renal production and sensitivity of no•. the beneficial action of no• in the blood vessel such as vasodilator of endothelium-dependent smooth muscle, inhibit the adhesion and platelet aggregation, inhibit the adhesion of leucocyte to activated endothelium, inhibit migration and proliferation of vascular smooth cell migration, decrease the oxidation macrophage to low density lipoprotein and inhibit the expression of endothelin and pdgf. endotel cell release no• as important regulatorfor tone blood vessel and vascular homeostasis vascular through its effect to platelet and smooth muscle cell. no• could decrease platelet activation without systemic effect (nong z et al., 1997). in his research, nong z (1997) has been evaluated the inhalation effect of no• to collagen which could stimulate platelet aggregation in vivo and c-gmp intrathrombocyte level and antithrombotic activity. 80 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 77-81 parameter of platelet aggregation (latent period, aggregation speed, aggregation index, aggregation procentage)in nonb group in this nonb group, we found the declination of aggregation speed (68,19 ± 3,37 become 62,81 ± 6,52), aggregation index (0,706 ± 0,118 become 0,625 ± 0,161) and aggregation procentage (73,94 ± 9,45 become 70,44 ± 9,86) from the first day and the fifth day value (before and after treatment). this could be cause by several factors such as exercise, stress and food diet. the exercise factor is still influenced by duration, the magnification (heavy or light exercise) and the level of fitness. in dm patient, there are also several other factors such as plasma insulin level, blood glucose level and the proportion of body fluid (sherwood l, 1994). in active muscles, the muscle need to glucose has been increased without the inclination of insulin level. this is caused by the inclination of receptor sensitivity in muscle and the addition of active insulin receptor. the active muscle is called non-insulin dependent tissue (stacy p, 1986; storlien h, 1993). exercise for niddm patient is beneficial to act as glycemic control, todecrease weight, to handle atherogenic complication, disturbance of blood lipid, the inclination of blood pressure and blood hypercoagulation (storlien h, 1993; wolfe rr, 1998). the aim of nutrient therapy in niddm patient are to control glucose level, lipid level and hypertension. the declination of the weight and hypocalory diet could impair short term glycaemic level and has the potency to increase long term metabolic control (soekardji k, 1999). emotional stress manifestation in dm patient oftenly appear as denied attitude, obsession, angry, frustration, fear, depression, anxiety and food problem. all the disorders could destruct the blood glucose level and would cause acute and chronic complication including rheology disorder (semiardji g, 1999). conclusion hbo 2,4 ata 100% o2, 3×30 minutes with interval 5 minutes inhale air, once a day, for 5 days subsequently could decrease latent period, aggregation speed, aggregation index, aggregation procentge in niddm patient. there is no correlation between age, weight, height, bmi, fasting blood glucose, 2 hours post prandial blood glucose, hba1c as moderator variable with dengan latent period, aggregation speed, aggregation index, aggregation procentage in niddm patient. references bridges jm, dalby am, millar jhd and weaver ja, 1965. an effect of d-glucose on platelet stickness. lancet 1, p. 75. colwell ja, halushka pv, sagel j, 1980. platelet and endotel function in diabetes mellitus. in: editors: stolz f, dronin p, doin. hemorheology and disease, paris, p. 501. ditzel j, 1967. the in vivo reactions of the small blood vessels to diabetes mellitus. acta med. scand. suppl 476, p. 123. epsthein i, 1998. diabetes mellitus. text book of hyperbaric medicine, 2nd ed., toronto, hogrefe and huber, p. 368–369. ersoz g, ocakcioglu b, bastug m, ficicilar h, yavuzer s, 1998. trombosit agregation and release function in hyperbaric oxygenation, ankara university faculty of medicine, department of physiology, turkey, undersea hyperb med, winter; 25(4): 229–32 foster dw, 1998. diabetes mellitus. williams text book of endocrinology, 9th edition, usa: wb saunders company, p. 1014–1015. foster dw, 1998. diabetes mellitus. in: harrison. harrison's principle of internal medicine, 14th ed, new york: mcgraw-hill, p. 2060–2070. hendromartono, 1997. status hemoreologi pada kualitas hidup penderita diabetes mellitus. dalam: editor: tjokroprawiro a, hendromartono, sutjahjo a, tandra h,pranoto a,murtiwi s, dkk. naskah lengkap surabaya diabetes update iii november 1997 .surabaya, hal. 14–15. ito t, yufu k, mori a, packer l, 1996. oxidative stress alters arginine metabolism in rat brain: effect of sub-convulsive hyperbaric oxygen exposure. neurochem int 29: p. 187–195. lacroix ka, davis gl, schneider da, lavoie p, kintzing e, waterfield da, 1990. the effects of acute exercise and increased atmpspheric pressure on the hemostatic mechanism and plasma catechol amine levels. thromb res 57: p. 717–728. mcmillan de, 1987. hematologic changes in diabetes. in: editors: andreani d, crepaldi g, diabetic complication early diagnosis and treatment. a willey medical publication, new york, p. 349. moncada s, higgs a, 1993. the l arginine nitric oxide pathway. n engl j med 329: p. 2002. muller r, lebrach f, 1986. hemorheology and diabetes mellitus. naskah lengkap konas i perkeni, jakarta, 22–25 november, hal. 1. nadler jl, natarajan r, 2000. oxidative stress, inflammation and diabeticoxidative stress, inflammation and diabetic complications. in (leroith d, taylor si, olefsky jm, eds.) diabetes mellitus, a fundamental and clinical text, 2nd edition, lippincott williams and wilkins, a wolters kluwer company, philadelphia, p. 1008–1016 nong z, hoylaerts m, van pelt n, collen d, janssens s, 1997. nitricnitric oxide inhalation inhibits platelet aggregation and plateletmediated pulmonary thrombosis in rats. circulation research vol. 81, no. 5 november, p. 865–869. oriani g, maroni a, wattel f, 1995. handbook on hyperbaric medicine. berlin: springer, p. 531–534. oury td, ho y, piantadosi ca, et.al, 1992. extracellular superoxide dismutase, nitric oxide and central nervous system o2 toxicity. proc natl acad sci 89: p. 9715–9719 radomsky mw, moncada s, 1993. the biological and pharmacological role of nitric oxide in platelet function. in: authi ks, et al. eds. mechanism of platelet activation and control. new york: plenum press, p. 251–261. schimdt h h, lohmann s, walter u, 1993. the nitric oxide and c-gmp signal transduction system. biochim biophys acta 1178: p. 153. semiardji g, 1999. stress emosional pada pasien diabetes. dalam:stress emosional pada pasien diabetes. dalam: penatalaksanaan diabetes mellitus terpadu, pusat diabetes dan lipid rsup nasional dr. cipto mangunkusumo fakultas kedokteran universitas airlanggadepartemen kesehatan republik indonesia-world health organization, cv aksara buana, jakarta, hal. 253–260. sherwood l, 1994. human physiology. west publishing company, p. 22–28. soeharjono s, 1985 . hemoreologi pada penderita diabetes mellitus. naskah lengkap simposium konsep baru penanganan gangguan pembuluh darah, surabaya, 14 desember 1985. stacy p, borushek a, 1986. cookbook for diabetics plus diet guide. western australia: west perth, p. 60. storlien h, 1993. obesity weight control and muscle metabolism. in: proceeding of 9th biennial conference: exercise, metabolism and nutrition, p. 6–12. 81widiyanti: the role of hyperbaric oxygen soekardji k, 1999. penatalaksanaan gizi pada diabetes mellitus. dalam: penatalaksanaan diabetes mellitus terpadu, pusat diabetes dan lipid rsup nasional dr. cipto mangunkusumo fakultas kedokteran universitas airlanggadepartemen kesehatan republik indonesia world health organization, cv aksara buana, jakarta, hal. 33–41. suyono s, 1996. masalah dm di indonesia,. buku ajar ilmu penyakit dalam. edisi 3, jakarta: balai penerbit fkui, hal. 597–600.edisi 3, jakarta: balai penerbit fkui, hal. 597–600. tjokroprawiro a, 1997. diabetes update 1997 a. naskah lengkap surabaya diabetes update-ii 1997, surabaya; pusat diabetes dan nutrisi rsud dr. soetomo-fk unair, hal. 1–21. wolfe rr, 1998. metabolic interactions between glucose and fatty acidsmetabolic interactions between glucose and fatty acids in humans. am j clin nutr vol. 67(3) suppl 5, p. 12–18. ijtid vol 1 no 2 may-aug 2010.25.pdf ijtid vol 1 no 2 may-aug 2010.26.pdf ijtid vol 1 no 2 may-aug 2010.27.pdf ijtid vol 1 no 2 may-aug 2010.28.pdf ijtid vol 1 no 2 may-aug 2010.29.pdf 65 vol. 1. no. 2 may–august 2010 research report mycobacterium leprae in daily water resources of inhabitants who live in leprosy endemic area of east java ratna wahyuni, dinar adriaty, iswahyudi, cita rosita s prakoeswa, indropo agusni, shin�o i�umiindropo agusni, shin�o i�umi leprosy study group, institute of tropical disease, airlangga university surabaya abstract leprosy still a health problem in indonesia, where many leprosy pocket areas still persists, especially in the eastern part of the country. although the program of who – multidrug therapy (mdt) regiment has been conducted elsewhere since 1980s, only the prevalence can be reduced but not the incidence of new leprosy cases. theoretically after the source of leprosy (the infectious leprosy cases) has been treated, no more transmission of the disease and should be no more new leprosy cases will be found. to explain this phenomenon, the non-human resource of m.leprae became a new topic of debates, especially the existence of bacteria in the environment. a field study of the existence of m.leprae in the environment of leprosy endemic area had been conducted in a leprosy endemic area of the northern part of east java. the aim of the study is to find any correlation of the existence of these bacteria in the environment with the presence of leprosy patients who live in that area, in order to study its role in the transmission of the disease. ninety water samples from wells in the house of inhabitants who live in one endemic sub district were collected. the owner of the well was interviewed whether any leprosy patients who routinely use the water for their daily life activities. water samples were examined by polymerase chain reaction (pcr) method to detect m.leprae dna, using the lpf-lpr and lp3-lp4 nested primers (99bp). the pcr results showed positive band for m.leprae in 22 out of 90 (24%) water samples. water samples from wells that used by leprosy patients showed positive pcr in 11/48 (23%), while 11 out of 42 (26%) water samples from wells that never been used by leprosy cases showed positive result. statistically there was no difference (p>0.05) in the positivity of m.leprae between the two groups. it was concluded that the existence of m.leprae in the daily water resource was not correlated with the present of leprosy cases in the area. possible symbiosis between protozoan and mycobacterium in the environment were discussed. key words: leprosy, m.leprae, environment, water resources introduction leprosy is a chronic infectious disease caused by mycobacterium leprae, which often cause disability of patients. the who-mdt program has been introduced since 1980s and the majorities of leprosy endemic countries have achieved the elimination era, which means the prevalence rate is <1/10.000 population. but at the present time indonesia still has a burden with around 17.000 new leprosy cases detected every year and become the 3rd highest number of the leprosy incidence in the world (depkes ri, 2008; who, 2008). theoretically, by treating all leprosy patients will eliminate the source of infection and no more transmission of the disease. but after more than 20 years of mdt program, the new case detection rate has not been reduced and relatively stable. many theories or opinions tried to explain this phenomenon, but the most possibility is the existence of non-human resource of m.leprae as mention in noordeen (1994) also in cree and smith (1998). up to present the bacteria could not be cultivated in artificial media and only growth by in vivo method using animal like mouse foot pad or armadillo. improvement of molecular biology techniques like polymerase chain reaction (pcr) method makes it is possible now to detect a small amount of m.leprae in clinical or environmental specimens. the aim of the study is to detect m.leprae in the daily water resources that used daily by inhabitants in leprosy endemic area using the pcr method, in order to find any correlation of the existence of m.leprae in the water environment with the present of leprosy cases in that area. 66 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 65-68 material and method water samples collection ninety water samples from wells in the house of inhabitants who live in leprosy endemic area of one sub district in the northern part of east java were collected. the prevalence of leprosy in this sub district was 8.02/10.000. based on the information data regarding the user of each well, 48 wells are used by leprosy patients and another 42 wells have never been used by leprosy patients who live in that area. using a sterile plastic bag, around 300 ml of water samples was collected from each well and kept in room temperature. before pcr examination, 50 ml water samples was filtered using millipore membrane filter 0.22 um. membrane washed with pbst 1.5 ml and vortexes 10 minutes. the suspension then centrifuged at 13.000 rpm, 4° c for 20 minutes and pellet formed was used for making a smear for ziehl neelsen (zn) staining and dna extraction. dna extraction the qiagen miniprep kit (research biolabs co) was used for dna extraction from the pellet, following the manual book, to obtain pcr template. pcr examination pcr examination was performed using nested primers: lpf-lpr (lpf: 5'tatcgatgcaggcgtgag tgt3', lpr: 5’ctaacacgatactgctgcac3’) and lp3-lp4 (lp3: 5'tgaggtgtcggcgtggtc3', lp4: 5'cagaaatggtgcaaggga3'). pcr procedure modify from donoghue and spigelman (2001) to detect m.leprae from the specimen. the amplified products were electrophoreses on 3% agarose gel and stained with ethidium bromide 0.1 ug/ml. positive result was presented by a band at 99 bp, as indicated by positive control (m.leprae thai 53 obtained from nude mouse culture in leprosy research centre japan). figure 1. water samples collection figure 2. ziehl neelsen staining of water samples 67wahyuni et al.: mycobacterium leprae in daily water resources results all of the sediments from water samples showed a positive acid fast bacilli (afb) after staining with zn method. some of these bacilli were found inside “amoeba-like” protozoas, some of them were moving microorganisms. positive results were found in 22 out of the total 90 water samples (24%), consists of 11/48 (23%) water that often used by leprosy cases and 11/42 (26%) water from wells that never been used by leprosy patients. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 figure 3. pcr of water samples (lane 1-14: samples, lane 15: negative control, lane 16: positive control, lane 17: 100 bp dna ladder) table 1. pcr results for m.leprae detection and the status of wells well status pcr m.leprae (+) pcr m.leprae (–) total leprosy cases (+) 11 (23%) 37 (77%) 48 (100%) leprosy cases (–) 11 (26%) 31 (74%) 42 (100%) total 22 (24%) 68 (76%) 90 (100%) chi square test: p = 0.909, df = 1, p > 0.05 discussion leprosy is still endemic in three big countries i.e. india, brazil and indonesia. the last two countries have similar climate condition of tropical area like warm temperature and rainy season. this situation causes a typical tropical diversity, where millions of micro organisms are live symbiotically in the environment. it has been known that m.leprae as the causal agent of leprosy, can survive in the soil up to 40 days (chakrabarty and dastidar, 2002). microbiological studies which indicate the existence of m.leprae in the water also has been reported by kazda et al. (1990). this bacterium is an obligate intra-cellular microorganism, which is only growth if the agent lives inside the host cell. since there are many mycobacterium in the environment, special primers needed, because the lp1-lp2 and lp3-lp4 coding 18 kda m.leprae antigen at region rlep3 repetitive element (x17153) recommended by donoghue and spigelman (2001) are to short. we create a new nested primer called lpf-lpr for outer primer (260bp) and used together with lp3-lp4 as inner primer (99bp) (izumi et al., 2008 unpublished). these primers were sensitive and specific for m.leprae. the first report on the existence of m.leprae in the public water resource in north maluku, was reported by matsuoka et al. (1999). using the pcr method, he found 21/44 of public water resources were contaminated by m.leprae. agusni et al (2004) reported the detection of m.leprae in some ponds that be used as water resource of inhabitants who live in leprosy endemic area in northern coast of east java. interestingly, positive results were found more in the root of water plants than in the water collected from the center site of the pond. this finding came to suspicion that the bacilli live in protozoan which are live in the root of many water plants. leprosy cases that often use the well for their daily activities could make the water contaminated with m.leprae. therefore if the bacteria came to water, the pcr examination will be positive. most of leprosy cases that live in this area have been treated by mdt regiment; some of them are already release from treatment (rft). the results of this study showed that 11 out of 48 water samples (23%) from wells that being used by leprosy patients were positive after pcr test to detect m.leprae. but interestingly, m.leprae were also positive in 11 out of 42 (26%) water samples collected from wells that never been used by leprosy cases. statistically, there was no significant difference on the pcr positive results for m.leprae. the daily water supply in this area mostly from the well that is belonged to the family. most of the owner said that the well only used by their family, so they know all the people who use their well. since the list of registered leprosy patients is available in the health centre, the possibility of water contamination in these wells was minimal. mudatsir et al. (2006) reported the genomic study of m.leprae isolates collected from leprosy patients and their environment, using the ttc repeat method. the result concluded that that m.leprae found from leprosy patients, daily water resources and nasal swabs from healthy inhabitants were originated from one population. this means that m.leprae in the environment is one of the links belonged to leprosy problem and not a separate entity. matsuoka (1999) also strongly suggests water as probable source of infection by showing significantly higher leprosy prevalence in water with m.leprae positive samples than in negative one. cirillo et al (1997) reported the survival of m.avium inside protozoa. while jadin (1975) found m.leprae inside amoeba. these finding creates a speculation that m.leprae could also be survived inside the cell of environmental protozoa. if it is true, the following question is “how they can be transmitted to human?”. many reports on the nose swab studies found high prevalence of m.leprae positive in the nasal cavity among people who live in leprosy endemic area. since they are healthy individuals, the bacteria were entered the nasal cavity from dust of the environment during breathing. this means that m.leprae was distributed generally in the environment of leprosy endemic area 68 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 65-68 and contaminates the dust or soil. study by lavania el al. (2006) showed the existence of m.leprae dna in soil of endemic leprosy area in india, continuing study by lavania et al (2008) found viable m.leprae (rna m.leprae). these puzzles should be uncovered by more investigations. conclusion the existence of m.leprae in the daily water resources of inhabitants in leprosy endemic area is not influenced by the presence of leprosy patients who live in the same area. more investigations are needed to find out the role of environmental m.leprae in the transmission of leprosy. acknoledgement this study support by tripujihastuti, dr. and the staffs, also all respondents of the sub district area in northern coast of east java. we also very thankful to lindawati alimsardjono, dr., m.kes., spmk; dr. florentina sustini, dr., ms; dr. e. bimo aksono, drh., m.kes; prof. dr. kuntaman, dr., ms., spmk from medical faculty airlangga university and sri wahjuni, dr., mph from health departement for supervising this study. references: agusni i., izumi s., adriaty d., iswahyudi. 2004. studi mycobacterium leprae dari alam lingkungan di daerah endemik kusta. maj kedokt ind. 58(8): 319–324. cirillo jd., falkow s., stomkins ls. 1997. interaction of mycobacterium avium with environmental amoebae enhances virulence. infect immun. 65: 3759–3767. cree ia., smith wc. 1998. leprosy transmission and mucosal immunity: towards eradication ?. lepr rev. 69: 112–121. departemen kesehatan ri. 2008. profil kesehatan indonesia 2006. depkes ri. jakarta. desikan kv., sreevatsa. 1995. extended studies on the viability of mycobacterium leprae outside the human body. lepr rev. 66: 287–295. donoghue hd., spigelman m. 2001. pcr primers that can detect low level of mycobacterium leprae dna. j med microbiol. 50: 177–182. izumi s., wahyuni r., adriaty d., iswahyudi, agusni i. 2008 unpublished. jadin jb. 1975. amibes limax vecteurs possible de mycobacteries et de m.leprae. act leprol. 59: 57–67. lavania m., katoch k., sachan p., dubey a., kapoor s., kashyap m., chauhan ds., singh hb., sharma vd., jadhav rs., katoch vm. 2006. detection of mycobacterium leprae dna from soil samples by pcr targeting rlep sequences. j commun dis. 38(3): 269–273. lavania m., katoch k., katoch vm., gupta ak., chauhan ds., sharma r., gandhi r., chauhan v., bansal g., sachan p., sachan s., yadav vs., jadhav r. 2008. detection of viable mycobacterium leprae in soil samples: insight into possible sources of transmission of leprosy. infection genetic and evolution. elsevier. 8: 627–631. matsuoka m., izumi s., budiawan t., nakata n., saeki k. 1999. mycobacterium leprae dna in daily using as a possible source of leprosy infection. indian j of lep. 71(1): 61–67. mudatsir. 2006. analisis pola distribusi pengulangan sekuens nukleotida ttc mycobacterium leprae dari penderita kusta dan sumber air penduduk di pulau poteran sumenep, madura. disertasi. program pasca sarjana universitas airlangga. surabaya. world health organization (who). 2008. weekly epidemiological report. 83(33): 293–300. ijtid vol 1 no 2 may-aug 2010.13.pdf ijtid vol 1 no 2 may-aug 2010.14.pdf ijtid vol 1 no 2 may-aug 2010.15.pdf ijtid vol 1 no 2 may-aug 2010.16.pdf 102 vol. 1. no. 2 may–august 2010 research report natural growth factor: platelet rich plasma stimulates proliferation of fibroblast cell culture ernie maduratna setiawati department of periodontics, faculty of dentistry, airlangga university abstract platelet rich plasma(prp) is a source of many natural growth factor that can modulate wound healing. prp has become a valuable adjunct in dentistry. fibroblast cell in ligament periodontal play on important role in periodontal regeneration. the study was performed to investigate the influence prp 10%–100% on the proliferation of fibroblast cell culture in vitro. a fibroblast culture was estabilished from baby hamster kidney (bhk-21) were tested on proliferation was measured with mtt assay after induced prp 10–100%. we showed that prp stimulate the proliferation of fibroblast, prp 80% as the optimal choice to a good enough proliferative stimulus. prp has proven to be effective at improving surgical results in periodontal surgery. prp also show promise in periodontal regenerative medicine. key words: prp, natural growth factor, proliferation of fibroblast introduction in recent years,scientific research and technology has provided a new perspective on understanding healing process to wards regenerative medicine and tissue engineering. growth factor play a crucial role in the healing process. the application of natural growth factor: platelet rich plasma (prp) has been safely used and documented in many fields including: dentistry, orthopedics, neurosurgery, cosmetic and maxillofacialsurgery. based on this principle platelets are introduced to stimulate a supra-physiologic release of growth factor in an attempt to jump start healing in chronic injuries. platelet also release many bioactive proteins responsible for attracting macrophages, mescenchymal stem cells and osteoblast.1,2 tissue repair normally begins with clot formation and platelet degranulation leading to release of various cytokines and coagulation factors, which modulate inflammatory response.to date, more than 30 different cytokines have been found in platelets including pdgfs,tgfs,egf and igf.3 the prp can be prepare by separating from fresh anticoagulated blood by simple centrifugation, which concentrates platelets up to six times the baseline count in whole blood.4 the prp play important role in repairing wounds by providing growth factors and extracellular matrix proteins that attract new cell. platelet rich plasma has become a valuable adjunct in dentistry such as periodontitis therapy. periodontitis is an inflammatory disease which manifest clinically as loss of supporting periodontal tissues including fibroblast periodontal ligament, gingival fibroblast and osteoblast. these changes often lead to an aesthetically and functionally compromised dentition. conventional open flap debridement falls short of regenerating tissues destroyed by the disease.5 clinicians and scientists in dentistry are investigate the use of prp as a way to enhance the body’s natural wound-healing mechanisms and towards attaining complete periodontal regeneration. however,controversies exist in the literature regarding concentration of prp stimulating fibroblast proliferation. the aim of this article are to investigate the influence of platelet rich plasma 10%–100% on the proliferation of fibroblast cell culture in vitro, to determine optimum prp concentrate for fibroblast proliferation. material and methods platelet rich plasma preparation platelet rich plasma was prepared from venous blood obtained from healthy volunteers after obtaining their signed informed consent. blood samples were collected in 103setiawati: natural growth factor 9 ml tubes with trisodium citrate anticoagulant. the blood was first separated into two layers by centrifugation at 272 g for 7 min. the upper layer was collected and centrifuged at 1.288 g for 7 min. prp was collected from the resulting 1–1,5 ml sediment.6 preparation of fibroblast cell culture cell bhk-21 were cultur under dulbecco’s modified eagle’s medium (dmem, cambrex, walkersville) with 4mm l-gluthamine, 0.05 units of penicilin per 0.05 ug of streptomycin served as the basal medium, which was supplemented with 10% fetal bovine serum. when cells reached confluent, cell culture were seeded in well plates. fibroblast proliferation assay fibroblast cell bhk-21 were seeded in 84 well plates in 0,5ml of the mem medium with 2% fbs. when cells reached 30% confluent growth, the medium was washed off and cells were incubated for 24 hours in the mem medium without serum. the medium refreshed before adding prp. twelve different growth condition were tested in the mem medium with prp 10%; 20%; 30%; 40%, 50%, 60%, 70%, 80%, 90%, 100%, positive control, negative control. the effect of different concentrations of prp on cell proliferation was also studied. after 4 to 5 days of incubation, the proliferation of fibroblast measured with an mtt assay (sigma-aldrich chemie). this is a colorimetric assay that measures the chemical reduction of mtt (3-4[4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide) into formazan, which is directly proportional to number of viable cells in the tested culture. cultures were incubated 2-3 hours in a culture medium with 0,5 mg/ml of mtt. the resulting formazan was then eluted with acidified isopropanol (0,04n hcl in isopropanol) and the optical density was measured at 570 nm. proliferation was measured in seven replication. optical densities were expressed as the means +standard deviation. differences between means were assesed by anova, value of p < 0,05 was considered to be statistically significant result 0 0,2 0,4 0,6 0,8 1 1,2 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% k + k od 3-d column 2 3-d column 3 figure 1. proliferation of fibroblast cell culture in the presence 10%–100% prp all component of prp 10%–100% significantly stimulated the growth of fibroblast when compared to the negative control ( figure 1, p < 0,05). fibroblast cell growth was enhanced in a dose dependent manner, but 90% and 100% prp proliferation decreased. prp 80% showed the strongest effect. discussion the wound healing process is a complex mechanism characterized by four distinct, phases: hemostasis, inflammation, proliferation and remodelling. all these events are coordinated by cell interactions and soluble gfs released by various cell type. platelet rich plasma is a biological source of various gfs which stimulate the proliferation of gingival fibroblast, osteoblast, periodontal ligament fibroblast, stromal stem cell, endothelial cell and enhance healing of ulcers. in our study we showed that prp stimulates the proliferation fibroblast cell culture (bhk21) in vitro, because prps contain approximately 7,9 times as many platelets as whole blood and it’s activation was associated with release a large amounts of pdgfab and tgf-beta 1. prp is a rich source growth factors including egf,tgf-beta, igf-1, ang-2 and pdgf. prp may suppress cytokine release,limit inflammation and thereby promote tissue regeneration. tgf-beta in prp stimulated undifferentiated mesenchymal cell proliferation, regulates mitogenic effect. pdgf stimulates chemotaxis and mitogenesis in fibroblast cell. 5 we showed that 80% prp are the strongest effect to stimulate proliferation cell, which is inconsistent with finding report by jeras et al 5 ,they showed that 20% prp have strongest effect in human dermal fibroblast, because they could not measure higher concentration with mtt. weibrich et al observed an advantageous with platelet concentrations, they state that higher concentrations might have a paradoxically inhibitory effect. schanabel evaluated collagen content, prp at 100% concentration stimulated the greatest collagen type 1, collagen type iii without increasing expression of the proinflammatory matrix metalloproteinases. prp led to significantly increased level of growth factors and suppressed inflammation by promoting secretion of lxa4. the use of natural and autologous growth factor reduces the risk of transmissible infection and alergic reaction. in our study, we did not observe any cytotoxic effect in high concentration of prp. prp is no plausible mechanism by which growth factor result in neoplastic development, because growth factor act on cell surface receptors, do not enter the cell and do not cause dna mutation. bieback6 show that prp have significantly higher proliferative effect on mescenscymal stem cell (msc) providing 1320–1400 fold expansion in 11 days compared table 1. proliferation of fibroblast cell culture in the presence 10%–100% prp prp 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% k+ k– od 0,61 0,688 0,55 0,82 0,96 o,67 1.1 1,15 0,74 0,66 0,58 0,28 104 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 102-104 with only 254-fold expansion with fetal calf serum. the fact that prp as alternative supplements trigger for msc expansion in clinical setting, since the large number of msc required for tissue engineering. this finding could be useful in preparing prp in advance and for storage in a –70° c for multiple apllications. in conclusion, we show that prp enhance proliferation of fibroblast cell culture. we promote 80% prp as the optimal choice to a good enough proliferative stimulus. therefore, we are currently adressing this issue in molecular and functional experiments. acknowledgments we thank to pusvetma surabaya, especially dr erna. this work was supported by a research fund of the phki faculty of dentistry, airlangga university and hibah bersaing referrence 1. roach r b and carlson n, 2002. platelet rich plasma: clinical applications in dentistry. jada.133: 1383–86. 2. sampson s, gerhardt m, mandelbaum b, 2008. platelet rich plasma injection grafts for musculoskeletal injuries: a review. curr rev musculoskelet med. 3. choung p, seo b, lee l. 2006.the effect of platelet rich plasma on proliferation of dental stem cells derived from human tooth. tissue eng and reg medicine, 3: 440–44. 4. gagnon g, martineau i, frechete jp.2005. platelet rich plasma: growth factor content and roles in wound healing. j dent res. 84: 434–39. 5. jeras m, svajger u, krasna m. 2007. platelet gel stimulates proliferation of human dermal fibroblast in vitro. acta dermatoven apa, 16: 105–9. 6. bieback k, kluter h, kern s. 2007. human ab serum and thrombinactivated platelet rich plasma are suitable alternatives to fetal calf serum for the expansion of mscs from adipose tissue. stem cells. 25: 1270–78 ijtid vol 1 no 2 may-aug 2010.50.pdf ijtid vol 1 no 2 may-aug 2010.51.pdf ijtid vol 1 no 2 may-aug 2010.52.pdf 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 90 vol. 1. no. 2 may–august 2010 antibiotic resistance control program in pediatric hematology and oncology patients at dr. soetomo hospital in 2006–2007 mia ratwita andarsini, i dewa gede ugrasena, bambang permono division of hematology and oncology department of child health,medical faculty,airlangga university-dr. soetomo hospital correspondence: mia ratwita andarsini, dr, spa, e-mail: miaratwita_spa@yahoo.com telp: 0315501688; fax: 0315501748. abstract antibiotic resistance has been increasing since the first years of the clinical usage. it caused by inappropriate usage and uncontrol of antibiotic drugs. therfore an antibiotic resistance control program (arcp) is needed to overcome the problem. the purpose of this study is to know microorganism pattern and evaluate antibiotic use. phase 1 (before arcp), retrospective study by medical record of pediatric hematology-oncology patients with suspision of infection and admitted at dr soetomo hospital from june–august 2006 was carried out. phase 2 (during arcp), a prospective observational study was done from november 2006 to january 2007. we were evaluated the isolated microorganism, quantity of antibiotic were determined by defined daily doses (ddd)/100 patients-days, quality of antibiotics usage were assessed with glyssen classification, and the cost calculation of antibiotic therapy. twenty seven patients were enrolled in phase 1 and 28 patients in phase 2. coagulase-negative staphylococci and acinetobacter sp as isolated microorganism was reported. phase 1, the most sensitive antibiotic was cefoperazone-sulbactam and the most resistant was penicillin g. phase 2, meropenem was the most sensitive antibiotic and cotrimoxazole was the most resistant antibiotic. the use of antibiotics were decreased 6 vs 12 and ddd/100 patients-days were 14.52 vs 45.04. there were improving of glyssen classification. the cost calculation of antibiotics therapy were decreased. arcp can improve antibiotic use in pediatric hematology-oncology patients. keywords: pediatric hematology-oncology, antibiotic resistance control program, antibiotic evaluation introduction antibiotic resistance is one of the health problem in the world. uncontrolled and inappropriate of antibiotic use can increase morbidity and mortality, and also give impact in the quality of health services.1,2 antibiotic resistance appear through selection process because of antibiotic overuse and quickly spreading through human contacts.3 today, there is many program in the world to overcome antibiotic resistance spreading.4 study of antimicrobial resistance in indonesia (amrin study) at dr. soetomo hospital was done in 2000– 2004. antibiotic resistance and inappropriate antibiotic use was found, on the other hand infection control has not been done properly.5 as follow up, antibiotic resistency control program (arcp) was performed with pediatric hematology oncology division as a pilot project. the purpose of this study is to know microorganism pattern and evaluate antibiotic use. methods the study consisted of 2 phase. in phase 1 (before arcp), retrospective study was done through medical record during june-august 2006. in phase 2 (during arcp), prospective study was done in november 2006–january 2007. pediatric hematology oncology patients who were admitted with suspicion of infection were subject of the study. inclusion criteria were body temperature >38o c or < 36o c, lekocyte count > 12000/cmm or < 4000/cmm, presence of takicardia and/or takipneu. patients who met the criteria were enrolled the study. in phase 2, will follow antibiotic guidance below (figure 1). data study were include patients’ characteristic and diagnosis, microorganism isolate from the cultures and result of antibiotic sensitivity tes. antibiotic usage were evaluated quantitatively with defined daily dose (ddd)/100 patients-days and qualitatively with gyssen case report 91andarsini et al.: antibiotic resistance control program classification.6 the ddd represent the average therapeutic dose for an adult for the standard indication. the cost analysis was also evaluated. results and discussions twenty seven patients were enrolled in the phase 1, with average age 101,22 (33–108) month-old and average length of stay 29,7 (8–69) days. in phase 2, 28 patients enrolled the study, average age 54,64 (8–69) month-old and average length of stay 29,5 (6–84) days. more than 50% were acute lymphoblastic leukemia patients. in phase 1, 27 blood cultures was collected, positive results were only found in 17 (37%) cultures. meanwhile in phase 2, 75 cultures were collected, consisted of 39 blood culture, 19 urine cultures and 7 fecal cultures. positive results found in 24 (32%) cultures. similarly study shiwed that positive culture only found in 11 put of 67 patients (16,4%) with febrile neutropenia.7 coagulase-negative staphylococci was found in 50% blood culture in phase 1 and 44,4% in phase 2. microorganism isolated in urine cultures were e coli esbl, klebsiella pneumoniae, enterobacter aerogenes, pseudomonas and acinetobacter sp. e coli pathogen serotype 1–11 were found in all of the fecal cultures. al-ahwal study showed that 5 out of 11 positive cultures were due to gram-positive microorganism (coagulasenegative staphylococci and staphylococcus aureus), 5 due to gram-negative microorganism (e coli, klebsiella and p aeruginosa).7 in malignancy patients, the primary anatomic site of infecton is the gastrointestinal tract, where mucosal damage from chemotherapy allows invasion fo microorganism. damage to the skin from invasive procedures, such as intravascular devices, similarly provides portals of entry for microbes. bacterial pathogens commonly implicated in neutropenic fever are gram-positive microorganism (staphylococcal sp, streptococcus sp, enterococcal sp and corynebacterium sp) and gram-negative microorganism (e coli, klebsiella sp, pseudomonas aeruginosa, enterobacter sp and acinetobacter sp).8 sensitivity tes was perfomed. in phase 1, sensitive antibiotics were cefoperazone sulbactam, netilmycin and gentamycin. in phase 2, the result were change into meropenem, ciprofloxacin, piperacillin tazobactam and amikacin. resistent antibiotics in phase 1 were penicilin g, erithromycin and cotrimoxazole. in phase 2, cotrimoxazole was still resistent followed by cefotaxime and ceftriaxone. quantitative and qualitative antibiotic evaluation were done in both phase. quantity of antibiotics usage were determined by counting ddd/100 patient-days (table 1). table 1. quantitative antibiotik evaluation antibiotics ddd/100 patient-days phase 1 phase 2 cefotaxime meropenem amikacin ceftazidime cloxacillin cefepime ciprofloxacin ceftriaxone clindamycin cotrimoxazole gentamycin cefoperazone sulb. 10.6 3.6 2.7 3,0 7.66 0.35 6.3 1.34 0.37 7.15 0.25 1.72 7.84 3.40 0.50 0.40 -0 -0 -0 -0 -0 1.50 -0 0.88 total 45.04 14.52 fiuture 1. antibiotic guideline during antibiotic resistance control program (arcp) new patients inclussion criteria temperature > 380c or < 360c leukocyte count > 12.000/mm3 or < 4.000/mm3 takikardi (hr > normal for age) takipnea (rr > normal for age) yes blood/urine culture empirical antibiotic cefotaxim 3 days evaluation no without antibiotic fulfill inclussion criteria improved (+) continued until 7 days improved ( ) meropenem or based on culture result 3 hari evaluation improved (+) continued until 7 days improved ( ) alternative antibiotic based on culture result 3 days evaluation improved (+) continued until 7 days perbaikan ( ) add antifungal (fluconazole) 3 days evaluation improved (+) continued until 10 14 days improved ( ) repeat blood/urine culture 92 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 69-68 quantity of antibiotics usage was decreased from 12 to 6 type of antibiotics. ddd/100 patient-days calculation was also decreased from 45,04 (phase 1) to 14,52 (phase 2). the main problem with ddd is that ddd was made for adults, therefore the result of this study can not be compared with other study in adults. table 2. quality antibiotic evaluation classification phase 1 phase 2 i (definitely appropriate) iia (improper dosage) iib (improper dosage interval) iic (improper route) iiia (excessive length) iiib (duration too short) iva (more effective alternative agent) ivb (less toxic alternative agent) ivc (less expensive alternative agent) ivd (less broad spectrum alternative agent) v (unjustified) vi (record insufficient for categorization) 22% 0% 0% 0% 46% 1.6% 4.7% 0% 9.6% 11.1% 5% 0% 38% 0% 0% 0% 30.2% 1.6% 11.1% 0% 11.1% 3.2% 4.8% 0% quality of antibiotic used were assessed with gyssen clasification (table 2). there was decreased of procentage were found in classification iiia, iiib, ivd and v. increased of procentage was found in clasification i, iva and ivc. similar study by gyssen in surgery ward showed improvement or quality antibiotics usage. there were increased of category i from 31% to 47% and was decreased of category v form 16% to 8%.9 the cost analysis was calculated including cultures and antibiotics usage. this study showed that implementation arcp could save around rp 13,135,000 for 27 patients. gyssen study also found total cost saving of 11% after intervention.9 conclusion from this study we conclude that antibiotic intervention trough arcp resulted in an improvement of the quantity and quality of antibiotic regimen and in term of costs. references 1. d. widodo, “kebijaksanaan penggunaan antibiotika bertujuan meningkatkan kualitas pelayanan pasien dan mencegah peningkatan resistensi kuman”, cermin dalam kedokteran, vol. 37, no. 1, 2010, pp. 7–10. 2. a. sadiumenge, e. diaz, a. rodriguez, l. vidaur, l. canadell, m. olona, “impact of diversity of antibiotic use on the development of antimicrobial resistance”, j antimicrob chemother, vol. 57, 2006, pp. 1197–204. 3. el. larson, d. quiros, t. giblin, s. lin, “relationship of antimicrobial control policies and hospital and infection control characteristic to antimicrobial resistance rates”, am j crit care, vol. 16, 2007, pp. 110–20. 4. le. nicholle, ïnfection control programmes to contain antimicrobial resistance”, available at www.wpro.who.int, accessed on july 2010. 5. u. hadi, do. duerink, es. lestari, nj. nagelkerke, s. werter, m. keuter, et al, “survey of antibiotic use of individual visiting public healthcare facilities in indonesia”, available at https://openaccess. leidenuniv.nl/bitstream/1887/13821/8/03.pdf, accessed on july 2010. 6. ic. gyssens, pj. van der broek, bj. kullberg, ya. hekster, jwm. van der meer, “optimizing antimicrobial therapy. a method for antimicrobial drug evaluation”, j antimicrob chemother, vol. 30, 1992, pp. 724–7. 7. ms. al-ahwal, “pattern of febrile neutropenia in solid tumor – a hospital based study”, park j med sci, vol. 21, no. 3, 2005, pp. 249–52. 8. s. kannangara, “management of febril neutropenia”, commun oncol, vol. 3, 2006, pp. 585–91. 9. ic. gyssen, iej. geerligs, jmj. dony, ja van der vliet, a. van kampen, pj. van den broek, et al, “optimising antimicrobial drug use in surgery: an intervention atudy in a dutch university hospital”, j antimicrob chemother, vol. 38, 1996, pp. 1001–1012. ijtid vol 1 no 2 may-aug 2010.38.pdf ijtid vol 1 no 2 may-aug 2010.39.pdf ijtid vol 1 no 2 may-aug 2010.40.pdf 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 118 vol. 1. no. 3 september–december 2010 urine specimen in detection of hepatitis a virus antibody in children bagus setyoboedi, maretha sukmawardani, sjamsul arief department of child health dr. soetomo hospital school of medicine airlangga university surabaya indonesia abstract hepatitis a is still a problem in developing country, especially in indonesia. twenty-three thousand cases per year in the world were reported by cdc in 1998. detection of hepatitis a virus (hav) antibody needs blood specimen, for which the drawing procedure is quite unpleasant for children. the purpose of present study is to examined the use of urine as an alternative specimen compared to blood in the detection of hav antibody. a cross sectional study on children age 5–12 years who lived in rusun sombo district simokerto surabaya was performed in 2007. blood and urine specimens were taken from all subjects, further tested with microparticle enzyme immunoassay (meia) method using axsym® havab® 2.0. a total 74 children were included in the study, 43 (58.1%) were boys. seropositive was found in 38 (51.4%) children and uropositive in 3 (4%) children. there are significant differences between blood and urine specimen, with sensitivity 7.9%, spesificity 100%, ppv 100% and npv 50.7%. urine could not replace blood specimen as the gold standart for the detection of hepatitis a antibody in children key words: hepatitis a, antibody, urine, blood specimen, meia case report background hepatitis a is an endemic disease in developing country and it has close relationship with hygiene, sanitation, and viral transmission factors. center for disease control and prevention reported 23,000 cases of hepatitis a every year worldwide, with peak age 5 to 14 year.[1] sulaiman (1993) found seroprevalence of anti-hav at papua was 100% in 4 year-old children. in jakarta, bandung, and ujung pandang, the seroprevalences were between 35–45% in 5 year-old children.[2] dr. soetomo hospital had 23 cases of hepatitis a during 1999–2004 (unpublished data). the process of taking blood sample from children who appeared healthy is somewhat hesitating especially in infant, considering that it needs agreement from parents and also qualified person to draw the specimen. therefore, people search for a non-invasive solution using alternative body secretion in antibody detection for many diseases. urine is an alternative solution, since it contains immunoglobulin even in low concentration and serologic assay could perform without centrifugation process with class-specific antibody capture assay. this assay has the ability to catch specific immunoglobulin in low concentration and to confirm diagnosis at the beginning of the disease.[3,4] total anti-hav detection (igg and igm) is used to define immune status. positive result of anti-hav indicates infection or long exposure of hepatitis a that stimulate immunity. this assay can determined immune status in natural infection or caused by vaccination.[5] this research was held at rusun sombo, a living location with very crowded population and low socialeconomic condition. kitchen and toilet were shared together among several families, causing easier transmission of hepatitis virus. clean water facility and waste product handling were not good. on the location, we tried to make a similar condition on social-economy status, hygiene and sanitation between samples. the purposes of this research are to study the use of urine specimen for non-invasive detection of hepatitis a antibody (anti-hav) and to study epidemiology of children with anti-hav positive in surabaya. sample and method this is a cross sectional study involving healthyappeared children age between 5–12 years in rusun sombo district simokerto surabaya. subjects were taken by 119setyoboedi et al.: urine specimen in detection of hepatitis a virus consecutive sampling. the research was held at septemberoctober 2007. we collected 2.5 cc of blood from cubiti vein and 2.5 cc of fresh urine in the morning. both specimens from every subject were tested with meia (microparticle enzym immunoassay) serologic assay to detect anti-hav level. result was analyzed with mcnemar test and kappa agreement. result we collected 86 subjects met inclusion criteria, 12 were dropped out due to sample damage, with a total of 74 subjects. from data analysis, 74 subjects consisted of 43 boys (58.1%) and 31 girls (41.9%), seropositive was found in 38 children (51.4%) and uropositive in 3 children (4%). thirty-one children (41.9%) age 10–12 years, 67 children (90.5%) were in elementary school. based on the education of their parents, 28 children (37.8%) had parents graduated from elementary school. socioeconomy status was dominated with middle-low status in 39 children (52.7%). sixty children (81%) had their parents worked as pedicab driver, “junkman”, market trader, etc). almost 68 children (91.9%) stated that they never had history of jaundice before, and 71 children (96%) had no contact with jaundice patients in last 6 months. sixty children (81%) regularly washed their hand before eat, and 68 children (91.9%) usually buy non-home-cooked meal or eating outside, 58 children (78.4%) liked to eat well-cooked meal. almost every child stated that they never had contact with jaundice patients or had that disease before. however, hepatitis a in children is an asymptomatic disease. the environment condition in rusun sombo may support transmission of viral infection because they usually shared a same kitchen and toilet together on public facilities among 3–5 families. all subjects stated that they never had hepatitis a immunization before. each family lived in a 30 m2 house with ceramic floor. about 33 children (44.6%) lived in a house with 4 persons with pdam as clean water facility. almost 73 children (98.6%) were nursed by their parents. some children from 12 families in this research live together in the same house and neighbored with 5 families which were seropositive. based on nutritional status, we found 57 children (77%) with well-nourished condition, 4 children (5.4%) with malnourished condition, 8 children (10.8%) with undernourished condition and 5 children (6.8%) in overweight condition. seropositive result was found commonly in higher age between 10-12 year-old (19 children), and according to education level, seropositive result was more common in elementary school children (35 subjects). based on parents� education level the most common seropositive result was found in family which their parents graduated from table 1. characteristic of subjects characteristic total subjects n % sex: boys girls 43 31 58.1 41.9 age: 5–7 years old 8–9 years old 10–12 years old 19 24 31 25.7 32.4 41.9 education: kindergarten elementary school junior high school 5 67 2 6.8 90.5 2.7 parents� education: no education elementary school junior high school senior high school university 3 28 21 20 2 4 37.8 28.8 27 2.7 income: < rp. 750.000 rp. 750.000-4.000.000 > rp. 4.000.000 25 39 10 33.8 52.7 13.5 history of jaundice: yes no 6 68 8.1 91.9 history of contact with jaundice patients: yes no 3 71 4 96 hand washing: regularly sometimes never 60 13 1 81 17.6 1.4 eating outside/non homecooked meal: yes no 68 6 91.9 8.1 uncooked meal: yes no 16 58 21.6 78.4 hepatitis a immunization: yes no 0 74 0 100 number of person living together: 4 persons 5–6 persons >7 persons 33 30 11 44.6 40.5 14.9 nutritional state: malnourished under-nourished well-nourished overweight 4 8 57 5 5.4 10.8 77 6.8 120 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 118-121 elementary school (20 children) and 11 children (28.9%) had parents graduated from junior high school. seropositive result were common in family with middle to low familyincome, about 14 children (36.8%) with income family < 750.000 rupiahs/month and 20 children (52.6%) had family income ranged from 750.000 rupiahs/ month up to 4 million rupiahs/month. based on history of jaundice in seropositive subjects, the majority of subjects never had the disease before (36 children or 94.7%) and all samples had no contact with jaundice patients before in last 6 months. good hand washing habits was found in 31 children (81.6%), with 35 (92.15) admitted usually buy food outside their house, only 7 children admitted like to eat unwell-cooked meal from sea product. seropositive result found commonly in well-nourished children, eventhough some literature stated that in children with malnourished condition usually had low level of immunoglobulin or antibody therefore seronegative result gained, but in this research we found 1 malnourished child with seropositive result. uropositive result was found only in 3 children, 2 girls and 1 boy. chi square test was performed and found only one variable (parents� income) with positive association with anti-hav level, where other variables had negative association. these results may be due to sampling process since we already make homogenous conditions of some characteristics such as socio-economy and environment which actually could influence hepatitis a viral transmission. in the other hand we must also consider the difficulties in objectivity of parents� disclosure in answering all questions which could interfere the result. mcnemar test was used to compare differentiation between two different independent tests in order to detect antibody level of hepatitis a, using anti-hav serum as gold standard compared with anti-hav level in urine. kappa value in this study was 0.53. table 2. statistical analysis of serology using with mc nemar test serology test anti hav serum total anti hav urine + + 3 0 3 35 36 71 total 38 36 74 the result from mcnemar test refused ho which meant that there were significant difference between this two tests in order to detect anti-hav level in children. based on kappa value we found that two tests had no good agreement. we had 7.9% of sensitivity and 100% specificity with ppv 100% and npv 50.7%, which described that only 7.9% urine samples could detect antibody to hav and that positive result from urine can 100% exclude the negative result. ppv 100% means that the probability of subjects to have anti-hav based on uropositive test result were 100% and npv 50.7% means that probability of subjects to have no anti-hav based on uronegative test result were 50.7%. discussion study about anti-hav level in rusun sombo district simokerto surabaya had found 51.4% children between 5–12 years old with seropositive result. this study tried to compare between blood (as a gold standart) and urine as specimen to detect anti-hav level in healthy-appeared children with the result a significant difference between those two specimens. some literatures mentioned that iga, igm, and igg can be found in external secretion of mucose of human body, such as in urine, saliva. it bounds to secretory component (sc), usually with fcrn. therefore antibody to hav can be found or can be detected too even in other body secretions like urine, even in low level compared to blood serum.[6-10] since the level in urine is very low, it needed a sensitive serologic test with elisa.[11,12] the natural infection of hepatitis a actually was not at mucosal site of the human bladder, but with homing mechanism of the body, secretion of antibody happened in other site of external body mucose.[6–10] this study was performed in healthy-appeared children, based on the theory that igm could be present or positive in external secretion until 3–6 months after infection and igg may persist for years.[3,13–16] the result of this study revealed 38 children with seropositive and 3 children with uropositive result. since level of antibody in mucosal secretion is very low, and even with very sensitive serologic test, the result revealed very low positif result and showed significant difference. therefore the use of urine as specimen for detecting antihav was not applicable. the result revealed sensitivity 7.9% and specificity 100% with positive predictive value (ppv) 100% and negative predictive value 50.7%, which means its sensitivity showed true positive result from all positive result was 7.9% and spesificity showed true negative result from all test that negative was 100%, therefore urine specimen which used as specimen showed big false negative result. ppv 100% showed probability of the sick from the positive result were 100%, with npv which showed probability of the health from the negative result were 50.7%. it means urine specimen can not replace blood specimen as gold standart for detecting anti-hav level in epidemiologic study. this result was different from the study by joshi (2002) and rodriguez (2003),[3,4] probably due to the very low level of antibody from past infection in body secretion that can not passed through level of detection of the serologic test resulting in negative result in our subjects. 121setyoboedi et al.: urine specimen in detection of hepatitis a virus summary based on the result of this study, the application of urine specimen in detection of anti-hav level can not be used. compared to blood specimen as gold standart, it showed a significant difference. references 1. rizzetto m. viral hepatitis. in: bircher j, benhamou jp, mcintyre n, rizzetto m, rodes j, editors. oxford textbook of clinical hepatology. 2nd ed. oxford: oxford university press, 1999. p. 827–37. 2. sulaiman ah, julitasari. hepatitis a. in: sulaiman ah, julitasari, editors. virus hepatitis a sampai e di indonesia. 1st ed. jakarta: musady, 1995. p. 1–15. 3. joshi ms, chitambar sd, arankalle va, et al. evaluation of urine as a clinical specimen for diagnosis of hepatitis a. clin diag lab immunol 2002; 10: 840–5. 4. rodriguez la, diaz ol, casanueva rm, et al. anti-hepatitis a virus immunoglobulin m antibodies in urine samples for rapid diagnosis of outbreaks. clin j diag lab immunol 2003; 10: 492–4. 5. lee c, ghadessi m, lei jd, hall g, sustarsic d, el shami as. detection of total antibodies to hepatitis a virus (hav) by immulite® and immulite® 2000. 2003. dpc scientific poster. 6. tomasi tb, larson l, challacombe s, mcnabb p. mucosal immunity: the origin and migration patterns of cells in the secretory system. postgraduate course presentation 1980; 1(65): 12–9. 7. roitt i. antibodies. in: roitt i, brostoff j, male d, editors. 6th ed. mosby, 2001. p. 65–85. 8. spiekermann gm, finn pw, ward es, durmont j, dickinson bl, blumberg rs, et al. receptor-mediated immunoglobulin g transport across mucosal barriers in adult life: functional expression of fcrn in the mammalian lung. j exp med 2002; 303–10. 9. mayer l. mucosal immunity. pediatrics 2003; 111(6): 1595–600. 10. woof jm, mestecky j. mucosal immunoglobulins. immunological reviews 2005; 206: 64–82. 11. lemon sm, binn ln. serum neutralizing antibody response to hepatitis a virus. j infect dis 1983; 148(6): 1033–9. 12. cole m, perry k. hepatitis a serological assay: a review of evaluation literature. http://www.pasa.nhs.uk/cep (accessed at march, 2006) 13. sherlock s, dooley j. virus hepatitis. in: sherlock s, dooley j, editors. diseases of the liver and biliary system. 9th ed. london: blackwell scientific publications 1993. p. 266–9. 14. bisanto j. permasalahan penyakit hepatitis virus di indonesia. in: zulkarnain z, bisanto j, pujiarto ps, oswari h, editors. tinjauan komprehensif hepatitis virus pada anak. 1st ed. jakarta: balai penerbit fk-ui 2000. p. 9–31. 15. el-kamary ss. in brief: hepatitis a. peds in rev 2003; 26(2): 75–6. 16. lemon sm. type a viral hepatitis: epidemiology, diagnosis and prevention. clin chem 1997; 43: 1494–9. ijtid vol 1 no 3 sep-dec 2010.16.pdf ijtid vol 1 no 3 sep-dec 2010.17.pdf ijtid vol 1 no 3 sep-dec 2010.18.pdf ijtid vol 1 no 3 sep-dec 2010.19.pdf 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 � vol. 4. no. 4 october–december 2013 research report comparative study on the intensity of mycobacterium leprae exposure to children who live in low and high altitude in low leprosy endemic area of south sulawesi rachmawati1, timurleng1 tonang mataallo1, safruddin adam1, a.m. adam1, safruddin amin1, farida tabri1, dinar adriaty2, ratna wahyuni2,iswahyudi2, indropo agusni2 1 dept, of dermato-venereology, hasanuddin medical faculty, makassar 2 leprosy study group, inst. of tropical disease, universitas airlangga, surabaya abstract background: the intensity of mycobacterium leprae exposure to people who live in leprosy endemic area could be measured by serological study and detection of the bacilli in the nose cavity. different geographical altitude might have some influences to this exposure since the bacilli prefer to live in warm areas. aim: a combined serological and pcr study of leprosy was conducted in selayar island, south sulawesi to 80 school children (40 from low land and 40 from highland altitudes) in order to compare the exposure intensity between the two areas. method: anti pgl-1 igm antibody (elisa) and pcr study to detect m.leprae in the nasal cavity were performed simultaneously from each person. result: seropositive cases were found in 23/40 children from low land compared to 16/40 children from high land, but statistically no significant difference (p>0.05). pcr positive for m.leprae in the nasal cavity only found in 1/40 children, both in low and high altitude. conclusion: it is concluded that although the existence of m.leprae in nasal cavity is minimal, the intensity of exposure to this bacilli still high as indicated by serological study. key words: leprosy, serology, pcr, children, low and high land abstrak latar belakang: intensitas paparan m.leprae terhadap penduduk yang tinggal di daerah endemik kusta dapat diukur dengan uji serologi dan deteksi kuman m.leprae di mukosa hidung. tujuan: perbedaan ketinggian geografis dapat memberikan pengaruh terhadap proses tersebut karena kuman kusta lebih menyukai daerah yang lebih hangat. metode: telah dilakukan studi serologi dan pcr di pulau selayar, sulawesi selatan terhadap 80 murid sekolah (40 anak dari dataran rendah dan 40 anak dari dataran tinggi), dengan tujuan untuk membandingkan intensitas paparan diantara kedua daerah. hasil: antibodi igm anti pgl-1 (elisa) dan studi pcr untuk mendeteksi m.leprae di mukosa hidung diambil secara bersamaan dari tiap-tiap anak. ditemukan hasil sero positif pada 23/40 anak dari dataran rendah dibandingkan 16/40 anak dari dataran tinggi, namun tidak ada perbedaan bermakna diantara keduanya (p > 0,05). pcr positif terhadap m.leprae di mukosa hidung hanya ditemukan 1/40 anak baik dari dataran rendah maupun dataran tinggi. kesimpulan: hal ini berarti walaupun eksistensi m.leprae di mukosa hidung sedikit, intensitas paparan kuman m.leprae tinggi ditunjukkan dari hasil serologi. kata kunci: kusta, serologi, pcr, anak-anak, dataran rendah dan dataran tinggi. � indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 1-4 introduction leprosy is still a public health problem in south east asia, including indonesia. although the elimination target of leprosy in indonesia has been reached in 2001, some pocket areas of leprosy still exist up till now.1 the selayar island district in south sulawesi is an area with a lower prevalence of leprosy, compared to the surrounding areas which have high prevalence of leprosy.2 based on the assumption that this area might has a low transmission of leprosy, a study of leprosy exposure to inhabitants of such area will show a low level. since m.leprae is known as a bacilli who prefer to live in relatively colder area of the body, a different geographical altitude might have also influence the exposure of the bacilli. after the m.leprae enter the body, an immunologic response will be developed and specific antibody to m.leprae will be produced (anti pgl-1 antibody). the level of this antibody is reflected the antigenic load of the bacilli.3. the leprosy bacilli enter the body via respiration tract and the detection of m.leprae in the nasal cavity could be performed by pcr study.4 the level of seropositivity and the presence of m.leprae in nasal cavity could be used as an indicator for leprosy exposure intensity in endemic area. the aim of this study is compare the exposure of m.leprae to inhabitants who live in low and high lands of pulau selayar area of south sulawesi, using specific serological indicators for leprosy and the presence of m.leprae in the nasal cavity by pcr method. material and methods fourty healthy school children from kahu-kahu village, selayar island, who live in low land and another 40 healthy school children from lembang matene village (high land) aged 9–12 years old were involved in the study (figure 1). leprosy contact history was taken to exclude contact individuals and clinical examination was conducted to exclude leprosy patients. blood and nose swab samples from these children were collected simultaneously. serological study from each children 100ul capillary blood was obtained by a needle puncture to finger tip, dried on a small filter paper and sent to leprosy lab of institute of tropical disease, airlangga university, surabaya. dried blood on filter papers were dissolved in 1 ml of bsa buffer and 10ul of the solution were taken for indirect elisa test to measure the level of anti pgl-1 antibody. using n dioctylbsa as an antigen, the level of igm anti pgl-1 antibody were measured follows the elisa procedure recommended by patil.5 the elisa results in optical density (od) were converted to unit/ml using the biolise computer software. three ml of peripheral blood were also collected from cubitous venes in 10 children, to measure the real blood level of anti pgl-1. after conversion to serum level, using cut off level 605 u/ml, sero-positive case was detected.6 figure 1. geographical area of selayar island, south sulawesi. figure 2. indirect elisa �rachmawati, et al.: comparative study on the intensity of mycobacterium leprae pcr study from nose swab samples nose swab sample was also taken simultaneously from the same children after collecting finger tips blood for serological sudy. the nose swab samples were kept in freeze condition until ready for pcr study. dna extraction was performed using the miniprep qiagen kit. using the lp1-lp4 nested primers, the m.leprae dna was detected by pcr, following the procedure recommended by plikaytis.7 positive pcr results is indicated by a band of 99 bp in agarose gel field, as pointed by positive control (figure 3) results clinical examination of all children revealed no sign of leprosy. based on the cut off value 605 u/ml for igm anti pgl-1, sero-positive cases was observed in 23 out of 40 (57,5%) children from low land compared to 16 out of 40 (40%) children from highland. statistically no significant difference between the two groups (p > 0.05). after pcr study from the nasal swabs samples, only one out of 40 samples from low land group show pcr positive, similar with the results of nose swabs samples from highland group (1 out of 40 samples or 2.5%). no statistical difference in the pcr results between the two groups. discussion this study was conducted in an area wich is reported as a “low endemic of leprosy” and start with an assumption that the transmission of the disease mainly from the environment. the humoral response to m.leprae is represented by specific antibody to cell wall of the bacilli which contains phenolic glycolipid-1 (pgl-1). this antibody is not protective to leprosy, but can be used as a marker of immune response to the presence of the bacilli.9 the level of this specific antibody is corresponded with the amount of antigen or bacilli in the body, which means high level of antibody indicates many bacilli inside the body.10 in this study, almost half of the school children have already showed sero-positive for leprosy. in this case, the cut off value (> 605 u/ml) used in the study was based on the previous serological study in east java.11 a new calculation for measuring the cut off value for south sulawesi is needed. although the selayar island is relatively low endemic for leprosy, it seems that the children who live in this area still exposed to leprosy bacilli quite often. the source of the bacilli might be from leprosy cases who are still not found by surveillance (“the back-log cases”) or from nonhuman resource of m.leprae in the environment.12,13 after tracing the sero-positive children by analyzed the history of contact with leprosy cases, it revealed that most of the children have no contact history with leprosy patients. high percentage of seropositivity to leprosy in south sulawesi area seems to be common in many parts of this province. since the island has a long coastal area, it would be possible also that many inhabitants use to go to other surrounding area in south sulawesi as a sailor and contact with many leprosy patients from other area. nasal swabs examination have been used to detect the port of entry of the lepra bacilli from inside or from outside or of the body.14,15 the pcr study for detection of m.leprae in the nasal cavity only showed positive in single case from both groups. this minimal results could be correlated with the season during collecting nose swab samples. the study was conducted during rainy season, probably this cause many lepra bacilli were cleaned up from the air and less bacilli aspirated by these children. this reason might explains why the nasal swabs mostly negative in the pcr study, beside the technical eror during sample collection or laboratory work. it is recommended to repeat the nasal swabs collection during the dry season, when the air might be more contaminated with lepra bacilli. the percentage of pcr positivity from nose swabs samples from this study was 2.5%, lower than previous study in other area of south sulawesi.16 the serological results of this survey shows that transmission of m.leprae is still intense, but pcr results from nose swabs does not support the hypothesis that transmission is occurred via the nasal cavity. why the percentage of leprosy sero-positivity among these children were high is still a question and more environmental investigation are needed. conclusion low endemic of leprosy does not means less intensity of exposure of m.leprae and need more environmental study of leprosy to find out the source of the transmission of the disease in this area. figure 3. pcr results for detection of m.leprae dna using lp1 – lp-4 nested primers sample no. : 1 2 3 4 5 6 7 nc pc ladder (100bp) � indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 1-4 references 1. smith wcs. 2011. epidemiology of leprosy. in (makino m eds) leprosy. science working towards dignity. tokai univ. press. pp 26–34. 2. dinkes prop. sulawesi selatan. 2009. profil kesehatan propinsi sulawesi selatan tahun 2009. dinkesprop. sulawesi selatan. 3. harboe m (1994). overview of host parasite relation. in hasting r (ed). leprosy. churchil livingstone. 4. gillis, t. p., and d. l. williams. 1991. polymerase chain reaction and leprosy. int. j. lepr. other mycobact. dis. 59: 311–316. 5. patil sa, ramu g, shinha s, senguta u. 1990. screening of anti m.leprae antibodies in the blood sample eluted from filter paper blood blots. int. jlepr; 58(1): 123–26. 6. prakoeswa crs, listiawan my, adraty d, wahyuni r, isahyudi, agusni i. 2007. the use of capillary blood dried on filter paper for measuring the anti pgl-1 antibodies in leprosy patients. annual scientific meeting perdoski, surabaya, 2007. 7. plikaytis, b. b., r. h. gelber, and t. m. shinnick. 1990. rapid and sensitive detection of mycobacterium leprae using a nested-primer gene amplification assay. j. clin. microbiol. 28: 1913–1917. 8. hartskeerl, r. a., m. y. de wit, and p. r. klatser. 1989. polymerase chain reaction for the detection of mycobacterium leprae. j. gen. microbiol. 35: 2357–2364. 9. harboe m (1994). overview of host parasite relation.in hasting r (ed). leprosy. churchil livingstone. 10. iskandar, f; amiruddin, md; maskur, z; tjahyadi, s. 1998. correlation of bacteriological and serological examination in leprosy. jurnal medika nusantara. 19 (1): 41–45. 11. smith wcs. 2011. epidemiology of leprosy. in (makino m eds) leprosy. science working towards dignity. tokai univ. press. pp 26–34 12. nurjanti l & agusni i 2002. some possibility sources of infection innleprosy. berkala ipkk 14: 288–98. 13. job c, jayakumar, kearney m. 2008. transmission of leprosy: a study of skin and nasal secretions of household contacts of leprosy using pcr. am soc of tropic med hyg 78(3), 518–21. 14. p, beers s, madjid b. 1993. detection of m.leprae nasal carriers in population for which leprosy is endemic. j clinmicrobiol 31(11): 2947–51. 15. sakamoto k. 2011.otorhinolaryngological findings of leprosy. (chapter 18). in (makino m eds) leprosy. science working towards dignity. tokai univ. press. pp 209–215. 16. jifanti f, amiruddin md, agusni i. 2010. the excistency of m.leprae in the nasal cavity of school children in majene district, south sulawesi. thesis. postgraduate study program. hasanuddin university, makassar. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 146 vol. 1. no. 3 september–december 2010 case report a c t i o n o f n a c e t y l c y s t e i n e o n a s y m m e t r i c dimethylarginine and albuminuria in stage 1-4 nondiabetic chronic kidney disease patients m. thaha*, widodo*, m. yogiantoro*, wenny**, m. yusuf***, y. tomino**** * division of nephrology, internal medicine department, airlangga medical faculty, surabaya, indonesia ** pharmacy faculty of airlangga university, surabaya, indonesia *** cardiology and vascular medicine department, airlangga medical faculty, surabaya, indonesia **** division of nephrology, department of internal medicine, juntendo university faculty of medicine, tokyo, japan abstract background: uremic patients are in a pro-oxidant state and show an increased level of asymmetric dimethylarginine (adma), which is due to increased prmt1 activity and reduced dimethylarginine dimethylaminohydrolase (ddah) as degradation enzymes. reactive oxidant species may play an important role in increasing the action of prmt1 and in inhibiting the action of ddah. albuminuria and adma are closely correlated with progression of cardiovascular disease in chronic kidney disease (ckd) patients as well as indicators for decreasing renal function. although aceis and/or arbs reduced albuminuria in ckd patients, the results are still conflicting. several factors in these patients may play important roles in the mechanism of albuminuria such as oxidative stress. the antioxidant n-acetylcysteine may prove to have beneficial therapeutic effect, because it can reduce oxidative stress as shown by evidence in humans, and subsequently increase adma. the objective of the present study is to explore the contribution of the antioxidant n-acetylcysteine (nac) to the decrease of adma and albuminuria in non-diabetic ckd patients. material and methods: patients with non-dm ckd stage 1–4 with albuminuria were randomized to receive acei and/or arb alone (control group) or with antioxidant nac 600 mg orally twice a day (treatment group). observations were performed for 3 months to measure adma and albuminuria before and after-treatment. 80 patients in total 40 in the control group and 40 in the treatment group were used. results: after oral treatment with nac, the plasma level of adma in the treatment group increased from 0.604 µmol/l to 0.689 µmol/l, whereas adma level in the control group exhibited a higher increase from 0.561 µmol/l to 0.743 µmol/l. the increases in these groups were significantly different (p < 0.02). moreover, the level of albuminuria was reduced from 148.12 µg/mg • cr to 132.7 µg/mg • cr in the treatment group, and from 75.25 µg/mg • cr to 71.85 µg/mg • cr in the control group. the difference was significant (p < 0.001). conclusion: the anti-oxidant n-acetylcysteine can be used as adjuvant therapy to inhibit the progression of ckd in patients by decreasing the adma level and albuminuria. key words: chronic kidney disease, reactive oxidant species, asymmetric dimethylarginine, albuminuria, n-acetylcysteine introduction chronic kidney disease (ckd) is highly prevalent with an estimated world wide prevalence of 10%. in ckd patients the main cause of death is cardiovascular disease (cvd).[1] the mechanism underlying this relationship is the occurrence of endothelial dysfunction due to reduced nitric oxide (no) bioavaibility associated with atherosclerosis.[2–5] impairment of no synthesis in ckd might be due to decreased substrates l-arginine or tetrahydrobiopterin (bh4) and/or inhibition of nitric oxide synthase (nos), which is required for synthesis of no. currently, several studies have revealed that the main cause of nos pathway disturbance is the presence of asymmetric dimethylarginin (adma).[6,7] adma is suspected to be a predictor risk of cvd in ckd. it is known that adma increases in ckd, even in ckd stage 1.[8-10] there are at least four mechanisms of adma increase as follows: i) increased protein methylation by prmt, ii) increase protein turnover, iii) decreased metabolism by dimethylarginine dimethylaminohydrolase (ddah) and iv) decreased kidney excretion, but it is assamed that increased protein methylation by prmt is the main mechanism. 147thaha et al.: action of n-acetylcysteine on asymmetric dimethylarginine although the molecular mechanisms of increased activity of prmt and ddah down-regulation remain unclear various studies indicate that oxidative stress is the main cause.[11-14] researchers showed that increased oxidative stress in patients with ckd is caused by increased ros (reactive oxygen species) and decreased antioxidants.[15-16] release of no a potent vasodilator, into the circulation from endothelial cells, regulates vascular resistance and blood flow into organ tissue. no can also inhibit the process of monocyte adhesion to endothelial cells, platelet aggregation and vascular smooth muscle cell proliferation.[17] if there is a decrease of no, endothelial dysfunction and glomerular damage characterized by proteinuria will occur. persistent proteinuria is generally a marker of kidney damage. various meta-analyse indicated that angiotensin converting enzyme inhibitors (aceis) and/or angiotensin ii receptor blockers (arbs) are able to inhibit serum creatinine increases, proteinuria and the progression to eskd. therefore, they are recommended as standard renoprotective and antiproteinuric therapy.[18] however, both aceis and/or arbs reduce proteinuria by only 23–32% within 1 to 4 months. thus, most ckd patients are still in a proteinuric state. since proteinuria reduces the gfr (ml/minute/10.54, 73m2/year) patients quickly become worse.[19] therefore, efforts are needed to discover alternative adjunctive therapies. the antioxidant n-acetylcysteine (nac) contains thiol groups as synthetic precursors of cysteine and glutathione. nac is officially indicated for prevention of mucolytic, acetaminophen poisoning and contrast-induced nephropathy. several studies have examined the effect of nac in patients with ckd. antioxidant nac when given to experimental animals was able to reduce homocysteine and pulse pressure, to increase no availability, and to decrease adma levels. hermansyah et al. (2008) reported that nac use in hemodialysis patients decreased adma levels by 31.9%.[20-23] based on those findings, the clinical trial in the present study was conducted to determine the effect of oral nac 600 mg bid for 3 months on plasma adma levels and albuminuria in non-diabetic ckd stage 1–4 patients with albuminuria who were receiving aceis/arbs. subjects and methods this study complies with the principles outlined in the declaration of helsinki. it was approved by the local ethics committee and all participants gave written informed consent. the study was an open-labelled randomized clinical trial for determining the effect of oral nac on serum asymetric dimethylarginine (adma) and albuminuria in patients who received acei or arb at dr. soetomo�s nephrology outpatient in surabaya clinic, indonesia. patients in this study showed albuminuria > 30mg/day, age of 21–65 years, hb > 10 g/dl, albumin > 2.5 g/dl and controlled hypertension. they had received an acei or arb for at least one month. research subjects were selected by a simple-random test to receive oral nac and ace inhibitor or arb for 3 months. serum adma levels and albuminuria were checked before and after administration of oral nac for 3 months. the exclusion criteria were as follows: (i) trigger factors of proteinuria; pregnant women, heart failure class ii-iv (nyha); (ii) trigger factors of ros; dyslipidemia, nephrotic syndrome, diabetes mellitus and smoking (in the past 2 weeks and during the study), (iii) risk factors for ckd, use of nsaids (more than 2 doses per week), (iv) folate therapy, vitamin b6, b12 or other antioxidants, (v) urinary tract infection (uti), (vi) steroid therapy or other immunosuppressive theraphy in at least the past 6 months, (vii) serum potassium > 5.1 meq/l, (viii) cardiac valvular disease and av block ii or iii without a pacemaker, (ix) history of hypertensive encephalopathy, cerebrovascular accident or transient ischemic cerebral attacks, (x) history of myocardial infarction, unstable angina pectoris, coronary bypass surgery or percutaneous coronary intervention, (xi) history of malignancy, including leukemia or lymphoma in the last 5 years, (xii) known or suspected contraindications or allergy to acei, arb or nac, (xiii) consumption of alcohol (last 2 weeks and during the study). the drop-out criteria we as follows: (i) uncontrolled blood pressure; systolic pressure > 130 mmhg, (ii) drug discontination, (iii) died during the study period or (iv) stopped participation in the study. data analysis and statistics the kolmogorov smirnov normality test was used to determine differences in distribution of albuminuria and adma in the treatment and control groups. correlation between glomerular filtration rate (gfr) and adma level was examined using the pearson correlation test. if the significance was greater than 0.05, the distribution was normal and vice versa. for normal group distribution, the parametric t-test was used, and the wilcoxon nonparametric test was used for the abnormal group. in the control and treatment groups, the wilcoxon test was used to determine whether there were differences in levels of albuminuria after 3 months treatment. in both tests, to determine whether differences were significant or not, table asymp. sig (2-tailed) was used. if the value was less than 0.05, differences was significant. if the value was more than 0.05, there was no significant difference. results characteristics of the sample data are listed in table 1. figure 1 shows the graphic correlation. from figure 1, the inverse correlation between the glomerular filtration rate (gfr) and adma level was moderate (r = –0.537). after 3 months of observation, the adma level in the control group was elevated by 0.182 µmol/l (p values; 0.001). adma levels in the nac treatement group were elevated by 0.086 µmol/l (p value; 0.001). adma level preand posttreatment comparisons in both groups are shown in 148 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 146-150 table 2. adma level elevation in both groups is shown in figure 2. table 1. clinical and demographic characteristics of treatme and control groups treatment control p number of samples 40 40 male/female 9/31 15/25 age 54.2 ± 52.5 ± 0.000**) staging of ckd i-ii/iii-iv 20/20 20/20 adma 0.000**) systolic blood pressure 0.000**) diastolic blood pressure 0.000**) figure 1. graphic correlation of gfr and adma level in ckd patients (n = 80). a comparison of pre and post treatment albuminuria in both groups is shown in table 3. figure 3. albuminuria lowering in both groups discussion in table 1, the distribution of ckd stages between the treatment group and control group did not differ significantly. every 10ml/min/1, 73 m2 decrease of gfr, will accelerate a gfr decline of 0.38 ± 0.08 ml/min/year,[19] so that if there is a difference among the groups, it can table 2. adma level comparison in the control group and treatment group adma (µmol/l) normality statistical significance before after test analysis (p < 0.05) control group 0.561 0.743 normal distribution paired-t 0.001 = s treatment group 0.604 0.689 normal distribution paired-t 0.001 = s table 3. albuminuria comparison in both groups albuminuria (µg/mgcreat) statistical significance before after analysis (p < 0.05) control group 75.25 71.85 wilcoxon 0.016 (significant) treatment group 148.12 132.7 wilcoxon 0.000 (significant) levels of albuminuria were examined before and after nac treatment. albuminuria was significantly decreased by 3 µg/mg • cr during 3 months of treatment in the control group (p value; 0.016), while it decreased by 15 mg/mg • cr in the treatment group (p value 0.000) figure 3. figure 2. elevated levels of adma in the control group and treatment group. 149thaha et al.: action of n-acetylcysteine on asymmetric dimethylarginine affect the outcome of therapy. however, since the two groups showed no differences, confounding factors can be minimized. the results obtained from this study showed a correlation between glomerular filtration rate (gfr) in patients with ckd stage 1-4 and adma levels (figure 1), with a moderate level (r = 0.537) of correlation. this result is similar to several previous studies. kielstein et al.[9,24] showed that adma plasma concentrations in non-diabetic ckd patients were significantly difference from those in patients without ckd (p < 0.001), even in the early stages of ckd. the study was also consistent with baylis et al.[7] that ckd patients with higher adma levels showed a high incidence of ckd progression. in research by yilmaz et al.,[25] there we increased levels of adma in non-nephrotic proteinuric patients. an nac dose of 600 mg bid for 3 months is expected to reduce oxidative stress, which will decrease adma levels through reduction of prmt1 activity, increase ddah activity and further improve endothelial function reflected by decreased albuminuria. so far, no study has mentioned the effective dose of nac as an antioxidant in ckd stage i-iv. the nac dose used in the prevention of contrast induced nephropathy is 1200 mg/day before and after the procedure. cases of contrast-induced acute kidney injury (ci-aki) have a similar pathogenesis as ckd. however aki, mostly occurs transiently, while ckd is a chronic process. extending the use of nac for 3 months, will decrease the level of oxidative stress. the results showed that in the control and treatment groups, increased adma levels were 0.182 µmol/l and 0.086 µmol/l. when compared, the adma level increase on the treatment group was owen (p = 0.021). these results suggest that nac therapy inhibits the increase of adma in ckd stage i-iv with albuminuria. table 3 revened that nac at 1200 mg/day for 3 months decrease albuminuria as much as 15.42 mg/mg • cr. in the control group, the decrease was only 3.42 mg/mg • cr. the decrease of albuminuria in the treatment group was significantly greater (p = 0.02). this evidence suggests that a decrease in proteinuria was very helpful in slowing the acceleration renal deterioration. a two-fold increase in proteinuria may accelerate a decline in gfr by 0.54 ± 0.05 ml/min/year. the decline in gfr at 10 ml/min/1.73 m2 will accelerate the decline in gfr by 0.38 ± 0.08 ml/min/year.[19] this study revealed that administration of nac antioxidant at 1200 mg/day can inhibit adma level increases and reduce albuminuria in ckd stages 1-4 patients with albuminuria who have received acei/arbs therapy. possible mechanisms underlying this correlation are decreased oxidative stress, decreased prmt activity and increased ddah activity. thus, decreased adma level improved endothelial function and reduced albuminuria. it appears that administration of nac at 1200 mg/day for 3 months in ckd stages 1–4 patients with albuminuria may inhibit adma level increases and reduced albuminuria. conclusion the anti-oxidant n-acetylcysteine can be used as adjuvant therapy to inhibit the progression of ckd in patients by decreasing the adma level and albuminuria. acknowledgements we wish to thank the dean of airlangga medical faculty, surabaya, prof muhammad amin, md; head of internal medicine department, chairul effendi, md, president of the indonesian society of nephrology (inash), and prof. suhardjono, md, phd. references 1. shah, et al. oxidants in chronic kidney disease. usa: american society of nephrology american society of nephrology. 2007; 18: 16–28. 2. cooke jp, dzau vj. derangements of the nitric oxide synthase pathway, l-arginine, and cardiovascular diseases. circulation. 1997; 96: 379–382. 3. harrison dg. cellular and molecular mechanisms of endothelial cell dysfunction. j clin invest. 1997; 100: 2153–2157. 4. cooke jp. does adma cause endothelial dysfunction? arterioscler thromb vasc biol. 2000; 20: 2032–2037. 5. ueda s, yamagishi s, kaida y, okuda s. asymmetric dimethylarginine (adma) may be a missing link between chronic kidney disease (ckd) and cardiovascular disease (cvd). nephrology. 2007; 12: 582–590. 6. cooke pj. asymetrical dimethylarginine, the uber marker? usa: american heart association; 2004. halaman 1813–1818. 7. baylis c. arginine, arginine analogs and nitric oxide production in chronic kidney disease. nature clinical practice nephrology. 2006; 2(4): 209–20. 8. caglar k, yilmaz mi, sonmez a, et al. adma, proteinuria, and insulin resistance in non-diabetic stage i chronic kidney disease. kidney int. 2006; 70: 781–787. 9. kielstein jt, martens-lobenhoffer j, vollmer s, bode-boger sm. l-arginine, adma, sdma, creatinine, mdrd formula: detour to renal function testing. j nephrol. 2008; 21: 959–961. 10. panichi v, mantuano e, paoletti s, et al. effect of simvastatin on plasma asymmetric dimethylarginine concentration in patients with chronic kidney disease. j nephrol. 2008; 21: 38–44. 11. boger rh, sydow k, borlak j, et al. ldl cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of s-adenosylmethionine-dependent methyltransferases. circ res. 2000; 87: 99–105. 12. leiper j, murray-rust j, mcdonald n, vallance p. s-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase. proc natl acad sci usa. 2002; 99: 13527–13532. 13. himmelfarb j, stenvinkel p, ikizler ta, hakim rm. the elephant in uremia: oxidant stress as a unifying concept of cardiovascular disease in uremia. kidney int. 2002; 62: 1524–1538. 14. zoccali c, mallamaci f, tripepi g. novel cardiovascular risk factors in end-stage renal disease. j am soc nephrol. 2004; 15 suppl 1: s77–s80. 15. locatelli f. oxidative stress in end-stage renal disease: an emerging threat to patient outcome. nephrology dialysis transplantation. 2003; 18: 1272–1280. 16. araujo m, and welch, j.m. oxidative stress and nitrite oxide in kidney function. current opinion nephrology and hypertension. 2006; 15: 72–77. 17. cooke jp. does adma cause endothelial dysfunction? arteriosclerosis thrombosis vascular biology. 2000; 20: 2032–7. 150 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 146-150 18. jafar. proteinuria as modifiable risk factor for the progression of non diabetic renal disease. kidney internal. 2001; 67: 2288–94. 19. lea. the magnitude between proteinuria reduction and risk end stage renal disease. archive internal medicine. 2005; 163. 20. ivanovski o, et al. the antioxidant n-acetylcysteine prevents accelerated atherosclerosis in uremic apolipoprotein e knockout mice. kidney int. 2005; 67: 2288–94. 21. schulze, et al. determination of asymetric dimethylarginine (adma) using a novel elisa assay. clin chem lab med. 2004; 42(12): 1377–1383. 22. thaha m, yogiantoro m, tomino y. intravenous n – acetylcysteine during hemodialysis reduces the plasma concentration of homocysteine in patients with end stage renal disease. clin drug invest. 2006; 26(4): 195–202. 23. yuli h. pengaruh pemberian n-asetilsistein intravena terhadap kadar adma pada pasien penyakit ginjal kronik stadium 5 selama tindakan hemodialisis. smf penyakit dalam, rsu dr. soetomo. 2006. 24. kielstein jt, boger rh, bode-boger sm, et al. smarked increase of asymmetric dimethylarginine in patients with incipient primary chronic renal disease. j am soc nephrol. 2002; 13: 170–176. 25. yilmaz, et al. adma levels correlate with proteinuria, secondary amyloidosis, and endothelial dysfunction. j am soc nephrol. 2008; 19: 388–395. ijtid vol 1 no 3 sep-dec 2010.44.pdf ijtid vol 1 no 3 sep-dec 2010.45.pdf ijtid vol 1 no 3 sep-dec 2010.46.pdf ijtid vol 1 no 3 sep-dec 2010.47.pdf ijtid vol 1 no 3 sep-dec 2010.48.pdf 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 49 vol. 5. no. 2 may–august 2014 management of hiv/aids infection in pregnancy endah dewati1, nasronudin1,2 1 infectious and tropical disease division department of internal medicine, dr. soetomo hospital airlangga university school of medicine 2 institute of tropical disease airlangga university abstract twenty years since identified for the first time, the disease of hiv/aids spread and cause greater damage than the previous prediction. according to the director general of p2m and environmental sanitation department of health by the end of 1999, there were 1066 people in indonesia who are infected with hiv even though this must be realized that the rate is still far lower than the actual numbers, because there are many cases of hiv infection reported in addition to energy awareness health of the possibility of hiv infection has not been evenly distributed. management of hiv infection/aids in pregnancy is done in time of antepartum, intrapartum and post partum, for mother and the baby, in general and specific. the important matters include the use of art, nutrition and psychological support. prevention and management of opportunistic infections to pwha are not different with that of non pregnant woman. however, it is not routinely advised because of drug toxicity. key words: hiv, aids, pregnancy, anti retroviral, plwhat abstrak dua puluh tahun sejak pertama kali diidentifikasi, penyakit hiv dan aids menyebar dan menyebabkan kerusakan yang lebih besar dari prediksi sebelumnya. menurut direktur jenderal pemberantasan penyakit menular (p2m) dan sanitasi lingkungan departemen kesehatan pada akhir tahun 1999, terdapat 1066 orang di seluruh indonesia yang terinfeksi hiv meski harus disaadari bahwa angka ini masih jauh lebih rendah daripada jumlah sesungguhnya, karena ada banyak kasus infeksi hiv yang dilaporkan disamping kesadaran tenaga kesehatan dari kemungkinan infeksi hiv yang belum merata. manajemen infeksi hiv dan aids pada kehamilan dilakukan pada saat antepartum, intrapartum dan post partum, untuk ibu dan bayi, secara umum dan khusus. hal yang penting adalah penggunaan terapi anti retroviral, dukungan gizi dan psikologi. pencegahan dan pengendalian infeksi oportunistik dari odha untuk tidak berbeda dengan yang non wanita hamil. akan tetapi, hal ini tidak dianjurkan secara rutin karena narkoba. kata kunci: hiv, aids, kehamilan, anti retroviral, ohdha introduction twenty years since identified for the first time, the disease of hiv/aids spread and cause greater damage than the previous prediction. this disease can affect men, women and children around the world. in 1999 it was noted that hiv/aids is the fourth leading cause of death in the world, while in africa is a cause of death terbanyak. dalam 2000, about 3 million people, including 500,000 children died of aids, and 5.3 million people, including 600,000 children received new hiv infections, most due to mothertochild transmission.1 the disease caused a crisis on various sectors including health. the majority of patients, ie 95 % are in developing countries, so far-reaching impact in the increase and population growth is even said to the population of a contracting state may decline not because of the family planning program but rather due to aids deaths1 according to the director general of infectious disease eradication and environmental sanitation department of health by the end of 1999, there were 1066 people in indonesia who are infected with hiv even though this must be realized that the rate is still far lower than the actual numbers, because there are many cases of hivmbers, because there are many cases of hiv literature review 50 indonesian journal of tropical and infectious disease, vol. 5. no. 2 may–august 2014: 49–55 infection reported in addition to energy awareness health of the possibility of hiv infection has not been evenly distributed.2 although various attempts have been made to control the increasing number of incidence and mortality due to hiv/aids but the transmission still continues until now. hiv transmission occurs through three main lines, namely: horizontal transmission is contact with blood (transfusions contaminated, use needles interchangeably in drug addicts, injury, etc.), vertical transmission (from mother to fetus during pregnancy, childbirth and breastfeeding), sexual transmission (homosexual or heterosexual) 3 management of infectious diseases of hiv/aids in pregnancy need to consider two important things, namely the impact of hiv/aids infection in pregnancy and the impact of pregnancy on the progression of infection than hiv/aids. various important things that need to be considered in the management of hiv/aids infection in pregnancy, namely the provision of antiretroviral therapy, nutrition, psychological support. here are discussed the management of hiv/aids infection in pregnancy with the hope to increase knowledge of the health workers, reducing the incidence of hiv infection, reduced mortality due to aids, and reduces transmission of hiv infection. pregnancy and hiv infection effect of pregnancy on hiv disease journey in normal pregnancy there is a decrease in the number of cd4 + fetus early in pregnancy to maintain. in women who do not have hiv +, cd4 + percentage will rise again started the third trimester to 12 months after birth; whereas in hiv decline persist during pregnancy and after childbirth.4 pregnancy is only slightly increase levels of virus (viral load) hiv. kehamilan also does not accelerate disease progression to aids. (dn burns, 1998; crombleholme wr md, 2002).4,5 effect of hiv infection in pregnancy research in developed countries before the era of antiretroviral showed that hiv does not cause an increase in prematurity, low birth weight, or intrauterine growth retardation. while in developing countries, hiv infection increases the incidence of abortion, prematurity, intrauterine growth retardation, especially at an advanced stage. this is because the mother ‘s physical condition is worse and the possibility of a higher perinatal transmission.1 vertical transmission of hiv without intervention, the risk of hiv transmission from mother to fetus are reported to range between 15–40%. 6,7 the risk of vertical transmission varies in different countries, depending on antiretrovirals that can be given. the risk of transmission is higher in developing countries than in developed countries. transmission can occur during pregnancy, intrapartum and post partum. most of the intrapartum transmission occurs.8 transmission mechanism in pregnancy is unclear, presumably through the placenta. pathological examination finding in the placenta in hiv-infected women hiv. sel lymphocytes or monocytes infected mother or hiv virus itself can be reached directly through the fetal syncytiotrophoblast layer, or indirectly through the trophoblast cells and infect placental macrophages (hofbauer cells) that have cd4 + receptor. according to the pediatric virology committee of the aids clinical trials gruoup (pactg), said in utero transmission/infection if the initial positive virological test within 48 hours after birth and the next test is also positive.9 in the intrapartum transmission, infection was diagnosed if a negative virological examination in the first 48 hours after birth, and the first test next week to be positive and the baby is not breastfeeding.9 as long delivery, the baby may be infected blood or servikovaginal f l u i d c o n t a i n i n g h i v t h r o u g h e x p o s u r e to tracheobronchial or ingested in the birth canal.9 infections associated with post partum lactation. virus particles can be found in the cell components and nonmilk cells ibu.konsentrasi highest virus in colustrum. kadar highest hiv in breast milk occurred from the first week to three months after delivery. hiv in low concentrations can still be detected in breast milk to 9 months after delivery.6,9 m a n a g e m e n t o f h i v/a i d s i n f e c t i o n i n pregnancy management of hiv/aids infection in pregnancy during antepartum, intra -partum and post partum for mother and baby, both generally and specifically.10 a. general treatment: • take a rest • adequate nutrition support • psychosocial therapy b. special treatment • provision of antiretroviral therapy (art) combination, by highly anti retroviral therapy (haart) • management of obstetric • treatment of opportunistic infections • treatment of malignant provision of antiretrovirus (art) art is recommended for all people with hiv who are pregnant to reduce the risk of perinatal transmission. the purpose of the provision of antiretroviral therapy in pregnancy is to maximize maternal health and reduce the risk of hiv transmission as low as possible. the advantage must be weighed against the potential toxicity, teratogenesis, and long -term side effects. side effects are expected to increase in the provision of a combination art. 51dewati e and nasronudin: management of hiv/aids infection in pregnancy namun, recent research by toumala, et al showed that, compared with monotherapy, combination antiretroviral therapy does not increase the risk of prematurity, low birth weight, or intrauterine fetal death.11 currently in indonesia, some of haart has been available in generic form at a lower price, such as zidovudine, lamivudine, nevirapine and stavudin. antiretroviral drugs are first examined to reduce perinatal transmission is zidovudine (zdv). in pactg protocol 076, zdv given orally start week 14 of pregnancy, followed by the time iv zdv intrapartum to the mother, followed by zdv syrup given to infants from the age of 6-12 hours to 6 weeks. in this study, infants are not breastfed. this method was effective at lowering the perinatal transmission of 25.5 % in the control group to 8.3 %.12 the longer the use of antiretroviral therapy, the more likely a reduced risk of transmission hiv. joao, et al revealed in infants who are not infected with hiv, the average duration of use of art in mothers compared with 16.63 weeks old maternal art use 6.28 weeks in the group of infants infected with hiv.13 explanation category b: there is no risk to the fetus in animal studies, but there has been no studies in pregnant women; or animal studies showed side effects according to controlled studies in pregnant women first trimester (and no proven risk in subsequent trimesters). category c: in animal studies found adverse effects on the fetus (teratogenic or embriosidal, or other), and there has been no controlled studies in pregnant women, there has been no research or drug side effects in animals or pregnant women. drugs in this category is given only if the benefits exceed the potential risk to the fetus. category d: there is positive evidence of fetal risk of side effects in humans, but the gain in pregnant women may be acceptable than the risks, especially for life-saving. perinatal hiv guidelines working group in the united states put forward the recommendations of antiretroviral with some scenarios.14 (table 2) nutritional support in hiv/aids nutritional management is important to prevent and cope with hiv infection. provision of antiretroviral therapy is still needed, but without adequate nutrition therapy intervention difficult to stem the negative effects of reactive oxygen species (ros), which induces cell death as well as disease progression. management of nutrition in pregnant women with hiv/aids, people live with hiv/ aids (plwha) can increase resistance to opportunistic infections and to improve the tolerance to the side effects of drugs, protection of cell viability, ensure continuity of organ function, improve the function of the immune system to prevent other microorganisms including viruses, bacteria, tumor cells and fungi. in pregnancies with hiv/aids infection is often accompanied by a deficiency of antioxidant vitamins and minerals, increased levels of ros which promotes apoptosis in immune cells and increased morbidity. ros can trigger the onset of the crisis scavenger enzyme-deficit micronutrient components such as fe, zn, selenium, vitamin c, vitamin b6, vitamin e, or an imbalance of some nutrients, such as essential amino acids can cause damage to components of the immune system. decreased antioxidant plwha very dangerous, because the more encouraging apoptosis in various cells and promotes the progression of hiv infection to aids.15,16 one complication that almost always accompanies hiv/aids patients is weight loss. when weight loss exceeds 10 % with chronic diarrhea over 1 month, and general weakness with fever or prolonged over 1 month called hiv/aids wasting syndrome.16 the cause of wasting in plwha is a decrease in the intake, malabsorption and increased metabolism. potential for more severe wasting in plwha. this is due to the higher nutritional needs with respect to pregnancy and emerging opportunistic infections, nutritional intake while on the other hand decreases with time and complexity of infections. immunocompromised immune system which can be inhibited by preventing through medical nutrition therapy (mnt).with mnt expected to reduce morbidity, improve quality of life, lower costs, shorten the hospital stay, tabel 1. categories food and drug administration (fda) antiretroviral for use in pregnancy (watts dh, 2002)10 division drug categories fda nucleoside reverse transcritase inhibitor (nrti) zidovudin/zdv/ azt c zalsitabin/ddc c didanosin/ddi b stavudin/d4t c lamivudin/3tc c abacavir/abc c tenofovir df b non-nucleoside reverse transcriptase inhibitor (nnrti) nevirapin c delavirdin c efavirenz c protease inhibitor (pi) indinavir c ritonavir b saquinavir b nelvinavir b amprenavir c lopinavir c others hidroksiurea d 52 indonesian journal of tropical and infectious disease, vol. 5. no. 2 may–august 2014: 49–55 improve the quality of life of patients with hiv/aids. ideal in the handling of involving doctors and nutritionists. mnt can increase the intake of energy, protein and micronutrient that can improve nutritional parameters. to obtain optimal effect of mnt, the nutritional therapy should be programmed so the diagnosis of hiv infection is established.17 according to the canadian dietetic association and the ada developed an hiv/aids medical nutrition therapy protocol on medical therapy interventions are promptly determine satus plwha nutrition, maintain and prevent the loss of body mass and nutritional deficiencies, support and improve the quality of life plwha. the protocol was evaluated two times a year in people living with hiv asymptomatic, and six times a year in patients who are symptomatic or aids. macronutrients 1. total calories, 35–40 kcal/kg weight/hr enough to fulfill the needs if patients in general 2. glucose, 30–70% of the total heat supplied in the form of glucose. 3. f a t , 2 0 – 3 0 % o f t h e t o t a l c a l o r i e s . o m e g a 6 polyunsaturated fatty acid (pufa) triglycerides should be given in doses sufficient to prevent fatty acid deficiency. 4. protein, 15–20% of total calories given as a protein or amino acids. micronutrients purposes of vitamins, minerals and trace elements need to be taken into account in the preparation of nutrient plwha every day. potassium, magnesium, fe, zn, phosphate is maintained in order to stay within normal levels in the blood. n u t r i t i o n a l s t a t u s a s s e s s m e n t d o n e b a s e d o n anthropometric, clinical performance, and bmi laboratory parameters should be monitored every week. every plwha need to be briefed about the weakness of the tabel 2. recomendation of antriretroviral (art) to reduce perinatal transmission (perinatal hiv guidelines working group, 2002)13 pregnancy condition recomendation hiv-positive pregnancy who had never used antiretroviral earlier pregnant hiv-positive who undergoing clinical examination, standard immunological and virology. consideration of art initiation and selection are the same as non-pregnant hivpositive people with consideration of the effect of the three-part zdv pregnancy. regimen recommended after the first trimester regardless of the levels of hiv of mother. regimen combination is recommended in hiv clinical status, immunological and its virology heavy or hiv levels > 1000 copies /ml. if people with hiv come in the first trimester of pregnancy, provision of art can be delayed until 10-12 weeks gestation. hiv-positive pregnancy who are getting pregnant and pregnant art if the pregnancy is known after the first trimester, prior antiretroviral therapy forwarded, preferably by including zdv. if in the first trimester of pregnancy is known, people with hiv are given counseling about the benefits and risks of antiretroviral therapy in first hiv-positive. if trimester pick off treatment during the first trimester, all drugs should be stopped and then given simultaneously after the first trimester to prevent drug resistance. without considering the previous regimen, zdv is recommended to be administered during the intrapartum and infants. hiv-positive pregnancy in childbirth and had never get art before there are several regimens are recommended : • single dose nevirapine during labor and a single dose to the infant at age 48 hours • oral zdv and 3tc during labor, followed by zdv / 3tc in infants during the week • intrapartum zdv, zdv in infants followed for 6 weeks • two-dose of nevirapine combined with zdv during labor followed iv zdv in infants for 6 weeks immediately after delivery, such as people with hiv undergoing cd4 + and hiv levels to determine whether the treatment will be continued. if infants of hiv-positive mothers came after childbirth, while the mother has not received antiretroviral therapy during pregnancy or intrapartum zdv syrup is given to infants for 6 weeks, starting as soon as possible within 6-12 hours after birth. some doctors may choose a combination of zdv with other antiretroviral drugs, especially if the mother is known to be resistant to zdv. however, the efficacy of this regimen is not yet known and the dose for children not yet fully known. immediately after delivery, hiv-positive undergo examination such as cd4 + and hiv levels to determine whether the treatment will continue. infant undergo early diagnostic tests that antiretroviral therapy can be given as soon as possible if it were hiv positive. 53dewati e and nasronudin: management of hiv/aids infection in pregnancy immune system that allows the transmission of disease through food. knowledge of the type, form, delivery and quality procedures is important to address these risks. counseling in people with pregnancy implemented since antepartum care. hiv-positive people are given information about the effects of pregnancy on hiv infection, the effect of hiv on pregnancy including perinatal transmission, the role of viral load monitoring, the use of drugs in pregnancy, use of zidovudine benefit in pregnancy and possible protection to sectio caesaria intrapartum.10,19 factors affecting hiv-positive woman‘s decision to continue or terminate a pregnancy, including stage of hiv infection, the use of antiretroviral and other drugs, pregnancy planning, the desire to have children, religion and belief, economic status, social support for children.18 tabel 3. recomendation of childbirth method to reduce hiv transmission from mother to child14 childbirth method recomendation hiv-positive pregnancy who come in pregnancies over 36 weeks, has not received antiretroviral therapy, and awaiting the results of the hiv and cd4 + levels are thought to exist before childbirth hiv-positive people should receive antiretroviral therapy regimens patcg (pediatric aids clinical trials group) 076. performed as like counseling about caesarean section for reducing the risk of transmis and the risk of post operative complications, the risk of anesthesia and surgery was decided others. if sc, sc planned at week 38 pregnancy. over sc, people living with hiv received zdv intravenous who started three hours earlier, and infants received zdv syrup for 6 weeks. decission will continue therapy after delivery or not depends on the results of virus levels and cd4 + hiv-positive pregnancy who came in early pregnancy, is being awarded a combination antiretroviral therapy, and hiv levels remained above 1000 copies/ml at week 36 of pregnancy anti retroviral therapy (art) regimens used are continued. people living with hiv should be counseled that hiv levels may not go down to less than 1000 copies/ml before delivery, so it is recommended to the sc. likewise, the increased risk of complications such as infection sc, anesthesia operation if disconnected sc, sc planned in the 38th week of pregnancy. during the sc, people living with hiv receive intravenous zdv initiated at least 3 hours in advance. another therapy be continued before and after childbirth. infants received zdv syrup for 6 weeks hiv-positive pregnancy who are on combination antiretroviral therapy, and hiv rna levels at week 36 of pregnancy people living with hiv were counseled that the likelihood of transmission if hiv rna levels may be less than 2% even in labor pervagina. selection of mode of delivery should weigh the benefits and risks of complications sc hiv-positive pregnancy who had planned elective sc, however came at the onset of labor or after rupture of membranes. intravenous zidovudine (zdv) given immediately. if the rapid progress of labor, people with hiv are offered to undergo a vaginal delivery. if cervical dilatation is minimal and supposedly will last long labor, can be chosen between intravenous zdv and do sc or provide pitosin to speed up delivery. if people with hiv decided to undergo a vaginal delivery, avoid the head electrode, invasive monitors and other tools. infants should receive zdv syrup for 6 weeks obstetric management perinatal hiv guidelines working group in the united states submit obstetric management recommendations to reduce vertical hiv transmission in some conditions. prevention and management of opportunistic infections during pregnancy prophylaxis therapy and therapy against infection with mycobacterium tuberculosis, pneumocystis carinii, m avium complex, toxoplasma gondii, and herpes simplex virus in pregnant hiv-positive people are no different from non-pregnant. however, primary prophylaxis against cytomegalovirus, candida and invasive fungal infections is not recommended routinely given drug toxicity. fluconazole for example, is known to cause skeletal and craniofacial deformities in long -term use during pregnancy.10 54 indonesian journal of tropical and infectious disease, vol. 5. no. 2 may–august 2014: 49–55 tabel 4. management of opportumistic inffections in pregnancy in women with hiv infection10 causing explanation mycobacterium avium complex azithromycin is the first choice for primary prophylaxis during pregnancy; clarithromycin teratgenik in animals; for maintenance therapy of azithromycin plus ethambutol mycobacterium tuberculosis isoniazid is preferred for prophylaxis during pregnancy; for tb that are resistant to combination therapy, consult to the experts herpes simplex virus prophylaxis and therapy as in nonpregnant pneumocystis carinii prophylaxis and therapy as in nonpregnancy; attention to neonate in women who received therapy sulfa cryptococcus neoformans primary prophylaxis is not recommended; abnormalities after long -term exposure; consider switching to amphotericin b in the first trimester for long-term suppression coccidioides immitis primary prophylaxis is not recommended; anomalies as mentioned above after long-term exposure to fluconazole; consider switching to amphotericin b in the first trimester when the long-term suppression continues histoplasma capsulatum primary prophylaxis is not recommended, for causing anomalies due to exposure to fluconazole and security is not yet clear from chronic itraconazole during pregnancy, consider switching to amphotericin b in the first trimester when the long -term suppression continues cytomegalovirus primary prophylaxis is not recommended; the management of long-term suppression should be consulted with the experts candida species prophylaxis is not indicated during pregnancy; craniofacial and skeletal abnormalities were reported in four infants after long-term exposure to fluconazole in utero toxoplasma gondii treatment and secondary prophylaxis (maintenance therapy) as in patients who are not pregnant; only primary prophylaxis with trimethoprim-sulfamethoxazole. vaccination against hepatitis b, influenza and pneumococcal remains can be given during pregnancy. the vaccination should be given after the levels dropped to undetectable hiv to prevent hiv-rna levels after vaccination.10 summary management of hiv infection/aids in pregnancy is was performed in time of antepartum, intrapartum and post partum, for mother and the baby, in general and specific. the important matters including the use of art, nutrition and psychological support. prevention and management of opportunistic infections to people live with hiv/aids (plwha) are not different with that of non pregnant woman. however, the administration of art is not routinely advised because of drug toxicity. references 1. usaid, 2000. leading the way: usaid responds to hiv/aids. the synergy project tvt associates, inc. washington dc, pp. 3–18. 2. djauzi s. 2001. aids. dalam: buku ajar ilmu penyakit dalam. editor: suyono s, waspadji s, lesmana l, dkk. edisi 3, balai penerbit fkui, jakarta, hal. 81–6. 3. royce ra, sena a, cates w jr, cohen ms, 1997. current concepts: sexual transmission of hiv. n engl j med. medical society. massachusetts, pp. 1072–1078. 4. burns dn, landesman s, minkoff h, et al., 1998. the influence of pregnancy on human immunodeficiency virus type 1 infection : antepartum and post partum changes in human immunodeficiency virus type 1 viral load. am j obstet gynaecol 178: 355–9. 5. crombleholme wr md. 2002. obstetrics. in: current medical diagnosis & treatment. 41th ed. lange medical books/mcgraw-hill. medical publishing division. new york, pp. 781–805. 6. khouri m, kovacks a. 2001. pediatric hiv infection. clin obstet gynaecol 44: 243–75. 7. bulterys m, fowler mg. 2000. prevention of hiv infection in children. pediatric clin north am 47: 241–60. 8. barbieri rl, repke jt. 2001. medical disorders during pregnancy. in: harrisons’s principles of internal medicine. 15th ed. mcgraw-hill medical publishing division. new york, pp. 25–30. 9. burgess t. 2001. determinants of transmission of hiv from mother to child. clin obstet gynaecol 44: 198–209. 10. watts dh. 2002. management of human immunodeficiency virus during pregnancy. n engl j med 346: 1879–91. 11. toumala re, shapiro de, mofenson lm, et al., 2002. antiretroviral therapy during pregnancy and the risk of an adverse outcome. n engl j med 346: 1863–70. 12. sperling rs, shapiro de, coombs rw, et al., 1996. maternal viral load, zidovudine treatment, and the risk of transmission of human immunodeficiency virus type 1 from mother to infant. n engl j med 335: 1621–9. 13. joao ec, cruz mls, menezes ja, et al., 2002. factors associated with vertical transmission in a cohort of hiv+ pregnant woman in rio de janeiro brazil. abstract of the 9th conference of retroviruses and opportunistic infections, washington, usa. 55dewati e and nasronudin: management of hiv/aids infection in pregnancy 14. perinatal hiv guidelines working group. 2002. public health service task force recommendations for use of antiretroviral drugs in pregnant hiv-1 infected woman for maternal health and interventions to reduce perinatal hiv-1 transmission in the united states, february 4, in: peiperi l, garbus l. ammann a, shannon m, editors. women, children, and hiv: resources for prevention and tretment, 2nd ed, san fransisco. 15. antelman g, msamanga gi, spiegelman d, urassa ejn. 2000. nutritional factors and infectious disease contribute to anemia among pregnant women with human immunodeficiency virus in tanzania. j nutr 130: 1950–7. 16. lee j, watson rr (2001). antioxidants in human aids. in: (watson rr, ed) nutrition and aids. 2nd ed, crc press, washington dc, pp. 15–22. 17. who. 2002. the use antiretroviral therapy: a simplified approach for resource constrained countries. who regional office for south-east asia, new delhi, pp. 1–49. 18. young js. 1997. hiv and medical nutririon therapy. j am diet assoc 97 (2): s 161–6. 19. mijch am, clezy k, furner v. 1997. women with hiv. in: managing hiv. ed: stewart g. australasian medical publishing co limited, sydney, pp. 128–130. �� vol. 4. no. 4 october–december 2013 awareness of using ringer lactat solution in dengue virus infection cases could induce severity soegeng soegijanto1,2, desiana w sari2, atsushi yamanaka1,3,5, tomohiro kotaki1,3, masanori kamoeka1,3,4, eiji konishi3,5 1 indonesian-japan collaborative research center for emerging and re-emerging infectious disease, institutes of tropical diseases, universitas airlangga, surabaya, indonesia. 2 soerya hospital, sidoarjo, indonesia 3 center for infectious diseases, kobe university graduate school of medicine, kobe, japan. 4 department of international health, kobe university graduate school of sciences, kobe, japan. 5 biken endowed department of dengue vaccine development, faculty of tropical medicine, mahidol university, bangkok, thailand abstract background: in 2012, serotype of dengue virus had changed from den-2 and den-3 to den-1. in 5–10 years ago, serotype of den-1 case showed a mild clinical manifestation; but now as a primary case it can also show severe clinical manifestation. one of indicator is an increasing liver enzyme, ast and alt, with level more than 100–200 u/l. aim: to getting a better solutions for this problem. method: obsevasional study had been done in medical faculty of airlangga university (dr. soetomo and soerya hospital) surabaya on mei–august 2012. there were 10 cases of dengue virus infection were studied, 5 cases got ringer acetate solution (group a) and 5 cases got ringer lactate solution (group b). the diagnosis was based on criteria who 2009. result: five cases of dengue virus infection had showed a liver damage soon after using ringer lactate solution; ast and alt were increasing more than 100–200 u/l; but the other 5 cases showed better condition. it might be due to use ringer acetate that did not have effect for inducing liver damage. by managing carefully, all of the cases had shown full recovery and healthy condition when being discharged. conclusion: using ringer acetate as fluid therapy in dengue virus infection is better to prevent liver damage than using ringer lactate. key words: ringer lactate, ringer acetate, dengue virus infection, pediatric cases, severity abstrak latar belakang: pada tahun 2012 serotype virus dengue telah mengalami perubahan dari den-2, den-3 ke den-1. dalam kurun waktu 5–10 tahun yang lalu, serotype den-1 menunjukkan manifestasi klinis yang ringan, tetapi sekarang serotype den-1 ini walaupun sebagai kasus primer ternyata dapat menunjukkan manifestasi klinis yang berat. salah satu indikator yang digunakan adalah kenaikan enzym ast dan alt lebih dari 100–200 u/l. tujuan: berusaha menemukan tatalaksana terbaru dengan hasil yang memuaskan. metode: studi observasional telah dilakukan di rsud dr soetomo surabaya & rs soerya sepanjang sejak bulan mei–agustus 2012. dari 10 kasus infeksi virus dengue yangditeliti, 5 kasus mendapatkan terapi cairan ringer acetate (kelompok a) dan 5 kasus mendapatkan cairan ringer lactate (kelompok b). diagnose infeksi virus dengue berdasarkan kriteria who 2009. hasil: lima kasus infeksi virus dengue telah menunjukkan gangguan fungsi hati setelah memperoleh infus cairan ringer lactate dan terbukti kadar ast dan alt meningkat lebih dari 100–200 u/l, tetapi 5 kasus yang memperoleh ringer acetate menunjukkan keadaan yang lebih baik. maka dapat dibuat kesimpulan bahwa terapi cairan pada penderita infeksi virus dengue lebih baik memanfaatkan cairan ringer acetate sebab metabolism cairan ini terjadi di otot ekstremitas dan tidak mengganggu fungsi hati. dan berbeda secara nyata apabila cairan yang digunakan larutan ringer lactate yang metabolism di dalam hati. hal ini sesuai dengan hasil penelitian terdahulu yang mengutamakan bahwa larutan ringer acetate yang diberikan kepada penderita infeksi virus dengue tidak meng ganggu fungsi hati penderita. kesimpulan: lebih baik memanfaatkan cairan ringer acetate sebagai terapi infeksi virus dengue daripada menggunakan cairan ringer lactate yang dapat mengganggu fungsi hati. kata kunci: ringer laktat, ringer asetat, infeksi virus dengue, kasus anak, kegawatan case report �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 35–41 patient 2 a nine years old girl was brought by his parents to soerya hospital, in emergency department with the main complaint of fever since 3 days before admission. she also suffered from nausea, and cephalgia. she lost her appetite and refuse to eat. she was in a weak condition. table 2. result of laboratory examination day of admission hb hct leukocyte trombocyte ast alt 1 11,7 35,5 2490 134000 36,1 15,8 2 11,4 34,6 4170 139000 3 11,3 33,9 3650 199000 31,1 16,4 4 11,1 33,5 3230 263000 5 11,4 34,6 4310 408000 patient 3 an eleven years old boy was brought by his parents to soerya hospital, in emergency department with the main complaint of fever since 6 days before admission. the fever had been subsided in the third day of fever, but the temperature raised in the forth day. table 3. result of laboratory examination day of admission hb hct leukocyte trombocyte ast alt 1 13,3 37,8 4420 34000 106,9 41,7 2 13,9 37,8 4420 30000 3 12,8 38,5 8230 32000 142,3 59,3 4 13,2 39,4 9200 80000 5 11,8 34,8 9460 166000 6 12 35,3 9610 150000 62,9 51,2 patient 4 a ten years old girl was brought by his parents to soerya hospital, with the main complaint of fever since 5 days before admission. she had been brought to the doctor before, but the fever stayed. she also suffered from vomit after eating, myalgia and cephalgia. table 4. result of laboratory examination day of admission hb hct leukocyte trombocyte ast alt 1 14,3 40,5 3610 36000 91,5 26,2 2 14,3 40,8 3640 36000 3 13 38,4 10780 35000 68,6 27,7 4 12,6 36,5 8110 46000 5 12,4 34,8 7770 94000 6 12,8 36,0 6630 196600 42,8 55,4 patient 5 a nine years old girl was brought by his parents to soerya hospital, in emergency department with the main introduction since 1968, dengue virus infection has always been found in indonesia. it is due to increasing the population of aedes aegypti and aedes albopictus that now transfer from one case to another case. it is supported by changing of summer to raining season. global climate can also affect all the event in the world. in 2009 some hospital in surabaya and sidoarjo had found the changing serotype on dengue virus from den-2 and den-3 to den-1. in 5–10 years ago, serotype of den1 showed a mild clinical manifestation, but now, it can also show severe clinical manifestation of dengue virus as a primary infection. it can be predicted by test marker of positive ns-1 dengue test. this virus can cause liver damage. based on this finding, we should be careful to treat dengue virus infection cases. firstly, we should look for the indicators of dengue virus infection suffering for liver damage, that are ast and alt titers in every case of dengue virus infection. if we find the indicators of liver damage (increasing ast and alt titers), please don’t use ringer lactate for treating dengue with shock case. to convince this, researcher want to present this papper to get a better solutions from many aspect. materials and methods observasional study had been studied in medical faculty airlangga university, surabaya, indonesia. on mei–august 2012, there were 10 cases of dvi, 5 cases were given ringer acetate (group a), and the other cases were given ringer lactate (group b). the diagnosis was made based on criteria who 2009. group a patient 1 a four years old boy was brought by his parents to soerya hospital, in emergency department with the main complaint of fever since 2 days before admission. she also suffered from nausea and cephalgia. table 1. result of laboratory examination day of admission hb hct leukocyte trombocyte ast alt 1 12,6 37,5 4860 280000 113,6 49,9 2 10,9 31,7 3020 222000 3 11,8 35,1 2090 188000 4 11,9 34,9 4110 134000 5 11,9 36,1 4110 126000 6 12,3 37,1 6870 187000 73,5 32,6 ��soegijanto, et al.: awareness of using ringer lactat solution in dengue virus infection cases complaint of fever since 2 days before admission. she also suffered from nausea, and cephalgia. table 5. result of laboratory examination day of admission hb hct leukocyte trombocyte ast alt 1 15,3 44 3140 91000 60,9 27,3 2 14,7 42 5050 60000 3 13 36,7 4210 42000 57,3 25,2 4 12,5 35,6 4970 53000 5 12,1 34,6 4840 120000 6 12,2 35,4 5990 213000 56,9 47,2 group b patient 1 a twelve years old boy was brought by his parents to dr. soetomo hospital, in emergency department with the main complaint of fever since 2 days before admission. he also suffered from heavy cephalgia. table 6. result of laboratory examination day of admission hb hct leukocyte trombocyte ast alt 1 12,6 39,5 3660 214000 36,8 18,9 2 11,5 35,9 3300 155000 3 13,1 40,8 3740 54000 809,1 382,6 4 15,2 46,4 5220 33000 5 12,9 39,6 8100 56000 6 11,5 35,1 7540 85000 179,4 178,1 7 12,1 36,2 11100 163000 137,1 147,7 patient 2 a nine years old boy was brought by his parents to dr. soetomo hospital, surabaya in emergency department with the main complaint of clammy extremities since 7 hours before admission. he suffered from acute and continues high grade fever for 5 days and subside suddenly. he also complained of headache, retroorbital pain, melena and myalgia. there was a history of contact to person with dengue hemorrhage fever in his neighborhood. table 7. result of laboratory examination day of admission hb hct leukocyte trombocyte ast alt 1 17,9 52,1 5400 29000 1360 566 2 16,8 43 2000 32000 3 10,6 27 7100 20000 7050 3541 4 5 12,5 35 7400 85000 1367 1037 patient 3 a three years old boy was reffered from mojokerto hospital with hepatic comme on dr. soetomo hospital in emergency department., with the main complain of fever since 5 days before. one day before admission he had clammy extremities. he also complained of nausea and vomitting and refuse to eat since 1 day before admission. no sign of bleeding was found in this child. table 8. result of laboratory examination day of admission hb hct leukocyte trombocyte ast alt 1 10,3 31,9 14600 15000 3154 1274 2 10,7 30 20500 85000 3 9,7 30,9 11000 75000 4 5 8,8 28,4 3500 100000 1254 1069 patient 4 a seven years old boy was brought to his parents in dr soetomo hospital emergency department with main complain of fever. he suffered from fever since 4 days before admission. the fever was suddenly continues and high grade. he also complained of headache, and difficult to sleep. there was no history of bleeding. he felt nausea and vomit which made he lost his appetite. there was no history of contact to person with dengue hemorrhagic fever in his neighborhood. table 9. result of laboratory examination day of admission hb hct leukocyte trombocyte ast alt 1 15,8 48 7400 26000 2824 1011 2 16,8 43 2000 32000 3 10,3 36,6 7600 22000 4 10,4 29,6 9700 73000 1107 537 5 8,8 26,7 9800 1122000 patient 5 an overweight-eleven years old boy had been reffered from bangkalan to emergency room in soetomo hospital, with maint complaint fever, cephlagia, headache, vomitting, dyspnea, and restless since 4 days before and had been managed in bangkalan hospital as dengue virus infection, and got ringer lactate solution for early recucitation, but the condition became worse, and reffered to soetomo hospital. table 10. result of laboratory examination day of admission hb hct leukocyte trombocyte ast alt 1 13 47 9400 200000 2 3 4 18 55 7000 40000 5 16 47 9100 80000 275 405 6 15,7 45 9800 29000 result in our cases, all of the patient were observed intensively in pediatric intensive care unit and treated conservatively. five cases (group a) had got ringer acetate since the first day came to the hospital. they showed naturaly clinical manifestation and recovered well. one case showed alt, �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 35–41 ast increase more than 100 i/u but in short time showed recovery. five cases (group b) had got ringer lactate in an early day of admission in hospital. three cases showed increasing ast and alt more than 1000 and showed encephalopaty and become severe. patient no 4 got transfusion of ffp which indicator were abnormal coagulation profile. patient no 2 had got prc transfusion to replace the blood loss from intetinal bleeding. all of the involved organ were recovered along with recovery process of the disease. discussion to manage dengue virus infection cases in our hospital, as a clinician or a doctor in charge should identify the cause of infection by doing the physical and laboratory examination. it should be correlated with criteria or sign & symptoms of the cases: 1. severe cases have to be handled in emergency room or intensive care unit. 2. early and mild case could be handled in general practitioner practice or pediatrician clinic. a primary or secondary antibody response can be observed in patients with dengue virus infection. in primary dengue virus infection, igm antibodies develop rapidly and are detectable on days 3–5 of illness, reach its peak at about 2 weeks post infection and then decline to indetectable levels over 2–3 months. anti dengue virus igg appears shortly afterwards. secondary infection with dengue virus results in the earlier appearance of high titers of igg before or simultaneously with the igm responses.2,4 everyday, one or two cases could be inpatient in our hospital, for making a proper diagnosis and well recovery. during three decades, the world health organization (who 1997) has recommended the classification of dengue virus infection in: dengue fever (df) and dengue hemorrhagic fever (dhf) with or without dengue shock syndrome (dss). in order to be regarded as a df (or classical dengue) case, the patient must present fever and two symptoms out of the following: headache, retroocular pain, osteomyoarticular pains, rash, leucopenia, and some kind of bleeding. the dhf requires the presence of the four following criterias: a) acute sudden onset of high fever for 2 to 7 days; b) some kind of spontaneous bleeding, usually petechiaes, or at least having a positive tourniquette test; c) thrombocytopenia lower than 100,000/mm3; and d) plasma leakage, evidenced by a 20% elevation of the hematocrite, or by a 20% decrease of the hematocrite after the critical stage, or by the verification of pleural leakage, ascites or pericardial leakage by means of image studies. the course of the dengue disease goes through 3 clinical stages: the febrile stage, the critical stage, and the recovery stage.6 (see figure 1) who 2009 has divided dengue virus infection into 3 grade of disease, 1) dengue virus infection without warning sign (we also can say probable dengue infection); 2) dengue virus infection with warning signs (such as: abdominal pain, persistent vomiting, bleeding, etc) and the worst is 3) severe dengue, which severe plasma leakage leading to hypovolemic shock and also it could be fluid accumulation leading to respiratory distress. severe bleeding and severe organ impairment also could be occurred. it could be evaluated by laboratory findings; increasing of ast and alt more than 1000.5 (see figure 2) clinical diagnosis of dhf was based on who criteria. hepatomegaly is a common but not constant finding. liver involvement in dengue virus infection occurred because dengue virus antigen has been found in liver; hepatocytes and kupffer cells support the viral replication. in some countries, however, hepatomegaly varies from one epidemic to another, suggesting that the strain or serotype of virus may influence liver involvement. elevated liver enzyme levels are usually happen. serum ast and alt were used as a measure of cellular injury. the elevation is usually mild, but in some patients, ast and alt level reach 500 to 1000 u/l. leucopenia is common; thrombocytopenia and hemoconcentration are constant findings in dengue virus infection.5 haemorrhagic manifestation in dengue virus infection patients are not common, and within mild to severe. skin hemorrhage, including petechiae and purpura, are the most common, along with gum bleeding, epistaxis, menorrhagia, and gastrointestinal bleeding. the pathogenesis probably derives from vasculopathy, platelet deficiency and dysfunction, and blood coagulation defects.5 figure 1. the course of dengue virus infection (who, 2009) ��soegijanto, et al.: awareness of using ringer lactat solution in dengue virus infection cases figure 2. level of severity of dengue virus infection cases (who, 2009) our study is focused in using ringer lactate as a fluid therapy. ringer lactate was usually used in health centre and hospital for dengue shock syndrome since 1969. but sometimes it could be found some cases that becoming more severe so that the patient had to be admitted in dr. soetomo hospital surabaya, as a top referral hospital in east java, indonesia. therefore if there was a severe case with liver damage, we try to evaluate the patient and change the fluid resuscitation with ringer acetate. since 1968, ringer lactate had been used for early treatment of cholera disease which the patient suffered from hypovolemic shock due to massive diarrhea. the result was proven good. from this experience, ringer lactate was also used to treat dengue shock syndrome, but after 30 years using this protocol for dss rescucitation, there were more cases become severe and difficult to manage.3 in 1997, in who course of dhf had been done in bangkok. i had followed the course as a fellowship doctor from east java, indonesia. i got information that don’t use ringer lactate in a case with liver damage. based on this experience, until now i always remind the statement of our who teacher there, that we have to be careful in using ringer lactate in a case with liver damage, especially ast and alt > 100–200. to make sure this information, we try again to study the new pathophysiology of dengue virus infection (see figure 3) focusing on pathogenesis of hemorrhage in dhf (see figure 3) and update pathogenesis of dhf (see figure 4). severity can occure due to virus replicate in hepatocyt and kupffer cells and induce necrosis as an apoptosis in liver and it’s function damage.1 this event can be promoted by using ringer lactate. it is due to ringer lactate is metabolized in liver so if this solution is used, liver damage can be occurred more severe. figure 3. pathogenesis of haemorrhage in dhf (malaysia ministry of health, 2003) �0 indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 35–41 figure 4. update pathogenesis of dengue virus infection in 2009 (who, 2009) summary ten cases of dengue virus infection had been observed intensively in pediatric care unit and treated conservatively. five cases as a group a got ringer acetate and 5 cases as group b got ringer lactate before refered or on the first day admission in dr soetomo hospital; recucitation start with 5–7 ml / kg bw / hour for 1–2 hours then reduce 3–5ml / kg bw / hour for 2–4 hours and then reduce to 2–3 ml / kg bw / hour or less. according to the clinical respond, if the hematocrite remains the same or rises only minimmaly continues with the same rate (2–3 ml / kg bw / hour ) for another 2–4 hours, but if vital signs are worsening and hematocrit is rising rapidly, increase rate to 5–10 ml / kg bw / hour for 1–2 hours. intravenous fluids usually needed only 24–48 hours, and should be reduced gradually which haematocrite level decreases. patient with warning signs should be monitored by health care provides until the period of critical phase is over. we prefer using ringer acetate to ringer lactate, due to ringer acetate has some benefit, such as: 1. ringer acetate is metabolized in muscle and could be tolerated in patient with liver dysfunction. 2. acetate metabolism is faster than lactate metabolism. conclusion using ringer acetate as fluid therapy in dengue virus infection is better to prevent liver damage than using ringer lactate. references 1. academy of medicine malaysia ministry of health. clinical practice guidelines; dengue infection in adults. dengue consensus 2003 2. gubler d. dengue and dengue hemorrhagic fever. clin microbiol rev 1998; 11: 480–96 3. kurane i, enis fa. immunopathogenesis of dengue virus infection. in: d.j. gubler dj. kun g (ed). dengue and dengue haemorrhagic fever. wallingfird uk: cab international, 2002: 273–90 4. pt otsuka. guidance of infus solution. revision edition viii. 2003 ��soegijanto, et al.: awareness of using ringer lactat solution in dengue virus infection cases 5. shu. p, huang j current advances in dengue diagnosis. clin diag immunol 2004; 11: 642–50 6. srichaikul t, nimmannitya s. haematology in dengue and dengue haemorrhagic fever. baillieres best pract res clin haematol 2000; 13(2): 261–76 7. wang s, he r, patarapotikul, j et al, (1995). antibody-enhanced binding of dengue virus to human platelets. j. virology. october 213: page. 1254–1257. 8. world health organization. dengue guideline for diagnosis, treatment, prevention, and control. geneva. 2009. 9. world health organization. dengue hemorrhagic fever: diagnosis, treatment and control. 2nd edition. geneva : who, 1997: p. 17–27. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 61 vol. 5. no. 3 september–december 2014 literature review manifestation of aids with diarrhea rahmat zainuddin,1 nasronudin1,2 1 tropical and infectious disease division department of internal medicine, dr. soetomo general hospital faculty of medicine universitas airlangga, surabaya, indonesia. 2 institute of tropical disease, universitas airlangga, surabaya, indonesia abstract infectious diseases hiv/aids is a global health problem. according to who (2000) reported that 58 million people in the world are infected with hiv, within the 22 million people died from aids or 7000 people die every day. hiv infection caused decrease and disorder of humoral and cellular immunity. intestinal mucosal normally shows a physiologic inflamation that account for intestinal mucosal integrity. diarhhea in hiv infection due to immune deficiency can caused by pathogen and non pathogen. acute and chronic diarrhea usually found in hiv infection patient, the latter is more frequent. hiv enteropathy cause chronic diarrhea without pathogen infection because intestinal mucous damage by hiv direct infection. treatment is characterized as causative supportive and symptomatic treatment causal, supportive and symptomatic. immunonutrient is very important within management patient hiv/aids. key words: hiv/aids, diarrhea, hiv infection, immunonutrient, symptomatic abstrak penyakit infeksiun hiv/aids hingga kini merupakan masalah kesehatan dunia. berdasarkan who (2000) melaporkan bahasa 58 juta jiwa di dunia terinfeksi hiv, dengan 22 juta jiwa meninggal karena aids atau 7000 jiwa meninggal setiap tahunnya. infeksi hiv disebabkan penurunan dan kelainan kekebalan sel dan humoral. mukosa usus normal menunjukkan suatu inflamasi fisiologis yang berperanan dalam menjaga dan memelihara integritas dari mukosa. diare pada infeksi hiv dikarenakan penurunan imun dapat disebabkan oleh patogen dan non patogen. diare akut dan kronis biasanya ditemukan pada penderita hiv, akhir-akhir ini lebih sering. enteropati hiv menyebabkan diare kronis tanpa patogen infeksius karena mukosa usus rusak akibat infeksi hiv. pengobatan ini bertujuan untuk mengetahui penyebab, suportif dan simptomatik. nutrisi untuk kekebalan tubuh sangat penting dalam manajemen pasien hiv/aids. kata kunci: hiv/aids, diare, infeksi hiv, nutrisi untuk kekebalan tubuh, simptomatik introduction infectious diseases hiv/aids until now is a global health problem. according to who (2000) reported that 58 million people in the world are infected with hiv, within the 22 million people died from aids or 7000 people die every day.1 in the united states until january 2000 found 724,000 cases of aids in adults, 425 of them died.2 in indonesia (1997) recorded in the moh are 555 cases, 423 cases of hiv and 132 aids cases. from a number of such cases 66 % of men, 30% women and 4% of indeterminate sex.3 although many advances have been achieved in the field of medicine and has been implemented secondary prevention efforts but nonetheless progression of hiv infection to aids with manifestation of diarrhea are found, of which more than 50% of aids cases with manifestations of diarrhea.2 factors driving the emergence of diarrhea in aids patients is a direct effect of hiv infection and the manifestation of secondary infection. most of the secondary infections that cause diarrhea are protozoa and cytomegalovirus infection.2 manifestations of diarrhea in aids patients need to get serious attention, because it is often fatal even encourage death.4,5 based on these various circumstances discussed aids with manifestation of diarrhea. 62 indonesian journal of tropical and infectious disease, vol. 5. no. 3 september–december 2014: 61-66 pathophysiology hiv infection causes a decrease and disruption of the function of the immune system, especially mobile. cellular immune system disorder characterized by a decrease in cd4 t-lymphocyte count to less than 1,000/ul. the mechanism of cd4 t-lymphocyte decline is not fully known. some theories suggest that the direct cause of the cytopathic effect (virulence) gp 41 on the outside of the hiv virion. while indirectly occur through the process of cell apoptosis, cell destruction caused by an autoimmune response, cell maturation barriers, destruction of cd4 memory t-lymphocytes. decreased function of other immune system includes barriers cd4 tlymphocyte interactions with mhc ii, resulting in decreased fungal antigen presenting cells (apcs). besides a decline in the function of macrophages and natural killer (nk) cells. as a result of a decrease in interferon-gamma, increased production of cytokines from t lymphocytes cells, resulting in cell proliferation and differentiation of b lymphocytes also decreased resulting in disruption humoral immune response.6,7 disease course of hiv infection is divided into stages based on clinical circumstances and the number of cd4 t lymphocytes that includes.3,6 1. primary infection; hiv is an acute phase that lasts 6-12 weeks after hiv enters the body. cd4 t limfositl decreased and then returned close to normal, the symptoms include fever, malaise, myalgia, arthralgia, enlarged lymph nodes and meningoencephalitis 2. e a r l y i m m u n e d e f i c i e n c y ( c d 4 > 5 0 0 / u l ) ; asymptomatic phase that lasts about 5 years, often no symptoms but can be guillain-barre syndrome symptoms, demielinating chronic neuropathy, idiopathic thrombocytopenia, reiter’s syndrome, bell’s palsy. 3. intermediate immune deficiency (cd4 200–500/ul); a replication phase that lasts between 5–10 years. in this phase, resulting in lysis of cd4 t-lymphocytes, are susceptible to secondary infections zooster herpes, tuberculosis, sarcoma and lymphoma nonhodgkins kapposi’s. clinical symptoms arise usually progressive weight loss, fever without apparent cause and diarrhea. 4. advanced immune deficiency (cd4 < 200/ul); is the last phase (aids), which took place 10–13 years after infection. in this phase of severe immune deficiency causing opportunistic infections and cancers. intestinal immunity consists of phagocytic cells, humoral and cell mediated. each component makes a specific contribution to the individual to an infection or inflammation of the intestine. normal intestinal mucosa showed a physiological inflammation in the lamina propria, with the number of neutrophils, macrophages, plasma cells, and lymphocytes, which play a role in protecting and maintaining the integrity of the mucosa. neutrophils are important in immunity, impaired neutrophil function when the defense mechanisms in the gut also disrupted so prone to infection of the gastrointestinal tract.5 pathophysiology diarrhea in hiv infection by: dirrect toxic effects of hiv this direct toxic effect of happens due to the influence of gp 41 on the outer surface of hiv virions to the mucosa, epithelial, nervous system, causing intestinal disturbances in intestinal motility and secretion. this occurs through a process:8  expression of gp 41 at the membrane and budding of virus particles can cause an increase in membrane permeability, calcium influx toxic or osmotic lysis of infected cells.  membrane of infected cd4 t lymphocyte virus fusing with other cells that are not infected by gp 120 causes the formation of multiple multinucleated giant cells or syncitia. this process is lethal to the infected cells and uninfected.  viral replication can interfere with the synthesis and expression of cellular proteins that result in cell death.  binding of gp 120 to cd4 + t-lymphocytes induces gp 41 that have toxic effects. this situation is known as aids enteropathy is due to the interaction of the hiv virus to the gastrointestinal tract. histological picture of the patient’s small bowel mucosal atrophy are lowgrade, with a decrease in the mitotic process that will lead to hyporegenerative state. expression of p24 antigen in the intestinal mucosa will cause the inflammatory process.7 this will lead to the opening of tight junctions between epithelial cells on cytokine stimulation of hiv causing diarrhea with leak flux mechanism.9 the condition is often associated with chronic diarrhea mechanism, due to the morphological changes of the intestinal mucosa, causing malabsorption and decreased intestinal absorption overall.10 the reaction is caused by the hiv hipersensitifiti slow type which results in increased permeability of the intestinal mucosa, causing disruption to absorption and increased secretion of the intestinal wall which will cause inflammatory diarrhea.11 due to pathogen infection a. infection from intraluminal intestinal intestinal microflora is a normal flora is very important role in the defense in the intestine. normal flora in the gut is important as a saprophyte which act to resist the colonization of pathogenic bacteria. because in hiv infection is immunodeficiency, it will change to the normal flora of pathogens that would induce the secretion of chemical mediators, cytokines and inflammation in the intestinal mucosa occurs that would cause an increase in intestinal secretion and absorption disorders. 63rahmat zainuddin and nasronudin: manifestation of aids with diarrhea the decline in the immune system in people with hiv/ aids led to the growth of other pathogenic bacteria in the gastrointestinal tract, such as bacterial infections, viruses, protozoa and fungi. classically pathogens in hiv/aids is cryptosporidia, isospora belli, mycrosporidia, cytomegalovirus and mycobacterium avium intraceluler (mai) which will give the manifestation in the form of chronic diarrhea.12 b. secondary infection extraluminal s y s t e m i c d i s e a s e s c a n g i v e g a s t r o i n t e s t i n a l manifestations such as nausea, vomiting and diarrhea, as in pneumocistis carinii pneumonia and sepsis caused by the release of endotoxins and toxins released by microbes. endotoxin shock is usually caused by bacterial products in sepsis can occur when there is excessive cytokines production. production of cytokines will stimulate the release of mediators such as bradykinin, prostaglandins and leukotrine that will increase motility and increased secretion of water and electrolytes from the intestinal wall.8,10 etiology of diarrhea in patients with hiv/aids hiv patients with diarrhea were not found in the intestinal pathogens found about 15–46% and the rest is caused by a pathogen invasion.11 diarrhea due to infection all pathogens can cause diarrhea, hiv/aids patients. classically pathogens in the gut in people with hiv/aids is cryptosporidia, isospora belli, and mycobacterium avium-mycrosporidia intraceluler (mai)13 bacteria: pathogenic bacteria found in people with hiv/ aids is as a opportunistic infection such as: – mycobacterium avium complex (mac) that gives non inflammatory manifestations include diarrhea, weight loss, night sweats and hot and usually occurs in patients with cd4 + t-lymphocytes < 50 u/l. mac bacteria are very distinctive with an overview granular nodules with a diameter of about 1-4 mm erythematous surface. complications arising from the mac infection is intestinal obstruction, perforation, fistula and gastrointestinal bleeding.5 – other pathogenic bacteria that are often found include salmonella, e. coli, campylobacter and shigella.11,13 parasit: intestinal opportunistic parasitic in infectious hiv patients which often cause diarrhea are cryptosporidi, 20% microsporidia, 4,9% giardia lamblia, 2,6% entamoeba histolitica and approximately 1,5% isoposa beli. cryptosporidium infection has secretoric diarrhea as an effect and mostly assumed that it is correlated with malabsorption. microsporidial is often correlated with atrophy villus, hipper, increasing lymphocyte intraepithelial and d-xylose malabsorption.14 virus: citomegalovirus (cmv) is common and its effect on a very serious intestinal infections in people with hiv/aids. symptoms of cytomegalovirus infection in gastrointestinal disease causing intermittent and persistent diarrhea and cause abdominal pain, tenesmus, heat and weight loss. cytomegalovirus infection is more frequent cause of infection in the colon with a picture of a diffuse erythematous mucosa and submucosa that fibrils with hemorrhagic and ulcerated mucosa. viral pathogens are also frequently found in people with hiv/aids, among others, the herpes simplex virus and adenovirus.5 fungi: histoplasma capsulatum and candida albicans is a two fungal pathogen that often cause colitis in patients with hiv/aids. candidiasis is a specimen that causes watery diarrhea and abdominal pain as well as the implications colonic ulcers.5 table 1. etiology agent of diarrhea in hiv infection based on the location in the intestine5 smal intestine large intestine cryptosporidia cytomegalovirus microsporidia cryptosporidia isospora belli mycobacterium avium complex mycobacterium avium complex shigella sonnei salmonella species clostridium difficille campylobacter species campylobacter jejuni giardia lamblia histoplasma capsulatum adenovirus herpessimplex pneumocytis carinii diarrhea caused by non infectious movement of non-infectious diarrhea in patients with hiv/aids need to be considered because of a neoplastic intestinal, drug reactions, lactose intolerance and pancreatic insuffisiensi secondary to pentamidine or didanosine therapy and therefore malabsorption and steatorrhea.9 diagnosis approach of hiv with diarrhea approach to the diagnosis of hiv/aids patients with diarrhea, an important consideration is to detect movement with a degree of immune deficiency and clinical symptoms.11 there is a term that is often used is the aids -related complex (arc). arc is diagnosed when there are symptoms/signs of constitutional aids without opportunistic infections or tumors. this concept uses for the benefit of the clinic, the alleged progression to aids and for prognosis.15 64 indonesian journal of tropical and infectious disease, vol. 5. no. 3 september–december 2014: 61-66 determination movement diarrhea noteworthy food history, history of medicine penggunaan, travel history and symptoms along with diarrhea (nausea, vomiting, fever, other systemic symptoms) and diet (lactose) which can give clues to the cause of diarrhea. furthermore, the determination of the degree of immune deficiency, decreased cd4 count and opportunistic infection is an indication of the decline of the immune system. evaluation of the degree of immune deficiency useful in determining the movement of diarrhea.5,9,11 examination support5,9,14,16 microscopic examination of feces, microbiological and culture is very important in determining the movement of the symptoms of diarrhea if the infection is still a suspect. examination is generally performed three times due to the growth of microorganisms in episodic. examination of stool culture can detect pathogens campylobacter, cytomegalovirus, adenovirus, mycobacterium avium complex and salmonella. acid fast staining bacil (afb) of the stool can detect pathogens mycoobacterium avium complex. antigen test to detect pathogens giardia and cryptosporidium are more sensitive than microscopic examination. endoscopic examination with biopsy be an option if you find a bloody diarrhea, tenesmus. by doing endoscopy can determine the specific location of pathogens in the gut. diagnosis of hiv enteropathy with histopathologic examination and abnormal function of the gut with no found any pathogens or malignancy. management hiv patients with diarrhea therapy intended to treat hiv infection and diarrhea. treatment of diarrhea in patients with hiv is essentially connected with a state of decreased immunity, opportunistic infections and hiv enteropathy. strategic treatment aimed at specific and general therapy to reduce complaints and general state of repair.8,13 specific therapy specific therapy aimed at the treatment of hiv infection (antiretroviral therapy), provision of antimicrobial, fluid rehydration and correction of electrolyte disturbances. provision of anti-retroviral therapy is not promptly given to patients suspected. for aids patients (stage iii and iv disease) is recommended immediately given antiretroviral therapy irrespective of cd4 cell count or total lymphocyte count. recommendation also be given to patients with stage ii and iii with number of cd4 < 200 cells/mm3.17 table 2. criteria arc: there are 2 or more of the clinical symptoms that have lasted 3 months or more plus 2 or more laboratory abnormalities.15 clinical symptoms laboratory abnormalities fever 38° c limfopeni/lekopeni bb decrease > 10% trombositopeni enlarged lymph nodes anemi diare intermitten/ continuou ratio cd4/cd8 decrease weak, physical activity decrease cd4 decrease night sweats decreasing blastogenesis, increasing globulin antiretroviral drugs recommended by who (2002)17 nucleosida reverse transcriptase inhibitors (nsrti) non nukleoside reverse trancriptase inhibitor (nnrti) protease inhibitors abacavir; tablet 300 mg, syrup 100 mg/5 ml efavirens; kapsul 50 mg, 100 mg, 200 mg indinavir; kapsul 100 mg, 200 mg, 333 mg, 400 mg didanosin; tablet 5 mg, 100mg, 150 mg, 200 mg nevirapine; tablet 200 mg, syrup 50 mg/5 mg ritonavir; kapsul 100 mg, syrup 400 mg/5 ml lamivudin; tablet 150 mg, syrup 50 mg/5 ml lopinavir + ritonavir; kapsul 133,3 mg + 33 mg, syrup 400 mg/5 ml + 100 mg/5 ml stavudin; kapsul 15 mg, 20 mg, 30 mg, 40 mg, syrup 5 mg/5 ml nelfinavir; tablet 50 mg, powder 50 mg/g zidovudin ; kapsul 100 mg, 250 mg, 300 mg saquinavir; kapsul 200 mg table 3. antiretroviral therapy17 if examination cd4 can be performed: – clinic stadium iv, without taking the number of cd4 – clinic stadium i,ii or iii with cd4 < 200/mm3 if examination cd4 can not be performed: – clinic stadium iv, without taking the number of lymphocyte – clinic stadium ii or with the number of lymphocyte ≤ 1200/mm3 65rahmat zainuddin and nasronudin: manifestation of aids with diarrhea therapeutic response sometimes is a tool in establishing a diagnosis of diarrhea caused by an infection. if the stool examination found no pathogenic organisms empirical therapy trial with a quinolone (ciprofloxacin) and metronidazole can be given for 1-2 weeks prior to the examination endoskofi. standard treatment regimens for movement of infection in hiv/aids patients with diarrhea.14,16 cryptosporidiosis were paromycin 500 mg every 8 hours for 14 days, azitromycin, and letrazuril. isosporiasis were trimethoprin – sulfamethosaxole 160/800 mg every 6 hours for 10 days, then thrice a day for 21 days, metronidazole, and pyrimethamin. microsporidiosis was albendazole 400 mg every 12 hours for 14 days. salmonella, shigella, enterocolitis were ciprofloxacin 500 mg every 12 hours for 14 days, cefotaxime, ceftriaxone, and chloramphenicol. campylobacter colitis was erytromycin 500 mg every 6 hours. mycobacterium avium complex was rifabutin 300 mg every days added ethambutol 400 mg every 12 hours dan claritromycin 500 mg every 12 hours. cytomegalovirus was ganciclovir 5 mg/kgbb every 12 hours for 14 days. candida albicans, histoplasma capsulatum were nystatin oral, amphotericin b, intraconazole, fluconazole used systematically in severe cases. lack of fluids and electrolytes cause serious problems in a patient with severe diarrhea. monitoring immediate fluid replacement especially for severe diarrhea so that the volume of fluid lost should be measured and the amount of fluid and electrolyte replacement should be done immediately.5 oral rehydration with fluids containing glucose, na, k, cl and bicarbonate is effective in patients with mild dehydration. parenteral fluid administration (ringer’s lactate) is given in the acute state of severe dehydration.12 general therapy therapy aimed at the general supportive and symptomatic treatment, nutritional support and counseling to people with hiv/aids. symptomatic treatment is very important for all patients with diarrhea in the case to prevent fluid loss and improve disturbance and functional status of patients. symptomatic therapy, among others:18 • luminal anti diarrhea such as bismuth subsalicylate or aluminum antacids, cholesttyramine, recommended especially in mild cases • antimotility agents including loperamide 2–4 mg given four times a day or diphenoxylate 2 tablets given four times daily. • octreotide 100–500 mcg administered subcutaneous or intravenously every 8 hours, especially given the severe cases that do not respond to oral medications. in hiv-infected patients often have impaired nutrient intake caused a decline in the body's biological functions. immunonutrient important to consider in the management of patients with hiv/aids, which contains a variety of materials that can meet the needs of patients who have an infection. immunonutrient contains the components necessary to meet basic metabolic needs are carbohydrates, proteins, fats and also contains 3 main imunonutrient namely arginine, gluthamin and fish oil that have a positive impact on the immunological function of the body. the supplement should also contain a variety of vitamins (vitamin c and vitamin e) and minerals (mn, cu, se and zn) that has the ability of anti -oxidants as well as exogenous triggers endogenous anti-oxidant potential and has anti apoptotic effects.19,20 nutrition given orally if the patient is still able to eat. oral nutritional supplements should be given in the form of a convenient and easily absorbed nutrient dense. enteral nutrition orally given if insufficient or limited by the presence of lesions in the mouth and esophagus until the patient is able to maintain sufficient oral intake. parenteral nutrition and oral performed when enteral nutrition can not be tolerated in considerable amounts. parenteral nutrition is given on a case by forceful vomiting and prolonged and severe diarrhea.21 summary hiv infection caused decrease and disorder of humoral and cellular immunity. intestinal mucosal normally shows a physiologic inflamation that account for intestinal mucosal integrity. diarhhea in hiv infection due to immune deficiency can caused by pathogen and non pathogen. acute and chronic diarrhea ussually found in hiv infection patient, the latter is more frequent. hiv enteropathi cause chronic diarrhea without pathogen infection because intestinal mucous damage by hiv direct infection. treatment is aimed to causal, supportive and symptomatic. immunonutrient very important within management patient hiv/aids. references 1. who. 2000. the world's health report: global burden disease 2000. world health organization. 2. zavasky dm, gerberding jl, md, sande ma, 2001. patients with aids. in: current diagnosis & treatments in infectious disease. editors: wilson wr, sande ma. international edition. new york, p. 315–327. 3. suseno ls, 1997. klasifikasi infeksi hiv/aids dan defenisi kasus surveilans aids. majalah kedokteran indonesia 47, 301–303. 4. verdier ri, 2000. trimethophrin-sulfamethoxazole compared with ciprofloxacin for treatment and prophylaxis of isospora belli and cylospora cayetanensis infetion in hiv patients. annals of internal medicine 132, 885–888. 5. scott gb. 2002. management of acute illness in hiv-infected children. in: hiv/aids primary care guide. editors: lawrence m, tierney. education and training university of florida, florida, p. 283–289. 6. bjarnason i, 1996. intestinal inflammation, ileal structure and function in hiv. aids 10, 1385–1391. 7. fauci as, lane hc, 2001. human immunodeficiency virus (hiv) disease: aids and related disorders. in: harrisons priciple of internal medicine. editors: isselbacher k, braunwald e, wilson jd, martin jb, fauci as, kasper dl. 15th ed. mcgraw-hill, inc. usa, p. 241–249. 66 indonesian journal of tropical and infectious disease, vol. 5. no. 3 september–december 2014: 61-66 8. kresno sb, 2001. imunodefisiensi. dalam imunologi diagnosis dan prosedur laboratorium edisi keempat. balai penerbit fakultas kedokteran universitas indonesia, jakarta, hlm. 233–258. 9. marriot dj, murchie mm, 1997. hiv and advanced immune deficiency. in: managing hiv.editor: stewar gj. australian medical publishing company limited, p. 15–16. 10. ahlquist da, camilleri m. 2001. diarrhea and constipation. in: harrisons priciple of internal medicine. editors: isselbacher k, braunwald e, wilson jd, martin jb, fauci as, kasper dl. 15th ed. mcgraw-hill inc. usa, p. 241–249. 11. mandell, 2000. diarhhea in patients with acquired immunodeficiency syndrome. in: princip les and practice of infectious disease. 5th ed. churchill livingstone inc, p.1103–1106. 12. kenneth r, 2001. alimentary tract. in: current medical diagnosis & treatment. editors: lawrence mt, stephen jm, maxine ap. 40th ed. lange medical boos/mcgraw-hill, medical publishing division, new york, p. 559–661. 13. poles ma, dieterich dt, cappel ms, 1999. gastrointestinal manifestations of hiv disease including the peritoneum and mesentry. in: textbook of aids medicine. editors: merigan tc, barlet jg, bolognesi d. 2th ed. williams & wilkins, baltimore, p. 542–546. 14. dieterich dt, wilcox cm, 1996. practice parameters committee of the american college of gastroenterology in diagnosis and treatment of esophageal diseases associated with hiv infection. am j gastroenterol 91, 2265–2269. 15. widodo j, 1992. gambaran klinis infeksi hiv. dalam seluk beluk aids. editor: aryatmo tjokronegoro, zubairi djoerban, corry s. matondan. fakultas kedokteran universitas indonesia. jakarta, hlm. 25–43. 16. simon d, weiss lm, brandt lj, 1992. treatment options for aids-related esophageal and diarrheal disorders. am j gastroenterol 87, 274–281. 17. who, 2002. principles of hiv theraphy. in: the use of antiretroviral theraphy: a simplifed of approach for resource constrained countries regional office for south-east asia, new delhi, p. 4–18. 18. jan z, 2002. nutrition for health and healing in hiv. acria update newyork 11 (2), 1–3. 19. evoy d, lieberman md, fahey tj, dall jm, 1998. immunonutrition: the role of arginin. nutrition 14, 611–617. 20. friss h, 2002. micronutrient and infection. in: micronutrients and hiv infection. editor: friss h. crc press washington dc,p. 2–21. 21. cimoch pj, 1997. treatment of nutrional health. prevention and hiv-ascociated malnutrition: a case manager guide. j am diet assoc 95, 428–432. vol. 9 no. 1 january–april 2021 ijtid, p-issn 2085-1103, e-issn 2356-0991 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ original article intestinal parasitic infection, the use of latrine, and clean water source in elementary school children at coastal and non-coastal areas, sumenep district, indonesia 1 medical student program, faculty of medicine, universitas airlangga, surabaya indonesia 2 tropical medicine program, faculty of medicine, universitas airlangga, surabaya indonesia 4 department of medical parasitology, faculty of medicine, universitas airlangga, surabaya indonesia 5 department of pediactrics, faculty of medicine, universitas airlangga, surabaya indonesia 6 laboratory of malaria, institute of tropical disease, universitas airlangga, surabaya indonesia received: 10th october 2020; revised: 15th october 2020; accepted: 5th february 2021 abstract inadequate latrine and water source cause transmission of intestinal parasitic infection, particularly in children. there is a lack information about it and it is needed to be investigated. this study aimed to compare the prevalence of intestinal parasitic infection, the use of latrine and clean water source in elementary school children at coastal and non-coastal areas in sumenep district, indonesia. an analytic observational study with cross sectional design was conducted in dasuk timur elementary school located at coastal area, and kolor ii elementary school at non-coastal area, sumenep district, in january 2020. intestinal parasites in students’ stools were identified by microscopic examination using wet direct smear stained with lugol. the use of latrine and water sources were analyzed with questionnaire. a total of 68 children stools were collected from both elementary schools. worm infections were not found. thirty-one children (31/44, 70.5%) from dasuk timur elementary school and eight children (8/24, 33.3%) from kolor ii elementary school were infected with intestinal protozoan and significant difference (p=0.003, chi-square test). blastocystis hominis was highly found in stools of dasuk timur elementary school’s students (31/44, 70.5%) and significantly different from kolor ii elementary school’s students (p<0.0001, chi-square test). three children (3/44, 6.8%) from dasuk timur elementary school were still practicing open defecation. dasuk timur elementary school’s students suffered from intestinal parasitic infection were mostly using non-piped water source (20/31, 64.5%) and were significantly differ ent between two elementary schools (p=0.015, fisher’s exact test). prevalence of intestinal parasitic infections in children was found higher in coastal than non-coastal area due to the commonly use of unclean water sources and inadequate latrine. keywords: intestinal parasitic infectio; clean water source; latrine, elementary children; coastal area abstrak jamban dan sumber air yang tidak layak menyebabkan transmisi infeksi parasit usus, terutama pada anak. sedikit informasi terkait jamban, sumber air dan infeksi parasit usus pada anak, sehingga perlu untuk diteliti. penelitian ini bertujuan untuk mengidentifikasi perbedaan prevalensi infeksi parasit usus, penggunaan jamban, dan sumber air bersih pada anak sekolah dasar di daerah pesisir dibandingkan dengan bukan pesisir di kabupaten sumenep, indonesia. penelitian observasional analitik dengan desain cross sectional dilaksanakan di sdn dasuk timur berlokasi di daerah pesisir, dan sdn kolor ii berlokasi di daerah bukan pesisir kabupaten sumenep pada bulan januari 2020. parasit usus dalam tinja anak sekolah dasar diidentifikasi dengan pemeriksaan mikroskopis dari sediaan hapusan tinja basah yang tercat dengan larutan lugol. penggunaan jamban dan sumber air dianalisis dengan kuesioner. sebanyak 68 tinja anak dikumpulkan dari kedua sekolah dasar. kecacingan tidak ditemukan. sebanyak 31 anak (31/44, 70.5%) sdn dasuk timur dan 8 anak (8/24, 33.3%) sdn kolor ii terinfeksi protozoa usus dan berbeda bermakna (p=0.003, chi-squ chisquare test). blastocystis hominis ditemukan banyak dalam tinja anak sdn dasuk timur (31/44, 70.5%) dan b * corresponding author: sukmab@fk.unair.ac.id open acces under cc-by-nc-sa share alike 4.0 3 dasuk timur elementary school, dasuk, sumenep, indonesia raden bagus yanuar renaldy1, m. ahda naufal aflahudin1, zukhaila salma2, sumaryono3, muhammad yasin fitriah4, sri wijayanti sulistyawati4, dominicus husada5, sukmawati basuki 4,6* r. bagus yanuar renaldy, et al.: intestinal parasitic infection, the use of latrine 17 ijtid, p-issn 2085-1103, e-issn 2356-0991 berbeda bermakna dengan anak sdn kolor ii (p<0.0001, chi-square test). tiga anak (3/44, 6.8%) dari sdn dasuk timur masih melakukan defekasi di tempat terbuka. anak sdn dasuk timur yang terinfeksi parasit usus kebanyakan menggunakan sumber air non-pipa (20/31, 64.5%) dan berbeda bermakna antara kedua sekolah dasar (p=0.015, fisher’s exact test). prevalensi infeksi protozoa usus pada anak ditemukan lebih tinggi di daerah pesisir dibandingkan di daerah bukan pesisir karena penggunaan sumber air tidak bersih dan jamban yang tidak layak. kata kunci: infeksi parasit usus; sumber air bersih; jamban; anak sekolah dasar; daerah pesisir how to cite: renaldy, rby., aflahudin, man., zukhaila., salma., sumaryono., fitri nmn., wijayanti, s., sulistyawati., husada, d., basuki, s., intestinal parasitic infection, the use of latrine, and clean water source in elementary school children at coastal and non-coastal areas, sumenep district, indonesia. indonesian journal of tropical and infectious disease, 9(1), 16–23 the intestinal helminth infections can cause iron deficiency anemia, impaired mental function and cognitive delevelopment that affect to children growth and development3. intestinal protozoan infections also impact growth and development in children2. children who consume contaminated food and water can lead to intestinal parasitic infections. moreover, enviromental and economy factors, such as poor sanitation, poverty, and lack of education, also contribute to intestinal parasitic infections. children have active period of playing and moving, then they forget to wash their hands that affect to the intestinal parasite transmission4. based on law number 27 year 2007 about management of coastal areas and small islands, coastal areas are transitional areas between land and marine ecosystems which are affected by changes on land and sea. indonesia is the largest archipelago in the world that consists of around 18,110 islands with coastline of 108,000 km5. this makes coastal areas in indonesia a hope for people to fulfill life necessities. however, the environd ment will be damaged as the development blooms in coastal area. the damaged environment can trigger health problems and makes the disease transmission easier, such as water pollution, littering, and defecate in the open place 6. the prevalence of parasitic infection in coastal area were reported by a study in tanawangko, tombariri sub-district, minahasa district found that 4.3% elementary school children were infected with ascaris lumbricoides 7. another study conducted in coastal area of wori sub-district, north minahasa district showed that 4.7% children suffered from intestinal helminth infections and 15.5% children were infected with with intestinal protozoa.8 in addition, previous research in coastal area of makassar city reported that the prevalence of intestinal helminth infections was 59.3% in children.9 therefore, the incidence of intestinal parasitic infection is still quite high in indonesia. sumenep regency is located at the eastern part of madura island and has a coastline length of 577.76 km. it can increase the risk of intestinal parasitic infections in coastal area.10 a study conducted in aeng merah iii elementary school, batuputih sub district, sumenep district showed that 55.6% of 14 children who defecated on the ground and 44.4% of 20 children who defecated in latrine were infected with intestinal nematodes in 2014.11 the prevalence of intestinal parasitic infection in elementary school children at sumenep district has not yet been open acces under cc-by-nc-sa share alike 4.0 introduction parasitic infections are caused by intestinal helminth and protozoa that are very common in developing country, such as indonesia. parasitic infections can cause high morbidity and mortality in endemic area1. intestinal parasitic infections are estimated to occur in 3.5 billion people around the world and the majority occur in children2. ijtid, p-issn 2085-1103, e-issn 2356-0991 18 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 16–23 materials and methods an observational analytic study with cross sectional design was conducted in coastal area, dasuk timur (dt) elementary school, and non-coastal area, kolor ii (kii) elementary school, of sumenep district, indonesia in january 6-13, 2020. sumenep district lies in the eastern part of madura island. the study sites is shown on figure 1. dasuk timur school is 0.07 km from seashore and kolor ii school is 6.32 km. the distance of dasuk timur school from kolor ii school is 16 km. elementary school children in 1-4 grade, who were willing and allowed by their parents, were included in this study. their stools and the questionnaires were collected. sterilized clean tool pots were distributed to children in both elementary schools. children, who submitted stools pots and also fulfilled the questionnaire, were analyzed. this study was declared by ethics committee of the faculty of medicine, universitas airlangga (number of 221/ec/kepk/fkua/2020). investigated further. thus, the identification of prevalence of intestinal parasitic infection with the use of latrine and clean water sources in elementary school children at coastal area and non-coastal area of sumenep district, indonesia were conducted in this study. the stool samples of elementary school children were collected and preserved by 10%formalin solution. stool samples were examined with direct smear using 1% lugol solution under light microscope with 200x magnifications for identifying intestinal helminth, and 1000x magnifications with immersion oil for detecting intestinal protozoa. the stool examination was performed in the laboratory of medical parasitology department, faculty of medicine, universitas airlangga, surabaya. the use of latrine and clean water sources were determined by using questionnaires. questionnaire was guided by researchers and teachers to the children. the latrine types consisted of household toilet, public toilet, and open air defecation in river, sea, or the bush. the clean water sources were composed by piped and non-piped water sources. the non-piped water sources consisted of river, sea, pond, and draw or artesian well water. the collected data were analyzed using 25.0 version spss program. the chi-square test were used when applicable. study site and population data collection ethical consideration figure 1. study sites are located in sumenep district, madura island, indonesia that are in dasuk timur elementary school (the blue circle) and kolor ii elementary school (the yellow circle). open acces under cc-by-nc-sa share alike 4.0 results r. bagus yanuar renaldy, et al.: intestinal parasitic infection, the use of latrine 19 ijtid, p-issn 2085-1103, e-issn 2356-0991 most samples were obtained from dasuk timur elementary school (44/68, 64.7%). the oldest participants (11-year old) were only found in dasuk timur elementary school. however, there was no significant difference between the distribution of age from both schools. the distribution of children infected with intestinal parasites according to sex and age did not show a significant difference between the two elementary schools (see on table 1). the collected stool samples were examined with a direct smear or wet mount. intestinal helminth infection was not found in children fotm elementary school children from grade 1st to 4th were voluntarily to participate this study by filling data using questionnaire and submitting their stools that were totally of 68 children (68/155, 43.9%) from both elementary schools (see on figure 2). figure 2. flow diagram of sample collection characteristics of subjects dasuk timur elementary school grade 1-4 kolor ii elementary school grade 1-4 5 children were absent 51 children received stool pots 86 children received stool pots 44 children participated 24 children participated intestinal parasites identification and questionnaire analysis 10 children were absent intestinal parasitic infections from both elementary schools (see on table 2). children infected with intestinal protozoan parasites were highly shown in dasuk timur elementary school (31/39, 79.5%). table 1. the characteristic of elementary school children study site characteristic dasuk timur elementary school n (%) kolor ii elementary school n (%) p value sex male 25 (56.8) 10 (41.7) 0.232* female 19 (43.2) 14 (58.3) age (y.o) 6 3 (6.8) 5 (20.8) 7 8 (18.2) 5 (20.8) 8 7 (15.9) 5 (20.8) 0.017 + 9 10 (22.7) 7 (29.2) 10 13 (29.5) 2 (8.3) 11 3 (6.8) 0 infected children sex male 15 (48.4) 5 (62.5) 0.695* * female 16 (51.6) 3 (37.5) age (y.o) 6 1 (3.2) 2 (25) 7 7 (22.6) 1 (12.5) 8 4 (12.9) 0 0.311+ 9 8 (25.8) 4 (50) 10 9 (29) 1 (12.5) 11 2 (6.5) 0 intestinal protozoan parasites were detected in 39 children stools (39/68, 57.3%) that distributed to 31 children (31/39, 79.5%) from dasuk timur elementary school and eight children (8/24, 33.3%) from kolor ii elementary school, and it was a significant difference found between two elementary schools (p<0,05, chi-square test). the infected stool samples consisted of 33 samples (33/39, 84.6%) with single infection and 6 samples (6/38, 15,8%) with mixed infection. b. hominis infection was found more frequently in dasuk timur elementary school children (31/31, 100%) compared to kolor ii elementary school children (5/8, 62,5%) and it was significantly different (p<0,05, chi-square test) (see table 2). seventeen children (17/36, 45.7%) were infected with b. hominis by density of 1/20 field (see on table 3). the intestinal protozoa were identified that were cysts of g. lamblia, cysts of e. coli, vacuolar type of b. hominis and oocyst of cryptosporidium spp. (see on figure 3). three children (3/44, 6.8%) from dasuk timur elementary school were still practicing open defecation in places such as a river, sea, or bushes, while all children from kolor ii elementary school were defecating in the latrine. however it was not statistically significant between both elementary schools (p=0.336, fisher exact test) (see on table 4). a total of 29 infected children from dasuk timur elementary school used household toilet (29/31, 93.5%), while all kolor ii elementary school children who suffered from intestinal parasitic infections used household toilet (8/8, 100%). it was no a significant difference found between two elementary schools (see on table 4). latrine open access under cc-by-nc-sa share alike 4.0 ijtid, p-issn 2085-1103, e-issn 2356-0991 20 indonesian journal of tropical and infectious disease, vol. 9 no. 1january–april 2021: 16–23 the intestinal protozoa were identified that were cysts of g. lamblia, cysts of e. coli, vacuolar type of b. hominis and oocyst of cryptosporidium spp. (see on figure 3). figure 3. the morphology of intestinal protozoa in children’s stool samples were (a) vacuolar type of b. hominis; (b) cyst of e. coli; (c) cyst of g. lamblia; and (d) oocyst of cryptosporidium spp (modified ziehl neelsen stain). minimal length is 1 micrometer. table 4. latrine usage of elementary school children study site latrine dasuk timur elementary school n (%) kolor ii elementary school n(%) p value household toilet 40 (90.9) 22 (91.7) public toilet 1 (2.3) 2 (8.3) 0.336* open defecation 3 (6.8) 0 infected children household toilet 29 (93.5) 8 (100) public toilet 1 (3.2) 0 1.000* open defecation 1 (3.2) 0 * p value is calculated using fisher’s exact test. p≤0.05 was significant. clean water sources non-piped water sources were mostly used by children from dasuk timur elementary school compared to children from kolor ii elementary school (28/44, 63.6% vs 2/24, 8.3%) and there was a significant difference between two elementary schools (p<0,05, chisquare test) (see table 5). non-piped water sources were mostly used by infected children from dasuk timur elementary school compared to infected children from kolor ii elementary school (20/31, 64.5% vs 1/8, 12.5%) and it was a significant difference between two elementary schools (p<0,05, chi-square test) (see on table 5). the non-piped water sources in dasuk timur village are open draw well, artesian well, river, a pond and sea water, while in kolor ii fga table 2. intestinal protozoan in stools of elementary school children study site intestinal parasitic infection dasuk timur elementary school n=44 (%) kolor ii elementary school n=24 (%) p value intestinal helminth positive 0 0 negative 44 (100) 24 (100) intestinal protozoan giardia lamblia single 0 0 0,536** mix 2 (4.5) 0 entamoeba coli single 0 1 (4.2) 0,413** mix 5 (11.4) 0 blastocystis hominis single 25 (56.8) 5 (20.8) <0,0001* mix 6 (13.6) 0 cryptosporidiu m spp. single 0 2 (8.3) 0,121** mix 0 0 negative 13 (29.5) 16 (66.7) 0,003* * p value is calculated using chi-square test. p≤0.05 was significant. ** p value is calculated using fisher’s exact test. p≤0.05 was significant. the infected stool samples consisted of 33 samples (33/39, 84.6%) with single infection and 6 samples (6/38, 15,8%) with mixed infection. b. hominis infection was found more frequently in dasuk timur elementary school children (31/31, 100%) compared to kolor ii elementary school children (5/8, 62,5%) and it was significantly different (p<0,05, chi-square test) (see table 2). seventeen children (17/36, 45.7%) were infected with b. hominis by density of 1/20 field (see on table 3). table 3. blastocystis hominis density number of blastocystis hominis* no. infected samples n (%) 1/20 fields 17 (47.2) 2/20 fields 4 (11.1) 3/20 fields 4 (11.1) 4/20 fields 1 (2.8) 5/20 fields 2 (5.5) 7/20 fields 5 (13.9) 10/20 fields 1 (2.8) 11/20 fields 1 (2,9) 40/20 fields 1 (2,9) total 36 (100) *number of b. hominis was counted by the field of 1000x magnifications with immersion oil in total 20 fields. open access under cc-by-nc-sa share alike 4.0 discussion r. bagus yanuar renaldy, et al.: intestinal parasitic infection, the use of latrine 21 ijtid, p-issn 2085-1103, e-issn 2356-0991 village is artesian well water (see on figure 4). figure 4. the non-piped water types are a) an open draw well, b) an artesian well, and c) a pond with outfall. table 5. clean water sources of elementary school children study site clean water source dasuk timur elementary school n (%) kolor ii elementary school n (%) p value piped water 16 (36.4) 22 (91.7) non-piped water 28 (63.6) 2 (8.3) <0,0001* infected children piped water 11 (35.5) 7 (87,5) non-piped water 20 (64.5) 1 (12,5) 0.015** * p value is calculated using chi-square test. p≤0.05 was significant. ** p value is calculated using fisher’s exact test. p≤0.05 was significant. due to the regular consumption of oral anthelmintic medicine. based on interview to teachers, anthelmintic medicine, albendazole, is regularly consumed by children in their schools twice per year.the albendazole was provided by public health service or local public health center. albendazole was last taken by children in september 2019 or 4 months before collecting the stools samples. previous studies also showed the reduction of intestinal helminth infection in elementary school students after providing regularly albendazole and health education such as in bunduduk elementary school central lombok14, and pagi paseban elementary school central jakarta15. the prevalence of intestinal helminth infections in elementary school children can be eliminated by regular consumption of oral anthelmintic and education of intestinal helminth infections. blastocystis hominis was found the most in children’s stools both from dasuk timur elementary school (31/31, 100%) and kolor ii elementary school (5/8, 62,5%) and significantly different (p<0,05, chi-square test). it indicates that dasuk timur elementary school children are more at risk to be infected with b. hominis. in addition, they were without symptoms and did not have a history of diarrhea. asymptomatic blastocystis infection might due to rare number of b. hominis in their stools. it was reported that finding >.5 parasites per highpower field (400 magnifications) is associated with the presence of gastrointestinal disease16. asymptomatic blastocystis infection occurred in elementary school children in coastal and non-coastal areas. they could be carriers who were able to contaminate b. hominis into environment, particularly in the poor personal hygiene and sanitation. most of dasuk timur elementary school children used non-piped water sources and they were carrying the b. hominis in their stools. in addition, people in dasuk timur village including elementary children used to the wells water for their drink. some of them used to without boiling wells water for drink (based on interviews with several children and teachers). b. hominis infection belongs to waterborne disease and transmitted by the fecal-oral route, the present study determined that sex and age had the same probability to be infected with intestinal parasites. molina12 and saputra13 stated that there was no statistical difference in intestinal parasitic infections based on age and sex. another study conducted in sanandaj city showed that sex and age did not have a significant difference in intestinal parasitic infection4. sex and age did not affect the incidence of intestinal parasitic infections at dasuk timur elementary school and kolor ii elementary school. the present findings found no intestinal helminth infections in children from both elementary schools. the zero prevalence of intestinal helminth infection in children might fa open access under cc-by-nc-sa share alike 4.0 ijtid, p-issn 2085-1103, e-issn 2356-0991 22 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 16–23 such as through food and water contaminated with feces containing b. hominis and poor sanitation in the community.17,18 a study in sanandaj city reported that a high prevalence of intestinal protozoan infection among school children occurred and the use of drinking water sources from unprotected wells was a risk factor of intestinal parasitic infection4. it was also found in lao pdr that people used the water sources from mountain and wells water were infected with either intestinal helminth or protozoa.19 this fact confirmed that unboiling non-piped water source for drink is potential to transmit the b. hominis into children living in dasuk timur village. poor sanitation facilities in coastal areas can also contribute to spreading the intestinal parasites infection, such as the inadequate supply of clean water, inadequate latrine, improper waste disposal, and littering.6,20 furthermore, a study in karangasem district, bali showed that 34% of elementary school children were infected with b. hominis and most of the children were still practicing poor sanitation, shared water with animals, and had a lack of household toilets21. our study showed that the most children stools carrying b. hominis used the house toilets for defecation. only two elementary children carrying b. hominis in their stools used to either open defecation or public toilet and they are living in dasuk timur. nevertheless, they could contaminate the water source. ironically, they still used unboiling non-piped water source for drink in dasuk timur village, so b. hominis transmission occurred more in dasuk timur than in kolor ii village. therefore, the nonpiped water source needs to be investigated further, whether contains b. hominis or not, in order to cut off the b. hominis transmission. thus, coastal areas with non-piped water sources, without boiling water for drink, and still doing the open defecation can increase the risk of b. hominis infection. our findings showed that g. lamblia and e. coli were found more frequently in dasuk timur elementary school children’s stools compared to kolor ii elementary school children’s stools. these protozoan parasites also belong to water borne disease. 22,23,24,25 it showed that dasuk timur elementary school children were more at risk to be suffered from intestinal protozoan infection because most of the children in the coastal area still used nonpiped water sources. gabbad et al revealed that difficulty accessing clean water in elengaz, khartoum, sudan caused children suffering from intestinal parasites.18 a study in a rural area of boyer-ahmad, iran represented that the prevalence of intestinal protozoan infection was 37.5% with 9 species of protozoa found in stool samples, including g. lamblia, b. hominis, e. coli, and endolimax nana. this high prevalence of intestinal protozoan infection might due to water shortages during decresed level of rainfall in iran, which caused poor sanitation.26 recent study in kenya stated that source of water for drinking was a major determinant for the risk of intestinal protozoan infections in children under 5 years with diarrhea.27 lack of access to clean water sources is one of the risk factors of intestinal protozoan infection. conclusion prevalence of intestinal parasitic infection was found higher in coastal than non-coastal area due to commonly use unclean water source and inadequate latrine. acknowledgement we would like to thank the students of dasuk timur elementary school and kolor ii elementary school who were willing to participate in this study to collect the stool sample and fill the questionnaire. we also thanked to the teachers of both elementary schools who assisted and coordinated the students to collect the stool samples and fill the questionnaires. this study was supported by research grant from universitas airlangga with number 2158/un3/2019. conflict of interest the authors declare that there is no conflict of interest. open access under cc-by-nc-sa share alike 4.0 references r. bagus yanuar renaldy, et al.: intestinal parasitic infection, the use of latrine 23 ijtid, p-issn 2085-1103, e-issn 2356-0991 1. farrar j, hotez p, junghanss t, kang g, lalloo d, white n. manson's tropical diseases, 23rd, elsevier saunders ltd. china, 2014 2. gelaw a, anagaw b, nigussie b , silesh b , yirga a , alem m, endris m, and gelaw b. prevalence of intestinal parasitic infections and risk factors among schoolchildren at the university of gondar community school, northwest ethiopia: a crosssectional study. bmc public health. 2013; 13:304. 3. world health organization, helminth control in school-age children, 2nd ed., who, geneve, 2011 4. bahmani p, maleki a, sadeghi s, shahmoradi b, ghahremani e. prevalence of intestinal protozoa infections and associated risk factors among schoolchildren in sanandaj city, iran. iranian journal of parasitology. 2017;12(1):108–116 5. uu no 27 tahun 2007. tentang pengelolaan wilayah pesisir dan pulau-pulau kecil. jakarta: republik indonesia. 2017 6. imroatus s, mulyadi, maryam l. gambaran sarana sanitasi masyarakat kawasan pesisir pantai dusun talaga desa kairatu kecamatan kairatu kabupaten seram bagian barat tahun 2014. higiene. 2015;1(2):75-83 7. luis r, tuda j, sorisi a. kecacingan usus pada anak sekolah dasar di tanawangko kecamatan tombariri kabupaten minahasa.jurnal e biomedik. 2016;4(2):70-75 8. tangel f, tuda j, pijoh v. infeksi parasit usus pada anak sekolah dasar di pesisir pantai kecamatan wori kabupaten minahasa utara. jurnal e-biomedik. 2016;4(1) 9. budiasri r, hadju v, sirajuddin s. infeksi kecacingan dan status gizi pada anak sekolah dasar di wilayah pesisir kota makassar. universitas hasanuddin. 2013 10. dinas perikanan kabupaten sumenep. laporan kinerja instansi pemerintah (lkjip) tahun 2017. sumenep: dinas perikanan kabupaten sumenep. 2017 11. ulfah a. hubungan antara kebiasaan defekasi dengan infeksi nematoda usus “soil transmitted helminthes” di sdn aeng merah iii kecamatan batuputih kabupaten sumenep. tesis, surabaya, universitas muhammadiyah surabaya. 2014 12. molina n, pezzani b, ciarmela m, orden a, rosa d, apezteguía m, basualdo j, minvielle m. intestinal parasites and genotypes of giardia intestinalis in school children from berisso, argentina. the journal of infection in developing countries. 2011;5(07):527-534 13. saputra i, sari m, gunardi w. prevalensi infeksi protozoa usus pada siswa sekolah dasar negeri papanggo 01 jakarta utara tahun 2016. j. kedokt meditek. 2017; 23(61):41-47 14. winita r, mulyati, astuty h. upaya pemberantasan kecacingan di sekolah dasar. makara, kesehatan. 2012; 16(2):65-71. 15. masniati, diarti m, fauzi i. pemberian obat cacing albendazol terhadap hasil pemeriksaan kecacingan golongan sth pada feses siswa sdn bunduduk lombok tengah. jurnal analis medika bio sains. 2018; 5(1):55-59 16. coyle cm, varughese j, weiss lm, tanowitz hb. blastocystis: to treat or not to treat. clinical practice. 2012; 54(1january):105-110. 17. de la cruz c, stensvold r. blastocystis. global water pathogen project. 2017 18. gabbad a, elawad m. environmental sanitation factors associated with intestinal parasitic infections in primary school children in elengaz, khartoum, sudan. iosr journal of environmental science, toxicology and food technology. 2014; 8(1):119-121 19. ribas a, jollivet c, morand s, thongmalayvong b,somphavong s,siew c, ting p, suputtamongkol s,saensombath v, sanguankiat s,tan b, paboriboune p, akkhavong k, chaisiri k. intestinal parasitic infections and environmental water contamination in a rural village of northern lao pdr. the korean journal of parasitology. 2017; 55(5):523-532 20. shobha m, bithika d, bhavesh s. the prevalence of intestinal parasitic infections in the urban slums of a city in western india. journal of infection and public health. 2013; 6(2):142-149. 21. diarthini n, swastika i, ariwati l, isyaputri r, fitri n m, hidajati s, basuki s. blastocystis and other intestinal parasites infections in elementary school children in dukuh village, karangasem district, bali. indonesian journal of tropical and infectious disease. 2018:7(3):57-61 22. adam ea, yoder js, gould lh, hlavsa mc, gargano jw. giardiasis outbreaks in the united states, 1971-2011. epidemiol infect. 2016;144(13):2790-2801. doi: 10.1017/s09502688 15003040 23. marshall mm, naumovitz d, ortega y, sterling cr. waterborne protozoan pathogens [published correction appears in clin microbiol rev 1998 apr; 11(2):404]. clin-microbiolrev.1997; 10(1):67-85. doi:10.1128/cmr.10.1.67-85 24. paniker c, ghosh s. paniker's textbook of medical parasitology. 7th ed. new delhi: jaypee brothers medical publ. 2013 25. bogitsh b, carter c, oeltmann tn. human parasitology. 4th ed. amsterdam: academic press. 2013 26. sarkari b, hosseini g, motazedian m, fararouei m, moshfe a. prevalence and risk factors of intestinal protozoan infections: a population-based study in rural areas of boyer-ahmad district, southwestern iran. bmc infectious diseases. 2016;16(703):1-5 27. caleb okeri ondaraα, benson omweri nyachong’i σ, wycliffe nyamwancha mogoaρ, vincent obino orucho. gastrointestinal protozoan infections and associated factors among children under 5 years with diarrhea in kisii county, kenya. global journal of medical research. 2020;20(1):33-40 open access under cc-by-nc-sa share alike 4.0 155 vol. 1. no. 3 september–december 2010 case report t h e m i g r a i n e v e r t i g o p e r i o d o n t a l d i s e a s e connection: evidence-based case and verification in an animal study haryono utomo dental clinic faculty of dentistry airlangga university surabaya indonesia abstract recently, two cns disorders, migraine and anxiety have been recognized as being commonly associated with dizziness (vertigo). these associations may be an expression of an etiological relationship, for example, dizziness caused by migraine, or dizziness caused by anxiety and termed as mard. chronic dizziness may become more disabling during the added stress of a migraine headache or panic attack. in addition, dizziness occurred comorbidly with both migraine headache and anxiety disorders. even though the etiology of migraine had been suggested from trigeminal nerve sensitivity and neurogenic inflammation, its linking to periodontal disease that innervated by the same nerve was still uncertain. however, an animal study revealed that porphyromonas gingivalis lipopolysaccharide stimulation was able to increase neurogenic inflammation. a male patient suffered with symptoms mimicking mard for years and concomitantly had chronic periodontitis. scaling and root planning combined with the assisted drainage therapy resulted in instant disappearing of most of the symptoms. this case report is to propose the mechanism of periodontal disease involvement in the etiopathogenesis of migraine and vertigo which could be treated with periodontal treatment. regarding to remarkable result, it was concluded that periodontal disease could be a source of neurogenic and immunogenic inflammation which if not treated periodically could perpetuate symptoms mimicking mard. key words: periodontal disease, migraine-anxiety related dizziness introduction migraine-anxiety related dizziness (mard) is a new term proposed by furman et al.[1] for a disorder which related to the co-morbidity of migraine, anxiety and dizziness (vertigo).the existing link between migraine and balance disorders and the link between anxiety and balance disorders suggests that a subgroup of such patients will manifest migraine, anxiety, and a balance disorder at the same time. treatments of mard were depended to the predominance and the treatment phases of the symptoms. the predominant symptoms could be vestibular (i.e meclizine), migraine (i.e triptan) or anxiety (i.e. clonazepam). the treatment phases of the symptoms were acute, preventive and maintenance.[1,2] the possible link between oral focal infection and non-oral diseases had been studied by li et al.[3] based on the evidence-based case reports. in addition, several case reports revealed that elimination of oral focal infection had beneficial effects to sinusitis[4] and headache[5,6] and symptoms mimicking chronic fatigue syndrome (cfs).[7] however, studies related to the link between periodontal disease and the etiopathogenesis of mard is still unclean. the successful result of periodontal treatment for mard symptoms is beneficial for minimizing drug abuse. a disorder which also had similar symptoms as mard is chronic fatigue syndrome (cfs). chronic fatigue syndrome (cfs) is the current name for disorders characterized by debilitating fatigue and several associated physical, constitutional, and neuropsychological complaints which lasting more than 6 months and coupled with 6 or more arbitrary symptoms.[8,9] there are various symptoms that frequently suffered by cfs patient i.e. difficulty in concentrating, headache, forgetfulness, sore throat, muscle aches, tender lymph nodes, feverishness, sleeping disturbances, psychiatric problems, allergy, dizziness, abdominal cramps, rapid 156 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 155-161 pulse, chest pain, night sweats, palpitations, premenstrual syndrome (pms) etc.[9,10] studies on stress-associated disorder or immune dysregulation have interested scientist and clinicians in the field of psychoneuroimmunology (pni). the field focuses on the interactions among central nervous system and the immune system, and the impact these interactions have on health.[11] it is also interesting that stress impaired periodontal disease.[12] a possible correlation of oral focal infection with mard could be predicted regarding to an object observation of a phenomenon that related to symptoms mimicking mard. scaling and root planning (srp) that had been conducted to a male patient suffered from symptoms mimicking mard was able to relief all of the symptoms. moreover, recent study by utomo[13] revealed that porphyromonas gingivalis lipopolysaccharide (pglps) stimulation increased neurogenic inflammation, via increasing the substance p (sp) and calcitonin gene-related peptides (cgrp) that had a correlation with migraine.[14] in this study, the “assisted drainage therapy”, a modification of srp which including massage of the subgingival tissue, was able to decrease systemic cgrp level in minutes.[13] the purpose of this case report is to reveal the possibility of the periodontal disease involvement in the etiopathogenesis of mard, based on the remarkable result of scaling root planning and the assisted drainage therapy to a patient suffered from symptoms mimicking mard. however, further researches should be done to support the validity of this successful clinical evidence-based case treatment. case a 44 years old male patient came to the dental clinic in the faculty of dentistry airlangga university surabaya, after reading about the connection between dental and systemic diseases in a local media. he suffered from several symptoms such as vertigo, headache, fatigue, pain and spasms of the neck and shoulder muscles, palpitations and blurred vision. the illnesses started two years earlier, he was a very active individual who spent most of his time traveling by plane to other islands in indonesian archipelago. during the journey, he often experienced the sensation of falling down, dizziness, heart palpitation and sometimes he felt as if his heart stopped for a while. these symptoms made him afraid of travelling by plane. treatment and medications had already been conducted by general practitioner, internist and cardiologist. several diagnostic procedures have been done, such as chest x-ray, electrocardiography (ecg) and treadmills, the results were all normal. the results of blood laboratory tests and urinalysis were mostly normal except for total cholesterol and ldl cholesterol. there were a lot of prescribed drugs such as, sodium diclofenac (nsaid), meloxicam (cox2 inhibitors), tizanidine-hcl (muscle relaxant), lecithin (liver function supplements), cinnarazine (anti vertigo), flunarizine (drug for migraine, cerebral and peripheral equilibrium disturbances), vitamins b and e, lanzoprazole (drug for gastric and duodenal ulcer), chlordiazepoxide + clinidium (anti-anxiety), clobazam and alprazolam (tranquilizer), bisoprolol fumarate (anti-hypertensive, angina pectoris) and acetyl salicylic acid (as an anti-coagulant). from physical examination, despite his stressful face, extra oral were normal, intra orally there were a lot of calculus deposits and gingivitis noted in all regions (fig. 1 and fig. 2). probing revealed that deep periodontal pockets (5–7 mm) existed in every region, especially in posterior teeth (fig. 3). no caries was found. figure 1. intra oral, right side. figure 2. intra oral, left side. case management visit #1 scaling and root planning with piezoelectric scaler was conducted, followed by the assisted drainage therapy. it consisted of srp with sickle-shaped scaler which simultaneously massaging the subgingival area for about 2–3 minutes (fig. 4 and fig. 5) in the left regions, because 157utomo: the migraine-vertigo-periodontal disease connection the patient felt that the left side had the worst symptoms i.e. headache and neck muscle spasm. figure 4. the assisted drainage therapy, scaling-root planing (srp) combined with subgingival massage (red arrow).15 figure 5. the assisted drainage therapy in patient. subsequently, in a few minutes after the periodontal treatments, the patient had already experienced a significant difference. he was able to stand without afraid of falling down; it could be from the diminished headache and vertigo symptoms. then he was scheduled for the next visit three days later, nevertheless it was cancelled since he had been flown to denpasar. visit #2. ten days later, the patient felt more comfortable, headache and vertigo symptoms and the heart palpitation were disappeared. the same periodontal treatments were done in the right regions. several minutes later the patient felt more comfortable and according to him, his eyes could see clearer than before, and amazingly, he was able to read without glassed, before that he had to wear (+) 2 eyeglasses. several days later, after examining his eyesight, his opticians confirmed that this result was true. visit #3. one week after visit #2, all symptoms were disappeared, he had been gone to irian several days before which supported the successful result of the periodontal treatments to the mimicking mard symptoms. at that time, selective grinding was conducted in order to eliminate traumatic occlusions, and to facilitate smooth anterior and lateral mandibular movements. after this procedure, the patient felt more comfortable. patient was expected to have his periodontal health checked every 6 months. evaluations were done one year later and the following six months, the symptoms did not reappear discussion according several literatures and studies, periodontal treatment and the assisted drainage therapy were able to reduce or eliminate several symptoms such as headache, sinusitis, fatigue, muscle pain or spasms4,5,7 and asthma.13 even though the etiopathogenesis is still unclear, the same result also occurred in this patient, who had no more dizziness and followed by diminish of headache after the treatment and also heart palpitations several days later. oral focal infection such as periodontal diseases were able to elicit systemic infection and modulate systemic immune response.3,15 one of the systemic effects of infection is sickness behavior; it refers to the coordinated set of behavioral changes that develop in sick individuals during an infection. at the molecular level, these changes are due to the effects of local pro-inflammatory cytokines such as interleukin-1b (il-1b) and tumor necrosis factor-a figure 3. panoramic radiograph. 158 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 155-161 (tnf-a) which may also affected the brain if produced in sufficient concentration.16,17 the cytokine-induced sickness behavior symptoms such as fatigue, malaise, headache, sleep disturbances, inability to concentrate and other symptoms are due to the brain action of pro-inflammatory cytokines17,18 and nitric oxide (no) which is produced by inflammation and infection.19 in addition, cfs is closely related with cytokine-induced sickness behavior.16,17 there is a possibility that mard also related to cytokine-induced behavior. nevertheless, to distinguish from a cfs patient, at least serum cortisol level should be measured.9,10 bacterial endotoxins (lipopolysaccharides, lps) are part of outer cell wall of gram-negative bacteria. lipopolysaccharide challenge upregulates the expression of endothelial cells adhesion molecules-1 and stimulate the release of high levels pro-inflammatory mediators by macrophages or monocytes such as il-1b, il-6, tnf-a, prostaglandin e2 (pge2) and no.3,12 other effects are mast cell degranulation which released mediators that indirectly stimulate afferent nerve endings.20,21 in order to recognize the effect of stress to immune response, the study of psychoneuroimmunology should also be understood.11 stress consisted of stress perception and stress response. stress perception is the product of learning process for selecting, organizing, interpreting and implicating the actual stressor. stress perception reflects cognitive alterations, whereas stress response reflects physiological or biological alterations.22 stress, mediated by cns, activates the hypothalamic-pituitary-adrenal axis (hpa-axis) and increases the cortisol secretion. at the same time, stress also activates the sympathetic-adrenal medullary axis (sam-axis) to produce more catecholamines (noradrenalin and adrenalin).11,16 peripheral blood monocytes from certain individuals with hyperinflammatory monocytes phenotype secrete are 3–10 folds greater than those with normal monocyte phenotype individuals, and this condition exist in patients with early onset periodontitis or refractory periodontitis. upon stressful condition, high-stress perception individuals also produce il-1b, tnf-a and il-6 that significantly higher compared to low-perception individuals.23,24 in this patient who had symptoms mimicking mard, the stress in his work was suspected as the main trigger of the existing symptoms. stress impaired body defense reaction to local infection. altered mood and emotional condition may be involved in the periodontal disease, stress is suggested to affect periodontal health by increasing the level il-1b, tnf-a and il-6.12 oral inflammation may propagate to distant targets could be through the interplay of immunogenic and neurogenic inflammation.20 interplay between immunogenic and neurogenic inflammation is termed “neurogenic switching.”21 immunogenic inflammation may initiated by mast cell degranulation which induced by antigens, bacteria,, proteoglycans lps, neuropeptides (i.e. substance p, sp; cgrp), chemokines, calcium ionophores and physical factors.25 degranulated mast cells release histamine and tryptase which may stimulate neurogenic inflammation by binding to a protease activated receptor (par) in afferent nerve fibers.20 the increased level of tnf-a, sp and cgrp was confirmed in a study in wistar rats injected with p. gingivalis lps (pglps).13 in chronic gingivitis, histopathologic alteration in gingival tissue facilitated gingival bleeding after innocuous stimuli i.e. tootbrushing, toothpicking. it was caused by 1) vasodilatation and engorgement of the capillaries and their closure to the surface; 2) thinning and ulcerated sulcular epithelium.15 therefore, it was logical that srp elicited bleeding and resulted in instant disappearing of the symptoms since it also decrease mediators concomitantly with the oozed blood. however, compared to the assisted drainage therapy, srp had a lesser effect to vasodilatation since piezoelectric scaler had water spray for cooling, this condition led to prevent the increase of gingival local temperature. subgingival massage enhanced the temperature and also more vasodilatation, thus also resulted in more drainage of the mediators. as a result, it was supposed to be the effect of the assisted drainage therapy to the existing pro-inflammatory mediators (cytokines, pge2, bradykinin, no, sp and cgrp) in the periodontal disease which then may immediately cut off the neurogenic switching mechanism.4,26 it was confirmed that assisted drainage therapy in rats reduced the tnf-a, and sp and cgrp level.13 since the chief complaint of the patient was vertigo, the headache types (i.e. migraine, tension or cluster headache) were not examined. nevertheless, there are several theories related to the etiopathogenesis of headache, such as the increase of pro-inflammatory cytokines level18,19,27 and no.27 the involvement of the trigeminal nerve (v2) was associated with the sphenopalatine ganglion (spg)26,29 and the neurogenic switching mechanism.29 headache symptoms in this case which accompanied by neck pain or spasm suffered by the patient according to several literatures are diagnosed as migraine.2,30 activated primary afferent neurons of trigeminal nerve sends impulses via trigeminus nucleus caudalis which acts as sensory relay center. neck pain may resulted from the excitation of trigeminus nucleus caudalis which may extend to dorsal horn for stimulation of c2, c3 and c4.2 most of the theories of migraine are the arterial concepts which have been focused on the enlargement of intracranial surface-of-the-brain arteries resulting from their exposure to nitric oxide.19,27 however, the effect of the release of peptides such as cgrp on the extra cranial and intra-nasal mucosa by their corresponding trigeminal nerve branches has been largely overlooked and considerably underrated.24 according to cady and schreiber,29 sinusitis cases often accompanied by migraine and vice versa which related to the “neurogenic switching” mechanism. periodontal ligament in the maxilla is also innervated by v2. stimulated c fibers from maxillary periodontal ligaments (v2) may antidromically release sp and cgrp, 159utomo: the migraine-vertigo-periodontal disease connection this mechanism is proposed to be the etiology of sinusitis and migraine.24,28 therefore, through the neurogenic switching mechanism, periodontal inflammation may also directly affects sinus inflammation (mucosa and artery) through the neuropeptides release of sp and cgrp by afferent nerve of nasal mucosa via the sphenopalatine ganglion.20,26,29 the trigeminovascular reflex, which is related to intracranial arterial vasodilatation due to increase no concentration or inflammation is a normal mechanism. neurons of the first division of trigeminal nerve (v1) reported this condition to the trigeminal sensory nucleus. however, in certain individuals with elevated sympathetic tone or pre-sensitized afferent nerves may trigger headache.26 according to furman et al.,1 there is connection between migraine, vertigo and anxiety. vestibular pathways can contribute to both central and peripheral migraine mechanisms. the reciprocal connections between vestibular nuclei and trigeminal nucleus caudalis suggest that vestibular and trigeminal information processing may be altered concurrently during migraine attacks, and that vestibular signals may directly influence trigeminovascular reflex pathways (fig. 6). in addition, central vestibular activation can affect activity in monoaminergic pathways through direct connections from the vestibular nuclei to the dorsal raphe nucleus, nucleus raphe magnus, locus coeruleus, and lateral tegmental region. these changes in monoaminergic activity due to vestibular activation may both trigger migraine related symptoms and modulate activity in both pain related and anxiety related pathways. conversely, regionally specialized noradrenergic and serotonergic inputs are potential substrates for altering central vestibular information processing during and between migrainous episodes. in addition, anxiety or hyperventilation may also reactivate a vestibular disorder by interfering with central compensation or by altering somatosensory input.1 there are three way interfaces among migraine, anxiety, and dizziness. the interactions between the balance–migraine and the balance–anxiety interfaces are shown schematically on the upper left. neuronal activity in the vestibular nuclei, particularly the superior vestibular nucleus, is a first major integrative site for the periodontal disease 1. lowered pain threshold by pge2, no, bradykinin 2. sensitization of the spg neurogenic switching mechanism lps pge2, tnf-a, sp, cgrp figure 6. pathogenesis model for migraine–anxiety related dizziness (mard) and the relationship with periodontal disease (source; furman et al., 2005). 160 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 155-161 balance–migraine–anxiety linkage. as this activity is a function of a) afferent input regarding head motion from the inner ear, somatosensation from the spinal cord (sc) and optic flow information from the accessory optic system (aos); b) trigeminal sensory inputs; and c) descending inputs from the neocortex, it has the potential to participate in the triggering, buildup, and perseverance of episodic dysfunction.1 the role of periodontal disease in neurogenic switching mechanism is shown on the lower right. abbreviations: 5-ht, 5-hydroxytryptamine (serotonin); aos, accessory optic system; drn, dorsal raphe nucleus; lc, locus ceruleus; ne, norepinephrine; pag (v and vl), periaqueductal grey (ventral and ventrolateral columns); sc, superior colliculus; smd, space and motion discomfort. periodontal disease is the source of lps, proinflammatory mediators including pge2, no and bradykinin3,12 that were able to lower pain threshold of the afferent nerve fibers of the trigeminal nerve31 (fig.5). it was also proposed to be involved in the sensitization of the sphenopalatine ganglion (spg) which related to migraine.4,26 vertigo in this patient may also caused by the release of sp from local sensory nerve fibers in the inner ear that stimulates expression of endothelium–leukocyte adhesion molecules from cochlear microvasculature. this mechanism decreases blood flow to cochlear sites resulting vestibular disorders.18 in animal study it was verified that pglps injection increase sp in nasal tissues which closely related to the vestibular system and the assisted drainage therapy also decrease sp level in nasal tissues.13 the instant relief of headache, improve of eyesight and other symptoms after scaling procedures may be caused by decreasing of the neurogenic switching mechanism. the oozed blood during scaling should contain proinflammatory mediators, bacteria and lps which may directly cut off the neurogenic switching mechanism.4 this phenomenon was verified by the decrease of tnf-a, sp and cgrp levels several minutes after the assisted drainage therapy in rat model.13 this case report is a retrospective study of patient suffered from symptoms mimicking mard according to the patient�s medical history and examined by a dental practitioner. further studies with the true mard should be done in collaboration with competent medical practitioners and comprehensive medical diagnostic procedures conclusion: based on the remarkable result of the periodontal treatments and supported by animal study related to this evidence-base case report, it is concluded that: 1) correlation oral focal infection, especially periodontal disease with illnesses mimicking mard symptoms should be exist; 2) periodontal treatments such as srp and the assisted drainage therapy are beneficial in mard management. nevertheless, further investigation should be done about the etiopathogenesis of periodontal – systemic related illnesses and increase the multidisciplinary approach in the scope of dentistry and general medicine to explore new interrelated cases. references 1. furman jm, balaban cd, jacob rg, marcus da. migraine-anxietymigraine-anxiety related dizziness (mard): a new disorder. j neurol neurosurg psych, 2005; 78: 1–8. 2. green mw. diagnosing and treating migraine: low tech diagnosis, high-tech treatment. available online at url: http://www.ama-assn. org / ama1/pub/upload/mm/31/24pres-green.pdf. accessed february 5, 2005. 3. li xj, kolltveit km, tronstad l, olsen i. systemic diseases caused by oral infection. clin microbiol rev 2000; 13(4): 547–58. 4. utomo h. sensitization of the sphenopalatine ganglion by periodontal inflammation: a possible etiology for headache and sinusitis in children. majalah kedokteran gigi fkg universitas airlangga. 2006; 39(2): 63–7. 5. utomo h, prahasanti c. periodontal disease in headache and menstrual pains. lustrum fkg universitas gadjah mada 2005; 14: 101–6. 6. utomo h, prahasanti c. periodontal disease as an etiology of orofacial and musculoskeletal pains in women. j dentistry 2006. 7. utomo h. elimination of oral focal infection: a new solution in chronic fatigue syndrome management? majalah kedokteran gigi surabaya 2005; 38(4): 169–72. 8. victor m, ropper ah. principles of neurology. 7th ed. new york: mc graw hill; 2001. p. 175–6. 9. strauss se. chronic fatigue syndrome. in: kasper dl, braunwald e, fauci as, hauser sl, longo d, jameson jl, eds. harrison�s principles of internal medicine. 16th ed. new york: mcgraw hill; 2005. p. 2545–6. 10. craig tj, kakumanu s. chronic fatigue syndrome: evaluation and treatment. am fam phys 2002; 65: 1083–90. 11. padgett da, glaser r. how stress influence the immune response. trends in immunology 2003; 24: 444–8. 12. kamma jj, giannopoulou c, vasdekis vds, mombelli a. cytokine profile in gingival crevicular fluid of aggressive periodontitis: influence of smoking and stress. j clin periodontol. 2004; 31: 894–902. 13. utomo h. immunoneuromodulatory effect of the assisted drainage therapy in allergic rats induced by porphyromonas gingivalis lipopolysaccharide. dissertation. doctorate program airlangga university. 2009. 14. bellamy jl, cady rk, durham p. salivary levels of cgrp and vip in rhinosinusitis and migraine patients. headache 2006; 46: 24-33. 15. newman mg, takei h, klokkevold pr, carranza fa, 2006. carranza�s clinical periodontology. 10th ed. elsevier-saunders. p. 146, 749–56. 16. licinio j, frost p. the neuroimmune-endocrine axis: pathophysiological implications for the central nervous system cytokines and hypothalamus-pituitary-adrenal hormone dynamics. braz j med biol res 2000; 33: 1141–8. 17. kiecolt-glaser jk, glaser r. depression and immune function: central pathways to morbidity and mortality. j psychosomatic res 2002; 53: 873–76. 18. dantzer r. cytokine-induced sickness behavior: where do we stand? brain behav immunity 2001; 15: 7–24. 19. stirparo g, zicari a, favilla m, lipari m, martelletti p. linked activation of nitric oxide synthase and cyclooxygenase in peripheral monocytes of asymptomatic migraine without aura patients. cephalalgia 2000; 20(2): 100–6. 20. lundy w, linden r. neuropeptides and neurogenic mechanism in oral and periodontal inflammation. crit rev oral biol med 2004; 15(2): 82–98. 21. meggs wj. neurogenic switching: a hypothesis for a mechanism for shifting the site of inflammation in allergy and chemical sensitivity. env health persp 1997; 105: s2 1–10. 22. putra st, asnar es. perkembangan konsep stres dan penggunaannya dalam paradigma psikoneuroimunologi. in: putra st, eds. psikoneuroimunologi kedokteran. 1st ed. surabaya: gramik; 2005. p. 12. (in indonesian language). 23. shapira l, wilensky a, kinane d. effect of genetic variability on the inflammatory response to periodontal infection. j clin periodontol 2005; 32 (s6): 72–86. 161utomo: the migraine-vertigo-periodontal disease connection 24. madianos pn, bobetsis ya, kinane df. generation of inflammatory stimuli: how bacteria set up inflammatory responses in the gingiva. j clin periodontol 2005; 32(s6): 57–71. 25. walsh lj. mast cells and oral inflammation. crit rev oral biol med 2003; 14(3): 188–98. 26. boyd jp. pathophysiology of migraine: and rationale for a targeted approach of prevention. available on line at url http://www. drjimboyd.com. accessed, nov 22, 2005. 27. jeong hj, hong sh, nam yc. effect of acupuncture in inflammatory cytokine production in patients with chronic headache. am j chin med 2003; 31(6): 945–54. 28. okeson jp. bell�s orofacial pain. 6th ed. carol stream. quintessence pub; 2005. p. 52–3. 29. cady rk, schreiber cp. sinus headache or migraine.sinus headache or migraine. neurology 2002; 58: s10–4. 30. unger j, cady rk, farmer-cady k. understanding migraine: pathophysiology and presentation. 2005. available online at url http://www.femalepatient.com. accessed march 20, 2006. ijtid vol 1 no 3 sep-dec 2010.53.pdf ijtid vol 1 no 3 sep-dec 2010.54.pdf ijtid vol 1 no 3 sep-dec 2010.55.pdf ijtid vol 1 no 3 sep-dec 2010.56.pdf ijtid vol 1 no 3 sep-dec 2010.57.pdf ijtid vol 1 no 3 sep-dec 2010.58.pdf ijtid vol 1 no 3 sep-dec 2010.59.pdf � vol. 4. no. 4 october–december 2013 case report problem of antibiotic use and antimicrobial resistance in indonesia: are we really making progress? usman hadi1, kuntaman2, mariyatul qiptiyah3, hari paraton4 1 department of internal medicine, dr. soetomo hospital-universitas airlangga; 2 department of clinical microbiology, dr. soetomo hospital-universitas airlangga; 3 department of pharmacy, dr. soetomo hospital-universitas airlangga; 4 department of obstetry and gynaecology, dr. soetomo hospital-universitas airlangga. abstract background: based on the results antimicrobial resistance in indonesia: prevalence and prevention-study (amrin-study), the ministry of health of indonesia in 2005 began a program antibiotic resistance control (ppra) in some government hospitals, and is currently developing to all government teaching hospitals in indonesia. aim: the core activities of the ppra are to implement standardized surveillance emergence of antibiotic resistant bacteria, and the surveillance of antibiotic use in terms of quantity and quality. method: our research in the years 2003 showed the proportion of antibiotic use 84% of patients in a hospital. the use of inappropriate antibiotics was very high, 42% no indication. result: in 2012 the results of surveillance showed decline of inappropriate use of antibiotic, but prevalence extended-spectrum b-lactamase (esbl)-producing k.pneumoniae (58%), and e.coli (52%) and methicillin-resistant s.aures (mrsa) (24%) were increasing. conclusion: it was needed to implement the most appropriate programs to prevent the growth and development of bacteria resistant to antibiotics. key words: indonesia, k.pneumoniae, e.coli, methicillin-resistance s.aureus, surveillance abstrak latar belakang: berdasarkan hasil penelitian antimicrobial resistance di indonesia: prevalensi dan pencegahan (amrin-studi), kementerian kesehatan republik indonesia tahun 2005 memulai program pengendalian resistensi terhadap antibiotik (ppra) di beberapa rumah sakit pemerintah, dan saat ini diperluas untuk semua rumah sakit pendidikan pemerintah di indonesia. tujuan: kegiatan inti dari ppra adalah untuk melakukan surveilance kuman kebal antibiotik, dan monitoring penggunaan antibiotik dalam hal kuantitas dan kualitas secara terstandar. metode: penelitian kami di tahun 2003 menunjukkan proporsi penggunaan antibiotik dari pasien di rumah sakit sebanyak 84%. penggunaan antibiotik yang tidak tepat sangat tinggi, 42% tidak ada indikasi pemberian antibiotik. hasil: hasil surveilance tahun 2012 menunjukkan penurunan penggunaan dari antibiotik yang tidak tepat, tetapi prevalensi extended-spectrum b-laktamase (esbl) k.pneumoniae (58%), dan e.coli (52%) dan methicillin-resistant s.aures (mrsa) (24%) meningkat. kesimpulan: untuk memperbaiki kondisi ini diperlukan program lain yang paling tepat untuk mencegah pertumbuhan dan perkembangan bakteri resisten terhadap antibiotik. kata kunci: indonesia, k.pneumoniae, e.coli, methicillin-resistance s.aureus, survailans introduction world health organization has announced that the issue of antibiotic-resistant bacteria is a global problem that threatens human being. this happens because of a deficiency in 6 issues: 1) lack of research, 2) lack of commitment, 3) lack of infection control, 4) irrational use of antimicrobials, 5) poor quality antibiotics, 6) weak oversight. to cope with these conditions, all countries should participate actively. efforts to suppress the development of antibioticresistant bacteria have been implemented throughout the world including indonesia. a scientific research on antimicrobial resistance in indonesia: prevalence and � indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 5–8 prevention (amrin) study was conducted between 2001 and 2005. it aimed to create a program of scientifically based guidelines for the assessment of antimicrobial resistance, the pattern of antibiotic use, infection control and the implementation of interventions in the home hospital in indonesia. the results of amrin study show that the problems also occur in indonesia. it means that integrated handling of the various stakeholders in the hospital is required (amrin study group, 2005). to overcome this problem, the ministry of health has taken an action by developing a program of antimicrobial resistance control program (amr-control program) for hospital in indonesia. the program was first implemented in teaching hospitals in indonesia and it is expected to further evolve to all hospitals in indonesia (amrin-study, 2005). the problem in indonesia is that not all hospitals have the facilities to conduct microbiological examination or culturing of bacteria, antibiotic resistant bacteria so that the surveillance can not be performed. amr-control program is expected to be able to make the use of antibiotic more rational, to improve the implementation of infection control measures, and to inhibit the development of antibioticresistant bacteria. hospitals that do not have a complete facility are expected to realize it because without good microbiology laboratory, the control of antimicrobial resistance is not possible to be done. this paper describes the implementation amrcontrol program so as to give an idea whether the program works well or whether it is still necessary to make some improvements to the program in order to achieve the program objectives. implementation method of amr-control program amr-control program was implemented in stages. first, identifying the readiness of the hospital management to implement this program. hospital is considered ready to execute this program when it has four supporting pillars, namely: 1) clinical microbiology, 2) clinical pharmacy, 3) pharmacy and therapy committee, 4) infection control committee. once the four pillars had been identified, they were then assessed to know whether they were already running as they should. it was then followed by performing training to the 4 pillars so as they had the same understanding of this program as shown in scheme 1. second, creating a team consisting of the four pillars responsible for the execution of amr-control program. each pillar has a function in accordance with its respective field. the role of clinical microbiology in the management of infectious diseases in indonesia most of clinical microbiology laboratories are not yet well developed. hence, the hospital management has to upgrade microbiology laboratory to meet the actual standard. the clinicians usually treat infectious disease patients with a clinical diagnosis and they give antibiotic empirically. to improve collaboration between clinicians and the clinical microbiologists, the two sides should enhance the quality of their work. we do the following recommendations to improve the cooperation between clinicians and microbiologists. 1. holding regular (weekly) meetings between clinicians and microbiologists to discuss current infectious disease cases in hospital. 2. holding routine evaluation of adherence to clinical guidelines by clinicians and giving feed back of adherence figures. 3. enhancing the involvement of the clinical microbiologist in patient care such as by instituting a 24 hour service for advice concerning diagnostics and treatment of patients with infectious diseases. clinical pharmacy clinical pharmacy has an important role to control antibiotic prescribed in the hospital. in addition, clinical pharmacists have to meet the needs of antibiotics prescribed by a clinician. they should have the signs made by the pharmacy and therapeutics committee to control the excessive use of antibiotic. pharmacy and therapeutics committee pharmacy and therapeutics committee is obliged to make the antibiotic guidelines and policies for the use of antibiotics. they have to revise guidelines for the use of antibiotics and antibiotic use policies when needed. another task of this committee is to conduct surveillance of antibiotic use quantitatively and qualitatively particularly in the ward where antibiotic-resistant bacteria emerge. infection control committee this committee is responsible for the prevention of amr organism spreading. besides, this committee also has to increase infection control implementation in hospital consisting of: scheme 1. the concept of amr-control program (amrin study group, 2005) �hadi, et al.: problem of antibiotic use and antimicrobial resistance in indonesia standard precautions isolation of patients source control surveillance of amr organisms implementation steps of amr-control program 1. forming a team of amr-c.p. in hospital. 2. choosing one department or unit as a pilot project, e.g. department of internal medicine or pediatric. 3. updating or developing antibiotics guideline. 4. socializing antibiotics guideline. 5. conducting baseline data collection for 1–3 months respectively. 6. implementing the antibiotics guideline. 7. conducting surveillance data collection post intervention. 8. evaluating and discussing. 9. presenting the result: e.g. workshop 10. getting antibiotics guideline updates. 11. conducting surveillance (monitoring and evaluating). 12. getting back to point number 3. results of amrin-study (period of study 2002–2005) our study of quantity and quality of antibiotic usw showed that the percentage of antibiotic prescribed in hospitalized patients was quite high (85%). as many as 90% of patients staying for 5 or more days in the department of surgery and pediatrics used antibiotics while in the gynecology & obstetrics department and internal medicine department, there were as many as 87% and 67% of the patients respectively using antibiotics (hadi, 2008). 53% out of 2058 prescriptions was classified as therapy, 15% as prophylaxis, and 32% as unknown indication. the quality of antibiotic use was assessed by two indonesian reviewers and one foreign reviewer. almost 60% of the assessed prescriptions was classified as incorrect either unjustified (42%) or inappropriate (15%) by at least two of the three reviewers (hadi, 2008). our study of bacterial resistance from normal flora gastro-intestinal tract showed that escherichia coli had been isolated from 781 hospitalized patients and all data were available for analysis. eighty-one percent of the hospitalized patients carried escherichia coli resistant to one or more antibiotics. ampicillin resistance was seen most frequently (570 isolates, 73%), followed by trimethoprim/sulfamethoxazole resistance in 434 isolates (56%), chloramphenicol resistance in 334 isolates (43%), ciprofloxacin resistance in 173 isolates (22%) and gentamicin resistance in 141 isolates (18%) (duerink, 2007). in the community group of 2996 individuals, 2494 information cases regarding carriage of escherichia coli were available. forty-three percents of the population carried resistant escherichia coli. ampicillin resistance was observed in 851 (34%) isolates, trimethoprim/sulfamethoxazole resistance in 716 (29%) isolates and chloramphenicol resistance in 369 isolates (15%) (duerink, 2007). surveillance of post implementation of amr-control program because of limited funds and facilities, surveillance could not be implemented fully in all hospitals in indonesia. we only present the results of surveillance in a hospital in surabaya. the results of surveillance of antibiotic use in the department of internal medicine in 2012 showed that 50.22% of patients was treated with antibiotics. it was lower compared to the percentage of antibiotic use in 2005 which was 67%. in terms of the quality of antibiotic use, there was also an improvement in which a more rational usage of antibiotics was found compared to the use of antibiotics in 2005. on the one hand, there was a decline regarding no indication of antibiotic therapy from 42% to 30.6%, and inappropriate antibiotic therapy from 15% to 7.3%. on the other hand, there was a change in the pattern of antibiotic use between 2005 and 2012. in 2005 the use of ampicillin was dominant whereas in 2012 3rd generation cephalosporin was more widely used. we conducted amr surveillance in the period from january to june 2010 by looking back to the medical records performed in the microbiology laboratory. 4359 bacteria were found consisting of 3115 negative gram and 1244 positive gram bacteria isolates. among these bacteria, 456 (22%) were esbl positive isolates and 45 (18%) were mrsa isolates from total of 250 s aureus isolates. of these esbl isolates, 107 specimens of e.coli esbl(+) (17%) were obtained from a total of 633 e. coli isolates and 196 isolates of k. pneumonia esbl(+) (23%) were obtained from a total of 196 k pneumonia. while the surveillance conducted in the period from july to december 2012 showed that there were k.pneumoni esbl(+) 202 (58%) specimens from a total of 351 k pneumonia specimens and e. coli esbl(+) 327 (52%) specimens from a total of 629 e.coli specimens. 63 (24%) mrsa specimens were obtained from a total of 259 s.aureus specimens while the prevalence of esbl was (53%). table 1. comparison of prevalence of esbl and mrsa between 2010 and 1012 � indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 5–8 surveillance of antibiotic use demonstrated an improvement compared to that between 2005 and 2012. yet, in terms of the development of antibiotic-resistant bacteria e.g. esbl producing bacteria, the prevalence increased. in addition, mrsa also increased. this could occur because the possibility of the use of antibiotics in the community outside the hospital was still very high or excessive. both improvement of health facilities especially microbiology laboratory and the addition of experts in the field of microbiology are required. references 1. amrin study group, 2005. antimicrobial resistance, antibiotic usage and infection control. a self-assessment program for indonesian hospitals. directorate general of medical care, ministry of health, republic of indonesia. 2. duerink do, lestari es, hadi u., et al. 2007. determinants of carriage of resistant eschericia coli in the indonesian population inside and outside hospitals journal of antimicrobial chemotherapy, 60(2): 377–84 3. hadi u, duerink do, lestari es, nagelkerke nj, werter s, et al. 2008a. survey of antibiotic use of individuals visiting public healthcare facilities in indonesia. international journal of infectious diseases: ijid: official publication of the international society for infectious diseases 12: 622–9 4. hadi u, duerink do, lestari es, nagelkerke nj, keuter m, et al. 2008b. audit of antibiotic prescribing in two governmental teaching hospitals in indonesia. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases 14: 698–70 5. who, 2001. who global strategy for containment of antimicrobial resistance. world health organization, switzerland 6. who. 2011. combat drug resistance, no action today, no cure tomorrow. available at www.who.int./world-health-day/2011/worldhealth-day2011-brochure-pdf (accessed 5th, november 2012) table 2. comparison between k pneumonia and e.coli esbl producing bacteria’s table 3. comparison of quality of antibiotic use between 2005 and 2012 table 2 shows that the quality of antibiotic use is improved in which there is an increase in the appropriate use of antibiotics and a decrease in the inappropriate use and no indication. conclusion this program could not be implemented fully because there were many limitations in many hospitals in indonesia. our study in surabaya showed that the results of 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 �� vol. 5. no. 1 january–april 2014 hiv and malaria ririek parwitasari1, nasronudin1,2 1 infectious and tropical disease division department of internal medicine, dr. soetomo hospital airlangga university school of medicine 2 institue of tropical disease airlangga university abstract hiv/aids is a global problem involving industrialized and developing country including indonesia. malaria has killed millions of human beings almost 3 million people each year, whereas since 1999, nearly 36 million people in the world infected with hiv and 3 million more have died (kakilaya, 2006). hiv infection increases the risk and aggravate malaria. in africa in the area of malaria transmission intensities high and low, hiv aggravate malaria and improve case fatality at any age (eline 2006). hiv is an rna viruses whose hallmark is the reverse transcriptation of its genomic. malaria is a protozoan disease transmitted by the bite of infected anopheles mosquito. infection malaria can stimulate hiv replication and may cause faster progression of hiv disease. key words: hiv, malaria, infection, rna, progression abstrak hiv/aids merupakan masalah global yang meliputi industri dan negara berkembang termasuk indonesia. malaria telah membunuh jutaan manusia yaitu sekitar 3 juta orang tiap tahunnya, dimulai tahun 1999, 36 juta orang di dunia terinfeksi hiv dan lebih dari 3 juta telah meninggal1. infeksi hiv dapat meningkatkan dan memperburuk malaria. di africa yang mana area transmisi malaria intensitasnya tinggi dan rendah, hiv memperburuk malaria dan meningkatkan kasus fatal di beberapa usia2. hiv adalah virus rna yang ditandai dengan reverse transcriptation gennya. malaria merupakan penyakit yang disebabkan oleh protozoa yang ditransmisikan oleh gigitan nyamuk anopheles yang terinfeksi. infeksi malaria dapat menstimulasi replikasi hiv dan menyebabkan percepatan progesi penyakit hiv. kata kunci: hiv, malaria, infeksi, rna, perkembangan introduction hiv/aids is a global problem involving industrialized and developing country including indonesia. the problem is growing increased rates pain and death. with respect to the decreasing immune from intervention hiv encourage micro growing organism one is a plasmodium malaria. with technological progress digit hiv medicine supposed decline in pain in fact the pain remains high. malaria has been known for centuries, while the hiv in the last 2 decades. malaria has killed millions of human beings almost 3 million people each year, whereas since 1999, nearly 36 million people in the world infected with hiv and 3 million more have died. both of these diseases is expected to infect and kill a lot of people in the world because hiv was increased dramatically in countries with malaria are not controlled.1 hiv infection increases the risk and aggravate malaria. in africa in the area of malaria transmission intensities high and low, hiv aggravate malaria and improve case fatality at any age.2 has known that hiv reduce the number of lymphocytes, especially cd 4, so the immune response decline consequently micro an organism grows with fertile, one is malaria. malaria while interference red blood cells and take glucose so happen deficiency nutrients that can result in immune deficiency. malaria burdensome travel hiv infection being aids. on the basis of various fyl above necessary knowledge deep, mutual the relatedness of hiv and malaria so as to be done steps to reduce the rate in pain and death. literature review ��parwitasari and nasronudin: hiv and malaria hiv hiv aids first known in 1981 when acquired are sufferers by infection has opportunity without predisposing disorder of the immune system (crowe, 2001; fauci, 2005).cause aids this is known as retrovirus divided into 2 groups namely human t lymphotropid viruses (htlv) i and htlv-ii as “transforming retrovirus and human immunodeficiency viruses (hiv)-1 and figure 1. hiv replication cycle. the cycle of hiv replication hiv is the rna virus that marked by a transcription inverted namely from rna to dna through enzyme reverse transcriptase. started from the bonds protein gp120 receptor surface host namely molecules cd 4. molecules cd4 is lymphocytes t that are responsible for helper t cells or induction the immune system. cd4 expressed also at monocytes/macrophages and cells dendritic/langerhans. after the bond between gp120 with cd 4, gp120 will facilitate bond with co-receptor namely ccr5 and cxcr4. bond with one or both these receptors will bring virus entering cells. dendritic cell expressing receptors lectin type c different one of them is called dc-sign on surface that will make proteins gp120 hiv. cell dendritic will facilitate binding virus t cell with cd 4+. after the ties it forms the surface virus will change, fusion to the cell membrane host would happen through penetration molecules gp 41 to a membrane plasma cells target. capsule rna viruses free and went into the cell target. enzyme resvers transcriptase then catalyze rna into double-strand dna. dna will hold translokation into the cell nucleus host and into the chromosome. integration hiv this reactivated gene and produce provirus that may be latent or manifest at several levels genes expressions to a virus active. next several levels activity cell host needed to start a transcription from proviral dna into rna or mrna. mrna hiv virus will be translation into protein were afterward subjected to modification through glicosilasion, miristilasion, fosforilasion, and division. the viral particles is formed of protein, enzyme, the genome rna to membrane plasma of the host. budding of virus happened through a special area in two layers of fat cell membranes host known as lipid rafts which will then be the outer layer of the virus.3 patofisiologi and patogenese of hiv sign of hiv is found immune deficiency caused by deficiency of lymphocytes t namely helper t cells or inductor t cells. cd4 that is part of t cells role as receptors of hiv. a co-receptor cd4 required to hold fusion and insert a virus hiv-1 into cells target for ccr5 and cxcr4. although some mechanism responsible on decline and dysfunction immune t cells cd4+ have been demonstrated in vitro, currently it is not clear mechanism which most responsible on decreasing and impaired function in in vivo. when the number of t cells cd4+ declining the hiv have risk suffer hierograms disease opportunistic, several infections and a neoplasm (fauci, 2005, o’neil, 2002). lysis of the virus and elimination directly caused by cellular immune response and humoral against viral is an important factor which contribute to the decreasing t cells are infected cd4 +. allegedly that hiv infection cause chronic conditions of activity immune cells that cause elimination cd4 + uninfected. mechanism is responsible for elimination cd4 + cell autoimmune and are not infected cell death.4 lymphoid tissue constitutes the main hiv replication of the virus is also resulting in place lesions specific occurring imunodefisensi.4 transmission of hiv hiv transmitted by contact heterosexual, homosexual, through blood and blood products, through mother to child is infected intra partum, perinatal or when breastfeeding. for research more than 20 years there is no evidence that hiv can be transmitted through the bite of an insect or propagated as mosquito.3,5 like a capsule virus other all retroviruses inactive, easily into shape and not, is transmitted via air dust or smoke. infection started having no direct contact through the tissue or body fluid from a source of infection.6 clinical symptoms and treatment the clinical and travel this disease correlated with cd4 number of cells. divided into four degrees primary, namely infection where the virus in blood and proliferation quickly limfonodi, early deficiency immune/early (cell number cd4 > 500/μl), intermediate deficiency immune (cd4: 200–500/μl) and immune deficiency further/advanced (cd4 < 200/μl).7 �� indonesian journal of tropical and infectious disease, vol. 5. no. 1 january–april 2014: 16–22 antiretroviral theraphy of hiv targets antiretroviral therapy (arv) that inhibits bond with cd4 (in research) enzyme inhibit reverse trancriptase (zidovudine, didanosine, zalcitabine, lamivudine, stavudine, foscamet) a non-nucleoside reverse transcriptase inhibitor (nevirapine) termination of dna (zidovudine, sintesa chain didanosine, zalcitabine, stop and budding assembly (viruses, interferon) hinder maturation protein virion core (a protease inhibitor: example saquinavir).8 it has proven that combination therapies more effective for viruses and impede progresivitation disease. therapy for patients without major complications is d4t-3tc-nvp namely stavudine-lamivudine-nevirapine.9 malaria malaria is a disease due to protozoa that are transmitted through the bite of an infected anopheles mosquito. there were four genus infecting humans, a plasmodium namely p. tertian, p. vivax, p. ovale, and p. malariae. infection began when a female an infected anopheles mosquito inoculation plasmodial sporozoites when suck the blood of humans. a microscopic malaria parasite would be taken quickly off the flow of blood to heart entering parenchyma liver and begin a period of asexual reproduction. through a process of amplification (known as intrahepatic or preerytrocityc schizogony or merogony) a sporozoites can produce 10,000 until > 30,000 & merozoites. liver swell will issue merozoites moving into the blood stream. then went into the red blood cells and multiplication 6 until 20 times every 48–72 hours. parasitic on sat reached–50/μl blood clinic symptom of infection this will seem (white, 2005). on p. vivax and p. ovale when phase intrahepatik not occurring cleavage immediately but is dormant between 3 weeks until a year or so before reproduction began, called hepaticae hypnozoit this condition allow parasitic adapted to climate change.11,12 upon entering blood flow merozoites entrance to erythrocytes and tropozoit be assisted by receptors surface erythrocytes specific during the early phases intra eritrosit notching the ring (ring forms) of four species parasitic looked same under a light microscope. as makin magnify tropozoit specific characteristic each species more real, pigment more visible, parasitic looked irregular or notching ameboid. after 48 hours the life cycle intraeritrosit (72 hours to p. malariae parasitic) has consume almost all hemoglobin and meet red blood cell (sdm), called schizont. occurring splitting the nuclei of multiple and sdm shatterlng make 6–30 merozoites female that can mengivasi sdm new and cycle as above will recurring again. this disease in humans caused by any indirect effect of his invasion sdm, destructived by parasites phase asexual and reaction host. after cycle asexual (p. falciparum) or immediately after out of liver (p. vivax. p. ovale, p. malariae) some parasitic form morphology different namely sexual phases (gametosit) can anything to malaria. after sucked by the bite of an infected anopheles mosquito female, gametosit male and female form a zygote in intestines of mosquitoes. which the zygote mature ookinet be entering and protected in the intestinal wall. ookcyst formed from cleavage asexual continue to cleave to a sporozoites moving then migrate to hemolimf further to the salivary glands mosquito waiting inoculation into another human.12 stadium 1 asimtomatik stadium 2 mild disease stadium 3 moderate disease stadium 4 severe disease (aids) weight tetap bb ↓ 5–10% bb↓ >10% hiv wasting syndrome a symptom of infection generally treatment and opportunistic infection according to the guideline and regulations  only limfadenopati  wound around lips (angula cheilitis)  itchy (seborrhoea/ prurigo)  infection up track breathing, as sinusitis or otitis.  sprue (hairy leukoplakia)  > 1 month • diarrhea • candidiasis vagina • fever  infeksion of heavy bactery(pneumoni, infeksion muscle)  tb lung in last year.  spure in esofagus  > 1 month • ulserasi herpes simpl  limfoma  kaposi sarcoma  ca cervix infasif  pneumocystis pneu  tb ekstrapulmoner  meningitiscryptococcal  ensefalopati arv therapy  only if cd4 < 200  only if cd4 < 200 or limfosit total < 1200/mm3  if cd4 not available start therapy. start from cd4 < 350  further evaluation of patient for starting arv. tabel 1. clinical stadium hiv who 2004 3 ��parwitasari and nasronudin: hiv and malaria e p i d e m i o l o g y a n d t r a n s m i s s i o n o f malaria malaria are found in tropic area. africa, new guinea, haiti dominant p. falciparum, p. vivax more numerous in central america and parts of the south north africa, middle east and zindian subcontinen. p. ovale rare outside west african while p. malariae found in many place.12 malaria transmitted by several species mosquito anopheles. transmission this is not happening in temperature under 160 c, or over 330 c and height of more than 2000 m. condition optimum is on moisture high with temperature between 20 and 300 c (white, 1996). an area that many obtained the gnat is marshes that deals with the high rate occurrence malaria. stagnant water into one that support mosquito reproduction.11 malaria can also occurring by sporadic in the non endemic. in example because of malaria latent relapse few months after traveling from the endemic. sufferers as is generally not getting therapy complete or got kemoprofilaksis inadequate.11 picture 2. the cycle of the transmission of malaria from the mosquito to humans. table 2. a manifestation of malaria clinics difficult becausea manifestation of malaria clinics difficult because p. falciparum3 signs manifestations major unarousable coma / cerebral malaria failure to localize or respond appropriately to noxious stimuli; coma persisting for > 30 min after generalized convulsion acidemia/acidosis arterial ph < 7.25 or plasma bicarbonate level of < 15 mmol/l; venous lactate level of > 5 mmol/ l manifests as labored deep breathing, often termed “respiratory distress” severe normochronic, normocytic anemia hematocrit of < 15% or hemoglobin level of < 50 g/l (< 5 g/dl) with parasitema of > 100,000/μl renal failure urine output (24h) of < 400 ml in adults of < 12 ml/kg in children; no improvement with rehydration; serum creatinine level of > 265 μmol/l (> 3.0 mg/dl) pulmonary edema / adult respiratory distress syndrome noncardiogenic pulmonary edema, often aggravated by overhydration hypoglycemia plasma glucose level of < 2.2 mmol/l (< 40 mg/dl) hypotension/shock systolic blood pressure of < 50 mmhg in children 1–5 years or < 80 mmhg in adults; core/skin temperature difference of > 10ºc bleeding / disseminated intravascular coagulation significant bleeding and hemorrhage from the gums, nose, and gastrointestinal tract and/or evidence of disseminated intravascular coagulation convulsions more than two generalized seizures in 24 h hemoglobinuriaa macroscopic black, brown, or red urine; not associated with effects of oxidant drugs and red blood cell enzyme defects (such as g6pd deficiency) other impaired consciousness obtunded but arousable extreme weakness prostration; inability to sit unaidedb hyperparasitemia parasitemia level of > 5% in nonimmune patients (> 20% in any patient) jaundice serum bilirubin level of > 50 mmol/l (> 3.0 mg/dl) if combined with other evidence of vitalorgan disfunction malaria also can be transmitted over blood transfusion, syringe or transplant organs. the incubation generally short having no stadium preeritrositik.12 20 indonesian journal of tropical and infectious disease, vol. 5. no. 1 january–april 2014: 16–22 symptoms clinic malaria is one of the causes of fever in tropical countries. early symptoms of malaria are non-specific, such as discomfort, headaches, tired, uneasy feeling in the stomach, muscle pain with heat is a symptom similar to some viral diseases. in some circumstances a very head aches, chest pain, abdominal pain, artralgia, myalgia or diarrhea may be due to other illnesses allegedly although headaches on malaria might be heavier. nausea, vomiting and ortostatik hypotension often occurs as well. the classic symptoms of malaria, such as high heat, chills and stiffness that occurs on a regular basis appropriate intervals, relatively rare and is thought to be caused by p. vivax or p. ovale. heat the irreguler on p. falciparum in patients with the declined immune and children often achieve above 40° c flutter, and sometimes accompanied by delirium. seizures can also be caused by p. falciparum and is signified the presence of cerebral malaria.12 found little physical examination teratology on patients without complication, malaria is hot malaise, anæmia light and in some cases spleen being palpable. anemia more often resulted in children living in the transmission stable place that is resistant to partially parasitic chloroquin or other drugs. enlarged spleen often resulted in patients in the endemic indicating the presence of infection repeated. enlargement of the liver mild also found chiefly on small children. mild jaundice associated with adults caused by p. tertian and generally recovered after 1 to 3 weeks.12 management when a sufferers of an area that get malaria having symptoms heat, hapusan blood with drops thick and thin must be done to diagnose and determine species parasite that infects. repet of hapusan blood to do at least every 12 to with 24 hours for two days, if hapusan first negative.as an alternative detection antigen or stick test assignment. some medicines can be used orally and it depends on the sensitivity parasite that infects. chloroquin is therapy options for malaria a tame p. vivax, namely p. ovale, table 3. medicine which recomendation for antimalaria12 drug uncomplicated malaria (oral) severe malariaa (parenteral) chloroquineb 10 mg of base/kg followed by 10 mg/kg at 24 h and 5 mg/kg at 48 h or by 5 m/kg at 12, 24, and 36 h (total dose, 25 mg/kg); for p. vivax or p. ovale, primaquine (0.25 mg of base/kg per day for 14 daysd) added for radical cure 10 mg of base/kg by constant-rate infusion over 8 h followed by 15 mg/kg over 24 h or by 3.5 mg of base/kg by im or sc injection every 6 h (total dose, 25 mg/kg)c amodiaquineb 15 mg of base/kg followed by 10 mg/kg per day at 24 and 48 h (total dose, 35 mg/kg) – sulfadoxine/ pyrimethamineb 25/1.25 mg/kg, single oral dose (3 tablets for adults) – mefloquineb 15 mg/kg followed 8-12 h later by second dose of 10 mg/kg – quinine 10 mg of salt/kg q8h for 7 days combined with tetracyclinee (4 mg/kg qid) or doxycycline (3 mg/kg once daily) or clindamycin (10 mg/kg bid) for 7 days 20 mg of salt/kg by iv infusion over 4 hf followed by 10 mg/kg infused over 2–8 h every 8 h quinidine gluconate – 10 mg of base/kg by constant-rate infusion over 1–2 h followed by 0.02 mg/kg per min, with ecg monitoringg artesunate in combination with 25 mg of mefloquine/kg, 12 mg/kg given in divided doses over 3–5 days (e.g., 4 mg/kg for 3 days or 4 mg/kg followed by 2 mg/kg per day for 4 days); if used alone or in combination with clindamycin or doxycycline, give for 7 days (usually 4 mg/kg initially followed by 2 mg/kg daily) 2.4 mg/kg iv or im stat followed by 1.2 mg/kg at 12 and 24 h and then daily (or 2.4 mg/kg once daily) artemether same regimen as for artesunate 3.2 mg/kg im stat followed by 1.6 mg/kg per day atovaquone-proguanil (malarone) for adults > 40 kg, each dose comprises 4 tablets (each tablet containing atovaquone 250 mg and proguanil 100 mg) taken once daily for 3 days with food – artemether-lumefantrine for adults ≥35 kg, each dose comprises 4 tablets (each tablet containing artemether 20 mg and lumefantrine 120 mg) at 0, 8, 24, 36, 48, and 60 h, taken after food – 2�parwitasari and nasronudin: hiv and malaria p. malariae. malaria on heavy anti arrhythmic quinidine gluconate replace quinine as malaria therapy in us. discharging quinidine must with the monitor tight if there disritmia and to prevent from happening hypotension. quinine safer than quinidine and has been much used in the world widely during malaria therapy heavy. in some area of chinese medicines derivable from artemesinin (artemether and artesunat) been first choice to malaria heavy.12 the influence of hiv for malaria currently two health problems in africa hiv and malaria. research on interaction between both still a little. hiv immunity so on could reduce malaria patients symptoms heavier. while malaria would accelerate hiv infection into aids (chandramohan, 1998, whitworth, 2005). immunologist mechanism will protect the infection. it can be achieved through destructive parasitic on phase preeritrositik in liver by of cytotoxic t cells and other mechanism that related. if this mechanism fail, parasitic will continue easily enter into the bloodstream, it will increase so that manifestation clinic was renewed and will overcome by an antibody in blood. t cells playing an important role in the immune response against malaria. cd4 cells will help production of antibodies against malaria and controls parasitemia by producing cytokines. on hiv with total cd4 decline will give effect on ability of body to form an immune response that is effective against malaria. resulting from infection hiv in malaria will improve incident pain than healing, increase incident symptoms clinic compared infection asimtomatik, malaria and increase the weight compared light.13 the influence of malaria for hiv there is a possibility that cellular aktifasi resulting from infection pathogenic microorganisms will increase replication of the virus. malaria is strong stimulators the immune system. someone who exposed to malaria would increase the level of serum imunoglobulin, and occurring igg acceleration of change. b cells activity excess this may be caused by some specific, the response to an antigen variant malaria and stimulation of non specific derivat a toxin or influential mitogen directly into b cells or through active t cells. the evidence active t cells that remains less strong than b cells but the inductions some immune response specific influence possible active helper t cells that recurs.13 there is evidence that function of t cells decline during acute phase episode malaria. response proliferative are squeezed during acute episode of malaria. malaria infections have an effect against hiv stimulated change t cells and and failure of function of cytotoxic t cells. malaria infections also can undermine the placenta to make way for the transmission of hiv in utero.13 p. falciparum stimulates hiv replication through production of cytokines (il-6 dan tnf-α ) that is activated by lymphocytes. a study in malawai, africa shows that plasmatic viral loads hiv sufferers with higher on malaria compared with only hiv.14 therapy respone and interaction of medicine therapy anti malarial will be effective on an individual that have had immunity against malaria. estimated that response therapy will decline in individuals whose immunosuppressive because hiv infection and living on the transmission malaria stable. recent observations estimate treatment with artemisin, sulfadoksin-pyrimetamine artemether-lumefantrine and less effective in people with with hiv. interaction between an anti malarial drug and arv most involving a protease inhibitor (pi) and nonnucleoside reverse transcriptase inhibitors (nnrti). an anti malarial drug halofantrine, artemether lumefantrine and should not given by those with that off drugs pi (or nnrti delavirdine) because will increase the risk toxicity. among who wears nnrti another (nevirapine or efavirenz) the lumefantrine and artemether concentration low will increase the risk failure therapy. acquired also potential interaction between quinine and nnrti or pi. however potential interaction between drugs is still needs far more research.14 chloroquin commonly used as anti malaria caped inhibits the activity of antiviral namely interferon and beta alpha on experiment animals. found also that chloroquine increase replication of the virus in mice tried. chloroquin often worn as chemotherapy malaria. ( k a k k i l a y a , 2 0 0 5 ) c h l o r o q u i n h a v e a n e f f e c t immunosuppression and only effective in the central america and middle east. prophylactic proper to malaria resistant chloroquin is mefloquin and doxycyclin (rulf at, 1997). in one research farmakokinetik obtained mefloquin norvir lower levels in the blood up to 30%.1 many patients hiv allergic to sulfonamide as pirymethamine-sulfadoxine (fansidar). granting doxycycline 100 mg/day for two days before traveling, during traveling and four after that have some benefits because besides can give prophylactic on malaria also in diarrhea and some other infections. prevent mosquito bite, and diagnosis rapid and on complaint heat especially during and after passing to the regions with risk malaria is important for the hiv who travel.14,15 combination therapy arv has a great potential to lower hiv dealing with malaria. prophylactic cotrimoxazol recommended for children and adults with hiv in africa and effective to relieve the symptoms clinic malaria.2 22 indonesian journal of tropical and infectious disease, vol. 5. no. 1 january–april 2014: 16–22 summary hiv is an rna viruses whose hallmark is the reverse transcriptation of its genomic. the hallmark of hiv disease is a profound immunodeficiency. primarily, deficiency of the subset t lymphocytes, cd4, which serves the us primaries celullar receptor of hiv. malaria is a protozoan disease transmitted by the bite of infected anopheles mosquito. four species of the genus a plasmodium cause malaria infection of human. these are p. vivax, p. falciparum, p. ovale and p. malariae. there are two cycles of a plasmodium life, sexual and asexual cycles which happen in human and female anopheles mosquito. the association between the two infection has important implications. malaria and hiv-1 are two most common infection in sub-saharan africa, to a lesser extent, in other developing countries. hiv-related immunosupression may increase malaria rates of infection and malaria clinical disease. infection malaria can stimulate hiv replication and may cause faster progression of hiv disease. there are some anti malarias and drug interactions between antiretroviral drugs (arv). refenreces 1. kakilaya bs, malaria dan hiv/aids, http://www.malariasite.com/ malaria/malariainaids.htm. accessed october 19, 2005. 2. eline l, et al., 2005. malaria attributable to hiv-1 epidemic, sub-saharan afrika, emerging infectious disease, vol. 11, no. 9, 1410–1417. 3. fauci as, lane hc, 2005. human immunodefficiency virus disease aids and related disorder. in: harrison internal medicine, 1076– 1138. 4. o’neil sp, shieh wj, zaki ar, 2002. pathology and pathogenesis of virus infection, in: imunology of infectious disease. asm press, washington dc, 307–309. 5. silvestri g, feinberg mb, 2002. immune intervention in aids in: immunology in infectious disease. asm press, washington dc, 453–470. 6. drew wl, 2001. hiv & other retrovirus, in diagnosis & treatmen in infectious disease. mcgraw-hill, 442–447. 7. stewart gj, 1997. strategies of care in managing hiv. in: managing hiv, 3–4. 8. lewin sr, crowe r, chambers de, cooper da, 1997. antiretroviral therapy in: managing hiv, 45–54. 9. who, 2004. chronic hiv care with arv therapy. 10. white 1996. malaria, in: manson's tropical disease, 21th edition, wb saunders, 1088. 11. procop gw, persing dh, 2001. malaria and babesia in: diagnosis & treatmen in infectious disease. mcgraw-hill, 793–803. 12. white nj, breman jg, 2005. malaria and babesiosis: diseases caused by red blood cell parasites in: harrison internal medicine, 1218–1232. 13. chandramohan, 1998. is there an interaction between human immunodeficiency virus and plasmodium falciparum. international journal of epidemiology, 27, 296–300. 14. whitworth j, 2005. malaria and hiv, http://hivinsite.ucsf.edu/ insite?page=kb-05&doc=kb-05-04-04. accessed february 26, 2006. 15. rulft at, 1997. travellers with hiv. in: managing hiv, 146–148. 16. crowe s, mills j, 2001. aids & other virus of the immune system. in: medical immunology, tenth edition. lange medical book, 636–648. � vol. 5. no. 1 january–april 2014 the photodynamic effect of led-magnetic exposure to photoinactivation of aerobic photosyntetic bacteria suryani dyah astuti department of physics faculty of science and technology university of airlangga abstract all photosynthetic bacteria have a major pigment of bacteriochlorophyl and accessor pigment e.g. the carotenoids, which both have an important role in photosynthesis process. this study aim to explore the exogenous organic photosensitizer from photosyntetic bacteria for photodynamic therapy application. this study is an experimental research aiming to test the potential illumination of led with wavelength 409, 430, 528 and 629 nm, and power optimization and time exposure led-magnetic for optimum photo activation rhodococcus growth. the reseach design use a factorial completely randomized design with factor of power and exposure time. the number of bacterial colonies grown measure using of total plate count (tpc) methods. the result of anova test shows that irradiation treatment with led 409 nm, 430 nm, 528 nm and 629 nm significantly affects on bacterial colony growth. led 409 nm exposure has the greatest potential to boost the growth of bacterial colonies by 77%. led exposure and the addition of 1.8 mt magnetic field increases bacterial colony growth by 98%. results of optimization of led and magnetic fields show power 46 mw and a 40 minute (energy dose 110 j/cm2) optimum growth of bacterial colonies increase by 184%. so led and magnetic illumination has potentially increased the viability of an aerob photosyntetic bacteria colonies. key words: photosynthetic bacteria, optimum energy dose, led-magnetic, rhodococcus abstrak semua bakteri fotosintetik memiliki pigmen mayor yaitu bakterioklorofil dan pigemen asesoris seperti karotenoid, yang memiliki peran penting dalam proses fotosintesis. penelitian ini bertujuan untuk mengeksplorasi eksogen fotosensitiser organic dari bakteri fotosintetik untuk aplikasi terapi fotodinamik. penelitian ini merupakan penelitian eksperimental bertujuan untuk uji potensi iluminasi led dengan panjang gelombang 409, 430, 528 dan 629 nm, dan optimasi daya dan lama waktu pemaparan led-magnet fotoaktivasi pertumbuhan rhodococcus. desain penelitian ini menggunakan desain acak lengkap pola faktorial dengan faktor daya dan waktu pemaparan. jumlah koloni bakteri yang tumbuh dihitung dengan menggunakan metode tpc. hasil uji anova menunjukkan bahwa perlakuan penyinaran dengan led 409, 430, 528 dan 629 nm berpengaruh signifikan terhadap pertumbuhan bakteri. pemaparan led 409 nm berpotensi terbesar untuk meningkatkan koloni bakteri 77% . pemaparan led-magnet meningkatkan pertumbuhan koloni bakteri 98%. hasil optimasi led-magnet menunjukkan daya 46 mw dan waktu 40 menit (dosis energi 110 j/cm2) optimum meningkatkan pertumbuhan bakteri sebesar 184%. jadi iluminasi led dan magnet meningkatkan viabilitas koloni bakteri fotosintetik aerob. kata kunci: bakteri fotosintetik, dosis energy optimum, led-magnet, rhodococcus research report � indonesian journal of tropical and infectious disease, vol. 5. no. 1 january–april 2014: 5–11 introduction all photosynthetic bacteria have photosynthetic pigments that are sensitive to light (photosensitizer). the main pigment in photosynthetic bacteria is bacteriochlorophyl and accessory pigments which one carotenoid, which both have an important role in photosynthesis process. bacteriochlorophyl have major role as a light harvesting which packaged in the form of first light harvesting (lh1) and second light harvesting (lh2) and as a charge separation in the form of the reaction center (rc)1. carotenoid also have major role as same as bacteriochlorophyl that is as an accessory light-harvesting pigment and as a triplet quencher to provide protection again photooxidative damage2. that is the difference between absorbance spectrum when photophysical process. exposure light will be absorbed by the photosensitizer molecules in photosynthesis bacterial, light photon energy absorbance will excite the photosensitizer molecules in the singlet excitation and triplet. excitation of photosensitizer molecules occurs only if of light photons spectrum correspond to the photosensitizer absorption spectrum. subsequent excitation energy is transferred and converted into electrochemical potential energy in the form of transmembrane charge separation as well as the synthesis of adenosine triphosphate (atp)3 for the activation of photosynthetic bacteria. amount of energy converted to atp (photoactivation) depends on the number of photosensitizer molecules and the number of photons of light absorbed. one of the photosynthetic pigment producing bacteria is rhodococcus. the rhodococcus include eubacteria subkingdom members, a group of bacteria autotrophs (able to make their own food from inorganic substances), have chlorophyl and is able to photosynthesize like plants. rhodococcus have chlorophyl pigment (green), carotenoids (orange) and pigment phicobilin consisting of phycocyanin (blue) and phicoeritin (red). combine of these pigments produce the color to be turquoise. cell wall contain peptides, hemicellulose and cellulose, and have a slimy membrane. these bacteria use two photosystems to split water and produce oxygen as a byproduct. these bacteria like to live in fresh water, but there are some that live in the sea.4 this study is an experimental research laboratory, aimed to determine the potential irradiation of led purple 409, led blue 430 nm, led green 528 nm and led red 629 nm and 1.8 mt magnetic field for rhodococcus growth activation as well as effectivel dose energy optimization of led-magnetic for photoactivation. giving of 1.8 mt magnetic field from a bar magnet aims to increase the biosynthesis of photosensitizer molecules, thereby increasing the amount of light-absorbing molecules. material the sample rhodococcus bacteria were isolated from water river mas, surabaya. the bacteria are grown on the photosynthetic media (pms). irradiation equipment irradiation equipment is a source led light instrument, microcontroller, cervo motors, temperature sensor and lcd display. the source led light is used i.e. 409nm purple, 430 nm blue, 528 nm green and 629 nm red for exposure the in vitro bacterial. the type avr 8535 microcontroller is used for setting an exposure time and power led. the paralax continous servo motors is used for rotating bacterial petri dish on holder for flatten irradiation. the temperature sensor type lm35 is used for controlling the room temperature remains constant. the lcd display is used for showing given the pulse width modulation of irradiation and equipped by the timer running according to the length of time a given input, and the room temperature is detected by the temperature sensor. before the led instrument used for irradiation, temperature and time exposure calibration were done before. methods this study are prepared using a completely randomized design (ral = fully randomized design) factorial5, which consists of two factors; a factor (irradiation power) with 4 levels (i.e. 17 mw, 28 mw, 34 mw and 46 mw) and b factor (exposure time) with 4 levels (i.e. 20 min, 30 min, 40 min and 50 min). each treatment is accompanied by the control group using 3 times replication. the bacterial growth the bacteria are grown on sterile pms medium (photosynthetic medium) for 48 hours at 37°c temperature on the shaker incubator until the absorbance values obtained (od) solution in 0.15 at 600 nm wavelength. the two ml of each bacterial sample is put in a sterile plastic dish diameter 3.5 cm and ready for irradiated. irradiation bacteria by led petri dishes containing bacteria are placed on the holder in the acrylic box above platform servo motors. the distance between the led and the cup is permanently made in 2 cm. the source led light are performed at various power and exposure time. subsequently, the bacteria in the treatment group and the control are grown in pms agar medium. counting the number of bacterial colonies samples are taken out from the incubator and counted the number of bacterial colonies growing by total plate count method using a quebec colony counter. next step, calculating the percentage would decrease the number of bacterial colonies that grow on each treatment using the equation: �astuti sd.: the photodynamic effect of led-magnetic exposure to photoinactivation ½(σ treatment colonies – σ control colonies)/ σ control colonies ½ 100% statistical analysis analysis of research data use spss statistical analysis (statistical package for social science) 13.0 for windows, i.e. factorial anova for determining the effect of each factor and the interaction between factors. to show couple of different treatment groups, the analysis use multiple comparison test, on spss using multiple comparison post hoc5. results and discussion the led instrument has a temperature gauge and a time duration of irradiation designed with precision calibrator. data of temperatur and time exposure calibration were showed in table 1 and 2. in led instrument performance measurement, the time duration and temperature of led exposure was set up by calibrator enko sport digital stopwatch timer and calibrators atech thermo l87ad (figure 1). the regression graph of time duration and temperature led instrument yield r2 = 1 and r2 = 0.9995 that means the led instrument has a good performance. table 3 and 4 showed the percentage of bacterial colonies growth after led exposure and with 1.8 mt magnetic field exposure. test results of the potential led irradiation and 1.8 mt magnetic field exposure can be summarized in table 4. the test results indicate that in led exposure has potentially increase the number of rhodococcus growth colonies at 77% and increase to 98% by the addition of 1.8 mt magnetic field exposure. in figure 3 and figure 4 show that potentialy percentage reduction in the number of rhodococcus colonies in 469 nm, 541 nm, 626 nm and 409 nm of led exposure and with 1.8 mt magnetic field exposure. table 4 showed the percentage of bacterial colonies growth after led exposure and with 1.8 mt magnetic field exposure in varying intensity and time exposure. figure 5 shows a graph of the percentage growth of rhodococcus colony on a variety of power and duration time in led irradiation and 1.8 mt magnetic field exposure. exposure to 409 nm violet led optimal rhodococcus increase growth by 119% in power 28 mw and 60 minutes duration time exposure (dose 101 j/cm2). results of exposure optimization in 409 nm purple led and 1.8 mt magnetic field shows the percentage of rhodococcus growth by 184% in the power of 46 mw and 40minute duration time exposure (dose 110 j/cm2). exposure to table 2. temperatur calibration data of led instrument temperature (ºc) temperature of led instrumen (ºc) t mean ∆t calibrator temperature 1 2 3 4 5 6 7 8 9 10 21 20 20 20 21 21 21 21 21 21 21 20,7 0,48 21 22 22 22 21 21 22 22 22 22 22 22 21,8 0,42 22 23 23 23 22 24 22 23 23 23 23 23 22,9 0,57 23 24 23 23 24 24 24 24 24 24 24 24 23,8 0,67 24 25 25 25 25 25 24 25 25 25 25 25 24,9 0,32 25 26 26 26 26 25 25 26 26 26 26 26 25,8 0,42 26 27 27 27 27 28 27 27 26 27 27 27 27 0,47 27 28 28 28 28 28 27 28 28 28 28 28 27,9 0,32 28 29 29 29 28 28 29 29 29 29 29 30 28,9 0,57 29 30 30 30 30 30 30 30 30 31 30 30 30,1 0,32 30 table 1. time exposure calibration data of led instrument time (s) time of led instrumen (s) tmean time of calibrator(s)1 2 3 4 5 6 7 8 9 10 300 300 300 300 300 300 300 300 300 300 300 300 300 600 600 600 600 600 600 600 600 600 600 600 600 600 900 900 900 900 900 900 900 900 900 900 900 900 900 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 � indonesian journal of tropical and infectious disease, vol. 5. no. 1 january–april 2014: 5–11 figure 1. regression graph of time duration led instrument irradiation with calibrator. figure 2. regression graph of temperature led instrument irradiation with calibrator. figure 3. the graph percentage of rhodococcus bacteria colonies growth in led exposure. figure 4. the graph percentage of rhodococcus bacteria colonies growth in led exposure and 1,8 mt magnetic field exposure. table 3. potential of led irradiation and 1.8 mt magnetic field exposure on the percentage growth of photosynthetic bacteria percentage of bacterial colonies growth (%) led exposure led 409 nm 77 led 527 nm 60 led 430 nm 40 led 629 nm 16 led and 1.8 mt magnetic exposure led 409 nm 98 led 527 nm 66 led 430 nm 51 led 629 nm 29 430 nm blue led and 1.8 mt magnetic field rhodobacteria optimal increase by 111% growth in power of 34 mw and 50 minute duration time exposure (dose 102 j/cm2). one way anova test results showed that there have influence of led irradiation treatment 409 nm, 430 nm, 528 nm and 629 nm against rhodococcus colony growth (table 5). statistical test results of exposure energy dose optimization purple led 409 nm and 1.8 mt magnetic field on rhodococcus data show significance (p) = 0.624 > α (0.05), which means the data i.e. the percentage growth of bacterial colonies on 409 nm purlple led irradiation normally distributed. the test results showed homogeneity (p) = 0.109 > α (0.05), which means homogeneous data. factorial anova test results showed that the power factor of 409 nm purple led irradiation has a significance level (p) = 0.043 < α 0.05, which means that the radiation has an effect on the percentage growth in the number of rhodococcus bacteria colonies. summary of statistical test results are shown in table 6 below. figure 5 shows a graph of the percentage growth of rhodococcus colony on a variety of power and duration time in led irradiation and 1.8 mt magnetic field exposure. exposure to 409 nm violet led optimal rhodococcus increase growth by 119% in power 28 mw and 60 minutes duration time exposure (dose 101 j/cm2). results of exposure optimization in 409 nm purple led and 1.8 mt magnetic field shows the percentage of rhodococcus growth by 184% in the power of 46 mw and 40 minute duration time exposure (dose 110 j/cm2). exposure to 430 nm blue led and 1.8 mt magnetic field rhodobacteria optimal increase by 111% growth in power of 34 mw and 50 minute duration time exposure (dose 102 j/cm2). �astuti sd.: the photodynamic effect of led-magnetic exposure to photoinactivation is packaged in first light harvesting (lh1) and second light harvesting (lh2) complex, but charge separation bacteriochlorophyll is packaged in the form of the reaction center (rc). reaction center is in the middle of the circle of lh1 and functionally very closely related, so that the unity between lh1 and rc often called rc-lh1 core complex. light harvesting is also carried by several major carotenoid pigments that give main pigmentation at visible spectrum area, between 450 to 550 nm.6 carotenoids are long chain isoprenoid, usually the amount of carbon is 40. carotenoids are synthesized from 8 isoprene units (c5), the bonds between the carbon system alternately (double and single). this double bond is able to absorb light.15 carotenoids have important roles as light-harvesting especially in environments with limited light conditions and photo-protector bacteriochlorophyll against excessive light. in addition, carotenoids also act to prevent photo oxidation, due to the presence of oxygen in photosynthesis.11 the mechanism of energy transfer on photosynthetic bacteria by using inductive resonance and delocation excited. in inductive resonance energy transfer occurred energy transfer. when the bacteria are exposed by appropriate spectrum to the light spectrum, photon absorption then occurs, followed by electron excitation of photosensitizer. the excited electron is unstable and will transfer its energy to another molecule before it fell to the ground state. on excited transfer energy mechanism, the excited electrons move from one molecule to another molecule. this movement is very short and it just occurred on adjacent molecules less than 2 nm at distance. according vermeglio and joliot12 photosynthesis of bacteria originated from the absorption of light by an antenna system which contains the chromophore such as bacteriochlorophyll and carotenoid polyenes. singlet excitation energy is quickly transferred between the antenna chromophores, and finally to the reaction center. the role of the reaction center is changing the excitation energy into table 4. the percentage of bacterial colonies growth after led 406 nm and magnetic exposure in varying led time and power exposure power time percentage of rhodococcus bacteria colonies growth (%) 25 mw 46 mw 68 mw 93 mw 20 minutes 102 90 109 111 30 minutes 111 123 98 85 40 minutes 116 148 112 114 50 minutes 126 117 95 81 figure 5. the graph percentage of increase rhodococcus bacteria colonies in 409 nm purple led irradiation and 1,8 mt magnetic field exposure. table 5. the result of one way anova tests for determining the effect of led irradiation 409 nm, 430 nm, 528 nm and 629 nm bacterial isolates group n percentage increase in bacteria colony (%) t test rate sd significance conclusion rhodococcus by led irradiation 409 nm led 5 76,8 5,5 p = 0,000 there is a significant difference 528 nm led 5 59,8 6,4 430 nm led 5 40,0 5,0 629 nm led 5 15,8 2,7 total 20 45,10 23,8 rhodococcus by 1,8 mt magnetic exposure 409 nm led 5 98,2 12,8 p = 0,000 there is a significant difference 528 nm led 5 66,8 12,8 430 nm led 5 51,4 10,1 629 nm led 5 28,4 7,7 total 20 60,95 27,9 all photosynthetic bacteria have main pigment such as bacteriochlorophyll (bchl) and accessory pigments namely carotenoids. both of these pigments have an important role in the process of photosynthesis. bacteriochlorophyll is magnesium porphyrin which has more saturated tetrapyrrole ring. this porphyrin causes bchl absorbs at a wavelength near-infrared around 620–700 nm2. the main role of bacteriochlorophyll is as a light-harvesting and charge separation. light-harvesting bacteriochlorophyll �0 indonesian journal of tropical and infectious disease, vol. 5. no. 1 january–april 2014: 5–11 electrochemical energy in the form of a trans membrane charge separation. the absorption of light is followed by electron transfer from bacteriopheophytin to bacteriochlorophylls, qa quinone, qb quinone and binds with hydrogen to form hydroquinone. hydroquinone which is produced diffuses to the cytochrome bc complex, which is a trans membrane proton pump.11 the next step is transferring electrons to cytochrome-c by realizing h+ ions. the energy released is used to transfer protons across the membrane and the resulting energy drives the synthesis of adenosine triphosphate (atp) from adenosine diphosphate (adp) and inorganic phosphate (pi) with catalysis by atp synthase. carotenoids are lipids so that this pigment is liposoluble (fat soluble) and soluble in nonpolar solvents.13 carotenoid pigments are very efficient in absorbing light at wavelengths (450–550 nm). when the carotenoid molecule is exposed by light, it will be excited to a certain energy level. opportunities excitation energy levels that occur can be divided into two kinds, namely the singlet and triplet state. the function of carotenoids as photo protector occurs on triplet-triplet (tt) energy transfer and singlet-singlet energy transfer mechanism.11 when the light absorption, carotenoid excited to singlet state and immediately transfers the excitation energy to bacteriochlorophyll by using singlet-singlet energy transfer.11 the process of singletsinglet (ss) energy transfer is more common than the triplet-triplet (tt) energy transfer.14 this is due to the energy transfer from carotenoids to bacteriochlorophyll does not take a long time and does not require too much energy to work in the process of photosynthesis so that the cycle of photosynthesis can take place. while the triplet-triplet energy transfer occurs at bacteriochlorophyll have excess energy so that the energy transfer to the carotenoid is happened. carotenoids can be excited to a triplet state, i.e. when it receives a transfer of energy from tripletbacteriochlorophyll via triplet-triplet energy transfer mechanism7. the energy transfer happens in order to the carotenoid makes photo protection to bacteriochlorophyll. photo protector carotenoids function is as a photo protector through suppression mechanism (quenching), either directly or indirectly. in the directly extinction process, carotenoids receive energy transfer from triplet bacteriochlorophyll directly and disposed the excess energy in the form of heat (energy dissipation). the process of extinction (quenching) is indirectly done by carotenoids in a manner involving singlet oxygen. singlet oxygen formed naturally from receiving oxygen triplet energy transfer from triplet bacteriochlorophyll. singlet oxygen is radical. in the process of extinguishing, carotenoid accepts energy transfer from singlet oxygen, so the carotenoid gets the transition to the triplet state. finally, the triplet carotenoids release the excess energy as heat. photophysical process also can be occurred in bacteriochlorophyl. but if bacteriochlorophyl absorb more photon energy, excitation state bacteriochlorophyl can be exchange to triplet state. an excited electron spin singlet sn can be reversed, leaving the molecule in the excited state triplet tn, called intersystem crossing 9. intersystem crossing probability increases if the lowest singlet of vibrational level experience overlap with one of the higher vibrational levels of the triplet state. the triplet state bacteriochlorophyl will interact with oxygen molecule so that produce reactive oxygen species (ros). ros is a toxic molecule so that make cell damage. in order to avoid such that carotenoids neutralize it with excitation singlet oxygen transfer energy to carotenoid so that carotenoid is at high vibrational levels of the triplet excited state and back to the ground state through transfer heat in environment, the mechanism is called triplet-triplet energy transfer. this energy is used to prevent photooxidation and photoprotection.1 exposure to magnetic fields significantly influence the growth of bacterial colonies. giving magnetic field causes stress on bacterial cells and activates genes ala dehydratase (alad).4 alad is a key enzyme of porphyrin biosynthesis.10 thus giving the magnetic field increases the synthesis of porphyrin photosensitizer in pigment-producing bacteria. exposure to light will be absorbed by the photosensitizer molecules in bacterial photosynthesis, light energy is absorbed photon will excite the photosensitizer molecules. further excitation energy is converted into electrochemical potential energy in the form of transmembrane charge separation and synthesis of adenosine triphosphate (atp).3 according to the result, wavelength light having increase of the number colonies is 409 nm purple led which it is accordance with maximum soret absorbance. photophysical process initiate the photochemical process.8 almost photophysical mechanism occurs in carotenoid. photon light absorption by carotenoids will excite the molecule from the singlet ground electronic vibrational levels s0 to one of the vibrational levels of the electronic excitation. excitation of the molecule to the higher energy state is likely to return to the ground state, either through tabel 6. statistical test results of exposure energy dose optimization purple 409 nm led and 1.8 mt magnetic field on rhodococcus colonies factor group n percentage increase in bacteria colony (%) anova means sd significance conclusion power 46 mw (a) 3 97,4 19,6 p = 0,000 there is a significant difference 34 mw (a,b) 3 103,3 20,8 17 mw (a,b) 3 113,7 22,1 28 mw (b) 3 119,2 25,6 total 12 108,4 23,1 ��astuti sd.: the photodynamic effect of led-magnetic exposure to photoinactivation chemical reactions or changes to the heat released into the environment in the process of internal conversion or vibrational relaxation. in the singlet excited state, the carotenoids transfer energy to bacteriochlorophyls via singlet-singlet energy transfer.6 conclusions as briefly described in this article, the research results indicate that in 409 nm purple led irradiation has potentially increased the number of rhodococcus growth colonies at 77% and increase to 98% by the addition of 1.8 mt magnetic field exposure. results of exposure optimization in 409 nm purple led and 1.8 mt magnetic field shows the percentage of rhodococcus growth by 184% in the power of 46 mw and 40 minute duration time exposure (dose 110 j/cm2). acknowledgement the authors gratefully acknowledge the grant research fron and contributions of the students (qisty wulandari and nike dwi gd), colleagues and collaborators who helped carry out this research and who are listed as authors of the cited references. this work was supported by grants from the general of indonesia higher education. references 1. papageorgiu, katsambas a, chu a. phototherapy with blue (415 nm) and red (660 nm) light in the treatment of acne vulgaris, british journal of dermatology , 2000; 142: 973–978. 2. ke, bacon, photosynthesis: photobiochemistry and photobiophysic. kluwer academic publisher, 2003. 3. gust d, moore ta, moor al, mimicking bakteri photosynthesis, pure & application chemistry, 1998; 70(11): 2189–2200. 4. sasaki k, watanabe m, tanaka t. biosynthesis, biotechnological, production and application of 5-aminolevulinic acid, appl. microbiology biotechnology, 2002; 58: 23–29. 5. kusriningrum rs, experimental design, perancangan percobaan, airlangga university press, surabaya, 2008. 6. macpherson an, et al. efficient energy transfer from the caretonoid s2 state in a photosynthetic light-harvesting complex. biophysical journal, 2001: 80: 923–930. 7. fuchino y and amao y. photochemical and photophysical properties of caretonoid immobilized on a surfactant micellar medium including chlorophyl as an artificial photosynthesis system. biophysics, 2006: 2(10): 57–61. 8. grossweiner li. the science of phototherapy: an introduction, springer: usa, 2005. 9. plaetzer k, krammer b, berlanda j, berr f. photophysics and photochemistry of photodynamic therapy: fundamental aspects, journal of laser medical sciences, 2009: 24: 259–268. 10. hamblin mr, hasan t. photodynamic therapy: a new antibakteri approach to infectious disease ?, j. of photochem & photobiol, science, 2003: 3, 436–450. 11. tugiman, rondonuwu s. ferdy, limantara l. mechanism of energy transfer from carotenoid to bacteriochlorofill (mekanisme transfer energi dari karotenoid ke bakterioklorofil). sigma, 2009: vol. 12, no. 3. 12. vermeglio a and joliot p. the photosynthetic apparatus of rhodobacter sphaeroides. elsevier science ltd. paris, france, 1999: vol. 7, no. 11. 13. gross j. pigment in vegetables: chlorophylls and carotenoids. new york; van nostrand reinhold, 1991. 14. hu, xiche. pigment organization and transfer of electronic excitation in photosyn-thetic unit of purple bacteria. j. phys. chem., 1997: 101: 3854–3871. 15. cogdell rj and gardiner at. “light harvesting by purple bacteria: a circular argu-ment.” microbiology today, 2001: 28: 120–122. �� vol. 4. no. 4 october–december 2013 the role of hyperbaric therapy in the growth of candida albicans prihartini widiyanti1,2 1 faculty of science and technology, universitas airlangga 2 institute of tropical disease, universitas airlangga abstract background: candida albicans is opportunistic pathogen fungi which cause many disease in human such as reccurrent apthous stomatitis, skin lesions, vulvavaginitis, candiduria and gastrointestinal candidiasis. aim: infection mechanism of c. albicans is very complex including adhesion and invasion, morphology alteration from khamir form cell to filamen form (hifa), biofilm forming and the avoidance of host immunity. method: the ability of c. albicans to adhere to the host cell which is act as important factor in the early colonization and infection. result: the phenotype alteration to be filament form let the c. albicans to penetrate to the epithelium and play important role in infection and separation c. albicans to the host cell. hyperbaric oxygen is the inhalation of 100 percent oxygen inside hyperbaric chamber that is pressurized to greater than 1 atmosphere (atm). conclusion: the organism was found to be inhibited within a pressure/time range well tolerated by human subjects, suggesting that hyperbaric oxygen might be used successfully in treating human candidiasis. key words: hyperbaric oxygen, candida albicans, infection, host cell, immunity abstrak latar belakang: candida albicans merupakan jamur patogen yang berpotensi menyebabkan beberapa penyakit di manusia seperti reccurrent apthous stomatitis, lesi kulit, vulvavaginitis, candiduria dan candidiasis pada gastrointestinal. tujuan: mekanisme infeksi c. albicans sangat kompleks meliputi adhesi dan invasi, perubahan morfologi dari bentukan khamir sel menjadi bentuk filamen (hifa), pembentukan biofilm dan penghindaran terhadap sistem imun tubuh. metode: kemampuan c. albicans untuk melekat di sel tubuh yang merupakan faktor penting pada kolonisasi awal dan infeksi. hasil: perubahan fenotip menjadi filamen menyebabkan c. albicans berpenetrasi masuk ke epithelium dan berperan dalam infeksi dan pemisahan c. albicans ke sel tubuh. hyperbaric oxygen merupakan terapi dengan menggunakan 100 percent oksigen di dalam ruang udara bertekanan tinggi (rubt)/hyperbaric chamber yang mendapatkan tekanan lebih dari 1 atmosphere (atm). kesimpulan: terjadi penghambatan organisme dalam tekanan dan waktu tertentu pada subyek manusia, menunjukkan bahwa hiperbarik oksigen mungkin dapat digunakan dalam terapi human candidiasis. kata kunci: oksigen hiperbarik, candida albicans, infeksi, sel tubuh, imunitas introduction candida albicans is the fourth most common hospital acquired infection.1,2 because c. albicans and other fungal pathogens are eukaryotes and therefore share many of their biological processes with humans, most anti-fungal drugs cause deleterious side effects and, at the doses used, are fungistatic rather than fungicidal. so, it is an important goal of candida albicans research to identify appropriate targets for anti-fungal technologies. literature review morphology candida albicans can exist in three forms that have distinct shapes: yeast cells (also known as blastospores), pseudohyphal cells and true hyphal cells. yeast cells are round to ovoid in shape and separate readily from each other. pseudohyphae resemble elongated, ellipsoid yeast cells that remain attached to one another at the constricted septation site and usually grow in a branching pattern that is thought to facilitate foraging for nutrients away from �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 23–25 the parental cell and colony. true hyphal cells are long and highly polarized, with parallel sides and no obvious constrictions between cells. actin is always localized at the tip of the growing hypha3.a basal septin band (green) forms transiently at the junction of the mother cell and the evaginating germ tube; the first true hyphal septum forms distal to the mother cell and well within the germ tube.4 candidiasis candidiasis or thrush is a fungal infection (mycosis) of any species from the genus candida (one genus of yeasts). candida albicans is the most common agent of candidiasis in humans.5 also commonly referred to as a yeast infection, candidiasis is also technically known as candidosis, moniliasis, and oidiomycosis.6 candidiasis encompasses infections that range from superficial, such as oral thrush and vaginitis, to systemic and potentially life-threatening diseases. candida infections of the latter category are also referred to as candidemia or invasive candidiasis, and are usually confined to severely immunocompromised persons, such as cancer, transplant, and aids patients, as well as nontrauma emergency surgery patients.4 superficial infections of skin and mucosal membranes by candida causing local inflammation and discomfort are common in many human populations.5,6 while clearly attributable to the presence of the opportunistic pathogens of the genus candida, candidiasis describes a number of different disease syndromes that often differ in their causes and outcomes.7,8 hyperbaric oxygen hyperbaric medicine is the fascinating use of barometric pressure for delivering increased oxygen dissolved in plasma to body tissues. hyperbaric oxygen therapy (hbo) is a form of treatment in which a patient breathes 100% oxygen at higher than normal atmospheric pressure that is greater than 1 atmosphere absolute (ata). therapy is given in special therapeutic chambers, which were earlier used primarily to treat illnesses of deep sea divers. in the sixties hbo went out of practice because of its use without adequate scientific validation. over the last two decades, animal studies, clinical trials and well-validated clinical experience has proved efficacy of hbo in many indications and there is recently a renewed interest in this field all over the world.9 the effects of hyperbaric oxygen on fungi many effort have been made for a number of years and various reasons to determine oxygen toxicity limits of yeast. cairney has reviewed this problems associated with studies on the effects of hyperbaric oxygen on the fungi.10 oxygen is the most prevalent and most important element for the human body. it passes from the ambient air to the alveolar air and continues through the pulmonary, capillary and venous blood to the systemic arterial and capillary blood. it then moves through the interstitial and intracellular fluids to the microscopic points of oxygen consumption in the perioxomes, endoplasmic reticulum and mitochondria.10 the interactions between oxygen and antimicrobial agents have important implications fot the therapy of infections, because oxygen tensions could influenced the static and cidal activity of human body against spesific fungies. increased oxygen tensions can stimulate changes in host tissues (e.g decreased reduction – oxidation potential and increased ph) that might affect the metabolism and/or activation of certain fungies. systemic fungal infections generally only occur in patients with other debilitating conditions like diabetes, severe burns or the immunocompromised. research has shown that there was no response to increase atmospheric pressure alone, but addition of 100% oxygen under pressure led to growth inhibition of po2 of 900 mmhg and killing of microorganism at a po2 value of 1800 mmhg. 11 the effect of hyperbaric oxygen on c.albicans the effects of hyperbaric oxygen at a steady level of 3 ata was possesed the same result with mcallister et al who reported total inhibition of c. albicans at 2 ata oxygen.12 gifford and pitchard describes responses of candida utilis to hyperbaric oxygen.13 cultures of organisms in an exponential growth phase did not undergo any further development when exposed to 10 ata oxygen. when the exposure was continued for several days, all cells died. in the study of gifford reported that exponentially growing populations were more sensitive than stationary phase populations. the study of cairney wj, 1978 showed significant result using 3 ata oxygen for 4,5 hours of 24 hours period is sufficient to cause inhibition of growth and ability to form pseudohyphae and chlamydospores. this work has confirmed that c. albicans is inhibited in vitro within a pressure range readily tolerated by human subjects. this suggests that hyperbaric oxygen treatment might be effective in treating human candidiasis and that exposure tables used for gas gangrene causing by clostridium ssp could be used with some expectation of success.14 one study of gottlieb sf et al, 1964 indicated that exposure of c. albicans to 10 ata of oxygen for 14 dayskilled the organism. it was possible of low oxygen tensions and shorter exposures times a large number of c. albicans could have been killed with only a few cells able to survive the exposure to oxygen. in this study they have designed quantitative approach to obtain information regarding fungicidal versus fungistatic effects of hbo on c. albicans. they investigated the effects of (i) pressure, (ii) 900 mmhg o2, (iii)1800 mmhg o2 on the growth of organisms.15 conclusion most studies of hyperbaric oxygen correlated with c. albicans have shown that the effect is inhibitory rather than cidal. ��widiyanti: the role of hyperbaric therapy in the growth of candida albicans references 1. beck-sague, c. & jarvis, w. r. secular trends in the epidemiology of nosocomial fungal infections in the united states, 1980–1990. national nosocomial infections surveillance system. j. infect. dis. 167, 1247–1251 (1993). 2. miller, l. g., hajjeh, r. a. & edwards, j. e. jr. estimating the cost of nosocomial candidemia in the united states. clin.infect. dis. 32, 1110 (2001). 3. hazan, i., sepulveda-becerra, m. & liu, h. hyphal elongation is regulated independently of cell cycle in candida albicans. mol. biol. cell 13, 134–145 (2002). 4. sudbery, p. e. the germ tubes of candida albicans hyphae and pseudohyphae show different patterns of septin ring localization. mol. microbiol. 41, 19–31 (2001).this paper shows that there are fundamental differences in cell-cycle organization between the switch from unbudded yeast cells to hyphae and to pseudohyphae. 5. walsh tj, dixon dm (1996). “deep mycoses”. in baron s et al. eds. baron’s medical microbiology (4th ed.). univ of texas medical branch. isbn 0-9631172-1-1. 6. james, william d.; berger, timothy g.; et al. (2006). andrews’ diseases of the skin: clinical dermatology. saunders elsevier. pp. 308–311. isbn 0-7216-2921-0. 7. fidel pl (2002). “immunity to candida”. oral dis. 8: 69–75. 8. pappas pg (2006). “invasive candidiasis”. infect. dis. clin. north am. 20 (3): 485–506. 9. sahni t, hukku s, jain m, prasad a, prasad r, singh k, 2004. medicine update, 14, the association of physicians of india, 632–639. 10. cairney wj, 1977. developmental effects of hyperbaric oxygen on selected human pathogenic fungi in culture. thesis. cornell university, chapt 1. 11. jain kk, 1996. textbook of hyperbaric medicine. 2nd revised edition. hogrefe and huber publishers, seattle, 190–191. 12. mcallister, ta, jm stark, jn norman, rm ross, 1963. inhibitory effects of hyperbaric oxygen on bacteria and fungi. lancet 2: 1040–1042. 13. gifford gd, gg pritchard, 1969. toxicity of hyperbaric oxygen to yeasts displaying periodic enzyme synthesis. j.gen. microbiol, 25: 111–152. 14. cairney wj, 1978. effect of hyperbaric oxygen on certain growth features of candida albicans. aviation, space and environmental medicine, august 1978, 49 (8): 956–958. 15. gottlieb sf, rose nr, maurizi j, lamphier ea, 1964. oxygen inhibition on growth of mycobacterium tubercolosis. j. bacteriol, 87: 838–843. 86 vol. 1. no. 2 may–august 2010 case report neonatal sepsis in low birth weight infants in dr. soetomo general hospital martono tri utomo division of neonatology, department of child health, faculty of medicine airlangga university dr. soetomo general hospital abstract infections of the newborn are a significant cause of mortality. preterm infant have a high risk sepsis.. the incidence of neonatal sepsis is 1 to 10 cases per 1000 live births and 1 per 250 live premature births. to describe the characteristics of neonatal sepsis in the low birth weight infant in the neonatal intensive care unit dr. soetomo hospital. retrospective analysis. the data were collected from the medical record of low birth weight infants who were diagnosed as sepsis in neonatal care unit of dr. soetomo hospital between january 2010 to june 2010 with purposive sampling. descriptive analysis of risk factor of sepsis and blood culture of the patient was calculated. chi-square analysis was performed in the laboratorium data. characteristics sample: male vs female 61% vs 39%, outcome of sepsis in lbw was death 69%, alive 25%, risk of infection: turbid amniotic fluid 21%, asphyxia 33%. laboratorium data leucopenia and thrombocytopenia (p < 0.05). blood culture: klebsiella pnemoniae. the incidence and mortality of neonatal sepsis in lbw infants was still high. asphyxia, turbid amniotic fluid, leucopenia and thrombocytopenia were associated with sepsis. pneumoniae was the most common organisms in the lbw sepsis infants. keywords: neonatal sepsis, low birth weight, premature introduction infections of the newborn and young infant are a significant cause of mortality and long term morbidity. preterm infant have a high risk sepsis and its sequelae. in united state showed that incidence of early onset sepsis in vlbw infants was 1.5% and that of late-onset sepsis was 25%.1,2 neonatal sepsis, sepsis neonatorum or neonatal septicemia is a clinical syndrome characterized by sistemic signs of infection and accompanied by bacteremia in the first month of life.1,3 the incidence of neonatal sepsis is approximately 1 to 10 cases per 1000 live births and 1 per 250 live premature births [1] the incidence rates of neonatal infection in several referral hospitals in indonesia is approximately 8.76%–30.29% with the mortality rate is 11.56%–49.9%. the incidence rates of neonatal sepsis in several referrals hospital in indonesia is 1.5%–3.72% with the mortality rate is 37.09%–80%.3 some conditions had been identified as risk faktor for developing a neonatal sepsis. these conditions are:3 1. maternal risk factors are premature rupture of membranes (especially more than 18 hours), infection and fever of the mother during labour, foul smell of amniotic fluid, turbidity and greenish amniotic fluid, and multiple gestation. 2. neonatal risk factors are prematurity, low birth weight, asphyxia, resuscitation during delivery, invasive procedure, congenital anomaly, parenteral nutrition, long hospital stay in neonatal intensive care unit. 3. other risk factors: more frequently found in male than female, in black neonate, and in low social economy neonate. attack rates of neonatal sepsis increase significantly in low birth weight infants and the presence of maternal (obstetric) risk factor or sign of chorioamnionitis such as prolonged rupture of membranes, maternal intrapartum fever (>37.5° c). host risk factors include male sex, developmental or congenital immune defect, congenital anomalies, omphalitis and twinning. prematurity is a risk factor for both early onset and late onset sepsis.1,2–4 in the collaborative perinatal research study sponsored by the national institutes of health, low birth weight infants acquired sepsis three times more frequently than did term infant who weighed more than 2500 gram.4 87utomo: neonatal sepsis in low birth weight infant the purpose of this study was to describe the characteristics of neonatal sepsis in the low birth weight infant who were delivered or referred in the neonatal intensive care unit dr. soetomo hospital between january 2010–june 2010 methods the study design was retrospective analysis. the data were collected from the medical record of low birth weight infants who were diagnosed as sepsis and delivered or admitted in neonatal care unit of dr. soetomo hospital between january 2010 to june 2010. technical sampling was purposive sampling. we reviewed data of all infants who had been diagnosed as sepsis and collected the data of samples characterisic such as sex, referral case, mode of delivery, birth weight, gestational age, and outcome. the risk factors that associated with sepsis such as maternal fever, premature rupture of the membrane, turbid amniotic fluid, and asphyxia were documented. the laboratorium data such as hemoglobin, wbc, platelet count, crp, and blood culture were also recorded. definitions – premature are liveborn infants delivered before 37 weeks from the first day of the last menstrual period – low birth weight infant are considered if infant who weight between 1,500 gram to 2,499 gram at birth. – very low birth weight infant are considered if infant who weight between 1,000 gram to 1,499 gram at birth – extremely low birth weight infant are considered if infant who weight less than 1,000 gram at birth – premature rupture of membrane is defined as the time from membrane rupture to onset of delivery was more than 18 hour. – maternal fever if mother suffered from fever which temperature > 37.5° c during delivery. – asphyxia is defined as apgar score in 5 minute is 3 or less – diagnosis of sepsis neonatorum based on clinical findings and supported by laboratory data ( routine blood examination, value of c reactive protein and culture). – anemia is defined as hemoglobine less than 13 g/dl – leucopenia if the wbc less than 4,000/cmm – leucositosis if the wbc more than 34,000/cmm – thrombocytopenia if the platelets less than 100,000/ cmm statitical analysis data are presented in distribution tabulation and data analysis was performed with a computer assisted statistical package (spss ver. 12.0). descriptive analysis of risk factor of sepsis, laboratorium data and blood culture of the patient was calculated. chi-square analysis was performed in the laboratorium data. results and discussion data from january 2010 until june 2010 revealed the low birth weight infant were 113 patient from total of 337 patient that admitted. diagnosis of sepsis in lbw infant were 36 (32% of lbw patient). the characteristics of the sample are listed in table 1. table 1. characteristic of low birth weight neonate patient charateristics sepsis (n = 36) non-sepsis (n = 77) sex male 22 (61%) 40 (52%) female 14 (39%) 37 (48%) location of delivery dr. soetomo 28 (78%) 71 (92%) referral 8 (22%) 6 (8%) mode of delivery spontaneous 16 (44%) 46 (61%) spontaneous bracht 4 (11%) 3 (4%) manual aid 1 (3%) 4 (5%) vaccum extraction 1 (3%) 0 (0%) forceps extraction 0 (0%) 1 (1%) caesarian section 13 (36%) 22 (29%) partus precipitatus 1 (3%) 0 (0%) birth weight < 1000 g 4 (11%) 7 (9%) 1000–1499 g 6 (17%) 11 (14%) 1500–1749 g 12 (33%) 10 (13%) 1750–1999 g 6 (17%) 10 (13%) 2000–2499 g 8 (22%) 39 (51%) gestational age < 28 weeks 2 (6%) 9 (82%) 29–32 weeks 14 (39%) 12 (16%) 33–36 weeks 12 (33%) 25 (33%) 37–42 weeks 8 (22%) 29 (37%) > 42 weeks 0 (0%) 2 (2%) outcome alive 9 (25%) 41 (53%) death 25 (69%) 24 (31%) discharge on request 2 (6%) 12 (16%) from table 1, in this study showed male infants were more suffered from neonatal sepsis, approximately 61% cases than female infants (39%) a predominance of male infant is apparent in almost all studies of sepsis in the newborn infant and the previous study in dr soetomo general hospital but not among infants infected in utero.4,5 the usual male predominance in neonatal sepsis has suggested the possibility of a sex–linked factor in host susceptibility. a gene located in x chromosome and involved with function of the thymus or with synthesis of immunoglobulins has been postuled.4,6 the female has 88 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 86-89 double the number of genes affecting these factors and thus might possess a greater resistance to infection. the immunologic basis for the superior survival of the female is reviewed by purtillo and sullivan.7 in the lbw sepsis group, the mode of delivery that frequently seen were spontaneous delivery (44%) and caesarian section (36%). this frequency of mode delivery in low birth infants was similar with the previous study.8 the spontaneous delivery of lbw and premature infant usually is waited for some hours to make the maturity of the lung by giving glucocorticoid to the mother but the other consequences is increasing the risk of infection from prematur rupture of the membrane.9 caesarian section may contribute the changes of normal flora in infant. the caesarian section infant have lower isolation rate of bifidobacteria and a much lower incidence of bacteroides spp.10 but from the other study showed there was no significant difference in the bowel flora between mode of delivery and feeding method in the seven day postnatally.11 the normal flora in infants have a role in the immunity system of the infant so the changes in the normal flora normal may lead to risk of sepsis condition. the sepsis in low birth weight infants in this study was 32% with mortality 69%. this condition is similar with the previous study done by simiyu, that incidence of sepsis in lbw was 37% with mortality 76%.8 the lbw infant and prematurity can increased the risk of sepsis by relatively immunodeficiency and may got some invasive, monitoring procedure, and longer duration of stay that may lead to nosocomial infection.12,13 the mortality of lbw infants with sepsis in this study was 69%, thas was similar with the previous study that mortality in the sepsis lbw was 76%.8 but in other study lower (46%).14 some condition that may contribute to the mortality of low birth weight infants are hypothermia, hypoglycemia, overcrowding and understaffing in nicu and apneic attacks beside the sepsis condition,8,14 but in this study the condition was not determined yet. table 2. risk factors of sepsis in lbw infants risk factors sepsis non-sepsis maternal fever 0 (0%) 1 (1%) prm > 18 hours 2 (6%) 5 (7%) turbid amniotic fluid 7 (21%) 7 (9%) asphyxia 11 (33%) 11 (15%) from table 2, showed that risk factor of sepsis in lbw were turbid amniotic fluid (21%) and asphyxia (33%). the turbid amniotic fluid can be caused by inflammation recation of infection in the choriamnionitis especially if it combined with foul smelling.1 from other study showe that meconeal or turbid amniontic fluid can increased the risk of infection. the chorioamnionitis was also involved in 28% of infection in lbw.13,15 in the asphyxia condition, the lbw infant was got the invasive procedure i.e resuscitation, intubation or prolonged of stay during stabilization.9 this condition can increase the risk of infection especially in the lbw infants3 but from the previous study in dr. soetomo hospital showed no significant of asphyxia as risk of infection16 in this study showed that asphyxia was found in 33% cases of sepsis in lbw, but we didn’t determined the risk of asphyxia in this study. table 3. laboratorium data of sepsis in lbw infants laboratorium data sepsis non sepsis p value anemia 10 (28%) 5 (17%) 0.38 leucopenia 8 (22%) 0 (0%) 0.006* leucocytosis 5 (14%) 3 (10%) 0.719 thrombocytopenia 14 (42%) 4 (13%) 0.015* positive crp 11 (48%) 8 (38%) 0.565 i n t h e t a b l e 3 i n d i c a t e d t h a t l e u c o p e n i a a n d thrombocytopenia were significantly correlated with sepsis. leucopenia in this study was higher than previous study.5 leucopenia condition was included in the scoring of sepsis to predict positive bacterial culture and correlated with the presence of bacterial infection.2,3 thrombocytopenia in sepsis can be caused by direct toxic injury to platelet and may be involved with immune mechanism.17 in this study from table 3 showed that thrombocytopenia was correlated with sepsis in lbw with p 0.015. thrombocytopenia in sepsis neonates was also found in the previous study and usually associated with gram negative or candida sepsis.18,19 table 4. result of blood culture blood culture microorganism positive klebsiella pnemonia 3 acinetobacter spp 1 candida spp 1 sterile 5 total 10 in this study, there were 10 (27%) patient from 36 lbwsepsis infants was obtained blood culture examination . blood culture should be done in the presence of suspected sespsis, but some condition may interfere this procedure such as, financial problem, antibiotic was already given, no media culture ready in the unit, and transportation of media culture to the microbiology. blood culture in the other study was less than in our study (only 14% blood culture done in the suspected sepsis patients)8 klebsiella pnemoniae klebsiella pnemoniae was the most common organism in this study and the sterile culture was found in 50% of the lbw sepsis infants. from previous study showed that klebsiella pnemoniae was as most common organism with high case fatality.21,22 89utomo: neonatal sepsis in low birth weight infant conclusions the incidence and mortality of neonatal sepsis in lbw infants was still high. some condition that associated with sepsis were asphyxia, turbid amniotic fluid, leucopenia and thrombocytopenia. klebsiella pnemonia was the most common organisms in the lbw sepsis infants. references 1. schelonka r, freij bj, mccracken gh. bacterial and fungal infection. in: macdonald m, mullett md, seshia mmk, editor. avery's neonatology pathophysiology and management of the newborn philadelphia: lippincott williams wilkins; 2005. p. 1235–73. 2. puopolo k. bacterial and fungal infections. in: cloherty j, eichenwald ec, stark ar, editor. manual of neonatal care. 5th ed. philadelphia: lippincott william & wilkins; 2008. p. 275–300. 3. rohsiswatmo r. kontroversi diagnosis sepsis neonatorum. in: hegar b, trihono pp, ifran eb, editor. update in neonatal infection. jakarta: departemen ilmu kesehatan anak fkui-rscm; 2005. p. 32–43. 4. klein j, marcy sm. bacterial sepsis and meningitis. in: remington j, klein jo, editor. infectious diseases of the fetus and newborn infant. 3rd ed. philadelphia: w.b. saunders co; 1990. p. 610–25. 5. jain n, jain vm, maheswari s. clinical profile of neonatal sepsis. kathmandu univ med j. 2003; 1: 117–20. 6. washburn t, medearis dn, childs b. sex differences in suceptibility to infections. pediatrics. 1985; 35: 57–60. 7. purtillo d, sullivan jl. immunological basis for soperior survival of females. am j dis child. 1989; 133: 1251–5. 8. simiyu e. morbidity and mortality of the low birth weight infants in newborn unit in kenyatta national hospital, nairobi. east african med j. 2004; 81: 367–74. 9. ringer s. care of the extremley low birth weight infant. in: cloherty j, eichenwald ec, stark ar, editor. manual of neonatal care. philadelphia: lippincott williams & wilkins; 2008. p. 78–85. 10. bennel r, nord ce. development of the faecal anaerobic microflora after caesarean section and treatment with antibiotics in newborn infants. . infection. 1987; 15: 332–6. 11. sung n, lee sg, kim mj, kim yh, yang s, hwang it, et al. the changes of intestinal normal flora in neonates for seven days postnatally. korean j pediatr gastroenterol nutr. 2006; 9: 162–8. 12. gupta r. care of low birth weight neonate. jk science. 2008; 10: 158–9. 13. shah g, budhathoki s, das bk, mandal rn. risk factors in early neonatal sepsis. kathmandu univ med journal. 2006; 4: 187–91. 14. bang a, reddy hm, bang ra, desmukh md. why do neonates die in rural gatchirolli, india? estimating population attirbutable risks and contribution of multiple morbities for identifying a strategy to prevent deaths. j perinatol. 2005; 25: s35–43. 15. janine m, jason md. infectious disease-related deaths of low birth weight infants, united states, 1968 to 1982 pediatrics. 1989; 84: 296–303 16. utomo m. risk factors of neonatal sepsis: a preliminary study in dr soetomo hospital. in j trop infec dis. 2010; 1: 23–6. 17. goorin a, cloherty jp. thrombocytopenia. in: cloherty j, eichenwald ec, stark ar, editor. manual of neonatal care. 5th ed. philadelphia: lippincott williams & wilkins; 2008. p. 455–62. 18. torkaman m, afsharpaiman sh, hoseini mj, moradi m, et al. platelets count and neonatal sepsis: a high prevalence of enterobacter spp singapore med j. 2009; 50: 482–5. 19. lee w, cho j, yoo s, lee c, et al. platelet count and mean platelet volume in low birth weight infants (≤2,000 g) with sepsis. korean j pediatr gastroenterol nutr. 2007; 50: 643–8. 20. waseem r, khan m, izhar ts, qureshi aw. neonatal sepsis. professional med j. 2005; 12: 451–6. 21. sundaram v, kumar p, dutta s, mukhophdhyay k, et al. blood culture confirmed bacterial sepsis in neonates in a north indian tertiary care centre: changes over the last decade. jpn j infect dis. 2009; 62: 46–50. ijtid vol 1 no 2 may-aug 2010.34.pdf ijtid vol 1 no 2 may-aug 2010.35.pdf ijtid vol 1 no 2 may-aug 2010.36.pdf ijtid vol 1 no 2 may-aug 2010.37.pdf 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 �� vol. 4. no. 4 october–december 2013 application of neural networks on blood serum image for early detection of typhus betty purnamasari1, franky chandra s.a2 , suryani dyah a3 1 bachelor of biomedical engineering study program, physics department, faculty of science and technology, universitas airlangga 2 bachelor of biomedical engineering study program, physics department, faculty of science and technology, universitas airlangga 3 bachelor of physics study program, physics department, faculty of science and technology, universitas airlangga contact person: bettysanchezh@yahoo.com abstract background: typhus is a disease caused by salmonella typhi, salmonella paratyphi a salmonella parathypi b, dan salmonella paratyphi c bacteria that attacks digestive tract and caused infection in small intestine. the common test that performed in the laboratory is widal test. the result reading of the widal test still processed manually with looking the turbidity caused by the agglutination. aim: the research was made to decrease human error by creating a program based on artificial neural network (ann) with learning vector quantization (lvq) method. method: input of this program is image of blood serum that has reacted with widal reagen. image procesing start with grayscaling, filtering, and thresholding. result: output of this program is divided into two classes, normal and typhus detected. conclusion: from this experiment result that using 24 testing data, gives the accuracy of this program 95.833% with 1 error result from 24 testing data. key words: artificial neural network, learning vector quantization, salmonella, typhus, widal abstrak latar belakang: penyakit typhus adalah penyakit yang disebabkan oleh bakteri salmonella typhi, salmonella paratyphi a salmonella parathypi b, dan salmonella paratyphi c yang menyerang bagian saluran pencernaan, sehingga terjadi infeksi saluran pencernaan tepatnya usus halus dan masuk ke aliran darah. pemeriksaan awal yang umum dilaksanakan di laboratorium adalah dengan melakukan pemeriksaan widal. pembacaan hasil pemeriksaan widal masih dilakukan secara manual dengan mengandalkan kemampuan manusia memeriksa kekeruhan yang timbul akibat terjadinya aglutinasi. tujuan: penelitian ini dibuat untuk mengurangi adanya human error yang terjadi pada pembacaan hasil tes dengan menggunakan program berbasis jaringan saraf tiruan (jst) metode learning vector quantization (lvq). metode: citra yang digunakan adalah citra serum darah yang telah direaksikan dengan reagen widal. proses pengolahan citra dilakukan dengan teknik grayscaling, filtering dan thresholding. hasil: keluaran dari program ini adalah deteksi citra serum darah typhus dan normal. kesimpulan: dari hasil penelitian ini dengan menggunakan 24 data uji, memberikan akurasi program sebesar 95,833% dengan 1 kesalahan uji dari 24 data uji. kata kunci: jaringan saraf tiruan, learning vector quantization, salmonella, typhus, widal introduction typhoid fever or commonly referred to as typhus is a disease caused by the bacterium salmonella typhi, salmonella paratyphi a salmonella parathypi b, and salmonella paratyphi c, which attacks the digestive tract. acute infectious disease is always there in society (endemic) in indonesia, ranging in age from toddlers, children and adults. according to who (world health organization) in 2003, each year there are approximately 17 million cases with 600,000 cases leading to death in the world. approximately 2% of patients with typhoid can be a carrier. in indonesia, there were 900,000 cases with 20,000 deaths case.1 a common initial examination was carried out in the laboratory by examining widal. widal tes is included in the class of serological test, which is done by reacting the blood serum of patients with widal reagents. widal test is the research report �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 53–58 examination steps are easy to do and the results are quickly obtained. widal test involving agglutination reaction that helps detect antibodies in the diagnosis of typhoid fever.2 positive widal test characterized by the appearance of turbidity caused by agglutination arising from the reaction of antibodies in the blood serum of patients with bacterial antigens present in widal reagents. widal test results can be used as a follow-up diagnosis of typhoid fever. diagnosis of typhoid disease from widal test must be supported by other examinations, such as checking the physical condition of the patient’s own or other laboratory tests such as examination of peripheral blood and blood cultures.3 the results of turbidity caused by agglutination widal examination read by relying on human capabilities so that errors can occur due to human error, because each medical staff have different possibility of reading the results of turbidity arising from the blood serum agglutination reagents.4 based on the above presentation, to reduce human error and to develop research using soft computing technologies it needs to make a program to solve it is by using a program based on artificial neural networks. artificial neural network is an information processing system that has characteristics similar to biological neural networks, which can be applied to one of them in pattern recognition or pattern recognition.5 typhus diagnostic research with widal test using artificial neural networks has been studied before with bacpropagation method.4 the procentage of the result is success detection for 93.75% positive typhoid and negative 90% for typhoid. the study was conducted by using the features in the form of a binary matrix pattern of image processing as input feature propagation. artificial neural networks have many methods that can be used. comparative research results using an artificial neural network classification methods bacpropagation and lvq (learning vector quantization) to obtain the result that the lvq training process faster and more accurate than backpropagation.6 due to the background, the author will make an application methode of artificial neural network in blood serum image for early detection of disease typhoid. the present study microscopic images of blood serum which has given widal reagents are used as inputs of the software, but the image is processed first using image processing methods. the output of image processing features value then processed using lvq neural network method and going through the learning phase. materials and methods sample data collection of blood serum samples is done by taking a blood serum sample data that has been diagnosed from clinical laboratory that consist normal blood serum sample and typhus blood serum samples. blood serum is then reacted with a reagent widal then conducted observations and image capture using a digital microscope. the whole image is obtained jpeg format, and done cropping on an object the size of 100 x 100 pixels. image of the blood serum was observed if there is agglutination in it, if it is exposed to typhus blood serum then there will be agglutination otherwise if there is no agglutination in the serum it is normal serum. agglutination occurs due to the reaction between serum by cellular antigen or cell body surface. the reaction between the reagent and serum observed under a microscope with magnification 4x10, the results of these observations in the form of images to be processed into the image processing and artificial neural networks. broadly speaking, software design schemes undertaken in this study is depicted in figure 1. figure 1. flow procedure typhus detection program blood serum samples processed image using a digital image processing techniques such as grayscale, namely, filtering, thresholding and clear border. first data of blood serum image was color image that consists of three layer matrix, namely r layer, g layer and b layer converted to grayscale images or images that represent the level of gray. grayscale process aims to alleviate the computational load while performing data processing. then the filtering process done to the image that has grayscale form. filtering is used to improve the quality of the processed image by smoothing noise contained in images of blood serum samples. the research will use median filter as the filter technique. median filter method serves as a nonlinear filter for the workings of this filter is not included into the category convolution operation. the next process is thresholding, thresholding is a simple and effective techniques for image segmentation of blood serum. this method can be used to extract objects from the background by selecting the threshold value t that separates the background and object and the result of thresholding process will produce a binery image or image with black and white colour. in this case the object needed is agglutination that caused by the reaction of serum with reagents. agglutination will be represented by the white pixels that result from thresholding ��purnamasari, et al.: application of neural networks on blood serum image process, so the feature image obtained in the form of the number of pixels that are white. clear borbeder process after thresholding process is used to eliminate unwanted image on the wall background. feature extraction is used to determine the characteristics of the image or pattern of positive and negative blood serum typhus before being put to be processed into the neural network. results of feature extraction is a number of white pixels, which is where white pixels represent the image in the image agglutination. results of feature extraction processing will be used as input to an ann using lvq models. the next stage is the determination of the network design. this stage will be determined the definition of the problem, namely the determination of input and output patterns for training and testing the ann. the next step taken is to initialize the network to be trained or tested. this research was conducted using 64 training sample data consist of 32 normal samples and 32 samples of typhus. the data used for testing amounted to 24 data consists of data from 12 normal and 12 data typhus. the flow chart of lvq itself can be shown in figure 2. result and discussion this research was conducted using 64 training sample data, where each consisting of 32 normal samples and 32 samples of typhus. the data used for testing amounted to 24 data consists of data from 12 normal and 12 data typhus. all training sample data processed with image processing and ann using lvq methode. for the first image processing is grayscale, grayscaling process is done for change the image color on the blood serum into gray image to reduce the computational burden. after grayscalling process, image has filtered with median filtering methode to reduce the noise. grayscalling image processing result is shown in figure 3. the next processed is thresholding. thresholding process in this study is done by taking 115 as the threshold value that obtained by observation the histogram blood serum images were used as training samples. objects needed in this case is the agglutination that caused by reaction with the reagent serum. agglutination will be represented by the white pixels caused by the thresholding process. image processing results for thresholding disaikan in figure 4. the last step of image processing that used in this study is clear border. clearborder process needed for eliminate unneeded image attached or contact with blood serum image to be processed. image processing results of the process is a clear border binary image like the result of the previous image processing wihich is thresholding. this binary image is used to deteminate the value of feature extraction. at this clearborder process used toolbox matlab syntax “cb = imclearborder (cb)”. results of image processing for clear border is shown in figure 5. figure 2. flowchart lvq (a) (b) figure 4. (a) threshold normal serum image (b) threshold typhus serum image (a) (b) figure 3. (a) grayscale normal serum image (b) grayscale typhus serum image �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 53–58 for feature extraction, object that needed in this case is the agglutination that caused by the reaction with the reagent serum. agglutination will be represented by a white pixel in the resulting binary image. binary image of blood serum results will be processed for image feature extraction process to count the number of white pixels as features that are used as input for training and testing process in ann. training process of neural network lvq method for detection of typhus used 64 data of blood serum image, which consists of 32 images of normal blood serum and 32 images of typhoid blood serum. training process is performed using some variations of learning vector quantization input parameters, as shown in table 1. the results of the training process is obtainment of final weight values that saved and used for the testing process. after the data passing through the training process will be performed test matches to the target data which is the result of a doctor’s diagnosis. the number of matches data with the target compared to the entire amount of data to get the accuracy rate of the training process. the accuracy obtained from each parameter changes is shown in table 2. table 2 shows the percentage level of accuracy of the results of the training process to some variations of the learning rate (a) and a reduction in the rate of learning (dec a), these variations affect the number of epoch that and the level of accuracy for the training. parameter variation indicates a accuray changes that does not necessarily(volatile). the most optimal level of accuracy in this study was the learning rate 0.01 with a reduction in the learning rate 0.5 and learning rate 0.001 with the reduction of learning rate 0.1, 0.25, 0.5 by 96.875% accuracy. display ann training program is shown in figure 6. the testing process used 24 data outside of the data that used for training process. 24 data consists of 12 images of normal blood serum, 12 images typhus blood serum. classification of testing data process performed by finding the minimum distance between features of testing data with the final weight values that obtained from the results of ann training process. the testing process is done by (a) (b) figure 5. (a) threshold image typhus serum (b) clearborder image typhus serum table 2. result accuracy testing value on training data a dec a epoch akurasi (%) 0.1 0.001 1146 93,75 0.1 110 93,75 0.25 41 93,75 0.5 17 93,75 0.01 0.001 917 93,75 0.1 88 93,75 0.25 33 95,3125 0.5 14 96,875 0.001 0.001 688 93,75 0.1 66 96.875 0.25 25 96.875 0.5 10 96.875 table 1. variation parameter of lvq amount of training data 64 amount of target classification 2 learning rate (α) 0.1 ; 0.01 ; 0.001 decrease of learning rate (dec α) 1.01 ; 0.1 ; 0.5 ; 0.25 minimum learning rate (min α) 0.0000001 maximum epoch 10000 figure 6. display of training program ��purnamasari, et al.: application of neural networks on blood serum image using a variation of the dec alpha and alpha which has the highest value accuracy of training data which is used alpha 0.01 and dec alpha 0.5. testing process results for testing data using the alpha value of 0.01 and the dec alpha value of 0.5 is obtained the accuracy of 95.833%. from 24 testing data are, there’s 1 testing data that does not match the target. display ann testing program is shown in figure 7. display program of early detection of typhus which thre are image processing, feature values and the image test results is presented in figure 8. in this page display there is a menu bar file, which is used to retrieve data from the directory. also in this page there are some buttons that have the functions of each. image processing button, a button that contains the command for perform the process image processing on the data tested. image processing process shown on this page are the original image, grayscale, binary image from thresholding process and clear border. this button also count the feature extraction value. test button, a button that contains the commands for perform testing process on the data tested. in this case the results of the test is information whether the data classified in “normal” or classified in “typhosa detected”. reset button, serves to reset or delete the previous data so that the page can be used for perform other testing data. close button, serves to close the page process the data and return to the home page. conclusion based on the results of the discussion, it concluded that early detection system design on blood serum image based on neural network methode, done by looking for the value of the number of features in the form of white pixels in the images of blood serum which has been processed using image processing. the most optimal parameter values for the typhus early detection design programs is using the value of the learning rate (a) of 0.01 and a reduction of learning rate (dec a) of 0.5 with the accuracy of the program 95.83%. references 1. crump, jhon a. 2004. stephen p, luby. eric d, mintz. 2004. the global burden of thypoid fever. buletin of world health organization 2. nigam, arty. ayyagary archana. 2007. lab manual in biochemistry: immunology and biotechnology. tata mcgraw-hill publishing company limited. new delhi. figure 7. testing program display figure 8. display of early detection of typhus program �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 53–58 3. djojodibroto, darmanto. 2001. seluk-beluk pemeriksaan kesehatan (general medical check up). pustaka populer obor. jakarta. 4. mak`ruf, muhammad ridha. 2006. identifikasi penyakit thypus dengan widal test menggunakan jaringan syaraf tiruan. teknik fisika, institut teknologi sepuluh nopember. surabaya. 5. siang, jong jek. 2009. jaringan saraf tiruan & pemrogramannya menggunakan matlab. penerbit andi. yogyakarta. 6. nurkhozin, agus, irawan, mohammad isa. mukhlas, imam. 2011. komparasi hasil klasifikasi penyakit diabetes mellitus menggunakan jaringan syaraf tiruan backpropagation dan learning vector quantization, fakultas mipa, universitas negeri yogyakarta. 7. sudibyo, akhmad. 2012. widal test (uji widal). fakultas kedokteran, universitas wijaya kusuma. surabaya. 8. putra, dharma, 2010, pengolahan citra digital, cv andi offset, yogyakarta 9. prasetyo, eko, 2011, pengolahan citra digital dan aplikasinya menggunakan matlab, yogyakarta: andi. isbn : 978-979-29 2703-0 10. kusumadewi, sri. 2004. membangun jaringan syaraf tiruan menggunakan matlab dan excellin., graha ilmu. edisi 1. jogjakarta 11. munir, rinaldi. 2004. pengolahan citra digital dengan pendekatan algoritmik. informatika bandung. 12. sumathi, s. paneerselvam, surekha, 2010. computational intelligence paradigms: theory & applications using matlab. taylor and francis group. llc. 13. sherwood, l. gorbach. dr john g bartlett, m.d., neil r. blacklow, m.d. 2004. infectious diseases, third edition. 60 vol. 1. no. 2 may–august 2010 research report the fluctuation of aedes aegypti in endemic area of dengue hemorrhagic fever in surabaya city, indonesia subagyo yotopranoto1,2, kusmartisnawat1, kris cahyo mulyatno2, and heny arwati1 1 parasitology department, medical faculty 2 entomology lab, institute of tropical disease airlangga university, surabaya abstract this research was aimed to describe the density pattern and the fluctuation of aedes aegypti larval based on the season in dengue haemorrhagic fever (dhf) endemic area in surabaya city. the population of the research was the house of people together with its breeding sites either inside and outside the house. the 55 houses were randomly chosen in 11 rws in nginden subdistrict. the sample of this research was aedes aegypti larva obtained from its breeding sites. the sample sizes were all of the larval in the containers existed in the chosen houses. the samples of the larval were taken and carried for identification and documentation of the species. this research was applied in the same houses in january, march, and may 2008 in rainy season. the results indicated that the highest larval index occurred in january with house index (hi) 76.8%, container index (ci) 40.5%, and breteau index (bi) 137.5%. there was a decrease in march with hi 63.3%, ci 31.3%, and bi 92.7%. it continued to decrease in may with hi 42.6%, ci 21.1%, and bi 57.4%. all of those larval indices in the three observed months showed a high percentage of larval which was much higher than the safe limit or the critical threshold (5%) as defined by who. it implied that people in nginden subdistrict were threaten by the infection of dhf. the three indices achieved the peak in january, which was also the peak of the rainy season. it gradually decreased in march and may as the rainy season decreased its intensity of rain. larval density index (ldi) also showed a high value. there were 173 larval/house in january, 187 larval/house in march, and 84.8 larval/house in may. the containers or breeding sites of aedes were mostly found in january then their existences gradually decreased in march and may. traditional bath tub was the most productive container inside the house, while drum and bucket were the most productive containers outside the house. other containers which contributed abundance of larval were refrigerator, flower pot, well, and pdam meter box. keywords: the fluctuation of aedes aegypti population, larval index, container, season introduction dengue hemorrhagic fever (dhf) has been one of serious health problems in indonesia. this disease was firstly reported in surabaya and jakarta in 1968 (sumarmo, 1987) then it spread to the entire cities and provinces in indonesia (suroso, 1996: suroso and umar, 1999). since the first spreading, surabaya has become an endemic area for dengue fever with several outbreaks which resulted many victims. from 1997 to 2004, there were 123 subdistricts as the endemic areas of dhf. three subdistricts were categorized as sporadic dhf and only seven subdistricts which were free from dhf (health office of surabaya, 2005). nginden subdistrict in sukolilo district was one of subdistrict and district with endemic dengue fever in surabaya. it was reported that there was one case of dhf in nginden subdistrict in 2004. it increased into seven cases in 2005 and nine cases in 2006. moreover, it was reported that from seven cases occurred in 2007, two out of seven patients died (health center of menur, 2007). the process of the spread of dhf is influenced by several factors such as host, agent, and environment. the host factor is a person who suffers from dhf. the agent factor is dengue virus which causes dhf and the environment factor is the surrounding environment which supports the life of mosquitoes. therefore, they remain living and breeding. it is also supported by the season or weather factor. efforts to prevent and eradicate the disease have been done either by the government of surabaya, other related institutions, and the society. one of the ways to break the dengue transmission chain is by eradicating the vectors 61yotopranoto et al.: the fluctuation aedes aegypty in pandemic area called aedes aegypti and ae. albopictus as the mosquito transmitter of dengue fever. various efforts have been performed but the results are not satisfied enough. it is indicated by the data of vector population density of dhf which are still high and occur in every year (yotopranoto et al., 2005; yotopranoto et al., 2007). the seasonal factor is also significant to the bionomics of ae. aegypti such as rain, air temperature, and air moisture. seasonal factor is a supporting factor in the reproduction and the distribution of dengue vector and it is very potential to cause outbreak (prasittisuk and andjapraridze, 1996; gubler, 1998, sukri et al., 2003). in this study, an observation towards the fluctuation of ae. aegypti and environmental factors supporting its existence during the rainy season had been performed in nginden subdistrict, sukolilo district, surabaya, in 2008. sample and method sample sample of this study was larval stage of ae. aegypti taken from breeding sites or containers both inside (indoor) and outside (outdoor) observed houses. types of containers were identified and measured based on their type and location either inside or outside the house. sample determination there were 11 rws in nginden subdistrict, sukolilo district, surabaya with regular cases of dengue fever in the last three years. in every rw, five houses were randomly chosen using cluster random sampling and used rw as the cluster. sampling method in the chosen houses, types and number of containers were identified and measured either from inside (indoor) or outside (outdoor) the houses. all of ae. aegypti larval were taken from the entire containers using a dipper and plastic pipette and counted to count the number of larval. then, larval were taken into a container with 5% formalin solution. each container was labeled then it was taken to entomology laboratory in institute of tropical disease (itd) airlangga university to identify and document the species. time of sampling sampling was done in january, march and may 2008 in rainy season. sampling was done in the same houses in every month of survey. data analysis the data were analyzed through the measurements of house index (hi), container index (ci), breteau index (bi), and larval density index (ldi). house index (hi) = number of ae. aegypti larval in infested houses × 100% number of inspected houses container index (ci) = number of containers with ae. aegypti larval × 100% n u m b e r o f i n s p e c t e d containers breteau index (bi) = number of containers with ae. aegypti larval × 100% number of inspected houses larval density index (ldi)= number of ae. aegypti larval × 100% number of inspected houses result and discussion dengue hemorrhagic fever incidences in nginden subdistrict annually occurred from 2004 to 2006. when this study was performed, there had been eight cases of dhf. it indicated that it continued to occur every year. in this study, 55 houses were randomly chosen. the containers which contained aedes larval were identified. the larval inside the containers were caught, counted, and identified regarding their species and number. the number of containers was used to measure hi, ci, and bi. beside that, the ldi also be measured in order to observe the density of aedes. the results of the measurements were stated in table 1, picture 1, table 2, and picture 2. this study was performed in the selected months in january, march, and may 2008 which rainy season occurred. therefore, the fluctuation of ae. aegypti population could be analyzed based on the time and season. table 1. house index (hi), container index (ci) dan breteau index (bi) aedes in nginden subdistrict, sukolilo district, surabaya 2008. month larval index house index (%) container index (%) breteau index (%) january 76.8 40.5 137.5 march 63.6 31.3 92.7 may 42.6 21.1 57.4 figure 1. house index (hi), container index (hi) and breteau index (bi) aedes di nginden subdistrict, sukolilo district, surabaya 2008. 0 20 40 60 80 100 120 140 160 january march may hi ci bi p e r c e n t (% ) 62 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 60-64 table 2. larval density index (ldi) aedes in nginden subdistrict, sukolilo district, surabaya 2008 month january march may number of larval/ house 173 187.8 84.8 0 20 40 60 80 100 120 140 160 180 200 january march mayn u m b e r o f la r v a l / h o u s e figure 2. larval density index (ldi) aedes in nginden subdistrict, sukolilo district, surabaya 2008 table 1 and figure 1 indicated that hi, ci, and bi in january were in the highest position compared to the other months. it decreased in march and may. ldi in january was lower than march (table 2 and figure 2) but it still showed a high level of larval population because it was the peak of rainy season in surabaya. usually, the peak of rainy season was in january and february. it was indicated through the high value of hi 76.8%. it implied that 76.8% inspected houses contained ae. aegypti. it was supported with the high value of bi 137.5% which meant that there were many containers containing ae. aegypti larval in the houses. rainy season made the empty containers outside the houses filled with water which were usually empty in dry season. they were used as breeding sites for ae. aegypti. moreover, during the rainy season, the air moisture was also high (95%) causing the air so moist and full of vapor. these conditions advantaged the life of mosquitoes because they could live longer in moist air than in dry air. they preferred warm temperature (25–30° c). if mosquitoes have longer time to live, they have more opportunity to breed that may cause the increase of their population. warmer weather also affected dengue virus inside the body of ae. aegypti. it would easily develop, the warmer the weather, the shorter the incubation period. general incubation period of dengue virus type 2 is about 12 days in 30° c, but it becomes 7 days in 32–35° c. the shorten of five days of incubation period of virus inside of mosquito would multiply the potency of transmission up to three times higher than in the normal temperature. it closely related to global warming issue (science daily, 1998; hales et al., 2002; kairi, 2007). in short, warm weather will cause the mosquitoes to be more dangerous and infectious with dengue virus inside them which are ready to infect human. in the following months, march and may, there was decreased hi, bi, and ci compared to january. the reason was because the rainy season had passed its peak causing decreased rain intensity. it was indicated in the less types and number of containers found in march and may as shown in table 3. however, ldi of ae. aegypti larva was higher in march than in january. it implied that the probability of dengue fever distribution was still high in march. on the other hand, people started to realize that the mosquitoes would probably infect them with dengue fever so that they voluntarily cleaned water containers that might contain the larval or pupae of ae. aegypti. table 3. types of breeding places of aedes larval in nginden subdistrict, sukolilo district, surabaya 2008 containers/breeding sites indoor outdoor january march may january march may traditional bath tub 37 31 10 8 2 4 gentong 8 3 1 8 6 5 refrigerator’s reservoir 3 1 bucket 2 1 water closed 1 3 1 aquarium 1 1 well 1 2 4 1 flower pot 3 1 drum 2 used tyre 1 used aquarium 1 pond 1 pdam meter box 1 total 52 40 15 26 14 10 63yotopranoto et al.: the fluctuation aedes aegypty in pandemic area the analysis result in may indicated that hi, bi, and ci decreased compared to january and march (figure 1) because of less intensity of rain. however, the case of dhf still occurred in the months after january, which were in march, april, and may. it indicated that unobserved houses might contain ae. aegypti larval. from these unobserved houses, the virus was started to be distributed by the mosquitoes. there were many types of breeding sites of ae. aegypti in nginden subdistrict located inside or outside the houses. overall, there were more breeding sites inside the houses in every observed month. it had higher potency to be the breeding sites of ae. aegypti. traditional bath tub was the most productive container inside the house because people mostly take a bath twice a day so they need to store the water in it. it was different with people who lived abroad and indonesian people of high community because they mostly used bath tub or shower when the water was automatically flushed or discarded after bathing. gentong or traditional water container made of earthenware was one of potential containers especially for the people who did not use pdam water or had stuck stream of pdam water. they needed to store the water for their daily needs. usually the water container mostly made of plastic but some of them made of earthenware or cement (tsuda et al., 2002). it was almost the same as what happened in laos when people provide water containers in front of their houses to wash their feet before entering the house also in vietnam (huber et al., 2003). this habit also existed in several regions in indonesia such as in bali, lombok, sumbawa, riau, and other places. in the south thailand, the type of container mostly used for the breeding sites was metal box, cement tube, and earthenware container both inside and outside the house, while ae. albopictus mostly bred in tree’s hole, coconut shell, rind, water tube, metal box, and flower pot (kittayapong and strickman, 1993; preechaporn et al., 2007). other breeding sites inside the house in nginden subdistrict were refrigerator, bucket, aquarium without any fish inside, wudlu point (water container for clean up body before pray) and well. the most productive container located outside the house was gentong or traditional water container and followed by traditional bath tub, well, flower pot, water reservoir, used tyre, used aquarium, pond, and pdam meter box. in this study, wells in both inside and outside the house had a significant contribution to the density of ae. aegypti population. the wall characteristic of well which made of cement and brick was preferable for attaching eggs. the mosquitoes also liked to attach their eggs in flower pot because it also made of cement and brick. in fact, there would be no egg in the pond, pool, or well which made of soil or contained soil. it seemed that they did not attach their eggs in the soil or a surface made of soil. hasyimi and soekirno (2004) stated that in their research location in tanjung priok, jakarta, certain subdistrict indicated several types of container of ae. aegypti. the most productive containers were crock and traditional bath tub in other subdistrict. it probably happened because in some areas, it was difficult to get water so they have to store the water to drink and cook, while they used the water from well to take a bath in a public bathroom. the result showed that the number of containers either inside or outside the house decreased month by month. one of the reasons was that people started to feel ashamed when aedes larval or pupae were found in their houses. therefore, the awareness to voluntarily clean their houses increased so there were no more larval found in their houses in the following months. it also might be caused by the decrease rain intensity affecting the decrease of containers especially which were located outside the house in march and may. there are some containers that must be carefully controled because many people don not aware that those places can be so potential for breeding sites such as water storage box of refrigerator. it always contains water but it is rarely replaced or emptied. while in outside the house, flower pot, well, and pdam meter box were the neglected water storages potentially used by the ae. aegypti to breed. the data of various containers were suitable with the study of yotopranoto et al. (2007) in sawahan district, and tambaksari district, surabaya which were endemic areas with the highest cases of dengue fever at that time. gionar et al. (2001) also stated that well was categorized as breeding site in gondokusuman, jogjakarta. eighty nine wells were observed and 35% wells were positive containing ae. aegypti larval in dry season and 51% wells positive in rainy season. most of the wells made of brick and cement. another factor causing the high values of hi, bi, and ci was the low awareness of people towards environment problem and the danger of dengue fever. many people had known about dengue hemorrhagic fever and its distribution including pkk members and leaders in rw but it was lack of implementation or direct action to prevent the disease (yotopranoto et al, 2005). continuous and sustainable efforts are needed to change the people’s behavior so that they are aware of dengue fever and actively prevent and eradicate the vector of the disease, ae. aegypti. conclusion and suggestion conclusion from the results of the research examining the larval of aedes aegypti in nginden subdistrict, sukolilo district, surabaya, during the rainy season in january, march, and may 2008, it can be concluded that: 1. the fluctuation of larval population of ae. aegypti occurs and follows the intensity of rain which falls in every observed month (january, march, and may). 2. larval indices (house index, container index, and breteau index) always exceed the safe level (5%). 3. larval density index of ae. aegypti is always high. 4. the most productive containers of ae. aegypti breeding site is traditional bath tub and followed by traditional water container and other water containers. 64 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 60-64 suggestion 1. it is necessary to examine the fluctuation of larva population of ae. aegypti in nginden subdistrict during the dry season. 2. it is important to observe the existence of ae. aegypti in upper-class houses in nginden subdistrict. 3. it is needed to perform continuous or sustainable counseling to the society to increase their awareness towards dhf and their active participation to prevent dhf especially the vector of the disease. reference dinas kesehatan kota surabaya, 2005. data jumlah inscidence rates and case fatality rates demam berdarah di kotamadya surabaya. gionar yr, rusmiarto s, susapto d, 2001. sumur sebagai habitat yang penting untuk perkembangbiakan nyamuk aedes aegypti l. buletin penelitian kesehatan, 29 (1): 22–31. gubler dj, 1996. world distribution of dengue. dengue bulletin. who the southeast asia and western pacific regions., 20:1–4. hales sn, de wet, maindonald i and woodward a, 2002. potential effect of population and climate changes on global distribution of dengue fever:an empirical model. lancet, 360: 830–834. hasyimi m and soekirno m, 2004. pengamatan tempat perindukan aedes aegypti pada tempat penampungan air rumah tangga pada masyarakat pengguna air olahan. jurnal ekologi kesehatan, 13 (1): 37–42. huber ku, luu ll, tran hh, tran kt, rodhain f and failox db, 2003. aedes aegypti in south vietnam: ecology, genetic structure, vectorial competence and resistance to insecticide. southeast asian j trop med public health, 34 (1): 81–86. kairi a, 2007. dengue fever on the rise of southeast asia; global warming partly to blame. http://www.associatedcontent.com/ article/277063.html kittayapong p and strickman d, 1993. distribution of container inhabiting aedes larvae (diptera: culicidae) at a dengue focus in thailand. j. med entomol, 30 (3): 601–606. preechaporn w, jaroensutasinee m, and jaroensutasinee k, 2007. seasonal prevalence of aedes aegypti and ae. albopictus in three topographical areas of southern thailand. world academy of science, engineering and technology, 36: 23–27. prasisttisuk c and andjapraridze ag, 1996. dengue haemorrhagic fever prevention and control activities in southeast asia region. dengue bulletin. who, the southeast asia and western pasific region, 20: 24–30. puskesmas menur surabaya, 2007. jumlah kasus dbd tahun 2004 sampai 2006 di nginden subdistrict sukolilo district, surabaya. science daily, 1998. global warming would foster spread of dengue fever into some temperate regions. http//www.sciencedaily.com/releases/ 1998/03/980310081157.html. sukri nc, laras k, wandra t, didi s, larasati rp, rachdyatmaka jr, 2003. transmission of epidemic dengue haemorhagic fever in easternmost indonesia. am j trop med hyg, 68 (5): 529–535. sumarmo, 1987. dengue haemorrhagic fever in indonesia. southeast asian j of trop med public health, 18(3): 45–51. sumarmo ps, 1999. masalah demam berdarah dengue di indonesia. demam berdarah dengue, naskah lengkap balai penerbit fkui, jakarta: 1–3. suroso t, 1996. dengue haemorrhagic fever in indonesia: epidemiological trend and development of control strategy. dengue bulletin. who, the southeast asia and western pacific regions, 20: 35–40. suroso t dan umar af, 1999. epidemiologi dan penanggulangan penyakit demam berdarah dengue di indonesia saat ini. demam berdarah dengue, naskah lengkap balai penerbit fkui, jakarta: 14–31. tsuda y, kobayashi j, nambanya s, miyagi i, toma t, phompida s and manivang k, 2002. an ecological survey of dengue vector mosquitoes in central lao pdr. southeast asian j trop med public health, 33 (1): 63–67. yotopranoto s, bendryman ss, rosmanida, salamun, soegijanto s, kawabata m, dachlan yp, 2003. vector situation in dengue haemorrhagic fever endemic areas of surabaya municipality, indonesia. indonesian journal of tropical medicine, 14 (2): 146–154. yotopranoto s, rosmanida, bendryman ss, 2005. peran serta kader pkk dalam pengendalian vektor penyakit dbd di subdistrict petemon, district sawahan, kota surabaya. majalah kedokteran tropis indonesia, 16 (1): 1–10. yotopranoto s, kusmartisnawati, machfudz, arwati h, sofaria r, suwandito, rehatta rm, 2007. vektor demam berdarah dengue di district tambaksari, surabaya. majalah kedokteran tropis indonesia, 18 (3): 62–68. ijtid vol 1 no 2 may-aug 2010.8.pdf ijtid vol 1 no 2 may-aug 2010.9.pdf ijtid vol 1 no 2 may-aug 2010.10.pdf ijtid vol 1 no 2 may-aug 2010.11.pdf ijtid vol 1 no 2 may-aug 2010.12.pdf vol. 9 no. 2 may–august 2021 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 research article increased interleukin-6 as infl ammatory response and magnesium defi ciency in pre-dialysis chronic kidney disease of indonesian children astrid kristina kardani, jusli aras, risky vitria prasetyo, ninik asmaningsih soemyarso*, mohammad sjaifullah noer department of child health, faculty of medicine, universitas airlangga – dr. soetomo general academic hospital, surabaya, indonesia th august 2020; revised: 27th th july 2021 abstract ammatory response in the body. one of the infl ammatory responses is an increase of interleukin-6 (il-6). study to analyze the correlation between mg and il-6 in pre-dialysis ckd children. the methods a cross sectional study was conducted in dr soetomo general academic hospital from november 2018 to april 2019. children with pre-dialyis ckd were included in this study. variables of serum mg level (mg/dl) and infl ammatory marker (il-6) were measured from the blood and analyzed by elisa method. the correlation between mg and il-6 was analyzed with spearman’s correlation test with p <0.05. result a total of 47 children (27 boys vs 20 girls) between 3 months to 18 years old, with pre-dialysis ckd and no history of magnesium supplementation were included. the primary disease that causes of ckd were lupus nephritis (38.3%), nephrotic syndrome (23.4%), urologic disorder (23.4%), tubulopathy (10.6%) and others (4.3%). the average il-6 level was 55.42±43.04 pg/dl and mg level was 2.06±1.54 mg/dl. there were no signifi cant correlation between il-6 level and mg level with staging of ckd and duration of illness (p>0.05), but there was a signifi cant correlation between serum mg level and il-6 level (r=-0.748; p<0.001). magnesium levels have a signifi cant inverse correlation with il-6 levels in pre-dialysis ckd children. the lower the mg levels in the blood, the higher il-6 levels and vice versa. keywords: chronic kidney disease, magnesium, interleukin-6, children, elisa method. abstrak penyakit ginjal kronik (pgk) merupakan masalah kesehatan yang serius pada anak, dengan angka kesakitan dan kematian yang terus meningkat di seluruh dunia. anak dengan ckd cenderung mengalami defi siensi magnesium (mg) yang dapat merangsang respon infl amasi dalam tubuh. salah satu respon infl amasi adalah peningkatan interleukin-6 (il-6). penelitian untuk menganalisis hubungan antara mg dan il-6 pada anak pgk pra-dialisis. metode penelitian cross sectional dilakukan di rumah sakit umum akademik dr soetomo dari november 2018 sampai april 2019. anak-anak dengan ckd pra-dialisis diikutsertakan dalam penelitian ini. variabel kadar mg serum (mg/dl) dan penanda infl amasi (il-6) diukur dari darah dan dianalisis dengan metode elisa. korelasi antara mg dan il-6 dianalisis dengan uji korelasi spearman dengan p<0,05. hasil total 47 anak (27 laki-laki vs 20 perempuan) antara 3 bulan sampai 18 tahun, dengan ckd pra-dialisis dan tidak ada riwayat suplementasi magnesium dimasukkan. penyakit utama penyebab pgk adalah lupus nephritis (38,3%), sindrom nefrotik (23,4%), kelainan urologi (23,4%), tubulopati (10,6%) dan lain-lain (4,3%). rata-rata kadar il-6 adalah 55,42±43,04 pg/dl dan kadar mg adalah 2,06±1,54 mg/dl. tidak terdapat hubungan yang bermakna antara kadar il-6 dan kadar mg dengan stadium pgk dan lama sakit (p>0,05), namun terdapat hubungan yang bermakna antara kadar mg serum dengan kadar il-6 (r=-0,748; p< 0,001). kadar magnesium memiliki korelasi terbalik yang signifi kan dengan kadar il-6 pada anak pgk pra-dialisis. semakin rendah kadar mg dalam darah, semakin tinggi kadar il-6 dan sebaliknya. * corresponding author: niniksoemyarso@yahoo.com received: 27 august 2020; accepted: 8 chronic kidney disease (ckd) is a serious health problem in children, with increasing morbidity and mortality rates throughout the world. children with ckd tend to experience magnesium (mg) defi ciency that can stimulate an infl 95astrid kristina kardani, et al.: increased interleukin-6 as inflammatory response ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 kata kunci: penyakit ginjal kronis, magnesium, interleukin-6, anak, metode elisa how to cite: kardani, ak., aras, j., prasetryo, rv., soemyarso, na., noer, ms. increased interleukin-6 as infl ammatory response and magnesium defi ciency in pre-dialysis chronic kidney disease of indonesian children. indonesian journal of tropical and infectious disease, 9(2), 94–101. introduction chronic kidney disease (ckd) is a serious health problem in adults and children, with increasing morbidity and mortality rates throughout the world1. the number of ckd patients globally in 2019 was 13.4% (11.7 15.1%) of the world’s population2 with the amount in pediatric patients is quite high3. moreover, the number of children with ckd in 2011 was 11-12 children per 1 million in ckd stages 3-5, .8 children per 1 million in ckd stages 3-5 (europe), 5.7 children per 1 million (america), and 38 children per 1 million (middle east and south asia)3. chronic infl ammation that occurs in ckd patients can increase the risk of disease becoming more severe and worsening of glomerular fi ltration rate1. children with ckd often have decreasing magnesium (mg) levels. about 65% of mg in the body is found in bones, 34% in smooth muscle and the remaining 1% is in plasma and interstitial fl uid4. magnesium defi ciency can stimulate an infl ammatory response and may infl uence the defense mechanism of human body5. the infl ammatory response in ckd patients is indicated by high levels of proinfl ammatory cytokines, including interleukin-6 (il-6) that are associated with morbidity and mortality6,7. interleukin 6 is a dissolved il-6 mediator with pleiotropic effect on inflammation, immune response, and hematopoiesis. interleukin 6 is produced quickly and temporarily in response to infection and tissue injury, contributes to host defense through stimulation of acute phase responses, hematopoiesis, and immune reactions8. dr. soetomo general academic hospital, surabaya, indonesia, is the fi rst referral hospital in east java, indonesia. the number of pediatric ckd patients in 2018 was 102 patients. most patients were often being admitted to treatment ward with infl ammatory problems as many as 72.4%. based on the description above and limited studies, the researchers focusing on correlation between mg and il-6 levels in pre-dialysis pediatric ckd patients in dr. soetomo general academic hospital, surabaya, indonesia. methods and materials participants participants in this study were children diagnosed with ckd9. the inclusion criteria were children aged 3 months to 18 years, diagnosed with ckd, in pre-dialysis. children having received mg supplementation and having an infection (fever/ temperature >37.5ºc and high leukocyte levels) were excluded. their parents were given an explanation regarding participant rights and obligations. all parents were also required to fi ll out an informed consent sheet. design an analytic observational study was conducted using cross sectional design. the research was carried out at dr. soetomo general academic hospital, surabaya, indonesia, from november 2018 to april 2019 (fi gure 1). this research had been declared to meet ethical requirements by the ethics commitee dr. soetomo general academic hospital, surabaya, indonesia (0835/kepk/ xii/2018). participants were chosen based on consecutive random sampling. the number of participants in this study were 47 participants. participants were fi rst identifi ed for characteristics and then measured for mg and il-6 levels. the examination of il-6 and mg levels were perform at clinical pathology laboratorium of dr. soetomo general academic hospital. measurement of il-6 levels used elisa test kits-quantikine hs human il-6 immunoassay (elabscience biotechnology co., ltd, wuhan, 96 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 94–101 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 hubei, china), during which participants were taken for venous blood. blood samples were centrifuged at 3000 rpm for 15 minutes. the serum samples were collected and stored at -80°c in the laboratory of dr. soetomo general academic hospital, surabaya, indonesia. the results of il-6 measurement were categorized into 3 groups: high (≥4.5 pg/dl), normal (1.8-2.3 pg/dl), and low (<1.8 pg/dl). magnesium levels were measured using photometry method with exl dimension analyzer (siemens healthcare diagnostics, erlangen, germany). the measurement results were categorized into 3: group high (≥1.6 mg / dl), normal (1.2-1.6 mg / dl), and low (<1.2 mg / dl)4. statistical analysis the results were displayed in figures and tables. the data were analyzed using ibm spss statistics software version 22.0 (ibm corp., armonk, ny, usa). analysis of correlation between mg and il-6 with staging of ckd and duration of illness, between mg and il-6 were carried out using spearmen correlation test. the p<0.05 showed a signifi cant correlation, while p ≥0.05 indicated no signifi cant correlation. figure 1. participant requitment process in this study results characteristics of participants table 1. demographics characteristics of pre-dialysis ckd children characteristics n (%) sex male female 27 (57.45) 20 (42.55) age (years) < 10 years 10-18 years 14 (29.79) 33 (70.21) duration of illness < 1 year 1-5 years >5 years 18 (38.30) 21 (44.68) 8 (17.02) etiology lupus nephritis nephrotic syndrome urological disorders tubulopathies hsp nephritis 18 (38.30) 11 (23.40) 11 (23.40) 5 (10.64) 2 (4.26) ckd stage stage i stage ii stage iii stage iv stage v 16 (34.04) 6 (12.77) 12 (25.53) 7 (14.89) 6 (12.77) mg low normal high 25 (53.19) 16 (34.04) 6 (12.77) il-6 low normal high 4 (8.51) 0 (0.00) 43 (91.49) ckd = chronic kidney disease most children were boys of about 27 (57.45%) children. there were 33 children belonged to age group of 10-18 years (70.21%) (table 1). the average age was 147.81±55.20 months, while mean age of boy and girl were 140.89±60.63 months and 157.15±46.76 months, respectively. there were 21 (44.68%) children suff ered from ckd for 1-5 years in, followed by children who experienced illness <1 year of about 18 (38.30%) children (table 1). the average time of experiencing ckd was 30.64±30.97 months. the average time of male and female participants duration of illness was 26.44±20.67 months and 36.30±40.97 months, respectively. the most common underlying disease in this study was lupus nephritis in 18 (38.30%) 97astrid kristina kardani, et al.: increased interleukin-6 as inflammatory response ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 children, followed by nephrotic syndrome and urological abnormalities, each occured in 11 (23.40%) children (table 1). the correlation between magnesium and il-6 levels with staging of ckd the mg level in ckd stage 1 was 1.75±0,4 mg/dl, ckd stage 2 was 1.71±0,4 mg/dl, ckd stage 3 was 2.79±2.93 mg/dl, ckd stage 4 was 1.7±0.5 mg/dl and ckd stage 5 was 1.92±0,7 mg/dl (fi gure 2a). the il-6 level in ckd stage 1 was 54±41.3 pg/dl, ckd stage 2 was 38.3±33.7 pg/dl, ckd stage 3 was 48.8±44.8 pg/dl, ckd stage 4 was 64.7±31.7 pg/dl and ckd stage 5 was 78.8±64.0 pg/dl (p=0.994, figure 2b). there were no signifi cant correlation between magnesium and il-6 levels with staging of ckd (figure 2a and 2b). figure 2a. magnesium level (mg/dl) based on staging of ck figure 2b. il-6 level (pg/dl) based on staging of ckd the average fi gure ff there were no signifi cant correlation between magnesium and il-6 levels with staging of ckd (figure 2a and 2b). the correlation between magnesium an il-6 levels with duration of illness the average mg level in <1 year duration of ckd was 1,8±0,53 mg/dl, 1-5 years was 2,15±2,06 mg/dl, and > 5 years was 1,8±0,34 mg/dl (figure 2c). the average il-6 level in < 1 year duration of ckd was 53,6±46,3 pg/dl, 1-5 years was 59,6±41,6 pg/dl, and > 5 years was 44,7±43,3 pg/dl (p=0.883, figure 2d). there were no signifi cant correlation between magnesium and il-6 levels with the duration of illness (figure 2c and 2d). figure 2c. magnesium level (mg/dl) based on duration of ckd figure 2d. il-6 level (pg/dl) based on duration of ckd correlation between magnesium and il-6 levels the average il-6 level was 55.42±43.04 pg/dl, that was categorized into high il-6 level. the average il-6 level in boys and girls were 63.89±46.82 pg/ dl and 43.98±35.30 pg/dl, respectively. most children had high il-6 levels in 43 (91.49%) children (table 1). the average mg level in boys and girls were 2.17±2.01 mg/dl and 1.90±0.46 mg/dl, respectively. most participants had low mg level in 98 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 94–101 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 25 participants (53.19%), followed by normal mg in 16 participants (34.04%; table 1). the distribution of data was abnormal. the results of statistical analysis using spearman correlation test showed a signifi cant relationship between average mg levels and average il-6 levels (p<0.001), with correlation coeffi cient value of -0.748. this indicated a strong negative correlation between average mg and average il-6 levels in the blood. the lower mg levels will further increase il-6 levels as a proinfl ammatory mediator and vice versa (figure 3a). there were no signifi cant correlation between average of mg (p=0.994) and il-6 levels (p=0,404) with staging of ckd (figure 3b and 3c), between average of mg (p=0,883) and il-6 (p=0,969) with duration of illness (figure 3d-e). figure 3a. correlation between mg (mg/dl) and il-6 (pg/dl) in pre-dialysis ckd children (r = -784; p = 0.001) figure 3b. correlation between mg (mg/dl) and staging of ckd (r = 0,01; p =0.994) figure 3c. correlation between il-6 (pg/dl) and staging of ckd (r = 0.125; p =0.404) figure 3d. correlation between magnesium (mg/dl) and duration of ckd (r = -0.22; p =0.883) figure 3e. correlation between il-6 (pg/dl) and duration of ckd (r = -0.006; p =0.969) 99astrid kristina kardani, et al.: increased interleukin-6 as inflammatory response ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 discussion this study found that most participants had low mg level <1,8 mg/dl. similar findings were obtained in swaminathan’s study [10], in which ckd patients with hypomagnesemia were higher than normal or high mg levels. in stage 1-3 ckd patients, there is an increase in fractional mg excretion as compensation for decreased or loss of kidney function to maintain normal serum magnesium levels in the blood. in ckd with glomerular filtration rate (gfr) <10ml/min/1.73m2, this compensatory mechanism is less effective (insufficient) to prevent an increase mg levels in the blood5. one of the causes of hypomagnesemia in this study might be caused by calcineurin inhibitors (cyclosporine) administration as a sparing agent for steroid therapy in children with resistant steroid nephrotic syndrome. visscer et al.’s study5 stated that hypomagnesemia can be caused by drugs such as thiazide group, proton pump inhibitors, antibiotics, aminoglycoside groups and calcineurin inhibitors. hypomagnesemia in ckd patients can also be caused by impaired intestinal absorption of mg due to vitamin d defi ciency, therefore routine vitamin d testing is needed in children with ckd11,12. in addition, hypomagnesemia is one of the early predictors of the risk of cardiovascular and cardiovascular disease in ckd patients5. the results of this study indicated that most participants experienced increasing il-6 levels as one of the proinfl ammatory cytokines. increased levels of il-6 in ckd patients are most often caused by increased oxidative stress activity, chronic infl ammation and fl uid overload13. the decreased clearance of il-6 results from impaired renal function14. their study stated that increasing il-6 levels in ckd patients were associated with the severity of metabolic acidosis and serum bicarbonate levels`15. in addition, high levels of il-6 are also caused by the activity of lupus nephritis and nephrotic syndrome14,16. patients with nephrotic syndrome also have increased il-6 levels. a study conducted by subandiyah et al. found a signifi cant increase in il-6 levels in patients with steroid-resistant nephrotic syndrome compared to steroid sensitive nephrotic syndrome17. jafar et al.’s study obtained similar fi ndings, stating that increasing il-6 levels were found in patients with idiopathic nephrotic syndrome that was related to the therapeutic response18. interleukin-6 expression in the urine and renal tissues was correlated with proteinuria in minimal changes disease rats 19. cunningham et al. found that the quantitative excretion of magnesium tends to decrease in ckd stage 4 dan 5 and cannot be compensated by an increased fractional excretion of magnesium12. in this study, there was no correlation between average magnesium level and staging of ckd, it might be due to several factors such as less of magnesium intake, calcineurin inhibitors (cyclosporine) administration and malabsorbtion in majority of children. study by magno et al. found the correlation of il-6 dependent on the type of kidney disease and overlapping conditions such as hypertension and diabetes, but not by duration and staging of ckd. the measurement of il-6 independently associated with mortality in patient with chronic kidney disease20,21,2220,21,22. it also can be used to explain that there were no correlation between average level of magnesium and il-6 with duration of illness. statistical analysis showed a significant relationship between decreased mg levels and increased il-6 levels. lower mg level will cause higher il-6 levels, which indicates a more severe inflammatory process. measurement of mg levels is aff ordable and can be used as an early predictor the severity of infl ammatory process in pre-dialysis children with ckd. magnesium is an important element that the body needs as a cofactor for >300 enzymatic reactions. magnesium is needed for biochemical functions of various body metabolism pathways. enzyme systems that involve magnesium include protein synthesis, muscle contraction, nerve function, controlling blood sugar, hormone receptor binding, regulating blood pressure, stimulating cardiovascular work, transmembrane ion fl ux, and connecting calcium. in addition, mg has an important role in energy production in the body such as having a crucial role in the atp metabolism (adenylate cyclase), oxidative phosphorylation, and glycolysis. 100 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 94–101 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 another function of mg is to play a role in the process of rna and dna synthesis23,24,253,24,25. whereas, il-6 is a dissolved il-6 mediator with pleiotropic effect on inflammation, immune response, and hematopoiesis8. kidneys have an important role in maintaining levels or concentrations of magnesium in the blood. the ability to regulate magnesium level will decrease along with decrease in kidney function. in addition, there is a decreased ability to absorb mg in the intestine in ckd children when compared to normal children. the use of drugs as proton pump inhibitors (ppi) in ckd also reduces the ability of intestine to absorb magnesium. in patients who have undergone both hemodialysis and peritoneal dialysis, hypomagnesemia often results from the use of low-magnesium dialysate (low-mg dialysate) fl uids5. magnesium levels have a signifi cant inverse correlation with il-6 levels in pre-dialysis ckd children. hypomagnesemia is associated with increased levels of il-6 proinfl ammatory cytokines. further research is needed to examine the role of magnesium with cardiovascular disease in children with ckd who do not yet have symptoms and signs. this research was presented as poster in international pediatric nephrology association congress on 17-21 october 2019 in venice, italy. astrid kristina kardani, jusli aras, risky vitria prasetyo, ninik asmaningsih soemyarso, and mohammad sjaifullah noer declare that they have no confl ict of interest this publication. 1. kaspar, c. d. w., bholah, r., & bunchman, t. e . a r e v i e w o f p e d i a t r i c c h r o n i c k i d n e y disease. blood purifi cation, 2016: 41(1-3), 211-217. .doi:10.1159/000441737 2. lv, j. c., & zhang, l. x. (2019). prevalence and disease burden of chronic kidney disease. renal fibrosis: mechanisms and therapies, 3-15. doi:10.1007/978981-13-8871-2_1 3. harambat j, van stralen kj, kim jj, tizard ej (2012) epidemiology of chronic kidney disease in children. pediatric nephrology. 2012; 27 (3):363-373. doi:10.1007/s00467-011-1939-1 4. floege j. magnesium in ckd: more than a calcifi cation inhibitor? j nephrol, 2015; 28 (3):269-277. doi:10.1007/ s40620-014-0140-6 5. van de wal-visscher er, kooman jp, van der sande fm (2018) magnesium in chronic kidney disease: should we care? blood purifi cation 45 (1-3):173-178. doi:10.1159/000485212 6. hénaut l, massy za. new insights into the key role of interleukin 6 in vascular calcifi cation of chronic kidney disease. nephrology, dialysis, transplantation : offi cial publication of the european dialysis and transplant association european renal association, 2018; 33 (4):543-548. doi:10.1093/ndt/gfx379 7. ferrè s, li x, adams-huet b, maalouf nm, sakhaee k, toto rd, moe ow, neyra ja. association of serum magnesium with all-cause mortality in patients with and without chronic kidney disease in the dallas heart study. nephrology, dialysis, transplantation : offi cial publication of the european dialysis and transplant association european renal association, 2018; 33 (8):1389-1396. doi:10.1093/ndt/gfx275 8. tanaka t, narazaki m, kishimoto t (2014) il-6 in infl ammation, immunity, and disease. cold spring harb perspect biol 6 (10):a016295-a016295. doi:10.1101/ cshperspect.a016295 9. kidney disease: improving global outcomes ckdmbduwg . kdigo 2017 clinical practice guideline update for the diagnosis, evaluation, prevention, and treatment of chronic kidney disease-mineral and bone disorder (ckd-mbd). kidney int suppl, 2011; 7 (1):1-59. doi:10.1016/j. kisu.2017.04.001 10. swaminathan r. magnesium metabolism and its disorders. clin biochem rev 24, 2003; (2):47-66 11. sakaguchi y, hamano t, isaka y. magnesium and progression of chronic kidney disease: benefits beyond cardiovascular protection?. advances in chronic kidney disease 25, 2018; (3):274-280. doi:10.1053/j.ackd.2017.11.001 12. cunningham j, rodríguez m, messa p. magnesium in chronic kidney disease stages 3 and 4 and in dialysis patients. clin kidney j. 2012; 5 (suppl 1):i39-i51. doi:10.1093/ndtplus/sfr166 13. su h, lei ct, zhang c. interleukin-6 signaling pathway and its role in kidney disease: an update. frontiers in immunology, 2017; 8:405. doi:10.3389/ fi mmu.2017.00405 acknowledgement conclusion confl ict of interest references 101astrid kristina kardani, et al.: increased interleukin-6 as inflammatory response ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 14. jones, s. a., fraser, d. j., fielding, c. a., & jones, g. w. interleukin-6 in renal disease and therapy. nephrology dialysis transplantation, 30(4), 564-574.2015; 30(4):564-574. doi:10.1093/ndt/gfu233 15. zahed ns, chehrazi s. the evaluation of the relationship between serum levels of interleukin-6 and interleukin10 and metabolic acidosis in hemodialysis patients. saudi journal of kidney diseases and transplantation : an offi cial publication of the saudi center for organ transplantation, saudi arabia, 2017; 28 (1):23-29. doi:10.4103/1319-2442.198106 16. cavalcanti a, santos r, mesquita z, duarte al, lucena-silva n. cytokine profi le in childhood-onset systemic lupus erythematosus: a cross-sectional and longitudinal study. brazilian journal of medical and biological research = revista brasileira de pesquisas medicas e biologicas, 2017; 50(4):e5738. doi:10.1590/1414-431x20175738 17. subandiyah k, ghofar hf, fitri le. diff erence of vitamin d and interleukin-6 levels in children with steroid-resistant, steroid-sensitive and idiopathic nephrotic syndrome. journal of tropical life science, 2019; 9 (2):179-187 18. jafar t, agrawal s, mahdi aa, sharma rk, awasthi s, agarwal gg. cytokine gene polymorphism in idiopathic nephrotic syndrome children. indian j clin biochem, 2011; 26 (3):296-302. doi:10.1007/s12291011-0126-2 19. kim sh, park sj, han kh, saleem ma, lim bj, shin ji. (2016) pathogenesis of minimal change nephrotic syndrome: an immunological concept. clin exp pediatr, 2016; 59(5): 205-211. doi:10.3345/ kjp.2016.59.5.205 20. magno al, herat ly, carnagarin r, schlaich mp, matthews vb. current knowledge of il-6 cytokine family members in acute and chronic kidney disease. biomedicines, 2019; (7):1-15. doi :10.3390/2019/7010019. 21. barreto dv, barreto fc, liabeuf s, temmar m, lemke hd, choukron g, massy za. (2010) plasma interleukin-6 is independently associated with mortality in both hemodialysis and pre-dialysis patients with chronic kidney disease. kidney international. 2010; 77:550-556.doi:10.1038/ki.2009.503 22. fasset rg, venuthurupalli sk, gobe gc, coombes js, cooper ma, hoy we. (2011) biomarkers in chronic kidney disease : a review. kidney international, 2011; 80:806-821. doi:10.1038/ki.2011.198 23. schwalfenberg gk, genuis sj. the importance of magnesium in clinical healthcare. scientifi ca (cairo) 2017; 4179326-4179326. doi:10.1155/2017/4179326 24. patel h, redkar v, kulkarni a, kale a. (2018) a study of serum magnesium level in patients with chronic renal failure at tertiary care hospital. international journal of contemporary medical research. 2018; 5(10);5-8. doi:10.21276/ijcmr.2018.5.10.21 25. bressendorf i, hansen d, schou m, silver b, pasch a, bouchelouche p, pedersen l, rasmussen lm, brandi l. (2017) oral magnesium supplementation in chronic kidney disease stages 3 and 4: effi cacy, safety, and eff ect on serum calcifi cation propensity, a prospective randomized double blinded placebo controlled clinical trial. kidney int rep, 2017; 2: 380-389. doi:10.1016/j. ekir.2016.12.008 ijtid vol 3 no 2 april juni 2012.indd 61 vol. 3. no. 2 april–juni 2012 research report hepatitis b serology profiles on children aged 1–13 years old in sumenep, madura edward m. putera1, dian marcia1, itja firdarini1, mochamad amin3,4, juniastuti2,3,4, priyo b. purwono2,4, takako utsumi3,5, maria i. lusida2,3,4* 1 dr. h. moh. anwar general hospital, sumenep, madura 2 department of microbiology, medical faculty, universitas airlangga, surabaya 3 indonesia-japan collaborative research center for emerging and re-emerging infectious diseases 4 institute of tropical disease, universitas airlangga, surabaya, indonesia 5 center for infectious diseases, kobe university graduate school of medicine, kobe, japan abstract background: hepatitis b virus (hbv) which was acquired during perinatal or childhood would promote hepatocellular carcinoma with even higher percentage than that which was acquired during adult age. that is why hbv represents a serious public health threat for children. hbv vaccination has been integrated into national expanded programme on immunization (epi) since 1997. the aim of they study is to investigate the prevalence of hbv among children who were born after 1997 in sumenep. material and methods: a total of 102 children who were born after 1997 were enrolled in this study. all children were admitted in the emergency room and pediatric ward of dr. h. moh anwar general hospital for some reasons. written informed consents were obtained from parents/ guardians of all the children. study protocol was reviewed and approved by the ethics committees. all of these cases were examined for hepatitis b surface antigen (hbsag), antibody to hbsag (anti-hbs), and antibody to hepatitis b core antigen (anti-hbc). result and discussion: overall, 6 (5.88%) of 102 samples were positive for hbsag, 51 (50.00%) of 102 samples were positive for anti-hbs, and 49 (48.04%) of 102 samples were positive for anti-hbc. all the children were born after 1997. conclusion: hbsag rate is still high even after universal vaccination program, acquired protective antibodies against hepatitis b surface antigen were sufficient, but there is a suspicion for occult hepatitis b infections (obi). a further study to confirm obi is needed. keywords: hbv, hbsag, anti-hbs, anti-hbc, immunization abstrak latar belakang: hepatitis b virus (hbv) yang diperoleh selama perinatal atau masa kanak-kanak akan menyebabkan karsinoma hepatoseluler dengan persentase lebih tinggi daripada apa yang telah diperoleh selama usia dewasa. itulah sebabnya hbv merupakan ancaman kesehatan masyarakat yang serius bagi anak-anak. hbv vaksinasi telah diintegrasikan ke dalam program imunisasi nasional yang telah diperluas sejak tahun 1997. tujuan: untuk menyelidiki prevalensi hbv antara anak-anak yang lahir setelah tahun 1997 di sumenep. bahan dan metode: total 102 anak yang lahir setelah tahun 1997 yang terdaftar dalam penelitian ini. semua anak-anak dirawat di ruang gawat darurat dan departemenen anak dari rsud dr. h. moh anwar untuk beberapa alasan. informed consent tertulis diperoleh dari orang tua/wali dari semua anak. studi protokol ditinjau dan disetujui oleh komite etika. semua kasus ini diperiksa untuk antigen permukaan hepatitis b (hbsag), antibodi terhadap hbsag (anti-hbs), dan antibodi terhadap antigen inti hepatitis b (anti-hbc). hasil: secara keseluruhan, 6 (5,88%) dari 102 sampel yang positif untuk hbsag, 51 (50,00%) dari 102 sampel yang positif untuk anti-hbs, dan 49 (48.04%) dari 102 sampel yang positif untuk anti-hbc. semua anak lahir setelah 1997. kesimpulan: tingkat hbsag masih tinggi bahkan setelah program vaksinasi universal, diperoleh antibodi pelindung terhadap antigen permukaan hepatitis b sudah cukup, tapi ada kecurigaan untuk okultisme infeksi hepatitis b (obi). sebuah studi lebih lanjut untuk mengkonfirmasi obi diperlukan. kata kunci: hbv, hbsag, anti-hbs, anti-hbc, imunisasi. * corresponding author. mailing address: maria inge lusida, institute of tropical disease, universitas airlangga, campus c mulyorejo, surabaya 60115, indonesia. e-mail: ingelusida@yahoo.com 62 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 61−64 introduction hepatitis b is an infectious disease caused by hepatitis b virus (hbv) that affects more than 400 million people worldwide, and 1.3 million die of decompensated cirrhosis and/or hepatocellular carcinoma (hcc) annually.1 hbv variants are currently classified into the human genotypes a to h.2 up to 90% of infected newborns develop chronic hbv infection, which gives a higher risk of hcc later in their adulthood, while 24% of adults chronically infected during childhood had either hcc or cirrhosis.2 a safe and effective vaccine against hepatitis b has been available since 1982. the introduction of a childhood immunization program in many countries has dramatically reduced the carrier rate of hbv and significantly decreased the incidence of hcc. in indonesia, hbv vaccination had been introduced since 1987, and has been integrated into national expanded programme on immunization (epi) since 1997. who recommends hbv vaccination to all infants, first at birth, then followed by two subsequent vaccinations with each minimum interval of 1 and 2 months respectively.1 however, the current serologic status of hbv in children has not been fully investigated in indonesia. the aim of this study was to investigate the prevalence of hbv among the children who were born after the national immunization program in sumenep, an area in east java, indonesia, which was with medium-to-high endemicity for hbv. materials and methods study subjects three ml of blood samples were taken from all the patients aged 1–13 who admitted in the emergency room (igd) and pediatric ward (zaal anak) of dr. h. moh anwar general hospital for some reasons. a hundred and two samples were collected in this study. serum samples were obtained during january-march 2012 and were stored at 20° c until further usage. written informed consents were obtained from parents/guardians of all the children. no individual hepatitis b vaccination records remained. the study protocol was reviewed and approved by the ethics committees of dr. h. moh. anwar general hospital. serological markers of hbv infection all refrigerated samples were tested for hbsag with enzyme-linked immunosorbent assay (elisa) (hepalisa hbsag) and for anti-hbs by enzyme-linked immunosorbent assay (elisa) (zhongsan anti-hbs elisa). in order to differentiate vaccine-induced antibody from naturally acquired antibody (and to identify the suspects of occult hbv infections), the prevalence of antibody to hepatitis b core antigen (anti-hbc) was assessed by enzyme-linked immunosorbent assay (elisa) (hepalisa anti hbc). results and discussion a total of 102 children were screened for serological markers of hbv infection. overall, positivity rates for hbsag and anti-hbs were 5.88% (6 out of 102) and 50.00% (51 out of 102), respectively, with the mean age of 5.76 years old. all the children (1–13 y.o.) were born after the introduction of the universal vaccination program. antihbc rates were 48.04% (49 out of 102). of 51 anti-hbs positive children, 23 were negative for anti-hbc. all six hbsag-positive children were negative for anti-hbs. similar study in borno state, nigeria showed that overall seroprevalence of hbsag among primary school pupils was 44.7%,3 while in those 439 children in moldova (mean age, 5 years), the prevalence of hbsag and antihbc were 6.8% and 17.1%, respectively.4 successful vaccination programs had been shown by several countries which previously belonged to high prevalence hbv countries, such as in a study in karachi, pakistan, among sixty five (1.8%) out of 3533 children (mean age 10±4 years old) were positive for hbsag.5 in taiwan, after 25 years of nationwide hbv universal vaccination program for infants, hbsag sero-prevalence sharply declined from 9.8% to 0.6% with hbv vaccination coverage as high as 97%.6 this study was unable to assess the actual coverage rate because no individual vaccination records remained. for this reason, efficacy of vaccination was not evaluated in this study. however, this study did show that acquired protective antibody against hbv infection was sufficient among children born after the universal vaccination program. the hbsag prevalence of 5.88% in this study was still considered high. a high coverage rate for hbv vacination is crucial for decreasing the prevalence of hbv infection. program for appropriate technology in health, a non governmental organization in united states of america, (path) stated that birth dose within seven days of birth was 65%, even though hbv 3 coverage was 80-85%.7 some of the first dose of hb vacccine in indonesia has been administered along with the first dose of dpt, which was generally 6 weeks to 2 months of age. delay in giving the first dose of hb vaccine would not prevent perinatal transmission.8 path worked with the indonesian ministry of health since the beginning of 1987 to launch a model immunization program on the island of lombok. the innovative program introduced a comprehensive system for delivering a vital birth dose of the vaccine and established a system for tracking and monitoring pregnancies and births. on october 2002, the government began an effort to ensure that every newborn is administered hepatitis b vaccines with prefilled, single use syringe and needle (uniject®) during the first seven days of life.7 hbsag rates on children who were born before 2002 and after 2002 were 0% (0 out of 17) and 7.05% (6 out of 83) respectively. this concludes that even after path uniject® programs in 2002, there had not been any effect in sumenep. 63putera, et al.: hepatitis b serology profiles on children aged 1–13 years old on the other hand, there was 0% hbsag rate in children aged 10–13 y.o., means before path uniject® program was promoted? while it was 7.05% in those born after. anti-hbc rate in previous groups was as high as 47.06%. this means that they had ever been infected before. our study showed that hbsag rate in children sumenep was still considered high. hepatitis b immunization coverage of 18.1% in sumenep, data from national basic references of ministry of health 2007, could be one of the factors which supported this fact.9 path’s uniject® program was one of the solutions to increase the hepatitis b immunization coverage of birth dose, but it had no significant impact in our study in sumenep. some other factors which might have played a role in this result should be searched and overcomed. the acip, the american college of obstetrics and gynecology (acog), the american academy of family practice (aafp), and the american academy of pediatrics (aap) recommend that all pregnant women receive prenatal testing for hepatitis b during each pregnancy by screening serum for the presence of hbsag, regardless of risk factors or immunization history.10 this routine hbv serological profile screening on pregnant women should be the target of the ministry of health of indonesia in the near future. on pregnant women with positive hbsag, hbv vaccination and hbig (0.5 ml) should be administered on their babies within 12 hours after birth.11 these efforts will lead to a greater control of hbv infection, and furthermore, liver diseases caused by hbv infection would be better controlled.12 the remaining challenges will be to minimize the rate of vaccine failure and to deal with potential vaccinerelated events, such as the emergence of escape surface mutants.13 further studies with larger samples in the future will accomodate better reflections of hbv immunology profile in children. hbv dna detection among those with hbsag negative, anti hbc positive, and or anti hbs positive or negative should be tested in order to detect occult hbv infections. in conclusion, hbsag rate among children born after the hepatitis b universal vaccination program is still high in sumenep, acquired protective antibodies against hbv infection were sufficient, and suspects of occult hepatitis b infections were found. continuation in path’s uniject® program, implementation of immunization programs, and routine hbv serological profile screening on pregnant women should proceed to eradicate hbv infection. some other aspects which play roles in the high hbsag rates should be explored, including molecular studies. table 1. seroprevalence of hepatitis b surface antigen (hbsag), anti-hbs, and anti-hbc among study population no. no. positive % hbsag 102 6 5.88 anti-hbs 102 51 50.00 anti-hbc 102 49 48.04 table 2. comparison of prevalence of hepatitis b markers in children born before and after path’s uniject® programs. age (years) hbsag anti-hbs anti-hbc no. pos. % no. pos. % no. pos. % 1–9 85 6 7.05 85 41 48.23 85 41 48.23 10–13 17 0 0 17 10 58.82 17 8 47.06 acknowledgments we are grateful to the japan initiative for global research network on infectious diseases (j-grid), the ministry of education, culture, sports, science and technology (mext) japan, head of badan kesatuan bangsa, politik, dan perlindungan masyarakat kabupaten sumenep (kesbanglinmas), director of moh. anwar general hospital, head of ethic committee of moh. anwar general hospital, head of information and evaluation department of moh. anwar general hospital, head of medicine department of moh. anwar general hospital, head of emergency room of moh. anwar general hospital, nurses of emergency room (igd) and pediatric ward (zaal anak) of moh. anwar general hospital, and medical analysts of laboratory moh. anwar general hospital for their great help and cooperation. references 1. wittet s (2001). hepatitis b vaccine introduction: lessons learned in advocacy, communication, and training. children’s vaccine program; 1–3. 2. simmonds p, midgley s (2005). recombinant in the genesis and evolution of hepatitis b virus genotypes. journal of virology; 79: 467–76. 3. bukbuk d, bassi a (2005). sero-prevalence of hepatitis b surface antigen among primary school pupils in rural hawal valley, borno state, nigeria. journal of community medicine and primary health care. june; 17(1): 20–3. 4. drobeniuc j, hutin y (1999). prevalence of hepatitis b, d, and c virus infections among children and pregnant women in moldova: additional evidence supporting the need for routine hepatitis b vaccination of infants. epidemiology of infectious disease june; 123(3): 463–7. 5. jafri w, yakoob j (2006). hepatitis b and c: prevalence and risk factors associated with seropositivity among children in karachi, pakistan. bmc infectious disease; 6: 101. 6. hsuan n (2009). hbv now in asia. hepatitis b vaccination in children. japan society of hepatology. japan; 42. 7. nelson c, widjaya a (2002). using uniject™ to increase the safety and effectiveness of hepatitis b immunization. program for appropriate technology in health (path) and path’s children’s vaccine program; 1–8. 8. levin c, moniaga v (2005). the cost of home delivery of a birth dose of hepatitis b vaccine in a prefilled syringe in indonesia. bulletin of the world health organization june, 83(6); 456–61. 9. soendoro t (2007). laporan riset kesehatan dasar tahun 2007 provinsi jawa timur. badan penelitian dan pengembangan kesehatan departemen kesehatan ri jakarta; 61–6. 10. mast ee, margolis hs, fiore ae, et al (2005). a comprehensive immunization strategy to eliminate transmission of hepatitis b virus infection in the united states: recommendations of the advisory committee on immunization practices (acip) part 1: immunization 64 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 61−64 of infants, children, and adolescents. mmwr. 2005; 54(rr-16): 1–31. 11. ranuh i, suyitno h (2011). pedoman imunisasi di indonesia. hepatitis b; viii.1; 256–63. 12. chang mh, chen cj, et al. 1997. universal hepatitis b vaccination in taiwan and the incidence of hepatocellular carcinoma in children. taiwan childhood hepatoma study group. new england journal of medicine; 336: 1855–9. 13. hsu y, chang h, et al. 1999. changes of hepatitis b surface antigen variants in carrier children before and after universal vaccination in taiwan. hepatology; 30: 1312–7. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 114 vol. 1. no. 3 september–december 2010 the clinical profiles of avian influenza in endemic and non-endemic regions in indonesia. hospital-based studies and its implication on clinical management in the future muhammad jusuf wibisono, resti yudhawati pulmonary department medical faculty of airlangga university – dr. soetomo teaching hospital surabaya indonesia abstract indonesia is a greatest burden country of h5n1 avian influenza (ai) virus infection in the world, since first outbreak in central java 2005 until august 2010 there was 168 confirmed cases and 138 dead cases. the incidence increasing rapidly in widespread area endemic in java, sumatera, bali and sulawesi, and sporadic outbreaks in other areas. the world health organization stated that ai still became a treat in the next pandemic. h5n1 ai virus infection spreads in almost all provinces, but its endemic in jakarta, tangerang and banten and in other area such surabaya, bali were sporadic outbreaks. there are 27 confirmed h5n1 ai infection cases in jakarta from 296 suspected cases, while in surabaya only 5 confirmed h5n1 ai infection cases from 12 suspected cases. the age of patient mean with h5n1 ai infection was 16.9 ± 11.6 yo in jakarta and 24 ± 8.51 yo in surabaya. there was no difference between male and female. mortality rate was 77.7% in jakarta and 60% in surabaya. a large number of case has indirect contact history, predominantly by visiting market or areas where outbreaks of poultry disease. the clinical feature h5n1 ai virus infection could manifest as mild until severe pneumonia that often progress rapidly to ards. in jakarta, 74% case showed abnormality chest radiography as bilateral pneumonia, while in surabaya showed lobar pneumonia and bilateral pneumonia. management patient of h5n1 ai infection is supportive therapy and antiviral, whereas a large number of cases needed mechanical ventilator support. key words: clinical profile, avian influenza, h5n1, epidemiology avian background highly pathogenic avian h5n1 influenza viruses (hpai) now appear to be endemic among bird and poultry populations in eurasia.[1,2] sporadic transmission to humans raises concern that the h5n1 virus may mutate or combine with genetic material from coinfecting human influenza viruses to generate a novel strain capable of sustained human-to-human transmission with pandemic potential.[2] the world health organization has described the threat from h5n1 as a "public health crisis", and declared that the world is as close as ever to the next pandemic.[3] avian influenza h5n1 — highly pathogenic avian h5n1 influenza viruses are endemic among bird and poultry populations in asian countries. the first association of avian influenza h5n1 with clinical respiratory disease were occurred in hong kong in 1997, when 18 human cases were occurred during a poultry outbreak of highly pathogenic h5n1 influenza in live-bird markets. this outbreak was associated with a high mortality rate (33 percent), a high incidence of pneumonia (61 percent), and a high rate of intensive care (51 percent).[3,4] in indonesia, the first human case of h5n1 avian influenza (ai) virus infection were reported in july 2005. on 19 september 2005, the ministry of health of indonesia confirmed an established outbreak of ai in humans in indonesia. this highly fatal infection has occurred across indonesia with a fatality rate of around 80%. until august 2010, there are 168 human cases h5n1 ai virus infection in indonesia with mortality 136 cases.[5] h5n1 hpai spreads endemic in java, sumatera, bali and sulawesi, and sporadic outbreaks in other areas. h5n1 hpai prevalence by village varies widely. only literature review 115wibisono et al.: the clinical profiles of avian influenza two of indonesia�s 33 provinces have never reported the occurrence of h5n1 hpai.[6] participatory disease surveillance and response (pdsr) is program that targets village poultry production systems (mainly backyard) and reports evidence of virus circulation in the village. during march 2010, pdsr officers visited 1.984 villages, of which 137 (6.9%) were infected (98 were newly found, while the remaining 39 carried over the infection status from the previous month). this infection rate was lower than the february 2010 infection rate of 16.6%, which was expected as indonesia emerged from the usual wet season peak. during the previous 12 months, pdsr officers visited 20 117 villages (30.0%) in the 349 districts under pdsr surveillance.[5] endemic and non-endemic region indonesia is consisting a thousand islands, the highest population density is in a java island. since first outbreak in 2005, h5n1 hpai spreads endemic in java and sporadic outbreaks in other area. the most h5n1 hpai human cases were found in dki jakarta, banten, west java whereas these areas were endemic region. while other areas as east java (surabaya), sumatera, bali and others were non-endemic region. pathogenesis of hpai influenza viruses are spherically or longitudinally shaped enveloped particles with an up to eight-fold segmented, single-stranded rna genome of negative polarity. influenza viruses hold generic status in the orthomyxoviridae family and were classified into types a, b or c based on antigenic differences of their nucleoand matrix proteins. avian influenza viruses (aiv) belong to type a.[1,8] influenza a and b viruses have two major antigenic surface glycoproteins embedded into the membrane, the hemagglutinin (ha) and neuraminidase (na) that induce antibody responses in humans. influenza virus strains were classified by their core proteins (ie, a, b, or c), species of origin (eg, avian, swine), geographic site of isolation, serial number, and for influenza a, by subtypes of ha and na.[1,4] influenza a is responsible for frequent, usually annual outbreaks or epidemics of varying intensity, and occasional pandemics; while influenza b causes outbreaks every two to four years. although 16 ha (h1-h16) and nine na (n1n9) virus subtypes has occured in their natural reservoir of aquatic birds, only three hemagglutinin subtypes have caused widespread human respiratory infection (h1, h2, and h3), suggesting a degree of host specificity.[1,3] human influenza h1 and h3 subtypes currently circulating continuously undergo variability or "antigenic drift." inefficient proofreading by influenza viral rna polymerase results in a high incidence of transcription errors and amino acid substitutions in the hemagglutinin or neuramidase, allowing new variants to evade preexisting humoral immunity and cause interpandemic outbreaks.[3] simultaneous infection of a cell by two influenza viruses may allow recombination of rna segments and result in the generation of a new "reassorted" virus with novel surface proteins to which there was little population immunity. pandemic influenza viruses arise by this process called "antigenic shift."[3] it has been hypothesised that the ha gene of the h5 and h7 subtypes harbour distinct secondary rna structures which favour insertional mutations (codon duplications) by a re-copying mechanism of the viral polymerase unit at a figure 1. location of human h5n1 avian influenza cases and animal outbreaks at 10 december 2008 adapted from reference no 5. 116 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 114-117 purine-rich sequence stretch encoding the endoproteolytic cleavage site of these ha proteins.[2,3] pigs may play an important role in the evolution of human pandemic strains. pig�s trachea contain receptors for both avian and human influenza viruses and the domestic pig supports the growth of viruses of both human and avian origin. thus, it has been proposed that genetic reassortment between avian and human virus may occur in pigs, leading to a novel strain.[4] the incubation periods for h5n1 avian influenza may be longer than normal seasonal influenza, which is around two to three days. current datas for h5n1 infection indicate an incubation period ranging from two to eight days and possibly as long as 17 days. however, the possibility of multiple exposure to the virus makes it difficult to define the incubation period precisely. who currently recommends that an incubation period of seven days be used for field investigations and the monitoring of patient contacts.[2,3] clinical manifestation initial symptoms include a high fever, usually with a temperature higher than 38o c, and influenza-like symptoms. diarrhoea, vomiting, abdominal pain, chest pain, and bleeding from the nose and gums have also been reported as early symptoms in some patients. watery diarrhoea without blood appears to be more common in h5n1 avian influenza than in normal seasonal influenza. the spectrum of clinical symptoms may, however, be broader, and not all confirmed patients have presented with respiratory symptoms.[3,9] case definition (who 2006) person under investigation is a person whom public health authorities have decided to investigate for possible h5n1 infection. suspected h5n1 case is a person presenting with unexplained acute lower respiratory illness with fever (> 38º c ) and cough, shortness of breath or difficulty breathing and > 1 of the following exposures in the 7 days prior to symptom: a. close contact with a person who is a suspected, probable or confirmed h5n1 case. b. exposure to poultry or wild birds or their remains or to environments contaminated by their faeces. c. consumption of raw or undercooked poultry products d. close contact with a confirmed h5n1 infected animal other than poultry or wild birds (e.g. cats or pigs). e. handling samples (animal or human) suspected of containing h5n1 virus in a laboratory or other setting. probable h5n1 case is a person meeting the criteria for a suspected case and one of the: a. infiltrates or evidence of an acute pneumonia on chest radiograph plus evidence of respiratory failure, or b. positive laboratory confirmation of an influenza a infection but insufficient laboratory evidence for h5n1 infection. confirmed h5n1 case is a person meeting the criteria for a suspected/probable case and one of the following positive results conducted in a laboratory whose h5n1 test results are accepted by who as confirmatory: a. isolation of an h5n1 virus b. positive h5 pcr results from tests using two different pcr targets c. a fourfold or greater rise in neutralization antibody titer for h5n1 based on an acute and a convalescent serum specimen. the convalescent neutralizing antibody titer must also be > 1:80 d. a microneutralization antibody titer for h5n1 of 1:80 or greater in a single serum specimen collected at day 14 or later after symptom onset and a positive result using a different serological assay. there were 27 confirm h5n1 case from 296 suspected cases managed in sulianti saroso infectious disease hospital in jakarta. twenty one cases had a fatal outcome with 6 cases survivors. while in dr. soeotomo general hospital surabaya, there are 5 confirm h5n1 case and 3 cases had fatal outcome and 2 cases survivors. the mean age of patient with h5n1 ai infection was 16,9 ± 11,6 years old in jakarta and 24 ± 8,51 years old in surabaya. whereas the youngest age of patient with h5n1 ai infection was 1 year. there was no difference beetwen male and female in h5n1 ai infection and mortality in both of jakarta and surabaya.[7,10] vital signs most patients have initial symptoms of high fever (typically a temperature of more than 38° c) and an influenza-like illness with lower respiratory tract symptoms. at presentasion the patiens had fever with temperature was more than 38o c in surabaya but in jakarta they were found the mean temperature at presentation was 37.5 ± 1.3° c with a median temperature of 37.8° c (range 35.8 to 40). the mean arterial pressure was 84.8 ± 11.6 mmhg with a median of 82 mmhg (range, 68 to 103). the mean respiratory rate at presentation was 36 ± 11/min with a median of 35/min (range, 15 to 60). the mean heart rate at presentation was 110 ± 24/min with a median of 104/min (range, 84 to 165). laboratory findings common laboratory findings have been leucopenia, p a r t i c u l a r l y l y m p h o p e n i a ; m i l d t o m o d e r a t e thrombocytopenia; and slightly or moderately elevated aminotransferase levels. in surabaya, all of cases were leucopenia. platelets had declining trend in most h5n1 ai infection cases. particularly patient showed a elevated aminotranferase levels. radiological findings radiologic findings of h5n1 hpai can show as a mild pneumonia until severe pneumonia or acute respiratory distress syndrome (ards). in jakarta, twenty of the 117wibisono et al.: the clinical profiles of avian influenza 27 patients had abnormal chest radiographs with the majority (19/20) showing evidence of bronchopneumonia or lobar pneumonia. of note, 4 patients had pleural effusions at presentation. while in surabaya, all of cases show a bilateral pneumonia. management based on information collected from publications as well as reports on a(h5n1) cases in affected countries, who gives a recommended guideline to h5n1 hpai infection 1. diagnosis 2. site of care 3. antiviral treatment a. oseltamivir b. other antiviral agent c. virological monitoring 4. other pharmacological intervention a. antibiotics b. immunomodulator c. h a e m o p h a g o c y t o s i s a n d i n t r a v e n o u s immunoglobulin 5. supportive therapy for critical ill care 6. special consideration 7. infection control condiseration oseltamivir remains the primary recommended antiviral treatment. treatment with oseltamivir is also warranted when the patient presents to clinical care at a later stage of illness (viral replication was more prolonged than with seasonal influenza, to last up to 15–17 days after illness onset. oseltamivir should be given 75 mg, twice a day dor 5 days for adult and 2 mg/kgbw (max 75 mg), twice a day for 5 days for children (age > 1 of age). when pneumonia was present, antibiotic treatment was appropriate initially for community-acquired pneumonia according to published evidence-based guidelines. when available, the results of microbiologic studies should be used to guide antibiotic usage for suspected bacterial coinfection.[11,12] monitoring of oxygen saturation should be performed: at presentation and routinely during subsequent care( pulse oximetry, arterial blood gases). supplemental oxygen should be provided to correct hypoxemia. oxygen therapy monitor oxygen saturation and maintain sao2 over 90% with nasal cannulae or face mask.[11,13] non invasive ventilatory is not recommended to support oxygen therapy for h5n1 hpai infection patient in ards state, instability hemodynamics and multiorgan failure, despite niv could increase risk for infectious aerosol to other patient. the recommended mode for mechanical ventilatory was inspiratory positive pressure ventilator.[11,13] summary h5n1 hpai infection in human was not different between endemic and non-endemic region include route of transmission, clinical severity, pathogenesis and respon to therapy. case detection was confounded by the nonspecificity of initial manifestations of illness, so that detailed contact and travel histories and knowledge of viral activity in poultry were essential. there was an urgent need for more coordination in clinical and epidemiologic research among institutions in countries with cases of influenza a (h5n1). reference 1. webster rg, govorkova ea. h5n1 influenza— continuing evolution and spread. n engl j med 2006; 355: 2174–7. 2. the writing committee of who consultation on human influenza a/h5, avian influenza a (h5n1) infection in humans. n engl jmed 2005; 353: 1874–85. 3. the writing committee of who consultation on human influenza a/h5, update on avian influenza a (h5n1) infection in humans. n engl jmed 2008; 358: 261–73. 4. schultsz c, dong vc, chau nvv, et al. avian influenza h5n1 and healthcare workers. emerg infect dis 2005; 11: 1158–9. 5. world health organization. avian influenza — situation in indonesia — update 16. 2010. (accessed august 20, 2010, at http://www.who. int/csr/don/2010_08_04/en/print.html.) 6. sumiarto b, arifin b, overview on poultry sector and hpai situation for indonesia with special emphasis on the island of java, 2007 available access on http://www.hpai-research.net/index.html. 7. sardikin et al, clinical and epidemiological features of patients with confirmed avian influenza presenting to sulianti saroso infectious diseases hospital, indonesia, 2005–2007, ann acad med singapore 2008; 37: 454–7. 8. de jong md, cam bv, qui pt, et al. fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma. n engl j med 2005; 352: 686–91. 9. to kf, chan pk, chan kf, et al. pathology of fatal human infection associated with avian influenza a h5n1 virus. j med virol 2001; 63: 242–6. 10. annual report database patient h5n1 ai infection in dr. soeotomo hospital. 11. world health organization. who interim guidelines on clinical management of humans infected by influenza a(h5n1). february 20, 2004. 12. yen hl, monto as, webster rg, govorkova ea. virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic a/vietnam/1203/04 influenza virus in mice. j infect dis 2005; 192: 665–72. 13. frederick g, hayden, antiviral resistance in influenza viruses – implications for management nad pandemic response. n engl j med 2006; 354: 785–8. 14. world health organization. summary of the second who consultation on clinical aspects of human infection with avian influenza a (h5n1) virus. (accessed august 20, 2010, at http://www. who.int/csr/ disease/avian_influenza/meeting19_03_ 2007/en/index. html.) ijtid vol 1 no 3 sep-dec 2010.12.pdf ijtid vol 1 no 3 sep-dec 2010.13.pdf ijtid vol 1 no 3 sep-dec 2010.14.pdf ijtid vol 1 no 3 sep-dec 2010.15.pdf 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 96 vol. 1. no. 2 may–august 2010 literature review tropical disease prevention and control: is it now knowledge management era? adithya sudiarno, arief rahman, sri gunani partiwi industrial engineering department industrial technology faculty sepuluh nopember institute of technology surabaya indonesia e-mail: adithya.sudiarno@gmail.com, adithya_sudiarno@ie.its.ac.id abstract indonesia is the part of developing asian countries, until now still not declared free from various types of tropical diseases. whereas, several years ago indonesia has declared free from various kinds of tropical diseases, but in fact, tropical diseases up to now still cannot be eliminated. moreover the prevalence rates of tropical diseases tended to increase from year to year. one of the reasons indonesia is a tropical climate, but in fact we are not able to control the climate. in this condition, what we can do is to raise public awareness so the spread of the disease can be controlled. it was time for an early awareness efforts conducted in a participatory manner by all stakeholders. in this case, stakeholders can be health practitioners, researchers, policy makers (official), and even the citizens. participatory awareness can be enhanced if we have an integrated system that can accommodate all knowledge about tropical disease. the knowledge consists of characteristic about disease, potential risk, how to cure, how to isolate disease in community, and absolutely important is how to prevent of illness, etc. this paper aims to propose an integrated system called tropical disease knowledge management system (tdkms) for enhancing the participatory awareness. key words: knowledge management system, tropical disease management existing phenomenon in indonesia infectious disease is a disease that transmitted through various media and a very high risk for humans, especially because of the rapid growth of human. example of infectious disease can be found in indonesia are leprosy, malaria, dengue fever, avian flu, swine flu, etc. because often appear in tropical area, these diseases commonly called as tropical diseases. these diseases was a big problem in almost developing country, because prevalence rate and mortality rate is high relative in short term (widiyono, 2005). in wide scale, tropical diseases can become endemic diseases, permanent disability, even death. based on long term national development of indonesia government (called rpjpk 2005–2025), tropical diseases is main priority to be eradicated (bappenas, 2005). indonesia is the part of developing asian countries, until now still not declared free from various types of tropical diseases. whereas, several years ago indonesia has declared free from various kinds of tropical diseases, but in fact, tropical diseases up to now still cannot be eliminated. moreover the prevalence rates of tropical diseases tended to increase from year to year. moreover the prevalence rates of tropical diseases tended to increase from year to year (riyadi, 2006). based on this phenomenon, indonesia’s health sector is currently in a situation transition of epidemiological, which must bear the burden of excess, commonly referred to as the triple burden (widiyono, 2005). the main cause of rapid spread of tropical diseases is the humid air that comes with the rainy season in tropical countries. humid air resulted disease-carrying agents, such as viruses, bacteria, fungi, and parasites grow more rapid. it is closely related to the indonesia’s tropical climate, and actually we are not able to control the climate. other factors that contribute to the rapid spread of tropical disease are poor physical environment, economy and social factor, culture, the high human mobility between countries, biological change of disease-carrying agents, etc. accumulation of factors above causes the emergence of new variants dangerous diseases. several systematic programs have been done by the government in order to eradicate tropical disease, such as “gerakan berantas kembali malaria” (gebrak malaria), “gerakan pemberantasan sarang 97sudiarno et al.: tropical disease prevention and control nyamuk” (psn), etc. in reality, application of the programs is not satisfy yet, because there is a gap between program and phenomenon happened. this gap proven by prevalence rate which is tend to increase from year to year (kompas, 2008). so, what should we do now? the keyword to solve this phenomenon is how to integrate knowledge about the nature of the agent, host, and environment. the integrated knowledge would be a base for designing prevention and control program, and also for early awareness efforts. with integrated knowledge, program proposed will be more realistic because it made from the real phenomenon that captured by integrated system. knowledge management system (kms) the knowledge means professional’s intellect such as know-what, know-how, know-why, self-motivated creativity, best practices, concepts, values, beliefs and method of working that can be shared and communicated among the professional. the knowledge can be in form of explicit knowledge or tacit knowledge. explicit knowledge means the knowledge is in a structured form and stored in knowledge bases (databases). the knowledge also can be in form of tacit, which means the knowledge is in a subjective (non-structured) form, so that needs to be structured before used. examples for tacit knowledge are ideas, individual insights, values, and judgments of individuals (bose, 2003). the goal of knowledge management is to convert tacit knowledge become explicit knowledge. below is illustration about explicit and tacit knowledge. until here, may be you will have question: “what is the differentiation between the commonly used terms information and knowledge?” the primary difference is in the degree of understanding of their underlying organizational data. information represents data endowed with meaning. meanwhile, knowledge represents information with experience, insight, and expertise (kebede, 2010). with this differentiation, we can say if the accumulation of data into a meaningful context called as information, and knowledge is the next degree of higher understanding toward information. kms deals with the strategies and processes for identifying, capturing, structuring, sharing and applying professional’s knowledge in order to extract competitive advantage and create sources of sustainable growth for future (bose, 2003). kms also deals with a class of information systems applied to manage professional’s (individual or organizational) knowledge. a lot of knowledge management initiatives rely on it (information technology) as a critical success factor and enabler. nowadays, progress in it has enhanced knowledge management capabilities that were not possible before (hsia, lin, wu, and tsai, 2006). in the future, with the it progress, knowledge management capabilities become more sophisticated and complex. hence, kms is it-based systems built to simplify and enhance the processes of knowledge acquisition, sharing, and utilization within the organization. idea for tropical disease prevention and control based on existing phenomenon explained above, it was time for an early awareness efforts conducted in a participatory manner by all stakeholders. stakeholders can be health practitioners, researchers, policy makers (official), and even the citizens. participatory awareness can be enhanced if we have an integrated system that can accommodate all knowledge about tropical disease from all stakeholders to be shared. the knowledge here means stakeholders intellect and wisdom, such as know-what, know-how, know-why, self-motivated creativity, best practices, concepts, values, beliefs and method of working that can be shared and communicated (bose, 2003). in this context, the knowledge for disease prevention and control can consist of characteristic about disease, potential risk, how to cure, how to isolate disease in community, and absolutely important is how to prevent of illness, etc. the idea for tropical disease prevention and control explained here also inspired from bose (2003) statement: well-managed information that is properly cataloged and structured, available and accessible by the right stakeholders and processes at the right time becomes knowledge. below figure 1. explicit and tacit knowledge (romy s.w. in saputri, 2010) km goal: converting tacit knowledge becoming explicit knowledge. 98 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 96-101 is illustration (architecture of integrated system) how to assure this idea: figure 2 above give a description of how the process of acquiring knowledge, how the knowledge can be shared, and also describe how the knowledge can be used. tdkms begins from the acquisition of knowledge then store it in the repository (knowledge collection database). repository built based on world wide web (www) technology. by using this technology user can share the knowledge easily. hence, knowledge can be utilized by stakeholders anytime and anywhere. while utilize knowledge, user can formulate new knowledge, with this integrated system user can update knowledge and store it to the repository. the most important issue in tdkms is knowledge acquisition, because in this acquisition we face problem how to externalize tacit knowledge become explicit knowledge. method used for externalizing the knowledge is cbr (case based reasoning). case based reasoning means to retrieve former, already solved problems similar to the current one and to attempt to modify their solutions to fit for the current problem. the underlying idea is the assumption that similar problems have similar solutions (schmidt, et al., 2001). figure 3 shows the cased based reasoning cycle: figure 3. cased based reasoning cycle developed by aamodt (1994) the following figure illustrating appearance of tdkms based on world wide web: figure 2. architecture of integrated system (tropical disease knowledge management system/tdkms) 99sudiarno et al.: tropical disease prevention and control figure 4. tdkms interface version 1.2 indonesian map to show where the tropical disease is appear. description of tdkms statistical data menu of tropical disease prevalence from year to year figure 5. simple knowledge captured in tdkms summary of tropical disease prevalence grouped by the disease simple knowledge contains common knowledge (but not mythos) menu for downloading result of research conducted by expert 100 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 96-101 figure 6. advanced knowledge captured in tdkms advanced knowledge collection contains know-what, knowhow, know-why about the tropical diseases (visualized with knowledge base method) advanced knowledge collection contains know-what, knowhow, know-why about the tropical diseases (visualized with inference method) knowledge about another endemic potential region 101sudiarno et al.: tropical disease prevention and control figure 7. discussion forum for facilitating knowledge acquisition and sharing conclusion it was time for an early awareness efforts conducted in a participatory manner by all stakeholders. participatory awareness can be enhanced if we have an integrated system that can accommodate all knowledge from the stakeholders about tropical disease. from explanation above, stakeholders can be health practitioners, researchers, policy makers (official), and even the citizens. the knowledge to be acquired, share, and utilized means stakeholders intellect and wisdom, such as know-what, know-how, know-why, self-motivated creativity, best practices, concepts, values, beliefs and method for disease prevention and control. with integrated knowledge, program proposed will be more realistic because it made from the real phenomenon that captured by integrated system (tdkms). so that nowadays is the era for knowledge management. references a. aamodt (1994) cased-based reasoning: foundation issues, aicom 7. 39–59 bappenas, (2005) rencana pembangunan jangka panjang 2005–2025. jakarta: bappenas. bose, r. (2003) knowledge management-enabled health care management systems: capabilities, infrastructure, and decision-support. expert systems with applications, 24, 59–71 hsia, t.l., lin, l.m, wu, j.h., and tsai, h.t. (2006) a framework for designing nursing knowledge management systems. interdisciplinary journal of information, knowledge, and management. volume 1. kebede, g. (2010) knowledge management: an information science perspective. international journal of information management. kompas (2008) penyakit tropis tidak teratasi, jumlah penderita tak kunjung turun. riyadi. (2006). laporan kebijakan penanggulangan penyakit menular. jakarta. saputri, e.m., (2010) perancangan prototype knowledge management menggunakan case based reasoning untuk meningkatkan efektifitas proses knowledge sharing antar perawat. schmidt, r., et al (2001) cased-based reasoning for medical knowledgebased systems. international journal of medical informatics 64, pp. 355–367 widiyono (2005) penyakit tropis epidemiologi, penularan, pencegahan, dan pemberantasnnya. jakarta: erlangga. discussion forum: “host factor” topic discussion forum: “environment factor” topic discussion forum: “agent factor” topic ijtid vol 1 no 2 may-aug 2010.44.pdf ijtid vol 1 no 2 may-aug 2010.45.pdf ijtid vol 1 no 2 may-aug 2010.46.pdf ijtid vol 1 no 2 may-aug 2010.47.pdf ijtid vol 1 no 2 may-aug 2010.48.pdf ijtid vol 1 no 2 may-aug 2010.49.pdf 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 vol. 9 no. 2 may–august 2021 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 * corresponding author: sukmab@fk.unair.ac.id research article soil-transmitted helminthes infection and nutritional status of elementary school children in sorong district, west papua, indonesia zukhaila salma1, fitriah2 3 , lynda rossyanti4 5 , soraya salle pasulu6, budiono7, i gusti made reza gunadi ranuh8 , dominicus husada8, sukmawati basuki 2,4* 1master of tropical medicine program, faculty of medicine, universitas airlangga, surabaya, indonesia 2 3bachelor of medicine program, faculty of medicine, universitas airlangga, surabaya, indonesia 4department of medical parasitology, faculty of medicine, universitas airlangga, surabaya, indonesia 5 puskesmas mayamuk, sorong-98421, west papua, indonesia 6 rsud kabupaten sorong, kampung baru, sorong, west papua, indonesia 7department of public health and preventive medicine, faculty of medicine, universitas airlangga, surabaya, indonesia 8department of child health, dr. soetomo hospital/faculty of medicine, universitas airlangga, surabaya, indonesia received: 10th december 2020; revised: 17th march 2021; accepted: 8th june 2021 abstract it is known that soil-transmitted helminths (sths) infection in children associates with growth and developed restriction in children, which is shown by nutritional status. however, the studies which are investigating this phenomenon is still limited in indonesia. this recent study aimed to compare students who infected and non-infected with sth towards their nutritional status. an analytic cross-sectional research design was conducted in two elementary school students at mayamuk sub-district, sorong district, in january 2020. sths infection was identifi ed by lugol stained wet mount smear from their stool under a light microscope. children nutritional status was determined by body mass index based on age. a total of 164 children (67.5%, 164/243) were voluntary to participate by informed consent and eligible. twenty-seven children (16.5%, 27/164) were infected with one or more sth species of ascaris lumbricoides, trichuris trichiura, hookworm, and strongyloides stercoralis. t. trichiura (81.5%, 22/27) was the most common species found, either in single or mixed infection. children nutritional status was observed as thinness, normal, overweight, and obese, that was 6.1% (10/164), 75% (123/164), 6.7% (11/164), and 12.2 % (20/164) respectively. sths infection occurred in children with nutritional status of thinness 3.7% (1/27), normal 74.1% (20/27), overweight 3.7% (1/27), and obese 18.5% (5/27). there was no signifi cant diff erence between sths infected children and non-infected children on their nutritional status (p=0.616, chisquare test). thus, it indicated that sths infection was not only the factor to induce the impairment of nutritional status in children at mayamuk sub-district. it needs further investigation to clarify the factors which are leading to the thinness, overweight, and obese in mayamuk children. keyword. soil-transmitted helminthes infection; nutritional status; children; elementary school, indonesia abstrak kecacingan yang ditularkan melalui tanah (infeksi sths) pada anak telah diketahui mempengaruhi pertumbuhan dan perkembangan pada anak, yang ditunjukkan dengan status gizi. penelitian yang membahas hal ini masih terbatas di indonesia. penelitian ini bertujuan untuk membandingkan anak yang terinfeksi sths dengan anak yang tidak terinfeksi sths terhadap status gizinya. desain penelitian cross-sectional analitik dilakukan pada murid dari dua sekolah dasar pada bulan januari 2020, di kecamatan mayamuk, kabupaten sorong. identifi kasi infeksi sths menggunakan pemeriksaan mikroskopis dari sediaan tinja anak dengan metode wet mount smear yang tercat oleh larutan lugol. status gizi anak ditentukan dari indeks massa tubuh menurut usia. sejumlah 164 anak (67,5%, 164/243) secara suka rela berpartisipasi , raden bagus yanuar renaldy , iwayan sarjana laboratory of malaria, institute of tropical disease, universitas airlangga, surabaya, indonesia 86 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 85–93 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 melalui informed consent dan sesuai kriteria. dua puluh tujuh anak (16.5%, 27/164) terinfeksi oleh satu atau lebih spesies sths, yakni ascaris lumbricoides, trichuris trichiura, hookworm, dan strongyloides stercoralis. t. trichiura (81.5%, 22/27) merupakan spesies yang paling banyak ditemukan baik dalam infeksi tunggal maupun ganda. status gizi anak yang didapatkan meliputi status gizi kurang (6,1%, 10/164), normal (75%, 123/164), gizi lebih (6,7%, 11/164) dan obesitas (12,2 %, 20/164). infeksi sths terjadi pada anak dengan status gizi kurang sebesar 3.7% (1/27), normal 74.1% (20/27), gizi lebih 3.7% (1/27), and obesitas 18.5% (5/27). tidak ditemukan perbedaan yang bermakna antara anak yang terinfeksi sth dengan yang tidak terhadap status gizinya (p=0.616, uji chi-square). hal ini menunjukkan bahwa infeksi sth bukan satu-satunya faktor penyebab gangguan terhadap status gizi anak di kecamatan mayamuk. kajian lebih lanjut perlu dilaksanakan untuk menentukan faktor penyebab status gizi kurang, gizi lebih, dan obesitas pada anak di kecamatan mayamuk. kata kunci: infeksi soil-transmitted helminthes; status gizi; anak; sekolah dasar, indonesia. how to cite: salma, z., fitriah., reynaldi, rby., rossyanti, l., sarjana, iw., pasulu, ss., budiono7, ranuh, igmrg husada, d., basuki, s. (2021). soil-transmitted helminthes infection and nutritional status of elementary school children in sorong district, west papua, indonesia. indonesian journal of tropical and infectious disease, 9(2). 85-93. introduction soil-transmitted helminthes (sths) infection is one of the neglected tropical infectious diseases which commonly occur in low-income countries and rural communities. helminths that cause sths infection in humans, are ascaris lumbricoides, trichuris trichiura, necator americanus and ancylostoma duodenale1,2. pullan et al estimated that 1.45 billion people worldwide were infected with at least one species of these helminths in asia3. globally, an estimated disability-adjusted life years (dalys) contributed by sths infection was 1.9 milion in 20174. sths infection is a chronic infection that tends to be asymptomatic, thus it is difficult to assess the morbidity, especially in endemic area5,6. symptoms and signs of sths infection are anorexia, anemia, dysentery, diarrhea, and intestinal obstruction which can aff ect the growth and development of the child. the presence of sths in the small intestine can interferes the absorption of nutrients and cause intestinal bleeding5–10. several studies showed that sths infection was significantly associated with a decrease of nutritional status indicators involving weight for age and height for age10,11. the severity of clinical manifestation is commonly performed by the infection with polyparasitism and heavy intensity of sths6,7,12. sths infection and stunting in children are public health problems in indonesia. the national survey showed that the average of sths infection prevalence of elementary school students between 2000-2011 was 28.7%13. several studies had indicated that sths infection in elementary school students in rural areas of indonesia were remained high14–16. the world health organization (who) reports that the cases number of under fi ve year old children who experience wasting and stunting in 2019 were 47 million and 144 million children, respectively, and most of them founded in africa and asia17,18. riset kesehatan dasar indonesia showed that the prevalence of wasting and stunting of children in 2018 were 10.2% and 30.8%, respectively19. in 2018, twenty out of thirty-four (58.9%, 20/34) provinces of indonesia were categorized as high stunting prevalence province20. west papua is one of the indonesian provinces, which is facing these two health problems. a study showed that the sths infection prevalence of elementary school children in the sorong ditrict was 30.6%21. a national nutritional status survey in 2018 reported that the prevalence of schoolage children and adolescents (5-12 year old) with stunting and wasting condition was 22.8% and 6.8%, respectively in west papua22. until now, it has not yet been studied the phenomenon of sths infection with nutritional status in west papua. our study aimed to compare between children infected and non-infected with sths towards their nutritional status. it would be meaningful for the control program of sths infection and stunting. 87zukhaila salma, et al.: soil-transmitted helminthes infection and nutritional status ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 materials and methods study area and population the study was conducted in two villages, where are located in mayamuk sub-district, sorong, west papua province, indonesia, where the average temperature of area was 27,90c and the humidity was 83,2%. geographically, most of the sorong area, a district, is directly adjacent to indonesian sea areas. it is bordered by the pacifi c ocean to the north; seram sea to the south and west; tambrauw district to the east and raja ampat regency to the west. sorong consists of 30 sub-districts and 115 islands with a total area of 13,075.28 km2 (figure 1). distribution of gross regional domestic product in 2019 based on sectors comprised of processing industri (42.54%), addition and excavation (15.95%), construction (14.65%), agriculture, forestry, and fi cheries (10,11%), and others (16.75%). the main production of the plantation sector in sorong are coconut, coff ee, and cocoa23. 0 3 6 km 0 200 400 km sorong district mayamuk sub-district klasmelek village inpres 14 makbusun village inpres 25 sorong district figure 1. study sites (source: arcgis.com24). mayamuk sub-district represents 4.4% (542.2 of 13,693.5 km2) of the total area of sorong. study was implemented in two public elementary schools, namely inpres 14 and inpres 25, in january 2020. inpres 14 is located in klasmelek village, while inpres 25 is in makbusun village. the distance between the two elementary schools is 3.1 kilometers. makbusun village is located ± 8.5 km from seashore, while klasmelek village is located ± 10.9 km from it. plantations, forest areas, and rivers are many in klasmelek village than in makbusun village. total of 3107 people are living in makbusun village and 674 people are in klasmelek village. sample and data collection an analytical cross-sectioal study design was conducted. elementary school students from grade one to grade six from both schools involved in this study. the minimum number of samples was determined by the proportion estimation formula added 10% to anticipate error result and total was 90 samples. a structured questionnaire which included information on general demographic data (name, date of birth, age, gender, and ethnic), history of sths infection, and antihelminthic drug was used. stool collection and sths identication children who participate in this research were given a stool tube (onemed, sidoarjo, indonesia) which had labeled according to the questionnaire number. they brought the tube back with as much as one knuckle of stool on the next day. the stools in tube were preserved with adding 10% formalin solution and checked the tube number based on the questionnaire data. sths was identifi ed by using wet-mount smear method stained with 1% lugol solution under light microscope with 100 and 400 magnifi cations (olympus© cx22, japan). it was repeated four times. stool examination was performed in the institute of tropical disease, airlangga university, surabaya. nutritional status measurement the body mass index according to age (baz) score was used to determine nutritional status of children. it is based on the body weight, height, and age. the children body weight and height 88 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 85–93 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 were measured to complete their questionnaire form. a calibrated needle scale (onemed, sidoarjo, indonesia) to the nearest 0.1 kg without shoes was used for measuring their body weight, and a microtoise (onemed, sidoarjo, indonesia) to the nearest 0.1 cm which attached to a vertical wall was applied for sizing their height with barefeet. their age was calculated in full month. nutritional status was classified as severely thinness (<-3 standard deviation (sd)), thinness (-3 sd to <-2 sd), normal (-2 sd to +1 sd), overweight (+1 sd to +2 sd), and obese (>+2 sd)25. statistical analyzes categorical variables were presented by number and percentage, while continuous variable was a mean value. the proportion diff erences of categoric variables were analyzed by chisquare test. mean comparison of continuous variables were carried out by t-test analysis on normal distribution data and by mann-whitney test on abnormal distribution data. a signifi cant comparison or diff erence was determined by p<0.05 value. all statistical analysis of this study was performed in version 22.0 statistical package for the social science (spss) (ibm, somers, ny). ethical clearance this study was approved by the health research ethics committee, faculty of medicine, universitas airlangga in number of 167/ec/ kepk/fkua/2020. results interview and anthropometric measurement were conducted into 194 children from two elementary schools, who were voluntary to participate in this study. a total of 164 children (84.5%, 164/194) were included and 30 children were excluded because they were without stools (figure 2). most of the children were non-papuan (79.9%, 131/164) (table 1). inpres 14 elementary school children (n=77) inpres 25 elementary school children (n=166) 63 children agreed to participate 15 children were absent or decline to participate 53 children were able to collect their stools 62 children completed the questionnaire and anthropometric measurement 111 children were able to collect their stools 132 children completed the questionnaire and anthropometric measurement 21 children were not able to collect their stools 1 child refused to be taken anthropometric measurement 9 children were not able to collect their stools 1 child refused to be taken anthropometric measurement 133 children agreed to participate 33 children were absent or decline to participate figure 2. diagram of participant involvement table 1. demographic characteristic of children in inpres 14 and inpres 25 elementary school at sorong district variable inpres 14 (n=53) (n, %) inpres 25 (n=111) (n, %) total (n=164) (n, %) age 6 – 7 13, 24.5 26, 23.4 39, 23.7 8 – 9 22, 41.5 46, 41.4 68, 41.5 10 – 11 16, 30.2 34, 30.6 50, 30.5 12 – 13 2, 3.8 4, 3.6 6, 3.7 >13 0, 0.0 1, 1 1, 0.6 sex girl 20, 37.7 56, 50.4 76, 46.3 boy 33, 62.3 55, 49.6 88, 53.7 ethnic papua 15, 28.3 18, 16.2 33, 20.1 non papua 38, 71.7 93, 83.8 131, 79.9 sths were detected in 27 children stools (16.5%, 27/164). t. trichiura was frequently found 89zukhaila salma, et al.: soil-transmitted helminthes infection and nutritional status ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 table 3. characteristic of antropometric and nutritional measurements in children either with or without sths infection at two elementary schools in sorong distric. antropometric and nutritional status elementary school p-valueα inpres 14 n=53 inpres 25 n=111 positive negative p-valueα positive negative p-valueα mean height (cm) 127.8 127.5 0.924 127 130.3 0.188 0.146 man weight (kg) 27.1 27,6 0.890 27.8 28.3 0.952 0.253 mean bmi 16.2 16.4 0.632 17 16.3 0.367 0.887 mean bmi/age (z-score) -0.1 -0,2 0.482 0.1 -0.2 0.587 0.808 thinness (n, %) 0, 0 2, 4.9 0.598 1, 6.6 7, 7.2 0.183 0.511 normal (n, %) 10, 83.4 33, 80.5 10, 66.7 70, 73 overweight (n, %) 1, 8.3 1, 2.4 0, 0 9, 9.4 obese (n, %) 1, 8.3 5, 12.2 4, 26.7 10, 10.4 α: mann-whitney test used for continous variable with abnormal data; t-test used for continous variable with normal data; chi-square test used for categorical data; positive means children with sths infection and negative is children without sths infection (13.4%, 22/164), then followed by hookworm (7.3%, 12/164) and ascaris lumbricoides (3.6%, 6/164). polyparasitized sths were observed in table 2. single and mix soil-transmitted helminthes infection cases among 29 infected children in inpres 14 and inpres 25 elementary school at sorong district. variable inpres 14 (n=12) (n, %) inpres 25 (n=15) (n, %) total (n=27) (n, %) single infection 4, 33.3 11, 73.3 15, 55.6 al 0, 0.0 1, 6.7 1, 3.7 tt 3, 25 7, 46.7 10, 37 hw 1, 8.3 1, 6.7 2, 7.4 ss 0, 0.0 2, 13.3 2, 7.4 mix infection 8, 66.6 4, 26.7 12, 44.4 tt + al 0, 0.0 2, 13.3 2, 7.4 tt + hw 6, 50 0, 0,0 6, 22,2 tt + hw + al 1, 8.3 2, 13,3 3, 11,1 tt + hw + ss 1, 8.3 0, 0.0 1, 3.7 al: ascaris lumbricoides, hw: hookworm, ss: strongyloides stercoralis, tt: trichuris trichiura 12 children stools (44.4%, 12/27) and dominated by t. trichiura with hookworm infection (50%, 6/12) (figure 3 and table 2). a b d e c figure 3. the morphology of soil-transmitted helminthes in children stools were (a) hookworm egg, (b) a. lumbricoides egg, (c) t. trichiura egg, (d) s. stercoralis larva and (e) hookworm larva under light microscope with 400 magnifi cations. minimum length is 1 micrometer. the majority of children had normal nutritional status (75%, 123/164). however, 41 children showed the abnormal nutriotional status that were 10 children with thinness (6.1%, 10/164), 11 children with overweight (6.7%, 11/164), and 20 children with obese (12.1%, 20/164). children with thinness in inpres 25 were higher than in inpres 14 (7.2%, 8/111 v.s 3.7%, 2/53) (table 3). 90 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 85–93 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 there was not signifi cant diff erence between children who infected and non-infected with sths towards their nutritional status (p>0.05, chi-square, test) (table 4). table 4. comparisson of antropometric measurement in elementary children with and without sths infection antropometric and nutritional status sth infection status p-valuepositive n=27 negative n=137 mean height (cm) 127.3 129.5 0.299 mean weight (kg) 27.5 28.1 0.957 mean bmi 16.6 16.3 0.326 mean bmi/age (z-score) 0.3 -0.19 0.397 thinness (n, %) 1, 3.7 9, 6.6 0.616 normal (n, %) 20, 74.1 103, 75.2 overweight (n, %) 1, 3.7 10, 7.3 obese (n, %) 5, 18.5 15, 10.9 α: mann-whitney test used for continouse variable with abnormal data; t-test used for continouse variable with normal data; chi-square test used for nominal data; positive means children with sths infection and negative is children without sths infection discussion school-age children living in a rural and a tropic area are vulnerable to sths infection due to their habits and inadequate sanitation. school-age children often play in the ground without using footwear, rarely cut their nails, and do not wash their hands after playing or defi cation26,27. the potential factors for sths infection in school-age children were due to their low hygiene practice. a low prevalence of sths infection was observed in this study (16.5%) based on who classifi cation and a decline prevalence compare to previous prevalence in 201721. both studies were conducted in mayamuk sub-district with different condition. the previous study was performed in 2017, a year before lymphatic fi lariasis mda implementation in sorong district that is every october since 201828, and the recent study was 3 months after administration and two-year implementation of lymphatic fi lariasis mda. it seemed that lymphatic fi lariasis mda is able to reduce the sths infection prevalence after 3 months administration and two-year implementation of lymphatic fi lariasis mda. therefore, it might need the follow-up study in order to clarify the eff ect of lymphatic fi lariasismda to reduce the sth prevalence. a single dose of combination diethyl carbamazine (dec) 100 mg and albendazole (alb) 400 mg, a lymphatic filariasis mda, is applied in indonesia, including sorong district29,30. this combination has been reported that impacted to sths infection, since the drugs have a broad range of anti-helminthic activity. it reduced 77% hookworm infection using the combination of ivermectine (ivm) and alb in côte d’ivore from 2014 to 201731. study by sunish et al showed 79% reduction of sths infection after 7 years administration the combination of dec and alb, and the highest reduction was for hookworm infection, followed by ascariasis, and trichuriasis32. our study demonstrated the decline prevalence of sths infections after 3 months administration and twoyear implementation of dec and alb, but it was not under 10% of prevalence and it was still 46% reduction. it suggested that the health education to improve the individual hygiene and sanitation needs to be implemented in these areas. it could be considered to administer an additional a single dose of alb at six months before lymphatic fi lariasis-mda in order to eliminate the sths infection in children. infection of t. trichiura was highly found in this study, either within mixed, mostly t. trichiura with hookworm, or single infection. the previous study conducted in sorong district reported similar results21. studies in côte d’ivoire31, tamil nadu state32, and congo33 resulted a low reduction of trichuriasis compared with hookworm infection and ascariasis after lymphatic fi lariasis mda administration by using respectively ivm-alb, dec-alb, and alone alb. it means that either those combinations or alb alone by a single dose are not enough eff ective to eliminate t. trichiura infection in human. the present study found no significant diff erence between sths infected children and non-infected children toward their nutritional status. it was similar with the previous studies, which had been conducted by suraweera et al. in 91zukhaila salma, et al.: soil-transmitted helminthes infection and nutritional status ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 kandy, sri lanka and kurniati et al. in madura, indonesia34,35. we found that the thinnes children mostly were not infected with sths infection (see on table 3 and 4). it indicated that nutritional status of children can be infl uenced by several factors, such as food intake, environment, ages, dietary habit and the type of food consumed, additional sths infection36,37. a study in surakarta showed that school-age children with stunting were influenced by their poor energy and protein intake. these intakes were signifi cantly related to the level of education and occupations of their mother and family income38. the prevalence of undernutrition in children from low socioeconomic family was found to be higher than those from middleto uppersocio-economic family (42.3% vs 19.28%)39. the factors that underlie the low nutritional status within lowincome family group are poverty, education of mother, number of family member, and also insecurity and safety of the food39,40. recently, the altered gut microbiota is associated with stunting and malnutrition in children41,42. thus, futher investigation is needed to clarify the factors, which contribute to children thinnes, overweight, and obese in mayamuk sub-district, such as socioeconomy, nutritient consumption, education, and gut microbiota, in order to overcome children nutritional status problem. children either with or without sths infection did not have a significant difference in their nutritional status in mayamuk sub-district. sths infection was not the only factor leading to nutritional status impairment of children in this study. thus, further research is needed to determine the factors, which aff ect to thinness, overweight, and obese in children living at mayamuk sub-district, sorong district, west papua province. we are grateful to the elementary school children, teachers and the head of elementary schools, staff s of primary health centre at mayamuk sub-district for their kindness, participation, and assistance in our study. our thanks are also addressed to airlangga university for supporting our study by a research grant with number of 2158/un3/2019. conflict of interest all authors stated that there is no confl ict of interest exists. references 1. who. soil-transmitted helminth infections, fact sheet updated march 2020. available from: https://www. who.int/news-room/fact-sheets/detail/soil-transmittedhelminth-infections, accessed on may 26, 2020 2. silver za, kaliappan sp, samuel p, venugopal s, kang g, sarkar r, ajjampur ssr, geographical distribution of soil-transmitted helminthes and the eff ects of community type in south asia and south east asia – a systematic review. plos negl trop dis 2018;12(1):e0006153 3. pullan rl, smith jl, jasrasaria r, brooker sj. global numbers of infection and disease burden of soiltransmitted helminth infection in 2010. parasite&vector 2014;7(37) 4. kyu hh, abate d, abate kh, abay sm, abbafati c, abbasi n, abbastabar h, abd-allah f, abdela j, abdelalim a, et al. global, regional, and national disability-adjusted life-years (dalys) for 359 diseases and injuries and healthy life expectancy (hale) for 195 countries and territories, 1990-2017: asystematic analysis for the global burden of disease study 2017. lancet 2018; 392:1859-922 5. usuanlele, mt. soil-transmitted helminth infection, nutrition and growth in school-age children from rural communities in honduras. thesis. 2012. master of science in applied health sciences, faculty of applied health sciences, brock university, st. catharines, ontario. 6. who. guideline: preventove chemotherapy to control soil-transmitted helminth infection in at-risk population groups. geneva: world health organization; 2017. licence: cc by-nc-sa 3.0 igo. 7. crompton dwt and nesheim mc. nutritional impact of intestinal helminthiasis during the human life cycle. annu. rev. nutr. 2002;22:35-59. 8. farhadi s and ovchinnikov rs. the relationship between nutrition and infectious diseases: a review. biomed biotechnol res j. 2018;2:168-72 9. echazu a, juarez m, vargas pa, cajal sp, cimino ro, heredia v, caropresi s, paredes g, arias lm, abril m, conclusion a c k n o w l e d g e m e n t s 92 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 85–93 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 gold a, lammie p, krolewiecki a. albendazole and ivermectin for the control of soil-transmitted helminthes in an area with high prevalence of strongyloides stercoralis and hookworm in northwestern argentina: a community-based pragmatic study. plos negl trop dis. 2017;11(10):1-20 10. sanchez al, gabrie ja, usuanlele mt, rueda mm, canales m, gyorkos tw. soil-transmitted helminth infections and nutritional status in school-age children from rural communities in honduras. plos negl trop dis. 2013;7(8): e2378 11. moncayo al, lovato r, cooper pj. soil-transmitted helminth infections and nutritional status in ecuador: findings from a national surveys and implication for control strategies. bmj open. 2018;8(4):1-9: e021319 12. mupfasoni d, karibushi b, koukounari a, ruberanziza e, kaberuka t, kramer mh, mukabayire o, kabera m, nizeyimana v, deville ma, ruzin j, webster jp, fenwick a. polyparasite helminth infection and their association to anemia and under-nutrition in northern rwanda. plos negl trop dis. 2009;3(9): e517 13. ditjen p2pl. profil: pengendalian penyakit dan penyehatan lingkungan. jakarta: direktorat jendral pengendalian penyakit dan penyehatan lingkungan. 2015. 14. mau f and mulatsih. prevalence and intensity of soiltransmitted helminth infections among elementary school students in west sumba and central sumba districts east nusa tenggara, indonesia. journal of medical science and clinical research. 2017;5(10). 15. pasaribu ap, alam a, sembiring k, pasaribu s, setiabudi d. prevalence and risk factors of soiltransmitted helminthiasis among school children living in an agricultural area of north sumatera, indonesia. bmc public-health. 2019;19(1):1066. 16. brahmantya iby, iqra hhp, hartawan ignbrm, anjani iaw, sudarmaja im, ryalino c. risk factors and prevalence of soil-transmitted helminth infections. open access macedonian journal of medical science. 2020;8(a):521-24. 17. who. malnutrition, fact sheet, updated april 2020. available from: https://www.who.int/news-room/factsheets/detail/malnutrition, accessed on october, 2020 18. unicef, who, worl bank group. levels and trends in child malnutrition: unicef/who/world bank group joint child malnutrition estimates, key fi nding of the 2020 edition. 2020. geneva: who. licence: cc by-nc-sa 3.0 igo 19. kementerian ppn. pembangunan gizi di indonesia. 2019. jakarta: direktorat kesehatan dan gizi masyarakat-kepedulian pembangunan manusia, masyarakat dan kebudayaan-kementerian ppn/ bappenas. 20. who. nutrition landscape information system (nlis), help topic: malnutrition in children, stunting, wasting, overweight, and underweight. available from: http://apps.who.int/nutrition/%0alandscape/help. aspx?menu=0&helpid=391&lang=en, accessed on october 2020. 21. yuwono n, pasulu ss, husada d, basuki s. prevalence of soil-transmitted helminthiasis among elementary children in sorong district, west papua. indonesian journal of tropical and infectious diseas. 2019;7(4):86-91 22. kemenkes ri. buku saku: hasil pemantauan status gizi (psg) tahun 2017. 2018. jakarta: direktorat gizi masyarakat-direktorat jendral kesehatan masyarakatkementrian kesehatan. 23. bps kabupaten sorong. kabupaten sorong dalam angka: 2020. 2020, kabupaten sorong: badan pusat statistik. issn: 2302-0512. publication number: 91070.2003. 24. arcgis. available from: https://www.arcgis.com/ home/signin.html?returnurl=https%3a//www.arcgis. com/home/item.html%3fid%3d92be9dc23fa14a2e8 3a8bc4a6f7caeba, accessed on october 2020. 25. peraturan menteri kesehatan ri. 2020. permenkes ri no.2 tahun 2020 tentang standar antropometri anak. 26. wiryadana ka, putra iwas, rahayu pds, pradnyana mm, purwanta mla, sudarmaja im. risk factors of soil-transmitted helminth infection among elementary school students. paediatrica indonesia. 2017;57(6): 295-302. 27. suryantari saa, satyarsa abs, hartawan ignbrm, parastuta iky, sudarmaja im. prevalence, intensity and risk factors of soil-transmitted helminths infections among elementary school students in ngis village, karangasem district, bali. indonesian journal of tropical and infectious disease. 2019;7(6):137-143. 28. budijanto, d. (maret, 2021). kebijakan program pencegahan dan pengendalian penyakit tular vektor dan zoonotik. slide dipresentasikan di seminar daring nasional p2ptvz kemenkes, jakarta. 29. arianto mf, kadir ar, maria il. pelaksanaan program eliminasi fi lariasis di kota sorong. tunas-tunas riset kesehatan. 2020;10(1). 30. kemenkes ri. peraturan menteri kesehatan republik indonesia nomor 94 tahun 2014 tentang penanggulangan filariasis. 2014. jakarta: kementerian kesehatan republik indonesia. 31. loukouri a, meite a, koudou bg, goss cw, lew d, weil gj, et al. impact of annual and semi-annual mass drug administration for lymphatic fi lariasis and onchocerciasis on hookworm infection in cote d’ivoire. plos negl trop dis. 2020;14(9): e0008642. 32. sunish ip, rajendran r, munirathinam a, kalimuthu m, kumar va, nagaraj j, tyagi bk. impact on prevalence of intestinal helminth infection in school children administered with seven annual rounds of diethyl carbamazine (dec) with albendazole. indian j med res. 2015;141:330-39. 93zukhaila salma, et al.: soil-transmitted helminthes infection and nutritional status ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 33. pion sds, chesnais cb, uvon npa, vlaminck j, abdou a, shako bk, simuna gk, tambwe jp, weil gj, boussinesq m. the impact of years of semiannual treatments with albendazole alone on lymphatic fi lariasis and soil-transmitted helminth infections: a community-based study in the democratic republic of the congo. plos negl trop dis. 2020;14(6): e0008322. 34. suraweera o, galgamuwa l, wickramasinghe s, iddawela d, nandasiri n. soil-transmitted helminth infections, associated factors and nutritional status in an estate community in sri lanka. sri lankan journal of infectious disease. 2018,8(2):100-14. 35. kurniati m, budiono, sulistyawati sw. intestinal protozoa infection in relation to nutritional status of the mandangin island elementary school 6 students in sampang regency. journal of aesculap medical science. 2019;10(1): 25-28. 36. stephenson ls, latham mc, ottesen ea. malnutrition and parasitic helminth infections. parasitology. 2000:121:s23-38. 37. ulijaszek sj. relationships between undernutrition, infection, and growth and development. human evolution. 1996;11:233-48. 38. utami ad, indarto d, dewi ylr. the effect of nutrient intake and socioeconomic factor toward stunting incidence among primary school students in surakarta. journal of epidemiology and public health. 2017;2(1);1-10. 39. babar nf, muzaff ar r, khan ma, imdad s. impact of socioeconomic factors on nutritional status in primary school children. j ayub med coll abbottabad. 2010:22(4):15-18. 40. kamiya y. socioeconomic determinants of nutritional status of children in lao pdr: eff ects of household and community factors. journal of health, population and nutrition. 2011:29(4):339-48. 41. kumar m, ji b, babaei p, das p, lappa d, ramakrishnan g, et al. gut microbiota dysbiosis is associated with malnutrition and reduced plasma amino acid levels: lessons from genome-scale metabolic modeling. metab eng. 2018; 49:128–42. 42. vonaesch p, randremanana r, gody jc, collard jm, giles-vernick t, doria m, et al. identifying the etiology and pathophysiology underlying stunting and environmental enteropathy: study protocol of the afribiota project. bmc pediatr. 2018; 18(1):236 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 138 vol. 1. no. 3 september–december 2010 m e c h a n i s m s o f p e r i o d o n t i t i s i n d u c e d atherosclerosis rikko hudyono1 and jenny sunariani2 1department of periodontology 2department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract nowadays cvd become the most common cause of death in us and worldwide. atherosclerosis plays an important role in cvds pathogenesis. atherosclerosis decreases the elasticity of the vascular. atherosclerosis shares the same risk factor as cvd, in which obesity, hyperlipidemia, hypertension and lack of physical activity may initiate it. however, 50% of all cvd patients are lack of the usual causes of cvd. the purpose of this review is to reveal the mechanism of periodontitis-induced atherosclerosis. inflammation and autoimmune disease might play an important role in initiate the cvd. periodontitis is one of the oral diseases which can cause systemic inflammation and may induce the atherosclerosis. porphyromonas gingivalis (pg) which is the major cause of periodontitis can induce it by expressing protein gp130 in its fimbriae. periodontics patients are prone to have bacteremia by daily routine oral hygiene activity. chronic bacteremia may alter the endothelial physiology, which is resulted in neointima formation, ec dysfunction, and lipid accumulation. it is concluded that periodontitis may play an important role in initiation and progression of atherosclerosis. key words: pg’s fimbriae, bacteremia, cytokines, endothelial dysfunction, atherosclerosis literature review introduction in the 20th century, due to developments and inventories in medical field, the human�s life expectancy was dramatically increased. there was a major shift in the causes of illness and death throughout the world. in 1950, infections were the most common causes of death. a century ago, cvd accounted for less than 10 percent of all deaths. nowadays, cvd become the most common cause of death in us[1] and worldwide.[2,3] it causes global epidemic worldwide.[4] approximately 30 percent of deaths worldwide are caused by cvd,[5] including nearly 40 percent in high-income countries and about 28 percent in lowand middle-income countries.[2] in 2006, it was reported that more than 81 million of us citizens got cvd.[6] driven by industrialization, urbanization, and associated life changes; e.c smoking, high calories and lipid intake, these ongoing transition are occurring around the world of all races, ethnic groups, and cultures at an even faster rate than last centuries and may lead to cause cvd.[7] the risk of having cvd will increase in obesity, hypertension and impairment of lipid metabolism.[2,7] atherosclerosis is mainly caused by decreasing in vascular elasticity and lumen.[8,9] atherosclerosis is known to share the same risk factors as cvd, which obesity, hyperlipidemic condition, hypertension and lack of physical activity may initiate it.[8] however, 50% of all patients with cvd were lack of known risk factors.[10] autoimmunity[11] and systemic inflammation[12-16] were supposed to be possible to play a role ini cvd�s initiation and progression. infection and bacterial products may play an important role in atherosclerosis pathogenesis.[17,18] immune response and inflammation factors, e.c. crp, interleukin, and chemokines were suggested to be the causes and markers in atherosclerosis lesions.[19,20] atherosclerosis had been proven to be induced by periodontopathogen from periodontitis.[21-28] inflammation in mouth may cause systemic inflammation, which is indicated by the elevation of crp in the body,[29,30] which is functioned as markers,[31,32] predictors of cvd,[33,34] and 139hudyono and sunariani: mechanisms of periodontitis-induced may induce atherosclerosis lesions.[35–38] focal infection in mouth may induce atherosclerosis lesions by initiate systemic and humoral immune responses.[39] periodontopathogen bacteria may widely spread to another part of the body. bacterial inoculation from atherosclerotic plaque have proven the presence of periodontopathogen bacteria such as porphyromonas gingivalis, actinobacillus actinomycetem comitans, dan bacteroides forsythus.[39,40] this article reviews the current state of knowledge concerning the direct role of periodontitis in developing atherosclerotic lesions. better understanding of these diseases and long-term research will be needed to rehabilitate these lesions in the future. literature review periodontitis is defined as an inflammatory disease of the supporting tissues of teeth caused by specific microorganism or group of specific microorganisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with pocket formation, recession or both.[41] chronic periodontitis is the most common form of periodontitis, which is associated with plaque accumulation. it generally has a slow to moderate rate of disease progression, but periods of more rapid destruction may be observed. systemic disease such as diabetes mellitus and hiv infection may influence the host defenses, and environmental factors such as cigarette smoking, and stress also may influence the response of the host to plaque accumulation.[41–44] although many factors may influence the onset of periodontitis, periodontopathogen bacteria play a key role in the onset and severity of periodontal diseases.[42,45,46] pg is an anaerob obligat,[47] non-motile,[48] pleiomorphic bacteria, and posses a capsul.[49] pg shows a strong proteolitic activity, grows in anaerobic environment, and shows dark pigmentation (brown, dark green, or black) on blood agar.[50] pg has fimbriae[51] which mediates adhesion.[52] the capsul serves a protection to prevent it from phagocytosis53 and triggers the secretion of il-1, il-6, dan il-8.[48] the end products are various kinds of amino acid and endotoxin, haemolysin, collagenase and proteases which may damage immunoglobulins, complements, and hemesequestering proteins: a protein which inhibits collagenase activities.[42,53,54] pg was shown to be able to invade epithelium,[51] soft tissue, inhibit pmn cell migration across the epithelium[55] and may cause cytokine degradation in mammal cells.[42,53] physiology of blood vessels arteries are strong, elastic vessels that are adapted for transporting blood away from the heart under high pressure to all parts of the body.[56] these vessels subdivide into progressively thinner tubes and eventualy give rise to the finer branched arteriolles.[56] the wall of an artery consists of three distinct layer, or tunics; tunica adventitia, tunica media and tunica intima as the innermost layer.[57-59] tunica adventitia is the outermost layer of the arterial wall. researches were conducted, as it was known to have a potential role in homeostasis and pathological effects on the artery.[8] this outer layer is thin and chiefly consists of irregular connective tissue[57] and collagenous fibers.[8] vasa vasorum and nerve endings are usually located on this outermost layer. this layer attaches the artery to the surrounding tissues.[57] cell populations in this layer are relatively rare compared with those in the other layer.[8] this layer mainly consists of fibroblast and mast cell.[8] in clinical research with animal, this layer was suspected to induce atheroma and aneurysm lesions.[8] tunica media, located between tunica intima and tunica adventitia, is the thickest layer of the arterial wall.[57] the artery, especially the aorta, is surrounded by tunica media, which has smc layer[57] and elastin as the extracellular matrix.[8] this structures make the artery very elastic and enable it to withstand against the force of blood pressure, and at the same time, tp stretch and accommodate the sudden increase in blood volume that accompanies ventricular contraction.[57] these structures are also important in maintaining the integrity of arterial branches.[8] on the smaller artery; where the smc in tunica media are not as strong as those in aorta; elastin are arranged in continuous layer, not in circular around the vessel wall.[8] on the capillaries, the tunica media becomes very thin, only single cell thick with some smc�s cells.[57] tunica intima is the innermost layer of the arterial wall.[8] the wall is covered with simple squamous epithelium attached to the basal lamina, fibrous connective tissue which is rich in elastic and collagen fibers,[57] known as endothelium.[56] in all newborn species, tunica intima is very thin.[8] however, in adult, its structure becomes more complicated and heterogen.[8] thin endothelial layer is attached to basalis membrane which is contain non-fibril collagen e.c collagen type iv,[8] proteoglycan (chondroitin and dermatan sulphate), elastin, protein plasma,[9] laminin, fibronectin, and the other extracellular matrixes. as becoming older, the intimal layer will be developed more complicated, where it will be contained smcs and fibril interstitial collagen, like tipe i and iii. the more complex intimal layer are known as intimal thickening, which is the common characteristic in adult�s vessels.[8] endothelial cells (ecs) ecs are the most important cells in tunica intima because they are fundamental to the maintenance of vessel wall homeostasis and normal circulatory function.[8,58] the endothelial lining of an artery provides a smooth surface that allows blood cells and platelets to flow through without being damaged.[57] ecs have five major role: 1) it is a metabolicaly active secretory tissue;[58] 2) to provide a smooth surface in the artery and secrete some anticoagulants and anti-thrombotic agents;[8,57] 3) as a barrier 140 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 138-145 to the indiscriminate passage of blood constituents into the arterial wall;[58,61] 4) to help in controlling growth and elasticity of the vessels;[61] and 5) to adjust the vascular tone by strictly regulating the paracrin and autocrine.[61] ecs may release vasoactive factors, which control the lokal vessel constriction and dilatation.[58] the vasodilator includes nitric oxide, prostacyclin, edrf, and edhf; and the vasoconstrictor includes endothelin, prostanoids and angiotensin ii.[58,59,61] ecs are also able to secrete some procoagulants agents as factor vii, factor va, factor von willebrand�s, tissue factor and pai-1;[58,61] and also some fibrinolitic agents as thrombomodulin, tissue plasminogen activator, heparin sulfate proteoglycan, which acts like heparin as a co-factor for antithrombin iii, a coagulationinhibitor by binding and inactivating thrombin.[8] no is known as a potent edrf[58] which inhibits vasoconstrictors� effects.[8] no is also inhibit platelets aggregation and adhesion, leukocyte infiltration and adhesion, and also smcs� migration and proliferation.[59] no is also able to prevent ldl oxidation.[61,62] no is produced in ecs by oxidating guanadino nitrogens larginine[58,61] which is catalyzed by enos enzyme in caveolae.[62] the presence of caveolin-1 proteins, which will bind the calmodulin, will inhibit the enos activity. chemically bond of ca2+ ion and calveolin will substitute caveolin-1 and thus increases no production.[59,62] some co factors like nadph,[59,62] bh4, [58,63] flavin nucleotide and oxygen molecule[64] are needed in no synthesis. bh4 is needed in electron transfer process from heme enzyme group in l-arginine to produce no.[59,62] in the atherosclerotic lesions, endothelium might be altered structurally and functionally. ecs may be more permeable to lipoproteins, hyperadhesive to leukocyte cells, and alter their homeostasis function in producing local pro and antithrombotic, growth factor stimulator and inhibitor, and also vasoactive enzyme. these alterations are known as endothelial disfunctions, which have a big impact in initiation, progression and complications of atherosclerotic lesions.[9,60] smooth muscle cell (smc) smcs have a lot of function in maintaining normal homeostasis vessels.[8] smc�s are responsible for vasoconstriction and dilation in response to normal or pharmacologic stimuli, homeostasis mechanisms to deliver blood to all parts of the body.[56,57] the arteries have more smcs than the veins do, which makes the arterial wall much thicker. smcs are innervated by simpathic nerves through adrenoreseptor with norepinepineprine as endogenous agonis. they also synthesize collagen, elastin, and proteoglicans; and elaborate growth factors and cytokines; which may alter morphology, proliferation rate and cell migration on the vessel�s wall.[65,66] smcs are involved in pathogenesis of atherosclerosis and become a target in cardiovascular management therapy.[8] in big arteries with atherosclerotic lesions, smc�s contraction will cause vasospasm and impede the blood flow.[8] normal smcs synthezise a lot of extracellular matrix to maintain normal homeostasis, and prevent atherosclerosis.[65] normally, smc�s rarely proliferate. the rate of cell proliferation and necrosis are very low under the normal condition. extracellular matrix will always be in homeostasis circumstances. synthesis and dissolution are always the same rate; there will never be cell accumulation or atrophy.[8] under the pathologic condition, the cells may proliferate and migrate; thus may induce hyperplastic lesions such as atherosclerosis and re-stenosis.[8] smcs� migration and proliferation are stimulated by pdgf, endothelin-1, thrombin, fgf, ifn-g, and il-1. on the contrary, no, heparin sulphate and tgf will inhibit this process.[66] atherosclerosis atherosclerosis is disease where the artery loses its elasticity.[9,55,56] atherosclerosis was defined as a chronic immunoinflamatory, fibroproliferative, disease which have been drived by lipids.[9] it affects primarily the intima of medium-sized and large arteries, resulting in intimal thickening, and may lead to luminal narrowing and inadequate blood supply.[9] endothelial disfunction is the main cause of this disease.[56] atherosclerosis forms atherosclerotic plaques,[56,66] which in turn, will cause lumen narrowing,[9] and obstruction;[66] weaken the big and medium-sized arterial structure;[9] impede the blood flow;[56] and reduce its elasticity.[56] as the name implies, mature atherosclerotic plaques consist typically of two main components: one is lipid-rich and soft (athére is greek for ‘gruel� or ‘porridge�) and the other is collagen-rich and hard (skleros is greek for ‘hard�).[9,66] the flow-limiting potential of an intimal plaque may be modified by reactive changes in the underlying media and adventitia that may be attenuate (positive remodeling) or accentate (negative remodeling) the luminal obstruction and consequent hemodynamic impact of the plaque.[9] furthermore, enhanced vasoconstriction and reduced vasodilator capacity associated with atherosclerotis can further contribute an additional dynamic component to luminal obstruction.[9] the aggregation of lipoprotein on the tunica intima is considered as the early step of atherosclerosis. on this early step, atherosclerosis will apparent as a fatty streak consists of foam cells filled with lipid.[8,9,60,66,67] lipoprotein will bind proteoglycan on the tunica intima where it will be stabilized on this layer. proteoglycan-binded lipoprotein will be oxydated easier and undergo chemically alterations which were believed to be the early pathogenesis of atherosclerosis.[8,9] the other researches showed that the increasing of endothelial permeability mainly caused ldl aggregation in the intima.[9] some factors, i.e nadh/nadph oxydase which is released by vascular cells; lypoxigenase that is released by infiltrating leukocytes; and myeloperoxidase may cause oxidative stress in the atheroma.[9] atherosclerosis lesions is also composed of leukocytes accumulation as a result of endothelial dysfunction.[8] normal endothelial cells are able to prevent leukocytes 141hudyono and sunariani: mechanisms of periodontitis-induced adhesion on their surfaces.[56] eventhough in the inflamed area, leukocytes infiltration is started in venous, not in the artery.[9] in hypercholesterolemic condition, leukocytes adhere on the endothel and have a diapedisis on the ec junction into the tunica intima, where these leukocytes start lipid accumulation and become foam cells.[9,66] besides the monocytes, lymphocytes t also tend to accumulate in atherosclerotic lesion in human and animals.[8,9] accumulation of monocyte and lymphocyte t were stimulated by leucocyte adhesion melocule secreted by ec surfaces.[8,9,66,68] discussion in many epidemiological studies, periodontitis was proven to play important roles in initiation and progression of cvd,[69,70] by chronic infection on the blood vessels[20–29] or by the elevation of body�s crp level.[29–39] the prevalence of chronic periodontitis was very high among populations, especially in chronic form.[41–44,69–72] chronic periodontitis is usually neglected and undetected, because it is lacked any clinical signs and symptoms.[41,42,71] in chronic gingival and periodontal infection, the cappilarries are more fragile, which make it possible for microorganisms in plaque and calculus to be spread along with blood flow.[73] chronic bacteremia from periodontitis may be easily happened from the daily activities e.c brushing, chewing,[73] and routine dental procedures like scaling and root planning, or the other treatments like endodontic, orthodontic and dental extraction.[74] many studies proved that atherosclerosis plaques contained numerous periodontopathogen bacteria,[3,40,76] especially pg.[39,40] researches have demonstrated that pg induction may invade endothel and may initiate atherosclerosis in pigs.[75] the presences of pg in atheroma and human carotid aorta had been detected by immunostaining and pcr.[76,77] pg was known to have fimbriae, which allowed it to invade[51] and stimulate host response to produce citokynes,[52,78-81] and may be in latent phase[82] to cause chronic infection in ec and smc.[75] chronic infection was known to be able to cause endothelial dysfunction.[21–38] pg�s fimbriae secretes protein, called gp130,[83] which facilitate pg to invade ec and trigger celluler immune response.[84] the host will secrete tnf,[85] il-1, il-6, il10, and il-12[86–88] by tlr�s stimulation.[26,85,88–90] tlr is part of immune system, which will respond to pamp.[91] protein in pg�s fimbriae may act as pamp which triggers immune response by activating tlr, which, hence stimulate the host to produce cytokines.[86,91] tunica intima thickening atherosclerosis may emerge from physiologic changes in ec. in early phase, atherosclerotic lesions is started by thickening of tunica intima (neointima).[8] epidemiologic studies reported a positive relationship between pg infection and the formation of neointima.[92] mechanism of pg infection and neointima thickening was remained unclear. it was supposed that tnf stimulation by protein gp130[83] might facilitate lymphocyte and monocyte adhesion[85] and also stimulate cytokines and growth hormone in host cells.[86–93] neointima formation is mainly caused by accelerating proliferation rate, inhibiting apoptotic process, and increasing smc migration to the neointima layer.[8] in ec, pg invasion may accelerate tnf[94] and il-6 synthesis,[68] which in turn may initiate atherosclerosis lesions by accelerating smc proliferation,[68] stimulating tissue factor,[95] increasing platelet aggregation[95] and increasing the level of fibrinogen in blood.[93] tnf may initiate neointima hyperplation through p55 pathway.[94] tnf was also known to induce fgf and nfkb secretion, smc proliferation and neointima formation.[96] nfkb may inhibit apoptotic process[97–99] by suppressing the activity of gen p53,[100] which is responsible to induce the apoptotic process.[101] its mechanism was remained unclear, but it was assumed to have the same mechanism as gen ie from cmv. gen ie was able to bind gen p53 and disturb transcription process by extracting this gen from nucleus by cytoplasmic sequestration process.[102] growth hormone, from tnf induction may increase proliferation rate.[96] rupture endothel will secrete mcsf,[103,104] which will increase fibroblast proliferation rate, increase the production of il-1,[105] induce the synthesis of vasoactive factors, growth factor, vascular adhesion molecules, and chemokines.[106] infection may increase the production of fgf and pdgf almost twice higher.[68] mcsf and il-1 induction in proliferation process of fibroblast and smc were supposed to be performed via cyclooxygenase pathway.[68,107] the formation of neointima mass is also caused by smcs migration from tunica media and tunica adventitia into the tunica intima.[8–10] infections may increase pdgf reseptors sensitivity which result in smc thickening in tunica intima. besides fgf and pdgf, there some factors are known to play roles in smc migration, they include endothelin-1, thrombin, ifn-g, tgf and il-1.[8,9,58,59,61,66] whereas no, heparat sulphate, and tgf-b will act as antagonists to inhibit smc migration.[8,9,61,62] injured ecs[68] and the presences of either tnf[108] or crp[109] may trigger the synthesis of cell adhesion molecules e.c vcam-1 and icam-1.[68] vcam-1 will interact with vla-4, which is exclusively sinthezised by monocyte, t cell, and leukocyte accumulated in atheroma.[8] vcam will facilitate monocyte adhesion[8] and infiltration into injured arterial wall and may increase smcs proliferation rate.[66,109] icam-1 is immunoglobulin secreted by ecs surfaces. the role of this molecule is remained unclear as it is produced only in very small amount, and the leukocyte which will be bound is remained unknown.[8] it was supposed that icam-1 will increase vcam-1 production.[110] once the leukocyte binds the ec, it needs a signal to penetrate into ec and enter the arterial wall.[8] leukocyte 142 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 138-145 migration would be impossible without the presence of protein molecules known as chemoattractant cytokine or chemokines. at the early phase of atherosclerosis, chemokines attract monocyte into the atheroma.[8] mcp-1 facilitate monocyte chemotactic into the arterial wall.[8,66] mcp-1 is a kind of chemokines produced by ecs as a response to crp,[111] mcsf,[68] and oxidized lipoprotein, and the other stimulus.[112,113] the disturbances in local blood flow may result in some physiologic changes, which are responsible for predilection lesion of atherosclerosis.[8,9,58,66] atheroma may act as an obstacle in local blood flow and causes local blood turbulances that will inhibit ecs to produce superoxide dismutase enzyme and enos, which are known to have a protective effect against the atherosclerosis.[58,62,66,101] superoxide dismutase enzyme is able to reduce oxidative stress by catalyzing catabolic reaction on reactive superoxide anion into oxygen and hydrogen peroxide, where this hydrogen peroxide will be converted to be water and oxygen.[58,101] enos produces no as an endogenous vasodilator.[62] besides as vasodilator, it also suppresses vcam-1 production from inflammation-induced ecs.[8,114] no may also act as anti-inflammatory agent by increasing ikba production,[115] an intracellular inhibitor, which will disturb the nfkb transcription process.[8,115,116] nfkb regulates various genes, which are responsible in inflammatory process and especially in atherosclerosis.[8,114-116] endothelial dysfunction prolonged infection in blood vessels may result in endothelial dysfunction.[8-10,17,59-62] normal ecs posses an antithrombotic effect, which make them able to release and synthezise substances as heparin sulphate pgi2 , no, plasminogen activator, and thrombomodulin.[8,9,59-62] infectious agent may change ecs phenotype, from anticoagulant in usual, to be procoagulant.[8-10,62,66,117] bacteria and its product, endotoxin, may cause endothel to produce tissue factor which in turn it will activate extrinsic blood clotting cascade,[117] increase thrombin formation, and platelets aggregation and at the same time these infections may suppress the synthesis of pgi2, and thrombomodulin.[8,9] inhibition of prostacyclin happened as the presence of crp which is suppressed pgi2 production which may cause disturbance in tbb2/pgi2 ratio. [35] the disturbance in the ratio of tbb2/pgi2 may facilitate platelets aggregation.[35] the other important role of ec is to have a local vasodilatation.[8-10] a pilot study had demonstrated that infection might disturb local endothelial vasodilatation response. this dysfunction is mainly caused by the disturbance in no and non-no pathways. disturbances in no pathways automatically will increase platelet aggregation, leucocyte adhesion and smcs proliferation.[61] decreased no production will increase ldl oxidation, where oxidated ldl may increase caveolin-1 production and inhibit no synthesis by inactivating enos.[62] macrophage is also able to produce ros10 which will inactivate no[118,119] and destroy bh4. [62] vascular damage was believed to induce atherosclerosis and be responsible for its progression.[8–10,66] in in-vitro study, it was found that pg was able to adhere and invade on vascular wall,[52,78–81] which was indicated that pg may cause vascular damages.[82] endothel damage may cause ecm, beneath it, become exposed. platelets may have a direct contact to ecm on that area which makes them active.[117] activated platelets will stimulate intrinsic pathway of coagulation cascade and activate fibrin-forming process.[117] lipid accumulation the presence of infection may facilitate lipid accumulation. infection was known to be able to reduce cholesterol ester hydrolytic activity[120,121] and increase the scavenger reseptor susceptibility.[122] infection on human smcs may increase ldl oxidation mediated by scavenger reseptor. foam cells accumulation and the level of cholesteryl ester will dramatically increase if infected macrophages are incubated in an area with a high ldl level.[122] ros may cause ldl oxidation in arterial wall, and then the oxidated ldl,[62] mediated by scavenger receptor, will be absorbed by macrophage and form foam cells.[123] mcsf may increase cholesterol uptake by macrophage and delayed the apoptosis process, which may cause foam cells forming.[68] it was summarized that atherosclerosis may be periodontically induced. pg, one of the periodontopathogens, was supposed to induce atherosclerosis via bacteremia. pg with its fimbriae may invade and stimulate various kinds of cytokines, which are caused neointima proliferation, endothelial dysfunction, and lipid accumulation. these were facilitated with endothelial physiologic switching that tends to be pro-thrombotic; lipoprotein accumulation, especially ldl; chemically altered ldl via oxidation; monocytes and platelets adhesion on the vessel�s wall; and also the inflammation factors released from platelets and macrophages. this review was not subjected to prove that periodontitis was the main cause of atherosclerosis. it was supposed to increase awareness of periodontitis as a risk and a predisposing factor for atherosclerosis. although this issue had not been clinically proven, but in my opinion it was important to reduce any oral-origin infection as our effort to maintain healthy mouth and reduce the risk factors for atherosclerosis, otherwise we might miss a chance to help our patients suffering from cardiovascular disease. references 1. neyer jr, greenlund kj, denny ch, keenan nl, labarthe dr, croft jb. prevalence of heart disease – united states. cdc 2007; 56(06): 113–8. 143hudyono and sunariani: mechanisms of periodontitis-induced 2. gaziano jm. fundamentals of cardiovascular disease. in: libby p, bonow ro, mann dl, zipes dp. braunwald�s heart disease: a textbook of cardiovascular medicine. 8th ed. usa: saunders; 2008. p. 1–21. 3. vasan rs, benjamin ej, sullivan lm, d�agostino rb. the burden of increasing worldwide cardiovascular disease. in: fuster v, alexander rw, o�rourke ra. hurst�s the heart. 11th ed. new york: mcgrawhill med publ; 2004. p. 15–6. 4. world health organization. the world health report 2002: reducing risks, promoting healthy life. geneva: who; 2002. 5. bonow ro, smaha la, smith sc. world heart day 2002: the international burden of cardiovascular disease: responding to the emerging global epidemic. circulation 2002; 106(13): 1602–5. 6. american heart association. cardiovascular disease statistics. accessed from http://www.americanheart.org on may 5th, 2010. 7. kristensen pl, wedderkopp n, møller nc, andersen lb, bai cn, froberg k. tracking and prevalence of cardiovascular disease risk factors across socio-economic classes: a longitudinal substudy of the european youth heart study. bmc public health 2006; 6:20 accessed from http://www.biomedcentral.com/1471-2458/6/20 on may 5th, 2010. 8. libby p. the vascular biology of atherosclerosis. in: libby p, bonow ro, mann dl, zipes dp. braunwald�s heart disease: a textbook of cardiovascular medicine. 8th ed. usa: saunders; 2008. p. 985–1002. 9. falk e, shah pk, fuster v. atherothrombosis and thrombosis-prone plaques. in: fuster v, alexander rw, o�rourke ra. hurst�s the heart. 11th ed. new york: mcgraw-hill med publ; 2004. p. 1123–40. 10. epstein se, zhou yf, zhu j. infection and atherosclerosis emerging mechanistic paradigms. j of am heart assc circulation 1999; 100: e20–e28. 11. shoenfeld y, gerli r, doria a, matsuura e, cerinic mm, ronda n, jara lj, abu-shakra m, meroni pl, dan sherer y. accelerated atherosclerosis in autoimmune rheumatic diseases. circulation 2005; 112: 3337–47. 12. kiechl s, egger g, mayr m, wiedermann cj, bonora e, oberhollenzer f, muggeo m, xu q, wick g, poewe w, willeit j. chronic infections and the risk of carotid atherosclerosis: prospective results from a large population study. circulation 2001; 103: 1064–70. 13. destefano f, anda rf, kahn hs, williamson df, russell cm. dental disease and risk of coronary heart disease and mortality. bmj 1993; 306: 688–91. 14. mattila kj, nieminen ms, valtonen vv, rasi vp, kesäniemi ya, syrjälä sl, jungell ps, isoluoma m, hietaniemi k, jokinen mj. association between dental health and acute myocardial infarction. bmj 1989; 298: 779–81. 15. stein jm, kuch b, conrads g, fickl s, chrobot j, schulz s, ocklenburg c, smeets r. clinical periodontal and microbiologic parameters in patients with acute myocardial infarction. j periodontol. 2009; 80(10): 1581–9. 16. paul a, ko kws, li l, yechoor v, mccrory ma, szalai aj, chan l. c-reactive protein accelerates the progression of atherosclerosis in apolipoprotein e–deficient mice. circulation 2004; 109: 647–55. 17. prasad a, zhu j, halcox jpj, waclawiw ma, epstein se, quyyumi aa. predisposition to atherosclerosis by infections: role of endothelial dysfunction. circulation 2002; 106: 184–90. 18. klein ec, rupprecht hj, blankenberg s, bickel c, kopp h, rippin g, victor a, hafner g, schlumberger w, meyer j. impact of infectious burden on extent and long-term prognosis of atherosclerosis. circulation 2002; 105: 15–21. 19. tzoulaki i, murray gd, lee aj, rumley a, lowe gd, fowkes gr. c-reactive protein, interleukin-6, and soluble adhesion molecules as predictors of progressive peripheral atherosclerosis in the general population: edinburgh artery study. circulation 2005; 112: 976–83. 20. fruchart jc, nierman mc, stroes esg, kastelein jjp, duriez p. new risk factors for atherosclerosis and patient risk assessment. circulation 2004; 109: iii-15-iii-19. 21. dietrich t, jimenez m, kaye eak, vokonas ps, garcia ri. agedependent associations between chronic periodontitis/edentulism and risk of coronary heart disease. circulation 2008; 117: 1668–74. 22. beck jd, garcia r, heiss g, vokonas ps, offenbacher s. periodontal disease and cardiovascular disease. j periodontol. 1996; 67(10 suppl): 1123–37. 23. beck jd, offenbacher s. relationships among clinical measures of periodontal disease and their associations with systemic markers. ann periodontol. 2002 dec; 7(1): 79–89. 24. beck jd, pankow j, tyroler ha, offenbacher s. dental infections and atherosclerosis. am heart j. 1999; 138(5 pt 2): s528–33. 25. mattila kj, asikainen s, wolf j, jousimies-somer h, valtonen v, nieminen m. age, dental infections, and coronary heart disease. journal of dental research 2000; 79(2): 756–60. 26. gibson, iii fc, hong c, chou hh, yumoto h, chen j, lien e, wong j, genco ca. innate immune recognition of invasive bacteria accelerates atherosclerosis in apolipoprotein e-deficient mice. circulation 2004; 109: 2801–6. 27. humagain m, nayak dg, uppoor as. periodontal infections and cardiovascular disease: is it a mere association? kathmandu university medical journal 2006: 4(3): 379–82. 28. friedewald ve, kornman ks, beck jd, genco r, goldfine a, libby p, offenbacher s, ridker pm, van dyke te roberts wc. the american journal of cardiology and journal of periodontology editors' consensus: periodontitis and atherosclerotic cardiovascular disease. journal of periodontology and the american journal of cardiology 2009. 29. pitiphat w, savetsilp w, wara-aswapati n. c-reactive protein associated with periodontitis in a thai population. j clin periodontol. 2008; 35(2): 120–5. 30. noack b, genco rj, trevisan m, grossi s, zambon jj, de nardin e. periodontal infections contribute to elevated systemic c-reactive protein level. j periodontol. 2001; 72(9): 1221–7. 31. ridker pm, cushman m, stampfer mj, tracy rp, hennekens ch. plasma concentration of c-reactive protein and risk of developing peripheral vascular disease. circulation 1998; 97: 425–8. 32. schillinger m, exner m, mlekusch w, sabeti s, amighi j, nikowitsch r, timmel e, kickinger b, minar c, pones m, lalouschek w, rumpold h, maurer g, wagner o, minar e. inflammation and carotid artery—risk for atherosclerosis study (icaras). circulation 2005; 111: 2203–9. 33. hommels mj, van der ven aj, kroon aa, kessels ag, van dieijenvisser mp, van engelshoven ja, bruggeman ca, de leeuw pw. c-reactive protein, atherosclerosis and kidney function in hypertensive patients. journal of human hypertension 2005; 19: 521–6. 34. schillinger m, exner m, amighi j, mlekusch w, sabeti s, rumpold h, wagner o, minar e. joint effects of c-reactive protein and glycated hemoglobin in predicting future cardiovascular events of patients with advanced atherosclerosis. circulation 2003; 108: 2323–8. 35. venugopal sk, devaraj s, jialal i. c-reactive protein decreases prostacyclin release from human aortic endothelial cells. circulation 2003; 108: 1676–8. 36. pasceri v, willerson jt, dan yeh eth. direct proinflammatory effect of c-reactive protein on human endothelial cells. circulation 2000; 102: 2165–8. 37. messer jv. c-reactive protein and cardiovascular disease. circulation 2006; 114: e253–e254. 38. khera a, de lemos ja, peshock rm, lo hs, stanek hg, murphy sa, wians fh, grundy sm, mcguire dk. relationship between c-reactive protein and subclinical atherosclerosis. the dallas heart study. circulation 2006; 113: 38–43. 39. haynes wg, stanford c. periodontal disease and atherosclerosis: from dental to arterial plaque. arterioscler thromb vasc biol 2003; 23: 1309–11. 40. ishihara k, nabuchi a, ito r, miyachi k, kuramitsu hk, okuda k. correlation between detection rates of periodontopathic bacterial dna in carotid coronary stenotic artery plaque and in dental plaque samples. journal of clin microbiology 2004; 42(3): 1313–5. 41. mueller hp. periodontology the essentials. germany: georg thieme verlag; 2005. p. 31–6, 56. 42. vernino ar. etiology of periodontal disease. in: vernino ar, gray j, hughes e. the periodontic syllabus. 5th ed. philadelphia: lippincott williams and wilkins; 2008. p. 15–25. 144 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 138-145 43. taggart ej, perry da in perry da, beemsterboer pl. periodontology for the dental hygienist. 3rd ed. missouri: elsevier pub; 2007. p. 124–53. 44. novak mj. classification of diseases and conditions affecting the periodontium. in: newman mg, takei hh, klokkevold pr, carranza fa. clinical periodontology. 10th ed. st. louis: saunders; 2006. p. 100–9. 45. torrungruang k, bandhaya p, likittanasombat k, grittayaphong c. relationship between the presence of certain bacterial pathogens and periodontal status of urban thai adults. j periodontol. 2009; 80(1): 122–9. 46. yano-higuchi k, takamatsu n, he t, umeda m, ishikawa i. prevalence of bacteroides forsythus, porphyromonas gingivalis and actinobacillus actinomycetemcomitans in subgingival microflora of japanese patients with adult and rapidly progressive periodontitis. j clin periodontol. 2000; 27(8): 597–602. 47. shah hn, gharbia se. progress in the identification of nonmotile bacteroidaceae from dental plaque. clin infectious disease 1994; 18(suppl 4): s287–92. 48. brunner j, scheres n, el idrissi nb, deng dm, laine ml, van winkelhoff aj, crielaard w. the capsule of porphyromonas gingivalis reduces immune response of human gingival fibroblast. bmc microbiology 2010;10(5). available at: fromfrom http://www. biomedcentral. com/1471-2180/10/5. 49. nelson ke, fleischmann rd, deboy rt, paulsen it, fouts de, eisen ja, daugherty sc, dodson rj, durkin as, gwinn m, haft dh, kolonay jf, nelson wc, mason t, tallon l, gray j, granger d, tettelin h, dong h, galvin jl, duncan mj, dewhirst fe, fraser cm. complete genome sequence of the oral pathogenic bacterium porphyromonas gingivalis strain w83. j bacteriol. 2003; 185(18): 5591–601. 50. holt sc, kesavalu l, walker s, genco ca. virulence factors of porphyromonas gingivalis. periodontol 2000; 20: 168–238. 51. sojar ht, sharma a, dan genco rj. porphyromonas gingivalis fimbriae bind to cytokeratin of epithelial cells. infection and immunity 2002; 70(1): 96–101. 52. graves dt, naguib g, lu h, desta t, amar s. porphyromonas gingivalis fimbriae are pro-inflammatory but do not play a prominent role in the innate immune response to p. gingivalis. journal of endotoxin research. 2005; 11(1): 13–8. 53. quirynen m, teughels w, haake sk, newman mg. microbiology in periodontal diseases. in: newman mg, takei hh, klokkevold pr, carranza fa. clinical periodontology. 10th ed. st. louis: saunders; 2006; p. 134–69. 54. tanamoto k, azumi s, haishima y, kumada h, umemoto t. endotoxic properties of free lipid a from porphyromonas gingivalis. microbiology. 1997; 143: 63–71. 55. andrian e, grenier d, rouabhia m. porphyromonas gingivalisepithelial cell interactions in periodontitis. j of dental research. 2006; 85(5): 392–403. 56. fox si. human physiology. 8th ed. new york: mcgraw-hill comp; 2004. p. 364–403. 57. shier d. hole�s human anatomy and physiology. 9th ed. new york: mcgraw-hill med publ; 2002. p. 608–11. 58. griendling kk, harrison dg, alexander rw. biology of the vessel wall. in: fuster v, alexander rw, o�rourke ra. hurst�s the heart. 11th ed. new york: mcgraw-hill med publ. 2004; pp. 135–54. 59. hajjar ka. the endothelium in thrombosis and hemorrhage. in: loscalzo j, schafer ai eds. thrombosis and hemorrhage. 3rd ed. philadelphia: lippincott williams & wilkins. 2003; pp. 207–19. 60. gokce n, vita ja. clinical manifestations of endothelial dysfunction. in: loscalzo j, schafer ai eds. thrombosis and hemorrhage. 3rd ed. philadelphia: lippincott williams & wilkins. 2003; pp. 685–701. 61. konkle ba, simon d, schafer ai. hemostasis, thrombosis, fibrinolysis, and cardiovascular disease. in libby p, bonow ro, mann dl, zipes dp. braunwald�s heart disease a textbook of cardiovascular medicine 8th ed. usa: saunders 2008; pp. 2049–75. 62. davignon j, ganz p. role of endothelial dysfunction in atherosclerosis. circulation 2004; 109: iii-27–iii-32. 63. antoniades c, shirodaria c, crabtree m, rinze r, alp n, cunnington c, diesch j, tousoulis d, stefanadis c, leeson p, ratnatunga c, pillai r, channon km. altered plasma versus vascular biopterins in human atherosclerosis reveal relationships between endothelial nitric oxide synthase coupling, endothelial function, and inflammation. circulation 2007; 116: 2851–9. 64. johns dg, cohen ra. endothelium-derived vasoactive factors. in: loscalzo j, schafer ai eds. thrombosis and hemorrhage. 3rd ed. philadelphia: lippincott williams & wilkins. 2003; pp. 278–93. 65. rensen ssm, doevendans pa, dan van eys gj. regulation and characteristics of vascular smooth muscle cell phenotypic diversity. neth heart j. 2007; 15(3): 100–8. 66. blood vessels. in: kumar v, abbas ak, fausto n. robins and cotran pathologic basis of disease. 7th ed. philadelphia: saunders. 2005; pp. 512–24. 67. leary mc, caplan lr. cerebrovascular disease and neurologic manifestations of heart disease. in: fuster v, alexander rw, o�rourke ra. hurst�s the heart. 11th ed. new york: mcgraw-hill med publ. 2004; pp. 2333–7. 68. ikonomidis i, andreotti f, economou e, stefanadis c, toutouzas p, dan nihoyannopoulos p. increased proinflammatory cytokines in patients with chronic stable angina and their reduction by aspirin. circulation 1999; 100: 793–798. 69. papapanou pn. periodontal diseases: epidemiology.papapanou pn. periodontal diseases: epidemiology. periodontal diseases: epidemiology. ann periodontol. 1996 nov; 1(1): 1–36. 70. irfan um, dawson dv, bissada nf. epidemiology of periodontal disease: a review and clinical perspectives. j int acad periodontol. 2001; 3(1): 14–21. 71. demmer rt, desvarieux m. periodontal infections and cardiovascular disease: the heart of the matter. j am dent assoc 2006; 137: 14s–20s. 72. research, science and therapy committee of the american academy of periodontology. epidemiology of periodontal diseases. j periodontol 2005; 76: 1406–1419. 73. geerts so, nys m, de mp, charpentier j, albert a, legrand v, rompen eh. systemic release of endotoxins induced by gentle mastication: association with periodontitis severity. j periodontol. 2002; 73(1): 73–8. 74. parahitiyawa nb, jin lj, leung wk, yam wc, dan samaranayake lp. microbiology of odontogenic bacteremia: beyond endocarditis. clin microbiology review 2009; 22(1): 46–64 75. brodala n, merricks ep, bellinger da, damrongsri d, offenbacher s, beck j, madianos p, sotres d, chang yl, koch g, nichols tc. porphyromonas gingivalis bacteremia induces coronary and aortic atherosclerosis in normocholesterolemic and hypercholesterolemic pigs. arterioscler thromb vasc biol. 2005; 25: 1446–1451. 76. haraszthy vi, zambon jj, trevisan m, zeid m, genco rj: identification of periodontal pathogens in atheromatous plaques. j periodontol 2000, 71(10): 1554–1560. 77. chiu b. multiple infections in carotid atherosclerotic plaque. am heart j. 1999; 138: s534–s536. 78. grenier d, tanabe s. porphyromonas gingivalis gingipains trigger a proinflammatory response in human monocyte-derived macrophages through the p38a mitogen-activated protein kinase signal transduction pathway. toxins 2010; 2(3): 341–352. 79. hajishengallis g, wang m, harokopakis e, triantafilou m,d dan triantafilou k. porphyromonas gingivalis fimbriae proactively modulate _2 integrin adhesive activity and promote binding to and internalization by macrophages. infection and immunity. 2006; 74(10): 5658–66. 80. ozaki k, hanazawa s. porphyromonas gingivalis fimbriae inhibit caspase-3-mediated apoptosis of monocytic thp-1 cells under growth factor deprivation via extracellular signal-regulated kinase-dependent expression of p21 cip/waf1. infection and immunity. 2001; 69(8): 4944–50. 81. wang m, shakhatreh ma, james d, liang s, nishiyama s, yoshimura f, demuth dr, dan hajishengallis g. fimbrial proteins of porphyromonas and exploit tlr2 and complement gingivalis mediate in vivo virulence receptor 3 to persist in macrophages. j. immunol. 2007; 179: 2349–58. 82. li l, michel r, cohen j, decarlo a, dan kozarov e. intracellular survival and vascular cell-to-cell transmission of porphyromonas gingivalis. bmc microbiology 2008; 8: 26. downloaded from: http://www.biomedcentral.com/1471-2180/8/26. 145hudyono and sunariani: mechanisms of periodontitis-induced 83. ho ys, lai mt, liu sj, lin ct, naruishi k, takashiba s, chou hh. porphyromonas gingivalis fimbriae-dependent interleukin-6 autocrine regulation by increase of gp130 in endothelial cells. j periodontal res. 2009; 44(4): 550–6. 84. chou hh, yumoto h, davey m, takahashi y, miyamoto t, gibson iii fc, dan genco ca. porphyromonas gingivalis fimbria-dependent activation of inflammatory genes in human aortic endothelial cells. infection and immunity 2005; 73(9): 5367–78. 85. davey m , liu x, ukai t, jain v, gudino c, gibson iii fc, golenbock d, visintin a, dan genco ca. bacterial fimbriae stimulate proinflammatory activation in the endothelium through distinct tlrs. the journal of immunology 2008; 180: 2187–95 86. jotwani r, cutler cw. fimbriated porphyromonas gingivalis is more efficient than fimbria-deficient p. gingivalis in entering human dendritic cells in vitro and induces an inflammatory th1 effector response. infection and immunity. 2004; 72(3): 1725–32. 87. kleemann r, zadelaar s, dan kooistra t. cytokines and atherosclerosis: a comprehensive review of studies in mice. cardiovascular research 2008; 79: 360–76. 88. zhang d, chen l, li s, gu z, dan yan j. lipopolysaccharide (lps) oflipopolysaccharide (lps) of porphyromonas gingivalis induces il-1b, tnf-a and il-6 production by thp-1 cells in a way different from that of escherichia coli lps. innate immunity. 2008; 14(2): 99–107. 89. yang rb, mark mr, gurney al, dan godowski pj. signaling events induced by lipopolysaccharide-activated toll-like receptor 2. the journal of immunology. 1999; 163: 639–643. 90. burns e, eliyahu t, uematsu s, akira s, dan nussbaum g. tlr2dependent inflammatory response to porphyromonas gingivalis is myd88 independent, whereas myd88 is required to clear infection. the journal of immunology, 2010, 184, 1455–1462. 91. hajishengallis g, sojar. h, genco rj, dan denardin e. intracellular signaling and cytokine induction upon interactions of porphyromonas gingivalis fimbriae with pattern-recognition receptors. immunological investigations 2004; 33(2): 157–172. 92. beck jd, elter jr, heiss g, couper d, mauriello sm, offenbacher s. relationship of periodontal disease to carotid artery intima-media wall thickness the atherosclerosis risk in communities (aric) study arteriosclerosis, thrombosis, and vascular biology. 2001; 21: 1816. 93. abbas a, lichtman ah, pober js. cellular and molecular immunology. 3rd ed. usa: wb saunders comp. 1997; pp. 250–76. 94. zhang l, peppel k, brian l, chien l, freedman nj. vein graft neointimal hyperplasia is exacerbated by tumor necrosis factor receptor-1 signaling in graft-intrinsic cells. arterioscler thromb vasc biol. 2004; 24(12): 2277–83. 95. paoletti r, gotto am, hajjar dp. inflammation in atherosclerosis and implications for therapy. circulation 2004; 109: iii-20–iii-26 96. chan j, lourenco lp, khachigian lm, bennett mr, dibartolo ba, kavurma mm. trail promotes vsmc proliferation and neointima formation in a fgf-2–, sp1 phosphorylation–, and nfkb-dependent manner. circulation research. 2010; 106: 1061. 97. plümpe j, malek np, bock ct, rakemann t, manns mp, dan trautwein c. nf-kb determines between apoptosis and proliferation in hepatocytes during liver regeneration. am j physiol gastrointest liver physiol 2000; 278: g173–g183. 98. bellas re, fitzgerald mj, fausto n, dan sonenshein ge. inhibition of nf-kb activity induces apoptosis in murine hepatocytes. american journal of pathology 1997; 151: 891–896. 99. ryan km, ernst mk, rice nr, dan vousden kh. role of nf-kb in p53-mediated programmed cell death. nature 2000; 404: 892–897. 100. schwarz em, badorff c, hiura ts, wessely r, badorff a, verma im, dan knowlton ku. nf-kb-mediated inhibition of apoptosis is required for encephalomyocarditis virus virulence: a mechanism of resistance in p50 knockout mice. j virol 1998; 72(7): 5654–5660. 101. cobb jp, hotchkiss rs, karl ie, dan buchman tg. mechanisms of cell injury and death. brit j of anaest. 1996; 77: 3–10 102. kovacs a, weber ml, burns lj, jacob hs, vercellotti gm. cytoplasmic sequestration of p53 in cytomegalovirus-infected human endothelial cells. am j pathol. 1996; 149: 1531–1539. 103. clinton sk, underwood r, hayes l, sherman ml, kufe dw, libby p. macrophage colony-stimulating factor gene expression in vascular cells and in experimental and human atherosclerosis. am j pathol. 1992; 140(2): 301–16. 104. eubank td, galloway m, montague cm, waldman wj, dan marsh cb. m-csf induces vascular endothelial growth factor production and angiogenic activity from human monocytes. the journal of immunology 2003; 171: 2637–43. 105. kaushansky k, lin n, adamson jw. interleukin-1 stimulates fibroblasts to synthesize granulocyte-macrophage and granulocytecolony stimulating factors. j clin invest. 1988; 81: 92–7. 106. chi h, messas e, levine ra, graves dt and amar s. interleukin1 receptor signaling mediates atherosclerosis associated with bacterial exposure and/or a high-fat diet in a murine apolipoprotein e heterozygote model: pharmacotherapeutic implications. circulation 2004; 110: 1678–168. 107. kleenmann r, zadelaar s, kooistra t. cytokines and atherosclerosis: a comprehensive review of studies in mice. cardiovascular research 2008; 79: 360–76. 108. henninger dd, panes j, eppihimer m, russell j, gerritsen m, anderson dc, dan granger dn. cytokine-induced vcam-1 and icam-1 expression in different organs of the mouse. j of immunology 1997; 158(4): 1825–1832. 109. ley k, dan huo y. vcam-1 is critical in atherosclerosis. j. clin. invest. 2001; 107(10): 1209–10. 110. lawson c, ainsworth m, yacoub m, dan rose m. ligation of icam1 on endothelial cells leads to expression of vcam-1 via a nuclear factor-kb-independent mechanism. the journal of immunology 1999; 162: 2990–6. 111. pasceri v, chang j, willerson jt dan yeh eth. modulation of c-reactive protein–mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerosis drugs. circulation 2001; 103: 2531–4. 112. liao f, andalibi a, lusis aj, fogelman am. genetic control of the inflammatory response induced by oxidized lipids. am j cardiol. 1995; 75: 65b–66b. 113. liao f, rabin rl, yannelli jr, koniaris lg, vanguri p, farber jm: human mig chemokine: biochemical and functional characterization. j exp med 182: 1301–1314, 1995. 114. lee sk, kim jh, yang ws, kim sb, park sk, park js. exogenous nitric oxide inhibits vcam-1 expression in human peritoneal mesothelial cells role of cyclic gmp and nf-kb. nephron 2002; 90: 447–454. 115. kawachi s, cockrell a, laroux fs, gray l, granger dn, van der heyde hc, dan grisham mb. role of inducible nitric oxide synthase in the regulation of vcam-1 expression in gut inflammation. am j physiol gastrointest liver physiol 1999; 277: g572–g576. 116. khan bv, harrison dg, olbrych mt, alexander w, dan medford rm. nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells. proc. natl. acad. sci. usa 1996; 93: 9114–9. 117. hemodynamic disorders, thromboembolic disease, and shock. in: kumar v, abbas ak, fausto n. robins and cotran pathologic basis of disease. 7th ed. philadelphia: saunders 2005; pp. 124–35 118. hua cai, david g. harrison endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. circulation research. 2000; 87: 840. 119. zhang p, hou m, li y, xu x, barsoum m, chen yj, bache rj. nadph oxidase contributes to coronary endothelial dysfunction in the failing heart. am j physiol heart circ physiol 2009; 296: h840–h846. 120. hajjar dp, falcones dj, fabricant cg, fabricant j. altered cholesteryl ester cycle is associated with lipid accumulation in herpesvirusinfected arterial smooth muscle cells. j of biological chemistry. 1985; 260(10): 6124–8. 121. hajjar dp, pomerantz kb, falcone dj, weksler bb, grant aj. herpes simplex virus infection in human arterial cells. implications in arteriosclerosis. j. clin. invest. 1987; 80(5): 1317–21. 122. de villiers wj, smart ej. macrophage scavenger receptors and foam cell formation. journal of leukocyte biology 1999; 66(5): 740–6. 123. zhao z, de beer mc, cai l, asmis r, de beer fc, de villiers wjs, van der westhuyzen wr. low-density lipoprotein from apolipoprotein e-deficient mice induces macrophage lipid accumulation in a cd36 and scavenger receptor class a-dependent manner. shouxi 2006; 10:26. downloaded from http://journal.shouxi.net/qikan/article. php?id=201401. ijtid vol 1 no 3 sep-dec 2010.36.pdf ijtid vol 1 no 3 sep-dec 2010.37.pdf ijtid vol 1 no 3 sep-dec 2010.38.pdf ijtid vol 1 no 3 sep-dec 2010.39.pdf ijtid vol 1 no 3 sep-dec 2010.40.pdf ijtid vol 1 no 3 sep-dec 2010.41.pdf ijtid vol 1 no 3 sep-dec 2010.42.pdf ijtid vol 1 no 3 sep-dec 2010.43.pdf 37 vol. 5. no. 2 may–august 2014 role of break cluster region (bcr) abelson murine leukimia (abl) examination in chronic myelogenous leukemia (cml) agung sosiawan molecular cancer division, human genetic laboratory institute of tropical disease, universitas airlangga surabaya, indonesia abstract chronic myelogenous leukemia (cml) is a clonal bone marrow stem cell disorder associated with a characteristic chromosomal translocation called the philadelphia chromosome which caused a proliferation of mature granulocytes (neutrophils, eosinophils and basophils) and their precursors, increasing unregulated growth of predominantly myeloid cells in the bone marrow and its accumulation in the blood. as myeloproliferative disease, chronic myelogenous leukemia or cml is a malignancy of the sixth-highest, reaching 15% of all blood malignancies in adults with an incidence of 1.1 per 100,000 population (ugroseno, 2012). the cml diagnosis is made based on a presence of philadelphia chromosome due to the existence of a reciprocal translocation of chromosomes 9 and chromosome 22 t (9.22), and raises the fusion of break cluster region (bcr) gene of chromosome 22 on band q11 by abelson murine leukemia (abl). the fused bcr-abl gene has bcr sequences of different length, so it produces a protein that has a different molecular weight. despite having different length of bcr sequences, however, the length of fuses abl gene sequence is constant. associated with this different bcr sequence length are the three variations of the bcr-abl gene fusion. the first variation is a major break cluster (m-bcr), the bcr gene break is found in exon 2 in e13-e14 region. this type of cml is the fusion of bcr exon b2 or b3 to abl exon a2, forming two major transcripts of the b2a2 or b3a2, which has a protein product with 210 kd weight or referred to as p210. the second variation is minor bcr (m-bcr), which has e1a2 fusion. cml with bcr-abl gene fusion of this type has a protein product with a molecular weight of 190 kda or called p190. the third variation is micro-bcr (m-bcr), with bcr gene break between exons e19 and e20b that form mrna transcripts e19a2, with bcr-abl protein p230. this fused gene can be detected with qualitative multiplex pcr. key words: cml, philadelphia chromosome, translocation t(9,22), fuse gene bcr abl, qualitative multiplex pcr abstrak chronic myelogenous leukemia (cml) adalah kelainan klonal dari stemsel sumsum tulang belakang terkait dengan adanya translokasi kromosom t (9,22) atau yang lebih dikenal dengan kromosom philadelphia. translokasi kromosom t (9,22) menyebabkan terjadinya proliferasi dari granulosit dewasa (neutrofils, eosinofils dan basofil). proliferasi sel granulosit ini menyebabkan terjadinya peningkatan dari pertumbuhan yang tidak terkendali pada sel mieloid di bone marrow dan terakumulasi dalam darah. sebagai suatu kelainan myeloproliferatif, chronic myelogenous leukemia atau cml merupakan malignansi urutan ke enam terbesar, di mana kelainan ini mencapai angka 15% dari seluruh malignansi darah pada orang dewasa dengan insidensi 1.1 per 100,000 populasi penduduk (ugroseno, 2012). diagnosis cml dibuat berdasarkan pada adanya kromosom philadelphia, yang terjadi sebagai akibat adanya translokasi resiprokal antara kromosom 9 dan kromosom 22 atau t (9.22). translokasi ini menyebabkan munculnya fusi gen bcr(break cluster region) pada chromosome 22 lengan q11 dengan gen abl atau abelson murine leukemia. fusi gen bcr-abl memiliki sekuen gen bcr dengan panjang sekuen yang berbeda, sehingga menghasilkan produk protein dengan berat molekul yang berbeda. terkait dengan perbedaan panjang sekuen gen bcr, menimbulkan adanya variasi fusi gen bcr-abl, yakni pertama major break cluster (m-bcr), fusi gen bcr ditemukan pada exon 2 di daerah e13-e14. tipe ini merupakan fusi gen bcr exon b2 atau b3 dengan gen abl exon a2, membentuk 2 jenis transkrip utama, yakni b2a2 atau b3a2, di mana produk proteinnya memiliki berat 210 kd atau biasa ditulis dengan p210. variaso kedua adalah minor bcr (m-bcr), yang memiliki fusi pada titik e1a2. cml dengan fusing memiliki fusi pada titik e1a2. cml dengan fusi case report 38 indonesian journal of tropical and infectious disease, vol. 5. no. 2 may–august 2014: 37–40 gen bcr-abl type ini memiliki produk protein dengan berat molekul 190 kda atau disebut p190. variasi ketiga adalah micro-bcr (m-bcr), di mana gene bcr terletak pada exons e19 dan e20b membentuk transkrip mrna e19a2, dengan produk protein p230. keseluruhan variasi tersebut dapat dideteksi dengan qualitative multiplex pcr. kata kunci: cml, kromosom philadelphia, translokasi t(9,22), fuse gen bcr abl, pcr multipleks kualitatif in cml, the form of the bcr-abl gene fusion will then be transcribed into bcr-abl fusion protein that has the ability to phosphorylate a substrate which further activates transduction cascade ras-signaling pathways2. a tyrosine kinase is an enzyme that can transfer a phosphate group from atp to a protein in a cell. the ras pathway is a signaling center that forwards signals both upstream and downstream, with 3 signaling pathway downstream of ras, namely ras-raf-map-kinase pathway, akt/pkb pathway and ral pathway. the substrate phosphorylation may activate ras/raf/mapk/erk jun kinase, myc, and jak-stat, and form pi3k (phosphatidylinositol-3 kinase)-akt protein, which plays a role in the inhibition of apoptosis, cell cycle progression, and dna repair, thus resulted in the deregulation of cell proliferation, through increased leukocytes proliferation.3 cml diagnosis cml diagnosis is made based on history, physical and laboratory examination, which includes a complete blood count, peripheral blood smear, cytogenetic examination to identify the presence of the philadelphia chromosome or the inspection of bcr-abl gene fusion.4 most of these are found adults. cml is rare in children. complete blood examination is intended to determine the amount of various kinds of cells in blood. in complete blood count examination, white blood cells often increase, typically > 25 109/l and could even above 100 ×109/l. differential cell shows granulocytes in all stages of maturation, ranging from the blast to the mature form. basophils also increases, but only at 10–15% of the patients who have basophils ? 7% in peripheral blood. eosinophils also increases lightly. platelets increases in 30–50% of the patients and only a few per cent were in excess of 1,000 × 109/l.1,4 cytogenetic examination of philadelphia chromosome or molecular examination of bcr-abl gene fusion are performed to confirm the diagnosis. in some cases, about 5% of philadelphia chromosome cannot be detected, so that it requires other methods, such as fluorescent in situ hybridization (fish) and molecular detection of bcr-abl gene fusion by examination of polymerase chain reaction (pcr) for establishing cml diagnosis.5 cml examination with polymerase chain reaction (pcr) pcr is an in vitro dna amplification technique in specific regions bounded by two oligonucleotide primer. primer is used as a barrier region is propagated singlestranded dna sequence with its complementary dna template. the process is similar to the process of dna replication in vivo, which is semi-conservative. pcr works introduction chronic myelogenous leukemia or cml is a malignancy of the sixth-highest, reaching 15% of all blood malignancies in adults with an incidence of 1.1 per 100,000 population1. specific characteristics of cml is the presence of philadelphia chromosome due to the existence of a reciprocal translocation of chromosomes 9 and chromosome 22 t (9.22). the presence of the translocation t (9; 22) raises the fusion of break cluster region (bcr) gene of chromosome 22 on band q11 by abelson murine leukemia (abl), which causes adult granulocytes proliferation grown without being interrupted by differentiation1. chromosomal translocation, the gene abl from chromosomes 9, which is replaced by the bcr gene on chromosome 22, is translated as bcr-abl fusion protein that has the ability to transform. the fused bcr-abl gene has bcr sequences of different length, so it produces a protein that has a different molecular weight. despite having different length of bcr sequences, however, the length of fuses abl gene sequence is constant. associated with this different bcr sequence length are the three variations of the bcr-abl gene fusion. the first variation is a major break cluster (m-bcr), the bcr gene break is found in exon 2 in e13-e14 region. this type of cml is the fusion of bcr exon b2 or b3 to abl exon a2, forming two major transcripts of the b2a2 or b3a2, which has a protein product with 210 kd weight or referred to as p210. the second variation is minor bcr (m-bcr), which has e1a2 fusion. cml with bcr-abl gene fusion of this type has a protein product with a molecular weight of 190 kda or called p190. the third variation is micro-bcr (m-bcr), with bcr gene break between exons e19 and e20b that form mrna transcripts e19a2, with bcr-abl protein p230. the transcript is even rarer, ie, with a clinical picture of netrophilia and or thrombocytosis.2 figure 1. bcr-abl gene fusion as a result of chromosomal translocation t (9,22) (goh et al., 2006) 39sosiawan a.: role of break cluster region (bcr) abelson murine leukimia (abl) to help speed up the diagnosis of malignant diseases such as chronic leukemia with bcr-abl gene fusion.1 stages of bcr-abl examination include rna isolation of peripheral blood samples of cml patients using the high pure rna isolation kit, cdna synthesis, using rna that had been isolated, converted into cdna using transcriptor first strand cdna synthesis kit, and examination of the bcr-abl gene fusion using multiplex pcr technique. multiplex pcr assay for the detection of bcr-abl gene fusion multiplex-pcr is a modification of pcr reaction in order to be faster in detecting gene deletions or duplications. this process amplifies genomic dna samples using multiple primers and a temperature-mediated dna polymerase in a thermal cycler. multiplex-pcr was first described in 1988 as a method to detect deletions in the dystrophin gene.5 it has also been used with the steroid sulfatase gene.6 in 2008, the multiplex-pcr was used for analysis of microsatellites and snps.7 multiplex-pcr consists of multiple primer sets within a single pcr mixture to produce amplicons of varying sizes that are specific to different dna sequences. by targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. cross et al, (1994) have used a multiplex-pcr for the identification of bcr-abl transcripts simultaneously from two or more genes in the same reaction. the goal is to qualitatively detect the bcrabl gene fusion. one example of the use of multiplex pcr examination is to identify for the presence of bcr-abl gene fusion by using the following primers: sense primers a1: caacagtccttcgacagcag (5’–3‘) on bcr exon 1 b1: gctacggagaggctgaagaa (5’–3‘) on bcr exon 11 anti-sense primer c1: cgtgatgtagttgcttggga (5’–3‘) on abl exon 3 primer sequences in nested rt-pcr sense primers a2: caacagtccttcgacagcag (5’–3‘) on bcr exon 1 b2: gtgcagagtggagggagaac (5’–3‘) on bcr exon 12 anti-sense primer c2: acaccattccccattgtgat (5’–3‘) on abl exon 3 the next phase is gel electrophoresis using 2% agarose. results of electrophoresis was stained with ethidium bromide (by soaking for 15–20 minutes). pcr results is positive when 627 bands are obtained for b3a2 transcript (e13a2), 552 for the b2a2 (e14a2), 378 for b2a3, and 580 for b1a1.8 examination of response to therapy using rq-pcr examination of response to therapy aims to quantitatively detect bcr-abl fusion gene transcript derived from mrna isolation of the patients’ blood samples, using real time pcr or better known as quantitative-pcr atau real time quantitative–pcr atau rq pcr. using rq-pcr the quantity of pcr products generated from each amplification cycle is done by measuring the strength/increase in fluorescence generated by the fluorescent molecules used in the reaction. rq pcr products can be quantified relative to control genes or house keeping gene. in particular, quantification process relies on a standard curve obtained from known concentration of plasmid dna. rq-pcr has many advantages over conventional pcr. rq-pcr allows amplification and detection process carried out simultaneously in one tube so that the process takes place more quickly and efficiently. in cml treatment management, real time quantitative pcr is used to evaluate molecular response of bcrabl therapy, namely by calculating residual bcr-abl transcripts within 2 weeks after reaching ccyr (complete cytogenetic response) and then performed every 3 months. molecular response can be divided into complete molecular response (cmr), in which bcr-abl transcript concentrations cannot be detected by rq-pcr, and mmr or major molecular response, in which bcr-abl decreases? 3-log or the ratio of bcr-abl/abl is < 0.1. resistance and examination of mutation detection with aso pcr according to national comprehensive cancer network (nccn) and leukemianet guidelines, resistance to tyrosine kinase inhibitors is defined as failure to achieve complete hematologic response (chr) in 3 months, cytogenetic response (cr) in 6 months, or major cytogenetic response (mcr) in 12 months. resistance to tyrosine kinase inhibitor of a drug can be divided into three, namely hematologic, cytogenetic and molecular resistance. failed therapy is defined as not achieving hematologic response after 3 months of therapy, or loss of complete hematological response at any time, or after 6 months not achieving cytogenetic response, or after 12 months not achieving major cytogenetic response, and or bcr-abl > 10%, or after 18 months not achieving complete cytogenetic response and or bcr-abl of > 10%.1 according to branford et al6 (2003), patients who fail to achieve a 1-log reduction in bcr-abl transcripts in 3 months or a decrease of > 2-log within 6 months, tend figure 2. example of electrophoresis results of bcr-abl qualitative examination.8. 40 indonesian journal of tropical and infectious disease, vol. 5. no. 2 may–august 2014: 37–40 not to give a significant response and at high risk for progressiveness. molecular resistance is defined as the lack or loss of complete molecular response (bcr-abl transcripts cannot be detected) using pcr rqor as a lack of major molecular remission (ie, a decrease of bcr-abl transcripts > 3-log or the ratio of bcr-abl/abl < 0.1%, respectively).9 according to corbin et al11 (2003), certain mutations, such as m244v, m351t, and phe311l also causes resistance to tyrosine kinase inhibitors (imatinib), which, using biochemical and cellular tests, the mutation will show decreased sensitivity to imatinib respectively of 1, 8 and 2.8-fold. slight shifts in terms of sensitivity causes kinase activity becomes sufficient to cause disease progression. however, in theory, the resistance caused by these mechanisms can be overcome with increased doses of imatinib. mutation found in t315i frequently causes of resistance to inhibitors of first-generation (imatinib) or second generation. this mutation is also associated with secondary imatinib resistance that usually occurs in the later stages of the disease and is associated with advanced age, receiving interferon previously, a high sokal score, and failure to reach the ccr in 12 months.10 detection of abl kinase domain mutations is performed when in chronic phase the cml patients, after treatment with tki drugs, provide inadequate therapeutic response (failure to achieve hematologic complete response in the first 3 months, minimal cytogenetic response within 6 months, or a major cytogenetic response within 12 months) or loss of therapeutic response (defined as hematologic relapse, cytogenetic relapse, 1-log increase of bcr-abl transcripts ratio, and the loss of major molecular response), or the disease becomes progressive (the occurrence of accelerated phase or blastic crisis).1 mutation analysis can be done in various ways. one is by using allele-specific olygonucleotide (aso)-pcr. this method is a technique that is highly sensitive and specific for the detection of known mutations1. this method is even more sensitive than mutation detection with sequencing method because dna sequencing method can only be useful for point mutation if the proportion of mutated cells is more than 30%. in cases where the number of mutated cells is less than 30% of total cells in the sample of the patients, at least 10 independent clones from patients should be analyzed for mutations detection, a quite expensive and time consuming procedure. in contrast, the aso-pcr is comparatively more sensitive, specific and “economical”, so it is a rapid method for mutation detection.12 conclusion the use of qualitative multiplex pcr to detect bcrabl fused gene becomes one of the methods to detect the chronic myelogenous leukemia (cml). it is easier to be performed while the use of karryotyping was complicated to detect this fused genes that called philladelphia chromosome references 1. ugroseno, 2012. analisis mutasi gen bcr-abl pada chronic myelogenous leukemia fase kronik kromosom philadelphia positif yang resisten terhadap inhibitor tyrosine kinase. disertasi. universitas airlangga. 2. sastre da, argaraña e, heller vb, gallo m, fernández en, rodríguez cm. 2007. an analysis of multiplex-pcr in the detection of bcr-abl transcripts in hematological disorders. genetics and molecular biology; 30; 3: 520–523. 3. di bacco alessandra, et al. 2000. molecular abnormalities in cml: deregulation of cell growth and apoptosis. oncologist: 5: 405–15. 4. cortes je, silver rt, kantarjian h. 2011. chronic myeloid leukemia. cancer management. 13th edition. http://www. cancernetwork.com/cancer-management/chronic-myeloid-leukemia/ article/10165/1802798 5. baccarani m, dreyling m. 2010. chronic mieloid leukaemia: esmo clinical practice guidelines for diagnosis, treatment and follow-up. annals of oncology 21 (supplement 5): v165–v167. 6. branford s, rudzki z, harper a, et al. 2003. imatinib produces significantly superior molecular responses compared to interferon alfa plus cytarabine in patients with newly diagnosed chronic mieloid leukemia in chronic phase. leukemia; 17: 2401–9. 7. chasseriau j, rivet j, bilan f, chomel jc, guilhot f, bourmeyster n, kitzis a. 2004. characterization of the different bcr-abl transcripts with a single multiplex rt-pcr. j mol diagn; 6 (4): 343–7. 8. goh hg, hwang jy, kim sh, lee yh, kim yl, kim dw. 2006. comprehensive analysis of bcr-abl transcript types in korean cml patients using a newly developed multiplex rt-pcr. translational research, 148: 249–256. 9. kantarjian hm, cortes j, guilhot f, hochhaus a, baccarani m, lokey l. 2007. diagnosis and management of chronic mieloid leukemia. cancer; volume 109, issue 7; 1365–1375. 10 ramirez p, dipersio jf. 2008. therapy options in imatinib failures. the oncologist 2008; 13: 424–434. 11. corbin as, la rosee pl, stoffregen ep, druker bj, deininger mw. 2003. several bcr-abl kinase domain mutants associated with inhibitor tirosin kinase mesylate resistance remain sensitive to inhibitor tirosin kinase. blood; 101: 4611–14. 12. iqbal z, rubina t. siddiqui rt, qureshi ja. 2004. two different point mutations in abl gene atp-binding domain conferring primary imatinib resistance in a chronic myeloid leukemia (cml) patient: a case reportbiol. proced. online; 6 (1): 144–148. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 vol. 9 no. 2 may–august 2021 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 review article covid-19 and endothelial dysfunction: biomarkers and potential drug mechanisms andrianto1, ronaldi rizkiawan1, primasitha maharany harsoyo1, syafira yasmine2 1 department of cardiology and vascular medicine, school of medicine, universitas airlangga dr. soetomo general academic hospital, surabaya, indonesia 2 school of medicine, universitas airlangga, surabaya, indonesia received: 15th february2021; revised: 4th april 2021; accepted: 11st april 2021 abstract since the fi rst report of pneumonia outbreak in wuhan by the end of 2019, coronavirus disease 2019 (covid-19) has become a global pandemic; causing millions of deaths globally and aff ecting the rest of worldwide population. the disease is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2), which enters hosts by inhabiting angiotensin-converting enzyme-2 (ace-2) receptors expressed in the endothelium layer of not only the respiratory tracts, but also various organs in the body. covid-19 has been reported to trigger multiple cardiovascular manifestations. since endothelial dysfunction plays an important role in cardiovascular events and the endothelium is heavily involved in covid-19 pathophysiology, it is important to investigate their associations and previously established drug potencies to improve endothelial functions as possible treatment options for covid-19. in this review, we summarize endothelial dysfunction biomarkers involved in covid-19 and drugs that have shown potential endothelial protective properties to better understand the incidence of endothelial dysfunction in covid-19 and its future treatment. we searched in pubmed, wiley online library, ebsco, sciencedirect databases for literatures containing following keywords: “endothelial dysfunction”, “covid-19”, and “biomarkers”. eligible publications were then assessed and studied to comprise our literature review. a total of 96 studies matched our criteria and provided scientifi c evidences for our review. materials were then compiled into a review summarizing endothelial biomarkers involved in covid-19 and potentially repurposed drugs targeting endothelium for covid-19.various endothelial dysfunction biomarkers were found to be elevated in covid19 and is found to be related to its severity, such as adhesion molecules, selectins, pai-1, and von willebrand factors. multiple drugs targeting the endothelium are also potential and some are under investigation for covid-19. keywords: covid-19, endothelial dysfunction, sars-cov-2, biomarkers abstrak sejak pertama kali pelaporannya sebagai wabah pneumonia di wuhan pada akhir 2019, corona virus disease 2019 (covid-19) telah menjadi pandemi global yang menyebabkan jutaan kematian dan memengaruhi populasi di seluruh dunia. penyakit ini disebabkan oleh sars-cov-2 yang menginvasi sel inang dan berikatan dengan reseptor angiotensinconverting enzyme-2 (ace-2) yang diekspresikan lapisan endotel saluran pernapasan serta berbagai organ lain dalam tubuh. covid-19 telah dilaporkan dapat menimbulkan berbagai manifestasi gangguan kardiovaskular. mengingat peran penting disfungsi endotel yang berperan dalam kejadian kardiovaskular dan patofi siologi covid-19, maka penting menyelidiki hubungan di antaranya. selain itu, perlu dilakukan telaah mengenai potensi obat yang sebelumnya telah terbukti memperbaiki fungsi endotel sebagai pilihan terapi yang digunakan untuk covid-19. pada studi ini, kami memaparkan beberapa biomarka disfungsi endotel yang terlibat pada covid-19 dan obat-obatan yang melindungi endotel sebagai terapi potensial covid-19 di masa mendatang. kami melakukan pencarian di database pubmed, perpustakaan online wiley, ebsco, dan sciencedirect untuk literatur yang berisi kata kunci berikut: “disfungsi endotel”, “covid-19”, dan “biomarker”. publikasi yang memenuhi syarat kemudian dinilai dan ditelaah untuk menyusun tinjauan pustaka. sebanyak 96 publikasi memenuhi kriteria dan memberikan * corresponding author: andrianto@fk.unair.ac.id 109andrianto, et al.: covid-19 and endothelial dysfunction ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 bukti ilmiah untuk tinjauan pustaka ini. materi kemudian disusun menjadi ulasan yang merangkum biomarker-biomarker endotel yang terlibat dalam covid-19 dan obat-obatan dengan target endotel yang potensial . berbagai biomarker disfungsi endotel ditemukan meningkat pada covid-19 dan terkait dengan tingkat keparahannya, seperti molekul adhesi, selektin, pai-1, dan faktor von willebrand. beberapa obat yang menargetkan endotel juga ditemukan memiliki potensi dengan beberapa tengah diinvestigasi lebih lanjut sebagai terapi covid-19. kata kunci: covid-19, disfungsi endotel, sars-cov-2, biomarka how to cite: andrianto., rizkiawan., harsoyo, pm., yasmine, s. covid-19 and endothelial dysfunction: biomarkers and potential drug mechanismsct. indonesian journal of tropical and infectious disease, 9(2), 108–119. introduction in december 2019, a novel corona virus was isolated from the respiratory tract epithelial layer of a patient with pneumonia of unknown cause in wuhan, china. by november 2020, the severe acute respiratory syndrome-coronavirus-2 (sars-cov-2), and its consequential disease, coronavirus disease 2019 (covid-19), has now infected more than 46 millions of people all over the world and caused more than 1.2 million global death.1 beside instigating substantial pulmonary disturbances such as pneumonia and acute respiratory distress syndromes, covid-19 is known to trigger extrapulmonary responses, including cardiovascular manifestations. several reported cardiovascular manifestations of covid19 include myocardial ischemia, arrhythmia, and even cardiogenic shock.1,2 endothelial dysfunction is a significant contributor of various cardiovascular diseases mechanism. endothelial cells play a major role in maintaining blood tissue non-thrombogenicity, regulating thrombosis, thrombolysis, thrombocyte adherence, vascular tone, and blood fl ow.1 any disturbance of endothelial function may trigger a progression of cardiovascular problems.3 s a r s c o v2 e x p l o i t s a n g i o t e n s i n converting-enzyme-2 (ace-2) as a receptor to attack human cells. ace-2, a main target component in various cardiovascular diseases mechanism and drugs, is widely expressed in both respiratory endothelial layers and vascular endothelial layers. therefore, it is important to learn of the endothelial involvement in covid-19 related cardiovascular function alterations as well as the changes of biomarkers it comprises.1,2 discussion sars-cov-2 related endothelial changes the innermost part of the vascular system is comprised of a single layer of endothelial cells called endothelium. a healthy endothelium maintains blood fl uidity through platelet reactivity regulation, coagulation, and smooth thrombolysis by synthesizing and responding to vasoactive molecules accordingly.2 the endothelium, alongside its primary immunoregulatory properties, also plays an important role in maintaining dynamic interactions between pro-coagulant and fibrinolytic factors within the vascular system. in its inactive state, the endothelium acts as a barrier that separates prothrombotic subendothelial layers and procoagulant factors carried within the circulation.3 sars-cov-2 directly aff ects the endothelial cells due to the high expression of ace-2 receptors and transmembrane protease, serine 2 (tmprss2) enzyme. after being bound by sarscov-2, ace-2 receptors are internalized such that the diminishing number of ace-2 in endothelial cells promotes infl ammation and thrombosis, triggered by subsequent hyperactivity of local angiotensin-ii (ang-ii). the decrease of ace-2 receptors also reduces a number of conversions it normally mediates, including angiotensin 1-7 from ang-ii, which acts as a vasoactive ligand of the mas receptor. this causes reduction of mas receptor activation and induction of pro-inflammatory phenotypes through the increase of type-1 angiotensin receptor (at1r) activation. furthermore, the decrease of ace-2 receptor prevents the degradation of des-arg-9110 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 108–119 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 bradykinin (dabk) into inactive peptides, which subsequently raises prothrombotic signals through bradykinin receptors (bkrs) activation.4,5 endothelial activation and subsequent d y s f u n c t i o n i s m a r k e d b y i m b a l a n c e o f endothelium-released vasomotor factors, expressions of inflammatory cytokines and chemokines, expression and secretion of selectins and adhesive molecules as well as modulation of local thrombotic pathway.6 the release of cytokines and pro-infl ammatory chemokines by activated macrophages augments the vicious circle of vascular integrity, coagulation, and thrombosis disturbances through reduction of endothelial glycocalyx, activation of coagulation system, and dampening of anticoagulation mechanism. endothelial cell adhesive phenotypes induced by pro-infl ammatory cytokines and chemokines promote neutrophil infi ltration, producing large quantity of histotoxic components such as reactive oxygen species (ros) and neutrophil extracellular traps (nets). endothelial activation is the transition of static phenotype to a specific phenotype involving responses to host immunity.5 endothelial cells activation causes an increase of infl ammatory cytokines and adhesion molecules, triggering releases of leukocyte, adhesion, and migration to subendothelial chambers, constituting fundamental process of atherosclerotic lesion initiation, progress, and destabilization.5 activated endothelial cells begin coagulation process by expressing p-selectin, von willebrand factor (vwf), and fi brinogen, causing massive platelet binding, formation of fibrin and clotting of red blood cells (rbc), which fi nally results in systemic thrombosis and disseminated intravascular coagulation (dic).4 measurable biomarkers of endothelial activation and dysfunction activated endothelial cells express increased levels of e-selectin, p-selectin, intercellular adhesion molecule 1 (icam-1), and vascular cell adhesion molecule 1 (vcam-1). upregulations of e-selectins, icam-1, and vcam-1 are mediated at transcription level. e-selectin induces the rolling of circulatory leukocytes. vcam-1 and icam-1 induces strong adhesion by binding very late antigen 4 (vla4) and leucocyte function antigen-1 (lfa-1). after strong adhesion, leukocyte migrates through endothelial cells into its underlying tissues. among these molecules, the adhesion molecules (icam-1 and vcam-1), selectins (e-selectin and p-selectins), plasminogen activator inhibitor-1 (pai-1), and figure 1. alterations of endothelial, systemic coagulation and thrombotic functions due to sars-cov-2 infection.6 111andrianto, et al.: covid-19 and endothelial dysfunction ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 von willebrand factor (vwf) are easily obtained from the circulation and well measured by commercial immunoassays.5 adhesion molecules endothelial dysfunction can be detected by the increase of circulating cellular adhesion molecules (cam), including icam-1 and vcam-1. icam-1 and vcam-1 are minimally expressed in inactive endothelial cells. however, these expressions can be augmented by activation of cytokines and endotoxin liposaccharides. thrombin and histamine stimulations selectively induce endothelial p-selectin expression, whereas cytokines and liposaccharides stimulations induce e-selectins expression.7,8 studies have shown that endothelial cellular adhesion markers, vcam-1 and icam-1, are increased in covid-19 patients compared to control group.9 these markers were also found signifi cantly higher in severe covid-19 infections, associating the severity of covid19 infection with increase of serum vcam-1 and icam-1.9 likewise, the recovery of severe covid-19 infections was also associated with a decrease of serum vcam-1 and icam-1 levels over time. therefore, it could be inferred that according to the study, the increase of endothelial cellular adhesion molecule expression is correlated with the presence of covid-19 and its severity, as well as indicating a contribution to patient’s coagulopathy state.9 to further support this, a previous postmortem study had demonstrated a marked endothelial dysfunction in covid-19 patients with signifi cant increase of icam-1.10 selectins besides vcam-1 and icam-1, endothelial cells also express p-selectin and e-selectin. p-selectin is a cellular adhesion molecule stored inside the endothelium and thrombocytes which would be rapidly deployed into plasma membrane upon activation.11 as infection progresses, p-selectin increases platelet aggregation and platelet-endothelium interaction. soluble p-selectin (sp-selectin) is produced through enzymatic release of mobilized p-selectins during infl ammation.12 experiments with genetically engineered rat in pro-coagulant state revealed high level of sp-selectin expressions. on the other hand, sp-selectin was also proven to be a signifi cant marker of several infl ammatory and pro-coagulopathy disorders, including systemic infl ammatory responses.11,14,15 most recent study showed increase of sp-selectin in covid-19 cases, further supporting the theory of covid-19 being an endothelial dysfunction disorder.16 e-selectin can also provide as markers of endothelial dysfunction. e-selectin (cd62e) is a leukocyte adhesion molecule that is expressed by activated endothelial cells. endothelial cells of normal skin and bone marrow or in the case of infantile hemangioma constitutively express e-selectin, in contrast with endothelial cells from other tissues that do not.18,19 however, these expressions are tightly regulated by infl ammatory cytokines. soluble e-selectins (se-selectin) are released during infl ammation and has long been proposed as a biomarker of endothelial dysfunction, especially in cases of sepsis.20 therefore, increased circulating se-selectin alongside mrna increase confi rm the presence of endothelial dysfunction in covid-19 as well as a linear correlation with its severity. smadja et al. found in their study that there was an increase of e-selectin level in covid-19 patients. this strengthens the theory of endothelial dysfunction in covid-19.21 plasminogen activator inhibitor-1 (pai-1) procoagulant condition formed by endothelial activation can also be measured from alterations of balance between tissue plasminogen activator (t-pa) and its endogen, plasminogen activator inhibitor-1.22 plasminogen activator inhibitor-1, also known as endothelial pai, is a serine protease inhibitor (serpin) which serves as main inhibitor of t-pa and urokinase type plasminogen activator (u-pa), a fi brinolytic agent with recent additional non-fi brinolytic properties reported.23-26 while also secreted by other tissues such as adipose tissue, pai-1is mainly produced by endothelium. increase of pai-1 is a risk factor of thrombosis and atherosclerosis.2,22 112 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 108–119 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 one characteristic of covid-19 is leukocyte sequestration, especially neutrophils, in the pulmonary microvasculature—which contributes to alveolar injury and infi nite infl ammation.27 l o c a l p r o i n f l a m m a t o r y e n v i r o n m e n t i s exaggerated by the formation of net which results in mass production of pro-infl ammatory cytokines.28 these cytokines trigger endothelial cells activations and possibly promotes release of t-pa and pai-1.28,29 activated endothelial cells expresses raised levels of pai-1, inhibiting t-pa and u-pa, further instigating hemostasis balance alteration to procoagulant state.17 in a study by zuo, pai-1 level is found to be raised in covid-19 patients.30 this raise has a significant correlation between pai-1 as well as circulating absolute neutrophil and calprotectin. this supports the presence of endothelial dysfunction in covid-19.30 besides endothelial activation, there is a possibility that direct infection and endothelial cells destruction by sars-cov-2 cause a potential release of t-pa and pai-1.31 a study by kang et al revealed that in advanced cases of covid-19 with severe respiratory dysfunction, the pai-1 level is signifi cantly higher than sepsis, acute respiratory distress syndrome (ards), or even burn cases.32 the signifi cant increase of pai-i in severe cases of covid-19, which are comparable to ards, had shown to induce vascular endothelium destruction. sars-cov-2 had also shown to directly infect the vascular endothelium, triggering endothelitis which indicates vascular endothelial destruction in patients with covid-19.31 collectively, this fi nding suggests that increased pai-1 level promotes endotheliopathy and coagulopathy in severe cases of covid-19.32 intensive care patients with severe conditions were reported to have signifi cantly increased level of pai-1 compared to non-intensive care patients. data from a study by nougier clearly demonstrated that balance between coagulation and fi brinolysis diminished in covid-19 patients in signifi cant hypercoagulability state related to hypofi brinolysis caused by high increase of pai-1 level.33 in a study by blasi, plasma pai-1 level was found 3.7 times higher in covid-19 patients compared to control.34 von willebrand factor von willebrand factor (vwf) is a major multidomain adhesive glycoprotein derived from endothelium, released into circulation by activated endothelial cells.35,36 vwf binds with platelet glycoprotein ib , iib 3 and endothelial collagen, which activates the platelets and commences platelet aggregation.35 as a carrier of blood clotting factor viii, vwf also has an important role in clotting cascade.37 vwf is synthesized by endothelial cells and megakaryocytes, which is then stored as ultralarge vwf multimers or multimers with large molecular weight within the endothelial weibelpalade bodies or platelet -granules. it is well established that pathological alterations of fibrinogen, d-dimer, vwf and p-selectin have important roles in abnormal coagulation and endothelial dysfunction. both infection and infl ammation can increase plasma vwf through activated endothelial cells.40,41 the majority of vwf originate from endothelial cells. endothelial cells vwf (ec-vwf), instead of platelet vwf, critically promotes thrombus formation. in accordance, ec-vwf contributes in vwf-dependent atherogenesis by raising platelet adhesion and vascular infl ammation.39 vwf is also a biomarker that is relatively easy to measure.42 in healthy individuals, ace2 transforms angiotensin-ii into angiotensin 1-7, which stimulates endothelial cells to produce nitric oxide (no). no aids blood vessels in vasodilation and suppressing platelet aggregation. in covid-19, sars-cov-2 occupies ace2, subsequently raising angiotensin-ii levels. this further enhance vasoconstriction and reduce blood fl ow. in this process, vwf stored inside weibel palade bodies, increasing formation of blood clots.44 activation of ec-vwf in relation of covid19 is known as acute phase protein, released from endothelial cells as infl ammatory response.45 in this case, its high level indicates a disturbance of endothelial function.46 several studies had been 113andrianto, et al.: covid-19 and endothelial dysfunction ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 performed to determine the role of vwf in covid19. covid-19 patients have a documented higher level of vwf, as shown in a study by blasi.34 an increased activity of vwf and vwf antigen (vwf:ag) was also found to be signifi cantly raised in a study by helms, which revealed a well-defi ned endothelial infl ammation with a very high level of vwf:ag.47 morici et al., in their study, reported a signifi cantly higher vwf in all covid-19 patients, supporting previous studies.48 katneni et al. and several other previous publications similarly reported that vwf level was higher in covid-19 patients.49-53 in a study by panigada, the increase of vwf was reported up to 863 u/dl, further supporting evidences of endothelial dysfunction in covid-19 patients and its potential use as a biomarker in covid-19.54 fraser, in his study, measured three thrombosis factors and fi ve markers of endothelial cell injury from the plasma using elisa, showing signifi cant higher levels in intensive care covid-19 patients compared to healthy individuals.16 a study by rauch has also shown evidences of associations between coagulation marker level on admission, amongst which are factor viii and vwf, with the severity of covid-19.55 in other words, increased levels of vwf in covid-19 patients is a potential biomarker associated with severity figure 2. thrombus formation in covid-19. of the disease. previous studies have shown the ability of pulmonary virus to promote plateletendothelium interactions through upregulation of endothelial icam-1, vwf, and fi bronectins, culminating ongoing pulmonary injury.56 state of hypoxia also triggers vwf expression and its following detachment from endothelial weibel-palade bodies.57,58 vwf upregulation induced by hypoxia has been associated with the presence of coronary and pulmonary vessels thrombus formation, as well as promoting leukocyte recruitment.59 accordingly, hypoxemia state observed in covid-19 patients induce prothrombotic condition through upregulation of t-pa inhibitor and stimulation of procoagulant endothelial synthesis, including tissue factor and vwf.57,60-62 vfw is an acute phase response protein released by activated endothelial cells as a reaction to infl ammatory stimuli. this increase in activity of vwf and its antigen level contribute to platelet aggregation. in another study, cases of covid-19 can cause up to 490% raise of vwf and vwf:ag level. vwf is a main determinant of platelet adhesion after vascular injury and its resulting blood clot. high vwf:ag level is an independent risk factor for ischemic stroke and myocardial infarction.64 thus, thrombocyte 114 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 108–119 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 inhibitors can be considered in prevention of cardiovascular events in covid-19.65 potential mechanisms of cardiovascular drugs in covid-19 related endothelial function repair various experimental and clinical studies have shown multiple currently used of investigated drugs currently can repair endothelial function, although they may have diff erent structures and mechanisms. among those drugs are angiotensin converting enzymes (ace) inhibitor (acei), angiotensin-ii receptor blocker (arb), statins and antioxidants. acei and arb evidences have established the role of ace inhibitors in repairing endothelial functions in animal models with heart failure and in animal models with coronary artery disease.66,67 ace inhibitors elevate endothelial functions by reducing angiotensin-ii, while regulating endothelial nitric oxide synthase (enos) and inhibiting production of reactive oxygen species (ros), giving the drug its protective properties for endothelium.68-70 in the trend study, quinapril, compared to placebo, repaired endothelial dysfunction in normotensive patients with coronary artery disease.67 administration of arb has shown to improve endothelial function and demonstrate an overall reduction of infl ammatory biomarkers, implying its importance in the pathogenesis of atherosclerosis.71 a previous meta-analysis showed that acei improves endothelial function in patients with endothelial dysfunction of multiple causes.72 however, despite available studies mentioned, there has not been any consensus related to covid-19 related endothelial dysfunction and acei or arb as its potential clinical approach. several available meta-analysis regarding eff ectivity of ace inhibitor or arb administration for endothelial dysfunction in covid-19 still have diff ering conclusions. 73,74 further investigation is necessary, especially considering that ace-2 receptor is a binding site of sars-cov-2. statins beneficial effects of statin in endothelial function involves multiple mechanisms. statins improve endothelial disfunction due to their ldl lowering properties, considering ldl and oxldl capability in reducing enos.75,76 statin improves no bioactivity by activating enos through pi3k/ akt signaling pathway.80 the benefi ts of statin for endothelial function is also related with its antiinfl ammation and antioxidant properties.77 statins provide direct antioxidant eff ects to ldl through reducing ldl electronegative forms.78,79 in 1995, randomized control trial stated that lovastatin can return endothelial function of a coronary artery.81 atorvastatin was shown to reduce pro-infl ammatory cytokines (tnf, il-1 and il-6), icam-1 and c-reactive protein (crp) in hypercholesterolemic patients.82 one study showed that simvastatin produces signifi cant reduction in endothelial dysfunction markers, infl ammation, oxidative stress and endothelial apoptosis; in this study, crp reduction seemed to have been in relation to the lipid lowering property of simvastatin.83 a meta-analysis showed that statin therapy is associated with signifi cant improvement of both coronary and peripheral endothelial functions.84 otherwise, statin elevate endothelial progenitor cells circulation, which contribute in the long-term eff ects of statin in endothelial function.85 the combination of ace inhibitors and statin therapy have also been demonstrating a relaxation eff ects in coronary vessels, which is largely dependent on endothelial function through no production.86 several studies reported benefi ts of statins in covid-19. masana et al. in their study revealed fewer deaths reported in statin-administered group compared to the non-statin-administered group. it has also been stated that statin therapy should not be halted in hospitalized patients due to lower death rate of sars-cov-2 infection patients who took statins before hospitalization.87 an observational study by omar et al. also reported that, in diabetic patients with covid19, statin users have a 12% lower chance of death during hospitalization compared to those who did not.88 a retrospective cohort in singapore 115andrianto, et al.: covid-19 and endothelial dysfunction ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 linked independent use of statin with lower icu admission rate.88 evidence by sophia et al suggested benefi cial use of continuous statin for hospitalized covid-19 patients, as it correlates with lower chance of invasive mechanical ventilation support.89 however, there has not been a randomized controlled trial which further supports the use of statins in covid-19. antioxidant several substances such as vitamin c, vitamin e and n-acetylcysteine provide an antioxidant eff ect through diff erent mechanisms. vitamin c protects the endothelium by eliminating superoxide, which in turn prevents no decomposition, lipid peroxidation, platelet and neutrophil activation, as well as upregulation of adhesion molecules.90,91 vitamin c scavenges reactive nitrogen species yielded by peroxidase and inhibits myeloperoxidase/h2o2.92 vitamin e acts as lipid soluble antioxidant, clearing radical hydroperoxyl in lipid environment.93 meanwhile, the eff ects of n-acetylcysteine in endothelial dysfunction are related to inhibitions of nadph oxidase expression, leukocyte adhesion and infl ammatory cytokine secretion.94 n-acetylcysteine also prevents platelet aggregation, which largely depends on vwf and collagen binding in human plasma, and inhibits upregulation of caveolin-1 as well as strengthening endothelial barrier function in rats.95 further investigations may provide useful information regarding antioxidants in the management of endothelial dysfunction related to covid-19. conclusion sars-cov-2 infection, which has caused a global pandemic, involved various clinical manifestations and underlying mechanisms. this virus invades hosts by occupying ace-2 receptors in endothelium, which signify the important role of endothelium in this disease. available studies have demonstrated the increase of several endothelial biomarkers such as icam-1, vcam-1, e-selectin, p-selectin, pai-1 and vwf in covid-19 patients, which supports the presence of endothelial dysfunction in covid-19, and could further provide helpful information for the detection of endothelial dysfunction in covid-19. these fi ndings may also explain the higher chance of individuals with comorbidity to contract covid-19, with increased severity. further studies investigating no levels in covid-19 is necessary to confi rm covid-19 related endothelial dysfunction. the use of drugs that has been established to improve endothelial functions may be useful as a baseline therapy in covid-19. it has also become a point of interest whether a cured covid-19 patient has an increased risk of cardiovascular diseases in the future due to their endothelial impairment. this topic is potential for future research, and may provide helpful insight in prevention of cardiovascular system events after covid-19. acknowledgement authors would like to thank department o f c a r d i o l o g y a n d va s c u l a r m e d i c i n e , airlangga university faculty of medicine for the support in conducting this study. the authors’ responsibilities were as follows—aa: determined and conceptualized the framework of studied topic, provided guidance in the design of manuscript and edit manuscript content; rr: conducted literature research and wrote the manuscript; pmh: translated, edited and had primary responsibility for the fi nal content of the manuscript; and all authors: read and approved the fi nal manuscript. conflict of interest none of the authors had a confl ict of interest in this study. references 1. galley hf, webster nr. physiology of the endothelium. br j anaesth [internet]. 2004;93(1):105–13. available from: http://dx.doi.org/10.1093/bja/aeh163 116 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 108–119 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 2. wang m, hao h, leeper nj, zhu l. thrombotic regulation from the endothelial cell perspectives. arterioscler thromb vasc biol. 2018;38(6):e90–5. 3. siddiqi hk, libby p, ridker pm. covid-19 – a vascular disease. trends cardiovasc med. 2020;(january). 4. perico l, benigni a, casiraghi f, ng lfp, renia l, remuzzi g. immunity, endothelial injury and complement-induced coagulopathy in covid-19. nat rev nephrol [internet]. 2020; available from: http:// dx.doi.org/10.1038/s41581-020-00357-4 5. deanfi eld je, halcox jp, rabelink tj. endothelial function and dysfunction: testing and clinical relevance. circulation. 2007;115(10):1285–95. 6. halcox jpj. endothelial dysfunction. prim auton nerv syst. 2012;319–24. 7. mclean rc, blumenthal rs. infl ammatory markers and the risk of coronary heart disease. cardiol rev. 2005;22(6):41–2. 8. ridker pm, brown nj, vaughan de, harrison dg, mehta jl. established and emerging plasma biomarkers in the prediction of fi rst atherothrombotic events. circulation. 2004;109(25):6–19. 9. tong m, jiang y, xia d, xiong y, zheng q, chen f, et al. elevated expression of serum endothelial cell adhesion molecules in covid-19 patients. j infect dis. 2020;222(6):894–8. 10. nagashima s, mendes mc, camargo martins ap, borges nh, godoy tm, miggiolaro afrds, et al. endothelial dysfunction and thrombosis in patients with covid-19 brief report. arterioscler thromb vasc biol. 2020;(october):2404–7. 11. furie b. p-selectin and blood coagulation: it’s not only about infl ammation any more. arterioscler thromb vasc biol. 2005;25(5):877–8. 12. ishiwata n, takio k, katayama m, watanabe k, titani k, ikeda y, et al. alternatively spliced isoform of p-selectin is present in vivo as a soluble molecule. j biol chem. 1994;269(38):23708–15. 13. andré p, hartwell d, hrachovinová i, saff aripour s, wagner dd. pro-coagulant state resulting from high levels of soluble p-selectin in blood. proc natl acad sci u s a. 2000;97(25):13835–40. 14. schrijver it, kemperman h, roest m, kesecioglu j, de lange dw. soluble p-selectin as a biomarker for infection and survival in patients with a systemic infl ammatory response syndrome on the intensive care unit. biomark insights. 2017;12. 15. katayama m, handa m, araki y, ambo h, kawai y, watanabe k, et al. soluble p‐selectin is present in normal circulation and its plasma level is elevated in patients with thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome. br j haematol. 1993;84(4):702–10. 16. fraser dd, patterson ek, slessarev m, gill se, martin c, daley m, et al. endothelial injury and glycocalyx degradation in critically ill coronavirus disease 2019 patients: implications for microvascular platelet aggregation. crit care explor. 2020;2(9):e0194. 17. shapiro ni, schuetz p, yano k, sorasaki m, parikh sm, jones ae, et al. the association of endothelial cell signaling, severity of illness, and organ dysfunction in sepsis. crit care. 2010;14(5). 18. silva m, videira pa, sackstein r. e-selectin ligands in the human mononuclear phagocyte system: implications for infection, infl ammation, and immunotherapy. front immunol. 2018;8(jan). 19. smadja dm, mulliken jb, bischoff j. e-selectin mediates stem cell adhesion and formation of blood vessels in a murine model of infantile hemangioma. am j pathol [internet]. 2012;181(6):2239–47. available from: http://dx.doi.org/10.1016/j.ajpath.2012.08.030 20. cummings cj, sessler cn, beall ld, fisher bj, best am, fowler aa. soluble e-selectin levels in sepsis and critical illness: correlation with infection and hemodynamic dysfunction. am j respir crit care med. 1997;156(2 i):431–7. 21. smadja dm, guerin cl, chocron r, yatim n, boussier j, gendron n, et al. angiopoietin-2 as a marker of endothelial activation is a good predictor factor for intensive care unit admission of covid-19 patients. angiogenesis [internet]. 2020;23(4):611–20. available from: https://doi.org/10.1007/s10456-020-09730-0 22. vaughan de. pai-1 and atherothrombosis. j thromb haemost. 2005;3(8):1879–83. 23. fay wp, korthuis rj. no sweetie pie: newly uncovered role for pai-1 in infl ammatory responses to ischemia/ reperfusion. physiol behav. 2019;176(3):139–48. 24. praetner m, zuchtriegel g, holzer m, uhl b, schaubächer j, mittmann l, et al. plasminogen activator inhibitor-1 promotes neutrophil infi ltration and tissue injury on ischemia-reperfusion. arterioscler thromb vasc biol. 2018;38(4):829–42. 25. vaughan de, rai r, khan ss, eren m, ghosh ak. plasminogen activator inhibitor-1 is a marker and a mediator of senescence. arterioscler thromb vasc biol. 2017;37(8):1446–52. 26. ji y, weng z, fish p, goyal n, luo m, myears sp, et al. pharmacological targeting of plasminogen activator inhibitor-1 decreases vascular smooth muscle cell migration and neointima formation. physiol behav [internet]. 2017;176(10):139–48. available from: fi le:///c:/users/carla carolina/desktop/artigos para acrescentar na qualifi cação/the impact of birth weight on cardiovascular disease risk in the.pdf 27. whyte cs, morrow gb, mitchell jl, chowdary p, mutch nj. fibrinolytic abnormalities in acute respiratory distress syndrome (ards) and versatility of thrombolytic drugs to treat covid-19. j thromb haemost. 2020;18(7):1548–55. 28. fox se, akmatbekov a, harbert jl, li g, brown jq, heide rs vander. pulmonary and cardiac pathology in african american patients with covid-19: an autopsy series from new orleans. lancet respir med 2020;(january):19–21. 117andrianto, et al.: covid-19 and endothelial dysfunction ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 29. meltzer me, lisman t, de groot pg, meijers jcm, le cessie s, doggen cjm, et al. venous thrombosis risk associated with plasma hypofi brinolysis is explained by elevated plasma levels of tafi and pai-1. blood. 2010;116(1):113–21. 30. zuo y, warnock m, harbaugh a, yalavarthi s, gockman k, lawrence da. plasma tissue plasminogen activator and plasminogen activator inhibitor-1 in hospitalized covid-19 patients. sci rep. 2020;1–13. 31. varga z, flammer a, steiger p, haberecker m, andermatt r, zinkernagel as, et al. endothelial cell infection and endothelitis in covid-19. ann oncol. 2020;(january). 32. kang s, tanaka t, inoue h, ono c, hashimoto s, kioi y, et al. il-6 trans-signaling induces plasminogen activator inhibitor-1 from vascular endothelial cells in cytokine release syndrome. proc natl acad sci u s a. 2020;117(36):22351–6. 33. nougier c, benoit r, simon m, desmurs-clavel h, marcotte g, argaud l, et al. hypofi brinolytic state and high thrombin generation may play a major role in sars-cov2 associated thrombosis. j thromb haemost. 2020;18(9):2215–9. 34. blasi a, von meijenfeldt fa, adelmeijer j, calvo a, ibañez c, perdomo j, et al. in vitro hypercoagulability and ongoing in vivo activation of coagulation and fi brinolysis in covid-19 patients on anticoagulation. j thromb haemost. 2020;18(10):2646–53. 35. löf a, müller jp, brehm ma. a biophysical view on von willebrand factor activation. j cell physiol. 2018;233(2):799–810. 36. da silva ml, cutler df. von willebrand factor multimerization and the polarity of secretory pathways in endothelial cells. blood. 2016;128(2):277–85. 37. butera d, passam f, ju l, cook km, woon h, apontesantamaría c, et al. autoregulation of von willebrand factor function by a disulfi de bond switch. sci adv. 2018;4(2). 38. verhenne s, denorme f, libbrecht s, vandenbulcke a, pareyn i, deckmyn h, et al. platelet-derived vwf is not essential for normal thrombosis and hemostasis but fosters ischemic stroke injury in mice. blood. 2015;126(14):1715–22. 39. doddapattar p, dhanesha n, chorawala mr, tinsman c, jain m, nayak mk, et al. endothelial cell-derived von willebrand factor, but not plateletderived, promotes atherosclerosis in apoe-deficient mice. 2019;38(3):319–35. 40. kayal s, jaïs jp, aguini n, chaudière j, labrousse j. elevated circulating e-selectin, intercellular adhesion molecule 1, and von willebrand factor in patients with severe infection. am j respir crit care med. 1998;157(3 part i):776–84. 41. meyer aa, kundt g, steiner m, schuff -werner p, kienast w. impaired flow-mediated vasodilation, carotid artery intima-media thickening, and elevated endothelial plasma markers in obese children: the impact of cardiovascular risk factors. pediatrics. 2006;117(5):1560–7. 42. mannucci pm. von willebrand factor a marker of endothelial damage? greece rome.?? arterioscler thromb vasc biol. 1967;14(2):188–98. 43. grobler c, maphumulo sc, grobbelaar lm, bredenkamp jc, laubscher gj, lourens pj, et al. covid-19: the rollercoaster of fi brin(ogen), d-dimer, von willebrand factor, p-selectin and their interactions with endothelial cells, platelets and erythrocytes. int j mol sci. 2020;21(14):1–25. 44. iba t, levy jh, connors jm, warkentin te, thachil j, levi m. the unique characteristics of covid-19 coagulopathy. crit care. 2020;24(1):4–11. 45. kawecki c, lenting pj, denis c v. von willebrand factor and inflammation. j thromb haemost. 2017;15(7):1285–94. 46. breakey n, escher r. d-dimer and mortality in covid19: a self-fulfi lling prophecy or a pathophysiological clue? swiss med wkly. 2020;150(21–22):1–7. 47. helms j, tacquard c, severac f, leonard-lorant i, ohana m, delabranche x, et al. high risk of thrombosis in patients with severe sars-cov-2 infection: a multicenter prospective cohort study. intensive care med [internet]. 2020;46(6):1089–98. available from: https://doi.org/10.1007/s00134-020-06062-x 48. morici n, bottiroli m, fumagalli r, marini c, cattaneo m. role of von willebrand factor and adamts13 in the pathogenesis of thrombi in sars-cov-2 infection: time to rethink. thromb haemost. 2020;120(9):1339–41. 49. bazzan m, montaruli b, sciascia s, cosseddu d, norbiato c, roccatello d. low adamts 13 plasma levels are predictors of mortality in covid-19 patients. intern emerg med [internet]. 2020;15(5):861–3. available from: https://doi.org/10.1007/s11739-02002394-0 50. huisman a, beun r, sikma m, westerink j, kusadasi n. involvement of adamts13 and von willebrand factor in thromboembolic events in patients infected with sars-cov-2. int j lab hematol. 2020;42(5):e211–2. 51. adam e, zacharowski k, miesbach w. a comprehensive assessment of the coagulation profi le in critically ill covid-19 patients. thromb res. 2020;(january). 52. latimer g, corriveau c, debiasi rl, jantausch b, delaney m, jacquot c, et al. cardiac dysfunction and thrombocytopenia-associated multiple organ failure infl ammation phenotype in a severe paediatric case of covid-19. lancet child adolesc health 2020;(january):19–21. 53. katneni uk, alexaki a, hunt rc, schiller t, dicuccio m, buehler pw, et al. coagulopathy and thrombosis as a result of severe covid-19 infection: a microvascular focus. thromb haemost. 2020; 54. panigada m, bottino n, tagliabue p, grasselli g , n o v e m b r i n o c , c h a n t a r a n g k u l v, e t a l . 118 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 108–119 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 hypercoagulability of covid-19 patients in intensive care unit: a report of thromboelastography fi ndings and other parameters of hemostasis. j thromb haemost. 2020;18(7):1738–42. 55. rauch a, labreuche j, lassalle f, goutay j, caplan m, charbonnier l, et al. coagulation biomarkers are independent predictors of increased oxygen requirements in covid-19. j thromb haemost. 2020;(august):1–12. 56. sugiyama mg, gamage a, zyla r, armstrong sm, advani s, advani a, et al. influenza virus infection induces platelet-endothelial adhesion which contributes to lung injury. j virol. 2016;90(4):1812– 23. 57. mojiri a, nakhaii-nejad m, phan wl, kulak s, radziwon-balicka a, jurasz p, et al. hypoxia results in upregulation and de novo activation of von willebrand factor expression in lung endothelial cells. arterioscler thromb vasc biol. 2013;33(6):1329–38. 58. pinsky dj, naka y, liao h, oz mc, wagner dd, mayadas tn, et al. hypoxia-induced exocytosis of endothelial cell weibel-palade bodies: a mechanism for rapid neutrophil recruitment after cardiac preservation. j clin invest. 1996;97(2):493–500. 59. mojiri a, alavi p, lorenzana carrillo ma, nakhaeinejad m, sergi cm, thebaud b, et al. endothelial cells of different organs exhibit heterogeneity in von willebrand factor expression in response to hypoxia. atherosclerosis [internet]. 2019;282(june 2018):1–10. available from: https://doi.org/10.1016/j. atherosclerosis. 2019.01.002 60. matsuura y, yamashita a, iwakiri t, sugita c, okuyama n, kitamura k, et al. vascular wall hypoxia promotes arterial thrombus formation via augmentation of vascular thrombogenicity. thromb haemost. 2015;114(1):158–72. 61. ogawa s, clauss m, kuwabara k, shreeniwas r, butura c, koga s, et al. hypoxia induces endothelial cell synthesis of membrane-associated proteins. proc natl acad sci u s a. 1991;88(21):9897–901. 62. fearns c, loskutoff dj. induction of plasminogen activator inhibitor 1 gene expression in murine liver by lipopolysaccharide: cellular localization and role of endogenous tumor necrosis factor-α. am j pathol. 1997;150(2):579–90. 63. gragnano f, sperlongano s, golia e, natale f, bianchi r, crisci m, et al. the role of von willebrand factor in vascular infl ammation: from pathogenesis to targeted therapy. mediators infl amm. 2017;2017. 64. andersson hm, siegerink b, luken bm, crawley jtb, algra a, lane da, et al. high vwf, low adamts13, and oral contraceptives increase the risk of ischemic stroke and myocardial infarction in young women. blood. 2012;119(6):1555–60. 65. hoechter dj, becker-pennrich a, langrehr j, bruegel m, zwissler b, schaefer s. higher procoagulatory potential but lower dic score in covid-19 ards patients compared to non-covid-19 ards patients. thromb res 2020;(january). 66. varin r, mulder p, tamion f, richard v, henry jp, lallemand f, et al. improvement of endothelial function by chronic angiotensin-converting enzyme inhibition in heart failure: role of nitric oxide, prostanoids, oxidant stress, and bradykinin. circulation. 2000;102(3):351–6. 67. mancini gbj, henry gc, macaya c, o’neill bj, pucillo al, carere rg, et al. angiotensin-converting enzyme inhibition with quinapril improves endothelial vasomotor dysfunction in patients with coronary artery disease: the trend (trial on reversing endothelial dysfunction) study. circulation. 1996;94(3):258–65. 68. fujii m, wada a, tsutamoto t, ohnishi m, isono t, kinoshita m. bradykinin improves left ventricular diastolic function under long-term angiotensinconverting enzyme inhibition in heart failure. hypertension. 2002;39(5):952–7. 69. bachetti t, comini l, pasini e, cargnoni a, curello s, ferrari r. ace-inhibition with quinapril modulates the nitric oxide pathway in normotensive rats. j mol cell cardiol. 2001;33(3):395–403. 70. mukai y, shimokawa h, higashi m, morikawa k, matoba t, hiroki j, et al. inhibition of reninangiotensin system ameliorates endothelial dysfunction associated with aging in rats. arterioscler thromb vasc biol. 2002;22(9):1445–50. 71. sola s, mir mqs, cheema fa, khan-merchant n, menon rg, parthasarathy s, et al. irbesartan and lipoic acid improve endothelial function and reduce markers of infl ammation in the metabolic syndrome: results of the irbesartan and lipoic acid in endothelial dysfunction (island) study. circulation. 2005;111(3):343–8. 72. shahin y, khan ja, samuel n, chetter i. angiotensin converting enzyme inhibitors eff ect on endothelial dysfunction: a meta-analysis of randomised controlled trials. atherosclerosis [internet]. 2011;216(1):7– 16. available from: http://dx.doi.org/10.1016/j. atherosclerosis.2011.02.044 73. hasan ss, kow cs, hadi ma, zaidi str, merchant ha. mortality and disease severity among covid19 patients receiving renin-angiotensin system inhibitors: a systematic review and meta-analysis. am j cardiovasc drugs [internet]. 2020;20(6):571–90. available from: https://doi.org/10.1007/s40256-02000439-5 74. liu x, long c, xiong q, chen c, ma j, su y, et al. association of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with risk of covid-19, infl ammation level, severity, and death in patients with covid-19: a rapid systematic review and meta-analysis. clin cardiol. 2020; 75. martínez-gonzález j, raposo b, rodríguez c, badimon l. downregulation by atherogenic levels of native ldls balance between transcriptional and 119andrianto, et al.: covid-19 and endothelial dysfunction ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 posttranscriptional regulation. arterioscler thromb vasc biol. 2001;804–9. 76. laufs u, fata v la, plutzky j, liao jk. upregulation of endothelial nitric oxide synthase by hmg coa reductase inhibitors .circulation. ulrich. 1998;1129– 35. 77. bonetti po, lerman lo, napoli c, lerman a. statin eff ects beyond lipid lowering are they clinically relevant? eur heart j. 2003;24(3):225–48. 78. aviram m, hussein o, rosenblat m, schlezinger s, hayek t, keidar s. interactions of platelets, macrophages, and lipoproteins in hypercholesterolemia: antiatherogenic eff ects of hmg-coa reductase inhibitor therapy. j cardiovasc pharmacol. 1998;31(1):39–45. 79. sánchez-quesada jl, otal-entraigas c, franco m, jorba o, gonzález-sastre f, blanco-vaca f, et al. eff ect of simvastatin treatment on the electronegative low-density lipoprotein present in patients with heterozygous familial hypercholesterolemia. am j cardiol. 1999;84(6):655–9. 80. kureishi y, luo z, shiojima i, bialik a, fulton d, david j, et al. the hmg-coa reductase inhibitor simvastatin activates the protein kinase akt and promotes angiogenesis in normocholesterolemic animals. 2010;6(9):1004–10. 81. anderson tj, meredith it, yeung ac, frei b, selwyn ap, ganz p. the eff ect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. j occup environ med. 1996;38(5):468. 82. ascer e, bertolami mc, venturinelli ml, buccheri v, souza j, nicolau jc, et al. atorvastatin reduces proinfl ammatory markers in hypercholesterolemic patients. atherosclerosis. 2004;177(1):161–6. 83. kirmizis d, papagianni a, dogrammatzi f, skoura l, belechri am, alexopoulos e, et al. effects of simvastatin on markers of infl ammation, oxidative stress and endothelial cell apoptosis in patients on chronic hemodialysis. j atheroscler thromb. 2010;17(12):1256–65. 84. reriani mk, dunlay sm, gupta b, west cp, rihal cs, lerman lo, et al. eff ects of statins on coronary and peripheral endothelial function in humans: a systematic review and metaanalysis of randomized controlled trials. eur j cardiovasc prev rehabil. 2011;18(5):704–16. 85. dimmeler s, aicher a, vasa m, mildner-rihm c, adler k, tiemann m, et al. increase endothelial progenitor cells via the pi 3-kinase / akt pathway. j clin inves 2001;108(3):365–6. 86. tiefenbacher cp, friedrich s, bleeke t, vahl c, chen x, niroomand f. ace inhibitors and statins acutely improve endothelial dysfunction of human coronary arterioles. am j physiol hear circ physiol. 2004;286(4 55-4):1425–32. 87. masana l, correig e, borjabad cr, anoro e, arroyo ja, al e. eff ect of statin therapy on sarscov-2 iinfection-related mortality in hospitalized partients. akrab juara [internet]. 2020;5(1):43–54. available from: http://www.akrabjuara.com/index.php/ akrabjuara/article/view/919 88. saeed o, castagna f, agalliu i, xue x, patel sr, rochlani y, et al. statin use and in‐hospital mortality in diabetics with covid‐19. j am heart assoc. 2020; 15(24): 1-12 89. song sl, hays sb, panton ce, mylona ek, kalligeros m, shehadeh f, et al. statin use is associated with decreased risk of invasive mechanical ventilation in covid-19 patients: a preliminary study. pathogens. 2020;9(9):1–9. 90. heitzer t, just h, münzel t. antioxidant vitamin c improves endothelial dysfunction in chronic smokers. circulation. 1996;94(1):6–9. 91. matsumoto t, d’uscio l v., eguchi d, akiyama m, smith la, katusic zs. protective eff ect of chronic vitamin c treatment on endothelial function of apolipoprotein e-defi cient mouse carotid artery. j pharmacol exp ther. 2003;306(1):103–8. 92. carr ac, mccall mr, frei b. oxidation of ldl by myeloperoxidase and reactive nitrogen species: reaction pathways and antioxidant protection. arterioscler thromb vasc biol. 2000;20(7):1716–23. 93. traber mg, stevens jf. vitamins c and e: benefi cial eff ects from a mechanistic perspective. free radic biol med. 2011;51(5):1000–13. 94. scioli mg, bielli a, agostinelli s, tarquini c, arcuri g, ferlosio a, et al. antioxidant treatment prevents serum deprivationand tnf-α-induced endothelial dysfunction through the inhibition of nadph oxidase 4 and the restoration of β-oxidation. j vasc res. 2014;51(5):327–37. 95. chen j, reheman a, gushiken fc, nolasco l, fu x, moake jl, et al. n-acetylcysteine reduces the size and activity of von willebrand factor in human plasma and mice. j clin invest. 2011;121(2):593–603. 96. shapiro, n.i., schuetz, p., yano, k. et al. the association of endothelial cell signaling, severity of illness, and organ dysfunction in sepsis. crit care 14, r182 (2010). https://doi.org/10.1186/cc9290 ijtid vol 3 no 2 april juni 2012.indd 83 vol. 3. no. 2 april–juni 2012 tuberculosis counter (tc) as the equipment to measure the level of tb in sputum fendy purwanda1, yufan fibriawan2, dyar sasmito3, fatkhunisa r4, prihartini widiyanti 5,6 1,4 student of biomedical engineering s1 study program, physics department, faculty of science and technology universitas airlangga 2 student of physics s1 study program, physics department, faculty of science and technology universitas airlangga 3 bachelor of automated system instrumentation vocation study program, physics department, faculty of science and technology universitas airlangga 5 biomedical engineering s1 study program, physics department, faculty of science and technology universitas airlangga 6 institute of tropical disease universitas airlangga abstract tuberculosis (tb) is an infectious disease caused by mycobacterium tuberculosis. this disease is the third killer disease after cardiovascular diseases and respiratory diseases, and is also he number one killer disease in a group of infectious diseases. this is partly due to the late handling and a non real time detection, both of which will inhibit the therapy which yields a large number of microorganisms in the body, and will eventually complicate the recovery. based on this phenomenon, we offered an alternative solution for detecting the sum of microorganism using tuberculosis counter, a tool used to count the number of tuberculosis bacteria in the patient's sputum. technically, the patient's sputum preparat was screened using the tcs230 color sensor that was able to filter the color of the preparat. tuberculosis bacteria in the stained sputum ziehl-niellsen preparat was colored red, while the other was colored blue. by utilizing these optical phenomena, the tcs230 color sensor was supposed to filter the red color in the preparat. by using regression equation measurement, we gained the equation which then correlated the bit value as an output of the sensor with the number of tuberculosis bacteria. then, the digitalization process yielded the real time and accurate data of tuberculosis bacteria. keywords: tuberculosis, ziehl-nellsen staining, tcs230 color sensor, counter of bacteria, sputum abstrak latar belakang: tuberkulosis (tb) adalah penyakit menular yang disebabkan oleh mycobacterium tuberculosis. penyakit ini adalah penyakit pembunuh ketiga setelah penyakit kardiovaskuler dan penyakit pernafasan, serta nomor satu penyakit pembunuh dalam kelompok penyakit menular. hal ini disebabkan sebagian karena penanganan yang terlambat dan tidak mampu mendeteksi masa inkubasi secara tepat hal tersebut dapat menghambat terapi, sehingga jumlah mikroorganisme dalam tubuh tinggi sehingga mengakibatkan sulitnya pemulihan. tujuan; menciptakan alat untuk menghitung jumlah bakteri tbc dalam dahak penderita. metode: secara teknis, para preparat dahak penderita akan diputar dengan menggunakan sensor warna tcs230 yang mampu menyaring warna preparat tersebut. tuberkulosis bakteri dalam preparat ziehl-niellsen dahak bernoda akan berwarna merah, sementara yang lain berwarna biru. dengan memanfaatkan fenomena optik, sensor warna tcs230 akan disaring merah dalam preparat menggunakan gejala optik tcs230 sensor warna akan menyaring warna merah di preparat tersebut. menggunakan pengukuran persamaan regresi, akan memperoleh nilai persamaan yang akan berkorelasi sebagai output dari sensor dengan jumlah bakteri tuberkulosis. kemudian proses digitalisasi akan menghasilkan real time (masa inkubasi) dan data yang akurat dari bakteri tuberkulosis. hasil: prototype alat penghitung jumlah bakteri tbc dalam dahak penderita. kata kunci: tuberkulosis, ziehl-nellsen pewarnaan, tcs230 sensor warna, counter bakteri, sputum research report 84 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 83−85 introduction tuberculosis (tb) is an infectious disease caused by mycobacterium tuberculosis. world health organization in 2005 estimated that the number of tb patients was 8,8 million and 1,6 million of them ended in death. tb itself is placed the third among diseases in indonesia after cardiovascular and airway canal diseases and the first among diseases which cause death in the type of infectious diseases. indonesia itself is the third country with most tb problems in the worlds, with the death number of one person every 5 minutes. in 2004, 211.753 new cases of tb in indonesia had been noted and approximately 300 deaths due to tb every day were also noted. new cases of tb in indonesia always rise to 250 million cases per year.1 based on the tb combat program in middle java, it has been shown that the number of tb case findings in 2006 had not reached the target which was 53%.2 many kinds of tb therapy have been used including routine standard therapy based on the ministry of health’s program. but somethimes the therapy do not work well due to a variety of reasons, one of the main reasons is people's poor knowledge of tb. it was considered that even though the therapy has been taken for a long time, the disease has not been cured yet.11 long therapy would pase a big physiological pressure to the patients. the patients would feel worried that their disease would never heal or become even worse. this excessive anxiety would also trigger other diseases to occur. based on the observation of the case handling which was focused on unspecified physical symptoms in patients, we offer tuberculosis counter (tc), a diagnostic tool which is capable of counting the number of tb bacteria in sputum in order to provide data of the tb degree of severity so that the clinicians could diagnose the disease in real-time condition. by using ziehl-nellsen staining in patient sputum, the red one produced by acid fast bacillus (afb) was filtered and processed digitally then and yielded real-time and up-date. material and method tuberculosis is on infectious disease which attacks lungs, and caused by mycobacterium tuberculosis. mycobacteria are aerob bacteria, rod-shaped, and have no capability to produce spores. they are difficult to be colored. if these bacteria have been colored, they will stand to laxative color such as acid or alcohol. there fore they are called acid-resistant bacteria or acid-resistant basil.3 tcs230 color sensor is a chip color sensor which works by conversing the acceptance of light emission from certain colors in the form of frequency. tcs230 color sensor is composed of 2 main parts which are acceptance light parts in the form of array photodiode and light converter to frequency. in general, tcs230 color sensor is a light sensor which is complemented with a light filter for basic color rgb (red-green-blue) and a light sensor without a filter in 8 bit scale in each sensor part. photodiode in tcs230 color sensor extracted the current of which the magnitude of the flow is comparable to basic color of light overwriting. this current then was converted into a pulse shape with a frequency comparable to the magnitude of the current. the output frequency of the tcs230 color sensor could be made as a scale by arranging data configuration s0 and s1 in selector pin s0 and s1 of tcs230 color sensor.4 figure 1. tcs230 figure 2. ic microcontroller microcontroller is a digital electronic equipment which has on input, on output and a controller with a program which could be written and deleted with special manners. microcontroller is a computer inside a chip which could be used to control electronic equipment so that it could be efficient and effective in cost. electronic system usually needs many supporting components such as ic, ttl and cmos. it could be reduced or minimized. it also has been focused and controlled by this “tiny controller” so that this electronic system could be compendious, faster, easy to modify and the distraction finding could be easier because the system is compact.5 result and discussion the making process of the tuberculosis counter equipment prototype took two months. in the preliminary study, the literature exploration and the experts' statements, indicated that it had been well known that tuberculosis 85purwanda, et al.: tuberculosis counter (tc) as the equipment to measure level of tb bacteria could be detected chemically using ziehl-niellsen staining. acid-resistant bacteria of tb was red in color and the others were be blue in color. from the optical symptoms, then the screening process using tcs230 color sensor had to be done to count and excute a certain calibration.6,7 based on the image, it would be difficult to differentiate tb bacteria from other artifact that it needs some special ability regarding sputum staining. from the instrumentation scheme, the sputum of tb patient which had been coloured by ziehl-nellsen method was previewed by the camera in the microscope and then previewed on an lcd. tcs230 color sensor was placed above the lcd to gain color bit value on the lcd.8 the result of output bit value was produced by the bit plot of the number of bacteria in each viewing area. the plot result was analyzed by regression to gain the regression equation which was converter bit formula of the number of bacteria in each viewing area. the formula was inserted into the chip as on analog data processor from the sensor.9,10,11 a conversion was performed to compare output bit sensor with the number of bacteria in the preparat. this conversion was performed by making a plot between two variables so that regression equation which would be used in the chip should be obtained. calibration test result of this equipment was compared the conventional test. the conventional test was performed by paramedical staff by observing the object inb the microscope and counting them per viewing area whereas this equipment technique had the same procedures as those with a conventional manner. the difference between the two methods was only the bacteria counting manner, which sused sensor and the length of detection time which was shorter than that of the conventional way. in conclusions, the tuberculosis counter (tc) equipment utilized the optical symptoms of tuberculosis bacteria by using ziehl-nielssen staining, and the processing utilized tcs230 color sensor. the benefit of the utilization of tb counter was to provide the real time and accurate data of the number of tuberculosis bacteria.12 reference 1. center data and information, 2008. ministry of health republic indonesia. 2. ronen basri, david jacobs, and ira kemelmacher , “photometric stereo with general, unknown lighting,” international journal of computer vision, 72(3): 239–57, 2007. preliminary version: ieee conf. on computer vision and pattern recognition (cvpr-01), kauai, vol. ii: 374–81, 2007. 3. tirtana bt, musrichan, 2011. faktor-faktor yang mempengaruhi keberhasilan pengobatan pada pasien tuberkulosis paru dengan resistensi obat tuberculosis di wilayah jawa tengah. artikel ilmiah. 4. graham. j., kryzeminski, m., and popovic, z, 2000.capacitance based thickness mapping of thin dielectric films. rev. sci. intrum., 71(5). 5. yoder, j.2000.coriolis effect mass flowmeters.in: mechanical variables measurement, j. webster, ed.crc press, boca raton, fl. 6. skubal, l.r., meshkov, n.k., and vogt, m.c.2001.detection and identification of gaseous organic using a tio2 sensor, j.photochem. photobiol. a: chem., 148, 103–8. 7. smith, j.a., polk b.j., kikas, t., and levermore, d.m. chemfets.2000. chemical sensor for the real world. www.bizoki. chemistry.gatech.edu/janata-chemical-sensor. 8. dybko, a. and wroblewski, w.2000. fiber optic chemical sensor. www.ch.pw.edu.pl/~dybko/csrg/fiber/operating. 9. del prete, z., monteleone, l., and steindler, r.2001.a novel pressure array sensor based on contact resistence variation: metrological roperties.rev.sci.instru. 72(3): 1548–58. 10 kawamura a, 2007. differential recursion, acm transactions on computational logic, vol. v, no. n, 20yy, pages 1–20. 11. philiph-chandy r., morgan r., and scully pj, 2000.drag force flowmeters. in: mechanical variables measurement, j. webster, ed. crc press.boca raton.fl. 12. pallas-areny, r. and webster, j. g.2001.sensor and signal conditioning.2nd edition.john wiley & sons.new york. 78 vol. 5. no. 3 september–december2014 research report update management concurrent infection between dengue viral and salmonella dyah wikanesthi1, desiana w sari1, eva chilvia1, oedojo soedirham1, lely kurniasari1, soegeng soegijanto1,2 1 soerya hospital, sidoarjo 2 indonesian-japan collaborative research center for emerging and re-emerging infectious disease, institute of tropical disease, universitas airlangga, surabaya, indonesia abstract since januari 2013, soerya hospital has found many cases with positive result of igm salmonella along with ns1 or igm & igg dengue. the clinical manifestations mostly are high fever, headache, vomiting, malaise and plasma leakage. some of them with convulsion and unconsciousness. therefore in order to get well of care management, this clinical phenomena should be studied carefully. the aim of this research is to get update management concurent dengue viral and salmonella infection. observational study had been done, since januari 2013 until juli 2013. purposive sampling in 30 case of concurent dengue viral and salmonella infection compared with 30 case of dengue viral infection alone. diagnosis has published based on who 2011 criteria. by using anti vomiting drug, anti pyretic, anti convulsion and antibiotic for salmonella infection and rehidration using ringer acetate, combining ringer asetat and dextrose 5% or combining ringer asetat saline 0,225% or solution of dextrose 5% and saline 0,45 during 4–5 days hospitalization. the result show that all cases were recovered and got well. there is no significant different between concurent dengue viral and salmonella infection compared with dengue viral infection alone. some cases showed that length time to stay in hospital become 1–2 days longer. it was due to delayed getting antibiotic for salmonella infection. all cases had got first drugs accurately in a clinical manifestation that has been daily showed. it was as a problem solving for saving all the cases. key words: concurrent infection, dengue viral infection, salmonella infection, care, ns1 abstrak sejak januari 2013, rumah sakit soerya menemukan banyak kasus pasien positif igm salmonella dengan ns1 atau igm & igg dengue. manifestasi klinis yang tampak ialah demam tinggi, pusing, mual, tidak enak badan, dan pecahnya plasma. beberapa di antara mereka ada yang mengalami gangguan hebat dan ada yang tidak. oleh karena itu untuk mendapatkan perawatan yang baik maka fenomena ini perlu dipelajari dengan teliti. tujuan dari penelitian ini ialah untuk mendapatkan pembaharuan manajemen virus dengue dan infeksi salmonella. observasi telah dilakukan sejak januari 2013 hingga juli 2013. sampel purposive pada 30 kasus virus dengue dan infeksi salmonella yang dibandingkan dengan 30 kasus infeksi virus dengue saja. diagnosa telah dipublikasikan oleh criteria who tahun 2011. menggunakan obat anti mual, anti penurun suhu tubuh, anti konvulsi (tidak enak badan), dan antibiotic untuk infeksi salmonella dan rehidrasi menggunakan ringer asetat saline 0,225% atau larutan dextrose 5% dan saline 0,45 selama 4–5 hari di rumah sakit. hasil penelitian menunjukkan bahwa semua kasus akan dipulihkan dan sembuh. tidak ada perbedaan yang signifikan antara virus dengue dan infeksi salmonella yang dibandingkan dengan infeksi virus dengue saja. beberapa kasus menunjukkan bahwa waktu untuk di rumah sakit menjadi 1–2 hari lebih lama. hal itu akan tertunda jika mengkonsumsi antibiotic untuk infeksi salmonella. semua kasus telah mendapatkan obat pertama pada manifestasi klinik yang mana ditunjukkan sehari-hari. hal tersebut merupakan solusi untuk semua kasus. kata kunci: infeksi konkuren, infeksi viral dengue, infeksi salmonella, perawatan, ns1 79wikanesthi d, et al.: update management concurrent infection between dengue viral and salmonella introduction on 2013 there are many cases dengue viral concurrent with salmonella infection. some of them showed a duration of clinical manifestation more longer than usual (see fig. 2 and 3). why this event occur, it might be due to late coming as the second infection occur. before discussing this event, we want to discussed a natural cause of dengue viral infection and salmonella infection. dengue viral infection are usually shown a clinical manifestation of fever as saddle back phenomena and followed by vomiting attack and headache.1,2,3,4 salmonella infection as usually shown the duration of infection need more time until four weeks, if the patient don’t get early antibiotic for bacterial of salmonella, patient showed clinical manifestation of gastritis, abdominal pain and concurrent with vomiting. in the past one decade, coincident cases rare to be concern by pediatrics, but in 2013 at soerya hospital has found more than 100 cases in 1 year. we thought why these case could happen and getting many more, these are the global changes season and population changes. in early rainy season, we found that dvi cases was increased in order to summer season. when the rainy season prolonged, it could cause many problem in environment such as worsening hygiene individu and environment, it could cause increased salmonella infection cases. that's why many cases coincident dvi and salmonella infection. materials and methods observational study had been done, since januari 2013 until juli 2013. purposive sampling in 30 case of concurent dengue viral and salmonella infection compared with 30 case of dengue viral infection alone. diagnosis has published based on who 2011 criteria. the result clinical manifestation of dvi patients including:1,2,3,4,5 a. fever: acute onset, high and continuous, lasting 2–7 days in most cases b. any of the following haemorrhagic manifestations including a positive tourniquet test (the most common), petechiae, purpura (at venepuncture sites), ecchymosis, epistaxis, gum bleeding and haematemesis and or melena c. enlargement of the liver (hepatomegaly) is observed at some stage of the illness in 90–98% d. shock, manifested by tachycardia, poor tissue perfusion with weak pulse and narrowed pulse pressure (20 mmhg or less) or hypotension with the presence of cold, clammy skin and or restlessness. clinical manifestation of concurrent dvi with salmonella infection patients including: a. fever b. nausea c. vomitting d. diarrhea and abdominal pain e. epistaksis these are clinical manifestations from 30 patients that we were observed in soerya hospital and 30 patients concurrent dvi with salmonella infection. table 1. clinical manifestation dvi patients and concurrent dvi with salmonella infection. symptoms dvi patients dvi + salmonella infection fever 100% 100% nausea 62% 83% vomitting 40% 63% diarrhea 6% 20% abdominal pain 36% 73% epistaksis 2% by using anti vomiting drug, anti pyretic, anti convulsion and antibiotic for salmonella infection and rehidration using ringer acetate, combining ringer asetat and dextrose 5% or combining ringer asetat saline 0,225% or solution of dextrose 5% and saline 0,45 during 4-5 days hospitalization. the result show that all cases were recovered and got well. there is no significant different between concurent dengue viral and salmonella infection compared with dengue viral infection alone. some cases showed that length time to stay in hospital become 1–2 days longer. it was due to delayed getting antibiotic for salmonella infection. discussion concurrent infection of dengue viral infection (dvi ) and salmonella in children. it is 2 kind of diseases that infect a child in a same time. how to know that these cases were caused by 2 agents (viral and bacteria), is we did anamnese, examined these patients and we used laboratory test (ns1 and igm salmonella) to support our diagnosa. it is very difficult for us to know whether dvi or salmonella infection that first infect to these children. we might try to study by identifying the agents that correlate with symptoms. we had analysed that concurrent dvi and salmonella infections patients may stay longer in the hospital, than patient with single infection, especially if they came late to the hospital. 80 indonesian journal of tropical and infectious disease, vol. 5. no. 3 september–december 2014: 78–81 there are the figure of length of stay patients with dengue viral infection (dvi) compared with concurrent dvi and salmonella infection. problem solving we try to study which one disease come first by doing observational study about clinical manifestation and symptom that occur,5,6,7 these are: 1. high fever (high fever curve) from the curve, we know that there was a different fever pattern of dvi patient and concurrent infection patients. dvi patients had a high fever in early day they admitted in the hospital, and then slowly go down in couple days.7 concurrent dvi and salmonella infection had an irregular fever pattern. 2. vomitting 3. nausea 4. abdominal pain 5. diarrhea 6. epistaksis figure 1. length of stay dvi patients. figure 2. length of stay concurrent dvi and salmonella infection patients. figure 3. body’s temperature of dvi patients (purple line) and heart rate (green line). figure 4. body’s temperature of concurrent dvi and salmonella infection patients (purple line) and heart rate (green line). treatment we use some drugs to these patients, such as: 1. crystalloid fluid we had use ringer acetate as a resuscitation fluid, because of its metabolism in muscle, not in liver so that it will not aggravate liver function.8 2. metoclopramide or ondansentron to solved the clinical manifestation of vomiting due to gastritis that can be given by oral or intravenous. based on the problem that had been ocur in concurent infection dengue virus and salmonella. the patient should be care in the hospital by giving infusion base on the age and body weight of cases, such as: 1. anti pyretic drop, per drop dossage 4-8 hours for high fever. from the figure 1 and 2 we know that patients with ns1 positive, stayed 5–6 days in the hospital. but the patients with concurrent dvi and salmonella infection might stay longer (7–9 days ) in the hospital. but in average they stayed for about 6 days in the hospital because we could diagnose the disease early and give therapy to them. 81wikanesthi d, et al.: update management concurrent infection between dengue viral and salmonella 2. anti convulsing drop such as dilantin, dossage is 5 mg/kgbb/24 hours giving per drip. 3. some cases who showed frequently vomiting try to give anti vomiting by drip per infussion such as ondancetron. beside this event, many cases showed plasma leakaged with could be identified by increasing hematocrit and decreasing trombocyte. the patient look pale of the face, foot and hands feel cold, high rate pulse of hand.5,6 for this case should be using cristaloid solution. such ringer acetat, phisiology sollution in 1–2 hours. if the condition of case still worse, give colloid solution. if the patient show bleeding such epistaxis, haematemesis or melena, please give blood transfusion from their own blood family transfusion.5,6,7,8 please awarness using ringer lactat could cause liver damage, so please choose ringer acetate, because ringer lactat metabolism in liver can make trouble of liver physiology and induce dic. but if we use ringer acetat that metabolism in muscle. conclusion all cases had got first drugs accurately in a clinical manifestation that has been daily showed. it was as a problem solving for saving all the cases. references 1. academy of medicine malaysia ministry of health. clinical practice guidelines; dengue infection in adults. dengue consensus 2003. 2. gubler d. dengue and dengue hemorrhagic fever. clin microbiol rev 1998; 11; 480–96. 3. wang s, he r, patarapotikul, j et al., 1995. antibody-enhanced binding of dengue virus to human platelets. j. virology. october 213: page: 1254–1257. 4. world health organization. dengue guideline for diagnosis, treatment, prevention, and control. geneva. 2009. 5. pt otsuka. guidance of infus solution. revision edition viii. 2003. 6. shu. p, huang j current advances in dengue diagnosis. clin diag immunol 2004; 11: 642–50. 7. srichaikul t, nimmannitya s. haematology in dengue and dengue haemorrhagic fever. baillieres best pract res clin haematol 2000; 13(2): 261–76. 8. kurane i, enis fa. immunopathogenesis of dengue virus infection. in: d.j. gubler dj. kun g (ed). dengue and dengue haemorrhagic fever. wallingfird uk: cab international, 2002: 273–90. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid vol 3 no 2 april juni 2012.indd 112 vol. 3. no. 2 april–juni 2012 the preliminary study of antioxidant activity from xylo-oligosaccharide of corncob (zea mays) hydrolysis product with endo-β-xylanase enzyme laura navika yamani1, alfinda novi kristanti2, ni nyoman tri puspaningsih1, 2 1 proteomic laboratory, institute of tropical disease, universitas airlangga, c campus mulyorejo, surabaya, east java 60115, indonesia. 2 departement of chemistry, faculty of science and technology, universitas airlangga, c campus mulyorejo, surabaya, east java 60115, indonesia. abstract xylo-oligosaccharide derived from corncob hemicellulose has been reported to possess antioxidant activity. in order to assess the effective scavenging of xylo-oligosaccharide, we conducted in vitro studies based on self-made xylo-oligosaccharide with dpph (2,2diphenyl-1-picrilhydrazil) method. xylo-oligosaccharide was prepared with enzymatic hydrolysis. the enzyme used for hemicellulose hydrolysis was endo-β-xylanase enzyme from pc-01 isolated bactrerium. pc-01 isolated bacterium used in this study was pacet hot spring which was isolated from east java. endo-β-xylanase enzyme is an extracelluler enzyme. there was about 0.199 u/ml after purification and dialysis process. hydrolisis product of hemicellulose a and b from corncob were analyzed with tlc (thin layer chromatography) and hplc (high performance liquid chromatography). this analysis showed that hydrolysis product of hemicellulose b had a lot of xylo-oligosaccharide hydrolysis product of hemicellulose than xylo-oligosaccharide hydrolysis product of hemicelluloses a. xylo-olygosaccharide was analyzed as on antioxidant activity. xylo-oligosaccharide hydrolysis product of hemicellulose b (ic50 = 48.96) has higher antioxidant activity than xylo-oligosaccharide hydrolysis product of hemicellulose a (ic50 = 92.302). the toxicity of xylo-oligosaccharide can be calculated by the value of lc50 (lethality concentration). lc50 of xylooligosaccharide derived from corncob hemicellulose was 400 ppm so that xylo-oligosaccharide has anti tumor activity because xylooligosaccharide has lc50 < 1000 ppm. keywords: hemicellulose, corncob, endo-β-xylanase, xylo-oligosaccharide, antioxidant activity, toxicity abstrak latar belakang: xilo-oligosakarida hasil hidrolisis hemiselulosa tongkol jagung dilakukan studi awal uji aktivitas antioksidan. tujuan: uji perendaman radikal bebas oleh xilo-oligosakarida, uji antioksidan dari xilo-oligosakarida ini dilakukan secara in-vitro dengan metode dpph (2,2-diphenyl-1picrilhydrazil). metode: xilo-oligosakarida diperoleh dari hasil hidrolisis secara enzimatis. enzim yang digunakan untuk proses hidrolisis ini adalah enzim endo-β-xilanase dari isolat bakteri pc-01. isolat bakteri pc-01 yang digunakan dalam penelitian ini adalah isolat dari sumber air panas pacet. enzim endo-β-xilanase adalah enzim ekstraseluler yang memiliki aktivitas 0,199 u/ml setelah proses pemurnian dan dialisis. produk hidrolisis hemiselulosa a dan b dari tongkol jagung dianalisis dengan klt (kromatografi lapis tipis) dan hplc (high performance liquid chromatography). analisis tersebut menunjukkan bahwa produk hidrolisis hemiselulosa b memiliki kandungan xilo-oligosakarida yang lebih banyak dibandingkan dengan produk hidrolisis hemiselulosa a dari tongkol jagung. hasil: xilo-oligosakarida hasil hidrolisis hemiselulosa tongkol jagung diuji aktivitas antioksidan. xilo-oligosakarida hasil hidrolisis hemi b (ic50 = 48,96) memiliki aktivitas antioksidan yang lebih tinggi dibandingkan xilo-oligosakarida hasil hidrolisis hemi a dari tongkol jagung (ic50 = 92,302). toksisitas xilo-oligosakarida dapat dihitung dari harga lc50 (lethality concentration). nilai lc50 dari xilo-oligosakarida hasil hidrolisis hemiselulosa b tongkol jagung adalah 400 ppm sehingga xilo-oligosakarida ini memiliki aktivitas antitumor karena nilai lc50 < 1000 ppm. kata kunci: hemiselulosa, tongkol jagung, endo-β-xilanase, xilo-oligosakarida, aktivitas antioksidan, toksisitas literature review 113yamani, et al.: the preliminary study of antioxidant activity introduction lately the medical world has been discussing free radicals that give bad effects to human health. these free radicals are physiologically produced by the cells due to the metabolic processes in the body. in addition, free radicals are also produced by other processes outside the body such as ionizing radiation, environmental pollutants (vehicle emission and industrial emissions, asbestos, cigarette smoke, etc.), alcohol, smoke and foods which contain high fat. free radicals can be easily formed by a compound that is ready to deliver a single electron, such as fatty acids. free radicals or oxidants in the body can be controlled by the body itself by forming endogenous antioxidants. on the situation of endogenous antioxidants that are not able to suppress free radicals that arise, it needs antioxidants from outside. antioxidants can be obtained from the synthesis or from natural compounds in plants.1 recently, it has been reported that the oligosaccharide c o m p o u n d s a l s o h a v e a n t i o x i d a n t a c t i v i t i e s . 1 9 oligosaccharide is an oligomer of hemicellulose which can be found in many agricultural waste. oligosaccharide is one example among other xilo-oligosaccharides (xos), galaktooligosaccharides, and frukto-oligosaccharides (fos).based on this background, the research was conducted as a preliminary study testing for oligosaccharides especially xylooligosaccharides obtained from enzymatic hydrolysis of corn cob as an anti-oxidant with several stages. these stages were hemicellulose isolation of corn cob and enzymatic hydrolisis of hemicellulose into oligimer which was xylooligosaccharides. the enzyme that was used for the hydrolysis of hemicellulose subtrate was endo-β-xylanase from bacillus subtilis pc-01.3 xilo-oligosaccharides derived was used to fix antioxidant tests and toxicity tests using shrimp fry. methods xilanolitik enzymes production inoculum of bacillus subtilis pc 01 was grown in 1 liter of media production and incubated for 8 hours at 60° c. cells were harvested after ± 8 hours at 4° c and centrifuged 10000 rpm for 10 minutes. cell pellet was discarded, while the supernatant (enzyme) was used for the enzyme presipitation process. xylanase enzyme precipitation using amonium sulfate (enzyme precipitation) to 100 ml of crude extract enzyme that had been soaked in an ice bath, some ammonium sulfate was added slowly, stirred frequently until the levels of ammonium sulfate saturation percentage reached 60%. ammonium sulfate saturation percentage was used based on ammonium sulfate saturation table.4 the enzymes were centrifuged at 6000 rpm for 10 minutes. precipitated enzyme was again precipitated and dissolved in 100 mm citrate phosphate buffer at ph 5, and then dialyzed. dialysis was performed, until the ammonium sulfate fraction-free enzyme was marked by the formation of a white precipitate when some buffer was poured into bacl2 solution. xilanolitik enzyme assay the standard reaction mixture, contained 100 μl of substrate and 100 μl of enzyme which was incubated at 70° c for 1 hour and finished by adding 600 μl of dns, after that, heated for 15 minutes together with the controls, and immediately cooled in ice water for 20 minutes. absorbance readings were analyzed at a wavelength of λ550 nm. the controls used were 100 μl of enzyme, 100 μl of substrate and 600 of μl dns. they were treated the same as those, above but without any incubation. isolation of corn cobs hemicellulose agricultural waste of corn cob powder weighed 5 grams. we put the two neck round bottom flask containing 2.0 m naoh solution up to 100 ml within a magnetic stirrer for heating for 4 hours. after the process was complete, cooled, and then filtered using buchner funnel, the filtrate was acidified with 4 n acetic acid to a ph of 5.5–6.0 for precipitating hemicellulose a and continued with dicentrifuging (10000 rpm, 20 min) to separate the sediment. the precipitate was freeze-dried to obtain hemicellulose that was of free water. the filtrate obtained was mixed with 96% ethanol to precipitate hemicellulose b. the precipitate obtained was washed with 96% ethanol and then powdered and freeze-dried to obtain a free of water hemicellulose b.5 the hemicellulose obtained could be used for further testing. hemicellulose enzymatic hydrolysis every 1% of hemicellulose a and b samples was taken as much as 100 μl and added with 300 μl of xylanase enzyme, incubated at 70° c for 24 hours. after centrifuging, the filtrate obtained was xilo-oligosaccharides and other sugar monomers were dried using a freeze drier.6 analysis of hydrolysis products thin layer chromatography (tlc) a number of oligosaccharide compounds contained in the hydrolysis products were analyzed by tlc with various comparison eluent. the eluent used n-propanol: ch3cn: water = 5: 3: 2; n-propanol: water: ammonia (70: 29: 1) and n-butanol: acetic acid: water = 2: 1: 1. the three systems of eluent were used to obtain the best separation and as a monitor in the subsequent separation process.7 the apparition stain used was sulfuric acid in methanol. high performance liquid chromatography (hplc) the hplc analysis used 2 different columns: the carbohydrates column (mikrobondapak, waters 2487) and a nh2si column, and 2 different detectors refractory index detector and elsd (evaporative light scaterring detector), as well as solvent methanol 80% in water and 83% acetonitrile in water, flow rate of 1 μl/min, injection volume of 20 μl. 114 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 112−117 anti free radical activity test the antioxidant activity xylo-oligosaccharide standards and the product of hydrolysis spectrometry were determined by measuring absorbance at a wavelength of 497 nm, 517 nm, and 537 nm. each sample of xylo-oligosaccharide standards and the product of hydrolysis was dissolved in water with various concentrations: 100, 80, 60, 40 and 20 ppm, taken as much as 1 ml, added with 1 ml of 0.4 m acetic acid buffer at ph 5.5 and 0.5 ml 10–4 m in ethanol and then incubated for 5 min at 20° c. after that, each solution absorbance was measured with uv-vis spectrophotometer at a wavelength of 497 nm, 517 nm, and 537 nm. observation of free radical activity of compounds against dpph reagent absorbance can be calculated as follows. ahit = a517nm – a497nm a537nm 2 anti free radical activity as % scavenging dpph was as follows. % scavenging dpph = [1 – (a count of test material)] × 100% a count dpph (comparator) determination of the inhibition ic50 (inhibitor concentration 50%) was based on the linear regression analysis of the concentration of % scavenging dpph. if ic50 is less than 100 ppm, the compound has the activity as an anti-free radical.8 toxicity test using bslt method bslt test was performed on the isolated pure compound or separation. sample weighed as much as 10 mg, and then was dissolved in 1 ml of water. after that, it was added with 99 ml of sea water and stirred until homogeneous to obtain a solution with a concentration of 100 ppm. from 100 ppm solution with concentrations of 100, 50, 25, and 12,5 ppm respectively, replication until 3 times was made. further into the sample solution and control, each shrimp fry 8–15 was added, thereafter left to stand for 24 hours. the number of dead shrimp fry was counted and recorded for each concentration of the sample solution and the control solution. good control data were obtamed when there was no dead shrimp fry. shrimp fry mortality data at each concentration was used for the analysis of lc50. 9 observations were made after artemia salina contact with the test solution for 24 hours. if the mortality in the control was more than 10 %, the test was canceled and re-tested. toxicity of xylo-oligosaccharides was determined by calculating the lc50. to determine lc50, data obtained from the test result bioactivity were processed using spss computer program to determine the lc50 value. the test result obtained, provided information about the toxicity of the hydrolisis product. results hemicellulose hydrolysis enzymatically the enzyme activity of crude endo-β-xylanase was as high as 0.119 u/ml. crude extract of endo-β-xylanase precipitated by ammonium sulfate showed that the enzyme activity of endo-β-xylanase had on optimum activity at 60% saturation of ammonium sulfate. it was based on the previous research.10 after the dialysis process was complete, the volume of endo-β-xylanase obtained was 15 ml from 1 liter of media production and the activity of endo-β-xylanase in total after ammonium sulfate precipitation and dialysis was 0.199 u / ml. in this study, hemicellulose a (hemi a) and hemicellulose b (hemi b) were produced. hemi a was a major hemicellulose whereas hemi b was hemi residual hemicellulose product. a hemi obtained was 7.6 grams, while the hemi b obtained was 6.4 grams. from the tlc results obtained, xylo-oligosaccharide from hemi b hydrolysis product had a retention factor (rf) of 0.36, while xylo-oligosaccharide from hemi a hydrolysis product had a value of rf as high as 0.41. based on these two rf. it was expected that xylo-oligosaccharide from hemi b hydrolysis product had a degree of polymerization (dp)which was higher than that from the hemi a. this can be seen from table 1. the results of hplc analysis for the hydrolysis products of corn cobs sample glucose (%) xylose (%) arabinose (%) xylo-oligosaccaride (%) ca ( xylo-oligosaccharides from hemi a of corncob) 5,0 0 0 4,6 cb (xylo-oligosaccharides from hemi a of corncob) 6,0 0 0,1 4,9 figure 1. tlc result of xylo-oligosaccharide compounds from hemi a and b. 115yamani, et al.: the preliminary study of antioxidant activity the spots on the tlc plates that the highest rf is the spot for the monomer-monomer sugars/monosaccharides (located around the upper limit of the plate). from table 1, we know that hemi b contains more xylo-oligosaccharides than hemi a. xylo-oligosaccharide from hemi a was 4.6% while from hemi b was 4.9%. anti free radical activity test free radical activity of each xylo-oligosaccharides can be determined based on regression equations derived from these curves of xylo-oligosaccharides (hemi a), y = 0.5406 x and xylo-oligosaccharides (hemi b), y = 0.8083x + 10.425. once the calculation was done by substituting the a value y with 50, it means that the ability was reduced to 50%, and the obtained x as ic50 values are as follows. based on the data in figure 2. ic50 value of xylooligosaccharides (hemi a) equaled 92.302 ppm, whereas the ic50 value of xylo-oligosaccharides (hemi b) was 48.96 ppm. the antioxidant activity of compounds oligosaccharides could be affected by dp of the compound. in this study, the antioxidant activity in xylo-oligosaccharide hydrolysis results hemi b with variations hydrolysis time were also tasted. figure 2. percentage of scavenging dpph curve vs. dpph concentration xylo-oligosaccharides. (a) hemia, (b) hemi b. table 2. the results of measurements and calculations % dpph scavenging by xylo-oligosaccharide from hemi b hydrolysis product with hydrolysis time variations xylo-oligosakarida (hemi b) xylo-oligosakarida concentration (hemi b) absorbance antioxidant activity a497 a517 a537 % scavenging ic50 (ppm) hemi b of corncob hydrolyzed for 6 hours 80 ppm 60 ppm 40 ppm 20 ppm control 0,081 0,080 0,090 0,083 0,081 0,082 0,085 0,099 0,104 0,100 0,081 0,087 0,104 0,115 0,103 87,5 % 81,25 % 75 % 37,5 % 24 hemi b of corncob hydrolyzed for 12 hours 80 ppm 60 ppm 40 ppm 20 ppm control 0,269 0,175 0,148 0,127 0,081 0,281 0,192 0,172 0,152 0,100 0,288 0,203 0,187 0,166 0,103 68,75 % 62,5 % 43,75 % 31,25 % 47,61 hemi b of corncob hydrolyzed for 24 hours 80 ppm 60 ppm 40 ppm 20 ppm control 0,482 0,398 0,508 0,477 0,516 0,541 0,474 0,607 0,567 0,635 0,565 0,475 0,619 0,540 0,594 78,125 % 53,125 % 43,225 % 26,875 % 48,96 table 3. observational data xylo-oligosaccharide toxicity tests with artemia salina l concentration of test solution (ppm) number of artemia salina larvae tested number of artemia salina larvae dead after treatment number of artemia salina larvae dead in control replication i replication ii replication i replication ii replication i replication ii 80 10 10 6 8 0 1 60 10 10 4 2 0 0 40 10 10 1 2 0 0 20 10 10 0 0 0 0 116 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 112−117 from table 2, the antioxidant activity of xylooligosaccharide was affected by the hydrolysis time. xylo-oligosaccharide from hemi b hydrolysis product was incubated for 6 hours and had a high antioxidant activity when compared with the incubations for 12 hours and 24 hours, while hemi b without hydrolysis had the lowest antioxidant activity. the antioxidant activity of xylooligosaccharide from hemi b hydrolysis product incubated for 12 hours and 24 hours were almost the same. toxicity test of brine shrimp lethality test (bslt) bslt method was performed by counting the number of dead larvae in each test solution. larvae mortality data were obtained and analyzed using a spss program to determine the relationship between the number of larvae mortality with the concentration of the test solution. test result was obtained in lc50 value. the calculation of lc50 value with spss obtained an average lc50 for xylo-oligosaccharide of 400 ppm. discussion the development and the advancement of agriculture and agricultural industry in indonesia have led to an increase in the agricultural waste that are largely a lignocellulosic biomass. lignocellulosic biomass has not been optimally utilized. most of biomass will only be destroyed by burning. continuous combustion process can lead to the accumulation of co2 in the air that will give an impact as global warming. when examined more deeply, lignocellulosic biomass is composed of organic materials such as hemicellulose, cellulose and lignin, and has a great potential as raw material for various industries. in addition, fractionation of this waste into its constituent components will increase its utilization in various industries. among lignocellulosic biomass, corncob is not optimally used. corncob fibers have a composition comprising starch (10–25% (b/b)), hemicellulose (40–50% (b/b)), cellulose (15–25% (b/b)) and phenolic acid (3–5% (b/b)), while the residual consists of protein and oil. natural antioxidant is an antioxidant that comes from nature or synthesized through a chemical reaction, and its structure is derived also from nature. the examples of natural antioxidants are poliphenol, flavonoid (flavonon, flavonol, katekin), vitamin e (tokoferol), vitamin c (asam askorbat), and β-karoten. synthetic antioxidants are antioxidants that are synthesized through a chemical reaction and their structure is derived from nature such as propyl galat, octyl galat, bha, bht and askorbil palmitat.11 research have recently observed that oligosaccharide compounds also have antioxidant activity.2 hemicellulose hydrolysis enzymatically has specific properties. the endo-β-xylanase can hydrolyze xylan as a constituent hemicellulose. the endo-β-xylanase (1,4 βd-xylanxylanohidrolase, ec.3.2.1.8) can hydrolyze xylan basic structure randomly into xylo-oligosaccharides. the antioxidant activity of a compound can be determined by various methods, such as by measuring the activity of dpph radical catcher, ftc measurement (ferry thiocianide), salt reduction method of fremy, teac measurement (trolox equivalent antioxidant capacity), etc. about the scavenging mechanism of dpph by the antioxidant, both xylo-oligosaccharide hydrolysis products from hemicellulose of corn cobs have antioxidant activity because they have ic50 values <100 ppm, 8 but these results suggest that xylo-oligosaccharide from hemi b hydrolysis product have a ic50 value greater than the that of hemi a (figure 2) that xylo-oligosaccharides from hemi b is more active as an antioxidant than the hemi a. the antioxidant activity of oligosaccharides compounds was affected by degree of polymerization (dp) of the compound. previous studies have succeeded in proving that the existence of the antioxidant activity from galactooligosaccharides obtained marine algae by acid hydrolysis, and are influenced by the degree of polymerization of is xylo-oligosaccharides.12 the higher the degree of the polymerization of xylo-oligosaccharides compound, is the higher the antioxidant activity will be. in this study, xylo-oligosaccharide from hemi b hydrolysis product based on the tlc has shown that they have a higher degree of polymerization than that from the hemi a (figure 1). figure 3. dpph scavenging mechanism with antioxidant. 117yamani, et al.: the preliminary study of antioxidant activity but the spots of both of the xylo-oligosaccharide were tailing although we could distinguish the rf value of xylo-oligosaccharide. this is because the tailing spots of xylo-oligosaccharides produced a mixture of xylooligosaccharides that have degrees of polymerization which are adjacent. we still have not been able to prove the influence of the degree of polymerization toward the antioxidant activity. in this study, the antioxidant activity in xylo-oligosaccharide from hemi b hydrolysis product with variations hydrolysis time (6 hours, 12 hours, and 24 hours) (table 2) was also tested. this is due to the very influential hemicellulose hydrolysis products and degree of polymerization of the product of hydrolysis. hydrolysis time can produce hydrolysis products with low degree of polymerization (dp) longer such as sugar monomers. hemi b was also used without hydrolysis as the polysaccharide production controller with a high molecular weight polysaccharide which was insoluble in water. from the calculation with spss, lc50 value was obtained, with the average lc50 for xylo-oligosaccharide of 400 ppm (table 3). it shows that xylo-oligosaccharide has antitumor activity since it has lc50 less than 1000 ppm. conclusion it can be concluded that hemicellulose of corn cobs hydrolysis product has higher antioxidant activity. hemi b without hydrolysis (polysaccharides) had no antioxidant activity, and had ic50 values > 100 ppm because of its very large molecular weight that the antioxidant activity is influenced by the steric effect of the polysaccharide in reducing free radicals. to figure out the toxicity of xylooligosaccharide as an antioxidant compound, bslt method was performed by counting the number of dead larvae in each test solution. references 1. cuendet m, hostettmann k, and potteral o, 1997. flavanoids with free radical scavenging from fagrae blumei, helvetica chimica acta, 80, 1144–52. 2. http://www.nutritionj.com/content/5/1/31, tanggal akses 4 januari 2008. 3. purwadi nm, 2006. identifikasi enzim-enzim xilanolitik dan analisis mikrobiologi isolat bakteri dari sumber air panas pacet jawa timur, skripsi-s1, fmipa-unair, surabaya. 4. pelczar mj dan chan esc, 1986, dasar-dasar mikrobiologi (diterjemahkan oleh anna poedjiadi), jilid 1, ui-press, jakarta. 5. anis m, mohamad h, http://www.ind.usm.my/ti/e-journal/ html-vol1/..%5chtml-vol1anis.htm., tanggal akses: 4 januari 2007.ohamad h., http://www.ind.usm.my/ti/e-journal/htmlvo..%5chtmvol1anis.htm., tanggal akses: 4 jan. 6. puspaningsih nnt, 2004. gen penyandi xilosidase dari bacillus thermoleovorans it-08, disertasi s3-ipb, bogor. 7. tjahjandarie ts, supriyanto g, pudjiastuti p, 2007. separation and purification of enzymatic degradation products from natural substrat xylans. spin progress report, netherland. 8. cos p, ying p, caromme m, hv, j.p., cimanga k, poel bv, pieters i, berghe dp, 1998. structure activity relationship and classification of flavanoids as inhibitor of xantine oxidase and superoxide scavengers, j. nat. prod., 61, 71–3. 9. meyer bn, nr. ferregni, je. putnam, lb jcobson, d.e. nichols l. mclaughin, 1982. brine shrimp: convenient general bioassay for active plant constituents, planta medica, 45, 31–4. 10. purwani nn, 2006. pemurnian parsial enzim xilanase asal isolat sumber air panas pacet, jawa timur, skripsi-s1, fmipa-unair, surabaya. 11. belitz hd & grosch w, 1999. phenolic compounds, food chemistry. berlin: springer, pp. 764–75. 12. chen h-l, hsiao s-c, lin t-l, yamauchi k, hasegawa h, hashimoto t. macromolecules 2001; 34: 671. 13. cano a and palet c., journal of membrane science, xylooligosaccharide recovery from agricultural biomass waste t r e a t m e n t w i t h e n z y m a t i c p o l y m e r i c m e m b r a n s a n d characterization of product with maldi-tof-ms. 14. coughlan mp and hazlewood gp, 1992. hemicellulose and hemicellulase, portland press, london and chapel hill. 15. gritter rj, james m. bobbit, dan arthur f. schwarting, 1991. pengantar kromatografi (diterjemahkan oleh kosasih padmawinata), terbitan kedua, penerbit itb, bandung. 16. lindsay s, 1992. analytical chemistry by open learning high performance liquid chromatography, second edition, john willey and sons, london. 17. nabarlatz d, ebringerova a, montane d, 2007. carbohydrat polymer, autohydrolysis of agricultural by-products for production of xylo-oligosaccharide, 69: 20–8. 18. scopes rk, 1987. protein purification principles and practice. edisi ke-2. new york: springerag. 19. subramaniyan s, prema p, 2002. biotechnology of microbial xylanases: enzymology, molecular biology, and application, critical rev biotechnol, 22: 33–64. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 �0 vol. 4. no. 4 october–december 2013 identification of influenza viruses in human and poultry in the area of larangan wet market sidoarjo-east java, indonesia edith frederika1, aldise mareta1, wilan krisna1, djoko poetranto1, laksmi wulandari2, retno asih setyoningrum2, lucia landia setyowati2, resti yudhawati2, gatot soegiarto2, masaoki yamaoka3 1 influenza study group institute of tropical disease, universitas airlangga, 2 rsud dr. soetomo surabaya departement of internal medicine 3 cellaboration research center emerging re-emerging infeetions disease, institute of tropical disease, universitas airlangga kobe university japan abstract background: influenza is a viral infection that attacks the respiratory system (nose, throat, and lungs) that commonly known as “flu”. there are 3 types of influenza viruses, such as type a, type b, and type c. influenza virus type a is the type of virus that can infect both human and animals, virus type b are normally found only in human, and influenza virus type c can cause mild illness in human and not causing any epidemics or pandemics. among these 3 types of influenza viruses, only influenza a viruses infect birds, particularly wild bird that are the natural host for all subtypes of influenza a virus. generally, those wild birds do not get sick when they are infected with influenza virus, unlike chickens or ducks which may die from avian influenza. aim: in this study, we are identifying the influenza viruses among poultry in larangan wet market. method: around 500 kinds of poultry were examined from cloacal swab. result: those samples were restrained with symptoms of suspected h5. the people who worked as the poultry-traders intact with the animal everyday were also examined, by taking nasopharyngeal swab and blood serum. conclusion: identification of influenza viruses was obtained to define the type and subtype of influenza virus by pcr. key words: subtype of influenza viruses, human, poultry, symptoms, pcr result abstrak latar belakang: influenza merupakan infeksi virus yang menyerang sistem pernafasan (hidung, tenggorokan, dan paru-paru), biasa dikenal sebagai “flu”. terdapat 3 tipe virus influenza yaitu tipe a, tipe b, dan tipe c. virus influenza tipe a adalah jenis virus yang dapat menginfeksi manusia dan hewan, virus influenza tipe b normalnya hanya ditemukan pada manusia, sedangkan virus influenza tipe c dapat menyebabkan sakit ringan pada manusia tanpa mengakibatkan kasus epidemi maupun pandemi. dari ketiga tipe virus influenza tersebut, hanya tipe a yang dapat menginfeksi hewan jenis burung, terutama burung liar yang secara alami merupakan host dari semua subtipe virus influenza a. secara umum, burung liar tersebut tidak akan menjadi sakit ketika terinfeksi virus influenza, tidak seperti pada ayam mapun bebek jika terinfeksi kemungkinan besar dapat mati. tujuan: dalam penelitian ini, kami mengidentiifikasi virus influenza pada unggas di pasar tradisional di larangan, sidoarjo. metode: sekitar 500 unggas diidentifikasi berdasarkan hapusan kloaka. hasil: pengambilan sampel tersebut disesuaikan dengan gejala yang muncul pada hewan itu terkait dengan gejala virus h5. selain itu, kami juga mengumpulan sampel dari responden yang merupakan pekerja di pasar larangan. sampel responden berupa hapusan hidung dan serum darah. kesimpulan: identifikasi virus influenza dilakukan untuk melihat tipe dan subtipe virus berdasarkan pcr. kata kunci: subtipe virus influenza, manusia, unggas, gejala, hasil pcr research report ��frederika, et al.: identification of influenza viruses in human and poultry introduction influenza virus type a can infect humans and animals. various subtypes of this influenza a virus which usually attack human are h1n1, h1n2, and h3n2 (rendell et al., 2006). meanwhile several other influenza a types of attacking animals like h7n9, h5n1, or h3n2. only this influenza a virus that attacks poultry which actually attacking domestic birds. human infections with avian influenza (ai or “bird flu”) are rare but occur most commonly after exposure to infected poultry (bird to human spread). hin1 is a flu virus that was first detected in 2009 called as “swine flu”, caused a world wide pandemic. currently the hin1 is a seasonal influenza virus found in humans and it is now also circulates among pigs. in 2010, even though world health organization announced that the pandemic was over, h1n1 flue virus is still circulating (corzo et al., 2013). recently, there is a new type of influenza virus. h7n9 is a new subtype of avian influenza that has been reported to be detected in poultry in china. however no cases of h7n9 outside china have been reported yet and no sustained person-to-person spread of the h7n9 virus has been found at this time. h5n1 is a highly pathogenic avian flu virus that caused serious outbreaks in domestic poultry in parts of asia and the middle east (who, 2012). although h5n1 does not usually infect humans, nearly 600 cases of human cases of h5n1 have been reported from 15 countries since 2003 in asia, africa, europe, and the near east. about 60% of these people died from their illness. in 2011, 62 human with h5n1 cases and 34 deaths were reported from five countries— bangladesh, cambodia, china, egypt, and indonesia. six countries— bangladesh, china, egypt, india, indonesia, and vietnam—have widespread and ongoing infections in their poultry. in 1997 an outbreak of h5n1 occurred in the farms and traditional markets in hong kong. for the first time reported that the h5n1 virus can infect human with the number of deaths of 6 to 18 cases. poultry outbreaks also happen in other countries recently as well. most human cases of “highly pathogenic“ h5n1 virus infection have occurred in people who had recent contact with sick or dead poultry that were infected with h5n1 viruses. however, unlike other types of flu, h5n1 usually does not spread between people and no further evidence discovers that this virus can spread easily between people. thus, the symptoms and possible complications of h5n1 in people can include fever, cough, shortness/difficulty breathing leads to respiratory failure, or pneumonia (iskander et al., 2013). markets in indonesia are the center of social and economic activities, but the market can also be a source of spread of diseases. a number of outbreaks of the disease at this time can even be transmitted through food products and living animals that are sold in the market. traditional animal market needs special attention due to the occurrence of direct contact between wild birds carrying the virus of avian influenza (ai) in poultry and human (the poultry traders or buyers). weak bio-security and poor hygiene and sanitation lead to the spread and transmission of ai virus in poultry markets. survey on wild birds around the larangan wet market sidoarjo has been conducted and showed that the wild birds infected with avian influenza virus h5n1 (poetranto, 2011). there were highly possibilities of transmission of h5n1 virus from wild birds to poultry sold in the market and to the people works in that market. therefore the influenza study group, institute of tropical disease airlangga university was planning to hold surveillance influenza virus in traditional community animal market, the larangan wet market sidoarjo. the aims of the study are to detect any potential transmission of ai virus in the traditional animal larangan wet market, and also to detect the presence of ai virus in poultry trade and wild birds around the traditional market. this study is only an identification project to obtain early detection of transmission of ai virus among wild birds to poultry and the impact to those who works as poultry traders. furthermore, the early detection could be useful for surveillance of influenza planning. materials and methods there were 3 sample activities in this study. the first one is the sampling on human. some general health assessment was carried out by checking the condition of 63 poultry traders. physical examination and nasal swab sampling was taken during the study. the second sampling was on the poultry. the types of poultry sell in larangan market was variety, such as chicken and duck. during the study, examination on the poultry in the market was carried out, especially those that showed the symptoms of influenza. nasal swab and cloacae swab was taken from around 350–400 poultry. and the last sampling was on wild birds and poultry around the market. the same examination was held, and cloacae swab was also taken from approximately 50–100 wild birds and poultry. the 63 nasal swabs were taken with cotton swab tube. each of the tube were given 2 ml 5% bsa-bhi (bovine serum albumin – brain heart infusion), and mixed by vortex twice. after all samples ready, the next step is filtration which were done inside the bio-safety cabinet (bsc), with 5 ml syringe and sterilized filter, then collect the filtrate in 1.5 ml tube. these filtrates samples were inoculated in monolayer of mdck cells. these samples were incubated at 33–35° c during 3–7 days. daily cpe (cytopathic effect) was observed. mdck cells were chosen considering their better virus sensitivity (possible positive samples). after that, the fluids were harvested for ha test. the positive samples were submitted to the serologic test such as the influenza rapid test, and also evaluated with haemagglutination test (ha) as described in who laboratory’s method (2007). around 500 animal samples with nasal and cloacae swab were collected. the samples were treated similar as human samples with the filtration procedures. after that, those samples were inoculated using 9–10 days old �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 30–34 embryonated hen’s eggs. the treatment of animal samples was performed in the bsl3 laboratory according to the cdc guidelines (who, 2011). after the samples has been harvested, and show ha test positive, the samples are extracted into rna and followed by cdna synthesis. the final product will be cdna, and ready to use or storage at -20° c until the polymerase chain reaction (pcr) techniques was performed. pcr was performed using primer forward (f) and reverse (r). because this is an identification study, the primers that we applied were: h1(f) – h1(r), h3(f) – h3(r), and h5(f) – h5(r). pcr reaction for each sample: 2.5 µl cdna were amplified in a volume of 25 µl containing 12.5 µl premix, 8 µl dilute water (dw), and 1µl primer (10 pmol) for each primer forward and primer reverse. this reaction mixture was then heated in the pcr machine for 3 hours 19 minutes, with the thermal cycler of 40 cycles as follows: 94° c for 2 minutes, 94° c for 30 second, 50° c for 40 second, 72° c for 2 minutes, and 72° c for 10 minutes. the mixture then held at 4° c for indeterminate period until the heat cools down. the amplified pcr product was analyzed by electrophoresis (elp) on 1.5% agarose gel at 100 v for about 40 minutes. the bands were stained with 2 µl/ml ethidium bromide, documented by gel documentation system. result and discussion human samples among 63 people who worked as a poultry traders in larangan market, several symptoms of influenza-likeillness (ili) was identified such as cough, heavy breathing, arthritis, and diarrhoea. the samples were categorized by age and symptoms. approximately around 36.5% samples were in the productive age between 31–40 years old, and only 6.3% were above 60 years old. several symptoms also have been identified. those symptoms are showed in the figure below. the most common symptoms were coughing and heavy breathing, which appeared in all age categories. however, only among people age 51–60 years old that have other symptoms as arthritis and diarrhoea. and surprisingly, those who are above 60 years old did not have any symptoms of ili even though they also working as poultry traders. this might relate on the length of working and how often they spent the time around the market, as well as how the antibody of the person who might resistant to the influenza virus. those are the limitation of this study, that the information on the length of working and the antibody serum were not collected. of these human samples, the serologic test has been taken using influenza rapid test. the results were identified as influenza type b. as the pcr reaction was conducted, only 2 samples (3.17%) were identified of positive h3 (with 1000 base pair), as showed below: animal sample about 500 poultry samples were collected from the market and the area near the market. several kinds of poultry and wild birds were identified with symptoms of influenza virus (particularly h5) are described below. the ha test was conducted and the titter results using chicken rbc (ha ck+) and guinea pig rbc (ha gp+), and also using the diagnostic kit (dx +) that showing possible positive outcome. the diagnostic kit result was identified as influenza type a however, based on the assessment on the egg conditions after inoculation, it showed a relatively high number of possible positive results especially on 24 and 32 hpi (post infection). further assessments were attained with pcr technique using h5 primer. there were two steps of pcr to identify the types of protein on the surface. the first one is to s12001 s12060 picture 1. the h3 result of larangan human sample table 1. age category of human sample and the symptoms age cough arthritis diarrhoea heavy breathing < 20–30 years old 21% 21% 31–40 years old 11% 11% 41–50 years old 9.7% 9.7% 51–60 years old 9% 4.5% 4.5% 9% > 60 years old table 2. poultry category with symptoms poultry category with symptoms broiler chicken 45.5% backyard chicken 46.5% duck 5.9% migrant bird 1.0% owl 1.0% total 100.0% ��frederika, et al.: identification of influenza viruses in human and poultry identify hemagglutinin (ha) by using h5 primers. and the second one is to identify neuraminidase (na) by using h5n1 primer. the h5 results are identified positive when the marks showed on 1.500 bp (base-pair). only 5 samples were identified positive result h5, specifically the types of h5n1. the pcr outcome of ha types are illustrated as follow. the pcr outcome of na types are described below. the na results are identified positive when the marks showed on 1.500 bp (base-pair). based on 5 positive sample of h5, we can identify the na result as positive, specifically the types of h5n1. picture 3. pcr result of na picture 2. pcr result of ha �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 30–34 conclusion there are some limitations on this study. for human sample, there are no information on the length of a person working in the market, the anthropometry measurement to check the nutrition aspect, the immune serum to identify the immune system and the antibody. as well as human sample, the animal sample also have are no specific assessments held, especially for the phylogenetic analysis to check any mutations occur. for the next study, it will better if the anybody serum also taken to check the level of immunity. the anthropometry measurement and nutrition assessment also needed to be carried out to identify the nutrition condition related to the immune system. furthermore, examinations such as sequencing need to be conducted, as well as the phylogenetic analysis. and last but not least, it is better to start developing a pandemic influenza planning as a result of the surveillance activity. references 1. corzo, ca., et al. (2013). active surveillance for influenza a virus among swine, midwestern united states, 2009–2011., emerging infectious disease, vol. 19 no. 6. 2. iskander, john. et al. (2013). pandemic influenza planning, united states, 1978–2008. emerging infectious disease vol.19 no. 6. 3. mancini, dap., et al. (2007). identification and characterization of influenza virus isolated from brazilian snakes., communicating current research and educational topics and applied microbiology. 4. palese, peter., schulman, jerome l., (1996). mapping of the influenza virus genome: identification of the hemagglutinin and the neuraminidase genes. proc. natl. acad. sci. usa 73. 5. poetranto, djoko,. et al. (2011). an h5n1 highly pathogenic avian influenza virus isolated from a local tree sparrow in indonesia. microbiology and immunology volume 55, issue 9, pages 666– 672. 6. rendell, e.g. (2006). “influenza virus types, subtypes, and strains”, submitted for the pandemic influenza preparedness on planning summit. 7. varough, m.d., et al. (2010). genomic signature-based identification of influenza a viruses using rt-pcr/electro-spray ionization mass spectrometry (esi-ms) technology. plosone volume 5 issue 10. 8. world health organization. (2007). recommended laboratory tests to identify avian influenza a virus in specimens from humans. geneva. 9. world health organization. (2011). molecular diagnosis of influenza virus in humans – update. 10. world health organization. (2012). avian influenza: food safety issue. 57 vol. 5. no. 3 september–december 2014 research report the anti-tb drug sensitivity of mycobacterium tuberculosis from cerebrospinal fluid and bone tissue biopsy specimens of patients suspected tuberculous meningitis and spinal tb in dr soetomo hospital indonesia ni made mertaniasih,1 deby kusumaningrum,1 eko budi koendhori,1 sugeng harijono,1 catur endra akry,1 jayanti putri,1 hanik urifah1 1 department of clinical microbiology, dr. soetomo general hospital faculty of medicine universitas airlangga, surabaya indonesia abstract tuberculous meningitis (tbm) is an infection of meningens which potentially life threatening with significant morbidity and mortality. spinal tb has the same problem with tbm, infection in bone and joint, the delayed diagnosis worsens the prognosis. the rapid and accurate diagnosis plus promt adequate treatment is essential for the good outcome. the aim of this research is to study the first line drug sensitivity of mycobacterium tuberculosis isolated from specimens of cerebrospinal fluid from suspected tuberculous meningitis patients and bone tissue biopsy from suspected spinal tb patients. the method of this research is tb laboratory examination in department of clinical microbiology – dr. soetomo general hospital, indonesia, using the gold standard liquid culture method mgit 960 system (becton dickinson) and solid culture method with lowenstein-jensen medium. the specimens csf from 50 tbm patients at january 2013 until may 2014. positive isolate detection of mycobacterium tuberculosis complex were 11 isolates (22%), which sensitivity 100% (11/11 isolates) to rifampin (r), pyrazinamide (z), ethambutol (e), and streptomycin (s); one isolate resistant to isoniazid, sensitivity to isoniazid 90,90% (10/11); and received 21 specimens of bone tissue biopsy which positive 5 isolates (23%), all isolates sensitive 100% (5/5 isolates) to rifampin and pyrazinamide, and 1 isolates resistant to isoniazid, ethambutol, and streptomycin, in which sensitivity 80% (4/5 isolates) to isoniazid, ethambutol, and streptomycin. the conclusion of this research is positivity detection 22% of csf specimens, and 23% of bone tissue biopsy were low. all isolates sensitive 100% to rifampin and pyrazinamide, and 80-90% sensitive to isoniazid. key words: first line anti-tb drug sensitivity, mycobacterium tuberculosis, tuberculous meningitis, spinal tuberculosis, cerebrospinal fluid abstrak meningitis tuberculosis (tbm) merupakan infeksi selaput otak/meningens, berpotensi mengancam kehidupan pasien dengan morbiditas dan mortalitas tinggi. spinal tb juga memiliki masalah yang sama dengan tbm, yaitu infeksi pada jaringan tulang dan sendi serta kelambatan diagnosis yang memperburuk prognosis. diagnosis akurat dan cepat, disertai segera pengobatan adekuat menentukan kesembuhan pasien. tujuan penelitian ialah studi kepekaan obat anti-tb lini i di antara mycobacterium tuberculosis complex isolat specimen cairan otak dari pasien diduga meningitis tb, dan biopsi jaringan tulang dari pasien diduga spinal tb. metode penelitian ini ialah pemeriksaan laboratorium tb di departemen mikrobiologi klinik/ rsud dr soetomo, indonesia, menggunakan metode gold standard metode kultur pada media cair mgit 960 system (becton dickinson) dan metode kultur pada media padat lowenstein-jensen. hasil penelitian ini ialah pada bulan januari 2014 sampai dengan mei 2014 diperoleh specimen cairan otak dari 50 pasien meningitis tb, terdeteksi 11 mycobacterium tuberculosis complex (22%), sensitivitas 100% terhadap rifampin (r), pyrazinamide (z), ethambutol (e), dan streptomycin (s) (11/ 11 isolat); satu isolat resisten terhadap isoniazid, sensitivitas sebesar 90,90% (10/11) terhadap isoniazid; pada 21 spesimen biopsi jaringan tulang ditemukan 5 isolat (23%), semua isolat 100% sensitif 58 indonesian journal of tropical and infectious disease, vol. 5. no. 3 september–december 2014: 57-60 introduction tuberculosis meningitis (tbm) is a common form of central nervous system infection in developing countries with high endemic tb. delayed diagnosis and therapy are major factors in determining outcome, death or severe disabilities. determining diagnosis of tbm based on the complementary standard examination of clinical manifestation, mri/ cranial ct, cerebrospinal fluid (csf) laboratory examination i.e. lymphocytes, glucose, protein, and microbes.1 the definitive diagnosis of tbm based on isolation and identification of mycobacterium tuberculosis from cerebrospinal fluid (csf). isolation and identification of mycobacterium tuberculosis based on the clinical microbiology examination using the gold standard method as follow: culture method and pcr.1,2,3 developed early diagnosis of tbm such as pcr, genxpert mtb/rif, interferon-gamma release assay (igras), tuberculostearic acid, and adenosine deaminase in csf. delayed diagnosis worsens the prognosis and increases morbidity. the microbiological diagnosis is crucial, despite surgical treatment always necessary anti-tb drugs (merino et al., 2012).4 method the 75 specimens or samples were csf from suspected tbm and 21 bone tissue biopsy from suspected spinal tb patients received in tb laboratory of department/ instalation of clinical microbiology-dr soetomo general hospital, surabaya, indonesia at january 2013 until juny 2014. laboratory examination of clinical microbiology using the gold standard: liquid culture method mgit 960 system (becton dickinson) and solid culture method with lowenstein-jensen medium; accurate specimens were centrifuged deposit of csf or processed tissue to microbiologic examination.5,6,7 result & discussion in tb laboratory of department of clinical microbiology dr soetomo hospital received 75 specimens csf at january 2013 until juny 2014. positive isolate detection of mycobacterium tuberculosis complex were 11 isolates (11/75 = 14,67%), which sensitivity 100% (11/11 isolates) to rifampin (r), pyrazinamide (pza), ethambutol (e), and streptomycin (s); one isolate resistant to isoniazid. sensitivity to isoniazid 91% (10/11) (table 1). table 1. positivity detection & first line anti-tb drug sensitivity of mycobacterium tuberculosis complex isolate from csf specimens of the suspect tb meningitis patients in dr. soetomo hospital indonesia, at january 2013-juny 2014 n specimen positive (%) sensitivity r i pza e s 75 liquor/ csf 11 (11/ 75= 14,67%) 11 (100%) 10 (91%) 11 (100%) 11 (100%) 11 (100%) r = rifampin, i = isoniazid, e = ethambutol, s = streptomycin, pza = pyrazinamide rifampin dan pyrazinamide, 1 isolat resisten terhadap isoniazid, ethambutol, dan streptomycin, dengan sensitivitas sebesar 80% (4/5 isolat). kesimpulan dalam penelitian ini ialah sensitivitas deteksi 22% dari spesimen cairan otak, dan 23% biopsi jaringan tulang, rendah, semua isolat sensitif 100% terhadap rifampin dan pyraziamide, 80-90% sensitif isoniazid. kata kunci: obat anti-tb lini i, mycobacterium tuberculosis, meningitis tb, spinal tb, cairan otak at january 2013 until march 2014, tb laboratorydepartment clinical microbiology dr. soetomo hospital received 21 specimens of bone tissue biopsy which positive 5 isolates (5/ 21 = 23.80%), all isolates sensitive 100% (5/ 5 isolates) to rifampin and pyrazinamide (pza), and 1 isolates resistant to isoniazid, ethambutol, and streptomycin. one isolate resistant to isoniazid, ethambutol, and streptomycin, in which sensitivity 80% (4/5 isolates) to isoniazid, ethambutol, and streptomycin (table 2). positivity detection in this study is very low 11/ 75 csf specimens (14,67%) positive isolate mycobacterium tuberculosis complex, and 5/21 bone tissue biopsy (23,80%) positive. many factors could influence the positivity detection of mycobacterium tuberculosis complex, i.e. decided the appopriate criteria of clinical diagnosis for suspected tbm or for suspected spinal tb; the accurate specimen for suspected tbm or for suspected spinal tb related to paucy bacilli in locally tissue specimens; and specimen handling. the sensitivity of mycobacterium tuberculosis complex in this study revealed all isolates 11 from csf and 5 from 59mertaniasih, et al.: the anti-tb drug sensitivity of mycobacterium tuberculosis bone tissue biopsy 100% still sensitive to rifampin and pyrazinamide, and the other advantage were sensivity 80 91% to isoniazid of isolates from csf or bone tissue biopsy; isolates from csf 100% sensitiv rifampin, pyrazinamide, ethambutol, and streptomycin; isolates from bone tissue biopsy 80% still sensitive to ethambutol and streptomycin, otherwise the number of isolate samples were very small that could be not significant to reveal the conclusion on the sensitivity, need the valid research with multy centre study. accurate definitive diagnosis for tbm or spinal tb start with the essential step i.e. to determine the appropriate criteria standard for suspected clinically diagnosis, accurate specimen collection and handling, accurate standard method on laboratory examination for isolation and identification of etiologic mycobacterium tuberculosis. accurate specimens for examination of mycobacteria from suspected tbm patients: aseptic collection of 2–3 specimens of csf with each volume 5–10 ml, because of paucy bacilli in csf specimens.2 accurate specimens for determine etiologic mycobacteria from suspected spinal tb is bone and joint tissue biopsy durante operation or percutaneous biopsy guided by ct or mri to obtain optimal tissues of destructive lesions, caseating granuloma or granulomatous inflammation or abscess in vertebral segments, 2 or more sites, on active cases could added blood aspirate around lesion with volume around 10 ml or more.8,9 conclusion determining tuberculous meningitis and spinal tuberculosis based on the gold standard that included the complementary of examination on clinical manifestation with the standard laboratory of the cns characteristic figure on mri/ ct; chronic inflammation or granulomatous or caseousus necotic on histo pathology; iflammatory reaction markers in blood, protein and glucose concentration, biochemical and pathological features in csf on clinical pathology; and isolation and identification of etiologic bacilli mycobacterium tuberculosis as definitive diagnosis. definitive diagnosis based on isolation and identification mycobacterium tuberculosis included the sensitivity to the first line anti-tb drug. the gold standard method for isolation, identification, and sensitivity tests of mycobacterium tuberculosis using the combined examination of standard culture method (solid and liquid medium) plus standardized pcr. positivity detection 14,67% of csf specimens, and 23% of bone tissue biopsy were very low. all isolates 100% sensitive to rifampin and pyrazinamide, and 80% sensitive to isoniazid, ethambutol, and streptomycin, with considered in very small isolate samples. the important strategy need for better outcome in management tbm or spinal tb could be starded by the research that included multy centre study to decide the standardized procedure on diagnosis, therapy, prevention and promotion. early accurate diagnosis and rapid appropriate therapy could be reached the better outcome, to ovoid disability sequele or mortality. acknowledgements thank to dr soetomo academic hospital for all kinds of supportings in the public services that could be study to improve the science and technology especially in medical. references 1. dandane t, madani n, zekraoui a, belayachi j, abidi k, zeggwagh aa, abouqal r. 2013. a simple method aid for tuberculous meningitis in adults in moroco by use of clinical and laboratory features. i.j.of infectious diseases: e461-e465. 2. bhigjee ai, padayachee r, paruk h, hallwirth-pillay kd, marais s, connoly c. 2007. diagnosis of tuberculous meningitis and laboratory parameters. i.j. of infectious diseases. 11: 348-354 3. takahashi t, tamura m, takahashi sn, matsumoto k, sawada s, yokoyama e, nakayama t, mizutani t, takasu t, nagase h. 2007. j. neurological sci.255: 69-76. 4. merino, p., candel, j.f., gestoso, i., baos, e., picazo, j. 2012. microbiological diagnosis of spinal tuberculosis. int orthop. 2012 feb; 36(2): 233-238. 5. brooks gf, butel js, morse sa. 2004. mycobacteria. in: jawetz, melnick & adelberg’s. medical microbiology. mc graw hill. boston – toronto: 319-329. 6. forbes ba, daniel fs and alice sw. 2007. mycobacteria. in: bailey & scott’s diagnostic microbiology. mosby. elsevier. st. louis, missouri: 478509. table 2. positivity detection & first line anti-tb drug sensitivity of mycobacterium tuberculosis complex isolate from bone tissue biopsy specimens of the patients suspected spinal tb in dr. soetomo hospital, indonesia, at january 2013 march 2014 n specimen positive (%) sensitivity r i pza e s 21 bone tissue biopsy 5 (5/21=23, 80%) 5 (100%) 4 (80%) 5 (100%) 4 (80%) 4 (80%) r = rifampin, i = isoniazid, e = ethambutol, s = streptomycin, pza = pyrazinamide 60 indonesian journal of tropical and infectious disease, vol. 5. no. 3 september–december 2014: 57-60 7. kim sj, frieden t, luelmo f, norval py, rieder h, valenzuela p, weyer k, 1998. laboratory service in tuberculosis control, culture part iii, who, geneva, switzerland: 11-85. 8. mc lain rf and isada ci. 2014. spinal tuberculosis deserves a place on the radar screen. cleveland clin. j. of medicine 71.7: 53-549. 9. weng cy, ho cm, dou hy, ho mw, lin hs, chang hl, li jy, lin th, tien n. 2013. molecular typing of mycobacterium tuberculosis isolated from adult patients with tubercular spondylitis. j. microbiol. immunol. and infection 46: 19-23. ijtid vol 3 no 2 april juni 2012.indd 104 vol. 3. no. 2 april–juni 2012 platelet rich plasma preparation protocols: a preliminary study hans kristian nugraha1, meiti muljanti2, yetti hernaningsih2, jusak nugraha2,3 1 medical faculty universitas airlangga surabaya hans.nugraha@yahoo.com, +62315940696, +62315927764 2 clinical pathology department, dr. soetomo hospital surabaya 3 institute of tropical disease, universitas airlangga surabaya abstract currently, therapy with platelet rich plasma (prp) has been widely used and continues to grow for various clinical applications. along with its development, there are various options in the method of obtaining prp, either automatic or manual, while one of the most reliable methods according to the literature is a double centrifugation method. the purpose of this research is to produce an optimization of the double centrifugation method. this study used experimental data obtained by conducting a research at the clinical pathology laboratory of dr. soetomo hospital, surabaya. experiments were conducted on stored blood obtained from the blood bag from indonesian red crossand fresh blood from healthy donors with cpd anticoagulant. results: prp with optimum platelet count could be made with sufficient personal laboratory skills and amounted to 4.11 times with the platelet count of 1.152 million using 1300 rcf for 5 minutes for the first centrifugation, and 2300 rcf for 7 minutes for the second centrifugation. keywords: platelet rich plasma, double centrifugation method, stem cell therapy abstrak latar belakang: saat ini terapi dengan menggunakan platelet rich plasma (prp) telah banyak digunakan dan menjadi pilihan dalam dunia klinis. adapun dalam perkembangannya terdapat beberapa pilihan metode, baik secara otomatis maupun manual, meskipun metode yang paling dipercaya adalah metode double centrifugation. tujuan: untuk menemukan optimalisasi yang paling tepat untuk metode tersebut. penelitian ini menggunakan data eksperimental yang dilakukan oleh peneliti dari laboratorium patologi klinik rsud dr. soetomo surabaya. metode: penelitian ini menggunakan plasma darah yang diperoleh dari palang merah, yang berasal dari donor dengan cpd anticoagulant. hasil: prp dengan perhitungan platelet optimum ditunjang dengan kemampuan laboratorium dapat dicapai hingga 4.11 kali dengan perhitungan 1.152 million, menggunakan 1300 rcf selama 5 menit pada putaran pertama dan 2300 rcf selama 7 menit pada putaran kedua. kata kunci: platelet rich plasma, double centrifugation method, stem cell therapy research report introduction as a relatively new subject, stem cells can be said to have tremendous potential, starting early in life and growing up as a sort of internal improvement system, proliferate and differentiate without definite limits to later form the other cells as long as the relevant person or animal is still alive . as one of the internal improvement system, the proliferation of stem cells can be stimulated in the presence of growth factors. growth factors are found in prp (platelet rich plasma) in large numbers. in indonesia, the current stem cell therapy is a field that currently emerged, and still not a lot of work of which the method is widely used and applicable. prp was highly rated in terms of potential for use as a treatment of chronic tendinitis, wound healing, regeneration of cartilage or discs, as well as cardiac applications. 105nugraha, et al.: platelet rich plasma preparation protocols there are various methods used for the manufacture of this prp, from sophisticated which can only be performed at hospitals using apheresis devices, up to a practical method that can be performed directly in clinics. from variously different methods and protocols, there are still no research in indonesia on what the most optimal method is. therefore, it is felt that there is a need to have a research and optimization on double centrifugation method. the method, according to the literature, is the most reliable one and relatively simple. so as to produce a protocol that prp produces reliable and proven quality. materials and methods this is a laboratory experiment which is intended to get the most reliable prp making method with platelet count as the indicator. the materials and tools were sterile tubes, syringes, sterile long needle, cpd anticoagulant, centrifuge, and sysmex automatic hematology analyzer. the experiment was conducted in the laboratory of clinical pathology department of dr. soetomo hospital, surabaya. sample collection the blood samples were obtained from a blood bag from a donor, and also from fresh blood from other donors. to get as much as 1 ml of prp, we took blood samples of 10 ml with 9:1 cpd anticoagulant. for the optimization experiment of centrifugation phase and separation phase, we performed 30 tests, hence the total experiment required a sample size of 300 ml of blood. prp isolation platelet count was conducted twice for a sample, one before, and another after the optimization process. the optimization process itself was a series of variation on velocity and time of centrifugation, twice on each sample. it varied from 200 to 2300 rcf, and 5 to 15 minutes on each centrifugation. separations were manually done with ± 1 mm of margin after each centrifugation, in order to purify as much as possible, the result was extracted from erythrocytes and platelet poor plasma (ppp). the visible buffy coat from the first separation was essential to be maintained until the end of the optimization process. the first separation was done in order to separate erythrocytes as much as possible, while table 1. platelet count result sample no. 1st centrifugation 2nd centrifugation platelet count 1 (x 103/μl) platelet count 2 (x 103/μl) rise force (rcf) time (min.) force (rcf) time (min.) 001 1200 5 2000 6 92 271 2.94 x 002 200 15 1500 15 87 194 2.23 x 003 300 10 1500 15 87 65 0.74 x 004 600 5 1500 15 87 106 1.22 x 005 900 5 1500 15 87 129 1.48 x 006 1200 5 1500 15 87 33 0.38 x 007 1500 5 1500 15 87 41 0.47 x 008 200 15 2000 6 87 90 1.03 x 009 300 10 2000 6 87 47 0.54 x 010 600 5 2000 6 87 23 0.26 x 011 900 5 2000 6 87 31 0.36 x 012 1200 5 2000 6 87 37 0.43 x 013 1500 5 2000 6 87 33 0.38 x 014 200 15 1500 15 170 62 0.37 x 015 200 15 600 15 104 109 1.05 x 016 600 30 1500 10 104 29 0.28 x 017 1300 5 2300 7 280 1152 4.11 x 018 900 5 2300 7 104 37 0.36 x 019 1300 5 2300 7 104 22 0.21 x 020 1200 5 2000 6 239 181 0.76 x 021 1300 5 2300 7 239 428 1.79 x 022 600 5 2000 6 234 140 0.59 x 023 1300 5 2300 7 234 186 0.79 x 024 600 5 2000 6 228 288 1.26 x 025 900 5 1500 15 228 292 1.28 x 026 1200 5 2000 6 228 431 1.89 x 027 1300 5 2300 7 228 898 3.94 x 028 600 5 2000 6 183 469 2.56 x 029 1200 5 2000 6 205 269 1.31 x 030 1300 5 2300 7 251 327 1.30 x 106 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 104−107 the second was to separate ppp leaving the bottom with 1 ml of liquid, which was considered to be the prp. each set of variation was repeated at least 2 times to assure the reliability of the current method. the resulting data were analyzed by using simple analytical statistics. the thrombocyte count of the prp obtained was measured by automated sysmex xe-2100 hematology analyzer which had a unique fluorescent flow cytometry and hydrodynamic focusing technologies to minimize the review rates and produce accurate results even when interferences or artifacts were present.1 hence, the result presented in this study was made to be as accurate as possible, with very less likely to be false positive. results and discussion experiments of optimization of double centrifugation method in platelet rich plasma gave the following result: due to the limited budget towards the experiment, a pdgf bb test was performed only as an additional infromation, based on the highest result produced by the protocol, to ensure that it would still be a useful prp after processed through the protocol. the result proved that it was still a useful prp with pdgf bb 3401 pg/ml after processing through this protocol. this amount could still increase after activation, which was not included and tested further in this current preliminary study due to some fund limitation. but, as defined by textor, the conventional human definition of prp is plasma containing resting platelets at a concentration of at least 106/ul. the prp may or may not be activated prior to use, hence the term “prp” does not indicate that activation has occurred.2 alone, platelet count itself is considered to be one of the key factors used to standardize research studies for the regenerative capacity of prp. maximum platelet concentration of prp with manual double centrifugation method by kakudo is 7.9 x.3 too, qualitative changes in the platelet may also affect the regenerative potential of prp. according to marx, a damaged or nonviable platelet will not release bioactive growth factors, thus the resulting prp may be disappointing. the prp for clinical treatment should be sought about 1,000,000 platelets per microliter. given that whole blood contains approximately 200,000 ± 75 000 platelets per microliter, then the therapeutic prp must have an average percentage of increase of about 400% in platelet count.4 therefore, this experiment indicates that the optimum result from this experiment might be a standard for prp making. but as we can see in table 1, there were quite significant inconsistencies in the correlation between force and time of centrifugation to increase the platelet count after the double centrifugation method in prp making. it seemed that it was very dependent on the fidelity of manual separation processes between the centrifugations. the later samples results were higher than the early ones since the researchers become more accustomed and skilled in the manual separation process. errors might also be found because of the absence of prp resuspension before the platelet was counted by the analyst. resuspension could be important because there were trapped platelets in the buffy coat, so resuspension will show the tangible number of platelets in prp.5 hence, it could be concluded that the factor of trained person in the whole process might be more significantly important rather than the centrifugation process itself. this might also explain the very wide spectrum and range in protocol in centrifugation force and time to this day, which ranges from 72 g for 15 minutes, 160 g for 20 minutes, 250 g for 10 minutes, 900 g for 5 min, 1300 g for 10 minutes, up to 1400 g for 4 minutes, for the first centrifugation. the second centrifugation itself has a variable speed from 400 g to 2000 g, and has a variable period of time from 6 minutes to 15 minutes. similarly, with temperatures varying from 4° c, 15° c, up to room temperature.5,6,7,8,9 anticoagulant acid citrate dextrose (acd) type a and low-speed centrifugation can be used to maintain the integrity of the platelet membrane.5 but according to the study of shimizu et al, prp is created with the anticoagulant citrate phosphate dextrose (cpd) had a 4% higher platelet count when using 5 ml of full blood, and 4.5% higher using 200 ml of full blood.10 hence, this experiment used cpd anticoagulant instead of acd. overall, the highest increment in platelet count was obtained using 1300 rcf for 5 minutes for the first centrifugation, and 2300 rcf for 7 minutes for the second centrifugation as we can see in samples 017 and 027. this is quite different with the finding of jo ch and colleagues from orthopedic surgery department of seoul national university boramae hospital, which stated that the platelet count would be optimum with double centrifugation method with the use of 900 g in the first centrifugation for 5 min and 1500 gin the second centrifugation for 15 minutes.9 besides the double centrifugation method, there are already several automated methods developed independently by many institutions. among these methods are curasan prp kit by curasan, kleinostheim, germany; method pccs prp system by 3i implant innovations, palm beach gardens, florida, united states; and methods smartprep by harvest technologies, plymouth, massachusetts united states. however, research by castillo showed no significant differences in the platelet concentration, in the number of erythrocytes, the tgfβ1, or in the fibrinogen levels between the various automated methods.11 and since there was no availability of any automated prp machines in both the medical faculty universitas airlangga and dr. soetomo hospital surabaya, there was no difference in the method used in this experiment. but, based on kurita et al experiment in 2008, double centrifugation method itself was described as the most accurate and rational choice for manual making experiment of prp, conformed by nagata in 2010, since it produced more platelets and more active substances.7,12 castillo also suggests that the usage of method and protocol should be based on associated rational choice instead of trying tremendous variability available 107nugraha, et al.: platelet rich plasma preparation protocols by automated machines which are described as “the jungle world of commercial proposals and products”. as the concentrated platelet, prp rich in basic growth factors and has potential various clinical applications with no virtual risks. to initiate the process of wound healing, there are platelet derived growth factor (pdgfαα, pdgfββ, pdgfαβ), transforming growth factors-β (tgf-β1 and tgf-β2), vascular endothelial growth factor (vegf), epithelial growth factor (egf). in addition prp also contains adhesion molecules necessary for bone grafting and the matrix of bone, such as osteocalcin, osteonectin, and bone morphogenic protein (bmp) -2 and bmp-4.4,13 platelets also have a platelet factor 4 (pf4), interleukin (il) -1, platelet-derived angiogenesis factor (pdaf), platelet-derived endothelial growth factor (pdegf), epithelial cell growth factor (ecgf), insulin-like growth factor (igf), vitronectin, fibrinogen, fibronectin, and thrombospondin (tsp) -1.14,15,16 prp also shows some potential for treatment of myocardial infarction in experiments using the musmusculus.17 in addition to the usefulness above, prp is also potentially used in a variety of other clinical applications such as muscle healing,18 peripheral nerve damages,19 and maxillofacial surgeries.20 suggestions this project is a preliminary study about the manual making of for prp and therefore its goal is to obtain a standard protocol to make prp according to the present definition. for further study such as therapeutic effect and usage safety, further investigation is encouraged by using both experimental and clinical trials to follow up the therapeutic effects of the prp in tissues or animal models by investigating the growth factors and the regeneration process from time to time there. acknowledgements we would like to thank the uppm medical faculty, universitas airlangga for the support to this research. references 1. sysmex. 2011. sysmex xe-2100™ automated hematology system: fast, accurate, dependable. https://www.sysmex.com/us/en/ products/hematology/xeseries/pages/xe-2100-hematologyanalyzer.aspx 2. textor, j. 2010. platelet-rich plasma: the rest of the story. american college of veterinary surgeons 2010 symposium. 3. kakudo n, kushidas, kusumoto k. 2009. platelet-rich plasma: the importance of platelet separation and concentration. plastic & reconstructive surgery: march volume 123 issue 3 pp. 1135–6. 4. marx re. 2004. platelet-rich plasma: evidence to support its use. j oral maxillofac surg; 62: 489–96. 5. gonshor, a. 2002. technique for producing platelet-rich plasma and platelet concentrate: background and process. int. j. periodontics restorative dent. 22: 547–57. 6. gimeno fl, gatto s, ferro j, croxatto jo, gallo je. 2006. preparation of platelet-rich plasma as a tissue adhesive for experimental transplantation in rabbits. thrombosis journal, 4: 18. 7. nagata mjh, messora mr, furlaneto fac, fucini se, bosco af, garcia vg, et al. 2010. effectiveness of two methods for preparation of autologous platelet-rich plasma: an experimental study in rabbits. eur j dent; 4: 395–401. 8. hemker hc, giesen pl, ramjee m, et al. 2000. the thrombogram: monitoring thrombin generation in platelet-rich plasma. thromb haemost. 83: 589–91. 9. jo ch, roh yh, kim je, shin s, yoon ks, noh jh. 2011. optimizing platelet-rich plasma gel formation by varying time and gravitational forces during centrifugation. journal of oral implantology. apr 11. 10. shimizu t, noda y, goto s, hasegawa i, fukuda t. 1984. high yield of platelet-rich plasma from cpd blood compared to acd blood. tohoku j exp med. sep; 144(1): 103–4. 11. castillo tn, pouliot ma, kim hj, dragoo jl. 2011. comparison of growth factor and platelet concentration from commercial platelet-rich plasma separation systems. am j sports med. 2011 feb; 39(2): 266–71. 12. kurita m, aiba-kojima e, shigeura t, et al. 2008. differential effects of three preparations of human serum on expansion of various types of human cells. plast reconstr surg. 2008; 122: 438–48. 13. eppley bl, pietrzak ws, blanton m. 2006.platelet-rich plasma: a review of biology and applications in plastic surgery. plastic and reconstructive surgery (2006) volume: 118, issue: 6: 147e–59e. 14. bhanot, s., and alex, j. c. 2002. current applications of platelet gels in facial plastic surgery. facial plastic surgery. 18: 27–33. 15. froum, sj., wallace, ss., tarnow, dp., cho sc. 2002. effect of platelet-rich plasma on bone growth and osseointegration in human maxillary sinus grafts: three bilateral case reports. int. j. periodontics restorative dent. 22: 45–53. 16. petrungaro, p.s. 2001. using platelet-rich plasma to accelerate softtissue maturation in esthetic periodontal surgery. compendium of continuing education in dentistry. 22: 729–32. 17. mishra a, vellota j, brinton tj, wang x, chang s, palmer o, et al. 2011. revaten platelet-rich plasma improves cardiac function after myocardial injury. cardiovascular revascularization medicine. may–jun; 12(3): 158–63. 18. borrione p, gianfrancesco ad, pereira mt, pigozzi f. 2010. platelet-rich plasma in muscle healing. am j phys med rehabil; 89: 854–61. 19. yu w, wang j, yin j. 2011. platelet-rich plasma: a promising product for treatment of peripheral nerve regeneration after nerve injury. int j neurosci. 2011 apr; 121(4): 176–80. 20. tamimi fm, montalvo s, tresguerres i, blanco jerez l. 2007. a comparative study of 2 methods for obtaining platelet-rich plasma. j oral maxillofac surg 2007; 65: 1084–93. 21. ehrenfest dmd, rasmusson l, albrektsson t. 2008. classification of platelet concentrates:from pure platelet-rich plasma (p-prp) to leucocyteand platelet-rich fibrin(l-prf). trends in biotechnology vol. 27 no. 3: 158–67. 72 vol. 5. no. 3 september–december2014 literature review a patient dengue hemorrhagic fever with spasms ulfa kholili1, nasronudin1,2 1 tropical and infectious disease division department of internal medicine, dr. soetomo general hospital faculty of medicine universitas airlangga, surabaya, indonesia. 2 institute of tropical disease, universitas airlangga, surabaya, indonesia abstract indonesia is one of the countries with the high endemic of dengue viral infection followed by thailand, myanmar, india and srilanka. for more 10-15 years, dengue viral infection/dhf has become a cause of patient who should be hospitalized and was the first cause of death children in south easthern asia.1,2 batavia was the first city of indonesia found dengue viral infection which had been written in journal by david bylon in the 1779. encephalopathy of dengue (ed) is one unusually complication of dengue viral infection which had been characterized by aberration the arrangement of nerves central (cns). this paper want to describe of a young teenage with suffer from dhf and seizure. beside it, pleural effusion and cerebral edema had been found. seizure most likely due to dengue encephalopathy associated with cerebral edema and was supported by positive igg and igm anti dengue. corticosteroid was given to improve cerebral edema. by good management as long as admission, she was discharged from hospital with a good condition. key words: dengue viral infection, encephalopathy endemic, pleural effusion, igm anti dengue test, igg anti dengue test abstrak tercatat negara-negara endemis tinggi kasus dd/dbd adalah indonesia diikuti oleh thailand dan myanmar, endemis sedang adalah india dan sri lanka. selama lebih 10-15 tahun terakhir, dd/dbd telah menjadi penyebab terbanyak indikasi opname dan menduduki peringkat pertama penyebab kematian anak-anak di asia tenggara.1,2 di indonesia, demam dengue endemik dilaporkan pertama kali di batavia oleh david bylon pada tahun 1779. ensefalopati dengue (ed) adalah salah satu komplikasi tidak lazim dari infeksi viral dengue yang ditandai dengan kelainan susunan saraf pusat (ssp). dilaporkan seorang anak remaja dengan dbd tingkat i dan kejang. bukti yang mendukung kebocoran plasma sebagai ciri dari dbd adalah efusi pleura dan edema logis. kejang diduga akibat dari demam berdarah ensefalopati berkaitan dengan edema otak dan diperkuat oleh igg positif dan igm anti demam berdarah, kortikosteroid diberikan untuk meningkatkan edema otak. dengan manajemen yang selama masuk, maka akan keluar dengan keadaan baik. kata kunci : infeksi viral dengue, endemik encepalopati, efusi pleura, uji anti dengue igm, uji anti dengue igg introduction today, dengue viral infection has been the main health problems. an estimated 2.5 billion people than 100 countries at risk exposed infection. reported a 10 million cases dengue fever and 500,000 dengue hemorrhagic fever was dengue shock syndrome with mortality 5% occurring every year. indonesia is one of the high endemic countries followed by thailand and myanmar, india and srilanka. for more 10-15 years, dengue fever/dengue hemorrhagic fever has become a cause most indication hospitalized and was the first rank cause of death children in southeastern asia.1,2 batavia was the first city of indonesia found dengue viral infection which had been written in journal by david babylon on 1779. on the next years, on 1968, dhf had been found in surabaya and followed by other city such as jakarta, medan, and so on. in indonesia adult cases trend to increase, since the 1993-1998 mostly of dhf case (60%) occurring at age group 5-14 years, then in 1997 and 1998 shift to age more than 15 years.3,4 73ulfa kholili and nasronudin: a patient dengue hemorrhagic fever with spasms encephalopathy of dengue (ed) is one of unusual complication dengue viral infection which had been characterized by aberration the arrangement of nerves central (cns). diagnosis encephalopathy had been identified after marker of diagnosis dhf with accompanied manifestation cns had been found. actually ed is a rare but more recently increased every year. at the intensive care child hospital ho chi minh had reported that rate of occurrence ed about 0.5% of the dhf by mortality of 22%; while at the child devision of rs cipto mangunkusumo jakarta obtained incident ed of 6.2% of the dhf that had been hospitalized. with encephalopathy, these cases were unusually occurred as a complication of prolonged shock with bleeding, but can also be occurred in dhf not be accompanied by shock.5,6 this paper would like to show on experience of a doctor which had found a dhf patient with spasms manifestation, and had been predicted as that this one had releated with primer disease, of dhf. case a patient nn r, 15 years old, address balon-cepu came to ird rsud dr. soetomo with firstly complain of spasms. the history of this case as followed: a patient got spasms since in cepu hospital or 5 hours before hospitalized. when she showed spasms attach she did not conscious two eyes of her foamed an appearance with hand and feet get stiff, no frothy mouth or bitten tongue, spasms occured about 5 minutes, then the patient conscious, she was bought to surabaya by ambulance. she showed a clinical manifest of spasms on the 7th day of fever she showed a better temperature than the days before especially on the first day of fever suddenly showed a high temperature, headache, nausea, painful muscle and joints. the temperature was becoming decrease after get a medicine but the temperature showed increase again especially on the 5th day, then she send to cepu hospital and was hospitalized for 2 days with diagnoses dhf with low trombosit. there was no nose bleeding, gum bleeding or manifestation of other bleeding. when there was no abnormality of intestine moving. on the last disease history there was no spasms history when the patient had been suffered from dhf in 2 years old and hospitalized in cepu hospital until recovered and discharged in better health condition. the physical examination of this case as followed: the patient was in good conscious (gcs 4-5-6) with tension 110/70, pulse 108/minutes (low), breath 22/minutes, and temperature 37,4°c. there was no anemia, icteric, cyanosis or dyspneu. did not found enlargement of the heart, normal heart sound and there is no murmur. vesikular breath sound, there was no ronchie and wheezing. move equipment in normal limit with result of rumpel leede test showed a positive. the laboratory examination showed: laboratory result: hb 11,6 g%, leukocytes 5.100 l/mm3, trombosit 83.000/mm3, pcv 33%, gda 90 mg/dl, sgot 87 u/l, bun 5 mg/dl, sc 0,53 mg/dl, k 3,78 meq/l, na 145 meq/ l. thorax x-ray showed a minimal right pleura effusion. result of head ctscan showed cerebral edema. the division of the lung xrays showed a minimal right pleura effusion cause dhf process due to extravasasion. result of neurology division concludedas a focal of secondary generalized seizure which might be caused by metabolic ensefalopaty (anoxiq of the brain can cause cerebral edema). there for the conclusion of the assesment she suffer from dhf with brain edema. diagnosis planning showed dl series ( total trombosit evaluation), igm and igg anti dengue, pleura fluid analisys (if trombosit > 100.000). the treatment had been done by given infus ringer acetat 2000 cc/24 hours, diet tktp 1900 calori, paracetamol 3  500 mg, ranitidine 2  1 ampoule, multivitamin 2  1 tablet, methylprednisolon injection 3  125 mg, and diazepam 1 ampoule dan fenitoin bolus 300 mg if spasms and dosis maintenance 3  100 mg. on the next period, the doctor in charge observed the clinical manifestation as followed: on the second day in dr. soetomo hospital there was no fever (in the 8th day) and the complaint of headache was decrease, painful of muscle and joint got better. nausea and vomit were still exist. there was no manifestation of bleeding and didn’t show a spasms. tension 110/50 mmhg, pulse 92/minutes, rr 20/minutes, and temperature 37°c. examination of head, neck, breast, abdomen, and member’s motion in normal limit. laboratory: hb 12.2 g%, leukocytes 4.200/mm3, diff count / / 3 / 55 / 39 / 3 , pcv 35.8% , trombosit 33.000/ mm3, albumin 3.4 mg/dl, total protein 6.2 mg/dl, sgot 67 u/l, sgpt 72 u/l, and also ig m and igg anti dengue showed positive result. physiologic coagulasion showed that ppt 12,1(c=13,9), kppt 25,7. assesment showed dhf with brain edema. therapy: infus ringer acetat 2000 cc and hes 500 cc/ 24 hours, methylprednisolon 3  125 mg (the 2nd day), fenitoin maintenance 3  100 mg. on the third day in dr. soetomo hospital the clinical manifestation of this case as following: there was no fever found and complaining of chemical manifestation was becoming decrease. there was no spasms and bleeding. tension 110/70 mmhg, pulse 88/minutes, rr 20/ minutes, temperature 37°c. laboratory result showed hb 12.6 g%, leukocytes 5.500/mm3, trombosit 149.000/mm3, pcv 36.9%, led 15-30/jam. the assesment showed as a case of dhf with brain edema. the treatment was to continue given methylprednisolon 3  125 mg until 3 days and together with other drug on the fourth day in dr. soetomo hospital hospital the clinical manifestation of this case as following: the patient could not defaecates for 3 days, but no fever, bleeding, or spasms. tension 110/70 mmhg, pulse 64/mnt, rr 18/mnt, temperature 36,8°c. meteorism occured with 74 indonesian journal of tropical and infectious disease, vol. 5. no. 3 september–december 2014: 72–77 normal noisy intestine. laboratory showed hb 11,7 g%, luekocytes 10.800/mm3, trombosit 201.000/mm3, pcv 36.7%. assesment showed constant and antasida 3 1 tablespoon, ranitidine 2  1 ampoule, multivitamin 2 1 tablet had been given, the condition of care more better. on the fifth day in dr. soetomo hospital the clinical manifestation of this case as following: there was no complain. general condition is good. stabil sign vital. hb 12 g%, leukocytes 8.720.000/mm3, trombosit 317.000/mm3. repeated breast x-ray showed no pleura effusion and the patient permitted to go home after the doctor control and the result better health. discussion dengue viral is infection a single stranded rna that was the family of flaviviridae and consists of 4 serotipe (den1, den-2, den-3, den-4 ).this viral rod-shaped, spatially termolabil, sensitive to inactivasion by dietileter and sodium dioksikolat, stable at a temperature of 70°c. fourth serotipe the viral has been found on patients in indonesia where den-3 serotipe is the dominant and has to do with cases heavy when this incredible happenings or outbrench of dengue viral infection.3,4,6 the length of the genome of dengue viral about the planning. the virions mature have three protein structure (core, associated membrane, envelope), and 7 (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5). principal biological function of these viruses associated with the envelope. it said that these proteins to bind with a receptor on a cell host and allowing the past. the envelope this is also related with the hemaglutinasion to erythrocytes, inducing an antibody netralising and who were protective immune response. principal of dengue vector in indonesia is the aedes aegypti, and aedes albopictus, aedes albopictus. the nest of vector is found in clearly water and as in the bathtube, drum chicken collectors water, canned the former, etc. the data dhf have been found in every province in indonesia and 200 city has been reported outbreak of dengue viral infection.6 there are many clinical spectrum of dengue infection forms. the differences of degree severity of clinical manifestation of dhf case it might be due to underlying pathophysiology of disease.6,7 clinical criteria: 1. fever ( suddenly high temperature for 2–7 days) 2. manifestation of bleeding (tourniquet test which is positive, petekie, purpura, ekimosis, epistaksis, gum bleeding, hematemesis or melena, hematuria) 3. enlargement of liver 4. shock, with high pulse and low and decrease tension, hypotension, foot and hand moist skin, with restless case. laboratory criteria 1. trombocytopenia (total trombosit 100.000/mm3 or less) 2. hemoconcentration (decrease hematokrit 20% or more) the diagnosis of dhf in this case based on the two clinical criteria and supported by thrombocytopenia and hemoconcentration. the presence an effusion of pleura and or hipoalbuminemia can strengthen the diagnosis.6 dhf diagnosis should be identified by laboratory test of igm and igg anti dengue or elisa method of dengue based on the severity of dengue viral infection.8 table 1. the severity of dengue viral infection8 grade symptoms grade i (lighter) suddenly fever (2-7 days) non specific constitunal symptoms bleeding manifestation on only if positive in tourniquet test grade ii (medium) same as level i spontan bleeding grade iii circulation failed/sign of early schock (pulse fast and low, tension decrease (20 mmhg or less), hypotension, cyanosis around mouth, skin cold and moist grade iv shock ( tension and pulse can not feel) this patient showed symptoms of muscular pains and manifestation bleeding form of a positive rumple leede, and obtained thrombocytopenia (83.000/mm3). the right lung of pleural effusion was improving of plasma leakage, so the diagnosed was dhf. it said that besides serotipe and virulence viral; other factors that influence the clinical manifestation dhf was age, sex, immune status and the background host. guzman et al said that a severity hospitalized cases dhf/dss the highest were found in group of babies and elderly, if these cases got secondary infection due serotipe den-2 it could cause a risk of mortality 15 times in children than adults. dhf had been reported more severity of women malnutrition dengue virus infection asymptomatic symptomatic fever without spesifik fever dengue dhf with plasma leakage criteria wthout with without shock with shock hemorrhagic hemorrhagic picture 1 : clinical manifestation of dengue virus infection8 clinical criteria : 1. fever ( suddenly high temperature for 2 – 7 days) 2. menifestation of bleeding ( tourniquet test which is positive, petekie, purpura, ekimosis, epistaksis, gum bleeding, hematemesis or melena, hematuria) 3. enlargement of liver 4. shock, with high pulse and low and decrease tension, hypotension, foot and hand moist skin, with restless case. laboratory criteria 1. trombositopenia (total trombosit 100.000/mm3 or less) 2. hemokonsentrasi (decrease hematokrit 20% or more) the diagnosis of dhf in this case based on the two clinical criteria and supported by thrombocytopenia and hemoconcentration. the presence an effusion of pleura and or hipoalbuminemia can strengthen the diagnosis6. dhf diagnosis should be identified by laboratory test of igm and igg anti dengue or elisa method of dengue based on the severity of dengue virus infection8. figure 1. clinical manifestation of dengue viral infection8 75ulfa kholili and nasronudin: a patient dengue hemorrhagic fever with spasms retrospective research of 152 dht cases that hospitalized in rscm-jakarta. acquired image clinical most prominent form of hyperpyrexia, change of consciousness and convulsions. examination laboratory showed, there were increased serum transaminase, hiponatremia and hypoxia. a neurological disorder in form of hemiparese, tetraparese and atrophy nervus the second.9 this event were followed by malaysia, reported the manifestation symptoms of weakness instrument motion and decline sensory on either side and bleeding the brain. there were also manifestation neurological form as stiff of the neck and seizure general in children supposedly caused by increased pressure intrakranial. the act of lumbar puncture (lp) have been done to exclusion possibility meningitis or ensephalitis. the result showed the all of the cases recovered perfect and leave no bad symptoms occured.10 in singapore ever reported a sufferers dengue virus infection with a complication cns disorder with manifestation of amnesia; obtained the result pcr and serologis a positive and results mri brain disorder showing the hippocampus.11 in thailand reported incidents encephalopathy related dhf about 34 the case of 1.465 cases dhf that hospitalized at the pediatrics rs petchabun for 3 years (2.3%). acquired 30 cases fall into encephalopathy during the shock 4 in while in the healing. factors the risk for the encephalopathy among other: shock conflict, bleeding gastroduodenal that profuse, impaired function severe heart and granting liquid excessive.12 in south vietnam the studies against 378 sufferers suspected infection exposed to the cns; infection acquired incident cns just because dengue viral as many as 4.2%. of the ed the 7 people exposed infection primary and 13 people exposed secondary infection. acquired isolation virus ( pcr positive ) on 10 sufferers while 3 patients of indicates the presence of antibodies in liquid his mind. manifestation main cns disorder of patients was in form of impairment of consciousness and convulsions.13 research of case-control and prospective study on the intensive pediatrics hospital in ho chi minh showed that the patients ed got an increase a liver enzyme and bilirubin with a significant result and one case produce pcr-rna virus den-3 from a liquid the brain, 14 cases showed a positive result of igm anti dhf and the majority mri showed edema of the brain.5 patient with manifestation cns disorder in form of spasm. she does not obtain bleeding gastroduodenal, disorder electrolyte, shock and granting liquid glasses. results ct-scan of head showed an barin edema. immunopathogenesis dhf and dss still controversial. the first theory which was professed was hypothesis secondary of heterologous infection or antibody-dependent enhancement.14 it said that if someone sinus infection for the second time with divergent (heterologous) serotipe dengue viral, it will happen cross reaction between antibodies serotipe viral from infection with the viral formerly without neutralization through the process so that the viral could enter the monocytes. the number of monocytes and t-cell infected was increase, it was describing increasing of antigens, frequency dengue viral t cells and activation and proliferation t memory. t cells also produces sitokins as ifn-γ, il-2 dan tnfα and also lysis of monocytes that infected dengue viral. the complement will be enabled by complex antigen-antibody by sitokins so that the discharge occurs c3a and c5a, which directly affect the permeability vascular. the synergistic effect of ifn-γ, tnf-α and complement who switched to would cause the occurrence of plasma leakage from the endothelial cells.6,7,15 dengue hemorrhagic fever can be stimulated a transcription and to secretions rantes and il-8, the establishment of an antibody hemorrhagic fever and the establishment of the complement non-lysis complex. dengue viral infection in endothelial cells in vitro can cause the occurrence of pothogenesis. it has been said that the complement, who switched to chemokin and mediate apoptosis causing the occurrence of leakage plasma membrane.16 a second hypothesis that the dengue virus could change genetic as resulting from pressure during a replication of the viral in human body and mosquitoes. a phenotype in a genome is a replication of the viral and could cause the viremia, increase and it is potential to cause virulence of the plague. it says that there the manifestations, and the dss is probably caused by a variant of dengue viral that has a different degree virulence. the epidemic in southeast asia support this hypothesis. it was reported that the risks may in thailand dss regarding the den-2. philippine den-3 role in the outbreak in indonesia den-3 a type virus existing related with severe cases presently occurring outbrench of dengue virus infection.6,15 typical patofisiologi of dengue fever is the leakage of plasma and disorder hemostasis. the data prove the existence of a leak plasma namely the increase in the hematokrit, an effusion of the pleural and ascites, hipoproteinemia and decrease in volume plasma. lost plasma that weight can cause the occurrence of shock hypovolemia and death. hemostasis an abnormality that occurs because by 3 the main factor of change namely: vascular, thrombocytopenia, and coagulopati. dissaminated intravascular coagulation (dic) can happen and cause bleeding great.7,17 pathogenesis the occurrence of encephalopathy hemorrhagic fever was still not clear. some research in indonesia thailand and other asian countries get that an abnormality of the cns occurs in dhf prolonged with or without the occurrence of shock. the hemorrhage brain is not caused directly by a virus that may be pierced blood brain barrier (bbb). gathered evidence in the form of the virus den-3 isolation from a liquid the brain on 4 cases, from 6 the remaining positive result pcr.18 imbert et al, 1994. stated that dengue viral which have the ability to infect broad neurons mice in vitro, viruses like has a specific receptor on the surface of neurons.19 chaturvedi et al found bbb damaged during infection dengue ( den-2 ) so happen leakage protein to the brain. 76 indonesian journal of tropical and infectious disease, vol. 5. no. 3 september–december 2014: 72–77 gubler et al suspected the genetic variation or due to changes in biological virus and also strains some viral.18 lum lc et al, 1996 reported that many factors who caused the encephalopathy among other: impaired function heart, disorder electrolyte, edema cerebri (caused by changes permeability vascular so extravasation a liquid); hipoperfusi (resulting from disruption circulation) and encephalitis week.20 in some patients encephalitis this obtained isolation virus from a liquid the brain.21 abnormality cns on df/dhf formerly thought due to nodular that causes penetrated bbb. the cognitive deficit is more because encephalopathy than encephalitis. although the mri by contrast show bbb who was whole, but partly expert confident that encephalitis caused by invasion virus directly to the brain through bbb, while other writers postulates that due to the occurrence of encephalitis process imunopatologis.11 due to an immune response that rises rapidly, difficult to get the isolation virus den a positive on blood sufferers. all the case indicating the presence of antibodies with titer high when examined. so clinical manifestation sufferers and the levels hi antibodies against flavivius, igm anti dengue a positive and neutralization a positive test had been considered that the virus as a cause.21based on patient who was 2 years old had gotten dhf, so it was really possible as the secondary heterologous infection. the ultimate principle management dbd aims to replace the liquid plasma during a period of leakage active in 24-48 the first hour because at that period would cause the occurrence of shock, anoxia, acidosis, and death. antipyretic can be given during phase of heat, avoid the use of aspirin. the critical period happen when a transition from phase of heat into free heat that can be started on the third day. a liquid used as a substitute for plasma shall spatially isotonic having the content of the electrolyte similar to plasma.3,6,17 on ed consciousness patient decreased to become on somnolen, it could accompanied by seizure. ed can happen to dbd/ssd. when patients shock,it will be found impairment of consciousness, and to ensure the ed shock to be overcome first. the act of lumbar punction (lp) done when shock has handled awareness but still declining (careful when platelets <50,000). on ed could be found elevated levels of transaminase (sgot/sgpt), ppt & kptt elongated, their blood sugar decline alkalosis on analysis gas blood, and hiponatremia.6 ed is one of complication dhf, its treatment more complicated. several points that should undertaken in management ed3: 1. replacement no liquid given in doses full, but fairly given 4/3 to 4 5/dose to prevent or make it worse edema brain during phase recovery shock 2. wearing a liquid crystalloids ringer acetic to avoid metabolism lactate in liver, when accompanied by impaired liver 3. a corticosteroid administered to reduce edema the brain, but is contra indication on dss with hemorrhage massive. on encephalopathy likely edema the brain and alkalosis, therefore when shock has handled, liquid replaced with a liquid containing no hco3and the amount of fluid must be reduced. prevent increased pressure an extern cranial by reducing the amount of fluid (when necessary with a diuretic), correction asidois and disorders electrolytes. try not giving drugs that not needed to reduce the burden detoxification medicine in heart. transfusion of blood fresh or components can be given over indications proper.6 main therapy with this replacement liquid to crystalloid and had given colloidal. ringer acetat was chosen because obtained increase transaminase, given 2 quarts in and colloidal (hes) 500 cc within 24 hours. methyl prednisolon 3  125 mg was given for 3 days, associated with edema cerebri. there was no shock; disorder diuresis, disorder electrolyte and lengthening faal hemostasis. a patient dbd could be discharged after to meet the criteria of clinical improvement, no fever for 24 hours), (without antipyretic cannot be found distress the breath (because effusion of the pleura or acidosis), hematocrit stable, the number of platelets tending to rise (> 50.000), three days after shock handled, and has been improving appetite.6 the patient was discharged after fulfill the criteria of those mentioned above. when a repeat photograph thoracic legs already does not obtain again an image of an effusion of plura. summary it has been reported a young teenage with dhf followed by seizure. evidences that supporting plasma leakage as hallmark of dhf are pleural effusion and cerebral edema. seizure most likely due to dengue encephalopathy associated with cerebral edema and strengthen by positive igg and igm anti dengue. corticosteroid was given to improve cerebral edema. by good management as long as admission, she was discharged from hospital with good condition. references 1. who (1996). management of dengue epidemic. report of technical meeting, searo, new delhi, november 28–30th pp. 1–40. 2. who (1999). guidelines for treatment of dengue fever / dengue hemorrhagic fever in small hospital. who regional office for south-east asia. 3. soewandojo e., (2002). tata laksana demam berdarah dengue pada orang dewasa, seri penyakit tropik infeksi, perkembangan terkini dalam pengelolaan beberapa penyakit tropik infeksi. airlangga university press, hlm 113–129. 4. hendarwanto (1996). dengue. buku ajar ilmu penyakit dalam jilid i, edisi ketiga. ketua editor: sjaefoellah noer. balai penerbit fkuijakarta, hlm 417–426. 5. cam bv, l fonsmark, nb hue, phuong nt, a poulsen, ed heegard (2001). prospective case-control study of encephalopathy in children with dengue hemorrhagic fever, am j trop med hyg, 65 (6), pp 848-851 77ulfa kholili and nasronudin: a patient dengue hemorrhagic fever with spasms 6. departemen kesehatan republik indonesia (2004). direktorat jendral pemberantasan penyakit menular dan penyehatan lingkungan, tata laksana demam berdarah dengue di indonesia. 2004 7. gubler dj (1998). dengue and dengue hemorrhagic fever. clinical microbiology reviews, july, vol 11; 3 : pp 480-496 8. who (1997). dengue hemorrhagic fever diagnosis, treatment prevention, and control. who, 2nd edition 9. hendarto sk., hadinegoro (1992). dengue encephalopathy, acta paediatr jpn. jun;34 (3): pp 350-357 10. george rabecca (1992). current status of the knowledge of dengue/ dhf/dss in malaysia: clinical aspect. 5th annual convention of philippine society for microbiology and infectious disease. november 28-30 11. yeo psd, l pinheiro, p tong, p l liem, yy sitoh (2005). hippocampal involvement in dengue fever, singapore med j 46 (11), pp 647-650 12. prasonk witayathawormwong (2004). dengue hemorrhagic fever encephalopathy/ fatality at petchabun hospital: a three-year prospective study (1999-2002), dengue bulletin volume 28, chapter 10. 13. solomon t., nguyen minh dung, david w vaughn, rachel kneen, le thi thu thao, boonyos r, et al (2000). neurological manifestation of dengue infection. the lancet, 355, march 25th, pp 1053-1059 14. supriatna, m., setiati, t.e., mairuhu, a.t., koraka, p., m.r. mac gillavry, d.p. brandjes, a.d. osterhaus, j., van der meer, e.c. van gorp, a. soemantri. 2007. dengue disease severity in indonesian children: an evaluation of the world health organization classification system. bmc infect. dis. 7:22. 15. lei huan-yao, trai-ming yeh, hsio-sheng liu et al (2001). immunopathogenesis of dengue virus infection, j biomed sci 8, pp 377-388 16. avirutnan p., prida malasit, barbara seliger, sucharit bhakdi and husmann m (1998). dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. immunology, 161 : pp 6338-6346 17. nimmannitya s., (2003). dengue and dengue hemorrhagic fever. manson’s tropical disease, 21st edition, editors: gordon cook and alimuddin zumla, elst with saunders, pp 765-772 18. vasconcelos, travassos, coelho, et al (1998). involvement of the central nervous system in dengue fever : three serologically confirmed cases from fortaleza, ceara, brazil, rev. inst. med. trop. s. paulo, vol 40 19. imbert, j.l., guevara, p., castaneda, j.r., sotelo, j. dengue viral infects mouse culture neurons but not astrocytes. j. med. virol., 42: 28-233, 1994. 20. lum l.c., lam s.k., choy y.s., et al. dengue encephalitis: a true entity? am j trop med hyg 1996. 54: 256-59. 21. malavige gn, fernando s, fernando dj and sl seneviratne (2004). dengue viral infection. postgraduate medical journal ; oct; 80 (948): pp 588-601 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid vol 9 no 3 september-desember 2021.indd vol. 9 no. 3 september–december 2021 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 original article analysis of hiv/aids health problems in pacitan district east java 2020 mohammad famil1* 1departemen of epidemiology, faculty of public health, universitas airlangga. surabaya indonesia received: 24th august 2021; revised: 18th october 2021; accepted: 29th november2021 abstract the implementation of health problem analysis is carried out to increase the eff ectiveness and effi ciency of solving health problems through the selection of health problems that become priority problems in a region. the purpose of this study was to analyze the problem and determine the priority of health problems in the work area of the pacitan district health offi ce, east java province. this research is a descriptive observational study conducted at the pacitan district health offi ce in january 2020. the type of data used is secondary data obtained from the 2016-2019 pacitan district health profi le and primary data obtained through interviews with related parties, namely the head of the fi eld. , section head and program holder. prioritization of health problems is carried out using the usg method based on the criteria of urgency, seriousness, growth and fi nding the root of the problem using the fi shbone diagram method. the increase in hiv/aids cases with an usg score of 128 has become a top priority health problem in pacitan district. an increase over the last 4 years with the highest number of cases in 2019, which was 39 cases. the fi shbine diagram shows the root of the hiv/ aids problem, namely the lack of public knowledge about hiv/aids, the lack of public knowledge about hiv/aids, the lack of awareness of people at risk for conducting an hiv test, this makes the community less aware of information about hiv/aids, causing public stigma. which results in people being closed / unwilling to check themselves at the puskesmas or hospital. the increase in hiv/aids cases is one of the problems in pacitan district. to reduce the incidence, health workers need to optimize the dissemination of information about hiv/aids, especially risk factors, causes, prevention, symptoms and treatment. increase the understanding of health workers and public awareness in conducting early detection. keywords: hiv/aids; urgency; seriousness; growth. abstrak pelaksanaan analisis masalah kesehatan dilakukan untuk meningkatkan efektivitas dan efi siensi penyelesaian masalah kesehatan melalui pemilihan masalah kesehatan yang menjadi prioritas masalah di suatu wilayah. tujuan dari penelitian ini adalah untuk melakukan analisis masalah dan menentukan prioritas masalah kesehatan yang ada di wilayah kerja dinas kesehatan kabupaten pacitan provinsi jawa timur. penelitian ini merupakan penelitian deskriptif obervational yang dilakukan di dinas kesehatan kabupaten pacitan pada bulan januari tahun 2020. jenis data yang digunakan yaitu data sekunder yang diperoleh pada profi l kesehatan kabupaten pacitan tahun 2016-2019 dan data primer yang diperoleh melalui wawancara dengan pihak terkait yakni kepala bidang, kepala seksi dan pemegang program. penentuan prioritas masalah kesehatan dilakukan dengan menggunakan metode usg berdasarkan kriteria urgency, seriousness, growth dan pencarian akar masalah menggunakan metode fi shbone diagram. peningkatan kasus hiv/aids dengan skor usg 128 menjadi masalah kesehatan prioritas utama di kabupaten pacitan. peningkatan selama 4 tahun terakhir dengan jumlah kasus tertinggi pada tahun 2019 yaitu sebanyak 39 kasus. diagram fi shbine menunjukan akar masalah hiv/aids yaitu kurangnya pengetahuan masyarakat terhadap hiv/aids, kurangnya kesadaran penderita berisiko untuk melakukan pemeriksaan tes hiv, hal ini membuat masyarakat kurang mengetahui informasi tentang hiv/aids sehingga menimbulkan stigma masyarakat yang buruk dan mengakibatkan masyarakat tertutup/tidak mau memeriksakan dirinya ke puskesmas ataupun rumah sakit. peningkatan kasus hiv/aids adalah salah satu masalah di kabupaten pacitan. untuk menekan angka kejadian, petugas kesehatan perlu mengoptimalkan penyebaran informasi mengenai hiv/* corresponding author: mohamad.famil2019@fkm.unair.ac.id ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 aids khususnya faktor risiko, penyebab, pencegahan, gejala yang timbul dan pengobatanya. meningkatkan pemahaman petugas kesehatan dan kesadaran masyarakat dalam melakukan deteksi dini. kata kunci: hiv/aids, urgency, seriousness, growth how to cite: famil, m. analysis of hiv/aids health problems in pacitan district east java 2020. indonesian journal of tropical and infectious disease, 9(3) introduction in an effort to improve health status and the implementation of health development in indonesia, there are various challenges, including the problem of inequality in public health status, access to health services, socio-economic level, and so on. in addition, new challenges arise as a result of socio-cultural, economic, and political changes as well as environmental changes. health as a human right is explicitly mandated by the 1945 constitution, which states that everyone has the right to live in physical and spiritual prosperity, to have a place to live, and to have a good and healthy living environment and have the right to health services. 1 the implementation of health problem analysis is carried out to increase the eff ectiveness and effi ciency of solving health problems through the selection of health problems that become priority problems in a region. by focusing on the selected health problems as a priority, it is hoped that the utilization of limited health resources can be carried out optimally in accordance with the leverage of the problem. hiv (human immunodeficiency virus) is a virus that attacks the immune system. the infection causes the patient to experience a decrease in immunity so it is very easy to be infected with various other diseases. aids (acquired immune defi ciency syndrome) is a collection of symptoms of reduced self-defense ability caused by the entry of the hiv virus. the hiv control program in indonesia aims to: 1.) reduce to eliminate new infections; 2) reduce or eliminate aids-related deaths; 3) reduce stigma and discriminationi. 2 according to who, 2019 hiv can be transmitted through the exchange of various body fl uids from an infected person, such as blood, breast milk, semen and vaginal fl uids. hiv can also be passed from a mother to her child during pregnancy and childbirth. people cannot be infected through everyday contact such as kissing, hugging, shaking hands, or sharing personal objects, food, or water. 3 hiv/aids is an infectious disease that occurs in the community for which there is no vaccine or effective drug for the prevention of hiv/ aids until now.4 according to world health organization (who) in 2018, there are 36.9 million people who have hiv/aids around the world.5 indonesia is one of the countries with the fastest addition of hiv/aids cases in southeast asia, with an estimated increase in the incidence of hiv infection by more than 36%. the hiv/aids epidemic in indonesia is growing the fastest among asian countries.4 indonesia occupies ranked third as a region with most people living with hiv/aids worldwide the world with a total of 5.2 million souls.6 as of december 2019, the number of aids cases reported in east java was 1,254 people, and 9,981 hiv cases. east java province is designated the results of the problem identification through a documentation study by comparing the program’s achievements against the mss targets, the strategic plan of the ministry of health and the rpmjd of pacitan district and looking at trends for three consecutive years found nine main problems, namely hiv/aids, leptospirosis, hepatitis a, dengue fever, diarrhea, pneumonia, larva free rate, complete basic immunization and tuberculosis. based on the results of data analysis and discussions with the head of the fi eld and program holders, the priority of the health problem that was taken was hiv/aids. indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 143–151 firda typewritten text 144 mohammad famil: analysis of hiv/aids health problems ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 as an area with concentrated hiv prevalence along with 5 (fi ve) other provinces, namely dki jakarta, papua, bali, riau and west java.7, 8 stated that hiv aids has become apandemic in sub-saharan africa. hiv aids pandemic slowly causes a decrease in energy employment, reduce agricultural productivity, increasing poverty, and changing the structure population pyramid in africa. almost half of all hiv cases had no known risk factors (51.0%). some of the highest risk factors are msm at 20.4%, heterosexual 19.6% and idu at 0.9%. while the highest aids cases were heterosexual at 73.4% and the lowest was transfusion at 0.3%. according to the type of work, the highest distribution of aids cases was among non-professional staff (employees) (26.4%), housewives (15.5%) and self-employed (12.6%). 2 based on the results of the problem analysis from the pacitan health offi ce, it was found that hiv/aids data in 2017 there were 36 cases then decreased although not so signifi cantly in 2018 to 25 cases then increased again in 2019 to 40 cases. the availability of health facilities in every sub-district in pacitan regency certainly makes it easier for the community to get access to better health services, if viewed from the distribution of sub-districts, there are already available puskesmas, an average of 2 units.9 the purpose of this study was to analyze the problem and determine the priority of health problems in the work area of the pacitan district health offi ce, east java province. materials and methods materials provide suffi cient detail to allow the work to be reproduced. methods already published should be indicated by a reference: only rele-vant modifi cations should be described. methods this research is a descriptive observational study conducted at the pacitan district health offi ce in january 2020. the type of data used is secondary data obtained from the 2016-2019 pacitan district health profi le and primary data obtained through interviews with related parties, namely the head of the fi eld. , section head and program holder. the types of data collected are data on health status, population aspects, health behavior, environmental data and data on morbidity and mortality. prioritization of health problems is carried out using the usg method based on the criteria of urgency, seriousness, growth. the steps taken in analyzing the health problems contained in the blitar district health offi ce are as follows: 1. establish program achievement indicators using national/regional standards. 2. comparing outputs on program achievements with indicators to look for gaps. 3. the method used for priority determination is the usg method. the usg method is one way to determine the priority order of problems using the scoring technique method. these are as follows: 1. urgency how urgently the problem must be discussed is related to the available time and how hard the time pressure is to solve the problem that caused the problem. 2. seriousness how serious the problem needs to be discussed is related to the consequences arising from delays in solving the problem that caused the problem or the consequences that cause other problems if the problem causing the problem is not solved. it should be understood that under the same circumstances a problem that can give rise to another problem is more serious than a separate problem. 1. growth how likely the problem is to develop is related to the possibility of the problem causing the problem to get worse if left alone. 2. there are many methods to fi nd out the root cause of problems that arise in the workplace, one of which is fi shbon. from the root of the problem found, then recommendations can be formulated for countermeasures that can be done to the problem. firda typewritten text 145 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 results and discussion overview of health problems in pacitan district based on the results of the identifi cation of health problems, examining the pacitan health profi le data for 2017-2019 and in-depth interviews and then comparing the program’s achievements against the mss targets, the ministry of health’s strategic plan and the pacitan district rpjmd, three main problems were found in pacitan regency as shown in table 1. table 2. determination of priority problems based on ultrasound criteria no health problems urgency seriuseness growth total prioritas 1 number of hiv/aids cases 45 45 38 128 i 2 number of leptospirosis cases 43 45 38 126 ii 3 hepatitis a 47 44 33 124 iii 4 dengue fever 44 42 33 119 iv 5 diarrhea 38 40 36 114 vi 6 pnemonia 36 38 29 103 vii 7 tubercolosis 41 37 38 116 v table 1. list of health problems in the pacitan district in 2017-2019 no problem 2017 2018 2019 tren target description 1 number of hiv/aids cases 36 23 39 increase when there is a decrease case hiv aids cases are still high 2 number of leptospirosis cases 52 42 54 increase when there is a decrease case the number of leptospirosis cases is still high 3 hepatitis a 0 0 1.314 increase hepatitis a outbreak occurs 4. dengue fever 72,1 48,3 121,9 rising bad the high number of dengue cases 5. diarrhea 54,8 56,4 3.26 rising bad rpjmd 100% haven’t hit the target yet 6. penemonia 90,67% 92,64% rising bad renstra 60% hope < 60% 7. tubercolosis 199 328 rising bad haven’t hit the target yet prioritize problems the method used is usg. the priority of the problem is determined by distributing the form based on the urgency, seriousness, and growth criteria. the fi lling is carried out by the head of disease prevention and control, the head of the infectious disease section, the head of the surveillance and immunization section, the head of the non-communicable diseases section, and all the staff of the disease prevention and control section. priority selection of health problems with ultrasound criteria, the score used is prone to 1-5 according to the provisions of the researcher. then the priority of the problem is scored, the greater the score indicates that the problem is becoming a priority problem. the following is a recap of the results of the problem assessment using the usg schoring technique in the work area of the pacitan district health offi ce which can be included in table 2. indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 143–151 firda typewritten text 146 mohammad famil: analysis of hiv/aids health problems ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 based on the results of the study documentation, table 2 explains that the results of the priority problem determination activities carried out in pacitan regency, hiv/aids is the fi rst priority problem. where the number of cases is increasing and the death rate is still there, as well as the consideration of the head of the fi eld through a discussion process, it is concluded that the main topic that is taken is the problem, namely the case of hiv/aids at the pacitan district health offi ce in 2019. figure 1. number of hiv/aids cases in 2017-2019 figure 1 shows that in 2019 the number of hiv/aids cases as many as 39 cases increased dramatically from 2018 with 23 cases. during the last 3 years cases of hiv/aids deaths at the pacitan district health offi ce have decreased but are still high, in 2018 cases deaths due to hiv/ aids were 6 cases and 2019 were 5 deaths. then the number who passed/moved from pacitan regency who were positive for hiv aids which was initially registered 0 cases in 2018 increased to 7 cases in 2019. table 3. distribution of hiv/aids cases by gender gender year 2016 2017 2018 2019 male 24 21 12 25 famale 16 15 11 14 number of men was higher than women namely 12 people, and in 2019 the number of men was still higher than women, namely 25 people. it can be concluded that the average number of hiv/ aids suff erers from 2016-2017 was more men. table 4. distribution of hiv/aids cases by age 2016 2019 number of cases category 2016 2017 2018 2019 age 0-9 month 0 0 0 0 1-10 years 3 0 0 5 11-20 years 0 0 0 0 21-30 years 10 6 8 8 31-40 years 14 9 5 13 >40 years 11 21 10 14 identifying the root of the hiv-aids problem after determining the priority of the problem using the usg method and discussing with the head of disease control and prevention and the section head for the infectious diseases section, hiv/aids is determined to be a priority problem, then proceed with compiling an ishikawa diagram (fi shbone diagram) to determine the root of the hiv/aids problem. in pacitan regency. the ishikawa diagram was prepared together with the head of division, head of the infectious diseases section and the hiv/aids program holder. based on table 4, the distribution of the frequency of hiv/aids cases by age in 2016 the highest was 31-40 years old and the lowest was 0-9 months and 11-20 years with 0 people, in 2017 the highest cases were age >40 year with 21 cases and the lowest was 0-9 months and 11-20 years with 0 people, in 2018 the highest number of cases was >40 years with 10 people and the lowest was 0-9 months and 11-20 years with 0 people, and in 2019 the highest cases were at the age of >40 years with 14 cases and the lowest cases were 0-9 months and 11-20 years with 0 people. it can be concluded that the most hiv/ aids suff erers are >40 years old. table 3 shows that the distribution of the frequency of hiv/aids cases by gender in 2016 the number of men was more than women, namely 24 people, in 2017 the number of men was more than women, namely 21 people, in 2018 the firda typewritten text 147 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 determination of the root of the problem in the priority of existing problems is done using 5m theory but the problems that exist in the hiv/ aids program in pacitan district are seen based on the infl uencing factors, namely man, method, and measurement. from the results of interviews that have been carried out with the head of the fi eld, the head of the infectious disease section and the holder of the hiv/aids program, the problem that causes hiv cases that are still high is the lack of public knowledge about hiv/aids, the lack of awareness of patients at risk for testing hiv/aids tests. this makes people less aware of information about hiv/aids, causing a bad public stigma and causing people to be closed/ not willing to go to the puskesmas or hospital to check themselves. this is in line with the article published by the pacitan pemkab which states that it is necessary to increase knowledge about hiv to all elements of society so that awareness arises to reduce the incidence of hiv.10 most of the cases found by the health offi ce are residents who work outside the city, have sexual relations not with partners, blood transfusions are unclear and drug users with injection needles.11 this is in line with the local regulations of pacitan regency, in the chain of hiv transmission there are vulnerable populations, high risk populations, and infected populations. vulnerable population is a group of people who due to their social environment, health status, resilience and family welfare, will be more easily infected with hiv. the population includes people with high mobility, teenagers, street children, and recipients of blood transfusions. 12 formulation of alternative problem solving based on the results of determining the root cause of the problem using the fish bone diagram, it is necessary to reduce hiv/ aids cases in pacitan regency: community participation as the management of various health eff orts for individuals, groups and communities by involving the community in a planned, integrated, and sustainable manner. the goal is that the community is able to take advantage of the various health services needed independently in order to achieve the highest level of public health. this community participation includes two elements: 1) the holding of coordination meetings by stakeholders and the community (for example, representatives of key populations), the availability of funds allocated to civil society in eff orts to combat hiv and aids, as well as capacity building (such as training and technical assistance) which is strategically followed as part of its planning, implementation and evaluation process (prites and posttests, 2) easy access to health services (both general health and hiv and aids services). discussion hiv or human immunodefi ciency virus is a type of virus that attacks/infects white blood cells which causes a decrease in human immunity. aids or acquired immune defi ciency syndrome is a collection of symptoms of diseases that arise due to decreased immunity caused by infection with hiv. due to decreased immunity, the person is very susceptible to various infectious diseases (oprtunistic infections) which are often fatal. people with hiv need treatment with antiretroviral (arv) to reduce the amount of virus arv to prevent opportunistic infections with various complications.13 acquired immune deficiency syndrome (aids) is a retroviral disease caused by the hiv virus characterized by a decrease in the body’s immune system, especially attacking t lymphocytes and a decreased cd4 count to less than 200 cells per l of blood or 14% of all lymphocytes regardless of clinical status. normal cd4 count is 800-1200 cells per l of blood.14 factors that are thought to influence the number of hiv and aids cases in east java are the ratio of pdp services, the ratio of sti services, the percentage of poor people, the percentage of people who use condoms and the ratio of kt services. 15 epidemiologically, the incidence of human immunodefi ciency virus (hiv) and acquired immunodefi ciency syndrome (aids) has increased the mortality and mortality rate of the population at a young age. in addition, the indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 143–151 firda typewritten text 148 mohammad famil: analysis of hiv/aids health problems ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 condition of hiv/aids can also damage the social economy, such as families can lose their livelihood arrangements, costs increase, and are a threat to national development and a challenge in managing the millennium development goals (mdgs) the rate of transmission of hiv and aids. (iakmi, 2013. 16 the incidence of hiv/aids is more common in risky sexual behavior as many as 16 cases (57.1 %). there is a signifi cant relationship between traditional stakeholders and the incidence of hiv/ aids, as evidenced by the p value of 0.014 (p < 0.05). odds ratio 4 and ci : 1, 284 – 12, 468 indicate that respondents who engage in risky sexual behavior are 4 times more likely to suff er from hiv/aids than respondents who do not engage in risky sexual behavior. 16 one of the root causes of the increase in hiv/aids is a lack of knowledge. on internal factors, information exposure can be infl uenced by age, social background, income level and education level of the respondent. this is in line with research, who is the ideal person to provide information about hiv/aids, most of them still answer friends or relatives (67% each) and 62 percent state that health workers are the ideal source of information.18 results showed that low knowledge about hiv/ aids can increase the vulnerability of young people to be infected. however, this study also found that the better the knowledge about hiv/ aids, the greater the attitude of not supporting (rejecting) premarital sexual behavior. from the root of the problem, one of them is the stigma of the community towards plwha. many factors infl uence the occurrence of stigma on plwha in society. health education that aims to increase knowledge about hiv/aids in many studies has been proven to be one of the most infl uential factors in reducing stigma.20 argued that the stigma against plwha which is infl uenced by the attitudes of family, neighbors, and community leaders is the source of certain perceptions (stigma) towards plwha, which is the most infl uential factor. diff erent things were stated by that the stigma given by people to plwha is infl uenced by age and education factors with educational factors having a greater infl uence. 21 23 prevention with an integrated approach is highly recommended to create knowledge, attitudes, and awareness to control the spread of hiv/aids among young people. in carrying out hiv/aids prevention and treatment actions are infl uenced by perceived costs, namely perceptions of negative costs/aspects that prevent individuals from taking health actions including conducting hiv/aids checks and counseling. the only table 3 shows that people with hiv/aids starting from 2016-2019 tend to be more male. related to the work done by men, more mobility outside the area. according to (17) an important aspect in the migration of indonesian workers from the perspective of the spread of hiv/aids is that it involves families such as wives/husbands, children and migrant workers individually for a long time in the destination area or usually with a group of their same-sex colleagues. this has created a situation where male migrant workers in the destination area are usually motivated to visit localization or commercial sex workers (psk). it is not a coincidence that the main concentration area for csws in indonesia is also a concentration area for male migrant workers. the results of the relationship or bivariate analysis using kai squared shows that there are four variables that has a signifi cant relationship with the stigma of plwha (p value < 0.05), namely the respondent’s perception of plwha, neighbor’s attitude factor towards plwha, factor family attitudes towards plwha, and the character’s attitude factors community towards plwha(22) based on research conducted in 24 puskesmas in 8 districts/cities, it shows that puskesmas offi cers are still not ready for activities related to sti and hiv-aids prevention services, both in terms of knowledge, skills and facilities that support these services. so it is necessary to conduct training to health workers about hivaids. thus, it becomes input for stakeholders to increase the role of offi cers in providing information about hiv/aids to the community. almost the same thing was stated about the level of knowledge of hiv/aids with pre-marital sexual behavior of students.19 the firda typewritten text 149 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 special attitude in internal medical personnel related to hiv/aids as an effort to prevent the chain of transmission is the aspect of selfprotection of medical workers and aspects of sterilization of medical devices. 13 what has been done by kab pacitan to prevent hiv-aids, communication, information and education is a process of delivering information (messages, ideas, ideas) about hiv and aids prevention and control from one party to another using information delivery media such as voice media. , print media and electronic media. 12 hiv/aids prevention and control eff orts are carried out through direct counseling at village meetings or at other meetings as well as through media such as radio broadcasts. another eff ort is to secure donor blood by screening donor blood samples.24 based on existing data, there were 3,169 teenagers who received counseling, consisting of 1,424 boys and 1,745 girls, spread throughout pacitan regency. 25 conclusions based on the identifi cation of health problems at the pacitan district health offi ce, the main problem in 2019 was hiv/aids with 40 cases and 5 deaths, an increase from the previous year. problems that are still a priority problem in 2019 at the pacitan district health offi ce are hiv/ aids, leptospirosis, dengue fever, hepatitis, diarrhea, peneumonia and tubercolosis. based on the analysis of the causes of the problem with the help of the fishbone diagram in pacitan regency, several causes of the problem were contain of there is still a lack of public knowledge about hiv/aids, lack of awareness of patients at risk for hiv testing. b a s e d o n t h i s h e a l t h p r o b l e m s , t h e recommendation of this research are first, increase and expand cross-sectoral and crossprogramme collaboration, both government, ngos, institutions, religious leaders, community leaders and existing communities in order to be able to prevent and control hiv/aids by conducting socialization. second, the recommendation for the health offi ce is to form a companion for hiv/aids sufferers in every puskesmas to control patients. optimizing training to increase health cadres. and kds to eliminate community stigma and discrimination so that plwha are expected to open up at least to their families so they can support their treatment. third, improving management systems, information, human resources, and health promotion. acknowledgement 1. depkes. sistem kesehatan nasional. 2009; 2. profi l kesehatan indonesia 2018. 2018. 3. pusat data dan informasi. infodatin hiv aids. pus data dan inf kementrian kesehat ri. 2020;1–8. 4. unaids. unaids scientifi c expert panel 2013-2015. 2015;1–46. 5. mccoy m. measurement and evaluation. public relations handb. 2018;(march):219–42. 6. dave s, peter t, fogarty c, karatzas n, belinsky n, pai np. which community-based hiv initiatives are eff ective in achieving unaids 90-90-90 targets? a systematic review and meta-analysis of evidence (2007-2018). plos one. 2019;14(7):1–18. 7. dinas kesehatan provinsi jawa timur. profi l kesehatan provinsi jawa timur 2019. dinas kesehat provinsi jawa timur [internet]. 2020;1–123. available from: www.dinkesjatengprov.go.id 8. oramasionwu cu, daniels kr, labreche mj, frei cr. the environmental and social infl uences of hiv/aids in sub-saharan africa: a focus on rural communities. int j environ res public health. 2011;8(7):2967–79. 9. p a c i t a n b p s k . k a b u p a t e n p a c i t a n . b p s . 2020;(8):1–9. 10. pacitan p. eling lan waspodo dengan hiv/aids. 2018; available from: https://pacitankab.go.id/tag/ hiv-aids/ the author is grateful for cooperation of head and all staff of heal offi ce of pacitan, east java and lecturer on departement of epidemiology, faculty of public health, universitas airlangga that facilitated this study. references the authors declare that they have no conflict of interest. conflict of interest indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 143–151 firda typewritten text 150 mohammad famil: analysis of hiv/aids health problems ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 14. amelia m, hadisaputro s, laksono b, anies a. faktor risiko yang berpengaruh terhadap kejadian hiv/ aids pada laki-laki umur 25 44 tahun di kota dili, timor leste. j epidemiol kesehat komunitas. 2016;1(1):39–46. 15. simanjuntak s, purhadi p. pemodelan jumlah kasus hiv dan aids di kota surabaya menggunakan bivariate generalized poisson regression. j sains dan seni its. 2017;6(2). 16. handayani, s. arman e. hubungan peranan lingkungan terhadap kejadian hiv / aids relationship of environmental role to hiv / aids private vocational school sri handayani *, eliza arman * , inge angelia * * sekolah tinggi ilmu kesehatan syedza saintika padang email : ririhermana3. j manaj kesehat yayasan rsdrsoetomo. 2018;04:134–43. 17. hugo g. mobilitas penduduk dan hiv / aids di indonesia. 2001; 18. herbawani ck, erwandi d. faktor-faktor yang berhubungan dengan perilaku pencegahan penularan human immunodeficiency virus (hiv) oleh ibu rumah tangga di nganjuk, jawa timur. j kesehat reproduksi. 2020;10(2):89–99. 19. rahayu i, rismawanti v, jaelani ak. hubungan tingkat pengetahuan tentang hiv aids dengan perilaku seksual pranikah pelajar jurnal metodelogi penelitian. j endur 2. 2017;2(june):145–50. 20. shaluhiyah z, musthofa sb, widjanarko b. stigma masyarakat terhadap orang dengan hiv/aids. kesmas natl public heal j. 2015;9(4):333. 21. haryanti t, wartini. perception of people living with hiv/aids on social stigma of hiv/aids in sukoharjo district. kesmas. 2019;13(3):132–7. 22. shaluhiyah z, musthofa sb, widjanarko b. stigma masyarakat terhadap orang dengan hiv / aids (public stigma to people living with hiv/aids). j kesehat masy nas [internet]. 2020;9(4):333–9. available from: http://journal.fkm.ui.ac.id/kesmas/ article/view/740 23. mujiati m, lestary h, sugiharti s. kecukupan tenaga kesehatan dan permasalahannya dalam pelayanan kesehatan anak dengan hiv-aids di rumah sakit pada sepuluh kabupaten/kota, indonesia. media penelit dan pengemb kesehat. 2017;27(1):1–8. 24. keuangan l. kabupaten pacitan tahun 2015. 2016;(031). 25. astuti si, arso sp, wigati pa. profi l statistik sosial 2019 kabupaten pacitan. anal standar pelayanan minimal pada instal rawat jalan di rsud kota semarang. 2015;3:103–11. 13. fitrianingsih, ersa cb, indriyani d, wirdayanti. gambaran karakteristik pasien hiv di poli rawat jalan rsud raden mattaher jambi. j ilm ilmu terap univ jambi. 2019;3(1):54–60. 12. pacitan pk. peraturan daerah kabupaten pacitan nomor 3 tahun 2018. 2018;1965. 11. indonesia t. temuan kasus baru hiv/aids di pacitan meningkat. 2019; available from: https://www. timesindonesia.co.id/read/news/242699/temuan-kasusbaru-hivaids-di-pacitan-meningkat firda typewritten text 151 �� vol. 4. no. 4 october–december 2013 analysis on whole blood, sgot, sgpt, and tnf-a examination in patients with non-dengue and positive dengue fever (df/dhf) rahayu anggraini1, nasronudin1 1 institute of tropical disease, universitas airlangga abstract background: in indonesia has four serotypes, the den-1, den-2, den-3 and den-4. the management of dengue virus becomes difficult because the patients were infected with different clinical profiles depending on the serotypes and genotypes of infecting dengue virus. consequently, the diagnosis and treatment becomes difficult. aim: the purpose of this study was to identify the difference between the results of laboratory tests between non-dengue fever and positive dengue fever. method: this study was an observational cross-sectional study. fifteen samples were diagnosed with dengue fever and fifteen samples with negative dengue fever on ns1, igm / igg-anti-dhf strip test results. laboratory tests comprising whole blood, sgot, sgpt, and tnf-a were first examined when the patient came to the hospital. the collected data were analyzed by chi-square test spss version 13 for windows. result: the results of the study in two groups regarding sex, age, days of fever, grade, hemoglobin levels, leukocytes count, platelet count, hematocrit percentage, sgot levels, and tnf-a level were not significantly different with p > 0.050, whereas the sgpt level in non-dengue increased 3 x of normal value of 66.7%, n = 10/15 and in positive dengue fever the sgpt level was within normal limits, found in 60%, n = 9/15, so there was significant difference with p = 0.022 (p < 0.05). conclusion: in non-dengue sgot and sgpt levels increased of 1–3 times the normal value. in positive dengue fever sgot levels increased 1–3 x normal value, but sgpt levels was within normal value, so sgpt levels can be used as a predictive factor for distinguishing the two types of fever. key words: dengue fever, whole blood, sgot, sgpt, tnf-a abstrak latar belakang: di indonesia memiliki empat serotype dengue yaitu den-1, den-2, den-3 dan den-4. aspek manajemen virus dengue menjadi sulit, karena orang terinfeksi hadir dengan profil klinis berbeda tergantung pada serotipe dan genotipe dari virus dengue yang menginfeksi, sebagai konsekuensi diagnosis dan perawatan menjadi sulit. tujuan: tujuan penelitian untuk mengetahui perbedaan hasil pemeriksaan laboratorium antara demam bukan dengue dan demam positif dengue. metode: penelitian ini bersifat observasional dengan bentuk cross sectional. lima belas sampel didiagnosis demam berdarah dan lima belas sampel negatif demam berdarah dari hasil ns1, igm/igg-anti dhf metode strip. pemeriksaan laboratorium adalah darah lengkap, sgot, sgpt, dan tnf-a diperiksa saat pertama kali penderita datang ke rumah sakit. data yang terkumpul dianalisis uji chi-square dengan spss versi 13 for windows. hasil: hasil penelitian pada jenis kelamin (sex), umur, hari demam, grade, kadar hemoglobin, jumlah lekosit, jumlah trombosit, persentase hematokrit, kadar sgot, dan kadar tnf-a pada kedua kelompok tidak berbeda bermakna dengan nilai p>0,050, sedangkan kadar sgpt pada demam bukan dengue meningkat 3x nilai normal sebesar 66,7%, n=10/15 dan demam positif dengue masih dalam batas normal sebesar 60%, n=9/15, sehingga hasil sgpt antara kedua kelompok terdapat perbedaan secara bermakna dengan p=0,022 (p<0,05). kesimpulan: pada demam bukan dengue kadar sgot dan sgpt meningkat 1–3x nilai normal. pada demam positif dengue kadar sgot meningkat 1–3x nilai normal, namun kadar sgpt masih dalam batas normal, sehingga kadar sgpt dapat sebagai faktor prediksi untuk membedakan kedua tipe demam tersebut. kata kunci: demam dengue, darah lengkap, sgot, sgpt, tnf-a. case report ��anggraini dan nasronudin, et al.: analysis on whole blood, sgot, sgpt, and tnf-a examination introduction dengue fever has become one of the world’s most important diseases caused by arthropods that develop in tropical and subtropical regions. approximately 100 million cases of dengue infection occur each year, and an estimated 2.5 to 5 billion people worldwide are at the risk of dengue virus infection (halstead sb, 2002). these four dengue virus serotypes (den-1, 2, 3 and 4) can transmit to humans through the bite of female mosquitoes of the genus aedes (chen lk, et al 2003). dengue hemorrhagic fever (dhf) are spread throughout southeast asia, western pacific, and the caribbean. indonesia is an endemic region with widespread distribution throughout the country. the incidence of dengue fever in indonesia between 6 to 15 per 100,000 population (1989–1995), and has risen sharply to 35 per 100,000 population in 1998, while the dhf mortality tends to decline until it reached 2% in 1999 (who, 2000). dengue fever is an acute febrile illness for 2–7 days, characterized by two or more clinical manifestations such as headache, retro-orbital pain, myalgia/arthralgia, skin rash, petechiae (hemorrhagic manifestations), and leukopenia. the diagnosis of dengue hemorrhagic fever (dhf) can be established when all above are met, and there is at least one hemorrhagic manifestations, such as petechiae, ecchymosis, purpura, epistaxis, gingival bleeding, melena, hemetemesis, thrombocytopenia (< 100.000/ul), and there is minimally one sign of pleural effusion, ascites, or hypoproteinemia, plasma leakage, such as increased hematocrit > 20% compared to a standard age and sex, decreased hematocrit > 20% after fluid therapy than previously hematocrit value (who, 1997). differences between dhf and df is the presence or absence of plasma leakage after a phase of fever, and the patient will experience critical phase for 2–3 days. during this phase, the patient has no fever, but at the risk for seizure/shock if not handled with adequate treatment. continuous abdominal pain is accompanied by vomiting, loss of consciousness, hypotension, restlessness, rapid and weak pulse and hypothermia are the signs and symptoms of dengue shock syndrome. dengue virus is a non-hepatotropic virus, but it can damage the liver because dengue infection is often unusual and has been described since the 1960s (burke t, 1968). according to hy lei et al. (2001), dengue virus infection affects immune system, such as the change of the cd4/cd8 ratio, cytokinesoverproduction can infect endothelial cells and hepatocytes with the result of apoptosis and dysfunction of these cells. coagulation and fibrinolysis systems are also activated during dengue virus infection. immune response disorder is not only disturbing viral clearing from the body, but cytokines overproduction can affect endothelial cells, monocytes and hepatocytes. platelet destruction is caused by cross-reaction of anti-platelet autoantibody due to overproduction of il-6 that plays a major role in the formation of anti-platelet autoantibodies and anti-endothelial cells, and increased levels of tpa and coagulation deficiency. the level of liver dysfunction in dengue infection varies from mild symptoms, identified through ast and alt examination, up to severity with jaundice and even fulminant hepatic failure (seneviratne sl, 2006, halstead sb, 2002). liver dysfunction could be a direct effect of viral infection that can be detrimental with consequences of dysregulated immune response to the virus in the host (seneviratne sl, 2006). however, clinical studies showed that liver involvement in dengue infection, especially in adults, is still rare. with such considerations, the liver function (sgot and sgpt) examination is worth to be investigated. materials and methods this was an observational cross-sectional study. the sampling process was done at inpatient wards, tropical infectious diseases division, department of internal medicine, dr. soetomo hospital, faculty of medicine, airlangga university, surabaya from july to december 2011. inclusion criteria were patients with df/dhf grade i-iv aged > 15 years, fulfilling the who criteria and positive on one of three tests: ns-1 dengue and or igm/igg anti-dengue. patients who showed positive results were classified as dengue positive patients, and patients with a negative result were classified as suffering from non-dengue fever. patients included in the study were willing to sign informed consent. sample size was 15 subjects of nondengue fever and 15 subjects of positive dengue fever. figure 1. scheme of dhf symptoms according to 1997 who criteria �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 46–52 research implementation venous blood of the subjects were taken as much as 5 ml, collected in a 2 ml edta tubes for whole blood count, and 3 ml was collected in plan tubes to obtain 1 ml serum for sgot, sgpt, and tumor necrosis factor a (tnf-a). whole blood count was performed with a sysmex 4020, while ast and alt examination was performed with a hitachi 902. examination of tnf-a was performed using an elisa method (biosource tnf-a easia kit). after all the data collected, the were processed and analyzed using a statistical test (chi square test) with spss for window version 13. results 1. sex distribution in groups of non-dengue and positive dengue fever. figure 2. sex distribution between the groups was not dengue fever and dengue fever positive. based on sex distribution, among 30 study subjects in both groups the higher prevalence was found in men (66.7%, n = 10/15). chisquare test results showed no significant differences, with p = 0.300 (p > 0.05). 2. age distribution in groups of non-dengue and positive dengue fever. figure 3. age distribution between in groups of non-dengue and positive dengue fever. based on age distribution, among 30 study subjects, higher prevalence was found between 15–24 years. chi square test results showed no significant differences, with p = 0.128 (p > 0.05). 3. fever distribution in groups of non-dengue and positive dengue fever. figure 4. fever incidence in groups of non-dengue and positive dengue fever. based on fever distribution among 30 study subjects, many in both groups come on days 5–6. chi-square test results showed no significant correlation, with a value of p = 0.096 (p > 0.050). 4. grade (disease severity) distribution in groups of nondengue and positive dengue fever. figure 5. grade (disease severity) distribution in groups of non-dengue and positive dengue fever. based on disease severity among 30 subjects, most visited the hospital in grade ii condition. chisquare test results showed no significant relationship with the value of p = 0.090 (p < 0.050). 5. hemoglobin levels in groups of non-dengue and positive dengue fever. figure 6. hemoglobin levels in groups of non-dengue and positive dengue fever. ��anggraini dan nasronudin, et al.: analysis on whole blood, sgot, sgpt, and tnf-a examination based on hemoglobin levels distribution, hemoglobin levels in both groups were still within normal limits. chi square test results showed no significant differences, with p = 0.434 (p > 0.050). 6. leukocytes count in groups of non-dengue and positive dengue fever. figure 7. leukocytes count in groups of non-dengue and positive dengue fever. the leukocytes count distribution in both groups was still within normal limits. chisquare test results showed no significant differences, with p = 0.274 (p < 0.050). 7. platelet counts distribution in groups of non-dengue and positive dengue fever. figure 8. platelet counts distribution in groups of non-dengue and positive dengue fever. based on platelet count distribution, both groups showed reduction less than normal value. chi-square test results showed no significant differences, with p = 0.338 (p > 0.050). 8. hematocrit percentage in groups of non-dengue and positive dengue fever. figure 9. hematocrit percentage in groups of non-dengue and positive dengue fever. based on hematocrit percentage distribution, both groups showed a decrease from normal value. chisquare test results showed no significant differences, with p = 0.161 (p > 0.050). 9. sgot levels in groups of non-dengue and positive dengue fever. figure 10. sgot levels in groups of non-dengue and positive dengue fever. based on sgot levels distribution, both subjects showed increase 1–3 times the normal value. chisquare test results showed no significant differences, with p = 0.143 (p > 0.050). �0 indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 46–52 10. sgpt levels in groups of non-dengue and positive dengue fever. figure 11. sgpt levels in groups of non-dengue and positive dengue fever. based on sgpt level distribution, in non-dengue group the sgpt level increased 1–3 times higher than normal (66.7%, n = 10/15) and in group with positive dengue fever it was still within normal limits (60%, n = 9/15). the results of chi-square test showed significant difference with a p value = 0.022 (p < 0.050). 11. tnf-a in groups of non-dengue and positive dengue fever. figure 12. tnf-a in groups of non-dengue and positive dengue fever. based on tnf-a distribution, the subjects in both groups were within normal limits. chisquare test results showed no significant difference, p = 0.155 (p > 0.050). table 1. laboratory analysis in groups of non-dengue and positive dengue fever on the first day of hospital admission no. variables fever n=15 dengue fever n = 15 p value 95% confidence interval 1 sex male: 66.7% (10/15) male: 66.7% (10/15) 0.300 0.219-4.564 2 age 15-24 years: 73.3% (11/15) 15-24 years: 40% (6/15) 0.128 3 fever day 5-6 53.3% (8/15) day 5-6 46.7% (7/15) 0.096 4 grade grade ii 60% (9/15) grade ii 86.7% (13/15) 0.090 0.708-26.531 5 hemoglobin normal 73.3% (11/15) normal 60% (9/15) 0.248 6 leukocyte normal 93.3% (14/15) normal 60% (9/15) 0.274 7 thrombocyte decrease 93.3% (14/15) decrease 86.7% (13/15) 0.388 0.174-26.672 8 hematocrit decrease 53.3% (8/15) decrease 73.3% (11/15) 0.161 0.090-1.918 9 sgot increase 1-3x normal 60% (9/15) increase 1-3x normal 53.3% (8/15) 0.143 10 sgpt increase 1-3x normal 66.7% (10/15) normal 60% (9/15) 0.022 11 tnf-a normal 86.7% (13/15) normal 66.7% (10/15) 0.155 0.519-20.370 12 vomiting vomiting > 3x 73.3% (11/15) vomiting > 3x 53.3% (8/15) 0.161 0.521-11.104 ��anggraini dan nasronudin, et al.: analysis on whole blood, sgot, sgpt, and tnf-a examination 12. vomiting incidence in groups of non-dengue and positive dengue fever. figure 13. vomiting incidence in groups of non-dengue and positive dengue fever. vomiting incidence in both groups was > 3 times. chi square test results showed no significant differences, with p = 0.161 (p > 0.050). discussion dengue fever (df) and dengue hemorrhagic fever (dhf) are diseases caused by dengue virus infection with clinical manifestations of fever, muscle aches and/or joint pain accompanied with leukopenia, rash, lymphadenopathy, thrombocytopenia and hemorrhagic diathesis. plasma leakage in dhf occurs characterized by hemoconcentration (increased hematocrit) or the accumulation of fluid in the body cavity. dengue shock syndrome (dss) is characterized by dengue shock (balmaseda a, et al 2006, nasronudin 2007, soegijanto s., 2006). ns1 is a glycoprotein with a molecular weight of 50 kda that is expressed in two forms of membrane-associated (mns1) and secreted (sns1) (paul y. 2004). ns1 protein has a high immunogenic properties compared with other nonstructural proteins, although its function has not been known. ns3 and ns5 proteins can stimulate humoral immunity, although the effect is very small when compared to the ns1 protein (earth c, 2006). upon the entry of dengue virus in the human body, the virus multiplies in the reticuloendothelial cells followed by viremia that lasts for 5-7 days. as a result of this emerging viral infection both humoral immune and cellular responses (antineutralization, antihemagglutination, anticomplementary). the appearing antibodies are generally igm and igg. in primary dengue infection, igm antibodies begin to form, whereas igg is not yet formed. in secondary infection igm levels are formed again and igg antibody levels that already exist will rise (booster effect) (chen lk, et al, 2003, soegijanto s. 2006). sgot, stands for serum glutamic oxaloacetic transminase or also called aspartate amino transferase (ast), is an enzyme that is always present in heart and liver cells. ast is released into the blood when the liver or heart is damaged. very high sgot levels in blood indicates liver damage due to viral hepatitis or possible occurrence of heart attacks. some medications can also raise sgot level. sgpt, stands for serum glutamic piruvic transaminase, or also called alt (alanine amino transferase), is an enzyme found in the liver cells as well as effective for diagnosing hepatocellular destruction. in small quantities this enzyme found in the heart muscle, kidney and skeletal muscle. generally, when acute liver parenchymal damage is present, alt level is higher than ast levels, whereas in chronic process the opposite condition is present. reference value of sgpt is 0–40 u/l. conditions that increase sgpt level > 20 times than normal are usually caused by acute viral hepatitis, liver necrosis (due to drug toxicity or chemicals). sgpt increase of 3–10 times more than normal is caused by mononuclear infection, chronic active hepatitis, biliary extra hepatic obstruction, reye ‘s syndrome, and myocardial infarction. sgpt increase of 1–3 times more than normal is caused by pancreatitis, fatty liver, cirrhosis laennec, and biliary cirrhosis. sgpt is more specific for liver damage. sgpt is an enzyme produced in the liver cells (hepatocytes), so it is more specific for liver disease compared with other enzymes. sgpt increase usually occurs when there is a damage liver cell membrane. any type of liver inflammation may cause an increase in liver cell membranes. inflammation of the liver can be caused by viral hepatitis, some medications, alcohol use, and diseases of the bile duct. sgot is a mitochondrial enzyme that is also found in heart, kidneys and brain. so this test is less specific for liver disease. in some cases of liver inflammation, an increase in sgpt and sgot will be similar (gowda, desai, hull, math, kulkarni, vernekar, 2009). the results of a study in india (itha s, 2005) revealed that sgpt and bilirubin levels rarely elevated in patients with dengue fever. according to souza lj (2002) and kalayanarooj (1997) sgot levels in dengue infections tend to be higher than sgpt, but also different from the pattern in patients with viral hepatitis, but similar to that in alcoholic hepatitis. the cause of sgot > sgpt in dengue patients is excessive sgot release from monocytes damaged during dengue infection. according to kuo ch (1992), the determination of sgot levels increasing 1-3 x than normal value and normal sgpt levels can be an early indicator of dengue virus infection. the findings are similar to the results of this study. the severity of liver dysfunction in dengue infections is associated with the severity of dengue disease. therefore, a good predictive factor for dhf severity is to identify the extent of liver damage (kalayanarooj s, 1997). according to rajoo singh chhina (2008) that the extent of liver damage (sgot levels) was higher in df and dhf group, not the sgpt levels, whereas in dss all liver function tests entirely revealed a rise. similar data have also been reported by seneviratne et al. (2006) and souza et al. (2004). the results of this study showed that whole blood count (hemoglobin level, leukocyte count, platelet count, hematocrit percentage, sgot levels, and tnf-a levels) and �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 46–52 symptoms (fever days, grade, time vomiting) were the same, except in non-dengue fever group the levels of sgpt was 1-3x higher than normal value, whereas in positive dengue fever the sgpt levels was within normal limits. conclusion in patients with fever for 4-5 days , which are grouped into non-dengue fever, sgot levels increased 1-3 x times and sgpt levels increased 1-3 x from normal value. in positive dengue fever sgot levels increased of 1-3 x, while sgpt level was still within the normal range (< 40 iu/l), so that sgpt levels can be used as a predictive factor for determining positive dengue fever (df and dhf). references 1. balmaseda a, hammond sn, perez l, et.al.,2006. serotype-specific differences in clinical manifestation of dengue, am. j. trop. med. hyg.; 74 (3), pp. 440– 456. 2. burke t, 1968. dengue haemorrhagic fever: a pathological study. trans r soc trop med hyg.; 62(5): 682–692. 3. bumi c, rantam fa., 2006. determinan virulensi virus dengue dalam demam berdarahdengue. airlangga university press. ed. 2. surabaya. pp. 239–245. 4. chen y, manguire t, hileman re, et.al. 1997. dengue virus infectivity depends on envelope protein binding to target cell heparan sulfat, nature; 3 (8), article, pp. 866–871. 5. chunlin z, 2004. problem encounterd in the molecular detection of dengue viruses i dengue diagnostics: proceeding of an international workshop. genewa switzerland. who. pp. 60–66. 6. clyde k, kyle jl, harris e, 2006. recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis. jvi. 80(23): pp. 11418–11431. 7. halstead sb, 2002. dengue. curr opin infect dis. 15(5): 471–476. 8. itha s, kashyap r, krishnani n, saraswat va, choudhuri g, aggarwal r. profile of liver involvement in dengue virus infection. natlmed j india. 2005; 18(3): 127–130. 9. lei hy, yeh tm, liu hs, lin ys, chen sh, liu cc., 2001. immunopathogenesis of dengue virus infection. j biomed sci. 2001 sep; 8(5): 377–88. 10. kalayanarooj s, vaughn dw, nimmannitya s, green s, suntayaorn s, kunentrasai n, viramitrachai w, ratanachu-eke s, kiatpolpoj s, innis bl, rothman al, nisalak a, ennis fa. 1997. early clinical and laboratory indicators of acute dengue illness. j infect dis.; 176(2): 313–321. 11. kuo ch, tai di, chang-chein cs, lan ck, chiou ss, liaw yf, 1992. liver biochemical tests and dengue fever. am j trop med hyg.; 47(3): 265–270. 12. lum lc, lam sk, george r, devi s, 1993. fulminant hepatitis in dengue infection. southeast asianj trop med public health.; 24(3): 467–471. 13. nasronudin, 2007.the prevalence of hypokalemia and hyponatremia in infectious disease hospitalized patients, dr soetomo hospital, surabaya, 2006 (tesis). universitas airlangga. 14. nguyen tl, nguyen nt, tieu nt, 1997. the impact of dengue fever on liver function. res virol, 148(4): 273–277. 15. seneviratne sl, malavige gn, desilva hj, 2006. pathogenesis of liver involvement during dengue viral infections. trans r soc trop medhyg. 100 (8): 608–614. 16. souza lj, gonçalves carnerio h, souto filho jt, souza tf, cortes va, neto cg, bastos da, siqueira ews, 2002. hepatitis in dengue shock syndrome. braz j infect dis.; 6(6): 322–327. 17. souza lj, alves jg, nogueira rm, gicovate neto c, bastos da, siqueira ew, souto filho jt, cezário tde a, soares ce, carneiro rda c, 2004. aminotransferase changes and acute hepatitis in patients with dengue fever: analysis of 1,585 cases. braz j infect dis.; 8(2): 156–163. 18. soegijanto, s. 2006. demam berdarah dengue edisi kedua. airlangga university press. surabaya. 19. wahid sf, sansui s, zawawi mm, ali ra, 2000. a comparison of the pattern of liver involvement in dengue hemorrhagic fever with classical dengue fever. southeast asian j trop med public health.; 31(2): 259–263. 20. world health organization. dengue hemorrhagic fever: diagnosis, treatment, prevention, and control, 2nd ed. geneva: who; 1997.p. 1–45. 21. world health organization: strengthening the implementation of the global strategy for dengue fever/dengue hemorrhagic fever prevention and control. geneva: who, 2000 ijtid vol 6 no 4 jan-maret 2017.indd 97 vol. 6. no. 4 january–april 2017 research report model of local capacity development for the tropical diseases handling in east java dwi windyastuti1a, dimas aryo wicaksono2, yulis setiya dewi3, puji srianto4 1 faculty of political sciences, universitas airlangga 2 faculty of psychology, universitas airlangga 3 faculty of nursing, universitas airlangga 4 faculty of veterinery, universitas airlangga a corresponding author: dwiwindyastuti@yahoo.com abstract indonesia is a tropical country with its all potential for tropical diseases that are vulnerable to attack its population. this study aims to identify the mechanisms of the tropical disease handling and the potentials that can be done to increase the capacity of tropical disease handling itself. the focus of this research is to increase the capacity of the tropical diseases handling existing in east java, more specifically in some regencies or cities, among others are bojonegoro, sampang and pacitan. the approach of the study was the qualitative approach which was characterized by the existence of an actual setting, researchers as a key instrument, emphasizing the process, and the data analysis is inductive. data were collected using in-depth interview has well as secondary data from health care institution and the internet. a focused group discussion was also occupied to enrich the results, the cases were illustrated and the models were structured more comprehensively in the handling of tropical diseases. participants of this study were health care workers who work at the health institutions including the health department, hospitals, the and public health centers. the findings were all analyzed qualitatively. the results of this study indicated that there are four dimensions of capacity, namely the capacity of the human resource, the capacity of the institution, the capacity of the system and the capacity of the community or the community itself. keywords: tropical diseases, capacity, health workers, community development abstrak indonesia adalah negara tropis dengan segala potensi penyakit tropis yang rentan sekali menyerang penduduknya. menyadari resiko yang ditimbulkan, maka mekanisme penanganan penyakit tropis perlu menjadi perhatian serius oleh pemerintah dan masyarakat. penelitian ini bertujuan untuk mengidentifikasi mekanisme penanganan penyakit tropis dan potensi yang dapat dilakukan untuk peningkatan kapasitas penanganan penyakit tropis itu sendiri. fokus dari penelitian ini adalah peningkatan kapasitas penanganan penyakit tropis yang ada di provinsi jawa timur, lebih spesifiknya di beberapa kabupaten atau kota yang menjadi focus penelitian antara lain bojonegoro, sampang dan pacitan. dengan menggunakan pendekatan kualitatif yang bercirikan adanya setting yang aktual, peneliti sebagai instrumen kunci, data yang ditampilkan adalah data yang bersifat deskriptif, menekankan kepada proses, dan analisis datanya bersifat induktif. focused group discussion juga digunakan untuk memperkaya hasil penelitian, kasus lebih tergambarkan dengan jelas dan model penanganan penyakit tropis dapat disusun dengan lebih komprehensif. partisipan dalam penelitian ini adalah tenaga kesehatan yang bekerja pada departemen kesehatan, rumah sakit dan puskesmas. penelitian ini dianalisis secara kualitatif. hasil penelitian ini menunjukkan adanya empat dimensi kapasitas dalam penanganan penyakit tropis yaitu kapasitas sdm, kapasitas institusi, kapasitas sistem dan kapasitas komunitas atau masyarakat itu sendiri. kata kunci: penyakit tropis, kapasitas, tenaga kesehatan, pengembangan masyarakat 98 indonesian journal of tropical and infectious disease, vol. 6. no. 4 january–april 2017: 97-103 introduction as a tropical country, indonesia is vulnerable to tropical diseases which are very specific, such as dengue fever, tuberculosis (tb) and leprosy. in east java, these diseases remain a serious problem. sampang is one of the regencies that is still considered to have many lepers, of which approximately 607 patients who have leprosy.1 besides, there are also many other tropical diseases that become the extraordinary events or outbreaks in east java, such as dengue fever and tuberculosis (tb). therefore, some concentration should be paid for developing mechanism for handling the diseases using some systematic approach, such as improving capacity of human resources, capacity of institution, some systems, and procedures and increasing community engagements.2,3 unfortunately, the efforts to improve the handling are still necessary even harder, since there are many common problems in health care, for example, the community assessment which only reached the criteria of “enough” for hospital services and noncompliance health workers in carrying out standard operations procedure (sop) on leprosy.1 the problems that are quite complete are also described as follows (1) the great number of health workers do not certainly improve the services, but there are still many “dual practices” without any adequate supervision, contributing to the weakness of the system to be applied; (2) the decentralization system of authority has not shown its potential to fix health care issues; (3) infrastructure and medical equipment are inadequate and not evenly distributed; (4) inefficiency makes less optimal health care, especially in the context of the utilization of medical equipment; (5) utilization of inpatient services is still low, because the aspects of cost, especially for the poor.4 one of the government’s efforts to reform the health sector is the decentralization of authority. however, the decentralization itself also still has many weaknesses.4 first, the difficulty of managing the fiscal decentralization in the beginning of the decentralization. when a transfer of budget allocation has been done to an area via the general fund allocation, the occurrence problem the failure of the health sector to get funding in the area happens. it has been responded by the central government by providing a great concentration fund. however, the limited ability of the central government’s financial and technical difficulties of the concentration fund distribution have caused a great difficulty in central government funding in 2006-2007 and early 2008. second, the implementation of the askeskin (health insurance for the poor) program has indicated a failure of central government to understand the meaning of decentralization in financing. in the beginning of the askeskin program, there was a tendency that the ministry of health did not pay attention to the area in funding and implementing the askeskin program. third, in the third year of decentralization, the health department issued a decree of the minister of health about surveillance, but it has not run. the local government has ignored those important technical guidelines. based on the problems, this research tries to create a model for the development of local capacity in tropical diseases handling. the area mentioned here is east java, which is also still known as a prone area of tropical diseases such as tuberculosis, dengue fever, and so on. the goals to be achieved from this research are: (1) to understand the problems faced in the tropical diseases handling; (2) to know specifically the scope of the policy and institution to implement and handle the tropical diseases in east java; (3) to know specifically the harmonization of policies and interaction among institutions in the tropical disease handling efforts; and (4) to arrange a model of action plan to improve services in the tropical diseases handling in east java. the capacity building in a simple way, the concept of capacity building is defined as the process of improving the ability of people, organizations, and systems to achieve the goals of the organization that have been set.5–9 although this definition is very simple, actually it contains extensive and very important meaning. specifically, the capacity can be seen as something that is specific to a particular task, and the limits of the capacity are specific as related to factors within an organization or a particular system for a particular period.10 the capacity building explains how far the staffs are able to show the real contribution to the development of personal, organization and community.11 the meaning has been extended and linked to the role of the civil/regional institutions, in which the capacity building is interpreted as an effort to improve the ability of people in developing nations to develop management skills and essential policies needed to build the cultural, social, politic, economic and human resources structures so that they are able to exist in the global world.12 an increase of the capacity-building in developing countries will also be able to affect changes in the cultural community, although the transformation of the changes is not so easy and not so quick to do. in fact, the development process of the capacity building can be seen as a political process, because it affects the elite decision-makers to create a policy based on adequate evidence. the development and capacity buildings are not only meant as an individual effort but also an institutional one.13 the capacity building does not only mean as an individual and an institutional effort but also as an effort to improve the community. six main domains to assess community capacity, namely: (1) a partnership in networking concerning the existence and functionality of a leadership role within the networking community; (2) the ability to formulate goals and to act collectively together with other community members; (3) the ability to identify and mobilize the organization and resources (both human and material), to implement a program, a knowledge 99windyastuti, et al.: model of local capacity development transfer related to the ability to develop programs; (4) the ability to transfer the information/ knowledge to other members, the ability to integrate those programs into the main agenda of the group, and problem solving to identify the key actor that influences for problem solving; (5) the ability to discuss and negotiate in problem solving with a good process; (6) the ability to identify problems followed by the correct solving.14 the capacity of decentralization system the implementation of decentralization requires the changing transformation towards the increasing of the local government ability in the aspects of the system, the management of the institution and the increasing of the quality and capacity of the personnel in the implementation of the development and public service process.15,16 osborne and gaebler has offered the idea of ‘reinventing government’, as an effort to carry out an entrepreneur transformation into a bureaucratic organization that has two goals at once, namely to improve the performance of the bureaucracy in running the role of public service and to create a bureaucratic efficiency aiming at overcoming the resources crisis faced by the government.17 meanwhile, the transformation will bring a change in the cultural aspects of the bureaucracy itself, namely from the bureaucratic to a governance model that involves community participation, from the command and control to the accountability of results achieved, from the reliance on internal systems to be competitive and innovative, from which are closed to the open, and from which does not tolerate the risk becomes open to the risks of success or failure.18 the main objective of the renewal is to do a planned change towards a better condition. on that ground, the renewal is called effective if, within the planned period, the better condition really happens. on the contrary, the renewal is called a failure if within the planned period the condition remains as usual and or even gets worse than before. in line with the view that it can be seen that the transformation dynamics of changes or renewals still need to be run within the scope of bureaucratic capacity building to improve the performance effectiveness of the bureaucracy itself.17 along with a number of policies that have been issued by the government in the health sector, there are still many health problems that cannot be handled optimally. indonesia, a country located in the tropics, has a positive or negative access to its people’s health, especially the rise and growth of tropical diseases, such as malaria, leishmaniasis, schistosomiasis, onchocerciasis, lymphatic filariasis, chagas disease, dengue fever, framboesia, and vector. those diseases are the major health problem in almost all developing countries because of the morbidity and death that are relatively high in a relatively short time.19 research method the approach of study is qualitative which is characterized by the existence of an actual setting, researchers as a key instrument, emphasizing the process, and the data analysis is inductive. researchers conducted the data exploration related to tropical diseases in three areas, namely bojonegoro, sampang and pacitan and the findings of both quantitative and qualitative data that are all analyzed qualitatively.20–22 there are several reasons for the selection of the research locations, including: first, pacitan is a relatively remote district from the central government of east java; second, bojonegoro is a region experiencing significant social changes in the presence of oil exploration; third, sampang has the lowest human development index in east java and the low category in indonesia. the location of research is health institutions including the health department, hospitals, the public health centers. the data collection in this study done was in 3 ways, namely in-depth interviews by using guided interview; collecting document data in hospital, health department and the internet; and focus group discussion with subjects of research. the process of inference interpretation of research associated with two dimensions: the text and the social contexts combined as a single unit of analysis. the next step is reconstructing the results of text analysis, the social cognition and social context with the theory framework within categorizations in order to obtain a new understanding of the phenomenon. result and discussion the capacity of human resources the discussion of this study is focused on how far the implementation of capacity-building efforts is. human resources in the health sector include health workers and nonhealth workers under the health act no. 36 of 2009 section 1, stated that: ”health worker is any person who is devoted to the health sector and owns knowledge and/or skills through education in the health field that for certain types require an authority to do health efforts”. this study related to the problems encountered in the management of human resources in the scope of health department in several regencies and cities in east java. problems faced by the health department in pacitan, relate to the number of inadequate human resources, especially the non-medical personnel. as stated by the head of public health center (puskesmas) of nawangan, pacitan, “functional officer concurs treasurer, one person does six programs, although only a few but all diseases exist. cooperation with the midwife, and 100 indonesian journal of tropical and infectious disease, vol. 6. no. 4 january–april 2017: 97-103 hiv, never isolates the hiv. the system of early awareness has been done in this public health center. the number of public health resources is more than the doctors. the health department that also has a shortage of human resources with administrative competence (for making letter of responsibility, handling financial affairs) should be added as well. there are only two accountants in the health department who control 24 public health centers. in almost every year, we propose it to the labor agency (bkd)” the fundamental problem of the analysis of human resources need is the insufficient amount of that, particularly in certain competencies. unfortunately, the need precisely is on the non-medical personnel. as a consequence, some medical personnel also concurrently function as financial staff. this will inevitably have an impact on the motivation of health workers themselves, because they are given the workload that is not in accordance with their competence, and in the end it will also have an impact on the services provided to the public because health professionals are less focused on their tasks; meanwhile, public have already got their protection on their rights over public services, as stipulated in act no. 25 of 2009 on public service. health personnel is the responsibility of the government, either the central government or the local one, as set in act no. 36 /2009 on health, article 25, paragraph 1, 2 and 3. the capacity building of human resources through various activities such as training, workshop, and education that hold a degree or not, should be facilitated by the institution, that is the health department either at the regency or provincial levels. the efforts for the development of individuals are facilitated through workshops on diseases handling such as hiv. the proportion of the budget for the capacity building covering 12% of the net-operational costs and it is focused on tropical diseases. in addition, the budgetary resources for the capacity building of human resources are also derived from general fund allocation (dau) in every field, according to the needs. an interesting mechanism of evaluation which becomes a model has been carried out by bojonegoro regency, in which the local regional head enforces an open communication to the public and local government unit to get input on the performance of the subordination, as stated, “then like handling, actually we have been helped by direct messages (short message system) to the regent, that was actually very helpful, there may be regarded as the disruption..” regent allowed people to send their complaints, including problems related to health care. furthermore, those messages will also be forwarded to the local government unit and personnel related, then hopefully it can improve their service. besides, bojonegoro regency also has regular meetings among local government unit personnel to improve their coordination, considering that the handling of cross-cutting issues related to tropical diseases is really needed. of the two earlier evaluation mechanisms, the positive impact is being felt by officers in the field. it becomes a boost for the officer to provide the best service for the people and also becomes a form of attention of the chief executive to the subordinates. the capacity of institutions the structure of organizations and procedures remain an important parameter in viewing the capacity of the institution. the emergence of a variety of health policy in indonesia will have implications on the development of various duty and authority of the district. surely, the presence of various regulations such as law, decree of health minister, district regulation and regent regulation becomes a lever for health service personnel to improve the program of their activities. in the tropical diseases handling, it has been structured in detail about the authority, duty and coordination flow among institutions through standard operating procedure (sop). it becomes the basis for the institution to act in the tropical diseases handling and has been provided by the health department. both the standard operating procedures (sop) and sop implementation mechanism have been structured, especially when the government would declare the conditions of extraordinary events (kejadian luar biasa/ klb) in a region. in the case of the extraordinary events, the referral mechanisms go from doctors, clinics, hospitals and the health department. preliminary examinations of a patient done by a doctor (public or private) to get the referral and from the examination the patient will be directed to a hospital depending on the grade of the patient. when the patient reaches the third grade, the patient will be referred to the hospital. then, based on the hospital examination results, the patient will be delivered by the public health center where the patient comes to be declared as an extraordinary events in a particular grade. the statement of the extraordinary events will be followed by the epidemiology research (er) with a random sample in the region. the observation was done at a distance of 100 meters from the houses surrounding the patient. finally, the government takes action in the form of fogging, as said by an informant that: “we face a case when sometimes the patients do not come to us, they may firstly check up his health toa private doctor. after being tested that they are positive, it shows clinically that the dengue fever is on grade 3. they are immediately referred to the hospital that the platelets counted drop significantly though not until 150.000. i have even experienced it when i was in ngumpak dalem, there was a feedback when a patient with dengue fever corrected by viper, epidemology evaluation (ee) on grade 3 or grade 4 or grade 2 and after that we do an epidemiology research (er) around the 100 homes of patients who have symptoms of fever or perhaps they show the same symptoms. we do an er, so if there are such cases they have 101windyastuti, et al.: model of local capacity development automatically followed the sop, they have known what they should do” the capacity of the institution is also determined by the coordination between health institution such as the public health center and the health department. one determination in coordination among institutions in the tropical diseases handling is the availability of health resources. often the availability of resources becomes an obstacle to meet the health services primarily. a few numbers of qualified doctors in handling hiv/aids has caused slow treatments to patients. an example is the incidence of a late handling of hiv disease despite the availability of two nurses and one specialist and one general practitioner who have the expertise to handle hiv/aids; but, it is not possible to always stay in the hiv room because they have to carry out their duties in other places as an additional task.the three districts under study (bojonegoro, sampang, and pacitan) have had a standard organizational structure, such as the existence of the general hospital and public health center and the institutions below it. nevertheless, only a few units of work has been certified. not all the financial managements use the pattern like public service agency. several legal institutions that shade health programs in five regencies are constitution of the republic of indonesia year 1945, presidential decree, health minister decree, decentralization laws, and regent regulation, also several articles such as: article 20, article 28h paragraph (1), and article 34 paragraph (3) of the constitution of the republic of indonesia year 1945 and the law of the republic of indonesia number 36 year 2009 on health. see the organizational structure in the health department. the capacity of systems the system capacity refers to the regulations issued by the ministry of health which involves the health sectors, local regulations as the implementation of national health policy, the decisions of governor and regent. the local government should synchronize the national policies and local policies. long before the decree of the health minister and the handbill has existed in east java, the east java provincial regulation no. 5 of 2004 on the prevention and control of hiv aids has been published in east java. referring to the handbill, east java provincial government has made efforts on health. the principal efforts expected to run well is strengthening a health promotion of prevention and expanding the hiv counseling and testing, care, support, and treatment. however, all the efforts that are written in this policy document are only as an appeal, not an obligation. it means that the health ministry cannot insist and ensure that the efforts will be done by local governments and hospitals. to support the health policy to achieve the millennium development goals (mdgs), the bojonegoro government tries to improve the health infrastructure so that it is able to provide a better health degree. in 2013, the health department of bojonegoro prioritized the improvement and repair of the infrastructure of public health centers in 28 districts. the increase includes the improvement of infrastructure, equipment and human resources of medical secretariat functionals subdivision subdivision subdivision programmer administration finance and equipment sector sector sector sector health service control of health problems development of health resources assurance and health facilities section section section section basic health service control and eradication of disease planning and utilization health insurance section section section section referral health service epidemic and disaster training and education facilities and medical equipment section section section section special health service environmental health registration and accreditation pharmacy technical service unit technical service unit technical service unit technical service unit public health pharmaceuticals regional health laboratories control agency of drug, food and beverages head of department s figure 1. organizational structure of provincial health department 102 indonesian journal of tropical and infectious disease, vol. 6. no. 4 january–april 2017: 97-103 personnel. this activity was budgeted at idr.16.2 billion, coming from the budget of bojonegoro governance. it is not only for the public health center (pusat kesehatan masyarakat), but for basic essential obstetric neonatal clinics/beonc (pondok kesehatan) and rural birthing clinics (poliklinik desa/ polindes). in indonesia, dengue fever was first discovered in 1958 in surabaya and now is spreading throughout the provinces in indonesia. the incidence of dengue fever was suspected by the existence of a correlation between strain and genetics, but recently there has been a tendency of different causing-agents of dengue in each area. the cases related to the epidemic of dengue fever that attacked east java during 2013 increased to 80 percent when compared to the previous year, i.e.8.257 cases increasing to be 14.837 cases. the data of the health department showed that the mortality rate declined. it means that the health department succeeded to reduce the mortality rate of patients due to dengue although cases found increased. the areas which the mortality rate increased include sampang and bojonegoro. bojonegoro government intends to carry out a program of preventing and overcoming infectious diseases, as informant said that, “there are 10 types of the diseases to be suppressed including acute respiratory infections/ari (ispa) attacking an 100.524 people (7.95%), diseases of the muscular system and connective tissue attacking 81.868 people (14.62%), gastric ulcer attacking 46.605 people (8.32%), high blood pressure attacking 46.099 (8.23%).then an observation on febricity attacking 28.212 people (5.04%), diarrhea attacking as many as 24.951 people (4.45%), skin diseases as many as 20 016 people allergic (3.57%), allergic skin disease attacking 14.469 people (2.94%), other diseases in the upper bronchial tube attacking 13.831 people (2.47%), asthma attacking 12.964 people (2, 31%)”. the capacity of community the public health efforts to involve the community are the establishment of rural health post (pos kesehatan desa/ poskesdes), the vigilant village (desa siaga, the health efforts on community based (usaha kesehatan bersama masyarakat/ukmb). in order to develop community participation, the government has encouraged the formation of poskesdes with the support of the social assistance of fund operational. the vigilant village (desa siaga) is one of the breakthroughs of the health development in empowering the community in east java. it is a village whose inhabitants have the readiness of resources, ability and willingness to prevent and solve health problems, disasters, and health emergencies independently. the desa siaga is developed through the preparation of the community, the introduction of the problem, the formulation of the achievement followup especially the agreement of poskesdes formation and the resources support. the government also develops the health efforts on community based (usaha kesehatan bersama masyarakat/ukmb) that has been formed in a village in order to bring/provide basic health services for rural communities that include activities of increasing healthy life (promotion), disease prevention, and treatment done by health workers (especially midwives) by involving cadre or other volunteers. the form of ukbm in a village includes posyandu involving community participation, and tiwisada cadre at school, who are scout members that care about health and are ready to provide assistance. this joint movement is built in the form of solidarity when encountering extraordinary events. various efforts for preventing diseases are carried out in a community movement, namely cooperation among local government unit (satuan kerja pemerintah daerah/skpd), police/ army and non-government organization/ngo (lembaga swadaya masyarakat/lsm). this cooperation fosters a spirit of solidarity to tackle tropical diseases such as in sampang.in the tropical disease handling, people are given an understanding of diseases such as lectospyrosis through socialization by giving pictures to all staff in public health centers (puskesmas) to be delivered to the public. to promote tropical disease prevention, a special program is made through a variety of media, as said by an informant from sampang: “the socialization has been done through the radio broadcast, mobile broadcast, spread pamphlets about how the symptoms and treatment. it has been spread to almost all districts, to rural areas, teenagers, schools; through cross-sector cooperation, collected to local government by involving the chairman of neighborhood association (rukun tetangga/rt) and administration unit (rukun warga/rw),and ngo agencies. they did the voluntary work, including in the traditional market, since it is the habitation of rats” these joint movements of community in bojonegoro start from a village, district and public health center, and school. one of the movements is in the form of implementation of communication information education (cie) and mosquito eradication, as stated: “nggayam shade 23 villages whose 3 villages are certainly dengue endemic. in those 3 dengue endemics, almost all in habitants, one or two of them, were certainly infected although not to die. we have triedto do cie to our community by crosssector cooperation, all villages and sub-districts completely moved together so that this mosquito problem could be fought. so before they become adults, they would later become our fekton to voluntary work together. we selected those three endemic villages, we all did the voluntary work and asked the head of sub-district for help and his officials to suppress the dengue fever, and we do 103windyastuti, et al.: model of local capacity development this attempt this year, so that one village was not affected by dengue fever”. the development of another community involvement is the formation of the association community. as occurred in sampang, there is an establishment of the association of former leprosy patients aimed to provide a reinforcement to the lepers and to empower leprosy patients. they become volunteers for other lepers. those volunteers are given the task as supervisors of swallowing drugs (pengawas menelan obat/pmo) besides giving skill assistance to lepers, so that they can be more independent and economically productive. another development of social networking is searching donation from corporate institutions. in tropical diseases handling, bojonegoro government received contributions from several institutions or mining investors such as pt.petrocina and pt. exxon mobile. those corporations provided infrastructure supports as a form of social responsibility (charity) for their existence. facilities provided by those corporations in the form of the building, training for health personnel and fund. conclusion to deal with tropical diseases, human resources such as health workers become an important dimension. it is not only supported by human resources that are adequate or have an appropriate professional field but also strengthened by experience in handling emergency situations of disease incidence. the good governance achievement in tropical diseases handling can only be done when the framework of the development of institutional networking between health care units is implemented properly. the cooperation ability within a working unit and among working units is a condition that sustains the success of diseases handling. coordination, communication, and synchronization are important prerequisites in strengthening the health institutional capacity. the pattern of vertical coordination between levels of government, namely between regency and province levels, and pattern of horizontal coordination among levels of government, namely the health department, hospitals and public health center become the key to success. the capacity of the system also becomes a dimension in the development of local capacities. the local policy in indonesian refers to the national policy. indonesia’s health policy is directed to one goal that is the achievement of the mdgs which has become an international agreement. various policy ratifications have been established by the indonesian government either in the form of laws or ministry regulations. in the context of preparing regulations at the local level, it still refers to the higher level’s regulations, it means that the regulations made by the local government do not disapprove the regulations of above level, but they are made in detail for implementation such as guidelines, regent regulations or local government regulations. a regulation consistency is a prerequisite for the implementation of the policy so that there is no debate in the implementation of disease handling. the involvement of health stakeholders contributes to the success of the capacity building. the development of community capacity on the incidence and prevention of health is one of the solutions to overcome limitations of health resources. the involvement of stakeholders in the search for donations either from corporations or foreign country in the tropical diseases handling is necessarily done by local governments in the concept of helping to resolve an incident. the reinforcement of non-institutional network, that is community resource development through volunteers or health cadres both of family or community, helps in the tropical diseases handling. acknowledgment we gratefully acknowledge members of health care workers in district of bojonegoro, sampang and pacitan who worked tirelessly to support our data collection by providing their experiences during the interview and fgd. we also thank ministry of higher education through rector of universitas airlangga who funds our research project. the opinions are the responsibility of the authors and do not necessarily reflect the views of indonesian government. refferences 1. hidayat t. analisa faktor yang memengaruhi kepatuhan petugas kusta dalam pelaksanaan sop pelayanan kusta di puskesmas kabupaten sampang analysis of factors affecting obedience officer leprosy in the implementation of leprosy sop services district puskesmas sampang. j adm dan kebijak. 2012;10(2):68–72. 2. morrison t. actionable learning: a handbook for capacity building through case based learning. asian development bank institute; 2001. 3. united nations development programme. capacity development: lessons of experience and guiding principles. wignaraja k, editor. new york: united nations development programme; 1996. 4. laksono. pelaksanaan desentralisasi kesehatan di indonesia 2000-2007: mengkaji pengalaman dan membahas skenario masa depan [internet]. 2010. available from: http://www. kebijakankesehatanindonesia.net/images/stories/fruit/desentralisasi kesehatan 2007_fix_tyo.pdf 5. brown l, lafond a, macintyre k. measuring capacity building. university of north carolina at chapel hill: carolina population center; 2001. 6. mondy rw, noe rm. human resources management. new york: prentice hall; 2005. 7. torrington d, hall l, taylor s. human resource management. essex: pearson education; 2008. 8. storey j. human resource management: a critical text. london: cengage learning; 2007. 104 indonesian journal of tropical and infectious disease, vol. 6. no. 4 january–april 2017: 97-103 9. armstrong m. armstrong’s essentials human resource management practice: a guide to people management. philadelphia; 2010. 10. milen a. what do we know about capacity building? : an overview of existing knowledge and good practice. 2001;(june). 11. berry ll. on great service: a framework for action. new york: the free press; 1990. 12. james vu. capacity building in developing countries: human and environmental dimensions. westport, connecticut london: praeger; 1998. 13. thompson j. participatory approaches in government bureaucracies : facilitating the process of institutional change. world dev. 1995;23(9):1521–54. 14. bush r, dower j, mutch a. community capacity index. cent prim heal care univ queensl. 2002;version 2. 15. buchan j. health sector reform and human resources: lessons from the united kingdom. oxford univ press. 2000;15(3):319–25. 16. lane j-e. new public management. int public manag j. 2001;4:115–8. 17. osborne d, gaebler t. reinventing government: how the entrepreneurial spirit is transforming the public sector (plume) paperback. new york: penguin group; 1993. 18. shah a, chaudhry tt. implementing decentralized local governance: a treacherous road with potholes, detours and road closures. 2004; 19. putra fi, rahman a, sudiarno a. perancangan knowledge management system dalam penanganan penyakit tropis dengan pemenuhan prinsip ergonomi kognitif. institut teknologi sepuluh nopember (its) surabaya; 2010. 20. miles mb, huberman am. analisis qualitative data. jakarta: ui press; 1992. 21. moleong lj. metodologi penelitian kualitatif. bandung. remaja rosdakarya; 2001. 22. denzin nk, lincoln ys. handbook of qualitative research. new york: sage publications; 2009. 29 vol. 5. no. 2 may–august 2014 colostrum-collagen-hydroxyapatite composite, an excellent candidate biomaterial for bone repair and bone infection management dio nurdin setiawan1, mirzaq hussein anwar1, kholifatul wanda putri2, nilna faizah fiddarain3, prihartini widiyanti4,5, heri purnobasuki6 1 bachelor of biomedical engineering study program, faculty of science and technology, universitas airlangga, surabaya, indonesia 2 bachelor of information system study program, faculty of science and technology, universitas airlangga, surabaya, indonesia 3 bachelor of pharmacy study program, faculty of pharmasy, universitas airlangga, surabaya, indonesia 4 institute of tropical disease, universitas airlangga, surabaya, indonesia 5 biomedical engineering study program, faculty of science and technology, universitas airlangga, surabaya, indonesia 6 department of biology, faculty of science and technology, universitas airlangga, surabaya, indonesia abstract in the case of bone fracture or defect after surgery, which is common in patients with bone cancer (osteosarcoma), it takes a long time for closure and it may cause an infection problem. the use of collagen-hydroxyapatite composite with a blend of colostrum as a scaffold is aimed to accelerate the process of osteoblast growth, inhibite the emergence of infections, and act as bone tissue repair material. the method used was the hydrogel formation process and freeze dry process to remove the solvent and to form pores. the composition of scaffold composite manufactured was 15% collagen, 75% hydroxyapatite and 10% colostrum. combination of scaffold collagen-hydroxyapatite-colostrum has quite reliable properties because sem test showed that scaffold could bind to both and could bind to both and could form sufficient pores to provide enough place for bone cells (osteoblats) to grow. the results of mtt assay revealed percentage of above 60%, which indicates that the material is not toxic. in conclusion, collagen-hydroxyapatite-colostrum combination is an excellent biomaterial candidate for bone repair and bone infection management. key words: collagen, hydroxyapatite, colostrums, osteoblasts, bone repair abstrak pada kasus fraktur atau defek tulang setelah operasi yang biasa terjadi pada penderita osteosarkoma (kanker tulang), membutuhkan waktu yang lama dan bisa menimbulkan problem infeksi. penggunaan komposit kolagen-hidroksiapatit dengan paduan colostrums sebagai scaffold diharapkan dapat mempercepat proses pertumbuhan sel osteoblast. metode yang digunakan yaitu dengan proses freeze dry untuk menghilangkan pelarut dan membentuk pori. perbandingan pembuatan komposit scaffold ini 15% kolagen, 75% hidroksiapatit, dan 10% colostrums. paduan colostrums scaffold kolagen-hidroksiapatit memiliki sifat cukup baik karena pada hasil uji sem scaffold dapat berikatan dengan baik dan dapat terbentuk pori yang cukup untuk tumbuhnya sel tulang (osteoblast). hasil mtt assay menunjukkan jumlah sel hidup diatas 60% yang berarti bahwa material tidak bersifat toksik. kata kunci: kolagen, hidroksiapatit, kolostrum, osteoblas, perbaikan tulang research report introduction according to world health organization (who), traffic accidents cause about 1.2 million deaths each year. losses due to traffic accidents, in addition to death, are physical damage as well. physical damage most often occours in an accident is fracture (broken bone). high accident rate results in high fracture incidence. fracture is a situation where bone disintegrating. the most common cause is accidents, but other factors, such as degenerative processes, can also affect the incidence of fracture. 30 indonesian journal of tropical and infectious disease, vol. 5. no. 2 may–august 2014: 29-31 scaffold is one component of tissue engineering applications that can be used as application in bone tissue repair.1 in producing scaffold, we require hydroxyapatite (ha). hydroxyapatite it self has osteoconductive and biocompatibility properties.2 however, ha also has characteristics of brittle and fatigue failure, so that in health applications ha is only used for unloading bearing repair and as a substitute.3 in scaffold formation, we need mixed materials for quality enchancement, and the collagen. approximately 25–35% of body proteins are composed by collagen.3–6, in the case of fracture or bone defect after surgery, which is common in patients with bone cancer (osteosarcoma), it takes a long time for closure. to overcome this problem, additional material other than ha and collagen is required to accelerate the regeneration of bone cells. regeneration of bone cells is also affected by immune quality of the human body. self-immunity is provide by many living things, including mammals. there is a fact in the society that drinks containing colostrums can accelerate healing, especially in adult to elderly whose healing process requires longer time. in this study, we added bovine colostrums to collagenha scaffold. bovine colostrums has content which is almost similar to that of human colostrums. colostrums itself has properties to stimulate body cells regeneration, peptide imunotheraphy, help fighting viruses, and so on.7 we expect that a combination of blend collagen-ha scaffolds and colostrums may accelerate cell regeneration, so that it may implicate the acceleration of bone grafting.2,4 materials and method materials that used in this research were hydroxyapatite of bovine obtained from tissue bank and biomaterial center dr. soetomo general hospital surabaya, east java, indonesia. collagen powder is derived from the skin of bovine, and colostrums powder is derived from dairy cattle. preparation of collagen-hidroxyapatite addition colostrums. 15% collagen dissolved in 0.5 mol/l cold acetic acid, then added with na2hpo4 0.02 mol/l and controlling ph up to 7.2 with aqueous naoh solution at temperature below 10oc. then collagen solution was added 75% hydroxyapatite are stirred in nh4oh for 2 hours, at ph 7. after dissolved, add 10% colostrums and they were stirred for 4 hours, and then incubated at a temperature of 35°c for 20 hours. the results scaffold obtained, then washed with aquadest, and then centrifuged, thus a mixture is obtained in the form of hydrogel and done printing. solid phase separation technique and liquid is done with composite cooling up to –20°c for 24 hours, while the solvent removed by freeze-drying. characterizations were carried out with scanning electron microscope (sem). biocompatibility was tested by mtt assay. results and discussion in collagen ha colostrums composite facilitate interaction between cells and implants material. it will cause the occurrence of osteogenic cells, adhesion, attachment and spreading phase which triggered the proliferation and differentiation of cells. attachment phase and physicochemical linkage formed appear at the same time with biomaterial implantation to bone cells.8 adhesion and spreading phase will occur when focal contacts and adhesion plaque between the surface of the implant material and cell membrane is formed. then actin filament reorganized cause adhesion process causes change cell and transmit signal transduction through proteins of the cytoskeleton to become nuclear matrix, changing the arrangement of the genes, and determine the number of cells for proliferation and differentiation.9 composite of collagen-ha as bone tissue engineering should have physichocemical binding and crystal structure to be recognized as good material. these will affect the characteristic of the osteogenic cells, determine the quality of them and success to perform new bone tissue. calcium phosphate powders is required on the average diameter 200–500 m, and if the size of particles less than 50 m then it will cause cytotoxicity.10 the pore size which are ideal for calcium phosphate is around 200-400 m. it provide the space for blood vessels and trigger migration, adhesion, proliferation, and differentiation of osteoblast in pores. the results of this research material products could be seen in figure 1. figure 1. scaffold collagen – ha – colostrums sem profile with edax in figure 2 below shows that the third main material in this research have been wellmixed. (a) 31dio nurdin s., et al.: colostrum-collagen-hydroxyapatite composite (b) (c) figure 2. result (a) sem with magnification 2000´, (b) sem with magnification 1000××´, (c) energy x-ray dispersion. sem results are showed shape of ha grain and collagen fibers. while the fiber shape of colostrum is already bind to ha. mtt assay is showed that the material biocompatibility exceeds 50% by the procentage of living cell.11,12 combination of colostrum on scaffold collagen hydroxyapatite is showed good physical properties. this can be evidenced from the test results the main material of the sem, result can bind to either and form a good pore as a condition the growth of osteoblast cells. interpretation of mtt assay result which exceed 60% could be considered that the material is not toxic. colostrum can actively support the body immunity, antibodies, and other protective proteins. colostrum provides ‘first immunity’ and the mechanism is to protect the body against many infections. the main immunity substances are immunoglobulin that can prevent and fight bacteria, viruses, fungi and toxins. immunoglobulin (iga) was act as the protectors in the area susceptible to bacteria commonly in the membranes of lung, colon and throat. colostrum contains white blood cells (leukocytes) which its function could fight microorganism. colostrum contains growth factors which could accelerate wound healing. colostrum which is rich in vitamins a and e will support to reduce infection. in addition, colostrum also contains vitamin b6, b12, c, d, k and minerals, especially iron and calcium. colostrum also contains several substances in such high quantities of sodium, potassium and cholesterol.13 based on the phenomena above, a blend of collagen-hydroxyapatite-colostrum is a candidate for an promising biomaterial for bone repair and bone infections treatment. conclusion the composition of scaffold composite manufactured was 15% collagen, 75% hydroxyapatite and 10% colostrums has quite reliable properties because sem test showed that scaffold could bind to both and could bind to both and could form sufficient pores to provide enough place for bone cells (osteoblats) to grow. the results of mtt assay revealed percentage of above 60%, which indicates that the material is not toxic. so that collagen-hydroxyapatite-colostrum combination is an excellent biomaterial candidate for bone repair and bone infection management. references 1. cahyanto a. 2009. biomaterial. departemen ilmu dan teknologi material kedokteran gigi. universitas padjadjaran. bandung. 2. feng, w. tang, k. zheng, x. yuanming. liu, j. 2009. preparation and characterization of porous collagen/hydroxyapatite/gum arabic composit. zhengzou university: cina. 3. rodrigues cvm. 2003. characterization of bovine collagenhydroxyapatite composite scaffold for bone tissue engineering. biomaterials, 2003; 24: 4987–4997. 4. gelse, ke. poschl, t. aigner. 2003. collagens-structure, function, and synthesis. advanced drug delivery, 2003; 55: 1531–1546. 5. lawson ac, czernuszka jt, 1998. collagen–calcium phosphate composites. proc instr mech eng, 1998; 212 (11): 413–438. 6. song, eun, so yeon kimb, taehoon chunc, hyun-jung byunc, young moo lee. 2006. collagen scaffolds derived from a marine source and their biocompatibility. biomaterials, 2006;27: 2951– 2961. 7. keech am. 2009. peptide imunotherapy colostrums. aks publishing; isbn 978-0-692-00242-1. 8. jie, wei, li yubao. 2004. tissue engineering scaffold material of nano-apatite crystals and polyamide composite. european polymer journal 2004; 40: 509–515. 9. park jb, bronzino jd, 2003. biomaterials principles and applications. crc press: boca raton. 10. kutz, myer. 2003. standard handbook of biomedical engineering and design. mcgraw-hill: new york. 11 tierney cm, haugh mg, liedl j, mulcahy f, hayes b, o’brien fj, 2009. the effect of collagen concentration and crosslink density on biological, structural and mechanical properties of collagen-gag scaffolds for bone tissue engineering. journal of the mechanical behaviour of biomedica materials, 2009; 2 (2): 202–9. 12. wahl da, czernuska jt. 2006. collagen-hydroxyapatite composites for hard tissue repair. european cells nd materials, 2006; 11: 43–56. 13. hurley wl. theil pk. 2011. perspectives on immunoglobins in colostrum and milk. nutrients, 2011; 3: 442–474. e issn 2356–0991 p issn 2085–1103 correlation of nutritional status with hookworm and strongyloides stercoralis infection in children under five years in kokar public health center, alor regency, east nusa tenggara the epidemiological pattern and risk factor of esbl (extended spectrum β-lactamase) producing enterobacteriaceae in gut bacterial flora of dairy cows and people surrounding in rural area, indonesia genexpert mtb/rif and mycobacterium tuberculosis sputum culture in establishing the diagnosis of pulmonary tuberculosis and rifampicin resistance in suspected childhood pulmonary tuberculosis in soetomo hospital c-reactive protein and hepcidin in non-dialysis chronic kidney disease gastric perforation associated with candidiasis and nsaids disseminated tuberculosis mimicking lung cancer with multiple bone metastasis: a case report effect of zinc(ii)-2,4,5-triphenyl-1h-imidazole complex against replication denv-2 in vero cell prevalence of methicillin-resistant staphylococcus aureus (mrsa) carrier in hemodialysis patients at dr. soetomo academic general hospital e-journal.unair.ac.id/index.php/ijtid vol. 8 no. 3 septmber-december 2020 indexed by: v l. 8 volume 8 number 3 september–december 2020 e issn 2356 0991 p issn 2085 1103 editorial team of indonesian journal of tropical and infectious disease editor in chief prihartini widiyanti, indonesia editorial board mark alan graber, united states kazufumi shimizu, japan masanori kameoka, japan hak hotta, japan fumihiko kawamoto, japan nasronudin nasronudin, indonesia maria inge lusida, indonesia puruhito puruhito, indonesia retno handajani, indonesia kuntaman kuntaman, indonesia soegeng soegijanto, indonesia bambang prajogo, indonesia ni nyoman sri budayanti, indonesia achmad fuad hafid, indonesia tri wibawa, indonesia irwanto irwanto, indonesia yulis setiya dewi, indonesia laura navika yamani, indonesia siti qomariyah khairunisa, indonesia siti churrotin, indonesia teguh hari sucipto, indonesia secretariat nur diana fajriyah zakaria pamoengkas secretariat office publishing unit of indonesian journal of tropical and infectious disease, institute of tropical disease universitas airlangga kampus c, jalan mulyorejo surabaya 60115, jawa timur – indonesia. phone 62-31-5992445-46 faximile 62-31-5992445 e-mail: ijtid@itd.unair.ac.id homepage: e-journal.unair.ac.id/index.php/ijtid mailto:ijtid@itd.unair.ac.id volume 8 number 3 september–december 2020 e issn 2356 0991 p issn 2085 1103 contents page ...................................................................................................... 137–143 2. the epidemiological pattern and risk factor of esbl (extended spectrum β-lactamase) producing enterobacteriaceae in gut bacterial flora of dairy cows and people surrounding in rural area, indonesia agusta reny soekoyo, sulistiawati, wahyu setyorini, k. kuntaman .............................................. 144–151 3. genexpert mtb/rif and mycobacterium tuberculosis sputum culture in establishing the diagnosis of pulmonary tuberculosis and rifampicin resistance in suspected childhood pulmonary tuberculosis in soetomo hospital berlian beatrix rarome, nurul aisah, retno asih setyoningrum, ni made mertaniasih ............. 152–160 4. c-reactive protein and hepcidin in non-dialysis chronic kidney disease edward muliawan putera, widodo, nunuk mardiana ..................................................................... 161–167 5. gastric perforation associated with candidiasis and nsaids febriana aquaresta, arthur pohan kawilarang,pepy dwi endraswari ......................................... 168–173 6. disseminated tuberculosis mimicking lung cancer with multiple bone metastasis: a case report laksmi wulandari, putri mega juwita .............................................................................................. 174–182 ilham harlan amarullah, harsasi setyawati, puspa wardhani, aryati, soegeng soegijanto................................................................................................................................ 183–188 8. prevalence of methicillin-resistant staphylococcus aureus (mrsa) carrier in hemodialysis patients at dr. soetomo academic general hospital eko oktiawan wicaksono, artaria tjempakasari, widodo widodo, kuntaman kuntaman, usman hadi ........................................................................................................................................... 189–194 printed by: universitas airlangga press. (rk 232/11.20/aup). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, fax. (031) 5992248. e-mail: adm@aup.unair.ac.id 1. correlation of nutritional status with hookworm and strongyloides stercoralis infection in children under five years in kokar public health center, alor regency, east nusa tenggara benaya y. onesiforus, indra e. lalangpuling, mahardika a.wijayanti, e. elsa herdiana murhandarwati 7. effect of zinc(ii)-2,4,5-triphenyl-1h-imidazole complex against replication denv-2 in vero cell aswandi wibrianto, fatimah martak, teguh hari sucipto, siti churrotin, mailto:adm@aup.unair.ac.id available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ original article * abstract elyana sri sulistyowati , septi dwi muningga, verarica silalahi quality and patient safety committtee of dr. kariadi hospital semarang, indonesia vol. 9 no. 1 january–april 2021 th th covid-19 is a communicable disease causing global pandemic. some factors inflict worse infection. this study aims to investigate risk factors of covid-19 confirmed died patients at dr. kariadi hospital semarang. it is a retrospective study with a total sample of all covid-19 confirmed patients involving died and healed patients from march to june 2020. data was gathered from screening forms and analysed with chi square (confidence interval of 95%). this study found sixteen risk factors of covid-19 confirmed died patients involving age (p= 0.000; or= 8.803; 95% ci 3.982-19.462), entrepreneur (p= 0.041; or= 14.894; 95% ci 1.12-198.65), farmer/trader (p= 0.029; or= 25.625; 95% ci 1.40-469.25), contact history (p= 0.000; or= 12.923; 95% ci 6.163-27.097), fever (p= 0.000; or= 4.877; 95% ci 2.647-8.984), dyspnea (p=0.000; or= 17.018; 95% ci 8.523-33.977), cough (p= 0.009; or= 2.178; 95% ci 1.205-3.935), lethargic (p=0.010; or= 2.282; 95% ci 1.205-4.323), cold (p= 0.002; or= 0.180; 95% ci 0.054-0.600), diabetes (p=0.000; or= 9.767; 95% ci 3.932-24.263), copd (p= 0.001; or= 6.360; 95% ci 2.164-18.690), hypertension (p= 0.043; or= 2.436; 95% ci 1.008-5.887), cancer (p=0.001; or= 9.647; 95% ci 2.413-38.579), heart disease (p= 0.000; or= 12.226; 95% ci 2.4-62.294), neurological disorders (p=0.008; or= 6.057; 95% ci 1.650-22.232), and immune disorders (p=0.031; or= 1.625; 95% ci 1.186-113.899). adequate handling is needed to prevent death. in patients with confirmed covid-19 who have risk factors. abstrak keywords: risk factor; confirmed patients; covid 19; died; retrospective covid-19 merupakan penyakit yang cepat menular sehingga menyebabkan pandemi di seluruh negara. ada beberapa faktor risiko yang menjadikan covid-19 menginfeksi seseorang menjadi lebih parah. penelitian ini bertujuan untuk mengetahui faktor risiko pasien terkonfirmasi covid-19 yang meninggal dunia di rsup dr. kariadi. penelitian ini merupakan retrospective study. populasi adalah seluruh pasien terkonfirmasi covid-19 di rsup dr. kariadi. sampel penelitian adalah seluruh pasien terkonfirmasi covid-19 yang terdiri dari pasien meninggal dan sembuh tercatat sejak bulan maret sampai juni 2020. data diperoleh dari formulir screeening dan dianalisis menggunakan uji chi square dengan tingkat kepercayaan 95%. hasil peneltian menemukan enam belas faktor risiko pasien terkonfirmasi covid-19 meninggal, yaitu umur (p= 0,000; or= 8,803; 95% ci 3,982-19,462), wiraswasta (p= 0,041; or= 14,894; 95% ci 1,12-198,65), petani/pedagang (p= 0,029; or= 25,625; 95% ci 1,40-469,25), riwayat kontak (p= 0,000; or= 12,923; 95% ci 6,163-27,097), demam (p= 0,000; or= 4,877; 95% ci 2,647-8,984), sesak napas (p=0,000; or= 17,018; 95% ci 8,523-33,977), batuk (p= 0,009; or= 2,178; 95% ci 1,205-3,935), lemah lesu (p=0,010; or= 2,282; 95% ci 1,205-4,323), pilek (p= 0,002; or= 0,180; 95% ci 0,054-0,600), diabetes (p=0,000; or= 9,767; 95% ci 3,932-24,263), gangguan paru kronik (p= 0,001; or= 6,360; 95% ci 2,164-18,690), hipertensi (p= 0,043; or= 2,436; 95% ci 1,008-5,887), keganasan (p=0,001; or= 9,647; 95% ci 2,413-38,579), penyakit jantung (p= 0,000; or= 12,226; 95% ci 2,4-62,294), gangguan neurologis (p=0,008; or= 6,057; 95% ci 1,650-22,232), and gangguan imunitas (p=0,031; or= 1,625; 95% ci 1,186-113,899). dibutuhkan penanganan yang adekuat untuk mencegah kematian pada pasien terkonfirmasi covid-19 yang memiliki faktor risiko. * corresponding author: elyana.ss@gmail.com ijtid, p-issn 2085-1103, e-issn 2356-0991 risk factors of covid-19 confirmed died patients in dr. kariadi hospital: a retrospective study open access under cc-by-nc-sa share alike 4.0 february 2021 received: 15 th january 2020; revised: 4 february 2020; accepted: 9 2 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 1–8 introduction kata kunci: faktor resiko; pasien terkonfirmasi; covid-19; meninggal; retrospektif how to cite: sulistyowati, es., muninggar, sd, silalahi, v. risk factors of covid-19 confirmed died patients in dr. kariadi hospital: a retrospective study. indonesian journal of tropical and infectious disease, 9(1), 1–8. ijtid, p-issn 2085-1103, e-issn 2356-0991 covid-19 is a communicable disease first reported in wuhan, china in december 2019. covid-19 known as novel coronavirus caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the first spread to other countries occured from 2020.1 in comparison with sars and mers, covid 19 is more contagious resulting in global pandemic.2,3 cases of pneumonia with unknown aetiology was announced as a public health emergency of international concern (pheic) in the end of january 2020 and declared as pandemic on 11 march 2020.4 covid-19 was predicted to have mortality rate lower than sars and mers, in fact its mortality rate is 2%2 2.3% or 20 times greater than common influenza.4 covid-19 cases is more prevalent among older people having comorbidities such as cardiovascular disease, diabetes, hypertension2, chronic respiratory disease and cancer.4,5,6 involving 1.159 healed cases and 150 death cases.9 it suggests the mortality rate of covid19 in indonesia and central java is about 5.1% and 3.9% respectively. it is significantly greater than global mortality rate (case fatality rate 2.3%) and south east asian mortality rate (cfr 2.1%).8 the high confirmed cases and mortality rate are a result of host and virus factors. the virus ability to defeat host immune systems serve as determinant of infection severity. inadequate host immune response results in virus mutation and more severe host tissue destruction. it is compounded with the virus ability to evade from host immune and replicate thus unrecognised by host immune.6 data indicate covid-19 is more prevalence among elderly and those with comorbidities. the mortality rate is higher among older people.10 the presence or absence of symptoms is not the sole hint as a study from china found 12.6% infection occurs pre-symptomatic and some cases confirmed without symptom (asymptomatic).11 covid-19 patients without symptom may transmit viruses to other people. people in group in certain environment are at high risk for infection such as dormitory, prison, other closed environment, and public facilities such as bus stop, bus station, airport and shopping centre.7 people having close contact with covid19 patients or taking care of covid-19 patients have heightened risk for covid-19 exposure.12 besides, smoking is associated with heightened disease severity and covid-19 related mortality.13,14 other factors such as vitamin d availability is considered playing important role for decreasing virus infection risk. it is known fd transmission of covid-19 can occur between people with or without symptoms. virus can survive for more than 72 hours resulting is fast contagion.3 so far, covid-19 spreads through air when performing aerosol-generating medical procedures where virus can survive for 3-16 hours. in this case, health practitioners such as laboratory technicians have greater risk for covid-19 exposure. outside medical facilities, covid-19 is found in the group of people in the crowded rooms such as restaurant and fitness centre without adequate ventilation.7 the fast spread of the virus results in fast growing of new cases. worldwide, it was confirmed 172.558 positive cases and 3.921 death on 30 june 2020.8 in indonesia, the first case was reported on 3 march 2020. it was then found 56.385 cases involving 24.806 healed cases and 2.876 death cases on 30 june 2020. in central java, it was confirmed 3.836 cases involv open access under cc-by-nc-sa share alike 4.0 elyana sri sulistyowati, et al.: risk factors of covid-19 confirmed died patients in dr. kariadi hospital 3 ijtid, p-issn 2085-1103, e-issn 2356-0991 it is a retrospective study. the population involves all covid-19 confirmed patients at dr. kariadi hospital. research samples involve all covid-19 confirmed patients both healed and died patients from march to june 2020. the inclusion criteria involved covid-19 confirmed g patients both died and healed patients confirmed based on swab test. the exclusion criteria involved patients both in inpatient and outpatient settings still waiting for swab test results. data was gathered from screening forms filled by patients or health practitioners interviewing patients on arrival (secondary data). data was analysed with chi-square with confidence interval of 95% using spss version 25. this research has gone through the review stage and has received approval from the health research ethics committee dr. kariadi hospital no. 561 / ec / kepk-rsdk / 2020. covid-19 confirmed patients are 277 patients involving 59 died and 218 healed cases from march to june 2020. research findings are shown at table 1. bivariate test shows sixteen risk factors of covid-19 confirmed died patients involving age (p= 0.000; or= 8.803; 95% ci 3.98219.462), entrepreneur (p= 0.041; or=14.894; 95% ci 1.12-198.65), farmer/trader (p= 0.029; or= 25.625; 95% ci 1.40-469.25), contact history (p= 0.000; or= 12.923; 95% ci 6.16327.097), fever (p= 0.000; or=4.877; 95% ci 2.647-8.984), dyspnea (p=0.000; or= 17.018; 95% ci 8.523-33.977), cough (p= 0.009; or= 2.178; 95% ci 1.205-3.935), lethargic (p=0.010; or= 2.282; 95% ci 1.205-4.323), cold (p= 0.002; or= 0.180; 95% ci 0.0540.600), diabetes (p=0.000; or= 9.767; 95% ci 3.932-24.263), copd (p= 0.001; or= 6.360; 95% ci 2.164-18.690), hypertension (p= 0.043; or= 2.436; 95% ci 1.008-5.887), cancer (p=0.001; or= 9.647; 95% ci 2.413-38.579), heart disease (p= 0.000; or= 12.226; 95% ci 2.4-62.294), neurological disorders (p=0.008; or= 6.057; 95% ci 1.650-22.232), and immune disorders (p=0.031; or= 1.625; 95% ci 1.186-113.899). in confirmed died patients we found that the cause of death was mostly caused by respirato ry failure 40 cases (67.80%) and the rest caused by cardiovascular / mod 19 cases (32.20%). furthermore, the days to develop critical all for deadly patient are 6-7 days with the shortest day that covid-19 cases are increasing during winter where 25-hidroxyvitamin d concentration is low in human body.15 conversely, countries in the south hemisphere with dry season tend to have low case numbers. race is also considered having relationship with percentage of covid-19 related cases and death cases.16 nutritional status data were secondar y data based on kartu menuju sehat/ children growth chart (kms/cgc) obtained from kokar health center. nutritional status data based on anthropometric measurements of body weight (kg) for age (month). subjects recruited in four categories nutritional status, in line with the proportion in the population, i.e. 7.7% severely underweight, 19.2% underweight, 70.5% normal and 2.6% overweight. dr. kariadi hospital is a referral hospital in central java having role and responsibilities for handling covid-19 patients involving those who are suspected, having history of close physical contact with patients, confirmed covid-19 patients and healed patients. the number of covid-19 patients is predicted to keep increasing and end in uncertain time. the higher death cases may occur as more cases are found, particularly when several risk factors present. covid-19 can impact significantly human life sustainability as supported by a study by trias-llimós dan bilal found covid19 lowers life expectancy 1.9 and 1.6 years for male and female respectively in madrid, spain.17 it encourages authors to investigate risk factors of covid-19 confirmed dead patients in dr. kariadi hospital. materials and methods results and discussion open acces under cc-by-nc-sa share alike 4.0 risk factors total (n=277) number of death cases (n=59) number of healed cases (n=218) pvalue or ( 95% ci) age ≥ 60 ≤ 59 38.99±14.954 32 (11.6) 245 (88.4) 53.27±13.249 20 (33.9) 39 (66.1) 35.12±12.925 12 (5.5) 206 (94.5) 0.000 8.803 (3.982-19.462) gender male female 145 (52.3) 132 (47.7) 36 (61.0) 23 (39.0) 109 (50.0) 109 (50.0) 0.133 occupation civil servant private sectors entrepreneur farmer/trader labour/driver/odd jobs unemployed/house wife/student n/a 169 (61.7) 30 (10.9) 23 (8.4) 8 (2.9) 6 (2.2) 38 (13.9) 3 11 (19.0) 7 (12.1) 12 (20.7) 7 (12.1) 5 (8.6) 16 (27.6) 1 158 (73.1) 23 (10.6) 11 (5.1) 1 (0.5) 1 (0.5) 22 (10.2) 2 0.922 0.265 0.041 0.029 0.199 0.126 14.894 (1.12-198.65) 25.625 (1.40-469.25) traveling history yes no n/a 30 (11.5) 232 (88.5) 15 7 (13.2) 46 (86.8) 6 23 (11.0) 186 (89.0) 9 0.813 contact history yes no n/a 180 (70.6) 75 (29.4) 22 12 (25.0) 36 (75.0) 11 168 (81.2) 39 (18.8) 11 0.000 12.923 (6.163-27.097) symptoms fever dyspnea cough lethargic nauseous vomit diarrhea shore throat headache cold loss of consciousness 97 (35.0) 64 (23.1) 132 (47.7) 60 (21.7) 28 (10.1) 22 (7.9) 58 (20.9) 39 (14.1) 53 (19.1) 3 (1.1) 38 (64.4) 40 (67.8) 37 (62.7) 20 (33.9) 9 (15.3) 8 (13.6) 7 (11.9) 6 (10.2) 3 (5.1) 2 (3.4) 59 (27.1) 24 (11.0) 95 (43.6) 40 (18.3) 19 (8.7) 14 (6.4) 51 (23.4) 33 (15.2) 50 (22.9) 1 (0.5) 0.000 0.000 0.009 0.010 0.139 0.072 0.053 0.330 0.002 0.054 4.877 (2.647-8.984) 17.018 (8.523-33.977) 2.178 (1.205-3.935) 2.282 (1.205-4.323) 0.180 (0.054-0.600) symptom duration (days) ≥ 6 ≤ 5 5.82 ± 6.899 55 (34.2) 106 (65.8) 5.63 ± 5.622 17 (29.3) 41 (70.7) 5.91 ± 7.502 38 (36.9) 65 (63.1) 0.330 comorbidities diabetes copd hypertension cancer heart disease neurological disorders immune disorders pregnancy/childbirth chronic kidney disease chronic liver disease others 24 (8.7) 15 (5.4) 24 (8.7) 10 (3.6) 8 (2.9) 10 (3.6) 4 (1.4) 6 (2.2) 1 (0.4) 2 (0.7) 6 (2.2) 16 (27.1) 9 (15.3) 9 (15.3) 7 (11.9) 6 (10.2) 6 (10.2) 3 (1.1) 1 (1.7) 0 (0.0) 0 (0.0) 1 (1.7) 8 (3.7) 6 (2.8) 15 (6.9) 3 (1.4) 2 (0.9) 4 (1.8) 1 (0.5) 5 (2.3) 1 (0.5) 2 (0.9) 5 (2.3) 0.000 0.001 0.043 0.001 0.000 0.008 0.031 0.779 0.602 0.460 0.779 9.767 (3.932-24.263) 6.360 (2.164-18.690) 2.436 (1.008-5.887) 9.647 (2.413-38.579) 12.226 (2.4-62.294) 6.057 (1.650-22.232) 11.625 (1.186-113.899) 4 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 1-8 ijtid, p-issn 2085-1103, e-issn 2356-0991 day 0 days and the longest day 28 days. treatment given to covid-19 confirmed patients consists of non-pharmacological and pharmacological therapy. non-pharmacological therapy includes chest x-ray examination, monitoring for signs such as tachypnea, oxygen saturation, lymphopenia, progressive crp and progressive lactic acidosis, and management of f critical cases, respiratory failure, hypoxemia and ards. pharmacological therapy are giving vitamin c, vitamin b1, zinc, azithromycin, antiniotics according to clinical conditions, chloroquine sulfate, hydroxycortisone injection, and antivirals (favipiravir, umifenovir, remdesivir, and oseltamivir). table 1. risk factors of covid-19 confirmed death and healed patients open access under cc-by-nc-sa share alike 4.0 elyana sri sulistyowati, et al.: risk factors of covid-19 confirmed died patients in dr. kariadi hospital 5 ijtid, p-issn 2085-1103, e-issn 2356-0991 discussion the research results are showed sixteen risk factors of covid-19 confirmed died patients involving age, occupation (entrepreneur, farmer/trader), contact history, symptoms (fever, dyspnea, cough, lethargic, cold), comorbidities (diabetes, copd, hypertension, cancer, heart disease, neurological disorders and immune disorders) (p<0.05). meanwhile, gender, traveling history and duration of symptoms were not risk factors for death in covid-19 confirmed patients (p>0.05). age is one of the death-contributing factors among covid-19 confirmed patients especially in the older age group. onder et al.18 found a higher case fatality rate in the age group over 80 years in italy and china (20.2% and 14%) compared to other age groups. nearly all studies have found a different mean age in patients with confirmed covid-19. zhou et al.19 found mean death ages of covid-19 patients is 69 years (63-76 years) and heightened along with age progression (p= 0.0043; or= 1.10; 95 % ci 1.03-1.17). harlem20 found most covid-19 positive cases in the age group of 25-44 years (28% and 31% respectively) and 45-64 years (26% and 27% respectively) in the high case country group (n= 178.469) and in the medium case country group (n= 178.196). nie et al.21 found mean ages of covid-19 confirmed patients is 43+15.09 years. they also found a relationship between ages and covid-19 severity (p= 0.003; or=:1.026; 95% ci 1.009-1.043). lai et al.22 state characteristic of the first 100 covid-19 cases in hong kong with the greatest proportions are among those aged 45 years (76%) and is increasing along with age progression (p<0.001). gemes et al.23 predicted one out of five people in sweden are at risk of severe covid-19 known from prognostic factors aged older than 70 years (14.1%). this study found most covid-19 confirmed patients are male, but it does not affect the numbers of death cases. these findings are similar with a study by nie et al. stating that most covid-19 patients are male accounting for 377 people (56.2%) and this result is not statistically significant (p>0.05).21 it is also in line with who that the percentage infection distribution in male is greater than female (51% vs 47%). who states the caused by different female against viruses and infections.24 alobuia et al. found female respondents were at least 85% more likely to have high practice scores compared to males (p < 0.001) in action against covid-19.25 the risky occupation during this study is farmer/trader and entrepreneur. in many cases, farmers are traders too. these findings are similar with that found in henan, china showing that the greatest cases occurred among farmers (21.2%) and labours (15.2%). death cases among farmers are around 0.3%.21 mutambudzi et al. categorize the entrepreneur as other essential workers that had a higher risk of severe covid-19 (rr= 1.60; 95% ci 1.052.45).26 furthermore, national statistics state that occupations demanding direct interaction with many people is very risky. farmers/traders and entrepreneur are types of occupations demanding direct interaction with many people without knowing whether they are infected by covid-19 or not.27 contact history in this study is defined as direct contact with a suspected, probable or confirmed covid-19 patients. findings show great number of died patients without any contact history. conversely, healed patients are found to have greater contact history. centers for disease control and prevention (cdc) stipulates close contact as a risk factor mainly among people living in the same house with a confirmed case without physical distancing.28 a study by nie et al. found a relationship between direct contact with infected patients and covid19 severity (p= 0.039; or= 0.456; 95% ci 0.213-0.976). in addition, nie et al. found visitation to crowded places such as hospitals and traditional markets augment positive cases. however, the sources of contagion of some covid-19 positive cases are still unknown.21 this study found the presence of symptoms serves as risk factors of covid-19. confirmed death cases show more symptoms than healed cases. from ten symptoms, seven symptoms show a higher percentage in death cases than healed that is fever (64.4 vs 27.1), dyspnea (67.8 vs 11.0), cough (62.7 vs 43.6), lethargic (33.9 vs 18.3), nauseous vomit (15.3 vs 8.7), diarrhea (13.6 vs 6.4), and loss of consciousness (3.4 vs 0.5). sanyaolu et al. showe open access under cc-by-nc-sa share alike 4.0 6 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 1–8 ijtid, p-issn 2085-1103, e-issn 2356-0991 showed the percentage of common symptoms similar with this study, that is fever (88.8%), dry cough (68%) and fatigue (33%), productive cough (28.5%), sob (17%), muscle pain (14.4%), sore throat (11.4%), headache (10.2%), diarrhea (4.4%), nausea and vomiting (4.1%), rhinorrhea (3.2%), abdominal pain (0.16%), and chest pain (0.11%).29 the high case of patients with symptoms is tightly related with ages and comorbidities. the risk for diseases severity increases along with age progression resulting in paediatrics tend to show no symptom (asymptomatic) compared with adults.24 although byambasuren et al. found 6-41% asymptomatic cases,30 but qu et al. found some symptoms such dyspnea (p<0.001), shortness of breath (p<0.01) and chest distress (p<0.05) were correlated with death.31 this study found no difference in symptom duration between death cases and healed cases. this indicates that the duration of symptoms is not a risk factor for death in confirmed covid19 patients. however, the duration of symptom in this study was about 6 days, both in death cases (0 to 12 days) and healed cases (2 to 13 days). this study similar with sanyaolu et al. found symptoms of covid-19 appear 5 days (2 to 14 days).29 the presence of comorbidities can worsen patient conditions particularly those indicated for covid-19. zhou et al. found 91 patients (48%) have comorbidities and significantly correlated to covid-19 related death (p<0.5). the comorbidities in death cases and healed cases are hypertension (48% vs 23%; p=0.0008), diabetes (31% vs 14%; p=0.0051), coronary heart disease (24% vs 1%; p=<0.0001), chronic obstructive lung disease (7% vs 1%; p=0.047), chronic kidney disease (4% vs 0%; p=0.024), and other (20% vs 8%; p=0.016).19 harlem found three most common chronic diseases involving hypertension (33% and 25 % respectively), obesity (28% and 15% respectively) and diabetes (15% and 8% respectively).20 not only adults, comorbidities also can be fatal among paediatrics. oualha et al. found 19 paediatrics (70%) aged between 1 month and 18 years with comorbidities (neurological, respiratory, and sickle cell disease conclusion disease). of five died paediatrics, two were died as a result of comorbidities.32 a meta-analysis by patel found 21% paediatrics has comorbidities such as asthma, immunosuppression and cardiovascular disease. the mortality rate of children that were hospitalized with covid-19 was 0.18%.33 there are so many risk factors that are likely to contribute to the occurrence of death in confirmed covid-19 patients while the risk factors examined in this study were only a small part. this is a limitation of this study. apart from the risk factors in this study, other risk factors such as education level, smoking behavior, nutritional intake, physical activity, socioeconomic, body mass index (bmi) category, lifestyle-related factors (alcohol consumption) and other factors needs to be researched. almost no confirmed cause of death for covid-19 patients who died was caused by only one factor. this is the same as that found by sanyaolu et al. that older patients with covid-19, especially those 65 years old and above, who have comorbidities, are more likely to develop a more severe course and increased admission rate into the intensive care unit (icu) and mortality from the covid-19 disease.29 conflict of interest the authors declare that they have no conflict of interest. age, occupation (entrepreneur and farmer/trader), contact history, symptoms (fever, dypsnea, cough, lethargic and cold), and comorbidities (diabetes, copd, hypertension, cancer, heart disease, neurological disorders, and immune disorders) were risk factors of covid-19 confirmed died patients in dr. kariadi hospital. meanwhile, gender, traveling history and duration of symptoms were not risk factors for death in covid-19 confirmed patients in dr. kariadi hospital. adequate handling is needed to prevent death in patients with confirmed covid-19 who have risk factors. open access under cc-by-nc-sa share alike 4.0 acknowledgement the authors are grateful for cooperation of head and all staff of centre of public health in kokar, and to all local authorities that facilitated this study. references elyana sri sulistyowati, et al.: risk factors of covid-19 confirmed died patients in dr. kariadi hospital 7 ijtid p-issn 2085-1103, e-issn 2356-0991 1. centers for disease control and prevention (cdc). novel coronavirus (2019-ncov) [internet]. 2020. [cited 2020 july 9] 2. peeri nc, shrestha n, rahman ms, zaki r, tan z, bibi s, et al. the sars. mers and novel coronavirus (covid-19) epidemics. the newest and biggest global health threats: what lessons have we learned? international journal of epidemiology. 2020; 1–10. doi: 10.1093/ije/dyaa033 3. kahn lh. commentary on: the sars. mers and novel coronavirus (covid-19) epidemics. the newest and biggest global health threats: what lessons have we learned? a one health approach to coronaviruses. international journal of epidemiology. 2020; 1–3 doi: 10.1093/ije/dya a071 4. the health ministry of indonesia. the decree of health ministry no hk.01.07/menkes/413/2020 about guidance for prevention of coronavirus disease 2019 (covid-19). jakarta; 2020 5. zhou f, yu t, du r, fan g, liu y, liu z. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan. china: a retrospective cohort study. lancet. 2020; 395(10229):1054-62. epub 2020/03/15 6. susilo a, rumende mc, pitoyo cw, santoso wd, yulianti m, herikurniawan, sinto r et al. coronavirus disease 2019: a literature review. jurnal penyakit dalam indonesia. 2020; 7(1): 45-67. doi: 10.7454/jpdi.v7i1.415 7. who. transmision of sars-cov-2: implication toward infection prevention awareness [internet]. 2020. [cited 2020 july 22]. available from: https://www.who.int/docs/defaultsource/searo/indone sia/covid19/transmisi-sars-cov-2implikasiuntuk terhadap-kewaspadaan-pencegahan-infeksi-pernya taan-keilmuan.pdf?sfvrsn=1534d7df_4 8. who. who coronavirus disease (covid-19) dashboard [internet]. 2020. [cited 2020 juli 9]. available from: https://covid19.who.int/ accessed on 9 juli 2020 9. health ministry. covid-19 cases in indonesia dashboard [internet]. 2020. [cited 2020 july 21]. available from: https://www.kemkes.go.id/ article/view/20031900002/dashboard-data-kasuscovid-19-di-indonesia.html 10. stand f, jöckel k, stang a. covid‑19 and the need of targeted inverse quarantine. european journal of epidemiology. 2020; 35:339–340. doi: 10.1007/s10654020-00629-0 11. du z, xu x, wu y, wang l, cowling bj, meyers al. serial interval of covid-19 among publicly reported confirmed cases. emerging infectious diseases. 2020; 26 (6): 1341-1342. doi: 10.3201/eid2606.200357 12. the health ministry of indonesia. coronavirus disease (covid-19) prevention and control guidance. jakarta: general directorate of disease prevention and control; 2020 13. guo rf. a flaw on a meta-analysis of smoking and the severity of covid-19: the association should have been endorsed. journal of public health. 2020; 42(3):653– 654. doi: 10.1093/pubmed/fdaa083 14. who. scientific brief: smoking and covid-19 [internet].2020. [cited 2020 july 22]. available from: https://www.who.int/newsroom/commentaries/detail/smo king-and-covid-19 15. ardiaria m. the role of vitamin d for influenza and covid-19 prevention. jnh (journal of nutrition and health). 2020; 8(2): 79-85.doi: 10.14710/jnh.8.2. 2020.79-85 16. mahajan uv, larkins-pettigrew m. racial demographics and covid-19 confirmed cases and deads: a correlational analysis of 2886 us counties. journal of public health. 2020; 1–4. doi:10.1093/pubmed/fdaa070 17. trias-llimós s, bilal u. impact of the covid-19 pandemic on life expectancy in madrid (spain). journal of public health. 2020; 1–2. doi: 10.1093/pubmed /fdaa087 18. onder g, rezza g, brusaferro, s. case-fatality rate and characteristics of patients dying in relation to covid19 in italy. jama. 2020; e1. doi:10.1001/jama .2020.4683 19. zhou f, yu t, du r, fan g, liu y, liu z et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan. china: a retrospective cohort study. lancet. 2020; 395: 1054-1062. doi: 10.1016/ s0140-6736(20)30566-3 20. harlem g. descriptive analysis of social determinant factors in urban communities affected by covid-19. journal of public health. 2020; 1–4. doi:10.1093/pub med/fdaa078 21. nie y, li j, huang x, guo w, zhang x, ma y et al. epidemiological and clinical characteristics of 671 covid-19 patients in henan province. china. international journal of epidemiology. 2020; 1–11. doi: 10.1093/ije/dyaa081 22. lai ckc, ng rwy, wong mcs, chong kc, yeoh yk, chen z et al. epidemiological characteristics of the first 100 cases of coronavirus disease 2019 (covid-19) in hong kong special administrative region. china. a city with a stringent containment policy. international journal of epidemiology. 2020; 1–10. doi: 10.1093/ije /dyaa106 open access under cc-by-nc-sa share alike 4.0 https://dx.doi.org/10.1093/ije/dyaa033 http://dx.doi.org/10.7454/jpdi.v7i1.415 https://www.who.int/docs/defaultsource/searo/indonesia/covid19/transmisi-sars-cov-2implikasiuntuk%20terhadap-kewaspadaan-pencegahan-infeksi-pernya%20taan-keilmuan.pdf?sfvrsn=1534d7df_4 https://www.who.int/docs/defaultsource/searo/indonesia/covid19/transmisi-sars-cov-2implikasiuntuk%20terhadap-kewaspadaan-pencegahan-infeksi-pernya%20taan-keilmuan.pdf?sfvrsn=1534d7df_4 https://www.who.int/docs/defaultsource/searo/indonesia/covid19/transmisi-sars-cov-2implikasiuntuk%20terhadap-kewaspadaan-pencegahan-infeksi-pernya%20taan-keilmuan.pdf?sfvrsn=1534d7df_4 https://www.who.int/docs/defaultsource/searo/indonesia/covid19/transmisi-sars-cov-2implikasiuntuk%20terhadap-kewaspadaan-pencegahan-infeksi-pernya%20taan-keilmuan.pdf?sfvrsn=1534d7df_4 https://covid19.who.int/ https://covid19.who.int/ https://doi.org/10.1007/s10654-020-00629-0 https://doi.org/10.1007/s10654-020-00629-0 https://doi.org/10.3201/eid2606.200357 https://doi.org/10.1093/pubmed/fdaa083 https://www.who.int/newsroom/commentaries/detail/smoking-and-covid-19 https://www.who.int/newsroom/commentaries/detail/smoking-and-covid-19 https://doi.org/10.14710/jnh.8.2.2020.79-85 https://doi.org/10.14710/jnh.8.2.2020.79-85 https://doi.org/10.1016/s0140-6736(20)30566-3 https://doi.org/10.1016/s0140-6736(20)30566-3 ijtid, p-issn 2085-1103, e-issn 2356-0991 8 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 1–8 with potential coronavirus disease 2019 (covid-19) exposures: geographic risk and contacts of laboratory-confirmed cases [internet]. 2020. [cited 2020 july 23]. available from: https://www.cdc.gov/ coronavirus/2019-ncov/php/risk-assessment.html 29. sanyaolu a, okorie c, marinkovic a, patidar r, younis k, desai p, hosein z, padda i, mangat j, altaf m. comorbidity and its impact on patients with covid-19. sn comprehensive clinical medicine. 2020. doi: 10.1007/s42399-020-00363 30. byambasuren o, cardona m, bell k, clark j, mclaws m-l, glasziou p. estimating the extent of true asymptomatic covid-19 and its potential for community transmission: systematic review and meta-analysis.medrxiv. 2020. doi:10.1101/2020.05.10. chen l, wang d, pei b. a quantitative exploration of symptoms in covid-19 patients: an observational cohort study. int. j. med. sci. 2021; 18(4): 1082-1095. doi: 10.7150/ijms.53596 23. gémes k, talbäck m, modig k, ahlbom a, berglund a, feychting aa et al. burden and prevalence of prognostic factors for severe covid‑19 in sweden. european journal of epidemiology. 2020;35:401–409. doi: https://doi.org/10.1007/s10654-020-00646-z 24. who. advocacy brief: gender and covid-19 [internet]. 14 may 2020. [cited 2020 july 22]. available from: https://www.who.int/publications/i/item/gender-andcovid-19 25. alobuia wm, dalva-baird np, forrester jd, bendavid e, bhattacharya j, kebebew e. racial disparities in knowledge. attitudes and practices related to covid19 in the usa. journal of public health. 2020; 1–9. doi:10.1093/pubmed/fdaa069 26. mutambudzi m, niedwiedz c, macdonald eb, leyland a, mair f, anderson j, celis-morales c, cleland j, forbes j, gill j, hastie c, ho f, jani b, mackay df, nicholl b, o’donnell c, sattar n, welsh p, pell jp, katikireddi sv, demou e. occupation and risk of severe covid-19: prospective cohort study of 120 075 uk biobank participants. occup. environ. med. 2020;0:1–8. doi:10.1136/oemed-2020-106731 27. office for national statistics. coronavirus (covid-19) related deaths by occupation, before and during lockdown, england and wales: deaths registered between 9 march and 30 june 2020. statistical bulletin. 2020;1-20 28. prevention cfdca. interim us guidance for risk assessment and public health management of persons wit open access under cc-by-nc-sa share alike 4.0 20097543 31. qu g, chen j, huang g, zhang m, yu h, zhu1 h, 32. oualha m, bendavid m, berteloot l, corsia a, lesage f, vedrenne m et al. severe and fatal forms of covid-19 in children. archives de pe´ diatrie. 2020; 27: 235–238. doi: 10.1016/j.arcped.2020.05.010. 33. patel na. pediatric covid-19: systematic review of the literature. am j otolaryngol. 2020; 41:1-9. doi: 10.1016/j.amjoto.2020.102573 https://www.cdc.gov/%20coronavirus/2019-ncov/php/risk-assessment.html https://www.cdc.gov/%20coronavirus/2019-ncov/php/risk-assessment.html https://doi.org/10.1007/s42399-020-00363 https://doi.org/10.1016/j.arcped.2020.05.010 https://doi.org/10.1007/s10654-020-00646-z https://www.who.int/publications/i/item/gender-and-covid-19 https://www.who.int/publications/i/item/gender-and-covid-19 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 55 vol. 6. no. 3 september–december 2016 research report the immunostimulan potential of tenggulun (protium javanicum) leaves towards t cell cd4+ and ifnγ secretion on pbmc chicken andi jayawardhana1a, heni puspitasari 2, dewa ketut meles 3 , setiawan koesdarto 4 1 nursery department faculty of health sciences merdeka university 2 toxoplasma study group institute of tropical disease universitas airlangga 3 basic medicine veteriner department faculty of veterinary medicine universitas airlangga 4 parasitology veteriner department faculty of veterinary medicine universitas airlangga a corresponding author: andi.jayawardhana@gmail.com abstract one of the plants with immunostimulant activity is tenggulun leaves which contain of flavonoid, like terpenoid. the aim of this research is to find the potential of tenggulun’s leaves extract to have the immunostimulant activities. the potential of immunostimulant activity is identified by the increasing the amount of t-cell cd4+ expression and ifnγ secretion. the research method is conducted through cultured chicken pbmc which is infected by nd virus; it is then treated with tenggulun’s leaves extract with immunostimulant. the result of immunocitochemistry examination cd4+ secretion on pbmc cultures shows how tenggulun is significantly different from the control in the secretion cd4+. the 10µg of tenggulun extract can modulate the t cell cd4+ secretion 68.8±0.83. it is significantly different from k (control) (p<0,05) and treatment group k+, p0, and p1. the examination of ifnγ level using elisa from tenggulun leaves extract of 10µg doses were inoculated after being infected by nd virus contained immonostimulant potential in increasing the secretion of ifnγ 120.91±6.44. it is significantly different from k-, k+, and p1, yet not significantly different from p0. the content of terpenoid can increase ifnγ secretion on the macrofag cells culture and limfosit cells. keywords: protium javanicum, imunostimulan, newcastle disease virus, cd4+, ifnγ abstrak salah satu tanaman yang memiliki aktivitas imunostimulan adalah daun tenggulun yang mengandung flavanoid seperti terpenoid. tujuan dari penelitian ini adalah untuk menemukan potensi ekstrak daun tenggulun memiliki aktivitas imunostimulan. potensi aktivitas imunostimulan dengan meningkatkan jumlah ekspresi sel t cd4 + dan sekresi ifnγ sekresi. metode penelitian dengan memberikan kultur pbmc ayam berbudaya dengan virus nd dan ekstrak daun tenggulun sebagai imunostimulan. hasil pemeriksaan immunocitochemistry sekresi cd4 + pada kultur pbmc yang diberikan ektrak daun tenggulun berbeda nyata dengan kontrol. ekstrak 10µg dari tenggulun mampu memodulasi sel t cd4 + sekresi 68,8 ± 0,83 yang secara signifikan berbeda dari k (kontrol) (p <0,05) dan kelompok perlakuan k +, p0, dan p1. pemeriksaan sekresi ifnγ menggunakan metode elisa dari ektrak daun tenggulun daun dosis 10µg yang diinokulasi setelah terinfeksi virus nd memiliki potensi immonostimulant dalam meningkatkan sekresi ifnγ yaitu 120,91 ± 6,44 yang secara signifikan berbeda dengan k-, k +, dan p1 namun tidak berbeda nyata dengan p0. kandungan terpenoid dapat meningkatkan ifnγ sekresi pada kultur sel macrofag dan sel limfosit. kata kunci: protium javanicum, imunostimulan, newcastle disease virus, cd4+, ifnγ 56 indonesian journal of tropical and infectious disease, vol. 6. no.3 september–december 2016: 55–58 introduction plants are the sources of herbal ingredients with various benefits, including for medicines against various diseases. disease prevention by increasing the body’s immune system maintenance is highly important, because high level of immunity enables us to face the attack of foreign substances, like pathogenic microorganisms. one way to maintain immune system is by administering immunomodulatory compound which can improve the immune function of the human body.1 immunostimulatory is a compound capable of boosting the immune system and reactivate immune system in various ways, such as by increasing the number and activity of t cells, nk cells and macrophages and by releasing interferon and interleukin.2 the use of immunostimulatory therapy is rather difficult as it is mostly imported and expensive. the incidence of immunosuppression is a problem mostly found a chicken farm. the cause is divided into two factors, namely non-infectious factors and infectious factors. the cases of immunosuppression in a farm are caused by one of those factors or combination of both factors. the combination of both factors may cause more severe immunosuppression. the research on the prevention of immunosuppression factor in farms and its application through vaccination and biosecurity to improve livestocks’ livelihood are conducted as a farming industry strategy nowadays. biosecurity aims to prevent disease and consists of isolation, traffic control, and sanitation.3 a research on the increase of cytokine secretion can be an alternative to cure various diseases through the immune system. one of the strategies to increase the expression of cytokines is to use herbal ingredients. tenggulun (protium javanicum) is used because it has certain bioactive compounds which influence the movement of the immune system or immunomodulator. the in-vitro and in-vivo research on medicinal plants have shown its potential to modulate cytokine secretion of various kinds, one of which is interferon gamma. divided into two major types, type i ifns are induced and act effectively in responses against viruses: ifn-α is secreted mainly by leucocytes, while ifn-β is produced by fibroblasts. type ii interferon, now referred to as ifn-γ, is synthesized mostly by t lymphocytes and nk cells after this cell is activated by immune and inflammatory stimuli, rather than by viral infection.4 the materials from the plants can modulate the performance of cytokines and spark a variety of materials from the original plants. they can also have the function of a spur of the various components of the immune system non-specific (phagocytes, nk cells) and specific immune system (the proliferation of t cells and b cells which produce antibodies) to improve the healing for various infectious diseases.5 tenggulun (protium javanicum) can be used as anti-inflammatory drugs. the plant is known from the experimental pharmacological research which explains that steroids and terfenoid contained in tengggulun can act as anti-bacteria, such as escherichia coli and staphylococcus aures.6 the carrageenan-induced peritonitis is known as acute inflammatory model in which fluid extravasation and leukocyte migration are involved in the inflammatory response. the anti-inflamatory activities of rat’s edema are induced by karagenan.7 the inflammatory process consists of diverse physiological and pathological activities.8 the anti-inflammatory activity and increased capacity can provide protuim hepthaphyllum in male mice.8 successful group issolation of terfenoid terpinene which is contained in the volatile constituent of protium heptaphyllum.9 terfenoid terpinene group can boost the immune response in-vivo and in-vitro.8 material and method the study group is divided into five groups, namely control group (k); meniran comparator control (k+); nd (newcastle disease) infection control (p0); treatment one (p1); and treatment two (p2). while the division of the treatment groups follows the control groups: pbmc (peripheral blood mononuclear cell) cultures are inoculated by 5μl of 0.01% (k) dmso (dimethile sulfoxide); pbmc cultures are exposed to newcastle disease virus which is inoculated by meniran extract of 5g (k+); p0: pbmc cultures are exposed to newcastle disease virus then inoculated by 0.01% dmso; p1: infection control group, pbmc cultures are infected by newcastle disease virus which is inoculated by 5μl of 0.01% dmso and tenggulun leaf extract of 5g; p2: pbmc cultures are infected by newcastle disease virus which is inoculated by 5μl of 0.01% dmso and tenggulun leaf extract (protium javanicum) of 10 mg. the production of pbmc cultures is conducted by taking peripheral blood aseptically from the brachial vein using a syringe of 15 ml of 3 broiler chickens aged 35 days. tenggulun dried leaf powder is then extracted using methanol. viral infections are performed with the suspension taken from newcastle disease virus titer of 1.1 x 105 eid50 as 50μl / ml per wells. it is subsequently inoculated cautiously in pbmcs cultured cells with a sterile pipette. the examination of t-cell expression is accomplished using immunocytochemistry techniques, while the ifnγ examination is conducted using elisa. result and discussion cell expression t cd4+ the immunocytochemistry assay results from the observation using fluorescent microscopy t cell expression of each control and treatment group are shown in figure 1. the potential of methanol extract from tenggulun leaves through the activation of specific immune system can be observed by t cell expression. the average and 57jayawardhana, et al.,: the immunostimulan potential of tenggulun (protium javanicum) the immunocytochemistry assay results from the observation using fluorescent microscopy t cell expression of each control and treatment group are shown in figure 1. figure 1 . the expression results in in pbmc cells per treatment. it is observedusing a fluorescent microscope magnification of 400x. it positively glowsgreen and is pointed by black arrow ( ). the potential of methanol extract from tenggulun leaves through the activation of specific immune system can be observed by t cell expression. the average and standard deviation of tenggulun leaves’ extracts are shown in percentage using immunocytochemistry method (table 1). table 1. the average and standard deviation of tenggulun leaves’ extracts (protium javanicum) on the expression and secretion of ifnγ no. treatment expression in % ifnγ levels in pg/ml 1 pbmc + dmso (k-) 5,60a ± 1,51 74,76a ± 10,99 2 pbmc + nd + 5 µ g meniran (k+) 25,60b ± 5,17 93,51b ± 18,06 3 pbmc + nd + dmso (p0) 12,40c ± 5,36 110,47 bc ± 1,62 4 pbmc + nd + dmso + tenggulun 5 µg (p1) 55,80d ± 1,64 100,25b ± 0,93 5 pbmc + nd + dmso + 68,80e ± 0,83 120,91c ± 6,44 tenggulun 10 µg (p2) description: different superscripts convey significantly different meaning the expression at the second day after adding the methanol extract for tenggulun leaves are analyzed using anova and duncan test (p ≤ 0.05). the results indicate that p2 groups record higher expression than the control group (k), p0, and p1. different p2 groups also express significantly higher than the control group (k), p0, and p1. the statistic results state that the secretion of cd4+ group which is not given immunostimuant and not inoculated by nd virus is lower than the treatment group (k+, p0, p1, p2). it is also stated that a viral infection of nd and extract meniran and tenggulun leaves can increase the secretion of cd4+. the nd virus infection which can increase the specific adaptive imunity are mediated by t lymphocytes.10 the infection itself is an important factor in the development and protection of chickens against viruses. cmi will be stimulated by cd4+ lymphocytes and mhc ii. the nd virus infection in-vitro induced by nitric oxide on heterophile chicken.11 pbmc also showed that ifnγ that ifnß in mrna can be detected in macrophages. the discovery of drugs derived from plants which modulate the function of cd4+ and cd8+ are important for the potential therapeutic of compounds.12 the average treatments group (k+, p0, p1, p2) are expressing cd4+ higher than the control groups due to the influence of nd viruses and the immunocytochemistry assay results from the observation using fluorescent microscopy t cell expression of each control and treatment group are shown in figure 1. figure 1 . the expression results in in pbmc cells per treatment. it is observedusing a fluorescent microscope magnification of 400x. it positively glowsgreen and is pointed by black arrow ( ). the potential of methanol extract from tenggulun leaves through the activation of specific immune system can be observed by t cell expression. the average and standard deviation of tenggulun leaves’ extracts are shown in percentage using immunocytochemistry method (table 1). table 1. the average and standard deviation of tenggulun leaves’ extracts (protium javanicum) on the expression and secretion of ifnγ no. treatment expression in % ifnγ levels in pg/ml 1 pbmc + dmso (k-) 5,60a ± 1,51 74,76a ± 10,99 2 pbmc + nd + 5 µ g meniran (k+) 25,60b ± 5,17 93,51b ± 18,06 3 pbmc + nd + dmso (p0) 12,40c ± 5,36 110,47 bc ± 1,62 4 pbmc + nd + dmso + tenggulun 5 µg (p1) 55,80d ± 1,64 100,25b ± 0,93 5 pbmc + nd + dmso + 68,80e ± 0,83 120,91c ± 6,44 tenggulun 10 µg (p2) description: different superscripts convey significantly different meaning the expression at the second day after adding the methanol extract for tenggulun leaves are analyzed using anova and duncan test (p ≤ 0.05). the results indicate that p2 groups record higher expression than the control group (k), p0, and p1. different p2 groups also express significantly higher than the control group (k), p0, and p1. the statistic results state that the secretion of cd4+ group which is not given immunostimuant and not inoculated by nd virus is lower than the treatment group (k+, p0, p1, p2). it is also stated that a viral infection of nd and extract meniran and tenggulun leaves can increase the secretion of cd4+. the nd virus infection which can increase the specific adaptive imunity are mediated by t lymphocytes.10 the infection itself is an important factor in the development and protection of chickens against viruses. cmi will be stimulated by cd4+ lymphocytes and mhc ii. the nd virus infection in-vitro induced by nitric oxide on heterophile chicken.11 pbmc also showed that ifnγ that ifnß in mrna can be detected in macrophages. the discovery of drugs derived from plants which modulate the function of cd4+ and cd8+ are important for the potential therapeutic of compounds.12 the average treatments group (k+, p0, p1, p2) are expressing cd4+ higher than the control groups due to the influence of nd viruses and the immunocytochemistry assay results from the observation using fluorescent microscopy t cell expression of each control and treatment group are shown in figure 1. figure 1 . the expression results in in pbmc cells per treatment. it is observedusing a fluorescent microscope magnification of 400x. it positively glowsgreen and is pointed by black arrow ( ). the potential of methanol extract from tenggulun leaves through the activation of specific immune system can be observed by t cell expression. the average and standard deviation of tenggulun leaves’ extracts are shown in percentage using immunocytochemistry method (table 1). table 1. the average and standard deviation of tenggulun leaves’ extracts (protium javanicum) on the expression and secretion of ifnγ no. treatment expression in % ifnγ levels in pg/ml 1 pbmc + dmso (k-) 5,60a ± 1,51 74,76a ± 10,99 2 pbmc + nd + 5 µ g meniran (k+) 25,60b ± 5,17 93,51b ± 18,06 3 pbmc + nd + dmso (p0) 12,40c ± 5,36 110,47 bc ± 1,62 4 pbmc + nd + dmso + tenggulun 5 µg (p1) 55,80d ± 1,64 100,25b ± 0,93 5 pbmc + nd + dmso + 68,80e ± 0,83 120,91c ± 6,44 tenggulun 10 µg (p2) description: different superscripts convey significantly different meaning the expression at the second day after adding the methanol extract for tenggulun leaves are analyzed using anova and duncan test (p ≤ 0.05). the results indicate that p2 groups record higher expression than the control group (k), p0, and p1. different p2 groups also express significantly higher than the control group (k), p0, and p1. the statistic results state that the secretion of cd4+ group which is not given immunostimuant and not inoculated by nd virus is lower than the treatment group (k+, p0, p1, p2). it is also stated that a viral infection of nd and extract meniran and tenggulun leaves can increase the secretion of cd4+. the nd virus infection which can increase the specific adaptive imunity are mediated by t lymphocytes.10 the infection itself is an important factor in the development and protection of chickens against viruses. cmi will be stimulated by cd4+ lymphocytes and mhc ii. the nd virus infection in-vitro induced by nitric oxide on heterophile chicken.11 pbmc also showed that ifnγ that ifnß in mrna can be detected in macrophages. the discovery of drugs derived from plants which modulate the function of cd4+ and cd8+ are important for the potential therapeutic of compounds.12 the average treatments group (k+, p0, p1, p2) are expressing cd4+ higher than the control groups due to the influence of nd viruses and figure 1. the expression results in cd4+ in pbmc cells per treatment. it is observed using a fluorescent microscope magnification of 400x. it positively glows green and is pointed by black arrow (→). table 1. the average and standard deviation of tenggulun leaves’ extracts (protium javanicum) on the expression and secretion of ifnγ no. treatment cd4+ expression in % ifnγ levels in pg/ml 1 pbmc + dmso (k-) 5,60a ± 1,51 74,76a ± 10,99 2 pbmc + nd + 5 μg meniran (k+) 25,60b ± 5,17 93,51b ± 18,06 3 pbmc + nd + dmso (p0) 12,40c ± 5,36 110,47 bc ± 1,62 4 pbmc + nd + dmso + tenggulun 5 μg (p1) 55,80d ± 1,64 100,25b ± 0,93 5 pbmc + nd + dmso + tenggulun 10 μg (p2) 68,80e ± 0,83 120,91c ± 6,44 description: different superscripts convey significantly different meaning standard deviation of tenggulun leaves’ extracts are shown in percentage using immunocytochemistry method (table 1). the expression at the second day after adding the methanol extract for tenggulun leaves are analyzed using anova and duncan test (p ≤ 0.05). the results indicate that p2 groups record higher expression than the control group (k), p0, and p1. different p2 groups also express significantly higher than the control group (k), p0, and p1. the statistic results state that the secretion of cd4+ group which is not given immunostimulant and not inoculated by nd virus is lower than the treatment group (k+, p0, p1, p2). it is also stated that a viral infection of nd and extract meniran and tenggulun leaves can increase the secretion of cd4+. the nd virus infection which can increase the specific adaptive immunity are mediated by t lymphocytes.10 the infection itself is an important factor in the development and protection of chickens against viruses. cmi will be stimulated by cd4+ lymphocytes and mhc ii. the nd virus infection in-vitro induced by nitric oxide on heterophile chicken.11 pbmc also showed that ifnγ that ifnß in mrna can be detected in macrophages. the discovery of drugs derived from plants which modulate the function of cd4+ and cd8+ are important for the potential therapeutic of compounds.12 the average treatments group (k+, p0, p1, p2) are expressing cd4+ higher than the control groups due to the influence of nd viruses and their immunostimulant. the cell mediated immunity (cmi) response to poultry may be obtained by nd vaccination. th-1 cells and the expression of il-18 can be detected at the third day of post-vaccination inactivated nd. il-18 cells can induce the expression of cytokines il-4 and ifn-γ plays an important role in the immune response of th-1.13 active immunization or active vaccination aims to give a specific antigen to animals to form a protective immunity against the disease.14 the secretion levels of interferon gamma (ifnγ) t h e e x a m i n a t i o n r e s u l t s o f t h e p o t e n t i a l immunostimulatory tenggulun leaf’s extract (protium javanicum) against ifnγ secretion by elisa can be seen in table 5.1. the extract (protium javanicum) can increase the secretion of ifnγ. it is indicated by the lower secretion of ifnγ for negative control group (k) than for the positive control group (k+); the treatment groups (p0, p1 and p2) also indicates significantly different secretions (p <0.05) in which p2 group records the highest value of ifnγ secretion. the data analysis results for the secretion of ifnγ showed that the control group + (k+), p0, p1 and p2 has a value secretion higher than the group control(k), because nd virus inoculation can increase the secretion of ifnγ.11,15 among the highest valued treatment group for the secretion of ifnγ, the treatment group 2 (p2) shows that the administration of 10μg dose tenggulun leaf extract is the most effective to increase the secretion of ifnγ. the increased secretion of ifnγ may be caused by tenggulun leaves’ terpenoids as the antibacterial and anti-inflammatory compounds. terpenoids is a substance which modulates the immune system by signaling pathway nuclear transcription factor kappa b (nf-ĸb).8 the modulated pathways of nf-ĸb lines include cytokines, limphotoxin, tnfα, and ifnγ-ifnß. terpenoids flavonoids can increase ifnγ secretion in cultured macrophages and lymphocytes.3,16 the treatment of k+ (inoculated nd and extract meniran) with p1 (inoculated with nd virus and tenggulun leaf extracts of 5μg) are not significantly different in 58 indonesian journal of tropical and infectious disease, vol. 6. no.3 september–december 2016: 55–58 secreting ifnγ, because the competition between the plant extracts with the viral antigen induces similar apc cells (dendritic cells); thus, the extract’s effect of 5μg dose is not enough to modulate the secretion of ifnγ post virus infection.12,17 conclusion based on this study we can conclude that the leaf extract of 10μg tenggulun has higher immunomodulatory potential than the 5μg dose in increasing the expression and secretion of cd4 + ifnγ in pbmc cultures which are infected by nd virus. references 1. devagaran t, diantini a. senyawa immunomodulator dari tanaman. students e-journals. 2012;1(1):1–2. 2. tjay th, rahardja k. obat-obat penting khasiat, penggunaan dan efek sampingnya. edisi keenam. jakarta: pt. elex media komputindo; 2007. 3. jain m, ganju l, katiyal a, padwad y, mishra kp, chanda s, et al. effect of hippophae rhamnoides leaf extract against dengue virus infection in human blood-derived macrophages. phytomedicine. 2008 oct;15(10):793–9. 4. thomson aw, lotze mt. the cytokine handbook (fourth edition). fourth edi. academic press; 2003. 5. baratawidjaja k. penggunaan herbal medisin untuk imunostimulator dan kemopreventif. 2006; 6. sanjaya ima. isolasi dan identifikasi senyawa atsiri yang memiliki aktivitas antibakteri pada daun tenggulun (protium javanicum burm. f.). universitas udayana; 2002. 7. sukmajaya igp. isolasi dan uji aktivitas antiinflamasi minyak atsiri daun tenggulun (protium javanicum burm. f.). universitas udayana; 2012. 8. amaral mpm, braga fa v, passos ffb, almeida frc, oliveira rcm, carvalho aa, et al. additional evidence for the anti-inflammatory properties of the essential oil of protium heptaphyllum resin in mice and rats. lat am j pharm. 2009;28(5):775–82. 9. zoghbi m das gb, maia jgs, luz air. volatile constituents from leaves and stems of protium heptaphyllum (aubl.) march. j essent oil res. 1995 sep;7(5):541–3. 10. lambrecht b, gonze m, meulemans g, van den berg tp. asessment of the cell mediated immune respons in chicken by detection of chicken interferron gamma in respon to mitogen and recall newcastle disease viral antigen stimulation. avian pathol. 2004 jun;33(3):343–50. 11. ahmed ka, saxena vk, ara a, singh kb, sundaresan nr, saxena m, et al. immune response to newcastle disease virus in chicken lines divergently selected for cutaneous hypersensitivity. int j immunogenet. 2007 dec;34(6):445–55. 12. patel k. a review on herbal immunoadjuvant. int j pharm life sci. 2012;3(3):1568. 13. rahman mm, uyangaa e, eo sk. modulation of humoral and cellmediated immunity against avian influenza and newcastle disease vaccines by oral administration of salmonella enterica serovar typhimurium expressing chicken interleukin-18. immune netw. 2013 feb;13(1):34. 14. islam m, parvin m, akhter j, islam m, siddique m, rashid m. clinical evaluation of hyperimmune serum for the treatment of newcastle disease in indigenous layer birds. bangladesh journals online (banglajol). 2014 jun 10;24(1–2). 15. sick c, schneider k, staeheli p, weining kc. novel chicken cxc and cc chemokines. cytokine. 2000 mar;12(3):181–6. 16. cruz gvb, pereira pvs, patrício fj, costa gc, sousa sm, frazão jb, et al. increase of cellular recruitment, phagocytosis ability and nitric oxide production induced by hydroalcoholic extract from chenopodium ambrosioides leaves. j ethnopharmacol. 2007 apr 20;111(1):148–54. 17. neves-souza pc, azeredo el, zagne sm, valls-de-souza r, reis sr, cerqueira di, et al. inducible nitric oxide synthase (inos) expression in monocytes during acute dengue fever in patients and during in vitro infection. bmc infect dis. 2005 dec 18;5(1):64. 113 vol. 5. no. 5 may–august 2015 research report relationship between clinical manifestations and antibody serum in outbreaks anthrax dhani redhono sub division tropical medicine & infectious disease faculty of medicine, sebelas maret university, moewardi hospital email of corresponding author : dhani_ipd@yahoo.com abstract introduction: anthrax is a zoonotic disease that often affects the grass-eating animals, which occurs due to the entry of spores into the bodies of animals and can be transmitted to humans. this disease often appear in certain seasons and occurs in endemic areas, including indonesia. cutaneous anthrax is the clinical manifestations that often arise on outstanding events in the area. this study aims to determine how the relationship between the clinical manifestations of the serum antibodies in people who are exposed to anthrax. material and methods: this study is an observational cross sectional analytic approach, in people exposed to anthrax to assess the clinical manifestations and antibody serum anthrax. results: obtained in this study respondents were 101 people with a history of contact with animals suffering from anthrax. the number of respondents with the highest age distribution was 31 to 40 years by 42%, and most were female gender, which is 57.7%, the highest level of education is 74% finished elementary school. forty-four percent of working as a housewife. risk factors are the most direct contact with and consume the flesh of animals as much as 34.6%. results of ig g antibody serum showed 50% negative, 15.4 borderline and 34.6% positive. clinical manifestations that occur in the skin as much as 13.5%, that is the eschar on all respondents and 92.8% showed positive ig g. while 86.5% did not show any clinical signs of anthrax, of that number 25.5% with ig g positive, 16.6% and 57.7% showed borderline negative with p 0.02. conclusion: there was a significant association between the clinical manifestation with antibody serum anthrax. but also found a positive ig g without the appearance of clinical signs in the skin. key words: clinical, manifestation, anthrax, serum antibody elisa, eschar abstrak pendahuluan. antraks adalah salah satu penyakit zoonosis yang sering menyerang pada hewan pemakan rumput, yang terjadi karena masuknya spora ke dalam tubuh hewan dan dapat ditularkan ke manusia. penyakit ini sering muncul pada musim tertentu dan terjadi di daerah endemi, termasuk indonesia. cutaneous anthrax merupakan manifestasi klinis yang sering timbul pada kejadian luar biasa di suatu daerah. penelitian ini bertujuan untuk mengetahui bagaimana hubungan antara manifestasi klinis terhadap serum antibodi pada orang yang terpapar antraks. bahan dan metode. penelitian ini merupakan observasional analitik dengan pendekatan cross sectional, pada orang yang terpapar antraks dengan menilai manifestasi klinis dan serum antibodi antraks. hasil. pada penelitian ini didapatkan responden sebanyak 101 orang dengan riwayat kontak dengan hewan yang menderita antraks. jumlah responden dengan sebaran umur tertinggi adalah pada 31 sampai 40 tahun sebanyak 42 %, dan jenis kelamin terbanyak adalah perempuan, yaitu 57,7 %, tingkat pendidikan terbanyak adalah lulus sd 74 %. empat puluh empat persen bekerja sebagai ibu rumah tangga. faktor risiko terbanyak adalah kontak langsung dan mengkomsumsi daging hewan sebanyak 34,6%. hasil pemeriksaan ig g antibodi serum menunjukkan 50% negatif, 15,4 borderline dan 34,6% positif. manifestasi klinis yang terjadi pada kulit sebanyak 13,5 % , yaitu adanya eschar pada semua responden dan 92,8% menunjukkan ig g positif. sedangkan 86,5% tidak menunjukkan adanya tanda klinis antraks, dari jumlah tersebut 25,5% dengan ig g positif, 16,6% menunjukkan borderline dan 57,7% negatif dengan p 0,02. simpulan. ada 114 indonesian journal of tropical and infectious disease, vol. 5. no. 5 may–august 2015: 113-118 hubungan yang bermakna antara manifestasi klinis dengan hasil serum antibodi antraks. namun juga didapatkan adanya antibodi ig g positif tanpa disertai munculnya tanda klinis di kulit. sehingga perlu dilakukan deteksi dini pada orang yang terpapar antraks. kata kunci: manifestasi, klinis, antraks, antibodi serum elisa, eschar introduction anthrax is one of the types of zoonotic diseases, which can be transmitted to humans, animals suffering from anthrax. the disease is caused by bacillus anthracis. anthrax commonly often attacked livestock such as cattle, sheep, goats and camels. transmission to humans occurs when there is direct contact of animals or animal products that suffer from anthrax, can be skin, blood and flesh.1,2 anthrax incident in indonesia in the last ten years has occurred five times the plague that is 1996 to 2000 in west java.3,4 since the outbreak of anthrax 15 years ago in indonesia, the patient sample should be sent abroad (usa) for the diagnosis of anthrax investigation. based on these events, the moewardi hospital cooperate with integrated biomedical laboratory of the faculty of medicine, university sebelas maret has been trying to develop anthrax test-based immunoassay using enzyme-linked immunosorbent assay (elisa) for the detection of proteins pa by using the anthrax protective antigen calbiotech (pa) igg elisa kit, as catcher agents that are sensitive to the elisa was able to detect pa (≥ 1 ng / ml pa) in the serum of patients with suspected anthrax.4,5 this problem is how is anthrax protective antigen serum antibodies (pa) ig g elisa in people who are exposed to anthrax and its relationship with clinical manifestations in outbreak area ? diagnosis approach anthrax diagnosis is made through history, clinical examination, laboratory and serology : 1. history: early diagnosis of anthrax is difficult to know because it does not show the typical signs and symptoms, usually preceded by the appearance of reddish nodule with pain and swelling. it needs to be asked is whether previously had contact with an animal that died of anthrax, either direct contact or eating meat or contact with animals or their products (skin, bone), how the employment status (farmers fields, ranchers, rph, tanners) and whether residence in endemic areas of anthrax.2,6 2. clinical manifestations: there are 3 clinical manifestations that may arise in people is cutaneous anthrax, gastrointestinal and inhalation: a. cutaneous anthrax most cases (95%) anthrax is happening in the world is cutaneous anthrax. patients usually have a history of contact with animals or their products, the anthrax bacteria or spores enter through the skin through a lesion on the skin, for example, when doing the slaughtering process (cutting, skinning or divide meat) cattle infected with anthrax. then came a low germination rate at the location where the entry of spores and cause lesions on the skin that itch, then papuler lesions arise and develop into vesicles accompanied by edema and pain. these lesions became necrotic eschar formation and accompanied local soft tissue edema. germination occurs within 1-3 hours after inoculation, but germination can not cause infection of the skin intact. endospores will undergo phagocytosis by macrophages and then be taken to the regional lymph nodes, causing lymphadenopathy and lymphangitis. hematogenous dissemination can occur, but with the provision of adequate spread of systemic antibiotics is quite rare. several case reports of infections caused by insect bites suspected of being infected (eating carcasses containing anthrax bacillus).4,5 common location is on the face, extremities or neck. endospores enter through skin abrasions or wounds. one to seven days after entry endospores, formed the primary skin lesions that are not painful and itchy papules. twenty-four to 36 hours later lesions forming vesicles containing clear fluid or serosanguineus, and contains many gram-positive bacteria. vesicles then undergo central necrosis, dry out and cause eschar (necrotic ulcers) blackish typical vesicles surrounded by edema and purple. edema usually occurs more severe on the head or neck than the body or limbs. lymphangitis and lymphadenopathy that pain can be found following systemic symptoms occur. although cutaneous anthrax can heal itself, but still need to be given antibiotics (to reduce systemic symptoms). in 8090% of cases the lesions healed completely without complications or scarring. malignant edema are rare, characterized by severe edema, induration, multiple bullae, and shock. malignant edema can occur in the neck and chest area that causes difficulty breathing, requiring corticosteroids or intubation.5,6 b. gastrointestinal anthrax gastrointestinal anthrax, although it can be fatal, has not been reported in the us. symptoms usually appear 2-5 days after eating raw or undercooked meat that is contaminated with germs. some cases may occur in the home. on pathological examination under a microscope can be found in 115redhono: relationship between clinical manifestations and antibody serum the mucosa and submucosa basil lymphoid tissue and mesenteric lymphadenitis. ulceration almost always be found. a large number of gram-positive bacteria can be found in the peritoneal fluid. mediastinal widening can also occur. clinical symptoms can include fever, diffuse abdominal pain, constipation or diarrhea. if there is ulceration of the bowel becomes blackish. ascites can occur with clear liquids until purulent.1,6 c. anthrax inhalation inhalation anthrax spores began with the entry into the alveolar cavity, then macrophages will fagocyt spores and some of the spores will lysis and broken. spores that survive will spread to the lymph nodes and mediastinal nodes. the process of change in vegetative forms occur approximately 60 days later. the slow process of change in shape is not known with certainty, but well-documented cases of anthrax in sverdlovsk that inhalation occurs between day 2 to day 43 after exposure. once germination has occurred, the disease will arise quickly and replication of bacteria causing bleeding, edema and necrosis. the term anthrax pneumonia is not used because it turned out after pathological examination abnormalities were obtained mainly in the form of thoracic lymphadenitis and mediastinitis hemorhagis without typical bronchopneumonia. however, in the event of inhalation anthrax in sverdlovsk, 25% of fatal cases was found bleeding focal necrosis and pulmonary lesions.1,2 anthrax meningitis a complication of cutaneous anthrax, inhalation and gastroitestinal, but is most common in inhalation anthrax (> 50%). often addressing bleeding and meningoencepalitis. anthrax death rate is over 90%.6,8 3. investigations : in the diagnosis of anthrax needed routine blood tests, culture swab the wound or blood (on the skin), sputum (on inhalation) chest x-ray (on inhalation), electrolyte (gastrointestinal) and serology using elisa (enzyme linked immunosorbent assay) and pcr (polymerasi chain reaction). samples were taken for laboratory examination of the above is the blood serum, rub the injured area, sputum and land near the cage or a dead animal.9,10 criteria of diagnosis the criteria used in the diagnosis of anthrax consists of three types, namely suspected (suspect), probable (possibility) and confirmed (confirmation).10,11 suspected, is clinically shown one form of anthrax and there is epidemiological evidence that exposure to anthrax environment, but there is no definitive laboratory evidence. probable, is in clinical symptoms of anthrax but do not meet the definition of confirmation, but shows one of the following : (1) in epidemiology, there are environmental exposures. (2) b. anthracis dna evidence collected from the lesion, usually sterile (such as blood or csf) or lesion of other affected tissue (skin, lung or digestive) (3) positive serology igg elisa anthrax lethal factor (lf) in the examination of the positive spectrometry. confirmed, is in clinical symptoms of anthrax with one of the following : (1) b. anthracis culture positive (2) demonstrate b. anthracis antigens of the network by immunohistochemical staining using the cell wall and capsule monoclonal antibody b. anthracis (3) proven 4x increase in antibody titer during the acute period and fixes the quantitative examination of anti-pa igg elisa testing (4) the presence of environmental exposure to anthrax and pcr test positive.10,11 method this study is an observational analytic with cross sectional approach, by screening immunoassay based on people exposed to anthrax outbreak in the area in 2011. the location of sampling is an area of outbreak anthrax, boyolali and sragen, central java. all the people who are exposed to dead animals suffering anthrax, a blood sample for examination igg antibodies in serum by elisa.3,4 intepretation elisa will get three categories: positive, borderline and negative, with inclusion criteria such as direct contact with infected animals anthrax, do not suffer from a disease that causes a decrease in the body’s immunity, there are currently using immune suppressant drugs, there are pregnant or breastfeeding and not in hormone therapy. there were exclusion citeria such as not willing to follow research and medium serious illness, such as sepsis. operational definitions of variables dependent variable: clinical manifestations is the result of the interview and physical examination in people who are exposed to anthrax animals. the independent variables: levels of ig g anthrax serology examination to assess the titer of anthrax protective antigen (pa) ig g elisa in order to confirm the presence of infection with bacillus antracis in human blood. interpretation of test results : <0.9 : no detectable igg antibodies against pa protein in elisa. 0.9 1.1 : borderline > 1.1 : detected the presence of igg antibodies to the protein pa, indicated patients were infected or infected with a never bacillus anthracis. scale: nominal 116 indonesian journal of tropical and infectious disease, vol. 5. no. 5 may–august 2015: 113-118 result in this study, 101 people with a history of contact with animals that died of anthrax. respondents in the youngest age is 6 years old (1%) and the oldest 80 years (1%). the distribution of the highest age at 21 to 40 years as much as 39.6%, and most are women sex, ie 57.4%. the education level of respondents at most 74.3% graduated from elementary school. subsistence farmers as much as 21.8%. the basic characteristics of the study subjects are shown in table 1. table 1. baseline characteristics of research subjects (n = 101) variable n % gender • male • female 43 58 42,6 57,4 age • 0 20 year • 21 – 40 year • 41 – 60 year • 61 – 80 year 2 40 37 22 1,9 39,6 36,6 21,7 pendidikan • elementary • junior high school • senior high school • university 75 15 5 6 74,3 14,9 5 6 profession • no work • farmer • civil • private 48 22 5 26 47,6 21,8 5,0 38,8 of the total sample, showed serum ig g antibodies showed negative 50.5%, borderline 15.8% and 33.7% positive. elisa serology results can be seen in table 2. table 2. results of elisa variable n % • positive • borderline • negative 34 16 51 33,7 15,8 50,5 in cross-table analysis results between risk factors contact with elisa serology results obtained at the same respondents who cook and eat the highest risk on positive serologic results 20.8%. serology results elisa risks associated with the contact can be seen in table 3. table 3. results of serology elisa risks associated with contact risk factors elisa positif border line negatif • wash the meat • eating • wash and eat • cooking and eating • slaughtering and eating • located near the cage 1 10 3 11 9 0 0 5 2 6 3 0 1 16 0 19 13 0 overall there are 11.9% of respondents who showed clinical signs of the appearance of the skin in the form of vesicles, accompanied by fever and ulcers which ended with eschar formation. the skin manifestations can be seen in table 4. table 4. distribution of clinical manifestation clinical manifestation n % • eschar • no eschar 12 89 11,9 88,1 at respondents with 10.9% positive serology results indicate a skin manifestation of the emergence of eschar, while only 1.0% with borderline serology showing the skin manifestations. there are as many as 22.8% with positive elisa results, but does not cause any skin manifestation in the form of eschar or other clinical signs (fever, myalgia, stone, spasms, nausea and vomiting). result of serology associated with skin manifestations such as eschar can be seen in table 5. table 5. results of serology elisha associated with skin manifestations in the form of eschar eschar elisa positive borderline negative • yes • no 11 (11%) 23 (23% ) 1 ( 1,0 % ) 15 ( 14,8 % ) 0 ( 0,0 % ) 51 ( 51% ) p 0.02 contact risk factor for the emergence of manifestations in the skin, especially on the respondents were slaughtered at once ate beef (6.0%), followed by the washing and eating meat, which is 3.0%, whereas only 1.0% wash and meating. the relationship between contact with the manifestation of the emergence of the eschar can be seen in table 6. 117redhono: relationship between clinical manifestations and antibody serum table 6. relationship between contact with the manifestation of eschar variable eschar yes no • wash the meat • eating • wash and eat • cooking and eating • slaughtering and eating • located near the cage 1 ( 1,0 % ) 2 ( 2,0 % ) 3 ( 3,0 % ) 0 ( 0,0 % ) 6 ( 6,0 % ) 0 ( 0,0 % ) 1 ( 1,0 % ) 29 (28,7 % ) 2 ( 2,0 % ) 36 (35,6 % ) 19 (18,9 % ) 2 ( 2,0 % ) discussion during the 2011 outbreak of anthrax in central java. initially obtained a cow belonging to one of the people who had collapsed and accompanied by seizures. the owner decided to slaughter cattle meat and sold to residents of 40 packs. beef meat and blood samples examined in laboratory of central java province and tested positive for anthrax. seven days later, seven people were complaining there are small bumps and itching, swelling and lesions accompanied wet in the area under the eyes, hands, legs or feet, then taken to the health center and declared suspected anthrax. then in may 2011 in sragen also occur the same thing and be some people who show symptoms of anthrax skin contact. clinical manifestations in the form of eschar present in 11.9% with cutaneous anthrax. respondents were taken from both locations, obtained 101 samples were then examined serological anthrax serum ig g antibodies. of these 50.5% negative and 33.7% positive, while 15.8% borderline. clinical manifestations in the form of a skin disorder that begins their edema or injury which led to edema and ends with eschar present in 10.9% of the respondents who ig g antibody positive and 1.0% of respondents with ig g borderline results. this is due to the emergence of antibodies against anthrax bacteria on respondents who had clinical manifestations in the skin, but that can not be explained is the result obtained antibodies also borderline clinical manifestations (see figure 1). twenty-two percent of respondents with a positive serum ig g antibody, did not lead to clinical manifestations. it might be due to the durability of the respondents, bacterial virulence factors and the amount of exposure that occurs may not be too much. but this can not be explained further, because of the endurance factor all pretty much the same condition, which probably is due to the virulence of the bacteria and germs that enter the number. the risk of direct contact, ie cooking and eating meat of infected animals showed 30.5% ig g positive results, but does not cause clinical manifestations with the advent of eschar (0%). this may be due to immune factors of patients as well as virulence b antrhacis that enters the body. risk factors eating only 32% of respondents showed positive results. while the risk factors slaughter and eat 24% manifested by the appearance of skin eschar. conclusions the conclusion of this study is the increase in serum antibody titer ig g anthrax does not occur in all of the respondents were exposed to the anthrax outbreak area, all respondents were obtained eschar followed by an increase in ig g antibody titers. researchers suggest screening anthrax using anthrax ig g antibodies can be done in areas that are outbreaks of anthrax and can proceed with dealing with how the eschar and its effect on ig g antibody titer anthrax. acknowledgements we thank to prof. guntur hermawan for helpful on the study and paramasari for advice on setting up the laboratory examination and sample analyses with elisa. figure 1. anthrax manifestations in the skin with the advent of eschar 118 indonesian journal of tropical and infectious disease, vol. 5. no. 5 may–august 2015: 113-118 reference 1. jeremy farrar, peter j. hotez, thomas junghanss, gagandeep kang, david lalloo and nicholas j. white; anthrax ; manson’s tropical diseases; 2014; 31, 395-398.e1. 2. fred f. ferri; anthrax; ferri’s clinical advisor 2015; mosby, an imprint of elsevier inc; 2014; 115.e2-115.e4. 3. redhono, paramasari. anthrax outbreaks in indonesia. proceeding in apsic 2011 the 5th international congress of the asia pacific society of infection control. melbourne ; 2011: 152. 4. redhono d, sumandjar t, hermawan g pemetaan antraks di jawa tengah. antraks: sebelas maret press; 2011: 11–17. 5. dirgahayu p pemeriksaan laboratorium deteksi antraks berbasis immunoassay. antraks : sebelas maret press; 2011: 18–26. 6. dixon tc, meselson bsm, guillemin j, hanna pc anthrax. n engl j med; (2005) vol. 341 p. 815–826. 7. pile jc, malone jd, eitzen em, friedlander am. anthrax as a potential biological warfare agent. arch intern med; 2005 vol 158 p. 429–34. 8. shafazand s, doyle r, ruoss s, weinacker a, raffin ta inhalation anthrax, epidemiology, diagnosis and management. chest ; 2005 vol 116 p. 1369–1376. 9. john e. bennett, raphael dolin and martin j. blaser; anthrax; mandell, douglas, and bennett’s principles and practice of infectious diseases; saunders, an imprint of elsevier inc; 2015; 209, 2391–2409. e2. 10. inglesby tv, henderson da, barlett jg anthrax as a biological weapon medical and public health management. jama; 2005 vol 281 p. 1735–1745. 11. holmes rk. diphtheria, other corynebacterial infection and anthrax. in : fauci as, braunwald e, isselbacher kj, wilson jd, martin jb, kasper dl, et al. eds. harrison’s principles of internal medicine. 16th ed. mcgraw-hill ; new york; 2009: 892–899. 129 vol. 5. no. 5 may–august 2015 management patient of swine influenza endra gunawan1, and nasronudin1,2 1 infectious and tropical disease division department of internal medicine, dr. soetomo hospital universitas airlangga school of medicine 2 institue of tropical disease universitas airlangga, surabaya, indonesia abstract influenza is an acute respiratory diseases caused by various influenza virus which infect the upper and lower respiratory tract and often accompanied by systemic symptoms such as fever, headache and muscle pain. influenza spreads through the air. swine influenza comes from swine and can cause an outbreaks in pig flocks. even this is a kind of a rare case but the swine influenza could be transmitted to human by direct contact with infected swine or through environment that already being contaminated by swine influenza virus. there are 3 types of swine influenza virus namely h1n1, h3n2 and h1n2. type h1n1 swine-virus had been known since 1918. avian influenza virus infection is transmitted from one person to another through secret containing virus. virus is binded into the mucous cells of respiratory tract before it is finally infecting the cells itself. management patients with h1n1 influenza is based on the complications and the risk. besides, it is also need to consider the clinical criteria of the patient. therapy medicamentosa is applied to the patients by giving an antiviral, antibiotics and symptomatic therapy. prevention can be done by avoid contact with infected animal or environment, having antiviral prophylaxis and vaccination. key words: influenza, swine, infection, h1n1, management patient abstrak influenza atau flu merupakan penyakit pernafasan akut yang disebabkan oleh bermacam virus influenza yang menginfeksi saluran pernafasan bagian atas maupun saluran pernafasan bagian bawah serta seringkali disertai gejala sistemik seperti demam, sakit kepala dan nyeri otot. flu dapat menular melalui udara. flu babi adalah jenis flu yang berasal dari babi dan dapat mewabah pada kawanan ternak babi. flu babi dapat menular ke manusia melalui kontak langsung dengan hewan babi atau lingkungan yang telah terinfeksi virus flu babi, meskipun hal ini terbilang jarang terjadi. terdapat 3 jenis virus flu babi yaitu h1n1,h2n3 dan h1n2. virus flu babi tipe h1n1 telah diketahui sejak tahun 1918. infeksi virus avian influenza ditransmisikan dari satu orang ke orang lain melalui pembawa virus tertentu. virus melekat pada sel mukosa pada saluran respirasi sebelum akhirnya menginfeksi sel tersebut. penatalaksanaan pasien dengan h1n1 didasarkan pada komplikasi dan risiko yang mungkin terjadi. selain itu, perlu juga ada pertimbangan mengeneai criteria klinis yang dimiliki pasien. terapi medikamentosa diterapkan kepada pasien dengan memberikan antivirus, antibiotik dan terapi sistomatis. tindakan pencegahan dilakukan dengan cara menghindari kontak langsung dengan hewan maupun lingkungan yang terinfeksi virus, serta melakukan profilaksis antivirus dan vaksinasi. kata kunci: flu, babi, infeksi, h1n1, penatalaksanaan pasien literature review introduction influenza is an acute respiratory diseases caused by various influenza virus that infect the upper and lower respiratory tract and often accompanied by systemic symptoms such as fever, headache and muscle pain. influenza spreads through the air. several epidemical evidence of influenza cases showed high morbidity rate especially for patient with higher risk caused by pulmonary complication.1,2 swine influenza comes from pig and could cause an outbreaks in pig flocks. clinical signs of this disease are fever, appetite loss, weight loss, sluggishness, cough, sneezes, cold and asphyxiate. pig incubation usually given 130 indonesian journal of tropical and infectious disease, vol. 5. no. 5 may–august 2015: 129-135 in 1 to 3 days. it has low mortality rate (1% to 3%) and the infected animals are usually getting better in 5 until 7 days. some pigs are showed a severe pneumonia symptoms as a major cause of death. secondary bacteria infection could occurs. the process of viral internalization could be happened within 24 hours after infection and become disease after 7 to 10 days.3,4,5 animal to human swine influenza virus transmission is rarely happen. but it can be transmitted through a direct contact between infected pig and human, also by living in environment that already being contaminated with swine influenza virus under special circumstances. once human are infected, they could transmitted the virus to one and another with the same way of influenza virus works through cough or sneeze.3 there are 3 type of swine influenza virus namely h1n1, h3n2 and h1n2. swine virus type h1n1 has been known since 1918, causing pandemic and death of 40-50 million people. this virus was dissapear until in year 1957 influenza a (h2n2) pandemic was infected 250.000 people in hongkong within 3 weeks before it abated. in 1968, another influenza pandemic caused by influenza a (h3n2) was named hongkong influenza. in 1977, epidemic swine influenza (h1n1) was re-emerge in fort dix, new jersey and infected more than 200 people, some of them were severe and one was dead.6,7 in march 2009, influenza a (h1n1) first emerged in mexico, then it was reported in united states and spread in some parts of other countries, including indonesia. on april 2009, who was announced the occurrence of the pandemic phase 5. until october 2009, 195 countries were reported their influenza a (h1n1) cases.5,8 influenza a virus influenza virus a is the rna virus which is the member of the orthomyxoviridae family. there are 3 type of influenza virus which are influenza virus a, b and c. rna nucleus consist eight segments of genee and surrounded by 10 layers of protein (influenza a) or 11 layers of protein (influenza b).2 influenza virus a is divided into subtype based on two protein on the surface, hemaglutinin (h) and neuraminidase (n). there are 16 kind hemagglutinin subtype and 9 neuraminidase subtype.virus type is based on these proteins, for example influenza a subtype h3n2 which express hemaglutinin 3 and neuraminidase 2. influenza a has 16 kind of h subtype and 9 kind of n subtype. geneome influenza a and influenza b have eight segments single stranded rna which coding the structural and non structural protein.3 influenza a is more pathogeneic than influenza b. influenza a is zoonosis infection and usually infect the most of birds, pigs, horse, dogs and humans. reasort genees segment can occur when influenza virus infects a cell simultaneously.2 h1n1 virus called as swine influenza because the laboratory test showed that several genees in this virus was equal to influenza virus that infect the swine in north america but further studies show that these two viruses are different. hini virus has 2 genees of the common influenza virus which usually possessed by european swine called gene na segment, gene ha segment, gene np segment and gene ns segment from the classical swine and triple reasortment swine. pb2 and pa derived by triple reasortment swine which come from avian species and pb1 derived by swine triple reasortment from human.9 pathophysiology avian influenza virus infection is transmitted from one person to another through secret containing virus. virus is binded into the mucous cells of respiratory tract before it is finally infecting the cells itself. influenza infection is occured after secret transfer in the exhalation from one to another. if it is not neutralized by antibodies, virus would strike the respiratory tract and the cells. in host cell, after cellular dysfunction and degeneeration happens, along with replication of the virus and release of viral replication. almost all cells in the respiratory could support replication of the virus. after the virus has led to respiratory tract infections, the virus infects many cell replication and damage epithelial cilia through direct cytopatic effect or apoptosis. the amount of virus in the respiratory tract is correlated with the severity of the disease and the degree of inflammatory cytokine pro-kemokin. increased levels of cytokines pro inflammatory such as interferon α, interleukin 6, tumor necrosis factor occur in the blood and respiratory secretions. and may contribute to systemic symptoms and fever. duration of viral replication depend on the age and the invulnerability status. shelding usually lasting during three to five days in the adult, often reaches the second week in children, and could survive for weeks on a host immunocompromised.1 clinical symptoms after the incubation period is complete 15 days, the onset of disease is tend to increase rapidly were actually causing symptoms. clinical symptoms divided into specific symptoms and non specific, based on cdc (2009) stricken with symptoms reported is: cough (98 %), febris (96%), weak (89 per cent), headache (82 %), cold (80 %), muscle pain (80 %), nausea (55 %), abdominal pain (50 %), diarrhea (48 %), shortness of (48 %), joint pains (46 %). the symptoms above are known as ili-fever (influenza like illnes) which has temperature more than 39,8oc accompanied by one or more symptoms of cough ingest, pain on swallowing, without any causes other the influenza.10 131gunawan and nasronudin: management patient of swine influenza there are mild criteria (outpatient with surveillance) such as without a symptom or symptoms at least, without fever tightness, no pneumonia, no comorbid and young age. middle criteria (isolation room) such as comorbid factor, asfixia, pneumonia, old age, pregnancy, diarrhea and vomiting. severe criteria (icu) such as pneumonia broad, breath failure, sepsis, shock, declining consciousness, artery respiratory distress syndrome (ards) and multiple organ dysfunction syndrome (mods).8 case identification definitin of cases a. cases of alleged (suspect): someone with symptoms ili accompanied by history: • contact with the confirmation influenza virus h1n1 2009, 7 days before sick. • visit where areas there is one or more cases of influenza virus with new confirmation h1n1 2009, 7 days before ili • reside in areas where there is one or more cases confirmation. b. probable case: someone with symptoms alleged (suspect positive laboratory result of influenza but cannot detect the subtype, with the ili in accordance with clinical symptoms who died because of failed cause of breath which could not be explained and related with epidemology with a case or confirm probable. c. must case (confirmation examination): someone from the laboratory is infected by a new influenza virus h1n1 2009 through one or more examination such as real-time (rt) pcr, virus culture and increasing 4 times in antibiotic specific influenza a h1n1 viruses with new tests in the neutralization.8 clinical findings patients with must case (confirmed) h1n1 virus 2009 with symptoms of the ili to pneumonia based on the degrees: 1. people with mild state (confirmed): must be the case of the symptoms of ili fever > 380c (cough without pain ingest) with ili symptom. 2. heavy state: someone with a must case be obtained (confirmed) clinical syndrome which is ili accompanied by respiratory infection is a low breath and pneumonia, failed a breath, dehydration and until death. occurring severe condition previously to be a chronic disease. weight degree criteria : fever > 38o c, rr> 24 x/ minutes, hipoxia (so2 < 90 %), disfungsi organ hipotensi (<90/60), declining conscious, tachicardia, hiperglikemia, photo thoraxabnormal.11 complication ili : 1. severe primary viral infection by fail of breath: happens when lung infection viruses infect with clinical symptoms, is not good in the thorax to infiltrate intersisial, and there’s a sign ards with severe hypoxia. 2. pneumonia bacteria, patients often complained of a fever and respiratory symptoms arise who had already died down.bacterial pathogenes to infect staphylocuccus aureus and are often steptococcus pneumonia and hemofilus influenza. 3. a bacterial virus pneumonia and pneumonia, often occurs in patients who had the disease; a chronic previous (ppok asthma, dm, ggk, heart failure etc) patients with ili having a high risk occurring complication such as children under 5 years, age above 65 years, child or adult who gets therapy aspirin long term, pregnant woman, patients with comorbid factors and immunodeficiency.4 laboratory examination virology examination a lot of tests available to detect influenza virus. this test has different sensitivity and specificity. including several examinations which can be seen in table i. specimens can be obtained through: nasopharyngeal swab, aspirate (wash or nasal swab) swab the endotracheal lavage, bronchoalveolar lavage (bla), combination of nasopharyngeal or oropharyngeal swab. oropharyngeal. tests should be done on the first week until healing (2 3 weeks after the onset of disease). examination of virology should be done in patients with an indication of in-patient medium and heavy.10,12 hematology examination dl examination (hb, platelets, leucocyte, count of leucocyte, a lymphocyte total). other examination depends on identification, such as chemicals analysis of the blood. to remove the diagnosis of appeals examined igg and igm opposed to culture week and gall salmonela thyposa. radiology examination examination of the lateral thoracic pa and their routines; if necessary you can be intensified ct scans in accordance with the indications. microbiology examination if in thought occurring co-infection by bacteria can detect by sputurn (smear and culture), culture blood and urine based on identification.10,12 132 indonesian journal of tropical and infectious disease, vol. 5. no. 5 may–august 2015: 129-135 management management new patients with influenza a h1n1 2009 was based on some new consideration of the degree of the sick the risk of complications and the results of laboratorium with the criteria consideration adjusted therapy the patient whether policlinic, in-patient, the icu. almost of influenza cases caused by a new influenza virus h1n1. there is no complications, short duration and the provision of anti virus tend to be needed for the majority of patients.13 management in general 1. mild case : supportive therapy such as providing an antipyretic rehidration 2. sufficient case : symptomatic therapy, rehydration, antiviral, antibiotic, if secondary infection was proven, fluids and nutrition therapy 3. severe case (care icu) : correction hypoxia with oxygene provision and ventilator assemblies for management strategy of ards, monitor hemodynamics for septic shock management, antiviral and antibiotic, parenteral and enteral nutrition table 1. comparison of sensitivity and specificity of influenza and diagnostic test the cdc, 2009. influenza diagnostic tests method availability typical processing time2 sensitivity3 for 2009 h1n1 influenza distingushes 2009 h1n1 influenza from other influenza a viruses ? rapid influenza diagnostic test (ridt)4 antigene detection wide 0.5 hour 10 – 70 % no direct & indirect immunofluorescence assays (dfa and ifa)5 antigene detection wide 2 – 4 hours 47 – 93 % no viral isolation in tissue cell culture virus isolation limited 2 – 10 days yes6 nucleic acid amplification test (including rrt – pcr)7 rna detection limited8 48 – 96 hours (6 – 8 hours to perform test) 66 – 100% yes table 2. the organisation antiviral drug use, organisation, 2009 population pandemic (h1n1) influenza virus 2009 multiple co-circulating influenza a sub-types or viruses with different antiviral susceptibilities sporadic zoonotic influenza a viruses including h5n1 mild to moderate uncomplicated clinical presentation at-riska population oseltamivir or zanamivir (04) zanamivir or oseltamivir plus m2 inhibitorb (10) oseltamivir or zanamivir otherwise healthyc need not treat (03) need not treat (09) oseltamivir a. infants and children aged less than 5, the elderly (>65years), nursing home residents, pregnant women, patients with chronic co-morbid conditions such as cardiovascular, respiratory or liver disease, diabetes, and those with immunosuppression related to malignancy, hiv infection or other disease. b. amantadine should not be used in pregnant women (recommendation 12) c. all those not covered by the at-risk definition above. severe or progressive clinical presentationd at-riska population oseltamivir (01) (zanamivir should be used where virus is known to be resistant to oseltamivir, or if oseltamivir unavailable) (02) oseltamivir plus m2 inhibitorb, or zanamivir (05,06,07) oseltamivir plus m2 inhibitor otherwise healthy d. see section 2 case description. would include all patients requiring hospitalization 133gunawan and nasronudin: management patient of swine influenza table 3. recommendations antiviral a dose of influenza a new h1n. oseltamivir oseltamivir is indicated for treatment of patients one year of age and older. for adolescents (13 to 17 years of age) and adults the recommended oral dose is 75 mg oseltamivir twice daily for 5 days. for infants older than 1 year of age and for children 2 to 12 years of age recommended doses are as follows : 15 kg or less 30 mg orally twice a day for 5 days 15 – 23 kg 45 mg orally twice a day for 5 days 24 – 40 kg 60 mg orally twice a day for 5 days 40 kg 75 mg orally twice a day for 5 days zanamivir zanamivir is indicated for treatment of influenza in adults and children (>5 years). the recommended dose for treatment of adults and children from the age of 5 years is two inhalations (2 × 5mg) twice daily for 5 days table 4. recommendation for giving antibiotics to influenza patients for influenza not complicated by pneumonia a. previously well adults with acute bronchitis complicating influenza, in the absence of pneumonia, do not routinely require antibiotics. b. antibiotics should be considered in those previously well adults who develop worsening symptoms (recrudescent fever or increasing dyspnoen). c. patients at high risk of complications or secondary infection should be considered for antibiotics in the presence of lower respiratory features. d. most patients can be adequately treated with oral antibiotics. e. the preffered choice includes co-amoxiclav or a tetracycline f. a macrolide such as clarithromycin (or erythromycin) or a fluoroquinolone active against streptococcus pneumoniae and streptococcus aureus is an alternative choice in certain circumstances. non-severe influenza-related pneumonia a. most patients can be adequately treated with oral antibiotics b. oral therapy with co-amoxiclav or a tetracycline is preffered c. when oral therapy is contraindicated, recommended parenteral choices include iv co-amoxiclav, or a secondor third geneeration cephalosporin (cefuroxime or cefotaxime). a macrolide (erythromycin or clarithromycin) or a fluoroquinolone active against s. pneumonia and s. aureus is an alternative regimen where required, e.g. for those intolerant of penicillins. currently, levofloxacin and moxifloxacin are the only recommended fluoroquinolones licensed in the uk. for severe influenza-related pneumonia a. patients with severe pneumonia should be treated immediately after diagnosis with parenteral antibiotics. b. an iv combination od a broad-spectrum β-lactamase stable antibiotics such as co-amoxiclav or a second-geneeration (e.g. cefuroxime) or third-geneeration (e.g. cefotaxime) cephalosporin together with a macrolide (e.g. clarithromycin or erythromycin) is preffered. c. an alternative regimen includes a fluoroquinolone with enhanced activity against pneumococci together with a broad-spectrum β-lactamase stable antibiotic or a macrolide. currently, levofloxacin is the only fluoroqionolone with an iv formulation licensed in the uk. the management of patients are not separated from the medikamentosa therapy provision: antivirus influenza virus a h1n1 sensitive to neuraminidase inhibitor (nals) namely oseltamivir and zanamivir, but resistant to amantadine or rimantadin. the antiviral could reduce the disease alleviate symptoms, for illness, progresivity at preventing disease and death and decrease hospitalization. based on the research the antiviral beneficial is given the maximum 48 hours after the onset of the disease, 5 days in therapy for patient hospitalization, patients with severe infections or in icu patients could be given more therapy.8,13 recommendations given of the antiviral that is hospitalized patients with the confirmation, probable or suspect, patient has a high risk, if there are complications and patients with chronic disease. antibiotics antibiotics can be given if the result of examination positive and secondary infection symptoms is caused by bacteria. recommendation for giving antibiotic to patients with influenza depend on british thoracic society (bts) can be seen in table 2 if based on clinical and radiological, influenza the assessment of the weight of pneumonia associated pneumonia can stamp use standard score as psi, crub 134 indonesian journal of tropical and infectious disease, vol. 5. no. 5 may–august 2015: 129-135 table 5. regimen antibiotics for infection by bacteria in patients after the flu.14 antibiotic regimen (doses provided are for adults) antibacterial activity ecological risk outpatients doxycydine 200mg once and then 100mg every 25h sp, strep, h, mc, mssa, mrsa, atypicals low co-amoxiclav 625mg every 8h sp, strep, mc, mssa, gneb moderate erythromycin 500mg every 6h or clarithromycin 500mg every 12h sp, strep, mc, mssa, atypicals moderate levofloxacin 500mg every 24h or moxifloxacin 400mg every 24h sp, strep, h, mc, mssa, gneb, atypicals high non-severe inpatients oral (as for outpatients [see above)] non-severe inpatients, iv (for non-severe inpatients unable to take oral therapy) benzylpenicillin 1.2g every 6h sp, strep low amoxicillin 500mg to 1g every 8h sp, strep, h moderate clarithromycin 500mg every 12h sp, strep, mc, mssa, atypicals moderate flucloxacillin 1g every 6h plus clarithromycin 500mg every 12h sp, strep, mc, mssa, atypicals moderate co-amoxiclav 1.2g every 8h sp, strep, h, mc, mssa, gneb moderate to high amoxicillin 500mg to 1g every 8h plus clarithromycin 500mg every 12h sp, strep, h, mc, mssa, atypicals moderate to high cefuroxime 750mg every 8h or cefotaxime 1g every 8h sp, strep, h, mc, mssa, gneb high levofloxacin 500mg every 24h sp, strep, mc, mssa, gneb, atypicals high severe inpatients (patients who require critical care, whether a bed is available or not; all regimens to be given iv initially) co-amoxiclav 1.2g every 8h plus clarothromycin 500mg every 12h sp, strep, h, mc, mssa, gneb, atypicals moderate to high cefuroxime 750mg every 8h or cefotaxime 1g every 8h plus clarithromycin 500mg every 12h sp, strep, h, mc, mssa, gneb, atypicals high levofloxacin 500mg every 24h sp, strep, h, mc, mssa, gneb, atypical high benzylpenicillin 1.8 – 2.4 g every 4h plus clindamycin 600mg every 6h for proven life-threatening lancefield group a, c or g streptococcal infection high mrsa (in the uk, only for patients with proven mrsa) linezolid 600mg iv / oral every 12h sp, strep, mssa, mrsa low other agenets that may be useful in specific circumstances (e.g. if microbiological results are available) trimethoprim 200mg oral every 12h h, some mssa/mrsa, some gneb low co-trimoxazole 960mg oral every 12h (iv; 960mg to 1.44g every 12h) sp, strep, h, mc, mssa, some mrsa, gneb low to moderate piperacillin/tazobactam 4.5mg iv every 8h sp, strep, h, mc, mssa, gneb moderate tigecycline 100mg iv once then 50mg is every 12h sp, strep, h, mc, mssa, mrsa, gneb, atypicals unclear, probably low to moderate – 63. regimen antibiotics for bacterial infection in patients with swine flu can be seen in table 5 cortikosteroid not give a corticosteroid routine in patients by a new, h1n1 influenza based on reports of mexico granting a corticosteroid unprofitable.a corticosteroid use higher doses will cause serious side effects due to rising replication of the virus and improve the germ opportunistic infection.11 prevention 1. be careful when contact with persons infected who is someone who can transmit h1n1 virus days before the onset of diseases such as 1 to 7 days, and after the onset of disease (24 hours no fever). an act done is cover nose and mouth or use a mask, wash hand soap and water or alcohol in the normal time 15 until 20 seconds, 135gunawan and nasronudin: management patient of swine influenza eat nutritious food, less migration to the epidemic and pandemics. 2. antiviral prophylaxis can be given to someone with the acts of close contact patients confirmation, case probable, infectious, suspect during the period of high risk patients experienced complications and health officers contact with confirmation, cases of patients probable, suspect infectious table during the period. 14 vaccination cdc identify priority to get population main vaccination are pregnant woman, all medical society, age 25 64 by the risk of complications and someone who care for infants under 6 months.13 summary influenza is respiratory infections caused by influenza virus a subtype h1ni. virus h1n1 could cause a new transmission between humans. it is because a mutation in the virus.virus h1n1 influenza totally different with the h1n1 virus seasonal.known for its clinical symptoms influenza like illness (ili) who clinically divide light being, weight. management patients with the h1n1 influenza a new year, in some degree was based on the consideration of the complications and the risk of a laboratory. consideration also according to this criteria of patients are policlinic, inpatient, icu. therapy medikamentosa be granted antiviral, antibiotics and therapy symtomatic. prevention is to avoid contact with an infected, the antiviral prophylaxis, and vaccination. table 6. antiviral prophylaxis drugs the who recommendations agenet age groups (yrs) duration 1 6 7 9 10 12 13 – 64 >65 oseltamivir begin as soon as exposure identified and continue for 5 -7 days after last known exposure weight adjusted doses : 30 mg/day for <15 kg 45 mg/day for <15 kg 60 mg/day for <15 kg 75 mg/day for <15 kg 75 mg/day 75 mg/day zanamivir begin as soon as exposure identified and continue for 5 -7 days after last known exposure 1 – 4 yrs : na 5 – 6 yrs : 10 mg (2 inhalations) once daily 10 mg (2 inhalations) once daily 10 mg (2 inhalations) once daily 10 mg (2 inhalations) once daily references 1. dolin r. (2008). influenza. in: harrison’s principles of internal medicine vol. i, 17th edition. editors:kasper dl,et al. mcgraw-hill medical publishing division .new york. 661–670. 2. derlet r. (2009). influenza. medicine infectious disease.cited on 2th may 2010.http://emedicine.medscape.com/article/overview 3. bronze ms. (2009). swine flu. emedicine infectious disease.cited on 12/17/2009. 4. cidrap. (2010). novelh1n1influenza.http://www.cidrap.wmn. edu/cidrap/content/influenza/swine flu 5. donnely ca. (2009). pandemic potential of a strain of influenza a (h1n1) : early finding. science.324 : 1557–1561. 6. kilbourn ed. (2006). influenza pandemics of the 20th century, emerging infectionus disease vol. 12: 9–14. 7. zimmer sm. (2009). historical prespective – emergenece of influenza a (h1n1) viruses. n eng j med 361: 279–285. 8. world health organization(who), (2009). clinical management of human infection with newinfluenza a (h1n1) virus: revised guidance. 9. garten rj. (2009). antigeneic and geneetic characteristic of swine – origin 2009 h1n1 influenza virus circulating in human. science 325: 197. 10. centers for disease control and prevention(2009).interim recommendation for clinical use of influenza diagnostic test during the 2009–2010 influenza season. http: //www.cdc.gov/h1n1flu/ guidance/diagnostictest.htm2009. 11. world health organization. (2009). who guidelines for pharmacological management of pandemic (h1n1) 2009 influenza and other influenza viruses. http://www.who.int/csr/resource/ publications/who.2009. 12. faix dj. (2009). rapid – test sensitivity for novel swine – origin influenza a (h1n1) virus in human. n eng j med 361: 728–734. 13. centers for disease control and prevention(2009). interim guidance for the use of antiviral medication in the treatment and prevention of influenza for the 2009–2010 season. http: // www.cdc.gov/h1n1flu/ recomendation. 14. barlow gd. (2009). swine flu and antibiotic. journal of antimicrobial chemotherapy 64; 889–894. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid vol 6 no 2 mei-agustus 2016_edit.indd 39 vol. 6. no. 2 mei–agustus 2016 clinical manifestation approach of dengue viral infection ganis tjahjono1, prihartini widiyanti1.3, nasronudin2a 1 infectious and tropical disease division department of internal medicine, dr. soetomo hospital school of medicine 2 institute of tropical disease universitas airlangga 3 faculty of science and technology universitas airlangga a corresponding author: nasronudindr@yahoo.com abstract currently by an estimated 50-100 million dengue fever cases per year in worldwide, 500.000 were in the form of a disease is heavy dengue hemorraghic fever (dhf) and dengue shock syndrome (dss). survey serology in indonesia show that den-1 and den-2 are the dominant serotipe virus until the end of the 1980s but the recent shift has occurred epizoötic where viruses den-3 dominant. (dos santos, 2004; malavige, 2004; stephenson, 2005). dengue virus infection induces transient immune aberrant activation of cd4/cd8 ratio inversion and cytokine overproduction, and infection of endothelial cells and hepatocytes causes apoptosis and dysfunction of these cells. the aberrant immune responses not only impaire the immune response to clear the virus, but also result in overproduction of cytokines that affect monocytes, endothelial cells, and hepatocytes. dengue-virus-induced vasculopathy and coagulopathy must be involved in the pathogenesis of hemorrhage, and the unbalace between coagulation and fibrinolysis activation, and prolonged duration of shock increase the likelihood of severe hemorrhage in dhf/dss. capillary leakage is triggered by the dengue virus itself or by antibodies to its antigen. to date, there are no effective strategies to prevent the progression of dhf/dss. the control of dengue will be possible only after an efficient vaccine has been developed. key words: aberrant immune, capillary leakage, hepatocytes, fibrinolysis, dengue shock syndrome abstrak saat ini diperkirakan sekitar 50-100 juta kasus demam berdarah per tahun di seluruh dunia, 500.000 adalah dbd. survei menunjukkan bahwa serologi di indonesia dan yang dominan adalah den-1 den-2 serotipe virus hingga akhir tahun 1980-an tapi akhir-akhir ini telah terjadi pergeseran epizootic di mana virus den-3 dominan (dos santos, 2004; malavige, 2004; stephenson, 2005). infeksi virus dengue yang aktif dapat menginduksi kekebalan tubuh dan rasio jumlah perbandingan cd4/cd8 dan kelebihan produksi cytokine, infeksi sel-sel endotel serta menyebabkan disfungsi hepatosit dan apoptosis sel-sel ini. penyimpangan respon imun yang terkait tidak hanya respons kekebalan tubuh untuk membersihkan virus, tetapi juga mengakibatkan kelebihan produksi sitokin yang memengaruhi monosit, sel endotel, dan hepatosit. virus dengue menginduksi vasculopathy coagulopathy dan terlibat dalam kegiatan patogenesis dari pendarahan, dan durasi shock sehingga dapat meningkatkan kemungkinan pendarahan yang parah pada dbd. kebocoran kapiler terjadi karena virus dengue itu sendiri atau oleh antibodi terhadap antigen dbd. tidak ada strategi efektif untuk mencegah perkembangan dbd, pengkontrolan dbd hanya mungkin setelah vaksin yang efisien telah berhasil dikembangkan. kata kunci: penyimpangan imun, kebocoran kapiler, hepatosit, fibrinolisis, dengue shock syndrome literature review 40 indonesian journal of tropical and infectious disease, vol. 6. no.2 mei–agustus 2016: 39−45 introduction dengue fever (df) is an acute infectious disease, caused by dengue virus that have four type of serotipe (den-1, den-2, den-3 dan den-4). with traits that is spatially biphasic, fever myalgia, headache, pain in some part of the body, rash, limphadenopati and leucopenia. in most case, df is self limited, but there is a risk of the development of being dengue hemorrhagic fever (dhf) or dengue shock syndrome (dss). dhf is a febrile disease with traits abnormality hemostasis and increase of vascular permeability as well as the development of progressive can be dss. dss is a condition shock hipovolemic, are clinically associated with hemoconcentration and can cause the death if adequate handling is not given. a mechanism that involved in pathogenesis hemorrhagic fever, a viral infection particularly regarding dhf/dss unresolved.1 currently, it has been estimated 50-100 million df cases per year in worldwide, 500,000 were in the form of a disease is heavy dhf and dss. survey serology in indonesia show that den-1 and den-2 are the dominant serotipe virus until the end of the 1980s but the recent shift has occurred epizoötic where viruses den-3 dominant.2,3,4 based on data cases report in september 1998, cases dhf in adults (≥ 18) most widely is jakarta (13,813), overtaken west java (10,730), east java (8,546), central java (6,879) and jogjakarta (3,257).5 who is categorized dengue as one of the main international public health problem because wider geographical distribution both virus and its vector, the increased frequency of epidemics, co-circulation of various serotypes of the virus and the emergence of dhf in new places.6 currently, there is no specific therapy against dhf. provision of adequate fluid, can decrease mortality due to dhf. control of the main vector (aedes aegypti) costly and often ineffective is the only method of prevention is still available to this day. therefore, the basic comprehension of the molecular pathogenesis dengue infection became very important for the development of diagnosis and therapeutic as well as facilitate the protective vaccine production, rather than aggravate the disease.4,7 the body response against infections hemorrhagic fever after infected mosquito is bite human, replication of the virus in regional lymph gland and spreading to the lymphatic system, blood and other tissues. replication in reticuloendotelial system and bark yield viremia.6,8 the immune system has a strong defense against bacteria or viruses invasion. the components that contribute to viral infections are the antibodies, phagocytes, ifn, nk cells and t cells. when a virus infects a cell, viral proteins are broken down into specific peptides were then expressed with the help of mhc-i molecules on the cell surface. then the peptide will be known by th1 cells which in turn activate effector cells or tc ctc can destroy virus-infected cells by direct (lethal hit). nk cells have fc receptors (fcγ r) plays a role in adcc.9 dendritic cells (dcs) (<1% of total blood cells in peripheral blood) derived from bone marrow, have a central role in the development of an immune response to both natural and adaptive. at least two subsets of myeloid dcs in humans, namely dcs (mdcs) and plasmacytoid dcs (pdcs). both have strong antigen presentation capacity and capability stimulate ag-specific t cell responses. pdcs are cd123+, with appropriate stimuli can migrate directly from the peripheral blood and lymphoid organs to limphonoduli. pdcs have the characteristics that the potential for a strong ag presentation, lymphoid morphology and strongly stimulate the secretion of ifn-α -mediated stimulation of cd40 as a virus. so, mdcs and especially pdcs assist in anti viral innate immunity and the formation of th1 adaptive immune response against viral pathogens.10 interferon response is the first line to inhibit viral infection. interferon (ifn) is a cytokine that is secreted by the immune system is the first virus to infect target cells. ifn binds to the ifn receptor on the cell surface and activates the jak target stat (transcription factors) signaling pathway to inhibit translation of viral rna and protein synthesis, thus the virus will be eliminated. however, the dengue virus has its own strategy for tackling defense mediated by ifn. dr. eva harris at the university of california, berkeley, showed that treatment with ifn-α and ifn-γ before infection can reduce the replication of dengue virus in human cells. same treatment when administered after infection have no effect at all, it shows that the dengue virus can suppress ifn signaling. dr. adolfo garcia sastre, mount sinai school of medicine, new york showed that the ns4b protein of dengue virus can inhibit the activation of stat1 in cell culture systems, so this explains some of the mechanisms that can be suppressed ifn signaling through stat1 barriers. whether the same mechanism also occurs in vivo in patients with dengue, is still unclear. although ifn and ribavirin combination therapy can be applied to hcv, which is classified with dengue virus (family flaviviridae), is not effective when it comes to the treatment of dengue infection.11,12 dengue virus infection causes lifelong imunity protective against the homologous serotype but only partial or temporary protection against subsequent infection by the other three serotypes. already a generally accepted concept that secondary infection or multiple major risk factors occurrence of dhf/dss as antibody dependent enhancement. other factors that play an important role in the pathogenesis of dhf have been formulated include the virulence of the virus, the genetic background host, activation of t cells and autoantibodies.4,13 41tjahjono, et al.: clinical manifestation approach of dengue viral infection characteristic of dengue virus dengue virus consists of a single strand of rna included in the family flaviviridae. the first discovered by albert sabin in 1944, there are four serotypes are classified according to biological and immunological criteria. the length of the viral genome is about 11 kb. mature virion consists of three structural (core, premembrane and envelope) and seven non structural proteins, namely ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns (figure 1). envelope protein required for various major biological functions for the virus, which binds to receptors on the surface of the host cell, allowing the virus to enter target cells. envelop proteins are also associated with erythrocyte hemagglutination, induces the formation of antibodies and protective immune response. non structural proteins (ns1 ns5) expressed as a membrane -associated and secreted forms also have an impact on the pathogenesis of severe disease. unlike other viral glycoprotein, ns1 does not form a component of the virion but is expressed on the surface of infected cells. levels of ns1 (sns1) secreted in plasma associated with viral titers, being higher in patients with dhf compared with df. furthermore, increased levels of free sns1 within 72 hours of the start of the illness, indicating the risk of patient become dhf. ns1 that very high levels of protein detected in the acute phase blood samples from patients with secondary dengue, rather than the primary infection. this suggests that ns1 has contributed to the formation of immune complexes in the circulation are likely to play an important role in the pathogenesis of severe dengue infection.2,3,7,14 figure 1. dengue virus genome, a protein produced and the location of the main targets of the immune response.7 latest hypotheses about the pathogenesis of dengue virus infection several hypotheses on the pathogenesis of dengue virus infection have been proposed. among these are the antibody-dependent enhancement (ade) of infection, which has long been considered an important role. ade hypothesis formulated to explain the finding that severe manifestations in dhf/dss occurs when an infection to dengue virus with two different serotypes of dengue virus infection before. antibodies are formed against a previous viral infection does not seem to be able to neutralize but rather aggravate the infection in vitro. serum that taken from children before an infection which then progress to dhf/dss further describe an ade than just being df. epidemiological studies support an association between dhf/dss with secondary dengue virus infections. thus, ade hypothesis is reinforced by the findings that: 1) prospective cohort study of dengue epidemic, indicating that the majority of dhf/dss occur during infection to two; 2) dhf/dss in infants less classical than 1 year in the first dengue infection, the presence of antibodies to dengue virus factor of maternal circulation; 3) in vitro, non neutralizing igg antibodies can bind to dengue virus and attached to the fc receptor and enhance dengue virus infection in monocytes or macrophages in the peripheral blood; 4) an increase in viremia was noted in vivo in animal models of rhesus macaques on secondary infection by den – 2; 5) in humans, high levels of viremia correlate with disease severity. it is not known how the addition of a viral infection caused by dengue enhancing antibodies can lead to dhf/dss, is the proliferation of dengue virus infects or signal amplification through fc receptors still require explanation. due to the absence of animal model for dhf/dss causal relationship between ade with dhf/ dss is still no verification.11,15,16,17 serotype crossreactive antibodies that resulting from previous infection binding with virions without neutralization and the increase of virus entry into monocytes, so that the number of virus-infected monocytes increased. as a result the number of activated t cells increased. this reflects the increased antigen presentation, increase in dengue virus -specific t cells on subsequent infection and activation and proliferation faster than memory t cells. the t cells produce cytokines such as ifn-γ, il 2 and tnfα, as well as lysis of dengue virus -infected monocytes. tnfα is also produced by activated monocytes. complement cascade is activated by a virus antibody complexes and the release of several cytokines through c3a and c5a are also having a direct effect on vascular permeability. the synergistic effect of ifn-γ, tnfα and activation complement proteins trigger plasma leakage from endothelial cells in secondary dengue virus infections. however, some problems still can not be explained by this theory. not all cases of dhf/dss is secondary infection. complement activation may be caused by severe disease and not the cause of dhf/dss.15,16 mongkolsapaya et al in 2003, did a study on the response of dengue virus specific t cells. in patients with dhf and dss found some cd8+ t cells are virus responsive most of undergoing apoptosis. this study is demonstrated the phenomenon of “original antigenic sin” that has been described a few years ago, the antibody responses to secondary viral infection is dominated by the activation of memory b cells cross reacting induced by primary infection. activation of memory b cells will produce low affinity antibodies against the virus that causes secondary infections. so the levels of activated t cells with rapid death and domination of cellular immune responses by cells with low affinity to viral infections may inhibit viral clearance 42 indonesian journal of tropical and infectious disease, vol. 6. no.2 mei–agustus 2016: 39−45 and lead to high viral loads and increase imunopatologis (mongkolsapaya, 2003; stephenson, 2004). in late 2004, a study of 48 vietnamese adults with secondary dengue virus infections showed that ns3 (epitopes targeted t cells) and t-cell responses cross-reactive responses do occur, but the magnitude was not significantly associated with clinical grading of disease.18 virulence of virus the virulence of the virus, the ability to cause disease in the host, is an alternative hypothesis on the pathogenesis of dhf/dss. different manifestations of df, dhf and dss may be caused by dengue virus variants with different degrees of virulence. the risk for the occurrence of dhf/dss higher in secondary infections with dengue virus serotype 2 when compared with other serotypes. structural differences of the virus also found in isolates of df and dhf patients. furthermore, it has been reported that in viremia with high titers of dengue virus increases the severity of disease. peak virus titer reached 100-1000 -fold higher in patients with dss than in df patients thai children infected with dengue virus. patients with secondary antibody response are likely to be dhf is twice than that has a primary antibody response. evidence available to date do not all support the hypothesis of viral virulence.15 the analysis of individual genotypes in a single population showed little evidence that there are differences between the isolates df patients with dhf/dss, although exceptions have been reported. so the evidence of a link between viral genotype with clinical manifestations is still weak.13 vulnerability host some researchers have proposed the existence of predisposing dhf or dss located on the human histocompatibility (hla) haplotypes, but which locci that linked is still unclear or other genetic factors as the cause of a severe form of dengue is also unclear. very few studies have been reported regarding the sensitivity of the host, especially regarding natural immunity. the latest scientific articles in nature genetics (sakuntabhai, nature genetics, 2005) have reported the existence of genetic variants in the dc -sign promoter region is associated with severe forms of dengue infection. however, the molecular mechanism which in the genetic variants that affecting viral replication or trigger an excessive immune reaction on dhf and dss still difficult to understand.13,17 clinical and pathological manifestations of dengue virus infection df symptoms include fever accompanied by headache, retroorbital pain, myalgia, arthralgia, race, mild leucopenia and thrombocytopenia. pharyngeal hyperemia is almost in 97% of patients with dhf. biphasic heat and the race was the most prominent characteristic of classical dengue fever. the symptoms will improve within 2–7 days. dhf is a syndrome of acute vascular permeability accompanied by abnormalities of hemostasis. clinical features include plasma leakage, bleeding tendency and liver involvement. capillary leak occurs rapidly in the period of a few hours, close to or at the time of the heat period ends when the classical df symptoms subside. pleural effusion, ascites, and hemoconcentration is indicative of a loss of intravascular volume. it can progress to shock if the patient did not receive fluid resuscitation. manifestation hemorrhagic cranging from a positive torniquet test until spontaneous bleeding from the nose or gastrointestinal tract. hemoconcentration and thrombocytopenia are the two main characteristic features of dhf/dss. liver involvement is common in dengue virus infection with a slight increase in serum transaminases. the liver is often enlarged, soft and slightly painful on palpation but usually no jaundice. generalized lymphadenopathy obtained approximately in 50% of cases. three organ systems (hematologic, vascular and hepatic) are involved in the pathological changes in dhf/dss. dengue virus infection causes dysfunction of the system either directly or indirectly, lead to the manifestation of dhf/dss.1,8,14 effects of dengue virus infection on blood cells immune aberrant activation during dengue virus infection in the analysis of blood samples collected during the outbreak of dengue virus serotype 3 from november to december 1998 in southern taiwan describe a disorder of immune status in patients with dengue. in the peripheral blood of uninfected persons, the number of cd4 + t cells more than cd8+. in the peripheral blood of patients with dengue, the number of cd8 + t cells more than cd4+, so the cd4/cd8 ratio decreased < 1. this phenomenon is not only found in patients with dhf/dss alone, but also on the patient df. of the 21 patients with df and 8 patients with dhf/dss, found cd4/cd8 ratio is inverted in 10 cases. the frequency of cd4/cd8 ratio is inverted is higher in dhf/dss (5/8) of the df (5/21, p < 0.05). kinetic analysis imunofenotip cd4 + t cells and cd8+, indicating that the cd4/cd8 ratio reverse occurs during acute infection (6-14 days after the onset of heat). cd4/cd8 ratio is slowly returning to normal after a day to 15. it also found cd4dim monocytes and cd8dim, the percentage in peripheral blood mononuclear cells (pbmcs) were higher or highest on days 6–7, then down to the lower level healing moment later. atypical lymphocytosis reached a peak on days 8-10, and then disappeared rapidly after day 12. initial activation of mononuclear cells was confirmed by expression of the early activation marker, cd69, on day 4 after the onset of heat. cd69 stained on the surface of lymphocytes, as well as on monocytes, but more widely expressed in cd8+ t cells of the cd4+. with the description of atypical lymphocytosis and dynamic changes in cd4/cd8 ratio, an indication of the occurrence of immune activation during dengue virus infection aberan.1,8 excessive cytokine production during dengue virus infection during acute dengue infection, mononuclear cells 43tjahjono, et al.: clinical manifestation approach of dengue viral infection activated excessively so expected that increased levels of cytokines can be found in the plasma. high levels of markers of t-cell activation such as soluble il-2 receptor, soluble cd4, soluble cd8, il-2 and ifn-γ, as well monokin eg tnfα, ifn-β and gm csf, all detected in children infected with dengue, and markers are higher in patients with dhf/dss than in df. high levels of cytokines in the serum inhibitors such as il-10 or soluble receptors (soluble tnf receptor) stnfri and stnfrii were also found in patients with dhf. in dengue patients in the study, cytokines such as rantes, il-8 and il-6 levels are increased after infection with dengue virus. levels of il-6 and il-8 in serum were higher in patients with dhf/dss than in df patients. il 6 has a dual role, as a mediator of pro-inflammatory or anti inflammatory. in the kinetic analysis showed large variations at points different times and in different individuals, but increased serum levels of il-6 high transient occurs either at day 7 or day 9-11 after the onset of heat. it is impressive when the host response to dengue virus infection through the formation of cytokines pro inflammatory cytokines also formed simultaneously inhibitor against inflammation. the end result depends on the balance between the two.1,19 thrombocytopenia and anti platelet pntibodies thrombocytopenia is common in df, and always found in dhf/dss. its pathogenesis is still poorly understood. impressed that the dengue virus triggers bone marrow suppression that reduces platelet production and resulting in thrombocytopenia. one group of studies found that dengue virus-2 can bind to platelets in the presence of specific viral antibodies and this supports the role of immune-mediated clearance of platelets. surprisingly, found igm (not igg) anti-platelet autoantibodies in patients with dengue. titer was higher in dhf/dss than in df patients. the presence of these autoantibodies is not only triggered by complement lysis of platelets but also inhibit adp-induced platelet aggregation. cross-reactions of antibodies against dengue virus proteins, particularly ns1 and platelets, suggesting a role for the pathogenesis of anti-platelet autoantibody during dengue virus infection.1,15 immune deviation caused dengue virus infection patients infected with dengue usually experienced leucopenia for a few days during the acute infection, with the characteristics of a decrease in the absolute number of neutrophils and monocytes. impaired t cell responses associated with pha-stimulated monocytes cd4dim or cd8dim deficiency. detection of early activation marker, cd69 on cd8+ t cells, nk cells and monocytes, and lymphocytes are atypical shapes showed activation of lymphocytes by dengue virus infection. dengue virus can infect langerhans cells or immature dendritic cells and can replicate more efficiently in these cells than in monocytes or macrophages. infected dendritic cells stimulate maturation cytokine production and tnfα and ifnα, but not il-6 or il-12. levels of il-12 in patients with higher df than in dhf. blunted blood pdc response to dengue virus infection associated with higher levels of viremia and is part of the natural immune response and changes cascade causes severe disease pathogenesis. in patients with dhf stage iii and iv, not detected the presence of il-12. production deficiency of il-12 can cause a shift to a th2 response and mismatch generation of cytotoxic t cells. dengue virus infection appears to strongly influence the immune response such as, monocytosis cd4dim or cd8dim early, inversion of cd4/cd8 ratio were temporary, atypical lymphocytosis with a large percentage and depressed t-cell proliferation. immune deviation is not only slow viral clearance, but also triggers excessive production of cytokines and antiplatelet autoantibodies that started the sequence of the pathogenesis of dengue virus infection.1,3,10 effects of dengue virus infection of endothelial cells vasculopathy due to dengue virus overview of the most distinctive and best indicator of disease severity is plasma leakage. plasma leakage due to increased capillary permeability in a diffuse manner and manifest as hemoconcentration, pleura effusion or ascites. it usually occurs on days 3-7, during the current easing of dengue fever. plasma leakage occurs systemically, developing progressive, but it will get better in 1-2 days in patients receiving fluid resuscitation adequately. no tissue or organ function abnormalities occur. although edema perivaskuler seems obvious, but no evidence of damage to vascular endothelial cells. dengue virus can infect endothelial cells in vitro led to the release of cytokines and chemokines il-6, il-8 and rantes. dengue virus infection can cause endothelial cell apoptosis in vitro, but the effect is directly dependent on the dengue virus isolates were used. endothelial cells were infected with dengue virus can activate complement and induce the expression of adhesion molecules such as icam-1. the expression of icam-1 along with the production of il-8 and rantes would invite polymorphonuclear cells, mononuclear cells, and then finally freed vasopermeability and trombomodulin, a marker of endothelial damage. increased levels of circulating trombomodulin in the acute stage of dhf/dss, indicating that the structural failure of endothelial cells in vivo. apparently, direct viral cytopathic effects and immune -mediated leukocyte recruitment and anti dengue virus antibodies, both of which cause structural damage to endothelial cells. vascular leakage can be caused by infection with dengue virus, both directly and indirectly. endothelial cells play an important role in maintaining hemostasis, therefore, endothelial cell damage due to dengue virus infection can affect either the balance of procoagulant or anticoagulant endothelium that increase the risk of bleeding. recruitment of platelets by activated endothelial cells may also cause thrombocytopenia.1,3 coagulopathy due to dengue virus hemorrhagic manifestations caused by the dengue virus, which is more common is vascular platelet abnormalities, but when severe bleeding can occur with 44 indonesian journal of tropical and infectious disease, vol. 6. no.2 mei–agustus 2016: 39−45 dic. hemostasis is maintained by a balance between coagulation and fibrinolysis. coagulation system can be activated by the intrinsic and extrinsic pathways to form thrombin converts fibrinogen to fibrin. fibrinolytic system on the other hand can break down fibrin. fibrinolytic system consists of plasminogen, a proenzyme that can be turned into an active enzyme plasmin by plasminogen activators several kinds. plasminogen activator is a major endogenous tissue -type plasminogen activator (tpa). plasminogen activator inhibitor (pai-1), which is produced by platelets, and endothelial liver, on the other hand is a major inhibitor of tpa. in general, secondary coagulation activation trigger fibrinolysis activation that inhibits the release of large amounts of pai-1 rapidly. during acute dengue virus infection, coagulation parameters such as platelet count, activated partial thromboplastin time (aptt), as well as fibrinolytic parameters tpa and pai-1 changes. aptt elongated, while tpa increased. both coagulation and fibrinolysis are both activated and this activation is more severe in patients with dhf/dss than in df. after healing increased levels of pai-1 and the platelet count in line with decreased levels of tpa and aptt returned to normal. the ratio of tpa/pai-1 was higher in patients with dhf/dss than in df patients. elongation of aptt and increased the ratio of tpa/pai-1 in the acute phase of dengue virus infection associated with disease severity and can be used as an early indicator of dhf/dss. aptt and prothrombin time is an indicator of intrinsic and extrinsic pathways of coagulation. only aptt, not prothrombin time that extending to dengue virus infection, suggesting that abnormalities in the intrinsic pathway. this can be caused by a decrease in the synthesis of specific factors or the increased use of specific factors. disorder of liver function is responsible for the reduction in the synthesis of specific factors in the intrinsic pathway. increased use of these factors as indicated by high levels of tpa is also associated with prolongation of the aptt but less meaningful. thus, both a decrease in the synthesis or increased consumption of coagulation factors, both involved in the lengthening of aptt. hiperfibrinolisis in the acute phase of dhf/dss, due to the increased production of tpa. correlation and linear regression analysis showed a significant association between il-6 serum with tpa in dhf, but not in the dengue fever. dengue virus infection stimulates endothelial tpa and to produce il-6. synthesis of tpa could be blocked by anti-il-6 antibody, suggesting that tpa production by endothelial cells is dependent il-6. furthermore, antibodies against dengue virus e protein, can bind to human plasminogen. it can inhibit the action of plasmin or plasminogen activation strengthens. so, both coagulation and fibrinolysis are experienced hyperactivation in the acute phase of dengue virus infection. imbalance between coagulation and fibrinolysis may cause bleeding in dhf/ dss. bleeding that occurs in dhf/dss is not directly due to the prolongation of the prothrombin time and partial thromboplastin time, nor is it just because of thrombocytopenia. the most powerful risk factor causes bleeding is the presence of prolonged shock. it shows that patient with a long shock, there has been a leakage of plasma and bleeding.20 effects of dengue virus infection in liver cells dengue virus is hepatotrophic. dengue virus antigen was detected in the liver cells and virus particles found in liver biopsy specimens from patients with dhf. dengue virus can infect the liver and cause hepatitis. increased levels of serum transaminases in patients with dengue and degree of elevated levels of aspartate aminotransferase (ast) associated with bleeding events. in hepatitis due to dengue virus, the ast level higher than alanin aminotransferase (alt) with a ratio of about 1-1.5, while hepatitis due to other viruses, alt is higher than ast. by using hepatoma cell lines, dengue virus can lead to apoptosis and chemokine rantes production through oxidative stress and activation of nf-κb. furthermore, rantes is typically caused by the dengue virus but not by enteroviruses in the liver cells. patients with dengue virus infection had serum levels of rantes were higher compared with other viral infections. rantes is a chemokine that can lead to the recruitment of lymphocytes and nk cells to sites of inflammation. liver is damage caused by dengue virus was due to a direct effect of viral replication or indirect effects of inflammation mediated by rantes, still require further investigation. the balance between virus elimination and tissue damage may result in disease severity. it is known that the liver is where most of the production of coagulation factors, the decreased levels of these factors is attributed to an increase in consumption, as well as impaired synthesis. the last thing is more likely due to injury to the liver. il-6 can lead to down regulation of the synthesis of factor xii, is the first factor that initiate clotting through the intrinsic pathway. elongation of activated partial thromboplastin time (aptt) in patients with dhf are caused by a deficiency in the intrinsic pathway is likely due to impaired synthesis in the liver factor xii.1,3,14 summary dengue virus infections causes dengue fever (df), dengue hemorrhagic fever (dhf) dan dengue shock syndrome (dss). current hypotheses antibody-dependent enhancement, virus virulence, and ifn-γ/tnfα –mediated immunopathogenesis are insufficient to explain clinical manifestations of dhf/dss such as thrombocytopenia and hemoconcentration. dengue virus infection induces transient immune aberrant activation of cd4/cd8 ratio inversion and cytokine overproduction, and infection of endothelial cells and hepatocytes causes apoptosis and dysfunction of these cells. the coagulation and fibrinolysis systems are also activated after dengue virus infection. the aberrant immune responses not only impaire the immune 45tjahjono, et al.: clinical manifestation approach of dengue viral infection response to clear the virus, but also result in overproduction of cytokines that affect monocytes, endothelial cells, and hepatocytes. platelets are destroyed by crossreactive antiplatelets antibodies. dengue-virus-induced vasculopathy and coagulopathy must be involved in the pathogesis of hemorrhage, and the unbalance between coagulation and fibrinolysis activation, and prolonged duration of shock increase the likelihood of severe hemorrhage in dhf/ dss. the overproduced il-6 might play a crucial role in the enhanced production of anti-platelet or anti-endothelial cell autoantibodies, elevated levels of tpa, as well as a deficiency in coagulation. capillary leakage is triggered by the dengue virus itself or by antibodies to its antigen. to date, there are no effective strategies to prevent the progression of dhf/dss. the control of dengue will be possible only after an efficient vaccine has been developed. references 1. lei hy, yeh tm, liu hs, lin ys, chen sh, liu cc, (2001). immunopathogenesis of dengue virus infection. j. biomed. sci. (8): 377–388. 2. dos santos fb, miagostovich mp, nogueira rmr, schatzmayr hg, riley lw, harris e (2004). analysis of recombinant dengue virus polypeptides for dengue diagnosis and 3. malavige gn, fernando s, fernando dj, seneviratne sl, (2004). dengue viral infections. postgrad. med. j. (80): 588–601. 4. stephenson jr, (2005). understanding dengue pathogenesis: implications for dengue vaccine design. bulletin of the world health organization, 83(4), pp. 308–314. 5. kosasih h, tan ri, porter kr, beckett cg, alisjahbana b, rudiman pjf, et al (2005). epidemiology of dengue and dengue hemorrhagic fever in a cohort of adult living in bandung, west java, indonesia. am. j. trop. med. hyg. 72(1): 60–66. 6. wilder-smith a, schwartz e (2005). current concepts: dengue in travelers. n. engl. j. med. 353: 924–32. 7. rothman al, (2004). dengue: defining protective versus pathologic immunity. j. clin. invest. 113(7), pp. 946–951. 8. nimmannitya s (2003). dengue and dengue hemorrhagic fever. in: cook g, zumla a (eds.). manson’s tropical diseases, 21st ed. rdc group, china, pp. 765–772. 9. baratawidjaya kg, (2002). imunitas terhadap virus. dalam: imunologi dasar, 5th ed. balai penerbit fkui, jakarta, hal. 198–202. 10. pichyangkul s, endy tp, kalanayarooj s, nisalak a, yongvanitchit s, green s, et al (2003). a blunted blood plasmacytoid dendritic cell response to an acute systemic viral infection is associated with increased disease severity. journal of immunology, (22): 5571–78. 11. halstead s (2005). host response to dengue infection. in: dengue digest. mica(p)(2):2. 12. bray m, (2005). pathogenesis of viral hemorrhagic fever. current opinion in immunology 17: 399–403. 13. shu py, huang jh, (2004). current advances in dengue diagnosis. clin. diagn. lab. immunol. 11(4): 642–8. 14. sheperd s, (2005). dengue fever. eds: wood mj, et al. available on: http://www.emedicine.com. accesed on january 5th, 2006. 15. lei hy (2005). thrombocytopenia and vascular leakage. dengue digest. mica(p)(2):2. 16. liu jw, khor bs, lee ch, lee ik, chen rf, yang kd, (2003). dengue hemorrhagic fever in taiwan. dengue bulletin 27, 19–24. 17. peters cj (2005). infection caused by arthropod and rodent-borne viruses. in: fauci as, braunwald e, isselbacher kj (eds.). harrison’s principles of internal medicine, 16th ed. mc graw hill, new york, pp. 1132–54. 18. simmons cp, dong t, chau nv, dung ntp, chau tnb, thao ltt, et al (2005). early t-cell responses to dengue virus epitopes in vietnamese adult with secondary dengue virus infections. journal of virology 79(9): 5665–75. 19. guzman mg, kourf g (2002). reviews, dengue: an update. the lancet inf. dis. 2(1):1-8. 20. see lum lc, golt ayt, chan pwk, el-amin alm, lam sk, (2002). risk factors for hemorrhage in severe dengue infections. journal of pediatrics, 140(5), pp. 1–3. e issn 2356–0991 p issn 2085–1103 indexed by: e-journal.unair.ac.id/index.php/ijtid vol. 9 ● no. 1 january-april 2021 plasmodium falciparum breath metabolomics (breathomics) analysis as a non-invasive practical method to diagnose malaria in pediatrics intestinal parasitic infection, the use of latrine, and clean water source in elementary school children at coastal and non-coastal areas, sumenep district, indonesia correlation between climate factors with dengue hemorrhagic fever cases in surabaya 2007 – 2017 genital tract infection during pregnancy and its association with preterm delivery risk factors of covid-19 confirmed died patients in dr. kariadi hospital: a retrospective study validity of method for mtbc and ntm detection in fnab specimens from tuberculous lymphadenitis using microscopy, xpert mtb/ rif and culture method influence of tlr-8 gene polymorphisms (rs3764880 and rs3788935) associated to pulmonary tuberculosis in kupang, indonesia editorial team of indonesian journal of tropical and infectious disease editor in chief prihartini widiyanti, indonesia editorial board secretariat secretariat office publishing unit of indonesian journal of tropical and infectious disease, institute of tropical disease universitas airlangga kampus c, jalan mulyorejo surabaya 60115, jawa timur – indonesia. phone 62-31-5992445-46 faximile 62-31-5992445 e-mail: ijtid@itd.unair.ac.id homepage: e-journal.unair.ac.id/index.php/ijtid e issn 2356 0991 volume 9 number 1 january–april 2021 p issn 2085 1103 mark alan graber, united states kazufumi shimizu, japan masanori kameoka, japan hak hotta, japan fumihiko kawamoto, japan yoshiharu matsuura, japan bimo ario tejo, malaysia nasronudin nasronudin, indonesia maria inge lusida, indonesia puruhito puruhito, indonesia retno handajani, indonesia kuntaman kuntaman, indonesia soegeng soegijanto, indonesia bambang prajogo, indonesia ni nyoman sri budayanti, indonesia achmad fuad hafid, indonesia tri wibawa, indonesia irwanto irwanto, indonesia yulis setiya dewi, indonesia laura navika yamani, indonesia siti qomariyah khairunisa, indonesia teguh hari sucipto, indonesia siti churrotin, indonesia nur diana fajriyah zakaria pamoengkas contents page printed by: universitas airlangga press. (rk 232/11.20/aup). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, fax. (031) 5992248. e-mail: adm@aup.unair.ac.id volume 9 number 1 january–april 2021 e issn 2356 – 0991 p issn 2085 1103 9–15 1–8 1. risk factors of covid-19 confirmed died patients in dr. kariadi hospital: a retrospective study elyana sri sulistyowati, septi dewi muninggar, verarica silalahi ............................................ 2. influence of tlr-8 gene polymorphisms (rs3764880 and rs3788935) associated to pulmonary tuberculosis in kupang, indonesia afandi charles, simeon penggoam, ani melani maskoen, edhyana sahiratmadja ............... 3. intestinal parasitic infection, the use of latrine, and clean water source in elementary school children at coastal and non-coastal areas, sumenep district, indonesia r. bagus yanuar renaldy, m. ahda naufal aflahudin, zukhaila salma, sumaryono, muhammad yasin fitri n, sri wijayanti sulistyawati, dominicus husada, sukmawati basuki................................................................................................................................................. 4. plasmodium falciparum breath metabolomics (breathomics) analysis as a non-invasive practical method to diagnose malaria in pediatrics ignatius ivan, maureen miracle stella, stella kallista, kevin tandarto, fanny budiman, freggy spicano joprang ........................................................................................................ .......... 5. validity of method for mtbc and ntm detection in fnab specimens from tuberculous lymphadenitis using microscopy, xpert mtb / rif and culture method herisa natalianan junus, ni made mertaniasih, soedarsono ............................................................ 6. correlation between climate factors with dengue hemorrhagic fever cases in surabaya 2007 – 2017 nadhilah putri ghaisani, sulistiawati, maria lucia inge lusida.............................................. 7. genital tract infection during pregnancy and its association with preterm delivery yohanes aditya adhi satria, tri nugraha susilawati.............................................................. 8. diagnosis based on detection of cxcl10 in urine as biomarker for the determining diagnosis of active lung tuberculosis i gede yogi prema ananda, ni made mertaniasih, soedarsono, deby kusumaningrum........ 16–23 24–32 33–38 39–44 45–56 57–65 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid vol 9 no 3 september-desember 2021.indd vol. 9 no. 3 september–december 2021 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 * corresponding author: andreaaprilia134@gmail.com research article correlation analysis between ratio of c-reactive protein/albumin and severity of dengue hemorrhagic fever in children agustin iskandar1,2, yuyun norwahyuni1, aryati3, andrea aprilia1* 1department of clinical pathology, faculty of medicine, universitas brawijaya/ saiful anwar general hospital, malang, east java, indonesia 2clinical pathology sub–speciality study program, faculty of medicine, universitas airlangga, surabaya, east java, indonesia 3department of clinical pathology, faculty of medicine, universitas airlangga dr. soetomo general hospital, surabaya, east java, indonesia received: 11st thseptember 2021; accepted: 26th october 2021 abstract dengue hemorrhagic fever (dhf) is a dengue infection which can cause shock and leads to mortality. hypoalbuminemia is a marker of plasma leakage in dhf and correlated with severity of infl ammatory response triggered by infection, including dhf. c-reactive protein (crp) is a proinfl ammatory marker that also increases in dhf. this study aims to determine a correlation of crp/albumin ratio with severity of dhf. cross sectional study on pediatric patients diagnosed as dhf at saiful anwar malang hospital was done in july-december 2016. crp levels were examined using immunoturbidimetry method, while albumin was examined by using bromocresol green (bcg) method. correlation of crp/albumin ratio with dhf severity was analyzed by using pearson correlation test.the result showed that there were signifi cant diff erences in crp levels and crp/albumin ratios in the dengue shock syndrome (dss) and non-dss group (p = 0.002, p = 0.001, α <0.05). there was no signifi cant diff erence in albumin level in the same group (p = 0.207, α <0.05). positive correlation found in crp and crp/albumin ratio (r = 0.46, r = 0.49, α <0.01). on the contrary the negative correlation was found in albumin (r = -0.21, α <0.01). this is presumably because albumin is an acute phase protein which will decrease along with the severity of infection. in contrast, crp will increase during the critical phase of infection. it can be concluded that the crp/albumin ratio was positively correlated with dhf severity, as well as crp levels, but not positively correlated with albumin. keywords: dengue hemorrhagic fever; dengue shock syndrome; severity; crp/albumin ratio; crp abstrak demam berdarah dengue (dbd) merupakan infeksi virus dengue (ivd) yang dapat menyebabkan syok dan berakhir dengan kematian. hipoalbuminemia merupakan salah satu penanda kebocoran plasma pada dbd sekaligus berkorelasi dengan intensitas respon infl amasi yang dipicu oleh infeksi, termasuk dbd. crp juga merupakan marker infl amasi yang juga meningkat pada dbd. penelitian ini bertujuan untuk mengetahui apakah rasio crp/albumin berkorelasi dengan keparahan pada dbd. penelitian cross sectional pada pasien anak dengan diagnosis dbd dilakukan di rs saiful anwar malang pada juli-desember 2016. kadar crp diperiksa menggunakan metode imunoturbidimetri, sedangkan kadar albumin diperiksa menggunakan metode brom cresol green (bcg). korelasi rasio crp/albumin dengan keparahan dbd dianalisis menggunakan uji korelasi pearson. hasil penelitian menunjukkan terdapat perbedaan kadar crp dan rasio crp/albumin yang bermakna pada kelompok dengue syok syndrome (dss) dan non dss (p= 0.002, p= 0.001, α <0.05). tidak didapatkan perbedaan kadar albumin yang bermakna pada kelompok yang sama (p=0.207, α<0.05). korelasi positif sedang ditunjukkan oleh crp dan rasio crp/albumin (r=0.46, r=0.49, α<0.01). sebaliknya korelasi negatif didapatkan pada albumin (r = -0.21, α<0.01). hal ini diduga karena albumin merupakan protein fase akut yang akan turun seiring dengan beratnya infeksi. sebaliknya, crp akan meningkat selama fase kritis. dapat disimpulkan august 2021; revised: 14 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 bahwa rasio crp/albumin berkorelasi positif sedang dengan keparahan dbd, demikian pula dengan kadar crp, namun tidak berkorelasi positif dengan albumin. kata kunci: demam berdarah dengue; sindrom syok dengue; keparahan; rasio crp/ albumin; crp how to cite: agustin , i., yuyun, n., aryati, andrea a. correlation analysis between ratio of c-reactive protein/albumin and severity of dengue hemorrhagic fever in children. indonesian journal of tropical and infectious disease, 9(3) introduction dengue virus (denv; family of flaviviridae, genus flavivirus) is transmitted by aedes aegypti mosquitoes and can cause relatively mild dengue fever (dengue fever-df); or more severe form of dengue (dengue hemorrhagic fever-dhf).1, 2 severe organ damage does not occur much but if it occurs, it can cause mortality because it is slowly detected. severe organ damage is one of the leading causes of mortality besides shock.3-6 therefore, it needs a marker which can predict organ damage. considering the clinical manifestations of dengue infection which vary from mild to severe and the result is diffi cult to predict, a predictor biomarker is needed to act as an early warning sign.7-12 suwarto in his study in 2016 has developed dengue scores to predict pleural eff usion and/or ascites in adults in dengue infection. the study showed that hemoconcentration was ≥15.1%, albumin concentration in the critical phase was ≤3.49g/dl, platelet count was ≤49,500/ μl, and high ast ratio was ≥2.5 had sensitivity and specifi city above 60%.4, 5, 13 another reliable biomarker when critical is c-reactive protein (crp). crp is an acute phase protein produced by hepatocytes, especially under il-6 control which has been proven as a sensitive prognostic indicator of infl ammation.14, 15 ranzani in his study (2013) on crp shows that crp can be used as a diagnostic tool for sepsis and for therapeutic monitoring. the measurement of crp level can also help clinicians in making decisions whether patients need an icu or not.16 grander (2010) has shown that crp level correlates with the level of infl ammation at the beginning of the diseases course. although some studies have shown that crp level when exiting from icu can be a reliable marker in monitoring but no studies have focused on dengue patients.17 not only for crp, but serum albumin can also be an important shortand long-term marker in determining prognosis. serum albumin is a negative acute phase protein, thus the level of hypoalbuminemia in critically ill patients correlates with the intensity of the infl ammatory response triggered by infection. therefore, crp and serum albumin level must be inversely proportional during the critical phase. the use of crp and albumin ratio will provide a variable which is able to combine information provided by crp and albumin. therefore, it can be an index which has a positive correlation with infection, a higher ratio indicates a higher infl ammatory status. crp/albumin ratio has been widely investigated in cases of malignancy. one of liu’s studies in 2015 showed that auc was 0.625, p < 0.001, for the role of the crp/albumin ratio as an independent prognostic marker in the preoperative of gastric cancer circumstance. the study underlines that the crp/albumin ratio not only refl ects infl ammation but also the nutritional status of cancer patients.16 based on the research background, infection is one of the strongest triggers for infl ammation, we hypothesized that crp and albumin level would be important markers of dengue severity. in addition, we also investigated whether the combination of information from crp and albumin through the crp/albumin ratio would improve the quality of the prognostic marker of dengue severity when compared to crp or albumin only. materials and methods this study was retrospective and was conducted on all pediatric patients with a diagnosis of dhf indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 136–142 firda typewritten text 137 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 who were treated in the pediatric ward of saiful anwar malang hospital during july-december 2016. the data were obtained from medical records then the data were carried out descriptive analysis. the population of the study subjects was divided into two groups of dengue severity: dengue shock (dhf grade 3,4) and dengue nonshock (dhf grade1,2). the inclusion criteria in this study were dhf pediatric patients who were hospitalized with positive ns-1 laboratory results and/or igm anti dengue immunoserology test and/or positive igg and examined serum albumin and crp on the same day during treatment. another inclusion criteria is the patients who diagnosed dhf and were <18 years old. the diagnosis of dhf was based on who 2011 criteria. the patients who were willing to be included in this study signed informed consents. while the exclusion criteria were the subjects who suff ered from another infection which could produce false positives on immunoserology dengue examination (e.g., malaria, typhoid fever). to provide suffi cient power in cross sectional study, at least 32 children were needed according to the sample size formula: n = z +zβ 2 + 3 0.5 ln [(1+r)/(1-r)] n = 1.64 +1.28 2 + 3 0.5 ln [(1+0.5)/(1-0.5)] = 32 sampel patients who became the sample were patients who came to the child polyclinic and emergency room of dr. saiful anwar malang general hospital, fulfilled the inclusion and exclusion criteria for clinical and laboratory examinations. the sample’s inclusion and exclusion criteria were determined by history, physical examination, completely blood laboratory examination, clinical chemistry, and immunoserology. patients’ serum were collected in laboratory and then stored at -80°c. when samples collection is completed, all serum were tested crp and albumin. this study was approved by the local medical ethical committee with ethical clearance number 400/196/k/3/302/2017. t h e d a t a a n a l y s i s c o n s i s t s o f s e v e r a l tests. shapiro-wilk test was used to see the data normality. mann whitney t-test was used to see the mean diff erences in the two groups. pearson test was used to determine the relationship of crp, albumin, and crp/albumin ratio with the severity degree of dengue infection/prognosis. roc curve was used to see the performance of crp single marker, albumin, and combined marker of crp/ albumin ratio. results characteristics of subjects thirty-nine pediatric patients infected with dengue virus were included in this study consist of 17 samples dengue non-shock and 22 samples dengue shock (table i, ii, iii). all patients were tested albumin and crp. table i. characteristic of subject based on age patients’ age prognosis total dengue without shock dengue with shock 0-1 year 5 5 10 1-5 years old 5 6 11 5-10 years 5 8 13 11-15 years old 2 3 5 15-18 years old total 17 22 39 table ii. characteristic of subject based on gender gender prognosis totaldengue without shock dengue with shock male 11 5 16 female 6 17 23 total 17 22 39 agustin iskandar, et al.: correlation analysis between ratio 138 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 table iii. characteristic of subject based on nutritional status nutritional status (z-score bb/tb) prognosis totaldengue without shock dengue with shock very thin (<-3sd) 1 0 1 thin (-3sd to <-2sd) 0 3 3 normal -2 sd to 2 sd 15 17 32 fat > 2 sd 1 2 3 total 17 22 39 data analysis the normality test showed the distribution of abnormal data for age, gender, and nutritional status. the results of the post-transformation normality test data also showed the data distribution which was not normal so that the diff erent test analysis used mann-whitney. the results of diff erent test showed that there were signifi cant diff erences in gender data in the shock and non-shock groups (table iv). table iv. diff erence tests based on age, gender, nutritional status in shock and non-shock groups (95% confi dence interval) diff erent test normality test p based on age (mann whitney) 0.004 0.136 based on gender (mann whitney) 0,000 0.009 based on nutritional status (mann whitney) 0,000 0.470 crp (t-test) 0.164 0.002 albumin (t-test) 0.653 0.207 crp/albumin ratio (t-test) 0.149 0.001 based on the normality test it was obtained the distribution of normal data for albumin (0.653), but there is an abnormal distribution of data (<0.05) for crp and crp/albumin level data, so that transformation needed to be done. the shapiro-wilk normality test showed the distribution of post-transformation normal data which 0.164 for crp and 0.149 for crp/ albumin ratio were so that data analysis could be continued using parametric tests. the results of diff erent marker tests showed only crp level which showed signifi cant diff erence in the shock and non-shock groups (table iv). the correlation tests showed positive correlations for crp and crp/albumin level, but there was a negative correlation for albumin level (table v). table v. pearson correlation test, with 99% confi dence interval pearson correlation tests r p dengue group and crp level 0.46 0.003 dengue group and albumin level -0.21 0.199 dengue group and crp/albumin ratio 0.49 0.002 furthermore, the data analysis was performed with receiver operating characteristic (roc) curve, auc (area under the curve) to see the performance of markers (table vi, table vii, table viii, table ix, and figure 1). table vi. area under the curve test variable area std. error asymptotic sig. asymptotic 95% confi dence interval lower limit upper limit crp 0.218 0.075 0.003 0.071 0.365 albumin 0.616 0.092 0.218 0.435 0.797 crp / albumin ratio 0.203 0.072 0.002 0.061 0.345 table vii. crp prognostic test shock non-shock total > 0.5 mg/dl 15 5 20 < 0.5 mg/dl 7 12 19 total: 22 17 39 sensitivity: 68.18% specifi city: 70.59% ppv (positive predictive value): 75% npv (negative predictive value): 63.16% rr (relative risk): 2.02 indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 136–142 firda typewritten text 139 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 table viii. albumin prognostic test shock non-shock total < 2.7 mg/dl 5 0 5 > 2.7 mg/dl 17 17 34 total: 22 17 39 sensitivity: 22.73% specifi city: 100% ppv (positive predictive value): 100% npv (negative predictive value): 50% rr (relative risk): 2 table ix. prognostic test for crp/albumin ratio shock non-shock total > 0.2 14 5 19 <0.2 8 12 20 total: 22 17 39 sensitivity: 63.64% specifi city: 70.59% ppv (positive predictive value): 73.68% npv (negative predictive value): 60% rr (relative risk): 1.83 figure 1. roc curve of prognostic test of crp, albumin, crp/albumin ratio against severity degree of dengue infection discussion in the baseline data, there were signifi cant diff erences in diff erent tests based on gender. in this case the female patients were signifi cantly (n=17) more than male patients (n=5) in the dengue group with shock. this is not the same as lovera’s study in 2016 which found that there was no gender preference in severe dengue manifestation.18 also this is not the same as anker’s study in 2011 in which his study looked at the incidence of dengue infection in children in asia, the data showed that the number of male cases was signifi cant in the age ≥ 15 years group. this diff erence based on gender was indeed not supported by specifi c pathophysiological mechanisms. the diff erence possibility related to gender in dengue fever was due to diff erence in exposure in the adolescent age group. the results of this study in asia were diff erent from those in south america, where there was a similar proportion of male and female patients in dengue fever cases or conversely the proportion of female cases were greater. the reason for this diff erence of incidence based on gender needs to be explored further.19 furthermore, there were no significant diff erence based on age and nutritional status. although most of the samples were form age 5 – 10 year old group which is similar to lam et al. study.20 our nutritional status was analyzed by z-score: weight/height (bb/tb). based on previous studies, moderate/severe malnutrition was associated with a signifi cant reduction in cellmediated immunity, as indicated by a reduction in the number of cd4+t cells, and a decrease in the cd4+/cd8+ ratio. there was also a decrease in secretory iga antibody production and various component supplements (c3, c4, and factor b) and decreased phagocytosis. the production of certain cytokines such as il-2 and tnf also decreases.21 the study done by kalayanarooj in 2005 study concluded that malnourished children had a lower risk of dengue infection, but if they were infected with dengue they had a high risk of dss. obese children had a higher risk of contracting dengue fever with a more unusual presentation; encephalopathy, related infections and complications of excess fl uid.22 widiyati’s study mentioned that obesity is not a risk factor for children with dengue infection to get dss.23 furthermore, in this study there was no signifi cant diff erence in the diff erent albumin tests (unpaired t test) 0.207, whereas for crp agustin iskandar, et al.: correlation analysis between ratio 140 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 and crp/albumin ratios there were signifi cant diff erences, 0.002, 0.001 respectively. albumin synthesis experienced signifi cant changes in the critical phase. as acute responses to trauma, infl ammation, and sepsis, it would improve the transcription process of acute phase proteins such as crp and would reduce transcription of albumin mrna and albumin synthesis. both il-6 and tnf-α could reduce gene transcription. based on liao’s study in 1986, the induction of infl ammation in mice was done to see changes in albumin levels. the study showed the lowest albumin levels were obtained at 36 hours and then rose again. a sustained infl ammatory response in critical illness could result in a long barrier to albumin synthesis as well.24 the fairclough’s study in 2009 mentions low albumin levels were most often associated with chronic diseases, also often associated with malnutrition.25 napoleon-tatura et al. found that low albumin levels can help predict shock in pediatric dengue.26 this study included an acute case study so that not all critical patients showed a decrease in serum albumin during treatment. fever days when samples taken, and nutritional status also varied so that the results obtained did not match the theory. based on the pearson correlation test, the same correlation was found between the relationship between crp (r = 0.46) and crp/albumin ratio with the friction of dengue infection (r = 0.49). this means there was a weak relationship. in accordance with the liao’s in 1986 that the day when sampling taken was very infl uential, and in the study indeed the data of the sampling taken was not homogeneous.24 while the correlation of the relationship between albumin and severity of dengue infection was r = -0.21. it is according to the theory that albumin is an acute phase protein that will decrease along with the severity of infection/infl ammation. but in this study a weak correlation was also found for albumin. based on fairclough’s study in 2009, nutritional status was very infl uential on albumin levels, while the nutritional status in this study was not homogeneous either.25 based on the prognostic test, the results were almost the same, both the sensitivity a n d s p e c i f i c i t y b e t w e e n c r p a n d c r p / albumin ratio were 68% and 70% respectively for crp performance; and 63% and 70% for crp/ albumin performance ratio. crp was at the cut off > 0.5mg/dl and the crp/albumin ratio was at the cut off > 0.2. the crp and crp/albumin ratio had similar auc (0.218; 0.203) with p < 0.05, whereas auc albumin was not signifi cant. menon’s study in 2005 using cut-off crp > 3 mg/l could be an independent marker of mortality risk factor in cardiovascular disease. the previous study on crp/albumin ratio used quite varied cut-off s.27 wei’s study in 2015 used cut-off > 0.095 which was associated with the size of esophageal tumor (squamous cell carcinoma).28 while xie’s study in 2011 used cut-off > 0.42 this was associated with mortality in aki patients.29 liu et al found that patients with pancreatic cancer that have crp/albumin ratio ≥ 0.18 have worse prognosis than those with crp/albumin ratio < 0.18.30 based on this study both crp and crp/albumin ratio were as good at predicting the severity of dengue infection. whereas serum albumin of cut-off < 2.7mg/ dl showed a low sensitivity of 22%, so it could not be used as a single marker of initial screening predictor of dengue infection severity. however, for the cut off , serum albumin had a specifi city of 100% which could specifi cally direct the severity that occurs in patients with dengue infection. based on this study the best relative risk (rr) was in crp (rr = 2.02) and albumin markers (rr = 2), in which the values were almost the same, followed by crp/albumin ratio (rr =1.83). however, all markers had a value of rr >1 so that they could be used to see the probability of dengue prognosis. from roc curve, only albumin that has good performance with auc 0.616 and p = 0.218, while crp and crp/albumin ratio has auc = 0.218 and auc = 0.203 with p = 0.003 and p = 0.002 respectively (figure 1). this study had limitations that must be considered. this study was conducted on patients without comorbidity, the sampling done on the varied fever days, the nutritional status was not indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 136–142 firda typewritten text 141 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 homogeneous. it was better if it was tested in populations with comorbidities, especially in people with comorbidities having potential to affect the levels/concentrations of predictive variables, such as kidney disease and liver disease. it was also best to do homogenization of sampling days and nutritional status. conclusion both crp and crp/albumin ratio are independent prognostic markers of the severity of dengue infection. the use of this ratio is easy, inexpensive, and has suffi cient availability, so it is very helpful for clinicians to identify high-risk dengue patients. single serum albumin cannot be used as a screening marker for the severity of dengue infection. further studies to predict the severity of dengue infection should include a population with comorbid kidney disease and liver disease, and collect more samples, including the participation of adult patients. hopefully the best predictor markers can be found. we would like to thank the dean of medical faculty of brawijaya university and general directorate of research, technology and higher education of indonesia, saiful anwar general hospital of malang, east java and our colleagues from department of child health, faculty of medicine, brawijaya university. reference 1. machain-williams c, mammen jr mp, zeidner ns, beaty bj, prenni je, nisalak a, et al. association of human immune response to aedes aegypti salivary proteins with dengue disease severity. parasite immunology. 2012;34(1):15-22. 2. guzman mg, gubler dj, izquierdo a, martinez e, halstead sb. dengue infection. nature reviews disease primers. 2016;2(1):1-25. 3. anders kl, nguyet nm, chau nvv, hung nt, thuy tt, lien lb, et al. epidemiological factors associated with dengue shock syndrome and mortality in hospitalized dengue patients in ho chi minh city, vietnam. the american journal of tropical medicine and hygiene. 2011;84(1):127. 4. huy nt, van giang t, thuy dhd, kikuchi m, hien tt, zamora j, et al. factors associated with dengue shock syndrome: a systematic review and meta-analysis. plos neglected tropical diseases. 2013;7(9):e2412. 5. jog s, prayag s, rajhans p, zirpe k, dixit s, pillai l, et al. dengue infection with multiorgan dysfunction:sofa score, arterial lactate and serum albumin levels are predictors of outcome. intensive care medicine experimental. 2015;3(1):1-2. 6. pothapregada s, kamalakannan b, thulasingham m, sampath s. clinically profi ling pediatric patients with dengue. journal of global infectious diseases. 2016;8(3):115. 7. sirivichayakul c, limkittikul k, chanthavanich p, jiwariyavej v, chokejindachai w, pengsaa k, et al. dengue infection in children in ratchaburi, thailand: a cohort study. ii. clinical manifestations. plos neglected tropical diseases. 2012;6(2):e1520. 8. arora m, patil rs. cardiac manifestation in dengue fever. j assoc physicians india. 2016;64(7):40-4. 9. suppiah j, ching s-m, amin-nordin s, mat-nor l-a, ahmad-najimudin n-a, low gk-k, et al. clinical manifestations of dengue in relation to dengue serotype and genotype in malaysia: a retrospective observational study. plos neglected tropical diseases. 2018;12(9):e0006817. 10. neeraja m, teja v, lavanya v, priyanka e, subhada k, parida m, et al. unusual and rare manifestations of dengue during a dengue outbreak in a tertiary care hospital in south india. archives of virology. 2014;159(7):1567-73. 11. pothapregada s, kamalakannan b, thulasingam m. clinical profile of atypical manifestations of dengue fever. the indian journal of pediatrics. 2016;83(6):493-9. 12. verma r, sahu r, holla v. neurological manifestations of dengue infection: a review. journal of the neurological sciences. 2014;346(1-2):26-34. 13. suwarto s, nainggolan l, sinto r, eff endi b, ibrahim e, suryamin m, et al. dengue score: a proposed diagnostic predictor for pleural eff usion and/or ascites in adults with dengue infection. bmc infectious diseases. 2016;16(1):1-7. 14. del giudice m, gangestad sw. rethinking il-6 and crp: why they are more than inflammatory biomarkers, and why it matters. brain, behavior, and immunity. 2018;70:61-75. the authors declare that they have no conflict of interest. conflict of interest acknowledgement agustin iskandar, et al.: correlation analysis between ratio 141 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 15. sproston nr, ashworth jj. role of c-reactive protein at sites of infl ammation and infection. frontiers in immunology. 2018;9:754. 16. ranzani ot, zampieri fg, forte dn, azevedo lcp, park m. c-reactive protein/albumin ratio predicts 90-day mortality of septic patients. plos one. 2013;8(3):e59321. 17. grander w, dünser m, stollenwerk b, siebert u, dengg c, koller b, et al. c-reactive protein levels and post-icu mortality in nonsurgical intensive care patients. chest. 2010;138(4):856-62. 18. lovera d, martinez de cuellar c, araya s, amarilla s, gonzalez n, aguiar c, et al. clinical characteristics and risk factors of dengue shock syndrome in children. the pediatric infectious disease journal. 2016;35(12):1294-9. 19. anker m, arima y. male–female diff erences in the number of reported incident dengue fever cases in six asian countries. western pacifi c surveillance and response journal: wpsar. 2011;2(2):17. 20. lam pk, tam dth, diet tv, tam ct, tien nth, kieu ntt, et al. clinical characteristics of dengue shock syndrome in vietnamese children: a 10-year prospective study in a single hospital. clinical infectious diseases. 2013;57(11):1577-86. 21. hung nt, lan nt, lei h-y, lin y-s, le bich l, huang k-j, et al. association between sex, nutritional status, severity of dengue hemorrhagic fever, and immune status in infants with dengue hemorrhagic fever. the american journal of tropical medicine and hygiene. 2005;72(4):370-4. 22. kalayanarooj s, nimmannitya s. is dengue severity related to nutritional status. southeast asian j trop med public health. 2005;36(2):378-84. 23. widiyati mmt, laksanawati is, prawirohartono ep. obesity as a risk factor for dengue shock syndrome in children. paediatrica indonesiana. 2013;53(4):18792. 24. liao w, jeff erson ls, taylor jm. changes in plasma albumin concentration, synthesis rate, and mrna level during acute infl ammation. american journal of physiology-cell physiology. 1986;251(6):c928-c34. 25. fairclough e, cairns e, hamilton j, kelly c. evaluation of a modifi ed early warning system for acute medical admissions and comparison with c-reactive protein/ albumin ratio as a predictor of patient outcome. clinical medicine. 2009;9(1):30. 26. napoleon tatura sn, kalensang p, mandei jm, wahyuni s, yusuf i, daud d. albumin level as a predictor of shock and recurrent shock in children with dengue hemorrhagic fever. critical care & shock. 2017;20(2). 27. menon v, greene t, wang x, pereira aa, marcovina sm, beck gj, et al. c-reactive protein and albumin as predictors of all-cause and cardiovascular mortality in chronic kidney disease. kidney international. 2005;68(2):766-72. 28. wei x-l, wang f-h, zhang d-s, qiu m-z, ren c, jin y, et al. a novel infl ammation-based prognostic score in esophageal squamous cell carcinoma: the c-reactive protein/albumin ratio. bmc cancer. 2015;15(1):1-11. 29. xie q, zhou y, xu z, yang y, kuang d, you h, et al. the ratio of crp to prealbumin levels predict mortality in patients with hospital-acquired acute kidney injury. bmc nephrology. 2011;12(1):1-8. 30. liu z, jin k, guo m, long j, liu l, liu c, et al. prognostic value of the crp/alb ratio, a novel infl ammation-based score in pancreatic cancer. annals of surgical oncology. 2017;24(2):561-8. indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 136–142 firda typewritten text 142 vol. 10 no. 1 january–april 2022 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ research article cat’s liver disease detection with sgot and sgpt evaluation as a gold standard diagnosis kurnia desiandura1*, asih rahayu1, freshinta jellia wibisono1 1faculty of veterinary medicine, wijaya kusuma surabaya university, surabaya, indonesia. received: 10th december 2021; revised: 9th january 2022; accepted: 10th january 2022 abstract sgot and sgpt are two enzymes found in the liver in large amounts. therefore, elevated levels of these two enzymes in the blood indicate liver disease. this study aims to identify liver disease in cats in surabaya through the levels of sgot and sgpt in the blood as the gold standard of diagnosis. samples came from stray cats and domesticated cats of random age, breed, and sex. the blood samples collected were 62 samples, consisting of 33 domestic cats and 29 samples from stray cats. this study showed that from 33 samples of domesticated cats, 19 samples had higher than normal levels of sgot, and from 29 samples of stray cats, 27 samples had higher than normal levels of sgot. for sgpt levels, from 33 samples of domesticated cats, six samples had higher than normal levels of sgpt, and from 29 samples of stray cats, six samples had higher levels of sgpt than average. data analysis used an independent sample t-test with spss for windows with a signifi cance level of 0.05. the data analysis results showed no signifi cant diff erence, which means that the high levels of cat sgot and sgpt enzymes did not signifi cantly aff ect the origin of the cat. therefore, it can be concluded that high levels of sgot and sgpt as the gold standard for detecting liver diseases can occur in all cats, including stray cats and domesticated cats. keywords: sgot, sgpt, blood chemistry, liver disease, cats abstrak sgot dan sgpt merupakan 2 enzim yang ditemukan pada organ liver dalam jumlah besar. peningkatan kadar kedua enzim tersebut dalam darah, merupakan salah satu indikasi adanya penyakit pada liver. penititian ini bertujuan untuk mengidentifi kasi adanya penyakit liver pada kucing-kucing di surabaya melalui kadar sgot dan sgpt dalam darah sebagai gold standar diagnosis. sampel berasal dari kucing liar dan kucing peliharaan dengan umur, breed, dan jenis kelamin acak. koleksi sampel darah yang didapatkan sebanyak 62 sampel, terdiri dari 33 sampel berasal dari kucing peliharaan, dan 29 sampel berasal dari kucing liar. hasil penelitian ini menunjukkan bahwa dari 33 sampel kucing peliharaan terdapat 19 sampel yang mempunyai kadar sgot lebih tinggi dari normalnya dan dari 29 sampel kucing liar terdapat 27 sampel yang mempunyai kadar sgot lebih tinggi dari normalnya. untuk kadar sgpt, dari 33 sampel kucing peliharaan terdapat 6 sampel yang mempunyai kadar sgpt lebih tinggi dari normalnya dan dari 29 sampel kucing liar terdapat 6 sampel yang mempunyai kadar sgpt lebih tinggi dari normalnya. analisis data menggunakan independent sample t-test dengan spss for windows dengan taraf signifi kasi 0,05. hasil analisis data menunjukkan bahwa tidak terdapat perbedaan yang nyata yang artinya tingginya kadar enzim sgot dan sgpt kucing tidak berpengaruh nyata terhadap asal usul kucing. dapat disimpulkan bahwa kadar sgot dan sgpt yang tinggi sebagai gold standar untuk mendeteksi penyakit liver dapat terjadi pada semua kucing, termasuk kucing liar dan kucing domestik. kata kunci: sgot, sgpt, kimia darah, penyakit liver, kucing * corresponding author: kurniadesiandura@uwks.ac.id ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 49kurnia desiandura, et al.: cat’s liver disease detection with sgot and sgpt how to cite: desiandura, k., rahayu, a., wibisono, f. j., cat’s liver disease detection with sgot and sgpt evaluation as a gold standard diagnosis. indonesian journal of tropical and infectious disease, 10(1), p. 48–54, apr. 2022. introduction the liver is the largest organ in the body.1 two enzymes synthesized in the liver and found in large amounts are sgot (serum glutamic oxaloacetic transaminase) and sgpt (serum glutamic pyruvic transaminase). sgot is an enzyme found in the cytosol of liver hepatocytes, but it is also present in the heart, skeletal muscle, kidneys and brain. therefore, the examination of sgot levels is a biochemical marker to determine the process of necrosis that occurs in the liver. sgpt is an enzyme whose direct synthesis is in liver tissue with the highest activity in the cytosol and mitochondria of hepatocytes. this enzyme is also found in skeletal muscle and cardiac cells, albeit in low concentrations.2 liver damage can cause sgot and sgpt to leak into the bloodstream.3 thus, sgot and sgpt can be better indicators than others to detect liver damage because these two enzymes will increase fi rst, and the increase is more signifi cant when compared to other enzymes. in general, cats’ lives are divided into two, that is, cats living by being kept by the community and cats living stray. stray cats are cats whose breeding is not controlled, the population continues to increase, there are no owners, and they live roaming and foraging in public places that provide food.4 cats are included among crepuscular mammals that have been associated with humans for more than 9,500 years.5 like humans, the cat’s body, is also composed of several systems to live everyday life, including the digestive system, musculoskeletal, nervous, endocrine, respiratory system, integumentary system, reproduction, secretory and urinary system, immune system, and circulatory system. six organs work well to carry out the functions of the system type as well. one of the most important organs is the liver. the liver is a central organ because of its essential function, such as playing a role in regulating and regulating metabolism, hormone and protein synthesis, and infl uencing the immune response and clearing toxins from the bloodstream. a problem that aff ects liver function is liver disease.7 the liver, with a similar role, also synthesizes the enzymes sgot and sgpt. thus, both stray cats and domesticated cats can be aff ected by liver disease. sgot and sgpt are considered the most eff ective because these enzymes will increase fi rst and more drastically when compared to other enzymes if the liver is damaged.8 a comparison is needed whether a cat that is properly cared for with a regular life, which we call a domesticated cat, will be at lower risk of liver disease than stray cats. so, this study aims to determine the levels of sgot and sgpt obtained from cat blood samples from stray cats and domesticated cats in surabaya through a blood chemistry laboratory examination. in addition, these results obtained evaluation materials to compare sgot and sgpt values from the two, which are then used as a standard gold diagnosis for liver disease in cats. materials and methods materials cat blood samples randomly selected were obtained from two different environments, a total 33 samples from domesticated cats with pet owners living in surabaya and 29 samples from stray cats from four different markets, namely: pacar keling market, pucang market, wonokromo market and keputran market. a sampling of domesticated cat blood was carried out at the physiological laboratory of the faculty of veterinary medicine, wijaya kusuma university, but for stray cat blood samples, it was directly carried out on the spot at the markets. pacar medical laboratory surabaya is a place to check the levels of sgot and sgpt samples. all the research procedures were conducted from january to february 2021. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 50 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 48–54 methods a. sample collection the sample was used for a blood chemistry test to determine cat sgot and sgpt levels in cat blood serum. blood was taken as much as 1-3 cc using an iv catheter or a 3cc syringe at the location of the anterior antebrachial cephalic vein, saphenous vein, or jugular vein. the blood taken was accommodated in a plain tube (non-edta) so that serum was obtained. b. laboratory test the tube containing the serum sample was then labelled and stored in a styrofoam box with icebox gel and then sent to the pacar medical laboratory surabaya for sgot and sgpt examination. c. data analysis the results of the data obtained were then tabulated, and compared with the reference values of normal cat sgot and sgpt, so that data were obtained for cats that experienced an increase in sgot and sgpt both in stray cats and in domesticated cats. then it continued with the independent t-test with the spss program.9 results and discussion the following are the levels of sgot and sgpt from the results of laboratory examinations of domesticated cats and stray cats shown in table 1. table 1. sgot and sgpt levels in domesticated cats no date cat’s name sgot (u/l) des sgpt (u/l) des 1 13/1/21 k.yuka 141 h 422 h 2 13/1/21 k.ciki 38 h 111 3 13/1/21 k.ninis 52 h 136 h 4 13/1/21 k.uprid 32 110 5 20/1/21 k.milo 42 h 89 no date cat’s name sgot (u/l) des sgpt (u/l) des 6 20/1/21 k.ibi 34 h 77 7 20/1/21 k.pesy 21 46 8 20/1/21 k.mila 179 h 477 h 9 20/1/21 k.meme 22 58 10 20/1/21 k.mochi 24 53 11 20/1/21 k.gembul 28 124 h 12 20/1/21 k.keke 25 175 h 13 20/1/21 k.onix 52 h 70 14 20/1/21 k.dudung 24 25 15 20/1/21 k.kecil 47 h 39 16 20/1/21 k.kumal 45 h 74 17 20/1/21 k.yello 28 56 18 20/1/21 k.haruka 22 50 19 20/1/21 k.coki 30 67 20 20/1/21 k.momo 25 47 21 20/1/21 k.gendut 33 h 63 22 20/1/21 k.doski 50 64 23 27/1/21 k. shawn 63 h 90 24 27/1/21 k. pipo 34 h 52 25 27/1/21 k. miu 71 h 69 26 27/1/21 k. kimmi 109 h 102 27 27/1/21 k. cici 32 49 28 27/1/21 k. moki 24 52 29 27/1/21 k. siko 40 h 59 30 03/2/21 k.moeza 91 h 73 31 03/2/21 k. tong 185 h 390 h 32 03/2/21 k.marvel 57 h 75 33 03/2/21 k. moi 36 h 94 * normal lab values feline from idexx lab sgot = 0.00-32.00 u/l (h= high) sgpt=12-115 u/l (h= high) table 2. sgot and sgpt levels in stray cats no date market name code sgot (u/l) des sgpt (u/l) des 1 21/1/21 pasar pacar keling surabaya pck 1 65 h 97 2 21/1/21 pck 2 20 40 3 21/1/21 pck 3 75 h 110 h 4 21/1/21 pck 4 34 h 76 5 21/1/21 pck 5 38 h 57 6 21/1/21 pck 6 43 h 78 7 21/1/21 pck 7 80 h 123 h 8 21/1/21 pck 8 45 h 68 9 21/1/21 pck 9 42 h 83 10 21/1/21 pck10 41 h 59 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 51kurnia desiandura, et al.: cat’s liver disease detection with sgot and sgpt no date market name code sgot (u/l) des sgpt (u/l) des 11 21/1/21 pasar pucang surabaya pc 1 70 h 85 12 21/1/21 pc 2 75 h 145 h 13 21/1/21 pc 3 32 48 14 21/1/21 pc 4 38 h 76 15 21/1/21 pc 5 128 h 238 h 16 21/1/21 pc 6 45 h 60 17 21/1/21 pc 7 58 h 67 18 21/1/21 pc 9 65 h 149 h 19 21/1/21 pc 10 35 h 67 20 04/2/21 pasar keputran surabaya k1 48 h 64 21 04/2/21 k3 60 h 59 22 04/2/21 k8 86 h 116 h 23 04/2/21 k9 65 h 47 24 04/2/21 k10 60 h 66 25 04/2/21 pasar wonokromo surabaya w1 42 h 66 26 04/2/21 w2 78 h 79 27 04/2/21 w5 50 h 55 28 04/2/21 w7 64 h 89 29 04/2/21 w8 68 h 93 * normal lab values feline from idexx lab sgot = 0.00-32.00 u/l (h= high) sgpt= 12-115 u/l sgot, sgpt, arginase, lactate dehydrogenase and gamma glutamyl transaminase are enzymes present in the liver. still, they are free to leave the cells and enter the blood vessels beyond their normal levels when damage to the liver parenchyma cell occurs as shown in table 2. however, sgot and sgpt are considered the most eff ective because these enzymes will increase fi rst and more drastically when compared to other enzymes if the liver is damaged.8 examining the levels of sgot and sgpt in domesticated and stray cats will be one indicator to detect the presence of liver disease in these cats. figure 1. sgot chart in domesticated cat figure 2. sgot chart in stray cat figure 3. sgpt chart in domesticated cat figure 4. sgpt chart in stray cat by comparing the expected values of sgot/ ast and sgpt/alt for cats the results of this study showed that, from 33 samples of ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 52 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 48–54 domesticated cats, 19 had higher levels of sgot than expected (58%) as shown in figure 1, and from 29 samples of stray cats, 27 had high levels of sgot. in addition, sgot was higher than expected (93%) as shown in figure 2. for sgpt levels, from 33 domesticated cats, six samples had higher than normal levels of sgpt (21%) as shown in figure 3, and from 29 samples of stray cats, six s had higher than normal levels of sgpt (18%) as shown in figure 4. table.3. mean value sgot in domesticated cat and stray cat cat mean signifi cance domesticated 107.212 t hit < t table 1.049 < 2.000 sig. (2 tailed) 0.299 > 0.05 not signifi cantly stray 84.827 table. 4. mean value sgpt in domesticated cat and stray cat cat mean signifi cance domesticated 52.606 t hit < t table 0,49 < 2,000 sig. (2 tailed) 0,626 > 0,05 not signifi cantly stray 56.896 analysis of independent t-test data sgot value of all samples sig. 2-tailed > 0.05 with a result of 0.626 > 0.05, indicating that the sgot value in domesticated and stray cats was not signifi cant as shown in table 3. this also applies to the sgpt value with 0.299> 0.05, which means no signifi cant diff erence between the sgpt value in domesticated and stray cats as shown in table 4. from these results, it is explained that both stray cats and domestic cats can experience liver problems. this is evident from the eff ects of increased levels of sgot and followed by levels of sgpt, which signifi cantly increased above the average experienced by some cats. the most drastic increase in domesticated cats occurred in yuka’s cat, with an sgot level of 141 u/l and an sgpt level of 422 u/l. meanwhile, the most drastic increase in stray cats was cat blood samples taken from the pucang market with an sgot level of 128 u/l and an sgpt level of 238 u/l. e v a l u a t i o n o f t h e s g o t a n d s g p t examinations results is one of the essential indicators for diagnosing liver disease in cats, just like humans. because when the liver is damaged, the enzymes sgot, sgpt, arginase, lactate dehydrogenase and gamma glutamyl transaminase are free to leave the cells to enter the blood vessels more than expected and their levels in the blood increase.10,11 although there are other enzymes, sgot and sgpt will increase first, and the growth is more extreme when compared to other enzymes.8 the markers of liver cell abnormalities (hepatocellular) are caused by changes in permeability or damage to liver cell walls, increasing sgpt or sgot.12 increased sgot can persist in the circulation between 2-5 days, and so it is used as a biochemical marker to determine the presence of necrosis in liver cells.2 although sgot and sgpt examinations from blood results are the gold standards to indicate liver disease, other supporting diagnoses are also needed, such as x-ray results, ultrasound, results of bilirubin, and gamma glutamyl transaminase enzymes level, etc. liver disease in cats has several factors that can cause sgot and sgpt enzymes to increase, for example, viral liver disease, liver ischemia caused by prolonged hypotension or acute heart failure, and heart damage due to drugs or toxins.13,14 for example, the toxic eff ects of paracetamol in cats, which can come from a single-dose or cumulative dose manifested in methemoglobinemia and liver problem.15 paracetamol is metabolized in the liver, and the rest is metabolized in the kidney. 16 cats with an amount of 10 mg/kg b.w. can cause symptoms of paracetamol poisoning. that matters because the cat cannot metabolize paracetamol due to the defi ciency of the enzyme glucuronyl transferase.17 paracetamol contains napqi compounds that cannot be detoxifi ed so that they form free radical toxic proteins and cause damage to cat liver cells.18 in addition, paracetamol overdose can also cause hepatic cell necrosis in the centrilobular area, which causes acute liver failure.19 an example of an organophosphate that farmers often use is diazinon.20 diazinon toxin will cause various damage in tissues, especially ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 53kurnia desiandura, et al.: cat’s liver disease detection with sgot and sgpt in organs that function as detoxifi cation, namely the liver.21 another factor that can lead sgot and sgpt enzymes to increase and cause liver damage is the entry of pathogenic microorganisms such as bacteria, viruses, fungi and parasites. a fungal microorganism that causes candidiasis, candida albicans causes cases of hepatitis in male domesticated cats. as a result of this infection, the cat develops progressive hyperbilirubinemia, a nearly 10-fold increase in sgot/ast and an sgpt/alt increase of more than 18-fold from ordinary.22 changes in sgot and sgpt levels are related to the rate of protein metabolism; the level of physical activity may also infl uence cell generation.23 protein metabolism is related to the liver because it can adjust protein production with the body’s protein requirements. the protein content in the blood is infl uenced by age and growth, nutrition intake, gender, hormones, pregnancy and lactation, stress, and fl uid loss.24 likewise, the age factor affects total protein levels; at a young age, total protein levels tend to be higher.25 strenuous physical activity can damage more muscle cells than the intermediate physical state acting correctly; it causes the occurrence of excessive circulation of sgot in blood, because large amounts of serum glutamate oxaloacetate transaminase (sgot) are found in muscle cells, liver cells, and heart muscle. and small amounts are found in other cells, such as cells of the kidney, pancreas, brain and erythrocytes.26 so, it is necessary to have other continuous parameters to determine the presence of liver necrosis, that is, to check the levels of sgpt which have been considered a sensitive marker of liver disease and hepatotoxicity compared to sgot levels.27 other factors such as obesity, genetics, and immune system disorders can also cause liver disease, driving an increase in sgot and sgpt in the blood. conclusions high sgot and sgpt as the gold standard for detecting liver diseases can occur in all cats, including stray cats and domesticated cats, because these two enzymes increase to the most extreme and earlier than other enzymes when the liver is impaired. other supporting diagnoses are also needed, such as x-ray results, ultrasound results, or results of bilirubin and gamma glutamyl transaminase enzymes level. acknowledgement this research is part of the community service activities for the 11th anniversary of the faculty of veterinary medicine, wijaya kusuma surabaya university. the researchers would like to thank the faculty of veterinary medicine, wijaya kusuma surabaya university, for supporting and assisting in carrying out this research activity. conflict of interest we declare that we have no conflict of interest. references 1. hayes, m.a. 2007. pathophysiology of the liver. saunder company, usa. 2. engelking, l. r. 2011. textbook of veterinary updated second edition. 3. fristiohady, a., w. wahyuni., m. i. yusuf., f. malik., l. o. m. j. purnama., m. bafadal., m. leorita., a. jabar., m. h. malaka., i. sahidin. 2020. hepatoprotective activity of etlingera elatior (jack) r.m. smith extracts against ccl4 -induced hepatic toxicity in male wistar rats. research j. pharm. and tech. 13(10). 4. sucitrayani pte, ida bmo, & made d. 2014. prevalensi infeksi protozoa saluran pencernaan pada kucing lokal (felis catus) di denpasar. buletin veteriner udayana. 6(2): 153-159. 5. farantika, r. 2016. eksplorasi dan prevalensi jenis telur cacing pada feses kucing liar dan kucing peliharaan di kawasan kampus universitas negeri semarang. fakultas matematika dan ilmu pengetahuan alam. universitas negeri semarang. 6. purba, d. j., s. k. widyastuti., m. s. anthara. 2020. laporan kasus: hemobartonella felis pada kucing lokal. indonesia medicus veterinus. 9(2): 157-167. doi: 10.19087/imv.2020.9.2.157 7. kassahun, a. n. hepatic diseases in canine and feline: a review. 2021. vet med open j. 6(1): 22-31. doi: 10.17140/vmoj-6-155 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 54 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 48–54 8. fathoni, f. 2008. studi kadar sgpt, sgot dan total protein pada serum darah anjing kampung (canis familiaris) usia 3 dan 6 bulan. fakultas kedokteran hewan. institut pertanian bogor. 9. sujarweni, v.w. 2014. spss untuk penelitian. yogyakarta : pustaka baru press. 10. amin i. 1995. pengaruh pemberian seduhan rimpang kunyit (curcuma domestica, val) terhadap aktivitas sgpt dan sgot ayam. fakultas matematika dan ilmu pengetahuan alam, institut pertanian bogor. bogor. 11. callbreath df. 1992. clinical chemistry. w.b. saunder company, usa. 12. rosida, a. 2016. pemeriksaan laboratorium penyakit hati. berkala kedokteran. 12(1): 123-131 13. hall p, johnny c. what is the real fungtion of the liver ‘function” test. ulster med j. 2012;81:30-36. 14. suryaatmadja m. pemeriksaan laboratorium uji fungsi hati. buletin abc.2009;11:2-8 15. mcconkey se, grant dm and cribb ae. 2009. the role of para-aminophenol in acetaminophen-induced methemoglobinemia in dogs and cats. j vet pharmacol ther. 32(6): 585-595. 16. steenbergen v. 2003. acetaminophen and cats – a dangerous combination. vet technician. 24(1): 4345. 17. denzoin-vulcano la, confalonieri o, franci r, tapia m.o and soraci al. 2013. effi cacy of free glutathione and liposomal glutathione in treating acetaminopheninduced hepatotoxicity in cats. open veterinary journal. 3(1): 56-63. 18. wang x, wu q, liu a, anadon a, rodriguez jl, martinez-larrañaga r, yuan z and maría-aránzazu m. 2017. paracetamol: overdose-induced oxidative stress toxicity, metabolism, and protective eff ects of various compounds in vivo and vitro. drug met rev. 49(4):81-83. 19. rafi ta, i. d., lisdiana., a. marianti. 2015. pengaruh ekstrak kayu manis terhadap gambaran histopatologi dan kadar sgot-sgpt hepar tikus yang diinduksi parasetamol. unnes journal of life science 4 (1): 29-37. 20. wu, h., c. evreux-gros, dan j. descotes. 1996. influence of cimetidine on the toxicity and toxicokinetics of diazinon in the rat. human & experimental toxicology. 15:391–395. 21. lari, p., k. abnous, m. imenshahidi, m. rashedinia, m. razavi, dan h. hosseinzadeh. 2013. evaluation of diazinon-induced hepatotoxicity and protective eff ects of crocin. toxicology and industrial health. 1–10. 22. palermo, s. m., a.w. newman., m. w. koch. 2 0 1 9 . c a n d i d a a l b i c a n s c h o l e c y s t i t i s w i t h associated hepatitis in a cat. journal of feline medicine and surgery open reports. 1-6. doi: 10.1177/2055116919854165 23. suarsana, n., suprayogi, a., ni nyoman, w. s., tutik, w. 2006. j. vet. penggunaan ekstrak tempe terhadap fungsi hati tikus dalam kondisi stres. 24. nagyova, v., c. tothova., o. nagy. 2016. the impact of colostrum intake on the serum protein electrophoretic pattern in newborn ruminants. journal of applied animal research. https://doi.org /10.1080/09712119.2016.1218886 25. lea dan febiger. 1986. veterinary hematology 4th ed. philadelphia. 26. benyamin, m. 1980. outline of pathology. 3rd ed. the low ames, lowa. usa. 27. kim, w.r., s. l. flamm, a.m. di bisceglie., h.c. bodenheimer. 2008. special article serum activity of alanine aminotransferase (alt) as an indicator of health and disease. 1363-1370. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 74 vol. 6. no. 3 september–december 2016 literature review the usage comparison of ceftriaxone and chloramphenicol for typhoid fever treatment: an evidence based case report jeffry adijaya susatyo1a 1 faculty of medicine, universitas indonesia a corresponding author: jeffryadijaya@yahoo.com abstract typhoid fever is a disease caused by the gram-negative bacterium salmonella typhi. since its introduction in 1949, chloramphenicol has become the first-line treatment of typhoid fever for decades. until now, chloramphenicol is still the first line treatment of typhoid fever in rural areas in indonesia, due to its low cost. however, in addition to the problem of bacterial resistance, chloramphenicol is known to cause some side effect such as bone marrow suppression. currently, many other antibiotics are used as the regimens for the treatment of typhoid fever, one of which is ceftriaxone. however, there are evidences on reemergence of chloramphenicol sensitivity in typhoid fever treatment. this report is created to answer the clinical question on whether ceftriaxone is more effective compared to chloramphenicol as the first-line treatment of typhoid fever. a structured search was performed on pubmed, ebsco, and sciencedirect and after a screening process and appraisal using the criteria from center of evidence based medicine at oxford university, only one article was selected. the article shows higher efficacy of ceftriaxone in term of defervescence rate (p = 0.0001). no other study that compares the efficacy of ceftriaxone and chloramphenicol for typhoid fever treatment during the last ten years could be found during article searching. in conclusion, ceftriaxone shows better efficacy in the treatment of typhoid fever compared to chloramphenicol but with the rise of microbial sensitivity to chloramphenicol in recent years, more studies on this topic are needed to support this conclusion. keywords: typhoid fever, enteric fever, ceftriaxone, chloramphenicol, effectiveness abstrak demam tifoid merupakan penyakit disebabkan oleh bakteri gram negatif salmonella typhi. sejak diperkenalkan pada tahun 1949, chloramphenicol selama puluhan tahun menjadi lini pertama pengobatan demam tifoid. hingga saat ini chloramphenicol masih merupakan lini pertama untuk pengobatan demam tifoid di daerah-daerah di indonesia terutama karena biayanya yang murah. namun, selain masalah resistensi kuman, chloramphenicol diketahui menimbulkan efek samping berupa supresi sumsum tulang, sehingga saat ini banyak digunakan antibiotik lain sebagai rejimen pengobatan demam tifoid seperti ceftriaxone. laporan ini dibuat untuk menjawab pertanyaan klinis apakah ceftriaxone lebih efektif dibandingan chloramphenicol sebagai lini pertama untuk pengobatan demam tifoid. pencarian artikel terstruktur dilakukan pada pubmed, ebsco, dan sciencedirect. setelah proses penyaringan dan appraisal menggunakan kriteria center of evidence based medicine dari universitas oxford, didapatkan satu artikel terpilih. artikel tersebut menunjukkan efektivitas ceftriaxone dalam menurunkan demam yang lebih baik dengan p = 0,0001. tidak ditemukan penelitian lain mengenai perbandingan efektivitas ceftriaxone dengan chloramphenicol dalam menangani demam tifoid pada pencarian artikel. kesimpulan yang ditarik adalah ceftriaxone menunjukkan efektivitas yang lebih baik dalam tatalaksana demam tifoid dibandingkan dengan chloramphenicol, namun dengan meningkatnya sensitivitas bakteri terhadap chloramphenicol dalam tahun-tahun terakhir, penelitian mengenai topik ini masih sangat diperlukan. kata kunci: demam tifoid, demam tipus, ceftriaxone, chloramphenicol, efektivitas 75susatyo: the usage comparison of ceftriaxone and chloramphenicol introduction typhoid fever is a disease which is caused by gram negative bacterium salmonella typhi. it is categorized as an endemic disease in indonesia. in 2006, there are 500 cases of typhoid fever reported out of 100,000 people, with 0.65% death rate.1 since it was introduced in 1949, chloramphenicol has been used as the first-line treatment for typhoid fever. it is still preferred in many areas in indonesia due to its relatively affordable price. in many other countries, the use of chloramphenicol has been less and less because many bacteria strains have already resisted it.2,3 however, a six years’ study conducted by moehario lh et al showed that 90% of bacteria were still susceptible to this drug.4 other studies in india also showed a reemergence of chloramphenicol sensitivity in typhoid fever treatment.5–9 the recommended dose of chloramphenicol is 2000 mg per day, divided to 4 dose orally or intravenous for at least 7 days. however, aside from bacteria resistance, chloramphenicol is known to induce bone marrow suppression. with that in mind, other antibiotics are often used as a therapy regiment for typhoid, one of which is a 3rd generation cephalosporin ceftriaxone.2 aside from avoiding the said side effect, the length of treatment using ceftriaxone is shorter than chloramphenicol and can improve a patient’s adherence to the treatment. the recommended dosage for ceftriaxone is 3-4 grams in 100 cc of 40% dextrose solution per day for 3 to 5 days.4 case a 18 years old female patient arrived with a chief complaint of fever for 1 week prior to the admission. the fever was accompanied with watery stool up to 3 times a day. serological widal examination showed a positive result, thus, the patient was treated with intravenous ceftriaxone antibiotic 3 grams per day. clinical question is ceftriaxone more effective than chloramphenicol as the first-line treatment for typhoid fever? material and method the method of this study is a systematic review on some articles relevant to the topic. a structured search was performed on three databases, namely pubmed clinical queries, ebsco medline, and sciencedirect, 3 2 1 screening of duplicate articles result: 1. islam et al: treatment of typhoid-fever with ceftriaxone for 5 days or chloramphenicol for 14 days a randomized clinical-trial 2. acharya et al: treatment of typhoid fever: randomized trial of a three-day course of ceftriaxone versus a fourteen-day course of chloramphenicol 3. hammad et al: ceftriaxone versus chloramphenicol for treatment of acute typhoid fever chloramphenicol and ceftriaxone and (typhoid or “enteric fever”) pubmed ebsco science direct 62 12 9 screening of titles and abstracts inclusion criteria: -typhoid fever treatment with ceftriaxone chloramphe nicol as control exclusion criteria: not in accordance with the clinical question figure 1. article searching method and result 76 indonesian journal of tropical and infectious disease, vol. 6. no. 3 september–december 2016: 74–77 table 1. keywords and filters for article searching keywords filter pubmed clinical queries chloramphenicol and ceftriaxone and (typhoid or “enteric fever”) therapy; broad human species, english language, full text available ebsco medline chloramphenicol [ab abstract] and ceftriaxone [ab abstract] and and (typhoid or “enteric fever”) [ab abstract] human, english, full text available science direct chloramphenicol and ceftriaxone and (typhoid or “enteric fever”) journal table 2. the critical appraisal of articles validity islam et al antimicrobial agents and chemotherapy (1993) acharya et al american journal of tropical medicine and hygiene (1995) hammad et al life science journal (2011) was the assignment of patients to treatments randomized? yes yes yes were all patients who entered the trial accounted for at its conclusion? yes yes yes were patients and clinicians kept “blind” to which treatment was being received? yes yes no aside from the experimental treatment, were the groups treated equally? yes yes yes were the groups similar at the start of the trial? yes yes yes using chloramphenicol and ceftriaxone and (typhoid or “enteric fever”) as the keywords (table 1). from those keywords, we found as many as 62 articles from pubmed, 12 articles from ebsco, and 9 articles from science direct. the title and abstract of those articles were then screened (as seen on figure 1) with inclusion criteria being: (1) a trial on typhoid fever treatment with ceftriaxone and (2) chloramphenicol as control. the articles found were as follows: (1) treatment of typhoid-fever with ceftriaxone for 5 days or chloramphenicol for 14 days a randomized clinical-trial, by islam et al; (2) treatment of typhoid fever: randomized trial of a three-day course of ceftriaxone versus a fourteen-day course of chloramphenicol, by acharya et al; and (3) ceftriaxone versus chloramphenicol for treatment of acute typhoid fever, by hammad et al.10 these articles were appraised using the criteria from center of evidence based medicine oxford university (table 2). articles by islam et al and acharya et al were published more than 20 years ago and therefore are not included in this review. result and discussion hammad et al did a study on 2007 to re-asses the effectiveness of chloramphenicol as typhoid treatment in response to the increase of multidrug resistance to the firstline antimicrobials in egypt for the last 30 years.10 fifty-two patients of acute typhoid fever with positive blood culture for salmonella typhi were divided into 2 groups. twenty-seven patients were randomly allocated to be treated with chloramphenicol (50 mg/kg bw/day orally or intravenously) which is given 6 times hourly until defervescence for further 5 days.10 twenty five patients were randomly allocated to be treated with ceftriaxone parenterally (80 mg/kg/day for children and 2 gm/day for adults) the treatment is given once a day for 7 days.10 clinical cure occurred on all patients. the mean time (mean±sd) of defervescence for ceftriaxone and chloramphenicol was 3.3±1.2 and 5.8±1.2 days respectively (p = 0.0001, 95% ci = 1.8-3.2). ceftriaxone treatment showed a shorter time of defervescence compared to chloramphenicol.10 we found only one article on pubmed clinical queries, ebsco medline, and sciencedirect using center of evidence based medicine oxford university criteria. a study by hammad et al showed that ceftriaxone has more efficacy than chloramphenicol in treating typhoid fever. ceftriaxone treatment had a shorter time o f d e f e r v e s c e n c e ( 3 . 3 ± 1 . 2 d a y s ) c o m p a r e d t o chloramphenicol (5.8±1.2 days). this study also showed an increased risk of bone marrow suppression in using chloramphenicol as a 77susatyo: the usage comparison of ceftriaxone and chloramphenicol treatment. it was showed by the decreased of hematocrit mean value compared to the ceftriaxone group. unfortunately, no other clinical trial that compares the efficacy of ceftriaxone treatment and chloramphenicol treatment in the last 10 years was found during article searching. although ceftriaxone showed better efficacy and less side effect, chloramphenicol treatment can still be considered effective in treating typhoid. all patients experienced clinical cure after being treated with either ceftriaxone or chloramphenicol. this can be considered an improvement from years ago when chloramphenicol was rendered ineffective as a treatment because of widespread microbial resistance.7 conclusion in conclusion, the use of chloramphenicol is still effective for the treatment of typhoid fever. however, ceftriaxone showed greater effectiveness in typhoid fever treatment as shown by shorter time of defervescence compared to chloramphenicol. the use of ceftriaxone also poses less risk on bone marrow suppression compared to cephalosporin. another advantage of using ceftriaxone as a treatment is the shorter length of treatment which can improve a patient’s adherence to the treatment. only one clinical trial was found from article searching and with the rise of microbial sensitivity to chloramphenicol in recent years5–9, more studies on this topic are needed to support this conclusion. references 1. herawati mh, ghani l. hubungan faktor determinan dengan kejadian tifoid di indonesia tahun 2007 (association of determinant factors with prevalence of typhoid in indonesia). 2009;xix(4):165–73. 2. longo dl, harrison tr, kasper d, jameson j, fauci a, hauser s, et al. harrison’s principles of internal medicine. 18th ed. /. united states: new york : mcgraw-hill, 2012; 2012. 1277 p. 3. department of vaccines and biologicals. background document: the diagnosis, treatment, and prevention of typhoid fever. world heal organ. 2003;19–20. 4. sudoyo aw, bambang setiyohadi, alwi i, simadibrata m, setiati s. buku ajar ilmu penyakit dalam edisi v. 2009. 2797-2801 p. 5. bhatia j, mathur a, arora m. reemergence of chloramphenicol sensitivity in enteric fever. med j armed forces india. 2007 jul;63(3):212–4. 6. harish bn, menezes ga. preserving efficacy of chloramphenicol against typhoid fever in a tertiary care hospital, india. who southeast asia reg reg heal forum. 2011;volume 15(number 1):92–6. 7. butler t. treatment of typhoid fever in the 21st century: promises and shortcomings. clin microbiol infect. 2011 jul;17(7):959–63. 8. jog s, soman r, singhal t, rodrigues c, mehta a, dastur fd. enteric fever in mumbai--clinical profile, sensitivity patterns and response to antimicrobials. j assoc physicians india. 2008 apr;56:237–40. 9. krishnan p, stalin m, balasubramanian s. changing trends in antimicrobial resistance of salmonella enterica serovar typhi and salmonella enterica serovar paratyphi a in chennai. indian j pathol microbiol. 52(4):505–8. 10. hammad om, hifnawy t, omran d, el tantawi ma, girgis ni. ceftriaxone versus chloramphenicol for treatment of acute typhoid fever. life sci j. 2011;8(2):100–5. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 2 may–august 2022 original article bacterial profile and antibiotic resistance pattern among children with urinary tract infections in dr. soetomo hospital, surabaya, indonesia fauziah adhima1 , manik retno wahyunitisari2,3* , risky vitria prasetyo4 , rebekah juniati setiabudi2,3 1medical program, faculty of medicine, universitas airlangga, surabaya, indonesia 2departement of medical microbiology, faculty of medicine, universitas airlangga − dr. soetomo general academic hospital, surabaya, indonesia 3clinical microbiology specialist program, faculty of medicine, universitas airlangga − dr. soetomo general academic hospital, surabaya, indonesia 4division of nephrology, department of child health, faculty of medicine, universitas airlangga − dr. soetomo general academic hospital, surabaya, indonesia received: january 14th, 2022; revised: february 10th, 2022; accepted: april 13th, 2022 abstract urinary tract infections (utis) are the most common infections in pediatric patients characterized by the growth of bacteria in the urine in significant numbers. antibiotics remain the primary treatment of uti in children. however, there has been an increase in antibiotic resistance to uropathogens worldwide due to their inappropriate and extensive uses. there is considerable geographical variation in the distribution of bacteria and antibiotic resistance pattern. thus, to prevent further resistance and provide empirical antibiotic options, this study aims to determine the profile of bacteria and antibiotics resistance pattern among uti pediatric patients in dr. soetomo hospital. this study was performed by collecting data from the urine culture logbook at the clinical microbiology laboratory of dr. soetomo hospital in july-october 2019. the sample was uti patients aged one day – 18 years due to bacterial infection with a colony count of ≥100,000 cfu/ml. in this study, 131 patients showed significant bacterial growth dominated by males and ages one month – 2 years. uti were caused by gram-negative bacteria (74%) and gram-positive bacteria (26%), with the most bacteria found in each group were escherichia coli and enterococcus faecalis. e. coli showed ≥70% resistance to ampicillin, cefazoline, piperacillin, tetracycline, and trimethoprim-sulfamethoxazole. comorbidities were dominated by hydronephrosis (10.98%), chronic kidney disease (9.79%) and hydrocephalus (8.09%). in conclusion, gram-negative bacteria were the leading cause of uti in children with e. coli as the most common uropathogen, highly resistant to ampicillin and cefazolin. grampositive bacteria were less frequent with varied resistance patterns. most common comorbidity was hydronephrosis. keywords: antibiotic resistance; bacterial pathogen; urinary tract infection abstrak infeksi saluran kemih (isk) merupakan penyakit infeksi yang banyak dijumpai pada anak ditandai dengan pertumbuhan bakteri urin dalam jumlah yang signifikan. pengobatan isk anak utamanya dengan pemberian antibiotik. namun, telah terjadi peningkatan resistensi antibiotik terhadap uropatogen di seluruh dunia akibat penggunaan yang kurang tepat dan terlalu ekstensif. variasi geografis dalam distribusi bakteri penyebab isk dan pola resistensi antibiotiknya juga cukup besar. untuk mencegah * corresponding author: manik-r-w@fk.unair.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-1649-4688 https://orcid.org/0000-0002-1649-4688 https://orcid.org/0000-0002-1649-4688 https://orcid.org/0000-0002-1649-4688 124 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fauziah adhima, et al. bacterial profile and antibiotic resistance pattern resistensi lebih lanjut dan memberikan pilihan antibiotik empiris, penelitian ini diperlukan untuk mengetahui profil bakteri dan pola resistensi antibiotik pada pasien anak isk di rsud dr. soetomo. penelitian ini dilakukan dengan menggunakan data sekunder berupa catatan hasil kultur urin di laboratorium mikrobiologi klinik rsud dr. soetomo pada bulan juli-oktober 2019 dengan sampel pasien isk anak berusia 1 hari – 18 tahun akibat infeksi bakteri dengan hitung koloni sebanyak ≥100,000 cfu/ml. dalam penelitian ini, 131 pasien menunjukkan pertumbuhan bakteri signifikan, yang didominasi oleh laki-laki dan usia 1 bulan – 2 tahun. isk disebabkan oleh bakteri gram negatif (74%) dan gram positif (26%) dengan bakteri terbanyak yang ditemukan pada masingmasing kelompok adalah escherichia coli dan enterococcus faecalis. e. coli menunjukkan resistensi ≥70% terhadap ampisilin, sefazolin, piperasilin, tetrasiklin, dan trimetoprim-sulfametoksazol. penyakit penyerta pada pasien isk anak didominasi oleh hidronefrosis (10,98%), penyakit ginjal kronis (9,79%), dan hidrosefalus (8,09%). sehingga dapat disimpulkan bahwa bakteri gram negatif merupakan penyebab utama isk anak dengan e. coli sebagai uropatogen yang paling sering dijumpai, yang resisten terhadap ampisilin dan cefazolin. sedangkan bakteri gram positif lebih jarang ditemukan dengan pola resistensi yang bervariasi. penyakit penyerta pasien terbanyak adalah hidronefrosis. kata kunci: bakteri patogen; infeksi saluran kemih; resistensi antibiotik how to cite: adhima, f., wahyunitasari, m, r. prasetyo, r, v., setiabudi, r, j. bacterial profile and antibiotic resistance pattern among children with urinary tract infections in dr. soetomo hospital, surabaya, indonesia. indonesian journal of tropical and infectious disease. 10(2). p. 123–136. aug. 2022. introduction urinary tract infection (uti) is the second most common infectious disease in children after respiratory tract infection characterized by the growth of bacteria in the urine in significant numbers.1,2 mostly, uti in children are caused by gram-negative bacteria with escherichia coli as the most common uropathogen.3 uti in children are often underdiagnosed due to their nonspecific signs and symptoms, especially in neonates and infants4, such as fever, decreased appetite, vomiting, diarrhea, jaundice, abdominal distension, weight loss, and failure to thrive.2 in addition, pediatric utis are commonly associated with various congenital abnormalities of the urinary tract, such as posterior urethral valves, ureteropelvic junction obstruction, neurogenic bladder, urethral stricture, and vesicoureteral reflux, which can lead to recurrent utis.5 if the patient is not treated promptly, complications such as renal scarring, hypertension, or chronic kidney disease, will develop progressively. thus, it is necessary to give empirical antibiotics based on local antimicrobial susceptibility patterns as initial therapy before the urine culture results are available.3 globally, uti in pediatric are estimated around 150 million cases annually.6 in the united states, there are an estimated 1.5 million cases of uti in pediatric outpatients.7 while at dr. soetomo hospital surabaya, indonesia, it obtained 94 urine samples among children with uti within two months.8 the incidence of uti in children is more common in girls (8%) than boys (2%).9 boys have a greater incidence of uti than girls with a ratio of 2:1 to 5:1 in the neonatal period and early infancy.3,10 in addition, the increasing prevalence of antimicrobial resistance among uropathogens over the past few decades also complicates uti management.3 the national healthcare safety network (nhsn) in the united states reported that an increase in multidrugresistant gram-negative bacteria was found in 2,039 hospitals.11 a study from south india demonstrated that extended-spectrum betalactamase (esbl) production was detected in 53% of isolates from patients with community-acquired bacteremia caused by e. coli and klebsiella spp.12 in the recent years, the increasing trend of bacterial uropathogen resistance against commonly used antimicrobials has become a major concern worldwide. antibiotic susceptibility patterns vary widely between different geographic areas. in a study in ethiopia showed that e. coli, as the most common isolated uropathogen, was resistant 125 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 123–136 to ampicillin (100%) and nitrofurantoin (78.6%) whereas sensitive to ciprofloxacin (71.4%), norfloxacin (71.4%) and ceftriaxone (57.1%).6 in nepal, the percentage of sensitivity for e. coli were high for nitrofurantoin, ceftriaxone, amikacin, gentamicin, and ofloxacin, while a high level of resistance was observed for ampicillin and cotrimoxazole.13 a study by patwardhan et al. in north india reported that the incidence of resistance to ampicillin, amoxiclav, nitrofurantoin, co-trimoxazole, and norfloxacin increased significantly over a five-year period. this situation is certainly very concerning because the complexity of uti treatment can increase the risk of longterm consequences in children.14 given the high prevalence of antibiotic resistance worldwide with the diversity of resistance patterns between geographic areas that change easily over time, continuous monitoring of uropathogens and local antibiotic resistance patterns is needed as a basic consideration in selecting empiric pharmacotherapy which is important to optimize the initial management of pediatric utis to reduce risk of unexpected complications.4 studies recommend that policies for uti treatment in children should be re-evaluated every five years according to local resistance levels.15 hence, this study was conducted to assess the prevalence of bacterial uropathogens and their susceptibility patterns to antibiotic agents amongst pediatric patients with uti in dr. soetomo hospital. materials and methods study design this descriptive retrospective study was conducted at the clinical microbiology laboratory of dr. soetomo hospital, surabaya, from september 2020 to june 2021. data on age, sex, urine culture, antibiotic sensitivity, and patient comorbidities were obtained from the urine culture logbook in july-october 2019. samples were collected using consecutive sampling techniques from pediatric patients aged one day – 18 years with uti (inpatient and outpatients). the diagnosis of uti was established when the result of the bacterial colony count was >100,000 colony-forming units per millilitre (cfu/ml).17 bacterial identification and antibiotic susceptibility test were carried out using the automatic microdilution method, bd pheonix and vitek, validated and interpreted by clinical laboratory standard international (clsi). patients with incomplete urine examination data and medical records were excluded from this study. statistical analysis the data were analyzed descriptively with statistical package for the social sciences (spss) 16.0 and microsoft excel resulted in the distribution of the number and percentage of each variable. ethical approval this research received ethical approval from the health research ethics committee of dr. soetomo hospital on november 26, 2020, with the letter number 0225/loe/301.4.2/xi/2020. results and discussion characteristics of pediatric uti patients based on the urine culture logbook in pediatric uti in july-october 2019, there were 211 data on patients aged one day – 18 years who performed urine culture examinations and antibiotic sensitivity tests at the clinical microbiology laboratory of dr. soetomo hospital. however, significant bacterial growth (≥100,000 cfu/ml) was found in 131 patients and was dominated by boys (54.2%). based on age, the results showed that uti in children mainly occurred in the age group of one month – 2 years. if we look at the distribution of age by sex (table 1), the results show that most boys are found in the age group of one month – 2 years, while most girls are found in the age group of 6–12 years. 126 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fauziah adhima, et al. bacterial profile and antibiotic resistance pattern table 1. age and sex distribution age sex total n (%) girl boy n (%) n (%) 0-30 days 0(0.00) 2(1.53) 2(1.53) 1 month 2 years 15(11.45) 23(17.56) 38(29.01) 2-6 years 8(6.11) 18(13.74) 26(19.85) 6-12 years 19(14.50) 15(11.45) 34(25.95) 12-18 years 18(13.74) 13(9.92) 31(23.66) total 60(45.80) 71(54.20) 131(100.00) bacteria isolation bacteria causing uti were dominated by gram-negative bacteria (74%) followed by gram-positive bacteria (26%). the most common gram-negative bacteria were e. coli (30.5%) while the most common grampositive bacteria were e. faecalis (8.4%). all the data are shown in figure 1. in this study, there were 17 isolates of e. coli and eight isolates of k. pneumoniae esbl-producing gram-negative bacteria. figure 1. distribution of bacteria causing uti gram-negative bacteria resistance pattern in this study, e. coli, p. aeruginosa, k. pneumoniae, e. cloacae, and a. baumannii, showed resistance to ampicillin and cefazolin. e. coli was found to be resistant to ampicillin, cefazolin, piperacillin, sulfamethoxazole, trimethoprimand tetracycline for about more than 70%. in contrast to p. aeruginosa which was resistant to more antibiotics such as ampicillin, cefazolin, amoxicillin-clavulanate, ampicillin-sulbactam, chloramphenicol, cefotaxime, nitrofurantoin, tetracycline, tigecycline, trimethoprim-sulfamethoxazole and ceftriaxone (table 3). in addition, the five most common gramnegative bacteria showed high sensitivity to amikacin, imipenem, meropenem, and piperacillin-tazobactam, as shown in table 3. e. coli was also sensitive to tigecycline, nitrofurantoin, gentamicin, and cefoperazonesulbactam, while p. aeruginosa was also found to be sensitive to piperacillin, gentamicin, and ceftazidime. 127 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 123–136 table 3. distribution of antibiotic resistance in gram-negative bacteria antibiotic e. coli (n=40) p. aeruginosa (n=14) k. pneumoniae (n=12) e. cloacae (n=8) a. baumanii (n=5) amikacin r (%) 0/40 (0.00) 1/14 (7.14) 2/12 (16.67) 4/8 (50.00) 0/5 (0.00) i (%) 0/40 (0.00) 1/14 (7.14) 1/12 (8.33) 0/8 (0.00) 0/5 (0.00) s (%) 40/40 (100.00) 12/14 (85.71) 9/12 (75.00) 4/8 (50.00) 5/5 (100.00) amoxicillinclavulanate r (%) 13/40 (32.50) 14/14 (100.00) 4/12 (33.33) 8/8 (100.00) 5/5 (100.00) i (%) 10/40 (25.00) 0/14 (0.00) 3/12 (25.00) 0/8 (0.00) 0/5 (0.00) s (%) 17/40 (42.50) 0/14 (0.00) 5/12 (41.67) 0/8 (0.00) 0/5 (0.00) ampicillin r (%) 36/39 (92.31) 13/13 (100.00) 12/12 (100.00) 8/8 (100.00) 5/5 (100.00) i (%) 0/39 (0.00) 0/13 (0.00) 0/12 (0.00) 0/8 (0.00) 0/5 (0.00) s (%) 3/39 (7.69) 0/13 (0.00) 0/12 (0.00) 0/8 (0.00) 0/5 (0.00) ampicillin-sulbactam r (%) 21/40 (52.50) 13/13 (100.00) 7/12 (58.33) 8/8 (100.00) 0/5 (0.00) i (%) 9/40 (22.50) 0/13 (0.00) 1/12 (8.33) 0/8 (0.00) 0/5 (0.00) s (%) 10/40 (25.00) 0/13 (0.00) 4/12 (33.33) 0/8 (0.00) 5/5 (100.00) aztreonam r (%) 17/40 (42.50) 8/14 (57.14) 8/12 (66.67) 4/8 (50.00) 5/5 (100.00) i (%) 3/40 (7.50) 3/14 (21.43) 0/12 (0.00) 0/8 (0.00) 0/5 (0.00) s (%) 20/40 (50.00) 3/14 (21.43) 4/12 (33.33) 4/8 (50.00) 0/5 (0.00) cefazolin r (%) 26/26 (100.00) 14/14 (100.00) 8/8 (100.00) 8/8 (100.00) 5/5 (100.00) i (%) 0/26 (0.00) 0/14 (0.00) 0/8 (0.00) 0/8 (0.00) 0/5 (0.00) s (%) 0/26 (0.00) 0/14 (0.00) 0/8 (0.00) 0/8 (0.00) 0/5 (0.00) cefepime r (%) 18/40 (45.00) 7/14 (50.00) 8/12 (66.67) 4/8 (50.00) 1/5 (20.00) i (%) 1/40 (2.50) 0/14 (0.00) 0/12 (0.00) 0/8 (0.00) 0/5 (0.00) s (%) 21/40 (52.50) 7/14 (50.00) 4/12 (33.33) 4/8 (50.00) 4/5 (80.00) cefotaxime r (%) 18/40 (45.00) 14/14 (100.00) 8/12 (66.67) 4/8 (50.00) 1/5 (20.00) i (%) 1/40 (2.50) 0/14 (0.00) 0/12 (0.00) 0/8 (0.00) 3/5 (60.00) s (%) 21/40 (52.50) 0/14 (0.00) 4/12 (33.33) 4/8 (50.00) 1/5 (20.00) gentamicin r (%) 8/40 (20.00) 2/14 (14.29) 5/12 (41.67) 4/8 (50.00) 2/5 (40.00) i (%) 0/40 (0.00) 1/14 (7.14) 0/12 (0.00) 0/8 (0.00) 0/5 (0.00) s (%) 32/40 (80.00) 11/14 (78.57) 7/12 (58.33) 4/8 (50.00) 3/5 (60.00) ceftazidime r (%) 17/40 (42.50) 2/14 (14.29) 8/12 (66.67) 4/8 (50.00) 1/5 (20.00) i (%) 2/40 (5.00) 1/14 (7.14) 0/12 (0.00) 0/8 (0.00) 0/5 (0.00) s (%) 21/40 (52.50) 11/14 (78.57) 4/12 (33.33) 4/8 (50.00) 4/5 (80.00) ceftriaxone r (%) 22/39 (56.41) 11/13 (84.62) 8/12 (66.67) 4/8 (50.00) 2/5 (40.00) i (%) 1/39 (2.56) 2/13 (15.38) 0/12 (0.00) 0/8 (0.00) 2/5 (40.00) s (%) 16/39 (41.03) 0/13 (0.00) 4/12 (33.33) 4/8 (50.00) 1/5 (20.00) chloramphenicol r (%) 1/3 (33.33) 12/12 (100.00) 1/1 (100.00) 4/4 (100.00) 5/5 (100.00) i (%) 0/3 (0.00) 0/12 (0.00) 0/1 (0.00) 0/4 (0.00) 0/5 (0.00) s (%) 2/3 (66.67) 0/12 (0.00) 0/1 (0.00) 0/4 (0.00) 0/5 (0.00) ciprofloxacin r (%) 12/39 (30.77) 3/13 (23.08) 2/12 (16.67) 4/8 (50.00) 1/5 (20.00) i (%) 1/39 (2.56) 1/13 (7.69) 2/12 (16.67) 0/8 (0.00) 0/5 (0.00) s (%) 26/39 (66.67) 9/13 (69.23) 8/12 (66.67) 4/8 (50.00) 4/5 (80.00) ertapenem r (%) 0/1 (0.00) i (%) 0/1 (0.00) s (%) 1/1 (100.00) fosfomycin r (%) 0/11 (0.00) 1/3 (33.33) 0/1 (0.00) 2/2 (100.00) i (%) 0/11 (0.00) 1/3 (33.33) 0/1 (0.00) 0/2 (0.00) s (%) 11/11 (100.00) 1/3 (33.33) 1/1 (100.00) 0/2 (0.00) imipenem r (%) 0/38 (0.00) 1/12 (8.33) 1/12 (8.33) 4/8 (50.00) 0/5 (0.00) i (%) 3/38 (7.89) 1/12 (8.33) 0/12 (0.00) 1/8 (12.50) 0/5 (0.00) s (%) 35/38 (92.11) 10/12 (83.33) 11/12 (91.67) 3/8 (37.50) 5/5 (100.00) levofloxacin r (%) 11/40 (27.50) 3/12 (25.00) 2/12 (16.67) 4/8 (50.00) 1/5 (20.00) i (%) 2/40 (5.00) 3/12 (25.00) 0/12 (0.00) 0/8 (0.00) 0/5 (0.00) s (%) 27/40 (67.50) 6/12 (50.00) 10/12 (83.33) 4/8 (50.00) 4/5 (80.00) meropenem r (%) 0/40 (0.00) 1/14 (7.14) 1/12 (8.33) 4/8 (50.00) 0/5 (0.00) i (%) 0/40 (0.00) 1/14 (7.14) 0/12 (0.00) 0/8 (0.00) 0/5 (0.00) s (%) 40/40 (100.00) 12/14 (85.71) 11/12 (91.67) 4/8 (50.00) 5/5 (100.00) 128 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fauziah adhima, et al. bacterial profile and antibiotic resistance pattern moxalactam r (%) 0/1 (0.00) i (%) 0/1 (0.00) s (%) 1/1 (100.00) moxifloxacin r (%) 13/38 (34.21) 2/12 (16.67) 4/7 (57.14) i (%) 0/38 (0.00) 2/12 (16.67) 1/7 (14.29) s (%) 25/38 (65.79) 8/12 (66.67) 2/7 (28.57) nitrofurantoin r (%) 4/39 (10.26) 14/14 (100.00) 7/12 (58.33) 6/8 (75.00) 5/5 (100.00) i (%) 1/39 (2.56) 0/14 (0.00) 3/12 (25.00) 0/8 (0.00) 0/5 (0.00) s (%) 34/39 (87.18) 0/14 (0.00) 2/12 (16.67) 2/8 (25.00) 0/5 (0.00) piperacillin r (%) 31/38 (81.58) 2/12 (16.67) 9/12 (75.00) 5/8 (62.50) 1/5 (20.00) i (%) 4/38 (10.53) 0/12 (0.00) 1/12 (8.33) 1/8 (12.50) 1/5 (20.00) s (%) 3/38 (7.89) 10/12 (83.33) 2/12 (16.67) 2/8 (25.00) 3/5 (60.00) piperacillintazobactam r (%) 4/40 (10.00) 3/14 (21.43) 1/12 (8.33) 4/8 (50.00) 1/5 (20.00) i (%) 1/40 (2.50) 0/14 (0.00) 1/12 (8.33) 0/8 (0.00) 0/5 (0.00) s (%) 35/40 (87.50) 11/14 (78.57) 10/12 (83.33) 4/8 (50.00) 4/5 (80.00) tetracycline r (%) 27/38 (71.05) 13/13 (100.00) 5/12 (41.67) 5/8 (62.50) 1/5 (20.00) i (%) 0/38 (0.00) 0/13 (0.00) 0/12 (0.00) 0/8 (0.00) 1/5 (20.00) s (%) 11/38 (28.95) 0/13 (0.00) 7/12 (58.33) 3/8 (37.50) 3/5 (60.00) tigecycline r (%) 1/39 (2.56) 14/14 (100.00) 1/12 (8.33) 2/7 (28.57) 1/5 (20.00) i (%) 1/39 (2.56) 0/14 (0.00) 1/12 (8.33) 2/7 (28.57) 1/5 (20.00) s (%) 37/39 (94.87) 0/14 (0.00) 10/12 (83.33) 3/7 (42.86) 3/5 (60.00) trimethoprimsulfamethoxazole r (%) 28/39 (71.79) 13/13 (100.00) 6/12 (50.00) 6/8 (75.00) 1/5 (20.00) i (%) 0/39 (0.00) 0/13 (0.00) 0/12 (0.00) 0/8 (0.00) 0/5 (0.00) s (%) 11/39 (28.21) 0/13 (0.00) 6/12 (50.00) 2/8 (25.00) 4/5 (80.00) sefoperazonsulbaktam r (%) 0/38 (0.00) 0/14 (0.00) 1/12 (8.33) 4/8 (50.00) 0/5 (0.00) i (%) 8/38 (21.05) 5/14 (35.71) 3/12 (25.00) 0/8 (0.00) 0/5 (0.00) s (%) 30/38 (78.95) 9/14 (64.29) 8/12 (66.67) 4/8 (50.00) 5/5 (100.00) gram-positive bacterial resistance pattern the five most common gram-positive bacteria, are e. faecalis, e. faecium, s. aureus, c. matruchotii, and s. pneumoniae showed varied resistance patterns. e. faecalis showed resistance to ceftriaxone, oxacillin, quinupristindalfopristin, tobramycin, trimethoprim, trimethoprim-sulfamethoxazole, gentamicin, clindamycin, cefotaxime, amikacin, cefoxitin, fusidic acid, tetracycline, and ciprofloxacin for about more than 70%. meanwhile, e. faecium was resistant to amikacin, ampicillin, cefotaxime, gentamicin, ceftriaxone, clindamycin, erythromycin, penicillin, trimethoprim-sulfamethoxazole, levofloxacin, ciprofloxacin, and nitrofurantoin. in contrast to s. aureus, c. matruchotii, and s. pneumoniae, which were only resistant to one or two types of antibiotics (table 4). for the sensitivity pattern, these five bacteria were sensitive to vancomycin and linezolid (table 4). e. faecalis is also sensitive to ampicillin, nitrofurantoin, and teicoplanin, while for e. faecium, another antibiotic sensitivity was only found in teicoplanin. in contrast to their resistance, s. aureus, c. matruchotii, and s. pneumoniae were found to be sensitive to many types of antibiotics. 129 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 123–136 table 4. distribution of antibiotic resistance in gram-positive bacteria antibiotic e. faecalis (n=11) e. faecium (n=9) s. aureus (n=3) c. matruchotti (n=2) s. pneumoniae (n=2) amikacin r (%) 6/7 (85.71) 4/4 (100.00) 0/1 (0.00) i (%) 0/7 (0.00) 0/4 (0.00) 0/1 (0.00) s (%) 1/7 (14.29) 0/4 (0.00) 1/1 (100.00) amoxicillinclavulanate r (%) 0/1 (0.00) 1/3 (33.33) i (%) 0/1 (0.00) 0/3 (0.00) s (%) 1/1 (100.00) 2/3 (66.67) ampicillin r (%) 2/11 (18.18) 6/6 (100.00) 3/3 (100.00) i (%) 0/11 (0.00) 0/6 (0.00) 0/3 (0.00) s (%) 9/11 (81.82) 0/6 (0.00) 0/3 (0.00) cefotaxime r (%) 7/8 (87.50) 9/9 (100.00) 0/2 (0.00) 0/2 (0.00) i (%) 0/8 (0.00) 0/9 (0.00) 0/2 (0.00) 0/2 (0.00) s (%) 1/8 (12.50) 0/9 (0.00) 2/2 (100.00) 2/2 (100.00) gentamisin r (%) 10/11 (90.91) 9/9 (100.00) 1/3 (33.33) 1/1 (100.00) 0/2 (0.00) i (%) 0/11 (0.00) 0/9 (0.00) 0/3 (0.00) 0/1 (0.00) 0/2 (0.00) s (%) 1/11 (9.09) 0/9 (0.00) 2/3 (66.67) 0/1 (0.00) 2/2 (100.00) cefoxitin r (%) 6/7 (85.71) 3/3 (100.00) 1/2 (50.00) i (%) 0/7 (0.00) 0/3 (0.00) 0/2 (0.00) s (%) 1/7 (14.29) 0/3 (0.00) 1/2 (50.00) ceftriaxone r (%) 9/9 (100.00) 8/8 (100.00) 0/2 (0.00) 0/2 (0.00) i (%) 0/9 (0.00) 0/8 (0.00) 0/2 (0.00) 0/2 (0.00) s (%) 0/9 (0.00) 0/8 (0.00) 2/2 (100.00) 2/2 (100.00) chloramphenicol r (%) 0/1 (0.00) 0/1 (0.00) 1/2 (50.00) i (%) 0/1 (0.00) 1/1 (100.00) 0/2 (0.00) s (%) 1/1 (100.00) 0/1 (0.00) 1/2 (50.00) ciprofloxacin r (%) 8/10 (80.00) 5/6 (83.33) 2/3 (66.67) 0/1 (0.00) i (%) 0/10 (0.00) 1/6 (16.67) 0/3 (0.00) 0/1 (0.00) s (%) 2/10 (20.00) 0/6 (0.00) 1/3 (33.33) 1/1 (100.00) clindamycin r (%) 10/11 (90.91) 8/8 (100.00%) 0/2 (0.00) 1/2 (50.00) i (%) 0/11 (0.00) 0/8 (0.00) 0/2 (0.00) 0/2 (0.00) s (%) 1/11 (9.09) 0/8 (0.00) 2/2 (100.00) 1/2 (50.00) erythromycin r (%) 6/9 (66.67) 9/9 (100.00) 0/2 (0.00) 1/2 (50.00) i (%) 2/9 (22.22) 0/9 (0.00) 1/2 (50.00) 0/2 (0.00) s (%) 1/9 (11.11) 0/9 (0.00) 1/2 (50.00) 1/2 (50.00) fusidic acid r (%) 6/7 (85.71) 3/3 (100.00) i (%) 0/7 (0.00) 0/3 (0.00) s (%) 1/7 (14.29) 0/3 (0.00) levofloxacin r (%) 6/9 (66.67) 7/8 (87.50) 2/3 (66.67) 0/2 (0.00) 1/2 (50.00) i (%) 1/9 (11.11) 1/8 (12.50) 0/3 (0.00) 0/2 (0.00) 0/2 (0.00) s (%) 2/9 (22.22) 0/8 (0.00) 1/3 (33.33) 2/2 (100.00) 1/2 (50.00) linezolid r (%) 2/11 (18.18) 1/9 (11.11) 0/3 (0.00) 0/2 (0.00) 0/2 (0.00) i (%) 6/11 (54.55) 1/9 (11.11) 0/3 (0.00) 0/2 (0.00) 0/2 (0.00) s (%) 3/11 (27.27) 7/9 (77.78) 3/3 (100.00) 2/2 (100.00) 2/2 (100.00) moxalactam r (%) 0/1 (0.00) i (%) 0/1 (0.00) s (%) 1/1 (100.00) moxifloxacin r (%) 2/3 (66.67) 0/2 (0.00) 0/1 (0.00) i (%) 0/3 (0.00) 0/2 (0.00) 0/1 (0.00) s (%) 1/3 (33.33) 2/2 (100.00) 1/1 (100.00) nitrofurantoin r (%) 2/11 (18.18) 7/9 (77.78) 0/3 (0.00) 1/2 (50.00) i (%) 0/11 (0.00) 1/9 (11.11) 0/3 (0.00) 0/2 (0.00) s (%) 9/11 (81.82) 1/9 (11.11) 3/3 (100.00) 1/2 (50.00) oxacillin r (%) 5/5 (100.00) 2/2 (100.00) 1/3 (33.33) i (%) 0/5 (0.00) 0/2 (0.00) 0/3 (0.00) s (%) 0/5 (0.00) 0/2 (0.00) 2/3 (66.67) penicillin r (%) 4/10 (40.00) 8/8 (100.00) 3/3 (100.00) 0/2 (0.00) 1/2 (50.00) i (%) 0/10 (0.00) 0/8 (0.00) 0/3 (0.00) 0/2 (0.00) 0/2 (0.00) s (%) 6/10 (60.00) 0/8 (0.00) 0/3 (0.00) 2/2 (100.00) 1/2 (50.00) 130 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fauziah adhima, et al. bacterial profile and antibiotic resistance pattern quinupristindalfopristin r (%) 11/11 (100.00) 1/6 (16.67) 1/3 (33.33) i (%) 0/11 (0.00) 3/6 (50.00) 0/3 (0.00) s (%) 0/11 (0.00) 2/6 (33.33) 2/3 (66.67) rifampin r (%) 0/3 (0.00) 0/2 (0.00) i (%) 0/3 (0.00) 0/2 (0.00) s (%) 3/3 (100.00) 2/2 (100.00) streptomycin r (%) 0/2 (0.00) 1/2 (50.00) i (%) 0/2 (0.00) 0/2 (0.00) s (%) 2/2 (100.00) 1/2 (50.00) teicoplanin r (%) 2/11 (18.18) 0/6 (0.00) 0/3 (0.00) i (%) 0/11 (0.00) 0/6 (0.00) 0/3 (0.00) s (%) 9/11 (81.82) 6/6 (100.00) 3/3 (100.00) tetracycline r (%) 9/11 (81.82) 2/6 (33.33) 2/3 (66.67) 0/2 (0.00) 0/1 (0.00) i (%) 0/11 (0.00) 0/6 (0.00) 0/3 (0.00) 0/2 (0.00) 0/1 (0.00) s (%) 2/11 (18.18) 4/6 (66.67) 1/3 (33.33) 2/2 (100.00) 1/1 (100.00) tigecycline r (%) 0/2 (0.00) i (%) 0/2 (0.00) s (%) 2/2 (100.00) tobramycin r (%) 7/7 (100.00) 3/3 (100.00) i (%) 0/7 (0.00) 0/3 (0.00) s (%) 0/7 (0.00) 0/3 (0.00) trimethoprim r (%) 6/6 (100.00) 3/3 (100.00) i (%) 0/6 (0.00) 0/3 (0.00) s (%) 0/6 (0.00) 0/3 (0.00) trimethoprimsulfamethoxazole r (%) 11/11 (100.00) 9/9 (100.00) 1/3 (33.33) 1/2 (50.00) 1/1 (100.00) i (%) 0/11 (0.00) 0/9 (0.00) 0/3 (0.00) 0/2 (0.00) 0/1 (0.00) s (%) 0/11 (0.00) 0/9 (0.00) 2/3 (66.67) 1/2 (50.00) 0/1 (0.00) vancomycin r (%) 2/11 (18.18) 1/9 (11.11) 0/3 (0.00) 0/2 (0.00) 0/2 (0.00) i (% 0/11 (0.00) 0/9 (0.00) 0/3 (0.00) 0/2 (0.00) 0/2 (0.00) s (%) 9/11 (81.82) 8/9 (88.89) 3/3 (100.00) 2/2 (100.00) 2/2 (100.00) co-morbidities in this study, children with uti were diagnosed with more than one disease. the patient's comorbidities were dominated by hydronephrosis, chronic kidney disease, and hydrocephalus (figure 2). figure 2. distribution of comorbidities 131 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 123–136 discussion uti is one of the most common bacterial infectious diseases in children with non-specific symptoms. epidemiologically, it is estimated that emergency department visits by children diagnosed with uti reach more than 500,000 visits and 50,000 hospitalizations.18 the incidence of uti is influenced by two important interrelated variables, namely age and gender.19 according to the american academy of pediatrics, the highest prevalence of uti in children is found at the age of two months two years, which is about 5% of children with fever complaints.20 similar to this study, which found that there were 54.2% of 131 children with uti were male, dominated by one month – 2 years. similar with mirsoleymani et al in their study in bandar abbas, south irian, uti incidence in boys reached 54.9%.21 the high incidence of uti in boys at this age may be due to their uncircumcised status, so that uropathogens colonize the foreskin and cause ascending infection.22 poor diaper hygiene during infancy is also an important predisposition to uti.23 in addition, the incidence of uti in boys in early life is also possible because males have a higher risk of congenital anomalies of the kidney and urinary tract (cakut) than females, so boys are more prone to uti.24 based on gender, the tendency of uti among children will change with age. the dominance of uncircumcised male of uti in infants will change to female preponderance in older children.17 at the age of 7 years, it is estimated that approximately 7.8% of girls and 1.7% of boys are diagnosed with uti.25 this study found that boys were most commonly found at the age of one month – 2 years, while girls were most commonly found at the age of 6-12 years. uti in girls is due to the relatively shorter urethral structure of girls so that bacteria more easily cause ascending infection to the bladder. it could also be due to heavy colonization of enteric bacteria in the perineal uropathogens.22 in the majority, utis in children are caused by gram-negative bacteria from the intestinal flora that colonize the perineum and cause ascending infection to the urinary tract. it is estimated that approximately 80% of pediatric utis are caused by e. coli.3 in concordance with this study, which found a predominance of gram-negative bacteria (74%) with e. coli as the most common gram-negative bacteria, followed by p. aeruginosa, k. pneumoniae, e. cloacae, and a. baumannii. e. coli has various virulence factors, namely p fimbriae, a type of surface fimbriae that induces attachment to host-specific receptors on the uroepithelium. in addition, flagella, lipopolysaccharide, capsule polysaccharide, and hemolysin are also important virulence factors in infecting the host. most uropathogenic escherichia coli (upec) can produce aerobactin, a high affinity ironbinding protein that causes acute pyelonephritis.2 while the gram-positive bacteria were only found in 26%, dominated by e. faecalis, followed by e. faecium, s. aureus, c. matruchotii, and s. pneumoniae. similar to benachinmardi et al in their study in india where 82.22% gram-negative bacteria were found, with e. coli (52.9%) as the most common bacterial isolate followed by k. pneumoniae (7.6%) while gram-positive bacteria were only found in 16% of isolates dominated by coagulase negative staphylococcus (9.8%) followed by enterococcus spp. (5.8%).4 currently, the management of uti is becoming more difficult as various resistance mechanisms emerge, such as members of the enterobacteriaceae family including e. coli and k. pneumoniae that produce esbl. kitagawa et al stated that esbl-producing e. coli and k. pneumoniae were found to be more dominant than non-esbl-producing isolates8, in contrast to this study which found non-esbl-producing e. coli and esbl-producing k. pneumoniae strains are more dominant. this difference can be attributed to risk factors for esbl infection including comorbidities, frequent use of health resources for a long time, previous use of antibiotics, experiencing recurrent uti, older age, and male gender.26 to reduce the risk of acute and chronic complications in pediatric utis, prompt and appropriate initial treatment with empirical antibiotics plays an important role. unfortunately, an increase in resistant strains 132 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fauziah adhima, et al. bacterial profile and antibiotic resistance pattern has been widely reported, especially in developing countries due to the habit of consuming over-the-counter antibiotics without a prescription and prior consultation.14 antimicrobial resistant pattern varies by geographic area. therefore, local antimicrobial susceptibility patterns are needed in selecting empirical antibiotics for initial treatment of pediatric utis considering potential side effect and economic consequences.4 this study showed that the most resistant antibiotics to e. coli, p. aeruginosa, k. pneumoniae, e. cloacae, and a. baumannii, were ampicillin and cefazolin, similar with kitagawa et al in their study of uti patients in surabaya.8 the high resistance to these two antibiotics may be due to their frequent use considering that uti management in indonesia generally uses ampicillin, cephalosporins, and fluoroquinolones.27 carbapenems are the broadest spectrum betalactam antibiotics that have become the gold standard for treating infections caused by esblproducing enterobacteriaceae. they have high stability against hydrolysis reactions by betalactamase enzymes28, however, its use should be limited to avoid irresponsible prescribing, resulting in the emergence of carbapenemresistant organisms.29 in contrast to amikacin, poey et al explain that amikacin monotherapy can be used as the first line of empirical treatment in febrile uti among pediatric patients so that amikacin may be a more appropriate empiric therapy option.30 however, this still requires further research in the form of randomized controlled trials (rct).31 this study showed that the five most common gramnegative bacteria were sensitive to carbapenems (imipenem, meropenem), amikacin, and piperacillin-tazobactam, similar to rahmadi in his research on uti patients at the department of internal medicine dr. soetomo hospital.32 in taiwan, wu et al reported that e. coli was resistant to ampicillin, piperacillin, trimethoprim-sulfamethoxazole, and sensitive to amikacin, imipenem, ceftazidime, ceftriaxone, and cefuroxime, gentamicin.33 similar to this study in which e. coli was also resistant to ampicillin, cefazolin, piperacillin, trimethoprimsulfamethoxazole, tetracycline exceeded 70%, and sensitive to amikacin, imipenem, meropenem, and piperacillin-tazobactam, tigecycline, nitrofurantoin, gentamicin, cefoperazone-sulbactam. a study in india stated that trimethoprim-sulfamethoxazole resistance increased significantly over a five-years period.14 the increasing resistance of trimethoprim-sulfamethoxazole in various regions has resulted in this antibiotic being no longer recommended as empiric therapy unless it is proven to be sensitive according to local antibiogram data.31 meanwhile, cefoperazonesulbactam showed a sensitivity of more than 90% in esbl-producing enterobacteriaceae.34 tigecycline is well tolerated in cases of serious extensively drug-resistant (xdr) gramnegative bacterial infections35, but should not be used as monotherapy in pediatric utis because of its limited excretion and some side effects, enamel hypoplasia.36 according to the american academy of pediatrics, nitrofurantoin is not recommended for febrile infants because serum and parenchymal concentrations may be insufficient to treat urosepsis or pyelonephritis. in addition, nitrofurantoin is contraindicated in cases of decreased renal function with creatinine clearance <60 millilitre per minute (ml/min).37 the second largest gram-negative bacteria, p. aeruginosa was also found to be resistant to amoxicillin-clavulanate, ampicillin-sulbactam, cefotaxime, chloramphenicol, nitrofurantoin, tetracycline, tigecycline, trimethoprimsulfamethoxazole, ceftriaxone and sensitive to piperacillin, gentamicin, ceftazidime. in previous studies, p. aeruginosa was reported to be highly resistant to trimethoprimsulfamethoxazole, nitrofurantoin, cefotaxime, ampicillin, amoxicillin/clavulanate, cephalexin, cefuroxime, ceftriaxone, nalidixic acid38,39 and more sensitive to piperacillin-tazobactam, ceftazidime, imipenem, ciprofloxacin, gentamicin, and tobramycin.40 it is different with gram-positive bacteria, which show high sensitivity to vancomycin and linezolid with varying resistance patterns. both of e. faecalis and e. faecium showed resistance 133 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 123–136 to ceftriaxone, trimethoprim-sulfamethoxazole, gentamicin, cefotaxime, amikacin, ciprofloxacin for about more than 70%, and sensitive to vancomycin, linezolid, teicoplanin. this is in accordance with hameed et al and benachinmardi et al which showed that enterococcus spp. resistant to trimethoprimsulfamethoxazole (100%), amikacin (71.43%), gentamicin (85%), erythromycin (76.92%), and ciprofloxacin (60%) and completely sensitive to vancomycin, linezolid, and teicoplanin.4,41 enterococci are resistant to antibiotics because they are naturally resistant to low levels of aminoglycosides, cephalosporins, clindamycin and trimethoprim/sulfamethoxazole. betalactams have also been reported to have limited clinical efficacy on enterococci due to the low affinity penicillin-binding proteins (pbps).42 in pediatric uti, urinary tract abnormalities contribute to increasing recurrent uti and resulting in the development of multi drug resistance organisms.43 in this study, comorbidities in pediatric uti patients were dominated by hydronephrosis (10.98%) followed by chronic kidney disease (9.79%), and hydrocephalus (8.09%). according to coelho et al, the increasing severity of hydronephrosis leads to an increased risk of uti due to urinary tract dilatation.44 hydronephrosis or dilation of the renal collecting system can be caused by partial or complete obstruction of urine flow caused by vesicoureteral reflux, posterior urethral valves, ureteropelvic junction obstruction, ureterocele, or duplication of the collecting system.45 the most severe long-term sequelae as a complication of uti is renal scarring that may progress to end-stage renal disease.46 on the other hand, chronic kidney disease can also be a contributing factor to uti due to oxidative stress and inflammatory cytokines, which can result in impaired immunity and increase susceptibility to various infections, especially uti.47 non-urinary disorders that can also increase the risk of uti is hydrocephalus. hydrocephalus is generally caused by myelomeningocele, the most common form of open spina bifida that can increase the incidence of uti in children.48,49 hydrocephalus has an additional effect at the central level on the micturition process controlled by the pons, brain stem, and cerebral cortex which can aggravate the neurogenic bladder which results in impaired bladder emptying and increases the risk of uti.50 the limitations found in this study are related to the instruments used. in this study, the researcher used secondary data in the form of a urine culture logbook, so that there could be bias because the researcher was not directly involved during the examination process and some of the data were found to be incomplete. however, this study is essential to evaluate the antibiotic resistance pattern among uropathogens in dr. soetomo hospital, who could be considered in selecting the appropriate empirical antibiotics to optimize initial uti therapy. conclusions this study revealed gram-negative bacteria isolates as the preponderance uropathogen, with e. coli as the most common bacteria found. gram-negative bacteria are highly resistant to ampicillin and cefazoline, while gram-positive bacteria showed varied antibiotics resistance. uti comorbidities are dominated by hydronephrosis, chronic kidney disease, and hydrocephalus. this research can be useful for health workers, especially in dr. soetomo hospital, surabaya, as an initial consideration in selecting empirical antibiotics before culture results are available. in addition, this study can be used as a reference for further research on children with uti in order to develop public health services. acknowledgement the authors would like to express my sincere gratitude to all staff of clinical microbiology laboratory of dr. soetomo hospital for giving permission to conduct this study and also for their assistance during data collection. 134 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fauziah adhima, et al. bacterial profile and antibiotic resistance pattern conflict of interest all authors declared that they do not have any conflict of interest. references 1. hanna-wakim rh, ghanem st, el helou mw, khafaja sa, shaker ra, hassan sa, et al. epidemiology and characteristics of urinary tract infections in children and adolescents. front cell infect microbiol. 2015;5(45):1–8. 2. hodson em, craig jc. urinary tract infections in children. in: avner ed, harmon w, niaudet p, yoshikawa n, emma f, goldstein s, editors. pediatric nephrology. 7th ed. springer; 2016. p. 1696–708. 3. goldberg b, jantausch b. urinary tract infection. in: kher k, schapner h, greenbaum l, editors. clinical pediatric nephrology. 3rd ed. crc press; 2017. p. 967–91. 4. benachinmardi k, padmavathy m, malini j, navaneeth b. microbiological profile and antibiogram of uropathogens in pediatric age group. int j heal allied sci. 2015;4(1):61. 5. taneja n, chatterjee ss, singh m, singh s, sharma m. pediatric urinary tract infections in a tertiary care center from north india. indian j med res. 2010;131(1):101–6. 6. belete y, asrat d, yimtubezinash w, gebeyehu y, addisu g. bacterial profile and antibiotic susceptibility pattern of urinary tract infection among children attending felege hiwot referral hospital, bahir dar, northwest ethiopia. infect drug resist. 2019;12:3575–83. 7. copp hl, shapiro dj, hersh al. national ambulatory antibiotic prescribing patterns for pediatric urinary tract infection, 1998-2007. pediatrics. 2011;127(6):1027–33. 8. kitagawa k, shigemura k, yamamichi f, alimsardjono l, rahardjo d, kuntaman k, et al. international comparison of causative bacteria and antimicrobial susceptibilities of urinary tract infections between kobe , japan , and surabaya , indonesia. jpn j infect dis. 2018;71(1):8–13. 9. kalantar ea, motlagh me, lornezhad h, reshadmanesh n. prevalence of urinary tract pathogens and antimicrobial susceptibility patterns in children at hospitals in iran. iran j clin infect dis [internet]. 2008;3(3):149–53. available from: https://www.sid.ir/en/journal/viewpaper.aspx?i d=143606 10. national collaborating centre for women’s and children’s health (uk). urinary tract infection in children: diagnosis, treatment and longterm management. rcog press; 2007. 11. sievert dm, ricks p, edwards jr, schneider a, patel j, srinivasan a, et al. antimicrobialresistant pathogens associated with healthcareassociated infections summary of data reported to the national healthcare safety network at the centers for disease control and prevention, 2009–2010. infect control hosp epidemiol. 2013;34(1):1–14. 12. abhilash kp, veeraraghavan b, abraham oc. epidemiology and outcome of bacteremia caused by extended spectrum beta-lactamase (esbl)producing escherichia coli and klebsiella spp. in a tertiary care teaching hospital in south india. j assoc physicians india. 2010;58(suppl):13–7. 13. singh sd, madhup sk. clinical profile and antibiotics sensitivity in childhood urinary tract infection at dhulikhel hospital. kathmandu univ med j. 2013;11(4):319–24. 14. patwardhan v, kumar d, goel v, singh s. changing prevalence and antibiotic drug resistance pattern of pathogens seen in community ‑ acquired pediatric urinary tract infections at a tertiary care hospital of north india. j lab physicians. 2017;9(4):264–8. 15. mortazavi f, shahin n. changing patterns in sensitivity of bacterial uropathogens to antibiotics in children. pakistan j med sci. 2009;25(5):801–5. 16. knoppert d, reed m, benavides s, totton j, hoff d, moffett b, et al. position paper : paediatric age categories to be used in differentiating between listing on a model essential medicines list for children. world heal organ. 2007; 17. lee sj. clinical guideline for childhood urinary tract infection ( second revision ). chil kidney dis. 2015;19:56–64. 18. spencer jd, schwaderer a, mchugh k, hains ds. pediatric urinary tract infections : an analysis of hospitalizations , charges , and costs in the usa. pediatr nephrol. 2010;25:2469–75. 19. schlager t. urinary tract infections in infants and children. microbiol spectr. 2016;4(5):1–7. 20. aap. urinary tract infection : clinical practice guideline for the diagnosis and management of the initial uti in febrile infants and children 2 to 24 months. pediatrics. 2011;128(3). 21. mirsoleymani sr, salimi m, brojeni ms, ranjbar m, mehtarpoor m. bacterial pathogens and antimicrobial resistance patterns in pediatric urinary tract infections : a four-year surveillance study ( 2009 – 2012 ). int j pediatr. 2014;2014. 22. chang sl, shortliffe ld. pediatric urinary tract infections. pediatr clin. 2006;53(3):379–400. 135 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 123–136 23. tullus k. fifteen-minute consultation: why and how do children get urinary tract infections? arch dis childhood-education pract. 2019;104(5):244–7. 24. li z, chen y, qiu l, chen d, hu c, xu j, et al. prevalence , types , and malformations in congenital anomalies of the kidney and urinary tract in newborns : a retrospective hospital-based study. ital j pediatr. 2019;45(50):1–7. 25. stephens gm, akers s, nguyen h, woxland h. evaluation and management of urinary tract infections in the school-aged child. prim care clin off pract. 2014;42(1):33–41. 26. yang ys, ku ch, lin jc, shang st, chiu ch, yeh km, et al. impact of extended-spectrum βlactamase-producing escherichia coli and klebsiella pneumoniae on the outcome of community-onset bacteremic urinary tract infections. j microbiol immunol infect. 2010;43(3):194–9. 27. lestari es, severin ja, filius pmg, kuntaman k, duerink do, hadi u, et al. antimicrobial resistance among commensal isolates of escherichia coli and staphylococcus aureus in the indonesian population inside and outside hospitals. eur j clin microbiol infect dis. 2008;27(1):45–51. 28. bassetti m, peghin m, pecori d. the management of multidrug-resistant enterobacteriaceae. curr opin infect dis. 2016;29(6):583-594. 29. mayers d, lerner s, ouellette m, sobel j. antimicrobial drug resistance volume 1: mechanisms of drug resistance. new york: humana press; 2009. 30. poey n, madhi f, biscardi s, béchet s, cohen r. aminoglycosides monotherapy as first-line treatment for febrile urinary tract infection in children. pediatr infect dis j. 2017;36(11):1104– 7. 31. lashkar mo, nahata mc. antimicrobial pharmacotherapy management of urinary tract infections in pediatric patients. j pharm technol. 2018;34(2):62–81. 32. rahmadi i. profil kuman dan sensitivitas kepekaan antibiotik pada pasien infeksi saluran kemih di smf penyakit dalam rsud dr. soetomo surabaya, indonesia. (doctoral diss fak kedokteran). 2018; 33. wu c, lee h, chen c, tuan p-l, chiu c-h. high prevalence and antimicrobial resistance of urinary tract infection isolates in febrile young children without localizing signs in taiwan. j microbiol immunol infect. 2016;49(2):243–8. 34. kuntaman k, santoso s, wahjono h, mertaniasih nm, lestari es, farida h, et al. the sensitivity pattern of extended spectrum beta lactamase-producing bacteria against six antibiotics that routinely used in clinical setting. j indon med assoc. 2011;61(12):482–6. 35. iosifidis e, violaki a, michalopoulou e, volakli e, diamanti e, koliouskas d, et al. use of tigecycline in pediatric patients with infections predominantly due to extensively drug-resistant gram-negative bacteria. j pediatric infect dis soc. 2017;6(2):123–8. 36. hsu aj, tamma pd. treatment of multidrugresistant gram-negative infections in children. clin infect dis. 2015;58(10):1439–48. 37. oplinger m, andrews co. nitrofurantoin contraindication in patients with a creatinine clearance below 60 ml / min : looking for the evidence. ann pharmacother. 2013;47:106–11. 38. rezaee ma, abdinia b. etiology and antimicrobial susceptibility pattern of pathogenic bacteria in children subjected to uti. medicine (baltimore). 2015;94(39):1–4. 39. marcus n, ashkenazi s, samra z, cohen a, livni g. community-acquired pseudomonas aeruginosa urinary tract infections in children hospitalized in a tertiary center : relative frequency, risk factors, antimicrobial resistance and treatment. infection. 2008;36(5):421–6. 40. rodríguez-lozano j, malet a de, cano me, rubia l de la, wallmann r, martínez-martínez l, et al. antimicrobial susceptibility of microorganisms that cause urinary tract infections in pediatric patients. enferm infecc microbiol clin. 2018;36(7):417–22. 41. hameed t, al nafeesah a, chishti s, al shaalan m, al fakeeh k. community-acquired urinary tract infections in children : resistance patterns of uropathogens in a tertiary care center in saudi arabia. int j pediatr adolesc med. 2019;6:51–4. 42. kristich c, rice l, arias c. enterococcal infection—treatment and antibiotic resistance. in: gilmore ms, clewell db, ike y, shankar n, editors. enterococci: from commensals to leading causes of drug resistant infection. boston: massachusetts eye and ear infirmary; 2014. p. 123–85. 43. wragg r, harris a, patel m, robb a, chandran h, mccarthy l. extended spectrum beta lactamase (esbl) producing bacteria urinary tract infections and complex pediatric urology. j pediatr surg. 2017;52(2):286–8. 44. coelho gm, bouzada mcf, lemos gs, pereira ak, lima bp, oliveira ea. risk factors for urinary tract infection in children with prenatal renal pelvic dilatation. j urol. 2008;179(1):284– 9. 45. gorelick mh. abdominal mass. in: zorc jj, alpern er, brown lw, loomes km, marino bs, mollen cj, et al., editors. schwartz‘s clinical 136 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fauziah adhima, et al. bacterial profile and antibiotic resistance pattern handbook of pediatrics. 5th ed. wolters kluwer lippincott williams; 2013. p. 100–1. 46. williams gj, hodson eh, isaacs d, craig jc. diagnosis and management of urinary tract infection in children. j paediatr child health. 2012;48(4):296–301. 47. ishigami j, taliercio j, i feldman h, srivastava a, townsend r, l cohen d, et al. inflammatory markers and incidence of hospitalization with infection in chronic kidney disease: the chronic renal insufficiency cohort study. am j epidemiol. 2019;189(5):433–44. 48. sacar s, turgut h, toprak s, cirak b, coskun e, yilmaz o, et al. a retrospective study of central nervous system shunt infections diagnosed in a university hospital during a 4-year period. bmc infect dis. 2006;6(43). 49. subandiyah k. infeksi saluran kemih sebagai komplikasi gangguan berkemih pada anak. in: soemyarso na, suryaningtyas w, prasetyo rv, editors. gangguan berkemih pada anak. surabaya: airlangga university press; 2015. p. 51. 50. suryaningtyas w. manifestasi neuro-urologi pada spina bifida dan tethered cord syndrome. in: soemyarso na, suryaningtyas w, prasetyo r v., editors. gangguan berkemih pada anak. surabaya: airlangga university press; 2015. p. 83 vol. 9 no. 2 may–august 2021 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 * corresponding author: dadik_tdc@yahoo.co.id research article antimicrobial activities of laurus nobilis leaves ethanol extract on staphylococcus aureus, salmonellae typhi, and escherichia coli. khawla abdullah sakran1, dadik raharjo2, *, ni made mertaniasih3 1immunology study program, post graduate school, universitas airlangga , surabaya, indonesia 2gastroenteritis and salmonellosis laboratory, institute of tropical disease, universitas airlangga, surabaya. indonesia 3department of clinical microbiology, faculty of medicine universitas airlangga received : 27th april 2021; revised : 28th august 2021; accepted : 29th august 2021 abstract laurus nobilis is one of the most well-known, most frequently used plants is from lauraceae family which contains up 2.500 species that grow in the subtropics and tropics of the mediterranean region and indonesia. this study was supposed to investigate the antimicrobial eff ect of l.nobilis leaves ethanol extract on staphylococcus aureus, salmonellae typhi, and escherichia coli. this preliminary study examined the antimicrobial eff ect of l.nobilis leaves ethanol extract. the method used agar-well diff usion for determination of the zone of inhibition and the minimum bactericidal concentration to investigate the activity of l.nobilis leaves ethanol extract at 100% concentration . the results revealed that extract of l. nobilis leaves had the antibacterial activity against staphylococcus aureus with a zone of inhibition (16.3 ±1.5 mm), staphylococcus aureus with (14.5±0.5 mm), and weak antimicrobial activity against escherichia coli (11.3±1.1mm). also, through the minimum bactericidal concentration experiment, the l.nobilis leaves ethanol extract had activity on staphylococcus aureus and salmonellae typhi, it’s killed the bacteria in all concentration start it from 5×107 to 5×104. but the activity on escherichia coli just weaken concentration 5×107 and 106. this research has concluded that the l.nobilis leaves ethanol extract exhibited a signifi cant antimicrobial eff ect against staphylococcus aureus and salmonellae typhi then escherichia coli that is considered a kind of multidrug-resistant bacteria. keywords: antibacterial activity; laurus nobilis leaves ethanol extract; staphylococcus aureus; salmonellae typhi; escherichia coli. abstrak laurus nobilis has been known for a long time as a plant that is effi cacious for preventing and treating several kinds of diseases. this study aims to determine the antimicrobial eff ect of l. nobilis leaves extracted using ethanol against staphylococcus aureus, salmonellae typhi, and escherichia coli. the research method used agar diff usion to measure the zone of inhibition against the test bacteria. gentamicin and dmso were used as positive and negative controls. the results showed that laurus nobilis extract had antibacterial activity against staphylococcus aureus with an inhibitory zone (16.3±1.5 mm), salmonellae typhi with a zone (14.5±0.5 mm), and against escherichia coli (11.3±1.1mm). kata kunci: antibakteri; laurus nobili etanol ekstrak; staphylococcus aureus; salmonellae typhi; escherichia coli. 121khawla abdullah sakran, et al.: antimicrobial activities of laurus nobilis leaves ethanol extract ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 how to cite: sakran, khawla a., raharjo, dadik., mertaniasih, ni made.antimicrobial activities of laurus nobilis bay leaves extract on staphylococcus aureus, salmonellae typhi, and escherichia coli. indonesian journal of tropical and infectious disease. 9(2), 120–125. introduction in 2018, the who global antimicrobial surveillance system reported that 500,000 people with suspected bacterial infections across the globe is attributed to the antibiotic resistance. staphylococcus aureus, salmonellae typhi, and escherichia coli are the most common human pathogens that are consistently causing diff erent sequelae of infection in both genders and all ages.1 these pathogens have also a signifi cant number of morbidities and mortalities, particularly in developing countries. the bacteria develop a resistance to antimicrobials by diff erent mechanisms whereby limiting uptake of the drug, enzymatic inactivation of the drug, modifi cation of the drug target, and active effl ux of the drug. depending on the antimicrobial involved, the bacteria may use one or several of these resistance mechanisms.2 pathogenic e. coli is resistant to various antibiotics and including the strain that extendedspectrum b-lactamase (esbl).3 where e. coli considered the most pathogenic bacteria that causes of diarrhea in humans and animals.4 increased resistant of the s. typhi to antimicrobial drugs was reported and may allow it to cross the intestinal mucosa to the bloodstream and infects deep organs such as the bones, joints, and meninges.5 methicillin-resistant s. aureus (mrsa) is a major pathogen associated with serious community and hospital-acquired disease where these strains showed resistance to a wide range of antibiotics, thus limiting the treatment options to very few agents such as teicoplanin and vancomycin.6,7 antibiotic resistance is an internationally recognized health problem. this problem, in recent years is greatly threatening because of emergence of multi-drug resistant organisms (mdro).8,9 new antibacterial agents from many sources including herbal products that are preferred over traditional medicines due to its wide biological activity, safety and lower cost. the herbal products contain groups of eff ective compounds that can be investigated for eff ectiveness as antimicrobials, antioxidants, antiseptic, and anti-infl ammatory. herbal products are increasingly used as a dietary supplement to fi ght against infection and lower the risk in population.10,11 l. nobilis is one of the most well-known and most frequently used plants and it is member lauracease family which contains up 2,500 species that grow in the subtropics and tropics of the mediterranean region include indonesia. most species possess aromatic stems, roots, leaves, and fruits.12,13 as a medicinal plant, its leaves and fruits have been known since long time ago as a species that can be used for therapy against rheumatism, skin rashes, earaches, stomachace, astringent, carminative, diaphoretic, stimulant, emetic, emmenagogue and abortifacient.12 in addition, its volatile oil is used by the cosmetic industry in creams, perfumes, and soaps. it has a lot of chemical properties that are useful in manufacturing of medicine, for instance, it represents a basic material in dentistry such as alkaloids, fl avonols, phenolic, fl avones (apigenin and luteolin), glycosylate fl avonoids, cysterpine and soliterpinat to fight against or prevent common diseases.14,15 several studies described and confi rmed that extracting phytochemicals and active ingredients of herbal remedies give medicinal benefi ts more than the use of the herb itself.16 many studies, for example yilmaz et al (2017) and aldhaher et al (2017) have found that the essential oil of l.nobilis leaves has strong antibacterial activity against gram negative and gram-positive bacteria.17,18 ozcan et al. (2016) found that the green synthesis of zinc oxide nanoparticles using the aqueous leaf extract of l.nobilis (ln-zno nps) were has antibacterial activity of ln-zno 122 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 120–126 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 nps was greater against gram-positive (s. aureus) bacteria than gram-negative (p. aeruginosa) bacteria.19 therefore, the main objective of this study was to evaluate the antimicrobial activity of l.nobilis leaves extract against s. aureus, s. typhi and e. coli. material and methods preparation of the l. nobilis leaves ethanol extract. fresh l. nobilis leaves ethanol extract (daun salam) weighing 5 kilograms (kgs) were collected from a farm in malang indonesia in october 2019. its were washed under running tap water, air dried and fi nely grinded with a blender. 500g of the fi nely grinded leaves were then soaked in 300mls of 70% ethanol in an airtight container for 24 hours. the mixture was fi ltered using fi lter paper 11μm and the solute was extracted with a rotary evaporator at 45°c were the fi nal volum of extract 100ml then stored in -20 °c before used.20,21,22 antimicrobial assay by agar-well diff usion method this study was an evaluation which was intended to assess the antimicrobial activity of l. nobilis leaves ethanol extract to s. aureus, s. typhi and e. coli. this research was conducted in the laboratory a bsl 3 universitas airlangga from november 2019 to december 2019. the used bacterial strains in this study are s. aureus (atcc 25423), e. coli (atcc 25922) and s. typhi (bsl 2 lab. collection). strains were overnight grown onto plates of muller-hinton agar (mha). antimicrobial activity was carried out using the agar well diffusion method according to clinical laboratory standards institute guidelines (clsi).23,24 three to five colonies of each bacterium were dissolved in 2 ml of physiological saline and the turbidity was adjusted to 0.5 mac farland’s turbidity which is equivalent to 0.5×108 bacteria per ml of solution. a swab use to spread the bacteria on surface of mha media and then applied 100μl of l.nobilis leaves ethanol extract at concentration 100% on labeled well and 100 μl of dimethyl sulfoxide put on another well as a negative control and gentamicin (10μg) disc was used as a positive control. the plate were incubated at 37°c for 24 h and the antibacterial activity determined by an inhibition zone (iz) that formed around the well. the iz of l.nobilis leaves ethanol extract was measure using calipers and compared with iz gentamicin.25 results and discussion through our experiments as shown in fig. (1) and table 1 the results of agar well diff usion assay showed the iz of l.nobilis leaves ethanol extract to s. aureus (16.3 ±1.5 mm), followed by s. typhi (14.5 ± 0.5 mm) and e. coli (11.3 ± 1.1 mm). this results were shown that the s. aureus were most sensitive against l.nobilis leaves ethanol extract. this fi nding was in tandem with the results published by al-ogaili (2020) which highlighted the great inhibition activity of l.nobilis leaves ethanol extracts to this grampositive bacterium.29 as reported by otsuka et al. (2008) the l. nobilis has antimicrobial activity against methicillin-resistant s. aureus (mrsa) through purifi ed two compound fl avonoids and kaempferol, that both compounds showed strong antimicrobial activity.32 table 1. antibacterial activity of l.nobilis leaves ethanol extracts microorganism diameter of growth of inhibition zones (mm) l.nobilis leaves ethanol extract gentamicin s. aureus 16.3 ± 1.5 25.6 ± 0.5 s. typhi 14.5 ± 0.5 20.6 ± 1.1 e. coli 11.3 ± 1.1 19 ± 0.5 *values, including diameter of the well (6 mm), are means of three replicates ± sd figure 1. antibacterial activity of l.nobilis leaves ethanol extract against bacteria (a) salmonella typhi (b) e. coli (c) s. aureus. the active compound was seen against s. aureus, s. typhi and e. coli. one from this flavonoid compound has antibacterial properties because it has the capability to produce transduction energy 123khawla abdullah sakran, et al.: antimicrobial activities of laurus nobilis leaves ethanol extract ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 that will aff ect the cytoplasm of the bacteria and slow down its motility, since it has an ability to interact directly with the deoxyribonucleic acid (dna) of the bacteria.14 type of solvent used for extracting l.nobilis leave has a major impact on their antibacterial activity. extraction of l.nobilis leave with ethanol resulted in a product with greater overall antibacterial activity. study of algabri that carried out on antibacterial activity of libya bay leave extracted with methanol and n-hexane, it was observed that the n-hexane extract showed no antibacterial activity but the methanol extract had good inhibitory activity against s. aureus.15 also, el malti and amarouch (2009) found that the bay leave extract has a signifi cant antimicrobial activity against wide range of human pathogen.28 therefore, the result that we found confi rmed that l. nobilis leaves ethanol extract has antimicrobial activity against microorganism, it’s that observed the antimicrobial activity during agar well diffusion and bactericidal activity experiment. these results concurred with the result of aldhaher that found aqueous extract had good inhibitory activity against streptococcus mutants with mbcs range 30-60mg/ml. also concurred with study of yilmaz who found that antimicrobial activity of the essential oil against the tested panel of food-spoiling bacteria and one yeast strain.14,17 also, the study of sırıken who demonstrated that the essential oil of l. nobilis had strong antibacterial activity against gram-negative and gram-positive food-borne pathogens.28 study of aljindan and alkharsah, (2020) show, the resistance of salmonella species to antimicrobial drugs increased from 24.6% in 2011 to 37.8% in 2018. the research study by in 2018 all salmonella isolates were completely resistant to cefalotin, cefuroxime, and cefoxitin, while they found some susceptibility to other cephalosporins and ciprofl oxacin.17 while study of patil and mule they found s. typhi sensitive to cefixime, ceftriaxone, and azithromycin and based on average minimal inhibitory concentration and mic breakpoints.30 through the experiment conducted on rats by qnais et al (2012) which found l. nobilis aqueous extract has antidiarrheal agent.31 study of nafi s et al. exhibited notable potency regarding antimicrobial activity of (eos) from l. nobilis leaves had the highest activity against e. coli, with mic: 22.2 mg/ml and iz 9.00 mm. while it had activity against s. aureus with iz 10.0 mm and moderate mic: 5.55 mg/ml.32 conclusions the result of this study demonstrated an antibacterial eff ects of l. nobilis leaves ethanol extract was proved a strong antibacterial activity against bacterial infections as they exhibited an antimicrobial eff ect against s. aureus, s. typhi and weak eff ect in e. coli, so that is considered a kind of drug development substance for multidrug resistant bacteria. acknowledgements the authors would like to thank prof dr. anwar ma’ruf, universitas airlangga, vice director of post graduate school at universitas airlangga, and dwi candra buana, also the laboratory staff of a bsl 3 universitas airlangga for their support. figure 1. antibacterial activity of l.nobilis leaves ethanol extract against bacteria (a) salmonella typhi (b) e. coli (c) s. aureus. 124 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 120–126 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 conflict of interest the authors declare that they have no confl ict of interest. references 1. van hoek ah, mevius d, guerra b, mullany p, roberts ap, aarts hj. acquired antibiotic resistance genes: an overview. frontiers in microbiology. 2011 sep 28;2:203. 2. yılmaz eş, aslantaş ö. antimicrobial resistance and underlying mechanisms in staphylococcus aureus isolates. asian pacifi c journal of tropical medicine. 2017 nov 1;10(11):1059-64. 3. al-talib h, al-khateeb a, hassan h. antimicrobial resistance of staphylococcus aureus isolates in malaysian tertiary hospital. international medical journal. 2015 apr 1;22(1):1-3. 4. garoy ey, gebreab yb, achila oo, tekeste dg, kesete r, ghirmay r, kifl ay r, tesfu t. methicillinresistant staphylococcus aureus (mrsa): prevalence and antimicrobial sensitivity pattern among patients—a multicenter study in asmara, eritrea. canadian journal of infectious diseases and medical microbiology. 2019 jan 1;2019. 5. alharbi ns, khaled jm, kadaikunnan s, alobaidi as, sharafaddin ah, alyahya sa, almanaa tn, alsughayier ma, shehu mr. prevalence of escherichia coli strains resistance to antibiotics in wound infections and raw milk. saudi journal of biological sciences. 2019 nov 1;26(7):1557-62. 6. torkan s, bahadoranian ma, khamesipour f, anyanwu mu. detection of virulence and antimicrobial resistance genes in escherichia coli isolates from diarrhoiec dogs in iran. archivos de medicina veterinaria. 2016;48(2):181-90. 7. aljindan ry, alkharsah kr. pattern of increased antimicrobial resistance of salmonella isolates in the eastern province of ksa. journal of taibah university medical sciences. 2020 feb 1;15(1):48-53. 8. bryce a, hay ad, lane if, thornton hv, wootton m, costelloe c. global prevalence of antibiotic resistance in paediatric urinary tract infections caused by escherichia coli and association with routine use of antibiotics in primary care: systematic review and meta-analysis. bmj. 2016 mar 15;352:i939. 9. odonkor st, addo kk. prevalence of multidrugresistant escherichia coli isolated from drinking water sources. international journal of microbiology. 2018 aug 19;2018. 10. tambekar dh, dahikar sb, lahare md. antibacterial potentials of some herbal preparations available in india. res j med med sci. 2009;4:224-7. 11. hidayat yw, widodo ad, dachan yp. the antimicrobial effect of+ oxivarea against methicillin resistance staphylococcus aureus and pseudomonas aeruginosa. internet journal of microbiology. 2019;16 (1):20010. 12. kaurinovic b, popovic m, vlaisavljevic s. in vitro and in vivo eff ects of laurus nobilis l. leaf extracts. molecules. 2010 may;15(5):3378-90. 13. harismah k. pemanfaatan daun salam (eugenia polyantha) sebagai obat herbal dan rempah penyedap makanan. warta lpm. 2017 feb 21;19(2):110-8. 14. aldhaher za, merza phd wm, almelan mf, shaker rm, yas ls. eff ectiveness of bay leaves aqueous extract on streptococcus mutans in comparision to chlorhexidine gluconate. j pharmacy and biological science. 2017:12-6. 15. algabri so, doro bm, abadi am, shiba ma, salem ah. bay leaves have antimicrobial and antioxidant activities. journal of pathogen research. 2018 aug 2;1(1). 16. klemow km, bartlow a, crawford j, kocher n, shah j, ritsick m. herbal medicine: biomolecular and clinical aspects. crc press. 2011;2(11):211-28. 17. yilmaz es, timur m, aslim b. antimicrobial, antioxidant activity of the essential oil of bay laurel from hatay, turkey. journal of essential oilbearing plants. 2013 feb 1;16(1):108-16. journal of microbiology. 2019 volume 16 number. 18. ozcan b, esen m, sangun mk, coleri a, caliskan m. eff ective antibacterial and antioxidant properties of methanolic extract of laurus nobilis seed oil. journal of environmental biology. 2010 sep 1; 31(5): 637-41. 19. diten pom. 1986. sediaan galenik. jakarta: departemen kesehatan republik indonesia. 20. sudjadi. 1986. metode pemisahan. yogyakarta: ugm press. 21. nugroho, agung.2017. teknologi bahan alam. banjarmasin: lambung mangkurat university press. 22. clsi c. performance standards for antimicrobial susceptibility testing. clinical lab standards institute. 2016;35(3):16-38. 23. balouiri m, sadiki m, ibnsouda sk. methods for in vitro evaluating antimicrobial activity: a review. journal of pharmaceutical analysis. 2016 apr 1;6(2):71-9. 24. alonso ca, domínguez c, heras j, mata e, pascual v, torres c, zarazaga m. antibiogramj: a tool for analysing images from disk diff usion tests. computer methods and programs in biomedicine. 2017 may 1;143:159-69. 25. mah tf. establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (mbc-p) and biofilm cells (mbc-b). jove (journal of visualized experiments). 2014 jan 2(83):e50854. 26. al-ogaili n, bilal r, younis h, khadim t. the examination of the water concentrates of laurus nobilis leaves antibacterial activity utilizing various strategies for extraction (in vitro). international journal 125khawla abdullah sakran, et al.: antimicrobial activities of laurus nobilis leaves ethanol extract ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 of research in pharmaceutical sciences. 2020 jan 6;11(1):66-9. 27. otsuka n, liu mh, shiota s, ogawa w, kuroda t, hatano t, tsuchiya t. anti-methicillin resistant staphylococcus aureus (mrsa) compounds isolated from laurus nobilis. biological and pharmaceutical bulletin. 2008 sep 1;31(9):1794-7. 28. el malti j, amarouch h. antibacterial effect, histological impact and oxidative stress studies from laurus nobilis extract. journal of food quality. 2009 apr;32(2):190-208. 29. sırıken b, yavuz c, güler a. antibacterial activity of laurus nobilis: a review of literature. medical science and discovery. 2018 nov 30; 5(11):374-9. 30. patil n, mule p. sensitivity pattern of salmonella typhi and paratyphi a isolates to chloramphenicol and other anti-typhoid drugs: an in vitro study. infection and drug resistance. 2019;12:3217. 31. qnais ey, abdulla fa, kaddumi eg, abdalla ss. antidiarrheal activity of laurus nobilis l. leaf extract in rats. journal of medicinal food. 2012 jan 1;15(1):51-7. 32. nafi s a, kasrati a, jamali ca, custódio l, vitalini s, iriti m, hassani l. a comparative study of the in vitro antimicrobial and synergistic eff ect of essential oils from laurus nobilis l. and prunus armeniaca l. from morocco with antimicrobial drugs: new approach for health promoting products. antibiotics. 2020 apr;9(4):140. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 2 may–august 2022 original article clinical profiles, laboratory, radiological and outcome of covid-19 elderly patients in waikabubak regional general hospital, west sumba lisbeth maria laurentia1 , rambu farah effendi2 1general practitioner, waikabubak regional general hospital, west sumba, indonesia 2department of internal medicine, waikabubak regional general hospital, west sumba, indonesia received: february 3rd, 2022; revised: march 2nd, 2022; accepted: june, 30th, 2022 abstract cases of covid-19 in the elderly show varied clinical characteristics. elderly patients tend to be easily infected with covid-19 and experience more severe conditions. this study aims to analyze the correlation between clinical characteristics of elderly covid-19 patients and severity of covid-19 disease at waikabubak regional general hospital. retrospective study from the medical records of elderly patients with confirmed covid-19 at waikabubak regional general hospital in march 2020 – september 2021. inclusion criteria were elderly patients with confirmed covid-19 who underwent treatment in covid isolation room and did rapid antigen examination. the data collected were demographics, clinical manifestations, laboratory, radiological features, comorbid, and outcomes. data analysis using spps for window 12.0 version. there were 33 patients with 18 men and 15 women. patients aged 65-74 are the most infected with covid-19 and experience more severe conditions. patients with no previous vaccine history were associated with the severity of covid-19. elderly patients with obesity tend to experience severe covid-19 symptoms. elderly patients with multi-comorbidities tend to experience severe covid-19 symptoms. dominant clinical symptoms in elderly patients were cough (33%), shortness of breath (25%) and fever (21%). hematologic parameters that correlated with severity were hemoglobin, platelets, nlr, alc and rbg. the most common radiological findings were bilateral infiltrates (92%). mortality rate of elderly covid-19 patients treated at the waikabubak regional general hospital still tends to be high (42%). age, vaccine history, obesity, shortness of breath, multi-comorbidities, laboratory and radiology significantly influence the severity of covid-19 infection in the elderly. keywords: clinical profile; covid-19; east nusa tenggara; elderly; severity abstrak kasus covid-19 pada lansia menunjukkan karakteristik klinis yang bervariasi. lansia mudah terinfeksi covid19 dan mengalami kondisi lebih berat. penelitian ini bertujuan untuk menganalisis korelasi antara karakteristik klinis pasien lansia covid-19 dengan tingkat keparahan penyakit di rsud waikabubak. penelitian retrospektif, data dari rekam medis pasien lansia dengan terkonfirmasi covid-19 di rsud waikabubak pada periode maret 2020 – september2021. kriteria inklusi pasien lansia dengan terkonfirmasi covid-19 yang dirawat di ruang isolasi covid, dan dilakukan pemeriksaan rapid antigen dengan spesimen swab. data yang dikumpulkan adalah demografi, manifestasi klinis, laboratorium, gambaran radiologis, penyakit komorbid, dan luaran. analisis data menggunakan spps for window 12.0 version. didapatkan 33 pasien dengan jumlah pria 18 orang dan wanita 15 orang. pasien yang berusia 65-74 merupakan pasien terbanyak terinfeksi covid-19 dan mengalami kondisi lebih berat. pasien yang tidak memiliki riwayat vaksin sebelumnya berkaitan dengan derajat keparahan covid-19. pasien lansia dengan obesitas cenderung mengalami gejala covid-19 berat. * corresponding author: lisbethlaurentia@gmail.com https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-0718-8945 70 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 lisbeth maria laurentia, et al. clinical profiles, laboratory, radiological and outcome of covid-19 pasien lansia dengan multikomorbid cenderung mengalami gejala covid-19 berat. gejala klinis yang dominan pada pasien lansia adalah batuk (33%), sesak napas (25%) dan demam (21%). parameter hematologik yang berkorelasi dengan derajat keparahan adalah hemoglobin, trombosit, nlr, alc dan gds. gambaran radiologis yang tersering didapatkan adalah inflitrat bilateral (92%). angka kematian pasien lansia covid-19 yang dirawat di rsud waikabubak masih cenderung tinggi, yaitu sebanyak 14 pasien (42%). karakteristik klinis seperti usia, riwayat vaksin, obesitas, sesak nafas, multikomorbid, laboratorium dan radiologis berpengaruh secara bermakna terhadap tingkat keparahan dari infeksi covid-19 pada lansia. kata kunci: covid-19; derajat keparahan; lansia; nusa tenggara timur; profil klinis how to cite: laurentia, l. m., effendi, r. f. clinical profiles, laboratory, radiological and outcome of covid19 elderly patients in waikabubak regional general hospital, west sumba. indonesian journal of tropical and infectious disease. 10(2). 69–75. aug. 2022. introduction the coronavirus disease (covid-19) pandemic is still happening today. the first case of covid-19 was reported in china's wuhan province in december 2019. the virus has infected more than 150 million people worldwide and caused more than five million deaths.1 indonesia is in the 16th position with the most covid-19 cases in the world based on data from gugus tugas penanganan covid-19 on january 31, 2022. confirmed covid-19 cases in indonesia have reached more than 4.3 million cases. currently, ntt covid-19 cases have reached 64,600 cases (1.5% of all cases in indonesia).2 among the confirmed cases at the end of october in indonesia, 11.8% were elderly patients aged 60 years and over. the elderly also contributed to 46.8% of covid-19related deaths nationwide. older age and comorbidities have been noted as major factors of susceptibility to covid-19.3 liu et al., stated that the mortality rate of covid19 patients aged 60 years and over (5.3%) was significantly higher than that of patients under 60 years (1.4%).4 unfortunately, data regarding the clinical profile of the elderly hospitalized with covid-19 in indonesia is still limited. the elderly are a population that is very at risk in a pandemic, so more attention is needed. waikabubak regional general hospital is one of the covid-19 referral centre in west sumba, which has integrated care and a special isolation ward for covid-19 patients. enforcing covid cases in remote areas is a challenge because of the limited support. the good news is that currently rapid antigen can be used to diagnose covid-19. aspects that need to be considered in reducing covid-19 cases are treating infected patients and avoiding the spread of the virus. this study aims to analyse the correlation between clinical characteristics of elderly covid-19 patients and the severity of covid-19 disease. we hope that by knowing a significant relationship between the clinical characteristics and the severity of covid-19 disease, patients will receive optimal therapy, thereby reducing mortality. materials and methods this retrospective study used secondary data from medical record data of elderly patients with confirmed covid-19 at waikabubak regional general hospital in the period march 2020 to september 2021. the inclusion criteria were elderly patients with symptoms of covid-19 based on the criteria of the indonesian ministry of health 2020, who underwent treatment in the ward isolation of covid and examination of nasal swab specimens using rapid antigen according to established standards. the data collected were age, gender, epidemiological history (contact with adult patients whether suspected, probable, or positive covid-19, and/or traveling or living in infected areas, red zone areas according to 71 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 69–75 the ministry of health), vaccination history, smoking history, body mass index, clinical symptoms, comorbidities, laboratories, and chest x-ray. images. laboratory examinations and x-rays were performed using the same equipment and protocol for all patients. patient outcomes were categorized as discharge with improvement and death. patients were excluded from the study if data were incomplete and patients aged <65 years. the collected data were analyzed using spps for window 12.0 version. results and discussion socio-demography during the study period, 33 patients were obtained who had complete data, consisting of 18 men and 15 women (table 1). table 1. demographics of elderly patients with covid-19 parameters severity p severe/ ards moderate bmi normal 5 (15%) 7 (22%) 0,251 overweight 8 (24%) 5 (15%) 0,201 obesity 8 (24%) 0 (0%) 0,001* early symptom fever 2 (6%) 5 (15%) 0,564 cough 8 (24%) 3 (9%) 0,153 shortness of breath 7 (22%) 1 (3%) 0,042* chest pain 2 (6%) 0 (0%) 0,274 malaise 0 (0%) 2 (6%) 0,274 nausea and vomit 0 (0%) 1 (3%) 0,553 anosmia 0 (0%) 1 (3%) 0,553 decrease of consciousness 1 (3%) 0 (0%) 0,553 comorbid hypertension 3 (9%) 6 (18%) 0,301 diabetes mellitus 1 (3%) 2 (6%) 0,352 coronary arterial disease 1 (3%) 0 (0%) 0,553 tuberculosis 2 (6%) 1 (3%) 0,401 hepatitis 0 (0%) 1 (3%) 0,553 absent 0 (0%) 3 (9%) 0,165 multicomorbid 13 (40%) 0 (0%) 0,001* the results showed that gender did not correlate with the severity of covid-19 (p=0.435). patients aged 65-74 years correlated with the severity of covid-19 (p=0.045). patients who were exposed to cigarette smoke were also not related to the severity of covid-19 (p=0.481). patients who had no previous vaccine history were associated with the severity of covid-19 (p=0.05). patients with a history of covid19 contact and travel history were not associated with the severity of covid-19 (p=0.515) and (p=0.737). clinical in the analysis of body mass index (bmi), obese patients tend to experience more severe symptoms (p=0.001) (table 2). patients with normal bmi and overweight did not correlate with severity (p=0.251 and p=0.201). table 2. clinical elderly patients with covid-19 on examination of clinical symptoms, fever did not correlate with the degree of severity (p=0.564). cough symptoms did not correlate with severity (p=0.153). patients with shortness of breath tended to experience severe symptoms (p=0.042). symptoms of chest pain did not correlate with severity (p=0.274). symptoms of weakness also did not correlate with the degree of severity (p=0.274). complaints of nausea and vomiting did not correlate with the degree of severity (p=0.553). symptoms of anosmia did not correlate with severity (p=0.553). symptoms of decreased consciousness did not correlate with severity (p=0.553). parameter severity p severe/ ards moderate sex men 12 (36%) 6 (18%) 0,435 women 8 (24%) 7 (22%) age 65-74 14 (43%) 8 (24%) 0,045* 75-84 5 (15%) 3 (9%) 0,873 ≥85 1 (3%) 2 (6%) 0,607 smoking exposed 19 (58%) 7 (22%) 0,481 not exposed 3 (9%) 4 (12%) vaccine history yes 3 (9%) 6 (18%) 0,05* no 17 (52%) 7 (22%) covid19 contact history yes 10 (31%) 5 (15%) 0,515 no 10 (31%) 8 (24%) travel history yes 4 (12%) 2 (6%) 0,737 no 16 (49%) 11 (34%) 72 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 lisbeth maria laurentia, et al. clinical profiles, laboratory, radiological and outcome of covid-19 patients with multi-comorbidity tended to have severe symptoms (p=0.001). patients with one or no comorbidities did not correlate with severity. table 3. laboratory results of elderly patients with covid-19 parameters severity p or (ci 95%) severe/ ards moderate blood routine test low hb level 11 (33%) 5 (15%) 0,049* 1,833 (0,29911.259) high wbc level 12 (36%) 10 (30%) 0,411 1,421 (0,8121,824) low wbc level 3 (9%) 1 (3%) 0,056 0,186 (0,0541,254) thrombo-cytopenia 6 (18,2%) 2 (6%) 0,011* 0,912 (0,7851,341) covid-19 support parameter nlr ≥3.13 20 (61%) 7 (21%) 0,001* 0,561 (0,421 1,125) <3.13 0 (0%) 6 (18%) 0,004* 0,462 (0,2081,022) alc ≥1500 0 (0%) 6 (18%) 0,001* 8,915 (1,51466,081) <1500 20 (61%) 7 (12%) 0,002* 0,814 (0,4561,208) blood glucose rbg ≥200 17 (52%) 2 (6%) 0,001* 0,441 (0,2150,822) <200 3 (9%) 11 (33%) 0,042 2,513 (1,1074,255) note. hb=haemoglobin; wbc=white blood cell; nlr=neutrophillymphocyte ratio; alc=absolute-lymphocyte count; rbg=random blood glucose. laboratory on laboratory results (table 3), patients with anemia tended to have severe symptoms (p=0.049; or=1.833; 95% ci=0.29911.259), as well as thrombocytopenia (p=0.011; or=0.912; 95% ci=0.785-1.341). patients with nlr ≥ 3.13 tend to experience more severe symptoms (p=0.001; or=0.561;95% ci=0.421-1.125), while patients with nlr <3.13 tend to experience milder symptoms (p=0.004; or=0.462; ci95%=0.208-1.022). patients with alc <1500 tended to experience milder symptoms (p=0.001; or=8.915; 95% ci=1.151466.081), while patients with alc <1500 tended to experience more severe symptoms (p=0.002; or = 0.814; 95% ci = 0.4561,208). patients with random blood glucose (rbg) ≥200 tended to have severe symptoms (p=0.001; or=0.441; 95% ci=0.215-0.822). radiological feature elderly patients with covid-19 had abnormal radiological features as shown in table 4, showing a tendency to experience severe symptoms (p=0.070). x-rays with bilateral infiltrates tend to have severe symptoms (p=0.024). the radiological features of pulmonary oedema were not related to the severity (p=0.530). table 4. radiological feature of elderly patients with covid-19 parameter severity p severe/ ards mode-rate x-ray abnormal 20 (61%) 11 (33%) 0,070* normal 0 (0%) 2 (6%) infiltrate unilateral 0 (0%) 3 (9%) 0,024* bilateral 20 (61%) 10 (31%) pulmonary oedema yes 3 (9%) 1 (3%) 0,530 no 17 (52%) 12 (37%) outcome patients with severe symptoms tend to have a worse outcome than patients with moderate symptoms as shown in table 5. table 5. outcome of elderly patients with covid-19 parameter severity p severe/ ards moderate outcome not survived 14 (42%) 0 (0%) 0,0002* survived 6 (18%) 13 (40%) discussion the results of this study showed several characteristics that correlated with the severity of the patient. based on sociodemography, ages 65-74 are the most infected patients with covid-19 and experience more severe conditions. aging is associated with ace-2 overexpression, immune dysregulation, decreased sex steroids, poor nutrition, vitamin d deficiency, mitochondrial dysfunction and oxidative 73 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 69–75 stress, comorbidities, and lower physical activity. these effects result in increased viral replication, cytokine storm, and poor lung protection against viruses.5 however, based on cdc data, 80% of deaths occur among adults aged 65 years with the highest percentage in older adults aged ≥85 years.6 in this study, 80% of patients were exposed to cigarettes, both active and passive smokers. however, patients exposed to cigarettes were not associated with the severity of covid-19. it is known that recent reviews show that nicotine exposure is associated with cardiopulmonary susceptibility to covid-19 and tobacco use is at risk of viral infection and more severe clinical symptoms.7, 8 from our study data, it was found that patients who had no previous vaccine history were associated with the severity of covid19. the covid-19 vaccine is highly effective in preventing covid-19-related hospitalizations in older adults. in the elderly aged 65-74 years, the effectiveness of 2 doses of the vaccine is 96%, while in the elderly aged ≥75 years it reaches 91%.9 based on clinical findings, patients with obesity tend to experience more severe symptoms. recent evidence suggests that obesity weakens the immune system, leaving the host vulnerable to infectious diseases.10 obesity is a risk factor for the development of severe covid-19 with the need for hospitalization and mechanical ventilation, especially in elderly patients. obesity causes changes in the microbiota, physiological and immune responses associated with poor viral responses.11 while the most common symptoms were cough (33%), shortness of breath (25%) and fever (21%). a systematic review of singhal et al, stated similarly that the most common symptoms in elderly patients were fever, cough and shortness of breath.12 on hematological examination, almost half of the patients were anemic (48%). this study is in accordance with bergamaschi et al, who stated that older people are at risk for anemia and it can affect their quality of life.13 in covid-19 patients, inflammation can cause changes in iron hemostasis and reduced iron absorption in the intestine, resulting in reduced metal availability in the process of erythropoiesis and hemoglobin (hb) production.14 several studies have focused on the relationship between anemia and the severity or mortality of covid-19, the results are still controversial.15, 16 in this study, the leukocyte count was increased (66%). the results of this study are similar to those of liu et al., the increase in white blood cell count was significantly more common in elderly patients, indicating that elderly patients infected with 2019-ncov were more likely to have bacterial infections.3 the increase in the number of white blood cells and neutrophils in elderly covid-19 patients was 30.64% and 33.33%.17 in particular, a high neutrophil count was an independent predictor of poor outcome. neutrophilia observed during a cytokine storm caused by viral infection.18 in the study of betsy et al, after autopsy they showed neutrophils infiltrating the lungs in the context of a cytokine storm triggering ards and causing organ damage and death in covid-19.19 in this study, the percentage of thrombocytopenia did not predominate, but patients with thrombocytopenia tended to experience severe symptoms. platelets tend to be activated in viral pneumonia causing lung damage by stimulating the respiratory inflammatory response. a tendency to thrombocytopenia in elderly covid-19 patients may indicate a worsening of the thrombotic state, which is associated with increased mortality.20 in elderly covid-19 patients, thrombocytopenia is associated with a 4.24-fold increased risk of death.21 inflammatory biomarkers describe immune status which is a predictor of covid-19 prognosis. hematological ratios such as neutrophil to lymphocyte ratio (nlr) and absolute lymphocyte count (alc) are markers of systemic inflammation that have 74 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 lisbeth maria laurentia, et al. clinical profiles, laboratory, radiological and outcome of covid-19 been extensively investigated as potential predictors of viral pneumonia. increased nlr and decreased alc predict poor outcome in elderly covid-19 patients.22 in this study, supporting examination for covid-19 showed an increase in nlr (82%) and a decrease in alc (82%). in our study, rbg value ≥200 mg/dl was present in most of the patients (58%). severe hyperglycemia is common in critically ill patients and is seen as a marker of disease severity. the study of li et al., examined covid-19 patients with hyperglycemia who were hospitalized, consisting of 21.6% of the elderly who had a history of diabetes, 20.8% were newly diagnosed with diabetes, and 28.4% were diagnosed with dysglycemia.23 the mechanism by which new-onset diabetes develops in the elderly with covid-19 remains unknown, but it is possible that a number of complex etiologies exist, including disturbances in glucose disposal and insulin secretion, stress hyperglycemia, diabetes preadmission, and steroid-induced diabetes.24 based on radiological results, 94% of chest x-rays had abnormal features with bilateral infiltrates (92%). our study is consistent with the neumann-podczaska study, which stated that the most common radiological features in the elderly were ground glass opacities (ggo) (28.6%), or ggo + consolidation (12.9%), affecting multiple lobes (62.2%) with a bilateral distribution (58.2%). although most studies did not specify a peripheral or central distribution specifically (93.0%), some studies tended to show a peripheral picture (4.5%) with some cases being peripheral and central (2.5%).25 the mortality rate for covid-19 elderly patients treated at the waikabubak regional general hospital still tends to be high, namely 14 patients (42%), which are included in the severe category. this figure is not much different from the national elderly mortality rate of 46% which was reported on january 31, 2022.2 conclusions the results of this study indicate that clinical characteristics such as age, vaccine history, obesity, clinical symptoms (shortness of breath), multi-comorbid, laboratory (hemoglobin, platelets, nlr, alc and gds) and radiologically have a significant effect on the severity of covid-19 infection in the elderly. by knowing the severity of the disease, patients will receive optimal therapy and reduce mortality. in addition, early diagnosis and supportive care are very important for elderly covid-19 patients. acknowledgement we would like to thank the covid-19 isolation team and the director of waikabubak regional general hospital for allowing us to conduct this study by using the hospital's medical records. conflict of interest all authors declared that they do not have any conflict of interest in both the research and also in the article writing process. references 1. zhu n, zhang d, wang w, li x, yang b, song j, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020. 2. satuan tugas penanganan covid-19. peta sebaran gugus tugas percepatan penanganan covid-19 [internet]. 2020 [cited 2021 nov 20]. available from: https://covid19.go.id/petasebaran 3. liu k, chen y, lin r, han k. clinical features of covid-19 in elderly patients: a comparison with young and middle-aged patients. j infect. 2020;80(6):e14-e8. 4. liu y, gayle aa, wilder-smith a, rocklöv j. the reproductive number of covid-19 is higher compared to sars coronavirus. j travel med. 2020. 5. farshbafnadi m, zonouzi sk, sabahi m, dolatshahi m, aarabi mh. aging & covid-19 susceptibility, disease severity, and clinical outcomes: the role of entangled risk factors. exp gerontol. 2021;154:111507. 75 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 69–75 6. centers for disease control and prevention. severe outcomes among patients with coronavirus disease 2019 (covid-19)—united states, february 12–march 16, 2020 [internet]. 2020 [cited 2021 des 17]. available from: https://www.cdc.gov/mmwr/volumes/69/wr/mm 6912e2.htm 7. gupta ak, nethan st, mehrotra r. tobacco use as a well-recognized cause of severe covid-19 manifestations. respir med. 2021;176:106233. 8. mohsin fm, tonmon tt, nahrin r, tithy sa, ame fa, ara i, et al. association between smoking and covid-19 severity: evidence from bangladesh. j multidiscip healthc. 2021;14:1923. 9. moline hl, whitaker m, deng l, rhodes jc, milucky j, pham h, et al. effectiveness of covid-19 vaccines in preventing hospitalization among adults aged≥ 65 years— covid-net, 13 states, february–april 2021. mmwr morb mortal wkly rep. 2021;70(32):1088. 10. mohammad s, aziz r, al mahri s, malik ss, haji e, khan ah, et al. obesity and covid-19: what makes obese host so vulnerable? immun ageing. 2021;18(1):1-10. 11. alberca rw, oliveira ldm, branco accc, pereira nz, sato mn. obesity as a risk factor for covid-19: an overview. crit rev food sci nutr. 2020:1-15. 12. singhal s, kumar p, singh s, saha s, dey ab. clinical features and outcomes of covid-19 in older adults: a systematic review and metaanalysis. bmc geriatr. 2021;21(1):1-9. 13. bergamaschi g, de andreis fb, aronico n, lenti mv, barteselli c, merli s, et al. anemia in patients with covid-19: pathogenesis and clinical significance. clin exp med. 2021;21(2):239-46. 14. dinevari mf, somi mh, majd es, farhangi ma, nikniaz z. anemia predicts poor outcomes of covid-19 in hospitalized patients: a prospective study in iran. bmc infect dis. 2021;21(1):1-7. 15. bellmann-weiler r, lanser l, barket r, rangger l, schapfl a, schaber m, et al. prevalence and predictive value of anemia and dysregulated iron homeostasis in patients with covid-19 infection. j clin med. 2020;9(8):2429. 16. tao z, xu j, chen w, yang z, xu x, liu l, et al. anemia is associated with severe illness in covid‐19: a retrospective cohort study. j med virol. 2021;93(3):1478-88. 17. li p, chen l, liu z, pan j, zhou d, wang h, et al. clinical features and short-term outcomes of elderly patients with covid-19. int j infect dis. 2020;97:245-50. 18. guo x-zj, thomas pg, editors. new fronts emerge in the influenza cytokine storm. semin immunopathol; 2017: springer. 19. barnes bj, adrover jm, baxter-stoltzfus a, borczuk a, cools-lartigue j, crawford jm, et al. targeting potential drivers of covid-19: neutrophil extracellular traps. j exp med. 2020;217(6). 20. işler y, kaya h. relationship of platelet counts, platelet volumes, and curb-65 scores in the prognosis of covid-19 patients. am j emerg med. 2022;51:257-61. 21. liu y, sun w, guo y, chen l, zhang l, zhao s, et al. association between platelet parameters and mortality in coronavirus disease 2019: retrospective cohort study. platelets. 2020;31(4):490-6. 22. aly mm, meshref ts, abdelhameid ma, ahmed sa, shaltout as, abdel-moniem ae, et al. can hematological ratios predict outcome of covid-19 patients? a multicentric study. j blood med. 2021;12:505. 23. li h, tian s, chen t, cui z, shi n, zhong x, et al. newly diagnosed diabetes is associated with a higher risk of mortality than known diabetes in hospitalized patients with covid‐19. diabetes obes metab. 2020;22(10):1897-906. 24. khunti k, del prato s, mathieu c, kahn se, gabbay ra, buse jb. covid-19, hyperglycemia, and new-onset diabetes. am diabetes assoc. 2021;44(12):2645-55. 25. neumann-podczaska a, al-saad sr, karbowski lm, chojnicki m, tobis s, wieczorowska-tobis k. covid 19-clinical picture in the elderly population: a qualitative systematic review. aging dis. 2020;11(4):988. 113 vol. 6 no. 5 may–august 2017 research report n o a s s o c i a t i o n o f r e c u r r e n t r e s p i r a t o r y p a p i l l o m a t o s i s a g g r e s s i v e n e s s a n d h u m a n papillomavirus type 6 and 11 rizka fathoni perdana1a, sri herawati1, bakti surarso1, eduardus bimo aksono h2 1 orl-hns departement, faculty of medicine, universitas airlangga – dr. soetomo general hospital 2 institute of tropical disease – faculty of vetenary, universitas airlangga a corresponding author: rizka.fathoni@gmail.com abstract recurrent respiratory papillomatosis (rrp) is the most common benign neoplasm of the larynx among children as a result of hpv infection mainly type 6 and 11. rrp is still considered as serious problem since papilloma in the airway can cause hoarseness and obstruction which later described as aggressive and non aggressive types. patients underwent multiple surgeries to keep airway patency and in order to communicate vocally. previous studies reported that hpv-11 was associated with a more aggressive disease course. this study aim is to determine the association of rrp aggressiveness with hpv-6 and 11. papilloma specimens were taken from patients who underwent surgical treatment of rrp patients and subjected hpv typing. all 17 patients with completed data and epidemiologic questionaires were defined for their staging to disease severity. informations in the database were analyzed to identity statistically significant relationship with hpv type and knowing the hpv type is to predict the disease course. 17 patients rrp (12 males and 5 females) with age at onset 3,15 + 2,28 years and age at diagnosis 3,81 + 2,51 years. surgery was performed 2,71 ± 1,16 times per year and 9 patients treated more than 3 surgeries per year. 10 patients had distal papilloma and 11 patients had tracheotomy. agrressive disease was show found in 14 patients among 17 patients. hpv-11 was detected in 10 patients, hpv-6 was detected in 7 patients. according to the statisitcal analysis (chi square test), there is no relationship between hpv type and disease aggressiveness. hpv-6 and 11 are not the only cause that affect the aggressiveness of recurrent respiratory papillomatosis. keywords: papilloma, recurrent papillomatosis, respiratory papillomatosis, human papilloma virus, juvenile papillomatosis, adult papillomatosis abstrak papiloma saluran pernapasan berulang (pspb) merupakan salah satu neoplasma jinak pada laring yang paling sering ditemukan pada anak sebagai hasil infeksi human papillomavirus khususnya tipe 6 dan 11. pspb masih dianggap sebagai masalah serius karena papiloma pada saluran pernapsan dapat menyebabkan suara parau dan lebih lanjut menyebabkan obstruksi jalan napas, sehingga dikatagorikan sebagai penyakit agresif dan tidak agresif. penderita dapat menjalani operasi beberapa kali untuk menjaga patensi jalan napas dan untuk memperbaiki kualitas suara. studi terdahulu melaporkan bahwa hpv-11 memiliki hubungan dengan perjalanan penyakit yang agresif. penelitian ini bertujuan untuk menentukan hubungan agresivitas pspb dengan hpv tipe 6 dan 11. diharapkan identifikasi tipe hpv dapat menjadi prediksi perjalanan penyakit. spesimen papiloma didapatkan saat penderita pspb menjalani pembedahan dan seluruhnya diperiksa tipe hpv. didapatkan 17 penderita memiliki rekam medik dan kuesioner yang lengkap sehingga dapat ditentukan derajat keparahan penyakit. informasi dikumpulkan dan kemudian dianalisis untuk mendapatkan hubungan yang bermakna dengan tipe hpv. 17 penderita pspb (12 laki-laki dan 5 perempuan) dengan usia saat onset gejala 3,15 + 2,28 tahun dan usia saat diagnosis 3,81 + 2,51 tahun. jumlah total operasi tiap tahun 2,71 ± 1,16 kali dan 9 penderita diantaranya menjalani lebih dari 3 kali pertahun. 10 penderita memiliki implantasi papiloma distal laring dan 11 penderita pernah menjalani trakeotomi. penyakit yang agresif didapatkan pada 14 dari 17 penderita, dan hpv-11 didapatkan pada 10 penderita. hpv-6 didapatkan pada 7 penderita. uji statistik menunjukkan tidak ada hubungan antara tipe hpv dengan agresivitas penyakit. hpv-6 dan 11 bukan menjadi satu-satunya faktor yang mempengaruhi faktor agresivitas penyakit kata kunci: papilloma, papillomatosis rekuren, papillomatosis pernapasan, virus papiloma manusia, papilomatosis remaja, papilomatosis dewasa 114 indonesian journal of tropical and infectious disease, vol. 6 no. 5 may–august 2017: 113–117 introduction human papillomavirus type 6 and 11 are the most important etiologies of rrp by expressing gene e6 and e7 within the cell and contribute in oncogenesis. protein e6 plays a role in binding and deactivating protein p53, whereas protein e7 will bind prb. these hpv types differ in affinity to bind protein p53 and prb.1,2 recurrent respiratory papillomatosis or rrp is a disease caused by hpv type 6 and 11 that manifest as papilloma growths in respiratory tract and characteristically recurrent. there are two types of rrp, child onset or juvenile type (juvenile onset rrp/jorrp) and adult onset or adult type (adult onset rrp/aorrp). juvenile has onset peak at age 2 – 4 years and grow aggressively, whereas adult type has peak incident at age 20 – 40 years.3 in united states, rrp incidents in child population estimated are 4,3 cases per 100 thousands children and 1,8 cases per 100 thousands adults. new incidents reported 80 to 1500 cases per year with prevalent rates of 700 to 3000 active cases in year 1999.4 at 1997, we have 57 new patients, 82% were below 10 years old. still in dr. soetomo hospital, we have 21 new patients of 51 total cases between 2006 to 2010, with common proportion on both gender. rrp is diagnosed by clinical symptoms, endoscopic examination and histopathology.5,6 stridor and persistent or progressive hoarseness accompanied by shortness of breath are the main symptoms.7 other rare symptoms are chronic cough, recurrent pneumonia, and failure to thrive.8,9 vocal cord is the initial and most common sites for papilloma growth thus hoarseness is the main symptom.10 endoscopy is the main examination method for definitive diagnosis.9 final confirmation is done by tissue histopathology examination.5 this disease can be both non aggressive which resulting a remission, or more aggressive that require repetitive surgery.3 aggressive type characterized by recurrent papilloma growth in more than one site. the most common sites are oral cavity, trachea and bronchus. the criterias according to buchinsky11 include total surgical histories, distal site of papilloma, and history of tracheotomy procedures. aggressive disease was classified based on its surgical requirement more than 10 times, three or more surgeries within a year, and papilloma involvement in subglottic area. criterias of non aggressive are based on surgical requirement less than 10 times, not exceed 3 surgeries within a year, and no subglottic involvement. this degree of severity will assist the identification of prognosis.10 yusuf3 reported 75% rrp patients that need tracheotomy due to upper airway obstruction. rachmawati6 found 11 of 51 patients (21,57%) had tracheal and bronchial involvements. thirty of 51 patients (70,59%) had tracheotomy histories, and 8 of 37 juvenile type rrp patients 21,62%) had 3 or more mls procedures (microscopic laryngeal surgery) within a year. material and method the study procedures are mention as followed. all rrp patients who visit the orl-hns opd and ward of dr. soetomo hospital for mls schedules were informed about the purposes and procedures of study (informing consent). patients/parents approval were asked by sign the informed consent. in ward they were interviewed by questionnaires and their medical records from opd were analyzed. patients then categorized become aggressive or non aggressive group. the tissue from mls is the study sample. the speciments were sent to lpt ua for hpv-6 and 11 examinations by pcr method. criterias of aggressive rrp consist of three or more papilloma removal surgeries within a year, history of tracheotomy, or papilloma implantation in distal part of larynx (trachea or bronchus) by rigid or flexible bronchoscopy.11 criterias of non aggressive rrp consist of less than three times papilloma removal within a year, never had tracheotomy, and no papilloma implantation in distal part of larynx (trachea and/or bronchus) by rigid or flexible laryngoscopy.11 inclusion criterias are rrp patients in all age with complete medical record data along medication, and agree to join this experiments (informed consent). exclusion criterias are patients with history ajuvan therapy within 30 days and not enough speciments biopsy for pcr examination. result and discussion there were total 17 juvenile type of rrp patients who had visit dr. soetomo hospital. all had completed data from when they were first diagnosed until their latest visit. aggressive papilloma found in 14 patients (82,4%), whereas 3 patients (17,6%) categorized as non aggressive rrp. the youngest onset was 0,5 year and the oldest was 8 years, with mean 3,15 ± 2,28 years and median 3 years. the ages when it were diagnosed ranged from 0,5 to 9 years with mean 3,81 ± 2,51 years and median 4 years. the range from onset of symptoms untill diagnosis establishment was 1 to 30 months with is mean 8,16 ± 10,88 months. clinical progress hoarseness the most common symptoms in patient’s first time visit a number of 9 patients (52,9%), whereas shortness of breath have been 8 patients (47,1%) as shown in table 1. the most frequent number of surgery within a year were 6 times and the less frequent number was 1 time, with mean 2,71 ± 1,16 times and median 2 times. distal laryngeal papilloma were found in 11 patients (64,7%), whereas 6 patients (35,3%) had no distal laryngeal papilloma. 115perdana, et al.,: no association of recurrent respiratory papillomatosis aggressiveness table 1. summary data for 17 patiens with rrp were included in this study gender male = 12 (70,6%), female = 5 (29,4%) birth order first = 11 (64,7%), the others = 6 (35,3) maternal condyloma yes = 2 (11,8%), no = 15 (88,2%) method of delivery pervaginam = 17 (100%) maternal age < 20 y = 1 (5,9%), ≥ 20 y = 16 (94,1%) age at onset symptom mean = 3,153±2,283 years, median = 3 year age at diagnosis mean = 3,812±2,512 years, median = 4 year gap between symptom and diagnosis mean = 0,659±0,907 years main complain at onset dsypnea = 8 (47,1%), hoarsness = 9 (52,9%) number of surgeries ≥ 3x = 9 (52,9%), < 3x = (47,1%) max number of surgeries in 12 months period median = 2x, mean = 2,71x, range = 1-6x agressiveness of course agressive = 14 (82,4%), indolen = 3 (17,6%) distal involvement yes = 11 (64,7%), no = 6 (35,3%) tracheotomy yes = 13 (76,5%), no = 4 (23,5%) hpv type 6 = 7 (41,2%), 11 = 10 (58,8%) table 2. crosstabulation hpv type and disease agressiveness hpv type agressivness p yes no 6 5 2 11 9 1 .537 tracheotomy histories were found in 13 patients (76,5%), whereas 4 patients (23,5%) had no tracheotomy histories. association hpv and number of surgery in 12 months, distal papillomas, and tracheotomy show no significan. human papillomavirus type 6 found in 7 patients (41,2%) and 10 patients (58,8%) were infected by hpv-11. aggressive rrp consist of 5 patients with hpv-6 (71,4% of total hpv-6 patients) and 9 patients with hpv-11 (90% of total hpv-11 patients). non aggressive rrp consist of 2 patients with hpv-6 (28,6% of total hpv-6 patients) and 1 patients (33,33%) of hpv-11 patients. fisher’s test resulted p value 0,537 as shown in table 2. the result of age of disease onset in this study is showed no significant different from the previous studies. wiatrak12 reported the age when they were diagnosed ranged from 0,33 to 12 years, with mean 44 moths (3,67 years). buchinsky11 reported 3 years median which range from 0,1 to 13,1 years. campisi13 was found mean 4,4 years which range from 0,1 – 14 years. it suggests that papilloma growth will reach peaks at age three years. hpv takes time to multiplicate and affect the infected cells tissue to grow larger. it also shows the difference between papiloma with other congenital laryngeal disease. congenital malformation of the larynx will cause symptoms immedieatly after birth. this study is showed the time break from onset of symptoms until when it was first diagnosed. the mean was 8,16 ± 10,88 months range from 1 to 30 months. patients were for examination when the symptoms first occurred and diagnosis were confirmed at the same time. but, it contrast to our country. patients often postponed seeking medical facility when hoarseness as early symptoms of laryngeal papilloma appeared. hence there were delayed diagnosis. good health facilities are still not evenly distributed in some areas in indonesia. this would postpone the therapy. shortness of breath due to airway obstruction and hoarseness are the main symptoms that cause patient to seek medication for the first time, it have almost equal percentages, hoarseness in 9 patients (52,9%) and shorness of breath in 8 patients (47,1%). these describe why the rrp patients visit in late phase hence patients often came with shortness of breath. the early symptom of laryngeal papilloma are hoarseness and shortness of breath. these symtoms are the late signs due to their growth in the larynx. these symptoms support the data that there is the time break between time of diagnosis and onset of symptoms. the patients excuse their delay to medical facility due to their assumption that hoarseness was not a dangerous anomaly. the parents decide to postpone to check their children. the other reason for this delay was patient seek for alternative medication, problem in financial and transportation to the refferal hospital. campisi13 was reported that the percentage of hoarseness as the most common symptoms when it first diagnosed (80%), while stridor and respiratory distress are 30% and 23% respectively. rrp have tri-symptoms that consist of progressive hoarseness, stridor, and airway obstruction. hoarseness is the common and early symptoms in young children that indicate structural and functional anomalies of larynx. vocal cord is the most common and initial sites of papilloma lesion. hoarseness could appeared due to small lesion and become the early symptoms, however in the distance lesion this hoarseness could appear as late symptom. in addition to cause hoarseness, papilloma also resulting stridor or obstruction, depend on size of lesion. generally, the surgical indication is to aim maintain airway patency in order to avoid airway obstruction. other indication including improving communication quality by preserving laryngeal structure to produce adequate voice. the mean ± sd was 2,71 ± 1,16, median 2 times which range from 1 to 6 times. buchinsky11 was reported the number of surgery within 12 months period with median 4 times which range from 1 to 52 times. this value was bigger compared to this study result with median 3 times which range from 1 to 6 times. as patients arrive in our hospital in late condition and requires a tracheotomy, the often postpone the laryngeal surgery to remove the papilloma. that causes longer operation interval in this study. definitive therapy to eradicate rrp effectively remains unavailable. the present standard therapy is micro laryngeal surgery (mls) to remove papilloma 116 indonesian journal of tropical and infectious disease, vol. 6 no. 5 may–august 2017: 113–117 mass for maintaining airway patency and normal laryngeal anatomy structure. the progresses of the disease including spontaneous remission, or constant which need repetitive surgeries, and or become more aggressive that yet need surgery every few days to weeks. aggressive disease need more frequent mls to avoid airway obstruction. the concept of disease aggressiveness involve some parameters, including papilloma growth, surgery frequency, total number of surgery, the need for adjuvant therapy and tracheotomy, and lower airway tract complications. these parameters related to some factors including onset in younger ages, hpv-11 infection, the need for tracheotomy, cigarette smoke exposure and poor response to interferon-α therapy. percentages of papilloma that grow in distal larynx is 64,7% and tracheotomy history is 76,5%. it showed that most of patients had aggressive type. that number identical with this reports were showed higher result compared with wiatrak12 who reported 11% tracehotomies and 23,3% distal papilloma growth. buchinsky11 was reported 10% tracheotomy histories and 25% with distal growth, while campisi13 was reported distal growth about 5,6% dan tracheotomies 2,8%. the more aggressive type of disease that found in this study seen by many distal papilloma growth incidents and tracheotomies. tracheotomy will induced the distal implantation of papilloma or spreading to the lower airway include the trachea or even bronchial tree. rrp is a rare disease with slow progressivity, some cases remain undetected until airway obstruction were occurred. the most common sites are oral cavity, trachea and bronchus. distal implantation also can be triggered by jet ventilation during mls. tracheotomy is one of the most frequent procedures in rrp patients in late condition although it could activate or trigger the spread of papilloma implantation in the distal larynx. continued tracheotomy and sub glottic papilloma during tracheotomy would increase the risk of tracheal spread. the experts had agreed that tracheotomy should be avoided unless it was extremely required, and immediate decanulation should be performed soon after mass removal via mls. children with prolonged endotracheal tube could also increase the risk of papilloma growth through lesion mechanism that disrupts the respiratory mucosal continuity thus cause papilloma dissemination. in our study, decanulation is still difficult to perform because patients have a low awareness to visit the doctor regularly. sometimes, tracheotomy must to be maintained until adolescent or adult so the risk of obstruction become smaller. percentage of the aggressive disease is 82,4%. some of aggressive rrps found by buchinsky11 about 81%. this study results match with those results. the high incidents of those aggressive rrps correlated with high occurrences of aggressive factors that were discussed previously. all of patients in this study categorized as juvenile type rrp, which is similar to buchinsky11 and maloney14. juvenile rrp tend to lead to aggressive disease caused by incompetent immune system to against the viral invasion, result in rapid cell growth and cell division. it will make the surgery more frequently performed. hpv-11 infection causes more stronger proliferative ability than hpv-6 which stimulate the disease course to be more aggressive.15 some literature findings by wiatrak12 were reported 53,5% of hpv-6 patients, 39,7% of hpv-11 and 6,9% of both types. buchinsky11 found 60% of hpv-6 infection and 40% of hpv-11. this study result similar to buchinsky findings which showed tendency of hpv-11 infection rather than hpv-6. maloney14 was report several children have coinfection with hpv type 6 and 11. buchinsky11 was found 43 aggressive rrp samples with hpv-11 infection and 52 patients with hpv-6, while non aggressive rrp with hpv-6 infection were 19 patients and 4 patients with hpv-11. the result showed that hpv11 tend to cause more aggressive compared to hpv-6 and that association was statistically significant with p = 0,02 (p < 0,05). it shows not only hpv that cause the aggressiveness of the disease but also, other aggressive aspect is host factor, particularly immune response and host suscepbility. one patient might have more aggressiveness type while others have mild type. the host susceptibility against viral infection is related with polymorphism of immune response regulator gene. effective immune responses against viral infection involve innate or adaptive immune response, th1 and th2 balances with certain chemokines and cytokines. this immune response can even control and predict the disease susceptibility and aggressiveness. we need more investigation to clarify the role of host respon to the hpv.14,15 there are several limitations on this study. first, we have a short of time to collecting samples and make small number of samples. second, all patients are from one single center, perhaps multicenter national study more reliable. conclusion this study presented that viral type of hpv have no association with aggressiveness of the disease and could not be used as a prognostic maker of the disease course. but the rrp patients need a regular follow up and proper treatment during the disease course, particularly for the aggressive disease. references 1. ganguly n, parihar sp. human papillomavirus e6 and e7 oncoproteins as risk factors for tumorigenesis. j biosci. 2009 mar;34(1):113–23. 2. ghittoni r, accardi r, hasan u, gheit t, sylla b, tommasino m. the biological properties of e6 and e7 oncoproteins from human papillomaviruses. virus genes. 2010 feb;40(1):1–13. 3. larson da, derkay cs. epidemiology of recurrent respiratory papillomatosis. apmis. 2010 jun;118(6–7):450–4. 117perdana, et al.,: no association of recurrent respiratory papillomatosis aggressiveness 4. derkay cs, darrow dh. seminar series recurrent respiratory papillomatosis. ann otol rhinol laryngol. 2006 jan 29;115(1):1– 11. 5. tasca ra, clarke rw. recurrent respiratory papillomatosis. arch dis child. 2006 aug;91(8):689–91. 6. andrus jg, shapshay sm. contemporary management of laryngeal papilloma in adults and children. otolaryngol clin north am. 2006;39(1):135–58. 7. derkay cs, faust ra. recurrent respiratory papillomatosis in cummings otolaryngology – head and neck surgery. 5th ed. philadelphia: mosby; 2010. 2884-95 p. 8. stamataki s, nikolopoulos tp, korres s, felekis d, tzangaroulakis a, ferekidis e. juvenile recurrent respiratory papillomatosis: still a mystery disease with difficult management. head neck. 2007 feb;29(2):155–62. 9. xue q, wang h, wang j. recurrent respiratory papillomatosis: an overview. eur j clin microbiol infect dis. 2010 sep;29(9):1051–4. 10. derkay cs, wiatrak b. recurrent respiratory papillomatosis: a review. laryngoscope. 2008 jul;118(7):1236–47. 11. buchinsky fj, donfack j, derkay cs, choi ss, conley sf, myer cm, et al. age of child, more than hpv type, is associated with clinical course in recurrent respiratory papillomatosis. kallas eg, editor. plos one. 2008 may 28;3(5):e2263. 12. wiatrak bj, wiatrak dw, broker tr, lewis l. recurrent respiratory papillomatosis: a longitudinal study comparing severity associated with human papilloma viral types 6 and 11 and other risk factors in a large pediatric population. laryngoscope. 2004 nov;114(11 pt 2 suppl 104):1–23. 13. campisi p, hawkes m, simpson k, canadian juvenile onset recurrent respiratory papillomatosis working group. the epidemiology of juvenile onset recurrent respiratory papillomatosis derived from a population level national database. laryngoscope. 2010 jun;120(6):1233–45. 14. maloney em, unger er, tucker ra, swan d, karem k, todd nw, et al. longitudinal measures of human papillomavirus 6 and 11 viral loads and antibody response in children with recurrent respiratory papillomatosis. arch otolaryngol neck surg. 2006 jul 1;132(7):711–5. 15. bonagura vr, vambutas a, devoti ja, rosenthal dw, steinberg bm, abramson al, et al. hla alleles, ifn-gamma responses to hpv-11 e6, and disease severity in patients with recurrent respiratory papillomatosis. hum immunol. 2004 aug;65(8):773–82. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 3 september–december 2022 original article characteristics of leptospirosis cases in pacitan district, east java province firman aji prasetio1 , muhammad atoillah isfandiari2 , agung nugroho3 1master program in epidemiology, faculty of public health, universitas airlangga, surabaya, indonesia 2division of epidemiology, department of epidemiology, biostatistics, population studies and health promotion, universitas airlangga, surabaya, indonesia 3east java provincial health office, surabaya, indonesia received: march 31st, 2022; revised: august 13th, 2022; accepted: september 16th, 2022 abstract leptospirosis is a disease that is still a public health problem in the world, however, these cases are rarely reported due to the difficulty of distinguishing clinical symptoms from other endemic diseases and the lack of appropriate laboratory diagnostic services. pacitan district is one of the districts in east java that reported leptospirosis cases for 3 consecutive years from 2017 to 2019. there were total 92 leptospirosis cases with case fatality rate (cfr) of 15.22% in pacitan. this study is a descriptive study with a cross sectional design that aims to describe the distribution of characteristics of leptospirosis cases in pacitan district based on person, place, and time. this study used secondary data from the pacitan district health office, east java province. the population in this study was all cases with leptospirosis cases as many as 92 cases recorded in the pacitan district health office data for 2017–2019. the sample of this study were all cases with leptospirosis as many as 92 cases.the results of the study obtained leptospirosis cases in pacitan district in 2017–2019 based on person occured most in the age group of 40–49 years old by 20.45%, in the male sex by 68.48%, and in the population who worked as farmers by 73.58%. based on the place where the most occured in tulakan sub district by 52.75%, while based on time, most occured in february, march and april, this is because february to april is the rainy season. therefore, based on the results of the study, it is necessary to educate the public, especially at risk groups, about the risk factors and prevention of leptospirosis. keywords: leptospirosis; pacitan; person; place; time abstrak leptospirosis merupakan salah satu penyakit yang masih menjadi masalah kesehatan masayarakat di dunia, namun kasus ini jarang terlaporkan karena sulitnya membedakan gejala klinis dengan penyakit endemik lainnya dan kurangnya pelayanan diagnostik laboratorium yang tepat. kabupaten pacitan merupakan salah satu kabupaten di jawa timur yang melaporkan adanya kasus leptospirosis selama 3 tahun berturut-turut dari tahun 2017 hingga tahun 2019. terdapat 92 kasus leptospirosis dengan case fatality rate (cfr) sebesar 15.22% di kabupaten. penelitian ini merupakan penelitian deskriptif dengan desain cross sectional yang bertujuan untuk menggambarkan distribusi karakteristik kasus leptospirosis di kabupaten pacitan berdasarkan orang, tempat, dan waktu. penelitian ini menggunakan data sekunder yang diperoleh dari dinas kesehatan kabupaten pacitan, provinsi jawa timur. populasi dalam penelitian ini adalah seluruh kasus leptospirosis sebanyak 92 kasus yang tercatat dalam data dinas kesehatan kabupaten pacitan tahun 2017–2019. sampel penelitian ini adalah seluruh kasus leptospirosis sebanyak 92 kasus. hasil penelitian diperoleh kasus * corresponding author: firmanajiprasetio@gmail.com https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0003-3711-9323 https://orcid.org/0000-0001-9010-0045 159 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license firman aji prasetio, et al. characteristics of leptospirosis cases in pacitan district leptospirosis di kabupaten pacitan pada tahun 2017–2019 berdasarkan orang paling banyak terjadi pada kelompok usia 40–49 tahun sebesar 20,45%, pada jenis kelamin laki-laki sebesar 68,48%, dan pada penduduk yang bekerja sebagai petani sebesar 73,58%. berdasarkan tempat paling banyak terjadi pada kecamatan tulakan sebesar 52,75%, sedangkan berdasarkan waktu paling banyak terjadi pada bulan februari, maret dan april, hal ini karena pada bulan februari hingga april merupakan musim penghujan. oleh karena itu berdasarkan hasil penelitian, perlu dilakukan edukasi untuk masyarakat terutama pada kelompok berisiko mengenai faktor risiko dan pencegahan penyakit leptospirosis. kata kunci: leptospirosi; orang; pacitan; tempat; waktu how to cite: prasetio, f. a., isfandiari, m. a., nugroho, a. characteristics of leptospirosis cases in pacitan district, east java province. indonesian journal of tropical and infectious disease. 10(3). 158–164. dec. 2022. introduction leptospirosis is a zoonotic disease that is a public health problem in the world. leptospirosis is common in tropical and subtropical developing countries and has high rainfall.1,2 the occurrence of leptospirosis is not only related to climate and environmental conditions, but due to contact with environments contaminated with leptospira bacteria such as agriculture, poor housing and waste disposal that can cause a source of infection. while in temperate countries, leptospirosis can occur locally and can also be transmitted by people who come from abroad, especially those who visit the tropics.2–4 annual incidence worldwide is estimated at >1 million cases, including approximately 59,000 deaths. the regions with the highest estimates of morbidity include south and southeast asia, oceania, the caribbean, parts of sub-saharan africa and parts of latin america. outbreaks can occur after heavy rains or floods in endemic areas, especially in urban areas in developing countries, where housing and sanitary conditions are poor. leptospirosis outbreaks have occurred in the united states after floods in hawaii, florida and puerto rico.5 the incidence of leptospirosis in subtropical countries is estimated at between 0.1–1 cases/100,000 inhabitants per year, while in tropical countries it is estimated at 10 cases/100,000 inhabitants per year and may increase to 100 cases/100,000 inhabitants in the event of an outbreak.2 in the united states, an estimated 100–200 cases of leptospirosis are identified each year, of which 50% occur in hawaii.1 cases of leptospirosis in humans are generally reported from india, indonesia, thailand and sri lanka during the rainy season. indonesia is a tropical country and some areas in indonesia are endemic areas of leptospirosis. leptospirosis can be a public health threat in the event of extraordinary events, this is because in indonesia there are several risk factors that affect the incidence of leptospirosis such as the high population of rats (rodent) as a reservoir of leptospirosis, poor environmental sanitation and flood areas are increasingly widespread.6 leptospirosis is a rarely reported disease, one of the causes of which is the difficulty of distinguishing clinical symptoms from other endemic diseases and the lack of appropriate laboratory diagnostic services.3,4 leptospirosis cases in indonesia are also not widely reported, only 9 provinces that report cases of leptospirosis are dki jakarta, west java, central java, yogyakarta, east java, banten, north kalimantan, south sulawesi and maluku.7 figure 1. the situation of leptospirosis in indonesia in 2017–20198 640 894 920 0 200 400 600 800 1.000 2017 2018 2019 cases 160 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 158–164 the figure 1 is showed an increase in leptospirosis cases in indonesia from 2017 until 2019 and there is a decrease in the case fatality rate from 2017 until 2019. the highest leptospirosis cases occurred in 2019 and the lowest leptospirosis cases occurred in 2017, while the highest case fatality rate occurred in 2017 and the lowest case fatality rate occurred in 2019. in 2017 there were 640 cases of leptospirosis with a cfr of 16.88%, in 2018 there were 894 cases of leptospirosis with a cfr of 16.55%, and in 2019 there were 920 cases of leptospirosis with a cfr of 13.26%.7 east java was one of the provinces that reported cases of leptospirosis from 2017 to 2019, which consisted of 106 cases in 2017 with a cfr of 17.92%, in 2018 as many as 128 cases with a cfr of 7.81% and in 2019 there were 147 cases with a cfr of 15.65%. figure 2. the situation of leptospirosis in pacitan district in 2017–20198 pacitan district is an endemic area of leptospirosis in east java province, the figure 2 is showed that leptospirosis cases and case fatality rate fluctuate. in 2017, there were 39 cases of leptospirosis and 32 cases of leptospirosis recovered with a cfr of 17.95%, in 2018 there were 11 cases of leptospirosis and 9 cases of leptospirosis recovered with a cfr of 18.18%, in 2019 there were 42 cases of leptospirosis and 27 cases of leptospirosis recovered with a cfr of 11.90%.8 this study aims to describe the characteristics of leptospirosis cases in pacitan district, east java province in 2017– 2019 based on person, place, and time. materials and methods this study was a descriptive study with cross sectional design to describe the distribution of characteristics of leptospirosis cases pacitan district based on person, place, and time. the population and sample in this study were all cases with leptospirosis with (92 cases) reported in the pacitan district health office in 2017 until 2019. the data used in this study were secondary data obtained from the pacitan district health office. results and discussion a. person 1. sex based on figure 3, the distribution of leptospirosis cases by sex in pacitan district in 2017–2019 is more common in men, namely 63 cases (68.48%), while in women as many as 30 cases (31.52%). figure 3. distribution of leptospirosis cases by sex in pacitan district in 2017–20198 in this study, it is stated that leptospriosis cases that occurred in pacitan district in 2017– 2019 were more common in men, namely as many as 63 cases (68.48%). this study is in line with research conducted prihantoro et al9, 80% of leptospirosis cases are male.9 this study is also in line with research conducted in boyolali, central java, leptospirosis cases occur most in men by 70%.10 men are 37.01 times more likely to be infected with leptospirosis than women.11 this can happen because men have jobs that are more often exposed to environments contaminated with leptospira bacteria.12 39 11 42 0 10 20 30 40 50 2017 2018 2019 cases 64.68% 31.52% male female 161 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license firman aji prasetio, et al. characteristics of leptospirosis cases in pacitan district 2. age figure 4. distribution of leptospirosis cases by age group in pacitan district in 2017–20198 based on figure 4 is showed that the number of leptospirosis cases in pacitan district in 2017–2019 was most prevalent in the age group of 40–49 years old by 20.45%, in the age group of 30-39 years old by 18.18%, then in the age group of 20–29 years old by 17.05%, age group 50–59 and age 60– 69 years old by 13.64%, in the age group >70 years old by 10.23%, and in the age group 10–19 years old by 6.82%, while in the age group 0–9 years old no cases. in the research that has been done, it is stated that leptospriosis cases that occurred in pacitan district in 2017–2019 occurred most in the age group of 40–49 years by 20%. while in the age group <10 years there are no reported cases of leptospirosis. cases of leptospirosis in children are rarely reported due to undiagnosed or different clinical manifestations with adults.6 this study is in line with that conducted by prihantoro et al9, leptospirosis cases occur at the age of more than 40 years old as many as 70 %.9 suprapto et al13 said that the most cases of leptospirosis in productive age (46–60 years).13 this can happen because men in productive age tend to do more activities outside the home. 3. job figure 5. distribution of leptospirosis cases by jobs in pacitan district in 2017–20198 based on figure 5, leptospirosis cases in pacitan district in 2017–2019 occurred most in people who worked as farmers (73.58%), while in students, private sector and workers leptospirosis cases amounted to 5.66%. in housewives, gardening and grazing the number of cases of leptospirosis amounted to 1.89% and others to 7.55%. job is one of the risk factors for the occurrence of leptospirosis. people who work in environments that contaminated with leptospira bacteria are at risk of developing leptospirosis.6 the risk of leptospirosis is higher in people who work outdoors or in contact with animals, such as farmers, planters, ranchers, slaughterers, veterinarians, veterinary nurses, mine workers, laboratory workers, fishermen, soldiers, fish traders, and traders in markets.14,15,16 0.00% 6.82% 17.05% 18.18% 20.45% 13.64% 13.64% 10.23% 0.00% 5.00% 10.00% 15.00% 20.00% 25.00% 0 9 years old 10 19 years old 20 29 years old 30 39 years old 40 49 years old 50 59 years old 60 69 years old > 70 years old 7.55% 5.66% 73.58% 5.66% 1.89% 1.89% 1.89% 1.89% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 162 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 158–164 residents of rural areas who work as farmers and ranchers are at risk of contracting leptospirosis.6 this study showed that leptospriosis cases that occurred in pacitan district in 2017–2019 were the most common in cases who worked as farmers, namely 73%. this study is in line with research conducted nuraini et al10, 44.7% of cases with leptsopsirosis occurs most in farmers.10 raharjo et al17 said that risky jobs have a 6,317 times higher risk of developing leptospirosis than non-risky jobs.17 while working as a farmer 2 times higher risk of leptospirosis.18 samekto et al19 stated that the habit of not wearing footwear is 4 times higher risk of developing leptospirosis.19 b. place figure 6. distribution of leptospirosis cases by sub districts in pacitan district in 2017–20198 based on figure 6, the most leptospirosis cases in pacitan district in 2017–2019 occurred in tulakan sub district by 52.75%, then punung sub district by 14.29%, kebonagung sub district by 9.89%, pringkuku sub district by 6.59%, pacitan sub district by 4.40%, and sudimoro sub district, arjosari sub district by 3.30% and donorojo sub district by 2.20%. however, in nawangan sub district, bandar sub district, ngadirojo sub district, there were no cases of leptospirosis. c. time figure 7. distribution of leptospirosis cases by time in pacitan district in 2017–20198 based on figure 7, the highest leptospirosis cases in pacitan district in 2017 occurred in february at 36.11%, in 2018 the highest leptospirosis cases occurred in march at 40.0%, in 2019 the highest leptospirosis cases occurred in april at 24.30%. the rainy season in pacitan district occurs in february–april and november– december, while the dry season in pacitan district occurs in may–october.20 in this study, it was stated that leptospirosis cases that occurred in pacitan district in 2017–2019 mostly occurred when rainy season occurs. one of the risk factors for leptospirosis is high rainfall. heavy rainfall can cause waterlogging up to flooding. leptospirosis can be transmitted through water contaminated with leptospira bacteria.24,25 rains and floods are one of the factors causing leptospirosis.16,25,26 the maniiah et al27 study showed that there was a relationship between the presence of standing water with the incidence of leptospirosis and cases who are around the house there is standing water has a risk of 3,385 times greater exposed to leptospirosis compared to respondents who are around the house there was no standing water.27 research conducted by suwanpakde et al28 in thailand showeds a relationship between flooding and the incidence of leptospirosis.28 36.11% 40.00% 24.32% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% ja n u a ry f e b ru a ry m a rc h a p ri l m a y ju n e ju ly a u g u st s e p te m b e r o c to b e r n o v e m b e r d e c e m b e r 2017 2018 2019 163 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license firman aji prasetio, et al. characteristics of leptospirosis cases in pacitan district conclusions the most cases of leptospirosis in pacitan district in 2017–2019 occurred in male, the age group 40–49 years old, farmers and occurred in the rainy season, from february to april. acknowledgement we would like to thank the pacitan district health office for the secondary data on leptospirosis reports. conflict of interest the authors state that they have no conflict of interest. references 1. mazhar m, kao jj, thomas d, md b. a 23year-old man with leptospirosis and acute abdominal pain. 2016;75:291. 2. world health organization. human leptospirosis: guidance for diagnosis, surveillance and control. world health organization; 2003. 3. world health organization. report of the first meeting of the leptospirosis burden epidemiology reference group. world health organization; 2010. 4. world health organization. leptospirosis prevention and control in indonesia [internet]. 2020 [cited 2022 aug 10]. available from: https://www.who.int/indonesia/news/detail/2408-2020-leptospirosis-prevention-and-controlin-indonesia 5. centers for disease control. leptospirosis chapter 4 2020 yellow book | travelers’ health | cdc [internet]. 2020 [cited 2022 aug 10]. available from: https://wwwnc.cdc.gov/travel/yellowbook/2020/ travel-related-infectious-diseases/leptospirosis 6. kementerian kesehatan republik indonesia. petunjuk teknis pengendalian leptospirosis [internet]. 2017 [cited 2022 aug 10]. available from: https://infeksiemerging.kemkes.go.id/download/ buku_petunjuk_teknis_pengendalian_leptospi rosis.pdf 7. kementerian kesehatan republik indonesia. profil kesehatan indonesia tahun 2020 [internet]. 2020 [cited 2022 aug 10]. available from: https://www.kemkes.go.id/downloads/resources/ download/pusdatin/profil-kesehatanindonesia/profil-kesehatan-indonesia-tahun2020.pdf 8. dinas kesehatan kabupaten pacitan. profil kesehatan kabupaten pacitan 2020. 9. prihantoro t, siwiendrayanti a, ilmu j, et al. karakteristik dan kondisi lingkungan rumah penderita leptospirosis di wilayah kerja puskesmas pegandan kota semarang. jhe (journal heal educ. 2017;2(2):178–184. 10. nuraini s, dian saraswati l, sakundarno adi m, setyawan bagian epidemiologi dan penyakit tropik hs, kesehatan masyarakat f. gambaran epidemiologi kasus leptospirosis di kabupaten boyolali, provinsi jawa tengah. 2017;5:2356– 3346. 11. ulfah m, anies a, adi ms, setyawan h, suwondo a. hubungan karakteristik demografi, faktor keselamatan dan kesehatan kerja (k3) dan lingkungan terhadap kejadian leptospirosis (studi pada pekerja sektor informal di kota semarang tahun 2013-2016). 2018;3(1):29. 12. ramadhani t, yunianto b. reservoir dan kasus leptospirosis di wilayah kejadian luar biasa. 2012;7(4):162–168. 13. suprapto ia, mahendrakrisna d, hudiyanti v, indianto w. gambaran kasus leptospirosis di rsud kota surakarta, 2015-2018. 2020;47(2):108–111. 14. andriani r, sukendra dm. faktor lingkungan dan perilaku pencegahan dengan kejadian leptospirosis di daerah endemis. 2020;4(3):471–482. 15. centers for disease control. principles of epidemiology | lesson 1 – overview [internet]. 2012 [cited 2022 aug 9]. available from: https://www.cdc.gov/csels/dsepd/ss1978/lesson1 /index.html 16. pereira mm, schneider mc, munoz-zanzi c, et al. a road map for leptospirosis research and health policies based on country needs in latin america. 2017;41:1–9. 17. raharjo j, hadisaputro s, litbang bp, jl selamanik no b, banjarnegara a, tengah j. faktor risiko host pada kejadian leptospirosis di kabupaten demak. 2015:105–110. 18. hinjoy s, kongyu s, doung-ngern p, et al. environmental and behavioral risk factors for severe leptospirosis in thailand. 2019;4(2). 164 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 158–164 19. samekto m, hadisaputro s, sakundarno adi m, widjanarko b, kesehatan kabupaten pati d, kesehatan masyarakat universitas diponegoro f. faktor-faktor yang berpengaruh terhadap kejadian leptospirosis (studi kasus kontrol di kabupaten pati). 2019;4(1):27–34. 20. badan pusat statistik kabupaten pacitan. dalam angka kabupaten pacitan [internet]. 2020 [cited 2022 aug 10]. available from: https://pacitankab.bps.go.id/publication/2020/04 /27/59b932e91b04de081ce4c48f/kabupatenpacitan-dalam-angka-2020.html 21. arsyad a, arsyad as, kusnanto h. pemetaan daerah kerawanan penyakit leptospirosis melalui metode geographically weighted zero inflated poisson regression. 2018;34(10):257– 262. 22. supranelfy y, hapsari ns, oktarina r, et al. analisis faktor lingkungan terhadap distribusi jenis tikus yang terkonfirmasi sebagai reservoir leptospirosis di tiga kabupaten di provinsi sumatera selatan. 2019;11(1):31–38. 23. yuliadi b, wahyuni, ristiyanto. distribusi spasial leptospirosis di wilayah provinsi jawa tengah tahun 2002-2012. 2013. 24. syakbanah nl, fuad a, kusnanto h. analisis temporal efek cuaca terhadap leptospirosis di kabupaten bantul, yogyakarta tahun 20102018. 2019;35(4):op1-12. 25. matsushita n, ng cfs, kim y, et al. the nonlinear and lagged short-term relationship between rainfall and leptospirosis and the intermediate role of floods in the philippines. 2018;12(4):e0006331. 26. lau cl, watson ch, lowry jh, et al. human leptospirosis infection in fiji: an ecoepidemiological approach to identifying risk factors and environmental drivers for transmission. 2016;10(1):e0004405. 27. maniiah g, raharjo m, astorina bagian kesehatan lingkungan n, kesehatan masyarakat f, diponegoro u. faktor lingkungan yang berhubungan dengan kejadian leptospirosis di kota semarang. 2016;4(3):792–799. 28. suwanpakdee s, kaewkungwal j, white lj, et al. spatio-temporal patterns of leptospirosis in thailand: is flooding a risk factor?. 2015;143(10):2106. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 3 september–december 2022 review article the curative innovation of novel triple-drug compared to double-drug regimen in lymphatic filariasis: a systematic review rivaldi ruby* , erlangga saputra arifin , charens faculty of medicine and health sciences, universitas katolik indonesia atma jaya, jakarta, indonesia received: april 20th, 2022; revised: may 10th, 2022; accepted: october 6th, 2022 abstract the world health organization has established a global program for the elimination of lymphatic filariasis by 2020; recent data has shown an impracticable result accomplishing it. therefore, this study aims to identify the efficacy and safety between triple-drugs (dec, alb, ivm) and double-drugs (dec & alb/ivm & alb) for lymphatic filariasis treatment. a systematic review was conducted with preferred reporting items for systematic reviews and meta-analysis (prisma) statement guidelines. the literature search was done using five databases: pubmed, proquest, sciencedirect, ebsco, and central until december 3, 2020 without any publication date range imposed. data collection was done by three independent reviewers and entered into a predesigned data extraction form. cochrane risk of bias tool 2.0 was utilized in the quality assessment of the studies. search strategies identified 209 studies. three relevant full-text articles met our inclusion criteria. overall studies had low risk of bias. the main findings are as follows: (a) administration of single dose of triple-drug regimen resulted in a total elimination of microfilaria 12 months after treatment whilst 91% participants given with double-drug remained microfilaremic (p=0.002); (b) in larger samples (n=182), triple drug cleared microfilaria in 96% of the participants and only 32% of the participants receiving double-drug regimen after 12 months observation; (c) statistically, the triple-drug safety has a lower degree than the double-drug regimen (p=0.02). the triple-drug treatment has a better efficacy compared to the double-drug regimen in treating lymphatic filariasis. furthermore, both regimens are proven safe with no serious adverse events elicited. keywords: albendazole; diethylcarbamazin; ivermectin; lymphatic filariasis; systematic review abstrak organisasi kesehatan dunia (who) telah menetapkan program global untuk mengeliminasi filariasis limfatik pada tahun 2020; data terbaru menunjukkan ketidakberhasilan pencapaian target tersebut. oleh karena itu penelitian ini bertujuan untuk mengidentifikasi efikasi dan keamanan antara terapi menggunakan tiga obat (dec, alb, ivm) dan terapi dua obat (dec & alb/ivm & alb) untuk pengobatan filariasis limfatik. telaah sistematis dilakukan dengan pedoman pernyataan preferred reporting items for systematic review and meta-analysis (prisma). pencarian literatur dilakukan melalui lima database: pubmed, proquest, sciencedirect, ebsco, dan central hingga 3 desember 2020 tanpa adanya batas rentang waktu publikasi. pengumpulan data dilakukan oleh tiga peninjau secara independen dan dimasukkan ke dalam formulir ekstraksi data yang telah dirancang sebelumnya. cochrane risk of bias 2.0 digunakan dalam penilaian kualitas studi.strategi pencarian mengidentifikasi 209 studi. tiga artikel yang relevan memenuhi kriteria inklusi studi. keseluruhan studi memiliki risiko bias yang rendah berdasarkan penilaian penulis. temuan utama adalah sebagai berikut: (a) pemberian dosis tunggal pada terapi tiga obat mengeliminasi seluruh mikrofilaria setelah 12 bulan pengobatan sedangkan 91% peserta yang diberi terapi dua obat masih mengalami mikrofilaremia (p=0,002); (b) dalam sampel yang lebih besar (n=182), mikrofilaria tereliminasi pada 96% peserta yang menerima * corresponding author: aldiruby@gmail.com https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-6609-8413 https://orcid.org/0000-0002-5094-9957 https://orcid.org/0000-0001-9202-3733 166 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 165–175 terapi tiga obat dan 32% peserta yang menerima terapi dua obat setelah 12 bulan observasi; (c) secara statistik, terapi menggunakan tiga obat lebih aman digunakan dibandingkan dengan terapi dua obat (p=0,02). terapi tiga obat memiliki efikasi yang lebih baik dibandingkan dengan terapi dua obat dalam menangani filariasis limfatik. kedua terapi juga terbukti aman tanpa adanya efek samping berat yang ditimbulkan. kata kunci: albendazol; dietilkarbamazin; filariasis limfatik; ivermektin; telaah sistematis how to cite: ruby, r., arifin, e. s., charens. the curative innovation of novel triple-drug compared to double-drug regimen in lymphatic filariasis: a systematic review. indonesian journal of tropical and infectious disease. 10(3). 165–175. dec. 2022. introduction lymphatic filariasis (lf) is a severe manifestation caused by a parasitic infection of worms belonging to the genus wuchereria and brugia, that is transmissible by means of a mosquito vector.1 this infection does not kill its host but significantly reduces their quality of life.2 latest data reported from the world health organization (who), which is in the year 2000, recorded over 120 million people were infected and about 40 million disfigured.3 in indonesia alone, the ministry of health reported over 14,000 people suffered from chronic filariasis (elephantiasis) in the year 2014.4 there is still an estimate of 893 million people worldwide remain at risk of getting lf.3 thus, this spectrum of disease is considered globally as one of the many neglected tropical diseases (ntd) requiring further interventions.5 the global programme to eliminate lymphatic filariasis (gpelf) was initially established by the world health organization (who) in 2000 with the aim to achieve global elimination of lf by 2020.6 the proposed strategy includes the interruption lf transmission using mass drug administration as well as managing morbidity and preventing disability (mmpd) by providing access to primary recommended care. latest reported data in 2019 on the progress of this program concluded a total of 37.3% are still at risk worldwide, which discourages the previous statement of it being completed in the year 2020.7 nevertheless, it is best to focus on the treatment rather than the prevention in order to minimize the damage being done in the meantime. currently, there are several regimens to treat lf.8 the well-known single-drug therapy is using diethylcarbamazine (dec) with a dosage of 6 mg/kg.9 other drugs, such as ivermectin (ivm) or albendazole (alb), rose to amplify the efficacy of dec when combined.10 however, some research found flaws in this double-drug therapy, a detection of microfilaria at one year posttreatment. a systematic review on the combination of alb with dec also yielded little or even no differences compared11 to using single-drug. in 2015, a pilot randomized controlled trial (rct) was done with the hypothesis that a triple-drug combination of dec, alb, and ivm might manifest a better result and coverage compared to only using two drugs.12 the downside is that in accordance with medical scientific theory, more drug consumption is equivalent to more adverse events (aes). through this hypothesis, the objective of our systematic review is to analyze the efficacy and safety between a triple-drug and double-drug regimen in lf curative management qualitatively. methods a systematic review was conducted with preferred reporting items for systematic reviews and meta-analysis (prisma) statement guidelines to identify the efficacy and safety between a triple-drug and double drug lymphatic filariasis treatment.13 population, intervention, comparison, and outcomes (pico) questions were also used to formulate the inclusion criteria of this systematic review. the answer of those questions consecutively: patients confirmed 167 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license rivaldi ruby, et al. the curative innovation of novel triple-drug suffering from lf with no prior antifilarial treatment; intervention with a triple-drug regimen consisting of dec, alb, and ivm; comparison with a double-drug regimen consisting of dec and alb or ivm and alb; outcomes measured are efficacy and safety of both regimens. differences between regimen doses and duration will be analyzed qualitatively based on the outcomes. the other inclusion criteria for this study is the study design and full-text availability. the study design had to be a rct with participants confirmed as lf patients, and all included studies must have full text. the literature search was done using five databases: pubmed, proquest, sciencedirect, ebsco, and central with “lymphatic filariasis,” “ivermectin,” “albendazole,” and “diethylcarbamazine” as the main keywords until december 3, 2020 without any publication date range imposed. no language restrictions were imposed. the complete keywords are listed in table 1 in the appendix. the result of the search was then imported to endnote x9, and the duplicates were removed. all authors participated through each phase of the review independently by screening the titles and abstracts, assessing the full text for eligibility criteria, then including the relevant studies. data collection was done by three independent reviewers (rr, ea, and c) and entered into a predesigned data extraction form. differences arising between the three reviewers regarding study eligibility were resolved by consensus. table 1. search keywords database keywords articles pubmed (((((((((elephantiasis, filarial[mesh terms]) or (elephantiasis, filarial[title/abstract])) or (lymphatic filariasis[title/abstract]))or (lymphatic filariases[title/abstract])) or (bancroftian filariasis[title/abstract])) or (malayi filariasis[title/abstract])) or (elephantiasis[title/abstract])) and ((((ivermectin[mesh terms]) or (ivermectin[title/abstract])) or (ivomec[title/abstract])) or(ivm[title/abstract]))) and ((((albendazole[mesh terms])) or (albendazole[title/abstract])) or (alb[title/abstract]))) and (((diethylcarbamazine[mesh terms]) or(diethylcarbamazine[title/abstract])) or ("diethylcarbamazine citrate"[title/abstract])) 105 proquest (ab(elephantiasis, filarial or lymphatic filariasis or lymphatic filariases or bancroftian filariasis or malayi filariasis or elephantiasis) or ti(elephantiasis, filarial or lymphatic filariasis or lymphatic filariases or bancroftian filariasis or malayi filariasis or elephantiasis)) and (ab(ivermectin or ivomec or ivm) or ti(ivermectin or ivomec or ivm)) and (ab(albendazole or alb) or ti(albendazole or alb)) and (ab(diethylcarbamazine or diethylcarbamazine citrate) or ti(diethylcarbamazine or diethylcarbamazine citrate)) 44 science direct (elephantiasis, filarial or lymphatic filariasis or lymphatic filariases or bancroftian filariasis or elephantiasis) and ivermectin and albendazole and (diethylcarbamazine or diethylcarbamazine citrate) 27 ebsco (ab(elephantiasis, filarial or lymphatic filariasis or lymphatic filariases or bancroftian filariasis or malayi filariasis or elephantiasis) or ti(elephantiasis, filarial or lymphatic filariasis or lymphatic filariases or bancroftian filariasis or malayi filariasis or elephantiasis)) and ( ab(ivermectin or ivomec or ivm) or ti(ivermectin or ivomec or ivm)) and (ab(albendazole or alb) or ti(albendazole or alb)) and (ab(diethylcarbamazine or diethylcarbamazine citrate) or ti(diethylcarbamazine or diethylcarbamazine citrate)) 98 central elephantiasis, filarial[mesh terms] and ivermectin[mesh terms] and albendazole[mesh terms] and diethylcarbamazine[mesh terms] 13 168 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 165–175 cochrane risk of bias tool 2.0 was utilized in the quality assessment of the studies which covers the following seven domains of risk. included study quality will be classified as low, unclear, or high risk of bias. 14 disagreements arising in the process of the evaluation were all resolved by discussion among the review team. results and discussion search results a literature search from electronic databases yielded 287 studies. after removing the duplicates, 209 remaining studies. screening through the titles and abstracts, the authors excluded studies with eight other studies that met the inclusion criteria. the result showed that three studies matched the criteria for inclusion.12,15,16 search flowchart and selection method was summarized in figure 1. figure 1. prisma flow diagram of the identification and selection of studies included in the analysis study characteristics all of the included studies were done using the rct design. inclusion and exclusion criteria across studies showed similarities. most of the study populations included were from papua new guinea12,15 and côte d’ivoire in west africa16 that showed small sample varieties. the regimen dosage and duration has slight differences amongst studies with efficacy and safety analysis. detailed characteristics of the included studies were summarized in table 2. quality assessment there were unclear risks for all studies from the domain of selection and reporting bias. concealment of the randomization done between the control and interventional group was unexplained.12,15,16 also, two studies excluded incomplete data from the statistical testing; thus can alter the results.12,15 two studies have an unclear risk in detection bias resulting from inadequate data.12,15 overall studies had a low risk of bias based on the author's judgement, was summarized in figure 2. figure 2. risk of bias summary: review authors’ judgements about each risk of bias item for each included study study characteristics the pilot study done by thomsen et al.12 included 24 participants with no chronic illnesses or prior lf infection. participants 169 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license rivaldi ruby, et al. the curative innovation of novel triple-drug also have not got the mda prophylaxis. included participants divided into two parallel groups with every 12 participants that received the triple-drug (dec + alb + ivm) or the double-drug regimen (dec + alb). the study yielded higher efficacy in the triple-drug regimen with total eradication until 12 months follow-up than the doubledrug regimen still resulting in 10 of 11 participants having positive microfilaremia (p=0.002). after two years of follow-up, both regimens showed complete eradication of microfilaremia.12 in 2018, another comparison of a rct study done by king et al. is conducted to evaluate the efficacy of a single dose of triple drug regimen in sustaining clearance being higher than a single dose of double-drug regimen for lf. a total of 182 participants were included, 60 of which were assigned to receive the triple-drug regimen administered once, and 61 for each double-drug regimen administered once and once a year for three years. among 182 participants, 172 (95%) were evaluated at 12 months, 165 (91%) at 24 months, and 158 (87%) at 36 months after trial initiation–several were excluded for similar reasons, including a withdrawal, moved from the area, died, took a second dose by mistake or merely lost to follow-up. however, despite the decrease of the number of participants, the outcome of triple-drug regimen still result in significantly greater microfilarial clearance at 36 months than a single dose of the doubledrug regimen, with a p-value lower than 0.025 (p=0.02), and was non-inferior to that with the double-drug regimen administered once a year for three years, with a one-sided p value for noninferiority lower than 0.025 (p=0.004).15 the most recent study was done in 2020 by bjerum et al. consisting of 97 participants and using a similar triple-drug regimen but a somewhat different double-drug regimen consisting of ivm and alb. the triple-drug group (45 participants) was given a single dose at the beginning of the research, while the double-drug group (52 participants) was given annually for three years. results measured were the clearance of microfilaremia at 6, 12, 24, and 36 months posttreatment. the triple-drug regimen was significantly better compared to the double drug regimen in clearing microfilaremia at 6 and 12 months (both with the value of p<0.001). however, superiority is the exact opposite in the 36 months (p=0.045). at the 24 months posttreatment, the triple-drug regimen was still better but with an insignificant value (p=0.53). this concludes that although the triple-drug group was only given once at the beginning of the study, it can maintain a better clearance up to 24 months posttreatment.16 170 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 165–175 table 2. characteristics of the included studies author study design samples interventions duration of follow-up result triple drug double drug effectivity adverse event king et al., 2018 randomized controlled trial n=182 this includes a total of 60 patients assigned ivm+dec+al b administered once, 61 to dec+alb administered once, and 61 to dec+alb once a year for 3 years dec + alb + ivm ivm 200 μg/kg plus dec 6mg/kg plus alb 400mg administered once at trial initiation dec + alb dec 6 mg/kg plus alb 400mg administered once at trial initiation follow-up was done at 12,24 and 36 months after trial initiation triple-drug regimen cleared microfilaremia in 55 (96%), 52 (96%) and 55 participants (96%) at 12, 24 and 36 months respectively. a single dose of double-drug regimen cleared microfilaremia in 18 (32%), 31 (56%), 43 (83%) participants at 12, 24 and 36 months respectively. moderate aes after the initial treatment were more frequent with the triple drug regimen than with the doubledrug regimen. thomsen et al. 2015 randomized controlled trial single blinded parallel group n=24 this includes a total of 12 patients assigned to dec + alb group and 12 patients assigned to dec + alb + ivm groups dec + alb + ivm single dose ivm 200 μg/kg; alb 400 mg; dec 6 mg/kg dec + alb single dose dec 6 mg/kg; alb follow up was done until 2 years posttreatment triple-drug regimen resulting total elimination of microfilaria after 36 h and 7 days treatments and remain so at 12 months posttreatment, than the double-drug regimen 10 of 11 participants remained microfilaremic at 12 months follow-up no serious ae was found in both treatment groups. most common aes elicited were fever, myalgia, pruritus, and proteinuria/hemat uria. adverse events were more on the triple-drug groups compared to the doubledrug groups (83% and 50%, respectively; p=0.02). bjerum et al. 2020 randomized controlled trial n=97 this includes a total of 52 patients assigned to ivm+alb group and 45 patients assigned to ivm+dec+al b group dec + alb + ivm ivm 200 μg/kg and alb 400 mg plus dec 6 mg/kg were given only once at the beginning of the study. ivm + alb ivm 200 μg/ kg and alb 400 mg were given in a 3 annual dose. follow-up was done at 6, 12, 24, and 36 months posttreatment triple-drug regimen resulted in a mf complete clearance of 89%, 71%, 61%, and 55% at a follow up time of 6, 12, 24, and 36 months, respectively. while double-drug regimen resulted in a mf complete clearance of 34%, 26%, 54%, and 79% at similar follow-up time, respectively. both groups resulted in similar aes, which are none in the severe (grade 3) category and a similar one at the mild (grade 1) category. however, triple drug therapy elicited more moderate (grade 2) aes compared to double-drug therapy. abbreviation: ae, adverse events; alb, albendazole; alt, alanine transferase; ast, aspartate transferase; dec, diethylcarbamazine; dl, deciliter; g, gram; ivm, ivermectin; kg, kilogram; mf, microfilaria; mg, milligram; ml. milliliter; μg, microgram; n, number of participants; n/a, not available safety regarding the adverse events all three studies reported that tripledrug therapy resulted in a much more ae compared to double-drug therapy. this is supported by the basic medical lessons that more drugs are equal to greater side effects. however, the aes reported are mild to moderate aes like fever, myalgias, headache, nausea, and the like. there are no serious or severe (e.g. grade 3) aes reported.12,15,16 this systematic review concludes a similar result found in all three studies.12,15,16 all things considered, the triple-drug regimen was revealed for being the more effective option compared to the double-drug regimen in treating lf. the clearance of 171 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license rivaldi ruby, et al. the curative innovation of novel triple-drug microfilaremia measures this effectiveness at their given posttreatment, follow-up time. study by bjerum et al., however, showed that the double-drug regimen was significantly superior at 36 months posttreatment.16 another important aspect is the therapy’s safety, which is measured by listing all aes elicited after drug consumption. both regimens are considered safe by means of no serious or severe aes obtained. mild to moderate aes like fever, nausea, headache, and the like are more commonly found in patients treated with the triple-drug compared to the double-drug regimen.12,15,16 pharmacological aspects between the regimen each single drug has variability in their pharmacokinetics profile. albendazole alone has poor absorption in gastrointestinal tract17,19 compared with ivermectin or dec that achieve peak plasma concentration faster than albendazole.20 well distribution is confirmed by each drug, but ivermectin has high lipophilicity,21,22 opposites with dec.23,24 metabolism of each drug occurs mostly in liver. only dec shows partial metabolism that slows down the elimination process. most of the drugs excreted through feces and bile, less in urine. nevertheless, lipophilicity profile that ivermectin has and partial metabolism in dec reflects higher half-time for both drugs.21,25 mechanism of action each drug also differs one to the other. active metabolites of albendazole (albendazole sulfoxide) causes degeneration of cytoplasmic microtubules helminths with the decrease of parasite’s glycogen stores.17,18 then, dec yields sensitization of macrophage to microfilaria that ease the clearance process.23,25 lastly, ivermectin can causes hyperpolarization, however other study shows that ivermectin has the ability to sterilize the adult filarial.26,27 combining of these drugs creates better pharmacokinetics and efficacy profiles because of uniqueness of each drugs. using triple regimen shows superiority rather than double regimen. half-life (t1/2) and concentration max (cmax) higher if we add ivermectin in standard double regimen (dec and alb). figure 3 illustrates slightly higher serum concentration of dec in initial times after consumed if combined as triple-drug regimen. similar profile also obtains from serum concentration of albendazole that increase in triple-drug regimen. those pharmacological profiles raise the efficacy of triple-drug regimen than double-drug regimen.12 figure 3. concentration of lymphatic filariasis regimen in correlation with duration. (a) mean serum plasma of dec (b) active metabolite of alb12 multimodal mechanism of actions also improves the efficacy of triple-drug regimen. different sites of parasites killing process increase the chances of microfilaria deaths. both albendazole and dec can directly kill microfilaria. addition to that, ivermectin will improves reduction of microfilaria with sterilization of adult worms that reflects lower microfilaremia level in longer times. 172 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 165–175 nevertheless, further research is needed to carry out in determining definitive reasons whether it is due to additive or synergistic effect between the triple-drug interactions.26 until now, each drugs using standard dose, even it already combined as triple-drug regimen. the fact that limitation arises from single drug with standard doses can be covered by other drugs. adverse effects with all standard doses also depict low or minimal events. nonetheless, specific research about dose adjustment still be needed. the high efficacy of triple-drug regimen can accelerate eradication of lf. this data is supported with a mathematical model that predicts for faster approaches to eliminate lf.28 difference in various aspects of the regimen although there are many similarities in the three included rcts, their differences are a requirement to be discussed. differences arise across studies yielded hazy analyzes in the overall results. the main distinguishable factor is the inconsistency in the drug combinations used. bjerum et al. use a different double-drug combination, which includes alb and ivm, compared with two other studies that used dec and alb.16 a clinical trial in ghana using a combination of alb and ivm showed the same efficacy and safety with a controlled group (dec and alb) but statistically incomparable.29 moreover, a model-analysis results in the same efficacy between both kinds of double drug regimen (ivm and alb/dec and alb).30 our analysis also considers ivm mechanisms of action that can be the main reason for dec substitution in lf. as mentioned previously, ivermectin causes paralysis and death of parasites by interacting with its chloride channel in the cell membrane leading to hyperpolarization.31,32 although there are differences among the double-drug regimens, it can be concluded that both showed the same efficacy and safety that can be compared with triple-drug based on the data analyses and each authors’ judgments. another difference comes from the regimen frequency in which the double-drug regimen is not only given once. this difference can be ignored since a higher frequency of the double-drug regimen still shows inferior outcomes compared to the triple-drug regimen.23 moreover, a study showed that the double-drug regimen has minor to none efficacious effect even with more frequency.33 however, in some studies, it can affect the effectivity outcome on the last day of the treatment follow-up.16 in the safety objectives, a longer duration of follow up showed an extension of the safety observation. although there are no serious aes, the triple-drug regimen contains more chemical agents compound which can cause more aes when compared to the double-drug regimen.34,35 risk vs benefit will be the primary consideration when it comes to lf treatment.11 the highest benefits, besides the side effects, can be found by using the triple drug regimen. however, in public situations, the doctor’s explanations about the triple-drug regimen’s potent benefits must be performed to prevent the misunderstanding of its aes. strength and limitation of each study included studies also shared some strengths and limitations. by referring to the risk of bias assessment, strengths such as the inclusion of using a random component had been performed, concluding a low risk of bias for random sequence generation, followed by the blinding of participants and the personnel or trial staff, specifically those who assessed the adverse events in the studies done by king et al. and bjerum et al.15,16 however, the blinding of outcome assessment was only low risk in the study done by bjerum et al. since it was specified that although it was an open label trial, the investigators and staff who evaluated the examinations were masked with respect to treatment arm assignments.16 meanwhile, the study done by king et al. gained the upper hand in incomplete outcome data, by conducting a sensitivity analysis (chi-squared analysis) to evaluate the potential effect of the missing data on the 173 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license rivaldi ruby, et al. the curative innovation of novel triple-drug primary outcome at 36 months.15 there were also no other biases present. several inherent limitations are the questionable allocation concealment, as these three studies did not indicate the method to conceal the possibility of foreseeing the assignments. as mentioned before, blinding of outcome assessment is also unclear in the studies done by thomsen et al. and king et al.12,15 another important caveat is the removal of participants which are lost to follow-up without performing a further analysis for the potential effects in the thomsen et al. and bjerum et al.’s study.12,16 king et al.’s study has also emphasized its limitations when assessing adverse events in the first 10 hours of follow-up since participants may have been aware of the treatment group assignments.12 although, the subsequent follow-up of the participants in their communities was performed in a blinded manner by different trial staff. also, the detection of microfilaremia at follow-up could have been due to reinfection. on the other hand, the limitations from bjerum et al.’s study include the unreliable outcome of higher infection rate in males due to its higher number of participants.16 this study is also involved in the retreatment of mf-positive individuals after 24 months following a single dose of triple-drug therapy; however, this number was later considered as a failure. summary the objective of this systematic review leads to the conclusion that the triple-drug regimen is superior in terms of efficacy, but resulted in the sacrifice of its safety. one of the downsides in these three studies is the small geographic variety in the included respondents. it is crucial that future studies consider and involve a wide variety of people in order for this research to be generalized. to conclude, based on this review, the presence of a triple-drug regimen can really aid in improving the course of lymphatic filariasis; thus, leading to much better outcomes. acknowledgement this endeavor would not have been possible without the generous support from our university. we would like to thank atma jaya catholic university of indonesia for the continuous encouragement, appreciation and keen interest in our research. conflict of interest the authors declare that they have no conflict of interest. references 1. cdc. lymphatic filariasis [internet]. centers for disease control and prevention. centers for disease control and prevention; 2018 [cited 2020 dec 14]. available from: https://www.cdc.gov/parasites/lymphaticfilariasi s/index.html 2. wynd s, melrose w. d, durrheim dn, carron j, gyapong m. understanding the community impact of lymphatic filariasis: a review of the sociocultural literature. bulletin of the world health organization. 2007;85:493-498. 3. world health organization. world health organization. lymphatic filariasis [internet]. [cited 2020 dec 14]. available from: https://www.who.int/news-room/factsheets/detail/lymphatic-filariasis 4. karun v, hotez pj, rosengart tk. global surgery and the neglected tropical diseases. plos neglected tropical diseases. 2017;11(9):e0005563. 5. infodatin. situasi filariasis di indonesia. pusat data dan informasi kementerian kesehatan ri [internet]. 2019 [cited 2020 dec 20] available from:https://www.kemkes.go.id/folder/view/01/ structure-publikasi-pusdatin-info-datin.html 6. ichimori k, king jd, engels d, et al. global programme to eliminate lymphatic filariasis: the processes underlying programme success. plos negl trop dis. 2014;8(12):e3328. 7. world health organization. world health organization. global programme to eliminate lymphatic filariasis: progress report [internet]. 2019 [cited 2020 dec 23]. available from:https://www.who.int/publications/i/item/w ho-wer9543 8. boniface pk, elizabeth fi. an insight into the discovery of potent antifilarial leads against lymphatic filariasis. curr drug targets. 2020;21(7):657–680. 174 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 165–175 9. rebollo mp, bockarie mj. toward the elimination of lymphatic filariasis by 2020: treatment update and impact assessment for the endgame. expert rev anti infect ther. 2013;11(7):723–731. 10. lourens gb, ferrell dk. lymphatic filariasis. nurs clin north am. 2019;54(2):181–192. 11. macfarlane cl, budhathoki ss, johnson s, richardson m, garner p. albendazole alone or in combination with microfilaricidal drugs for lymphatic filariasis. cochrane database syst rev. 2019;1(1):cd003753. 12. thomsen ek, sanuku n, baea m, et al. efficacy, safety, and pharmacokinetics of coadministered diethylcarbamazine, albendazole, and ivermectin for treatment of bancroftian filariasis. clin infect dis. 2016;62(3):334–341. 13. page mj, mckenzie je, bossuyt pm, et al. the prisma 2020 statement: an updated guideline for reporting systematic reviews. bmj. 2021;372:n71. 14. sterne jac, savović j, page mj, et al. rob 2: a revised tool for assessing risk of bias in randomised trials. bmj. 2019;366:l4898. 15. king cl, suamani j, sanuku n, et al. a trial of a triple-drug treatment for lymphatic filariasis. n engl j med. 2018;379(19):1801– 1810. 16. bjerum cm, ouattara af, aboulaye m, et al. efficacy and safety of a single dose of ivermectin, diethylcarbamazine, and albendazole for treatment of lymphatic filariasis in côte d'ivoire: an open-label randomized controlled trial. clin infect dis. 2020;71(7):e68-e75. 17. schulz jd, neodo a, coulibaly jt, keiser j. pharmacokinetics of albendazole, albendazole sulfoxide, and albendazole sulfone determined from plasma, blood, dried-blood spots, and mitra samples of hookworm-infected adolescents. antimicrob agents chemother. 2019;63(4):e02489-18. 18. abongwa m, martin rj, robertson ap. a brief review on the mode of action of antinematodal drugs. acta vet (beogr). 2017;67(2):137–152. 19. pawluk sa, roels ca, wilby kj, ensom mh. a review of pharmacokinetic drug-drug interactions with the anthelmintic medications albendazole and mebendazole. clin pharmacokinet. 2015;54(4):371–383. 20. brunton ll, hilal-dandan r, knollmann bc. goodman & gilman’s the pharmacological basis of therapeutics. 13th edition. new york: mcgraw-hill; 2018. 21. crump a. ivermectin: enigmatic multifaceted 'wonder' drug continues to surprise and exceed expectations. j antibiot (tokyo). 2017;70(5):495–505. 22. schulz jd, coulibaly jt, schindler c, wimmersberger d, keiser j. pharmacokinetics of ascending doses of ivermectin in trichuris trichiura-infected children aged 2-12 years. j antimicrob chemother. 2019;74(6):1642–1647. 23. bennett je, dolin r, blaser mj. mandell, douglas, and bennett’s principles and practice of infectious diseases volume 1. philadelphia: elsevier; 2020. 24. john ln, bjerum c, martinez pm, et al. pharmacokinetic and safety study of coadministration of albendazole, diethylcarbamazine, ivermectin and azithromycin for the integrated treatment of neglected tropical diseases [published online ahead of print, 2020 aug 20]. clin infect dis. 2020;ciaa1202. 25. ritter j, flower rj, henderson g, yoon kong loke, rang hp. rang and dale’s pharmacology. 9th ed. endinburgh: elsevier; 2020. 26. weil gj, jacobson ja, king jd. a triple-drug treatment regimen to accelerate elimination of lymphatic filariasis: from conception to delivery. international health. 2020;13:s60–4. 27. walker m, pion sds, fang h, et al. macrofilaricidal efficacy of repeated doses of ivermectin for the treatment of river blindness. clin infect dis. 2017;65(12):2026–2034. 28. stolk wa, prada jm, smith me, et al. are alternative strategies required to accelerate the global elimination of lymphatic filariasis? insights from mathematical models. clin infect dis 2018; 66:260–6. 29. dunyo sk, simonsen pe. ivermectin and albendazole alone and in combination for the treatment of lymphatic filariasis in ghana: follow-up after re-treatment with the combination. trans r soc trop med hyg. 2002;96(2):189–192. 30. thomsen ek, sanuku n, baea m, et al. efficacy, safety, and pharmacokinetics of coadministered diethylcarbamazine, albendazole, and ivermectin for treatment of bancroftian filariasis. clinical infectious diseases. 2016;62(3):334–341. 31. laing r, gillan v, devaney e. ivermectin-old drug, new tricks?. trends parasitol. 2017;33(6):463–472. 32. degani-katzav n, klein m, har-even m, gortler r, tobi r, paas y. trapping of ivermectin by a pentameric ligand-gated ion channel upon opento-closed isomerization. sci rep. 2017;7:42481. 33. dubray cl, sircar ad, beau de rochars vm, et al. safety and efficacy of coadministered diethylcarbamazine, albendazole and ivermectin during mass drug administration for lymphatic filariasis in haiti: results from a two-armed, 175 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license rivaldi ruby, et al. the curative innovation of novel triple-drug openlabel, cluster-randomized, community study. plos negl trop dis. 2020;14(6):e0008298. 34. edi c, bjerum cm, ouattara af, et al. pharmacokinetics, safety, and efficacy of a single co-administered dose of diethylcarbamazine, albendazole and ivermectin in adults with and without wuchereria bancrofti infection in côte d'ivoire. plos negl trop dis. 2019;13(5):e0007325. 35. weil gj, bogus j, christian m, et al. the safety of doubleand triple-drug community mass drug administration for lymphatic filariasis: a multicenter, open-label, cluster-randomized study. plos med. 2019;16(6):e1002839. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 �� vol. 1. no. 1 january–april 2010 research report �’utr polymorphism of nramp� gene and susceptibility to lung tuberculosis among patients and nurses in surabaya, indonesia rahayu anggraini division of tuberculosis, institute of tropical disease, airlangga university abstract the objectives of this study was to evaluate a potential role for natural-resistance-associated macrophage protein 1 (nramp1) gene in the human homologue using four single base pair polymorphisms (d543n, 3’utr, int4, 274c/t) for susceptibility to tuberculosis infection in surabaya, indonesia. the study population were 69 lung tuberculosis patients and 43 healthy nurses were genotyped with the polymerase chain reaction (pcr) and the product amplified from their genomic dna were subjected to restriction enzyme digestion (rflp) and were analysed using agarose gel electrophoresis. results of this study showed only the homozygous tgtg deletion allele at the 3’untranslated region (3’utr) of the nramp1 gene i.e. the tgtgdel/del genotype was more frequently found in lung tuberculosis patients (20/69=29%) compared to that found in nurses (2/43=4.7%). the odds ratios (ors) were 8.37 (95% confidence interval [ci], 1.85 to 37.94; p=0.002). this finding shows that polymorphism 3’utr of nramp1 gene increased the risk of lung tuberculosis in surabaya, indonesia. key words: nramp1 gene, pulmonary tuberculosis, surabaya introduction despite the availability of a number of anti-mycobacterial drug, lung tuberculosis is still difficult to eradicate. in 1990 the incidence of tuberculosis (tb) world-wide approximately 1.7 billion or about one third of the world population at that time. the incidence of tb in 1997, is about 8 million and mortality amount 3–4 million cases per annual. therefore who started that tb is one of the more important infectious disease to notice and to tackle.1 in indonesia, tb re-appears as a prominent cause of death. the result of family health survey held in 2005, showed that tb is the third leading cause of death after cardiovascular and respiratory tract disease in all age and is the first leading cause of death in infectious disease.2,3 lung tb arises when tb bacilli present in air droplets enter the respiratory tract, finally reaching lung alveoli. upon which it will evoke an inflammatory response causing in accumulation of macrophage and neutrophyl. these phagocyte cells will than migrate to regional lymph nodes crossing swelling of these lymph nodes, thus forming the so called primary complex. the tb bacilli in lung tissue and lymph node were engulfed by macrophages. if the host immune response is adequate, tb bacilli will be killed by macrophage causing healing of the primary complex. how ever when the host immune response not adequate, tb bacilli survive and multiply within macrophage which upon leaving these cells, finally enter lymph and blood circulation, spreading to other organs.4 the nramp1 (natural resistance associated macrophage protein-1) gene is expected in macrophages and also in blood cells (peripheral blood mononuclear cells/ pbmc). the gene encodes a protein that function as a divalent ion channel including fe++ ions. fe++ ions are known to inhibit growth and ultimately kill m. tuberculosis. when a mutation in the nramp1 gene results in a non functional nramp1 protein or the protein is rapidly degraded, then the inhibition action of the nramp1 protein is lost or lessened and the mycobacteria will multiply freely in macrophages.5–7 based on that above facts it can be concluded that in certain cases susceptibility to lung tuberculosis is under genetic control. that certain genetic mutation in nramp1 gene leads to susceptibility been reported by several �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 17-22 authors.8–17 the present study is the first known study done in indonesia involving the influence of nramp1 gene polymorphism in acquiring susceptibility to lung tuberculosis involving the following genotype d543n, 3’utr, int4, and 274c/t polymorphism which have been previously investigated by other author outside indonesia. the d543n polymorphism (g/g, g/a, a/a genotype), 3’utr polymorphism (tgtg+/+, tgtg+/del, tgtg del/del genotype), int int4 polymorphism (g/g, g/c, c/c genotype) and 274c/t polymorphism (c/c, c/t, t/t genotype) has been found to show a significantly different distribution frequencies between tuberculosis patients and healthy subjects (nurses), thus showing differences in susceptibility between these two groups toward acquiring tuberculosis.8–17 field experience have been shown that nurses serving lung tuberculosis patients in hospital wards for years often do not acquire the disease. likewise, only 10% of people infected by m. tuberculosis actually acquire the disease.11,13 materials and methods participant subjects participating in this study are 69 lung tuberculosis patients (38 male and 31 female) treated in dr. soetomo general hospital and karang tembok hospital at surabaya, they had normal blood and serum analysis (hb, creatinine, bun, glucose, and hiv), and abnormal x-ray and positive culture and acid-fast bacilli in sputum and 43 healthy nurses (18 male and 25 female) with normal blood and serum analysis (hemoglobin, creatinine, bun, glucose, and hiv) as well as normal x-ray and negative cultur and acid-fast bacilli in sputum, but positive ppd. patient ranged in age between 18 and 50 years, with average between 25 and 45 years. ethical clearance and informed consent approval for the study was obtained from the dr. sutomo general hospitals at surabaya. dna preparation pbmc isolations, 2 ml whole blood were obtained by vena puncture using edta as anticoagulant (whole blood edta) is then centrifuged at 3000 rpm for 30 minutes. the resulting buffycoats containing pbmc is then pipette and stored frozen for further use. dna is isolated using high pure pcr preparation kit (roche, applied science; mannheim, germany). the isolated dna is then stored at 4o c for immediate use. nramp1 genotyping nramp1 genotyping method was performed according to the ceillier method. pcr-rflp analysis, to distinguish the different genotypes of each nramp1 gene (d543n, 3’utr, int4, and 274c/t), pcr-rflp (polymerase chain reaction-restriction fragment length polymorphism) analysis were done. the principle of this procedure is as follow: using a pair of specific primers, part of the nramp1 gene is amplified by pcr and the amplified fragment is then cut by a specific restriction enzyme so that either the normal or mutated allele is cut. pcr (polymerase chain reaction) using fast start pcr master (roche, applied science; mannheim, germany) was done using a thermal cycler (model 9600; parkin elmer; branchburg, nj) as follows: addition of 5 µl dna solution, primers, and other reagents needed for dna fragment amplification, activation of thermal cycler 95oc for 5 minutes, denaturation 95o c for 30 seconds, annealing 60o c for 30 seconds, extension 72o c for 3 minutes, steps 3,4,5 repeated for 35 cycles and final extension 72o c for 7 minutes. the temperature of the thermal cycler is than chilled to 4o c and the resulting amplified dna fragment is stored 4o c. the amplified dna fragments (often called ”amplicon”) is than identified by agarose gels electrophoresis, visualized by adding ethidium bromide to the gel. if the procedure is success full a single green color bands will appear when expressed to uv radiation. d543n polymorphism, forward primer: 5’– gca tct ccc caa ttc atg gt-3’and reverse primer: 5’aac tgt ccc act cta tcc tg -3’. restriction enzyme avaii (roche, applied science; mannheim, germany) with normal allele (g): 3 fragments: 126bp, 79bp, and 39bp and mutant allele (a): only 1 fragments: 244bp. numbers of bands seen after agarose gels electrophoresis, genotype g/g: 126bp, 79bp and 39bp (3 bands), genotype g/a: 244bp, 126bp, 79bp and 39bp (4 bands), and genotype a/a: 244bp (1 bands). 3’utr polymorphism, the primer same as used for d543n mutation, forward primer: 5’– gca tct ccc caa ttc atg gt-3’ and reverse primer: 5’aac tgt ccc act cta tcc tg -3’. restriction enzyme: foki (roche, applied science; mannheim, germany) with normal allele (tgtg+): 2 fragments: 211bp, and 33bp and mutant allele (tgtg del): only 1 fragments: 244bp. numbers of bands seen after agarose gels electrophoresis, genotype tgtg+/+: 211bp and 33bp (2 bands), genotype tgtg+/del: 244 bp, 211bp, and 33bp (3 bands), and genotype tgtg del/del : 244bp (1 bands). int4 polymorphism, forward primer: 5’tct ctg gct gaa ggc ctc tcc-3’ and reverse primer: 5’-gag gct caa act gat agc aca3’. restriction enzyme apai (roche, applied science; mannheim, germany) with normal allele (g): 1 fragments: 624bp and mutant allele (c): 2 fragments: 455bp and 169bp. numbers of bands seen after agarose gels electrophoresis, genotype g/g : 624bp (1 bands), genotype g/c : 624bp, 455bp, and 169 bp (3 bands), and genotype c/c : 455bp and 169bp (2 bands). 274c/t polymorphism, forward primer: 5’tgc cac cat ccc tat acc cag-3’ and reverse primer: 5’-tct cga aag tgt ccc act cag3’. restriction enzyme mnli (roche, applied science; mannheim, germany) with normal allele (c): 4 fragments: 102bp, 65bp, 37bp, and 12bp and mutant allele (t): 3 fragments: 167bp, 37bp, and 12bp. numbers of bands seen after agarose gels electrophoresis, genotype c/c : 102bp, 65bp, 37bp, ��anggraini: 3'utr polymorphism of nramp1 gene and 12bp (4 bands), genotype c/t : 167bp, 102bp, 65bp, 37bp, and 12bp (5 bands), and genotype t/t : 167bp, 37bp, and 12bp (3 bands). statistical analysis for each polymorphism, allele and genotype frequency differences in each group were examined using pearson’s chi-square or fisher exact test. odds ratios (ors) and 95% confidence intervals (ci) were calculated to quantitatively assess the degree of association between these polymorphism from patient and nurses. results from an amplification genomic dna with pcr were analyzed by electrophoresis on 2% agarose gel showed in figure1 the d543n and 3’utr polymorphism nramp1 gene have 244 base pair and int4 polymorphism have 624 base pair. figure 1. pcr result from genomic dna amplification, d543n and 3’utr (a) polymorphism nramp1 gene with 244 base pair and int4 (b) polymorphism nramp1 gene with 624 base pair. from an amplification genomic dna 274c/t polymorphism with pcr were analyzed by electrophoresis on 2% agarose gel showed have 216 base pair can showed in figure 2. figure 2. pcr result from genomic dna 274c/t polymorphism nramp1 gene amplification with 216 base pair. the pcr-rflp of d543n polymorphism from lung tb patients and nurses g/g genotype showed 3 bands of 126bp, 79bp and 39bp, genotype g/a showed 4 bands of 244bp, 126bp, 79bp, 39bp and genotype a/a showed 1 band of 244bp who show in figure 3. figure 3. the pcr-rflp of d543n polymorphism from lung tb patients (a) and nurses (b). lane1: marker, and showed 1 band was genotype a/a, 2 bands were genotype g/g and 3 bands were genotype g/a. �0 indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 17-22 the pcr-rflp of 3’utr polymorphism from lung tb patients and nurses. tgtg +/+ genotype showed 2band of 211bp and 33bp, tgtg +/del genotype showed 3 bands of 244bp and 211bp and 33bp and tgtg del/del genotype showed 1 band: 244bp who show in figure 4. figure 4. the pcr-rflp genotyping polymorphism 3’utr from lung tb patients (a) and nurses (b). lane 1 (a, b): marker; showed 1 band was genotype tgtg del/del, 2 bands were tgtg +/+; 3 bands were tgtg +/del. the pcr-rflp of int4 polymorphism from lung tb patients and nurses. g/g genotype showed 1 band of 624bp, g/c genotype showed 3 bands: 624bp, 455bp, and 169bp who show in figure 5. figure 5. the pcr-rflp genotyping polymorphism int 4 from lung tb patients (a) and nurses (b). lane 17 (upper) and lane 1 (lower): marker; showed 1 band was g/g and showed 3 bands were g/c genotype. the pcr-rflp of 274c/t polymorphism from lung tb patients and nurses. t/t genotype showed 3 band of 167bp, 37bp and 12bp, c/c genotype showed 4 band of 102bp, 65bp, 37bp and 12bp, and g/c genotype showed 5 bands: 167bp, 102bp, 65bp, 37bp and 12bp who show in figure 6. figure 6. the pcr-rflp genotyping polymorphism 274c/t from (a) nurses and lung tb patients (b). lane 4 (upper) and lane 5 (lower): marker; c/c genotype were showed 3 band not 4 bands, c/t genotype were showed 4 bands not 5 bands, and t/t genotype were showed 2 bands not 3 bands, because 12bp band was disappear. the frequencies of the d543n, 3’utr, int4, and 274c/t polymorphism nramp1 gene mutation in lung tuberculosis patients and healthy nurses were compared using fisher exact test for frequencies d543n because certain cell is zero, and chi-square statistical method for d543n, 3’utr, int4, and 274c/t polymorphism nramp1 gene see table 1. the above table revealed that only allele tgtg del genotype tgtg del/del of 3’utr polymorphism showed a frequencies were statistically different (p=0.002), that was pointed to susceptibility of tuberculosis infection. discussion our study shows that allelic frequencies in the d543n, int4, and 274c/t showed no allelic association was identified different between the nramp1 alleles and tuberculosis susceptibility, except the 3’utr of nramp1 differ between tuberculosis patients and healthy nurses in surabaya, it was suggesting that nramp1 gene could be associated with susceptibility to tuberculosis. we assume that genetic variants 3’utr polymorphisms locating in nramp1 are responsible for the allelic difference in our study. ��anggraini: 3'utr polymorphism of nramp1 gene it is not yet clearly whether the 3’utr polymorphism directly affect nramp1 function, or whether another functional polymorphism exists in the nramp1 gene. further studies are necessary to answer this question. the 3’utr polymorphism allele associated with susceptibility to tuberculosis has been reported to be very uncommon in caucasians, but is present in many cases in west african.9,11 as well as in surabaya. these observations may explain in part why african americans and asians have greater susceptibility to tuberculosis than caucasians. the nramp1 gene has been identified as a critical factor for host defense against some mycobacterial species among inbred mouse strains.18 the protein encoded by the nramp1 gene is exclusively expressed in the macrophage/monocyte, and it is assembled onto the subcellular membrane of the lysosome / endosome and phagolysosome.19 it is likely to restrict mycobacterial replication by influencing the transmembrane transportation of divalent cations, which are essential for the survival of mycobacteria.20,21 in human mycobacterium tuberculosis is found within phagosomes present in macrophages, because the bacteria is phagocyte by macrophage. when phagosome fuse with late endosome, the bacteria will then be present in late endosome. late endosome contains a fe2+ (divalent) ion channel encoded by the nramp1 gene. if the nramp1 protein is functional, then fe2+ ions will enter late endosome. late endosome contain h2o2 (hydrogen peroxidase) which upon reacting with fe2+ ions will then give rise to the highly reactive hydroxyl radical (oh*) through a chemical reaction called the fenton reaction: fe2+ + h2o2  oh* + oh + fe3+ the highly reactive oh* radical will kill the mycobacteria by causing membrane demage.15 in human with a mutated nramp1 gene giving rise to a non functional nramp1 protein, the above reaction will not be killed and still survive within macrophage. several case-control study9,10,12,20,22 have indicated that polymorphism of the human nramp1 gene (ie, d543n, 3’utr, and 274c/t polymorphism) modify the susceptibility of the host to tuberculosis, with the group affected including africans and asians. in addition, one interesting point is the fact that the proportion of tgtg del/del genotype in surabaya patients is higher than that in the healthy nurses, whereas the proportion of tgtg del/del in a west african control group was slightly higher than that of west african patients, as reported in the study of bellamy et al. the different proportions of tgtg del/del in surabaya and west africans in the case-control study seem to indicate that the 3’utr polymorphism might not be the direct cause of susceptibility, but rather that another functional polymorphism exists in nramp1. the polymorphism might be strongly linked to the genotypes. the question is why the 3’utr mutation can result in susceptibility to tuberculosis? it was found that the mutant tgtg del allele produce a normal nramp1 protein, but in a much lower amount compared to the normal tgtg+ allele. the majority of eukaryote mrna posses a poly-a tail about 250 nucleotide long.23,34 including the nramp1 mrna. when eukaryote mrna enters cytosol, the poly-a tail is gradually degraded by cytosolic nuclease. cytosol contains poly-a polymerase (pap) which can results the poly-a tail, but it can not outcompete. the destruction due to the cytosol nuclease that the net effect is the gradually, but inescapably shortening of the poly-a tail with time. when the poly-a tail is completely destroyed then the cytosol nuclears will eventually degrade the coding sequenes of the now tailless mrna and protein production will stop. the tgtg del allele produce a much shorter. shorter poly-a tail so that its mrna will be degraded much eailier and resulting in a lower amount of nramp1 protein produced as compared to the tgtg+ allele with its longer poly-a tail. why the tgtg del allele produce a much shorter tail is not enterely clear. apparently the 3’utr region contain certain sequences needed to produce a normal length polya tail. these sequences is an aataa sequence followed 23–24 nucleotides down stream with a gt rich region. a table1. the frequency of d543n, 3’utr, int4, and 274c/t polymorphism nramp1 gene in lung tb patients and healthy nurses group polymorphism genotype lung tb patients (n=69) healthy nurses (n=43) risk estimate (95% confidence interval [ci]) p value d543n g/g g/a a/a 35 (50.70%) 28 (40,60%) 6 (8.7%) 28 (65.10%) 15 (34.90%) 0 (0%) 0.551(0.252-1.209) 1.275(0.579-2.809) 1.095(1.018-1.178) 0.171 0.558 0.080 3’utr tgtg+/+ tgtg+/del tgtgdel/del 35 (50.70%) 14 (20,30%) 20 (29.0%) 28 (65.10%) 13 (30.20%) 2 (4,7%) 0.551(0.252-1.209) 0.587(0.245-1.411) 8.367(1.846-37.937) 0.135 0.232 0.002 int4 g/g g/c c/c 67 (97,10%) 2 (2.9%) 0 (0%) 40 (93,00%) 3 (7.00%) 0 (0%) 2.513(0.402-15.687) 0.398(0.064-2.485) 0.370 0.370 274c/t c/c c/t t/t 68 (98,60%) 1 (1,40%) 0 (0%) 40 (93,00%) 2 (4.70%) 1 (2.30%) 5,100(0.513-50.697) 0.301(0.026-3.430) 0.977(0.933-1.023) 0.296 0.557 0.384 �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 17-22 loss of a tgtg sequence within the gt rich region may possibly desturb the formation of a normal poly-a tail resulting in a much shorter poly-a tail.23,24 this cross sectional study showed that homozygotes at tgtg del/del genotypes of 3'utr polymorphism in nramp1 gene were observed at significantly higher frequencies in patients with lung disease than in healthy nurses. this is the first study to indicate a possible genetic risk factor associated with isolated lung disease. additional studies with patients from diverse ethnic backgrounds will be required to further investigate the relationships underlying these preliminary findings. acknowledgment we thank all of the subject in this study for their participation. we also thank to the health department republic indonesia and moloculer biology eijkmann institute who has the research provided funds. references 1. who report, global tubeculosis control: surveillance, planning, financing, whocdstb ; 295 : 21, 2002. 2. p2m plp, pedoman penyakit tuberkulosis dan penanggulangannya. depkes ri, 1997. 3. who report, global tuberculosis control, 2007. 4. schaible, u, collins, h. & kaufmann, she, confrontation between intracellular bacteria and the immune system. adv. immunol. 71, 267–377, 1999. 5. blackwell jm, goswani t, carlton awe, dean s, natalie p, jacqueline kw, susan s, e nancy m, christopher sp, hiba m, and muntaser i, slc11a1 (formerly nramp1) and disease resistance. cellular microbiology. vol.3 issue 12 page 773, 2001. 6. camstock gw, tuberculosis in twins: a re-analysis of the prophit study. am rev resp dis; 117:621, 1978. 7. buu nt, cellier m, gros p, schurr e, identification of a highly polymorphic length variant in the 3'utr of nramp1. immunogenetics 42:428–429 [pubmed] ,1995. 8. cellier m, govoni g, vidal s, et al., human natural resistancehuman natural resistance associated macrophage protein: cdna cloning, chromosomal mapping, genomic organization, and tissue-specific expression. j exp med; 180:1741,, 1994. 9. bellamy r, ruwende c, corrah t, mcadam kpwj, whittle hc, hill avs, variation in the nramp1 gene is associated with susceptibility to tuberculosis in west africans. n engl j med 338:640–644, 1998. 10. gao, ps, fujishima, s, mao, xq, et al genetic variants of nramp1 and active tuberculosis in japanese populations. clin genet;58,74–76, 2000. crossrefmedlineweb of science 11. kim jh, lee sy, lee sh, sin c, shim jj, in kh, yoo sh, kang kh, nramp1 genetic polymorphisms as a risk factor of tuberculous pleurisy. int j tuberc lung 7(4): 370–375, 2003. 12. liu, j, fujiwara, tm, buu, nt, et al., identification of polymorphisms and sequence variants in the human homologue of the mouse natural resistance-associated macrophage protein gene. am j hum genet; 56,845–853, 1995. medlineweb of science 13. zhang wh, shao l, weng x, hu z, jin a, chen s, pang m, and chen zw, variants of the natural resistance-associated macrophage protein1 gene (nramp1) are associated with severe forms of pulmonary tuberculosis. clinical infection disease; 40:1232–1223, 2005. 14. delgado jc, baena a, thim s, and goldfeld ae, ethnic-specific genetic associations with pulmonary tuberculosis. the journal of infectious disease : 186:1463–8, 2002. 15. ma x, dou s, wright ja, et al 5' dinucleotide repeat polymorphism of nramp1 and susceptibility to tuberculosis among caucasian patients in houston, texas. int j tuberc lung dis;6,818–823, 2002. medlineweb of science 16. soborg, c, andersen, ab, madsen, ho, et al natural resistanceassociated macrophage protein 1 polymorphisms are associated with microscopy-positive tuberculosis. j infect dis;186,517–521, 2002. crossrefmedlineweb of science 17. liaw ys, tsai wu jj, wu ch, hung cc, lee cn, yang pc, luh kt, kuo sh, variation in the nramp1 gene and susceptibility of tuberculosis in taiwanese. int j tuberc lung d15 6(5):454–460, 2002 18. vidal, s, tremblay, ml, govoni, g, et al the ity/lsh/bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the nramp1 gene. j exp med;182,655–666, 1995. abstract/free full text 19. gruenheid, s, pinner, e, desjardins, m, et al natural resistance to infection with intracellular pathogens: the nramp1 protein is recruited to the membrane of the phagosome. j exp med;185,717–730, 1997. abstract/free full text 20. canonne-hergaux, f, gruenheid, s, govoni, g, et al the nramp1 protein and its role in resistance to infection and macrophage function. proc assoc am physicians;111,283-289, 1999.crossrefmedlineweb of science 21. blackwell, jm, searle, s, mohamed, h, et al divalent cation transport and susceptibility to infectious and autoimmune disease: continuation of the ity/lsh/bcg/nramp1/slc11a1 gene story. immunol lett;85,197203, 2003. crossrefmedlineweb of science 22. ryu, s, park, yk, bai, gh, et al 3'utr polymorphisms in the nramp1 gene are associated with susceptibility to tuberculosis in koreans. int j tuberc lung dis; 4,577–580, 2000. medlineweb of science 23. weaver rf, molecular biology, 474–490, mc graw-hill, new york, usa, 2002. 24. suryohudoyo p, kapita selekta kedokteran molekuler, 17–30, cv sagung seto jakarta, 2000. ijtid vol 1 no 1 jan-apr 2010.19.pdf ijtid vol 1 no 1 jan-apr 2010.20.pdf ijtid vol 1 no 1 jan-apr 2010.21.pdf ijtid vol 1 no 1 jan-apr 2010.22.pdf ijtid vol 1 no 1 jan-apr 2010.23.pdf ijtid vol 1 no 1 jan-apr 2010.24.pdf 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 11 no. 1 january–april 2023 original article germ tube induction test comparing total of six liquid and three solid media in candida albicans rivaldi ruby1 , erlangga saputra arifin1 , sandy vitria kurniawan2 , sem samuel surja3* 1school of medicine and health sciences, universitas katolik indonesia atma jaya, jakarta, indonesia 2department of pharmacology and pharmacy, school of medicine and health sciences, universitas katolik indonesia atma jaya, jakarta, indonesia 3department of parasitology, school of medicine and health sciences, universitas katolik indonesia atma jaya, jakarta, indonesia received: march 3rd, 2022; revised: november 6th, 2022; accepted: february 27th, 2023 abstract invasive candidiasis (ic) has a high mortality rate of 70%, thus diagnosis should be established without delay. given its fast result, serological test such as β-d-glucan (bdg) test is one alternative diagnosis modalities. however, it lacks specificity. candida albicans germ tube antibody (cagta) test is an alternative serological test which has a high sensitivity of 76.2% and specificity of 80.3%. manufacturing cagta serological test requires provision of specific germ tube antigen. in this study, various culture media were tested to find the best media for germ tube induction. this study was an experimental in vitro study. the number and length of the germ tube were recorded in twoand three-hour incubation periods. a total of six samples containing one c. albicans atcc 90028, four c. albicans wild type strains, and one c. krusei wild type strain were used. nine media were tested to induce germ tube formation: human and sheep serum, fetal bovine serum, mueller hinton agar and broth, tryptic soy agar and broth, brain heart infusion agar and broth. at both incubation periods, the medium with the highest number of germ tube was human serum (p=0.001 and p=0). the longest germ tube was found in sheep serum at two-hour incubation period (p=0.005). mueller hinton broth (mhb) showed comparable results with human and sheep serum (p>0.05). human serum is a superior inducer of morphogenesis. however, the use of mhb is recommended in this study, since provision of fresh human and sheep serum on a regular basis is impractical. keywords: candida albicans; germ tube; human serum; mueller hinton broth; sheep serum highlights: several media could induce not only numbers of germ tube, but also its length. therefore, they could benefit for easier diagnosis and also higher amounts of germ tube protein. how to cite: ruby, r., arifin., e. s., kurniawan, s. v., surja, s. s. germ tube induction test comparing total of six liquid and three solid media in candida albicans. indonesian journal of tropical and infectious disease. 11(1). 18–26. apr. 2023. doi: 10.20473/ijtid.v11i1.34097 * corresponding author: sem.samuel@atmajaya.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-6609-8413 https://orcid.org/0000-0002-5094-9957 https://orcid.org/0000-0002-1707-0166 https://orcid.org/0000-0001-5981-0014 19 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 8–26 introduction candidiasis is a disease with a high prevalence rate globally. this disease generally affects the skin and mucosal tissue, causing mild conditions such as oral and vulvovaginal candidiasis.1 at the systemic level, it has high mortality and morbidity, referred to as invasive candidiasis (ic). it is associated with prolonged intensive care unit (icu) admission and immunocompromised conditions such as acquired immune deficiency syndrome (aids).2 globally, candidiasis occupies the top three incidences of diseases caused by fungi in the year 2017—the first is oral candidiasis with an incidence rate of 2,000,000, followed by esophageal candidiasis with 1,300,000, and then ic with 750,000 incidences.1 invasive candidiasis yields a high mortality rate of 70%.3 invasive candidiasis is caused by candida spp. with the most common etiology being candida albicans.4 in humans, this fungus is a normal flora of the skin, oropharynx, digestive, and urogenital tract.5 infection occurs when there is hyphal growth and biofilm formation in the tissue. these mechanisms also allow resistance of c. albicans to traditional antifungal agents.6 timely diagnosis is required in order to reduce mortality. currently, ic is diagnosed through the findings of hyphae on microscopic examination or through a timeconsuming culture.1 serological tests provide relatively faster and easier way to diagnose ic. β-dglucan (bdg) test is one widely used candida serological test. however, it lacks specificity due to cross reaction with other fungi.7 a serological test detecting antibody against germ tube could be used as an alternative to diagnose ic, namely c. albicans germ tube antibody (cagta) test.8 germ tubes are formed by c. albicans in a number of conditions such as starvation, presence of serum or n-acetylglucosamine, physiological temperature, and co2. 9 it has high sensitivity of 76.2% and specificity of 80.3% since morphological transition from yeast to germ tube and hyphae is important for pathogenicity of c. albicans.8,10 combination of bdg and cagta serological tests is recommended for ic early diagnosis.11 the first step in manufacturing cagta serological test is isolation of the germ tube antigen. it is important to seek the best medium for c. albicans since this antigen is obtained by inducing its growth in a suitable environment. various media can be used for induction of germ tube, each with unique compositions and function. human serum is the most used medium for germ tube test.12 its main limitation is the requirement of fresh human serum on a regular basis. for this reason, this study aims to find the best media in inducing germ tube formation of c. albicans. while previous studies mainly assessed the sensitivity of each medium for germ tube test, this study also measured the number and length of germ tube formed after certain incubation period. materials and methods study design this experimental in vitro study was conducted in the parasitology laboratory, school of medicine and health sciences, atma jaya catholic university of indonesia from august 2020 to october 2020. ethical clearance was obtained from the atma jaya ethical committee with the number 01/06/kep-fkuaj/2020. fungi strains candida albicans wild type, c. albicans atcc 90028, and c. krusei wild type were used in this study. all c. albicans wild type were obtained from patient’s sputum in microbiology laboratory, school of medicine and health sciences, atma jaya catholic university of indonesia, while c. albicans atcc 90028 and c. krusei wild type were obtained from the collection of department parasitology, school of medicine and health sciences, atma jaya catholic university of indonesia. each strain 20 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license rivaldi ruby, et al. germ tube induction test was identified and confirmed through macroscopic and microscopic examination. macroscopic identification was made using chromagar (oxoid, united kingdom) to analyze the color and characteristics of the fungal colonies. candida albicans was characterized by the formation of green colonies, in contrast with c. krusei which appeared as pink colonies.13 microscopic identification was made through lactophenol cotton blue (lpcb) staining and germ tube test. light microscope (olympus cx21) is used to identify the morphologies. ovoid and spherical yeast cell shapes are a characterization of c. albicans, distinguished from c. krusei that commonly appear as a more elongated (long grain rice) shape. a positive germ tube test is also only found in c. albicans.14,15 a total of six isolates containing one c. albicans atcc 90028, four c. albicans wild type, and one c. krusei wild type were used. candida albicans atcc 90028 and c. krusei were used as the positive and negative control, respectively. medium the media used for induction of germ tube were serum, broth, and agar. sera used were human serum, sheep serum, and fetal bovine serum (fbs, biowest, france). the fbs used was not diluted with 100% concentration. human serum was prepared by centrifugating blood from a healthy donor.16 the broth and agar media used were mueller hinton agar (mha, oxoid, united kingdom), mueller hinton broth (mhb, conda, spain), tryptic soy agar (tsa, oxoid, united kingdom), tryptic soy broth (tsb, merck, germany), brain heart infusion agar (bhia, oxoid, united kingdom), and brain heart infusion broth (bhib, oxoid, united kingdom). all media were obtained from the department of microbiology, parasitology, and pharmacology, school of medicine and health sciences, atma jaya catholic university of indonesia. broth and agar media were prepared by combining 1 l of aquadest with 38, 21, 45, 30, 53, and 37 g of mha, mhb, tsa, tsb, bhia, and bhib, respectively. these suspensions were heated until completely dissolved, followed by sterilization using an autoclave at 121°c with a pressure of 15 psi for 15 minutes. broth medium was poured into the 1.5 ml test tube, while agar medium was poured into a petri dish. for all media, ph was adjusted at 7.4 which is confirmed by ph meter. gem tube induction candida albicans and c. krusei were inoculated in sabouraud dextrose agar (sda, oxoid, united kingdom) for 48 hours at room temperature (25°c). two hundred μl of 3 mcfarland fungi suspension was added into 800 μl of each serum and broth medium. the mixture was incubated for 24 hours at 37°c.12 the number and length of the germ tube were recorded in two-, three-, and 24-hour incubation periods. ten μl of the mixture was dripped into an improved neubauer counting chamber and the germ tube was observed under the microscope (figure 1a).17 all processes were performed in duplicate. germ tube induction in agar media was conducted by dripping 10 μl of 0.5 mcfarland fungi suspension into 1 x 1 cm2 agar.18 cover slip was placed to facilitate easier examination. the agar was then incubated for three hours at 37°c. the number and length of germ tube induction were recorded in twoand three-hour incubation periods using the microscope (figure 1b). all processes were performed in duplicate. germ tube calculation this study measured the number and length of germ tubes formed. the number of germ tube was calculated in five small squares of the counting chamber using standardized formula (figure 2).19 germ tube length measurement was conducted by comparing the length of the germ tube and the counting chamber small squares. the longest germ tube in the five small squares was 21 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 8–26 recorded. germ tube length measurement was done only in serum and broth media. there are no difficulties in performing the measurement methods. figure 1. germ tube appearance under the microscope in different preparations. (a) improved neubauer counting chamber – germ tube (red circle); (b) slide culture 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔𝑒𝑟𝑚 𝑡𝑢𝑏𝑒 / 𝜇𝐿 = 𝑛 𝑉 𝑉 = 𝑖𝑚𝑝𝑟𝑜𝑣𝑒𝑑 𝑁𝑒𝑢𝑏𝑎𝑢𝑒𝑟 𝑐𝑜𝑢𝑛𝑡𝑖𝑛𝑔 𝑐ℎ𝑎𝑚𝑏𝑒𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 = 1 50 𝑚𝑚3 𝑛 = 𝑡𝑜𝑡𝑎𝑙 𝑜𝑓 𝑔𝑒𝑟𝑚 𝑡𝑢𝑏𝑒 𝑖𝑛 5 𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 figure 2. improved neubauer counting chamber calculation of the number of germ tube on agar media was carried out by taking three representative images using the high-power field (hpf) microscope. then the number of germ tubes was grouped into several categories (table 1). table 1. germ tubes counts categories using agar media inductions categories germ tube count/hpf 1+ 1-10 2+ 11-20 3+ 21-30 4+ 31-40 5+ 41-50 6+ >50 hpf – high-power field data analysis data analysis was done using statistical product and service solution (spss) version 22. data on serum and broth medium was analyzed using a one-way anova statistical test or kruskal-wallis test, depending on data normality. it was then followed by a bonferroni post hoc test if significant results were found. in agar medium, fisher-exact or chi-square test were used depending on the terms and criteria of the statistical test. a significant value was yielded if p<0.05. results and discussion fungi identification macroscopic identification of all samples was done using chromagar. it is depicted in figure 3. moreover, the morphologies found in microscopic identification using lpcb confirmed the samples’ species (figure 4). figure 3. macroscopic characterizations on chromagar. the green colonies are c. albicans and the pink colony is c. krusei (a) (b) figure 4. microscopic characterizations of candida species grown in sda. (a) ovoid appearance of c. albicans (black circle) (b) elongated appearance of c. krusei (red circle) 22 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license rivaldi ruby, et al. germ tube induction test germ tube induction in serum and broth all candida samples were used in germ tube induction. candida albicans atcc 90028 and c. krusei function serve as a positive and negative control, respectively. in all media, c. albicans atcc 90028 showed positive germ tube results, while c. krusei had negative results. candida albicans wild type was used for the evaluation of each media’s ability to induce germ tube formation. several media could facilitate germ tube formation. results were variable between media at twoand three-hour incubation period. at two-hour incubation period, it was found that the medium with the highest number of germ tube was human serum. sheep serum also showed comparable results in germ tube induction. roughly, the order of media that could facilitate germ tube induction was as follows: human serum, sheep serum, fbs, mhb, tsb, and bhib; while at three-hour incubation period, the order was as follows: human serum, mhb, sheep serum, fbs, tsb, and bhib as shown in table 2. table 2. number of germ tubes formed on serum and broth media (number/μl) time fungi media p-value human serum sheep serum fbs mhb tsb bhib 2 hours ca 1 38125 40625 26875 11250 5625 5000 0.001 ca 2 23125 20625 10000 22500 11250 3750 ca 3 25000 23125 8750 15625 13750 0 ca 4 21250 15000 18125 11250 0 625 3 hours ca 1 42500 16250 13750 15625 3750 0 0 ca 2 26250 15000 5000 37500 15625 6875 ca 3 32500 20625 7500 25000 16250 0 ca 4 31875 13125 18750 15000 3750 0 *post hoc (2 hours) human serum vs. bhib p=0.003; human serum vs. tsb p=0.024 **post hoc (2 hours) sheep serum vs. bhib p=0.006 ***post hoc (3 hours) mhb vs. bhib p=0.004 ****post hoc (3 hours) human serum vs. bhib p=0; human serum vs. sheep serum p=0.03; human serum vs. tsb p=0.001; human serum vs. fbs p=0.003 no difference was shown between mhb and human serum in post hoc analysis (p>0.05). the number of germ tubes between broth media were highest in mhb, compared to tsb and bhib (p=0.015 and p=0.009 at twoand three-hour incubation periods, respectively) as shown in table 3. at the 24hour incubation period, the fungi experienced rapid growth into hyphae. therefore, further analysis was not performed in this incubation period. table 3. number of germ tubes formed on broth media (number/μl) time fungi media p-value mhb tsb bhib 2 hours ca 1 11250 5625 5000 0.115 ca 2 22500 11250 3750 ca 3 15625 13750 0 ca 4 11250 0 625 3 hours ca 1 15625 3750 0 0.009 ca 2 37500 15625 6875 ca 3 25000 16250 0 ca 4 15000 3750 0 *post hoc (2 hours) mhb vs. bhib p=0.015 **post hoc (3 hours) mhb vs. bhib p=0.009 23 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 8–26 table 2. number of germ tubes formed on serum and broth media (number/μl) time fungi media p-value human serum sheep serum fbs mhb tsb bhib 2 hours ca 1 14 28.89 22.22 17.775 24.445 2.22 0.005 ca 2 26 38 37.78 22.22 26 17.78 ca 3 20 33.335 26.665 23.33 31.115 0 ca 4 30 55.555 38 33.335 0 10 3 hours ca 1 26.665 53.33 26 31.115 14 0 0.155 ca 2 57.78 50 42 77.775 95.555 48.89 ca 3 28.89 36 34 37.78 57.78 0 ca 4 77.78 44.665 22 73.335 26.665 0 *post hoc (2 hours) bhib vs. sheep serum p=0.003 *post hoc (2 hours) bhib vs. fbs p=0.039 in the measurement of length, the longest germ tube was found in sheep serum at twohour incubation period, followed by fbs, mhb, tsb, human serum, and bhib. no difference was found at the three-hour incubation period as shown in table 4. no broth media was found superior to the other in terms of the germ tubes length as shown in table 5. table 5. length of the germ tube formed on broth media (μm) time fungi media p-value mhb tsb bhib 2 hours ca 1 17.775 24.445 2.22 0.998 ca 2 22.22 26 17.78 ca 3 23.33 31.115 0 ca 4 33.335 0 10 3 hours ca 1 31.115 14 0 0.132 ca 2 77.775 95.555 48.89 ca 3 37.78 57.78 0 ca 4 73.335 26.665 0 germ tube induction in agar the number of germ tubes formed was highest in mha. however, results were not significant both in twoand three-hour incubation periods as shown in table 6. table 6. length of the germ tube formed on agar media (μm) time fungi media p-value mha tsa bhia 2 hours ca 1 +3 +1 0 0.408 ca 2 +6 +1 +2 ca 3 +4 +1 +1 ca 4 +1 0 0 3 hours ca 1 +1 0 0 1 ca 2 +6 +1 +1 ca 3 +2 +1 +1 ca 4 0 0 0 hyphal morphogenesis is one of the most investigated virulence attributes of c. albicans. the ability of c. albicans to undergo reversible morphological transition could be triggered by variety of environmental condition.20–22 serum, especially human serum, contains several important components that promote germ tube formation; therefore, it accounts as strong inducer for yeast-to-hyphae formation. burch et al. (2018) stated that human serum fraction revealed signs of bacterial peptidoglycan (pgn)-like molecules which highly active for hyphae induction.23 glucose could also act as morphogen, which in the certain amount could stimulate morphogenesis of c. albicans.24,25 human serum, sheep serum, and fbs have approximately 1 mm to 10 mm of glucose.26– 28 this amount of glucose is optimal for germ tube formation according to previous study.24 moreover, combined with exposure of 37°c and neutral ph environment, serum could inhibit nrg1 transcription, a potent inhibitor for hyphal formation (su et al. 2018).29 this exposures to 37°c and neutral to alkaline ph, could induce hyphal growth through the cek1 mitogen-activated protein kinase pathway (mapk pathway) and the rim101-ph sensing pathway, respectively.21 studies by hilmioglu et al. (2007) found that human serum was superior with the highest number of positive germ tube.30 in concordance to previous data, present study found that human and sheep serum were capable of inducing the highest number and longest germ tube, respectively, compared to other 24 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license rivaldi ruby, et al. germ tube induction test media. this study also found that extended incubation period on serum led to an increasing number of hyphae. long incubation period causes deprivation of nutrient and energy which leads to more effective hyphal growth.31,32 utilization of human serum for germ tube induction has few drawbacks despite its superiority to other media. serum has to be fresh otherwise stored serum could decrease germ tube production.12 some serum could have biological inhibitor present in it. wich et al. (2021), found that human serum antibodies have the capability to inhibit adherence of c. albicans to epithelial cells.33 moreover, ding et al. (2014) stated that germ tube formation in rpmi 1640 medium was delayed in the initial stage of the culture (within 90 min) in the presence of serum, although the number of hyphae was gradually increased to normal after extension of incubation period (from 2h to 3h).31 presence of these biological inhibitor could cause inconsistent result in different batches of serum. lastly, serum preparation poses possible risk of biohazard.12 in this study, several standardized media were studied to find comparable alternative of serum. application of commercially available media also facilitates further culture and production of germ tube antigen for serological test. broth medium also have essential substances for the growth and morphogenesis of c. albicans. mhb, tsb, and bhib were tested in their ability to promote germ tube formation. as stated above, elevated temperature and neutral ph were the one main inducer for morphogenesis.29 moreover, all media contains nutritive compounds that are necessary for fungal growth. amino acids are one potent inducer present in the medium. it promotes yeast-tohyphal transition through the camp-pka pathway.21 in this study, mhb, tsb, and bhib contain amino acids which further facilitate hyphal formation.21,22 furthermore, in mhb, c. albicans showed the highest number of germ tube formation. one possible explanation is that it contained starch components with protective colloid roles against toxic compounds in the medium.34 bhib is the most nutritious medium with combination of brain and beef infusion (a total of 17.5 g/l), protease peptone (10 g/l), and dextrose (2 g/l) which provide carbon, nitrogen, amino acids, and other nutrients.34 however, c. albicans showed lowest number of germ tube in bhib. an exact explanation of this phenomenon is unknown. it seems that high nutrient provision is not an inducer of morphogenesis. according to mba et al. (2020), c. albicans exhibits metabolic flexibility and filamentous growth in the condition of nutrient starvation.34 it is difficult to identify which media facilitate better morphogenesis based on previous studies, since results were mostly conflicting. hilmioglu et al. (2007) showed that bhib surpassed tsb as morphogenesis inducer, while yakasiri et al. (2020) concluded that tsb exceeds mhb and bhib.30,35 different results could be attributed to different strain, media quality, and research conditions. interestingly, atalay et al. (2017) found that mha was the best media for germ tube induction compared to human serum.18 distinct factor affects the yeast-to-hyphal transition in agar media. villa et al. (2020) stated that c. albicans hyphal growth in agar is influenced by the embedded surroundings or conditions. this is achievable through the upregulation of czf1 transcription factor when the fungi are inoculated within the agar matrix.9 in present study, higher number of germ tube was found in mha compared to other agar media although the result was not significant. strength and limitation to our knowledge, this is the first study that compares several media by measuring the number and length of the germ tubes. however, this study has several limitations. lack of fungal strain prevents generalization of the result to other strain of c. albicans. 25 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 8–26 wider variety of culture medium could be used, since rpmi 1640 and yepd broth also showed potential in previous studies.12,31 conclusions this study showed that certain media in a specific environmental condition could facilitate hyphal growth that initially appears as germ tube formation. human serum is a strong inducer of morphogenesis. incubation of c. albicans in standardized medium such as mhb and tsb, coupled with 37°c environmental temperature and neutral ph, is also adequate to facilitate such phenomenon. those media are preferred to human serum because it is readily available, routinely used in daily microbiology laboratories, and it provides stable result between batches. acknowledgement we would like to thank linda, yasir, and riska, laboratory workers in the department of parasitology and department of microbiology, school of medicine and health sciences, atma jaya catholic university of indonesia for assisting all laboratory procedures in this study. etchical clearance ethical clearance was obtained from the atma jaya ethical committee with the number 01/06/kep-fkuaj/2020. funding this research was funded by the school of medicine and health sciences, atma jaya catholic university of indonesia. conflict of interest the authors declare that they have no conflict of interest. author contribution designed the study, collected and analyzed the data, and also prepared the manuscript: rr and eas. a scientific adviser in the field of mycology: ss and svk. all authors read and approved the final manuscript. references 1. bongomin f, gago s, oladele ro, denning dw. global and multi-national prevalence of fungal diseases—estimate precision. journal of fungi. 2017;3(4):57. 2. clancy cj, nguyen mh. diagnosing invasive candidiasis. journal of clinical microbiology. 2018;56(5):e01909-17. 3. loreto es, tondolo jsm. fungal infection. london, uk: intechopen; 2019. 4. lockhart sr. current epidemiology of candida infection. clin microbiol newsl. 2014;36(17):131–6. 5. yapar n. epidemiology and risk factors for invasive candidiasis. therapeutics and clinical risk management. 2014;10:95105. 6. henriques m, silva s. candida albicans virulence factors and its pathogenicity. microorganisms. 2021;9(4):704. 7. hage c, carmona e, epelbaum o, evans s, gabe l, haydour q et al. microbiological laboratory testing in the diagnosis of fungal infections in pulmonary and critical care practice. an official american thoracic society clinical practice guideline. american journal of respiratory and critical care medicine. 2019;200(5):535-550. 8. parra-sánchez m, zakariya-yousef breval i, castro méndez c, garcía-rey s, loza vazquez a, úbeda iglesias a, et al. candida albicans germ-tube antibody: evaluation of a new automatic assay for diagnosing invasive candidiasis in icu patients. mycopathologia. 2017;182(7– 8):645–52. 26 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license rivaldi ruby, et al. germ tube induction test 9. villa s, hamideh m, weinstock a, qasim m, hazbun t, sellam a et al. transcriptional control of hyphal morphogenesis in candida albicans. fems yeast research. 2020;20(1). 10. mayer fl, wilson d, hube b. candida albicans pathogenicity mechanisms. virulence. 2013;4:2 119-128. 11. pini p, colombari b, marchi e, castagnoli a, venturelli c, sarti m et al. performance of candida albicans germ tube antibodies (cagta) and its association with (1 → 3)-β-d-glucan (bdg) for diagnosis daof invasive candidiasis (ic). diagnostic microbiology and infectious disease. 2019;93(1):39-43. 12. mehta a, kumar m, bhumbla u, vyas a, dalal as. comparison of different media for germ tube production by candida albicans: a retrospective study. int j curr microbiol appl sci. 2018;7(6):819– 23. 13. wohlmeister d, vianna d, helfer v, calil l, buffon a, fuentefria a et al. differentiation of candida albicans , candida glabrata , and candida krusei by ft-ir and chemometrics by chromagar™ candida. journal of microbiological methods. 2017;141:121-125. 14. silva s, negri m, henriques m, oliveira r, williams d, azeredo j. candida glabrata, candida parapsilosisandcandida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance. fems microbiology reviews. 2012;36(2):288-305. 15. gómez-gaviria m, mora-montes hm. current aspects in the biology, pathogeny, and treatment of candida krusei, a neglected fungal pathogen. infect drug resist. 2020;13:16731689. published 2020 jun 10. doi:10.2147/idr.s247944 16. procedure for serum processing from whole blood. brd.nci.nih.gov.https://brd.nci.nih.gov/brd/sop/d ownload-pdf/148. published 2020. accessed january 28, 2021. 17. audreylia e, budiman y, surja ss. mentha piperita extract, a potential antifungal agent against candida albicans and candida krusei. curr res environ appl mycol. 2020;10(1):236–41. 18. atalay ma, koc an, parkan om, aydemir g, elmali f, sav h. can serums be replaced by mueller-hinton agar in germ tube test? niger j clin pract. 2017;20(1):61–3. 19. barbedo jga. automatic object counting in neubauer chambers. brazilian telecommunications. 2013. 20. tsui c, kong e, jabra-rizk m. pathogenesis of candida albicans biofilm. pathogens and disease. 2016;74(4). 21. chen h, zhou x, ren b, cheng l. the regulation of hyphae growth in candida albicans. virulence. 2020;11(1):337-348. 22. garbe e, vylkova s. role of amino acid metabolism in the virulence of human pathogenic fungi. current clinical microbiology reports. 2019;6(3):108-119. 23. burch j, mashayekh s, wykoff d, grimes c. bacterial derived carbohydrates bind cyr1 and trigger hyphal growth in candida albicans. acs infectious diseases. 2018;4(1):53-58. 24. hudson da, sciascia ql, sanders rj, norris ge, edwards pjb, sullivan pa, et al. identification of the dialysable serum inducer of germ-tube formation in candida albicans. microbiology. 2004;150(9):3041–9. 25. van ende m, wijnants s, van dijck p. sugar sensing and signaling in candida albicans and candida glabrata. frontiers in microbiology. 2019;10. 26. seo d, paek sh, oh s, seo s, paek sh. a human serum-based enzyme-free continuous glucose monitoring technique using a needle-type biolayer interference sensor. sensors (switzerland). 2016;16(10):1581. 27. al-hadithy ha, badawi nm. determination of serum proteins and glucose concentrations in clinically normal and anemic awassi sheep. world s veterinary journal. 2015;6(1):01. 28. branzoi i, iordoc m, branzoi f, vasilescu-mirea r, sbarcea g. influence of diamond-like carbon coating on the corrosion resistance of the nitinol shape memory alloy. surface and interface analysis. 2010;42(6-7):502-509. 29. su c, yu j, lu y. hyphal development in candida albicans from different cell states. current genetics. 2018;64(6):1239-1243. 30. hilmioglu s, ilkit m, badak z. comparison of 12 liquid media for germ tube production of candida albicans and c. tropicalis. mycoses. 2007;50(4):282– 5. 31. ding x, liu z, su j, yan d. human serum inhibits adhesion and biofilm formation in candida albicans. bmc microbiol. 2014;14(1):80. 32. mba i, nweze e. mechanism of candida pathogenesis: revisiting the vital drivers. european journal of clinical microbiology &amp; infectious diseases. 2020;39(10):1797-1819. 33. wich m, greim s, ferreira-gomes m, krüger t, kniemeyer o, brakhage a et al. functionality of the human antibody response to candida albicans. virulence. 2021;12(1):3137-3148. 34. becton dickinson and company. manual of microbiological culture media db (2nd edition). difco & bbl manual manual of microbiological culture media. 2009. 35. yakasiri hp, siddabathuni a. utility of nonserum liquid media against conventional human serum in germ tube production test. ip international journal of medical microbiology and tropical diseases. 2020;6(1):54-57. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 �� vol. 1. no. 1 january–april 2010 research report correlation between soluble urokinase plasminogen activator receptor with cd� t lymphocyte and who clinical staging of hiv infection shinta oktya wardhani, niniek burhan, gatoet ismanoe, and tri yudani mr tropical and infectious disease division, department of internal medicine, brawijaya university abstract the urokinase-type plasminogen activator (upa) and its receptor play a key role in pericellular proteolysis, cell migration and signal transduction. previous study showed that supar could be used as an independent prognostic marker of disease progression in hiv-1 patients.1,17 immune status of hiv patient and progressivity of disease are important parameters used as clinical concideration before initiating anti retroviral treatment and for monitoring treatment effectivity. recently immune status of hiv patients is determined by cd4 t lymphocyte counting which represents the remaining healthy lymphocyte t expressing cd4 that very expensive and need special laboratory equipment. destruction and shedding of t lymphocyte, macrophage and natural killer cell will deliver soluble urokinase plasminogen activator receptor, a surface protein which is expressed by those cells and can be measured by elisa8,9,11. this study objective is to determine correlation between supar plasma concentration and cd4 t lymphocyte and who clinical stagging of hiv infection. study subjects. fifty four naieve hiv-1-infected patients (32 males, and 22 females) are participant in a cross sectional study enrolled on 22 november 2007 until 31 july 2008 at the department of infectious disease saiful anwar hospital, malang, indonesia. blood sampling. two blood samples were drawn before treatment, cd4 counts were measured with an epics xl-mcl coulter flowcytometer. edta plasma for supar measurement was stored at -80°c. data are presented as mean±standart deviation. p<0.05 is considered significant. statistical calculations were done using ssps 15. patients (n = 54) enrolled and clustered according to who clinical stage ( i iv) at inclusion. all hiv-infected patients had measurable levels of plasma supar with a median value of 8,9 ng/ml(range 1,65-29,7 ng/ml). pearson correlation demonstrated a weak but significant negative between supar and cd4 t lymphocyte count (p=-0.634, p<.0005). supar level positively correlated with the who-defined clinical stages (p< .0005, spearman correlation test, r=0,87). there were significant difference between each stage i.e i(1,6± o,61ng/ml), ii(3.04±1.03 ng/ml), iii (10.53±7.1ng/ml) and iv (20.42±10.81ng/ml) (p< .0005, spearman test). in addition pearson correlation demonstrated a weak but significant negative correlation between supar and cd4 count (p=-0.66; p<.0005). there were negative significant correlationthere were negative significant correlation between cd4 count and supar level, suggested that supar could provide as a complementary biological marker for hiv-1 although it can not replace the cd4 count. supar plasma concentration and clinical stage give significantly correlation with who clinical staging of hiv infection. key words: supar, hiv, cd4 t lymphocyte, who clinical stage introduction hiv infection/aids is a global pandemic with cases reported from virtually every country. approximately 40.000 individuals are newly infected each day1. progression of hiv infection is largely dependent on the interaction between tha viral factors and host factors. hiv primarily infect cells which expressed cd4 receptor such as monocyte-macrophage, t lymphocyte, dendritic cell, langerhans and nk cell.1,2,3,4 it brings about the destruction of those cell through multiple mechanism including apoptosis.5,6,7,8,9 the loss of cd4 cell population ultimately leads to the inability of infected persons to deal with opportunistic organism.5,6 the hallmark of hiv/aids infection is to identify immunodeficiency status (stage), because these stage will predict the progression of the hiv infection and treatment response. imunodefficiency status may be measured through cd4 t lymphocyte count.3,4 immune activation in hiv infection is known to be linked positively to hiv��wardhani, et al.: correlation between soluble urokinase plasminogen activator receptor with cd4 t lymphocyte 1 replication and negatively to cd4 t-cell depletion.5,6,7,8 supar is a component of the plasminogen activation system, which comprises urokinase-type plasminogen activator (upa) and its receptor (upar).10 upar is expressed on a variety of different immune cells such as macrophage, t lymphocyte, nk cells, dendritic cell and langerhans cells.10,11 supar is generated by either proteolytic cleavage or shedding from cells.10,11 supar concentrations are increased and prognostic in a variety of inflammatory including hiv infection.10 the blood level of the soluble urokinase-type plasminogen activator receptor (supar) is increased in untreated human immunodeficiency virus-1 (hiv-1) infection and decreases in hiv-1-infected patients after initiation of highly active antiretroviral therapy (haart).12,13,14,15 the plasma concentration of soluble urokinase-type plasminogen activator receptor (supar, cd87) is a strong independent predictor of mortality in untreated patients with hiv-1 infection. 15 plasma concentrations of this immune marker can be quickly and inexpensively measured using a simple enzyme-linked immunosorbent assay (elisa), which requires much less sophisticated laboratory infrastructure than that needed for cd4 cell count or plasma viral load measurement. such an assay might therefore be potentially useful in resource-limited settings.15,16 this study try to determine correlation of supar plasma consentration with cd4 t lymphocyte to identify immunodeficiency state of hiv/aids infection based on who 2006 criteria. material and methods study subjects fifty four naieve hiv-1-infected patients (32 males, and 22 females) are participant in a cross sectional study enrolled on 22 november 2007 until 31 july 2008 at the department of infectious disease saiful anwar hospital, malang, indonesia. all patients enrolling in studies fulfill the inclusion criteria (provide written informed consent and this study was approved by the research ethics committee of the university of brawijaya; age between 15-50 years old, not pregnant and already confirmed diagnosed suffered from hiv infection). clinically, we grouiping all the participant based on who 2006 criteria. blood sampling two blood samples were drawn before treatment, cd4 counts were measuresed with an epics xl-mcl coulter flowcytometer.17 edta plasma for supar measurement was stored at -80°c. plasma supar concentrations were measured using a commercially available enzyme-linked immunosorbent assay (elisa) (suparnostic,™ virogates, lyngby, denmark) following the manufacturer’s instructions. this is a simple double monoclonal antibody sandwich assay that measures total supar, including both full-length and cleaved forms of the receptor. in brief, a standard control curve (range 0.6 – 19.3 ng/ml), positive control, and test samples were incubated in duplicates in a 96-well plate pre-coated with anti-supar antibody. following further incubation with a secondary peroxidase-conjugated antibody, the assay was developed by addition of a tetramethylbenzidine (tmb) chromogenic substrate. the reaction was terminated by addition of sulphuric acid and absorbance at 450 nm was determined using a microtitre plate reader. the linear standard curve was used to determine concentrations in positive control and test samples. samples with concentrations exceeding the highest standard (19.3 ng/ml) were reanalysed using a further 5-fold sample dilution.18 data analysis data were analysed using spss for windows release 15.0. as the frequency distribution of values table 1. patients baseline characteristic variable who clinical stagging pstage 1 (n=16) stage 2 (n=10) stege 3 (n=13) stage 4 (n=15) sex 10 % and 6 & 4 % and 6 & 9 % and 4 & 9 % and 6 & 0.841 age 30.13±6.97 30.50±6.70 29.62±5.22 29.67±5.31 0.983 total lymphocyte 1232.50±337.12 1628±674.68 1101.54±501.51 719.33±413.74 0.000 * hb 11.79±1.43 12.42±1.57 10.32±1.86 10.52±1.78 0.007 * albumin 3.84±0.85 3.71±0.95 3.37±0.73 2.82±0.92 0.011 * bmi(kg/m2) 20.32±2.23 18.67±1.67 17.56±1.84 16.45±1.75 0.000 * cd4 (sel/ul) 330.63±113.06 195.40±102.51 128.62±132.14 57.60±94.11 0.000 * supar(ng/dl) 1.65±0.61 3.04±1.03 10.53±7.13 20.42±10.81 0.000* source of infection • c o n t a m i n a t e d n e e d l e / d r u g abuse • sexual intercourse 10 6 4 6 8 5 8 7 0.801 hb = hemoglobin, bmi = body mass index, cd4 = cd4 t limfosit,, supar = soluble urokinase plasminogen activator receptor. �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 32-35 was highly right-skewed, the supar values were log10transformed for bivariate analyses (based on the mann whitney u or kruskall wallis tests to compare medians) correlation between supar consentration and clinical who staging assessed with spearman analysis and correlation between supar consentration and cd4 t lymphocyte count assessed with pearson analysis, significant if p<0,05.19,20 results patient baseline characteristics there were 54 patient full fill inclusion criteria enrolled in our study. these patients had a median age of 32 (59,3%) years, 32(59%) males dan 22 (41%)females (table 1). after assessed clinical who staging, there were 16(29%) patient stage i, 10(18%) stage ii, 13(24%) stage iii and 15(27%) stage iv. there were gradual cd4 t lymphocyte count depletion in every stage of who staging in our patient. cd4 t lymphocyte count 330.63±113.06 in stage i, 195.40±102.51 in stage ii, 128.62±132.14 in stage iii and 57.60±94.11 in stage iv. all patient showed decreased of bmi (body mass index) especially in stage iv. mean of bmi 20,32 ± 2,23 kg/m2 stage i, stage ii 18,67 ± 1,67 kg/m2, stage iii 17,56 ± 1,84 kg/m2 and stage iv 16,45 ± 1,75 kg/m2. most of patient infected from using contaminated needles for injecting drugs and sexuall intercourse. plasma supar concentrations detectable levels of supar were measured in plasma samples from all 54 patients. the standard curves for each run were linear (mean r2 = 0.995; sd = 0.004) and all positive control readings were consistent with the expected value. the mean supar concentration in the patient plasma samples was 1.65±0.61 ng/ml in stage i, 3.04±1.03 in stage ii, 10.53±7.13 in stage iii and 20.42±10.81 in stage iv. references 1. who/unaids, summary country profile for hiv/aids treatment scale up: indonesia june 2005. 2. hammer scott, management of newly diagnosed hiv infection, n enl j med, 353; 16; 2005. 3. musey luwy, james hughes, timothy schacker, theresa shea, lawrence corey, and juliana mc elrat, cytotoxic-t-cell responses, vial load and disease progession in early human immunodeficiency virus type 1 infection, n engl j med; 337: 18, 1997. 4. langford simone e, jintanat ananworanich and david a cooper, predictor of diesease progression in hiv infection: a review, aids research and therapy 2007, 4: 11. 5. fauci as, pantaleo g, stanley s. immunopathogenic mechanisms of hiv infection, ann intern med, 1996; 124: 654–653. 6. calles n.r, evans d, terlonge delouis, pathophysiology of the human immunodeficiency virus, weill medical college of cornell university. available at: http://edcenter.med.cornell.edu/cumc_ pathnotes/hiv_infection/hiv_infection_04. di akses september 2008 7. paranjape rs. immunopathogenesis of hiv infection. indian j med res 2005; 121: 240–55. 8. kilbi j.michael, eron joshep, novel therapies based on mechanism of hiv-1 cell entry, n engl j med 348; 22, 2003 9. nasronudin. the effect of hiv/aids infection diagnosis to tcd4 lymphocytes apoptosis mechanism in hiv/aids patients, psychoneuroimmunoligical approach. thesis. airlangga univ. surabaya: 2005. 10. blasi f, carmeliet p. upar: a versatile signalling orchestrator. nat. rev.mol.cell biol. 2002; 3: 750–54. 11. montuori nunzia, maria vincenza carriero, alvatore salzano, guido rossi, and pia ragno, the cleavage of tha urokinase receptor regulates its multiple functions, jbc.org, 2002. 12. murali rama, joshua h.wolfe, rebecca erber, seto m. chice, m.r.murali, helen g durkin, petr zach and dominick l.auci, altered levels of urokinase on monocytes and in serm of children with aids; effects on lymphocyte activation and surface marker expression, j.leukbio; 64, 1998. 13. ostrowski. s.r, t. l.katzenstein, g.hoyer-hansen, j.gerstoft, b.k.pedersen, h.ullum, plasma level of intact and cleaved urokinase receptor decrease in hiv-1-invected patients initiating highly active antiretroviral therapy, scandinav j immunol 2006; 63, 478–486. 14. sidenius n, sier cf, ullum h. serum level of soluble urokinase-type plasminogen activator receptor is a strong and independent predictor of survival in human immunodeficiency virus infection, blood 2000; 96: 4091–95. 15. lawn sd, myer l, bangani n, vogt m, wood r, plasma levels of soluble urokinase-type plasminogen activator receptor (supar) and early mortality risk among patients enrolling for antiretroviral treatment in south africa, bmc infect dis 2007; 7: 41. 16. schneider uffe, nielsen rl, pedersen court, olsen je, the prognosis value of the suparnostic tm elisa assay in hiv-1 infected individuals is not affected by upar promoter polymorphisms, bmc infectious disease 2007, 7: 134. 17. bd tritest cd3/cd4/cd4 reagent for flowcytometer equipped (bd catalog no.340385) 18. missionpharma, suparnostic elisa kit, www.missionpharma.com, 2007. 19. santoso,s. buku statistik non parametrik. jakarta:penerbit pt elex media komputindo, 2003. 20. dajan, a, pengantar metode statistik, jilid i, pustaka lp3es indonesia, jakarta, 1995. 21. kofoed kristian, ove andersen, gitte kronborg, mchae tvede, janne petersen, jasper eugen-olsen and klaus larsen, use of plasma creactive protein, procalcitonin, neutrophils, macrophage migration inhibitory factor, soluble urokinase-type plasminogen activator receptor, and soluble triggerring receptor expressed on myeloid cells-1 in combination to diagnose infections: a prospective study, critical care 2007, 11: r38. 22. mangione cm, gerberding jl, cummings sr. occupational exposure to hiv: frequency and rates of underreporting of percutaneous and mucocutaneous exposures by medical housestaff. am j med. 1991; 90: 85–90. 23. nasronudin. pencegahan penularan infeksi hiv dan aids melalui universal precaution. hiv & aids: pendekatan biologi molekuler, klinis dan sosial. airlangga university press, surabaya; 2007.airlangga university press, surabaya; 2007. 24. nasronudin. hiv & aids: intervensi hiv dan peran mitochondria. pendekatan biologi molekuler, klinis dan sosial. airlanggaairlangga university press, surabaya; 2007. 25. andersen ove, eugen-olsen, kofoed kristian, iversen johan, haugaard steen b; soluble urokinase plasminogen activator receptor is a marker of dysmetabolism in hiv-infected patients receiving highly active antiretroviral therapy. journal of medical virology, 2008. 26. friis hendrik, gomo exnevia, et al. hiv and other predictors of serum folate, serum ferritin and hemoglobin in pregnancy: a cross-sectional study in zimbabwe, am j clin nutr; 73: 2001. 27. djoba siawaya jf, ruhwald m, eugen-olsen j, walzl g, correlates for disease progression and prognosis during concurrent hiv/tb infection; int j infect dis. jul; 11(4): 289–99. 2007. 28. ditjen pp dan pl 2005, laporan triwulan pengidap infeksi hiv dan kasus aids sampai desember 2005, jakarta ditjend pp dan pi, depkes ri, 2005. ��wardhani, et al.: correlation between soluble urokinase plasminogen activator receptor with cd4 t lymphocyte 29. kpa 2003, strategi nasional penanggulangan infeksi hiv/aids 2003–2007. kementrian koordinator bidang kesejahteraan rakyat, komisi nasional penanggulangan aids, 2003. 30. ostrowski sr, katzenstein tl, piironen t, gerstoft j, pedersen bk, ullum h. soluble urokinase receptor levels in plasma during 5 years of highly active antiretroviral therapy in hiv-1 infected patients. j acquir immune defic syndr 2004; 35: 337–42. 31. ostrowski sr. the soluble urokinase receptor in inflammation-with focus on hiv-infection and malaria. ph.d.diss. copenhagen univ. denmark; 2004. 32. ostrowski. s.r, t. piironen, g.hoyer-hansen, j.gerstoft, b.k.pedersen, h.ullum, reduced release of intake and cleved urokinase receptor in stimulated whole-blood cultures from human immunodeficiency virus-1-infected patients, scandinav j immunol 2005; 61, 347–356. 33. eugen-olsen j, gustafson p, sidenius n, fischer tk, parner j, aaby p, gomes vf, lisse i, the serum level of soluble urokinase receptor is elevated in tuberculosis patients and predicts mortality during treatment: a community study from guinea-bissau, int j tuberc lung dis. aug; 6(8): 686–92, 2002. 34. kronborg, n. weis, h. nielsen, n. obel, s. s. pedersen and j. eugenolsen, the plasma level of soluble urokinase receptor is elevated in patients with streptococcus pneumoniae bacteraemia and predicts mortality, clin microbiol infect 2004; 10: 409–415. 35. ostergaard c, benfield t, lundgren jd, eugen-olsen j soluble urokinase receptor is elevated in cerebrospinal fluid from patients with purulent meningitis and is associated with fatal outcome; scand j infect dis. 36(1):14–9 2004. ijtid vol 1 no 1 jan-apr 2010.34.pdf ijtid vol 1 no 1 jan-apr 2010.35.pdf ijtid vol 1 no 1 jan-apr 2010.36.pdf ijtid vol 1 no 1 jan-apr 2010.37.pdf ijtid vol 6 no 4 jan-maret 2017.indd 84 vol. 6. no. 4 january–april 2017 research report antiviral activity of copper(ii)chloride dihydrate against dengue virus type-2 in vero cell teguh hari sucipto1,2a, siti churrotin1, harsasi setyawati3, tomohiro kotaki4, fahimah martak2, soegeng soegijanto1 1 dengue study group, institute of tropical disease, universitas airlangga, indonesia 2 departemen of chemistry, faculty of mathematic and natural science, sepuluh nopember institute of technology, indonesia 3 departemen of chemistry, faculty of science technology, universitas airlangga, indonesia 4 center for infectious disease, kobe university graduate school of medicine, japan a corresponding author: teguhharisucipto@gmail.com abstract infection of dengue virus (denv) was number of globally significant emerging pathogen. antiviral dengue therapies are importantly needed to control emerging dengue. dengue virus (denv) is mosquito-borne arboviruses responsible for causing acute systemic diseases and grievous health conditions in humans. to date, there is no clinically approved dengue vaccine or antiviral for humans, even though there have been great efforts towards this end. copper and copper compounds have more effective in inactivation viruses, likes an influenza virus and human immunodeficiency virus (hiv). purpose in this project was investigated of copper(ii) chloride dihydrate antiviral compound were further tested for inhibitory effect on the replication of denv-2 in cell culture. denv replication was measures by enzyme-linked immunosorbent assay (elisa) with selectivity index value (si) was determined as the ratio of cytotoxic concentration 50 (cc50) to inhibitory concentration 50 (ic50) for compound. the maximal inhibitory concentration (ic50) of copper(ii)chloride dihydrate against dengue virus type-2 was 0.13 μg/ml. the cytotoxic concentration (cc50) of compound against vero cell was 5.03 μg/ml. the si values for copper(ii)chloride dihydrate 38.69. result of this study suggest that copper(ii) chloride dihydrate demonstated significant anti-denv-2 inhibitory activities and not toxic in the vero cells. copper mechanisms play an important role in the prevention of copper toxicity, exposure to excessive levels of copper can result in a number of adverse health effects, as a result increased reactive oxygen species and oxidative damage to lipid, dna, and proteins have been observed in human cell culture models or clinical syndromes of severe copper deficiency and inhibition was attributed to released cupric ions which react with cysteine residues on the surface of the protease. keywords: antiviral, dengue virus type-2, copper(ii)chloride dihydrate, inhibitory, cytotoxity abstrak infeksi virus dengue (denv) adalah patogen yang muncul secara global. terapi antivirus dengue penting diperlukan untuk mengontrol muncul dengue. dengue virus (denv) disebabkan oleh mosquito-borne arboviruses yang menyebabkan penyakit sistemik akut dan kondisi kesehatan pada manusia. sampai saat ini, tidak ada vaksin dengue klinis disetujui atau antivirus bagi manusia, meskipun telah ada upaya besar menjelang akhir ini. tembaga dan senyawa tembaga memiliki efektivitas dalam inaktivasi virus, seperti virus influenza dan human immunodeficiency virus (hiv). tujuan dalam proyek ini adalah menyelidiki senyawa antiviral copper (ii) klorida dihidrat yang kemudian diuji lebih lanjut untuk efek penghambatan pada replikasi denv-2 dalam kultur sel. replikasi denv diukur dengan enzyme-linked immunosorbent assay (elisa) dengan nilai indeks selektivitas (si) ditentukan sebagai rasio konsentrasi toksisitas 50 (cc50) ke konsentrasi penghambatan 50 (ic50) senyawa. maksimal konsentrasi penghambatan (ic50) tembaga(ii)klorida dihidrat terhadap virus dengue tipe 2 adalah 0,13 μg/ml. konsentrasi toksisitas (cc50) senyawa terhadap sel vero adalah 5.03 μg/ml. si nilai untuk tembaga(ii)klorida dihidrat 38.69. hasil penelitian ini menunjukkan bahwa tembaga(ii) klorida dihidrat signifikan anti-denv-2 dan tidak toksik dengan sel vero. mekanisme tembaga berperan penting dalam pencegahan toksisitas tembaga, paparan kadar tembaga yang berlebihan dapat mengakibatkan sejumlah efek kesehatan yang merugikan, akibatnya 85sucipto, et al.: antiviral activity of copper(ii)chloride dihydrate peningkatan spesies oksigen reaktif dan kerusakan oksidatif pada lipid, dna, dan protein telah diamati pada model kultur sel manusia atau sindrom klinis defisiensi tembaga berat dan penghambatan dikaitkan dengan ion cuprik yang dikeluarkan yang bereaksi dengan residu sistein pada permukaan protease. kata kunci: antivirus, virus dengue tipe-2, tembaga(ii)klorida dihidrat, penghambatan, toksisitas introduction infection of dengue virus (denv) was number of globally significant emerging pathogen. it is member of flaviviridae family, with the genus flavivirus. denvs were distributed in the tropical and sub-tropical areas and transmitted to humans by aedes agypty and aedes albopictus.1 dengue virus (denv) is mosquito-borne arboviruses responsible for causing acute systemic diseases and grievous health conditions in humans. more than 2.5 billion cases of dengue infection occurred in the worldwide.2 indonesia is one of the largest counties in the dengue endemic region worldwide. dengue was occurred for the first time as an outbreak in surabaya and jakarta in 1968.3 to date there are not effective vaccine and antiviral treatment for denv, patient supportively-treated without any specific treatment measures.4 antiviral dengue therapies are importantly needed to control emerging dengue. effective antiviral therapies, currently unavailable for any type of denv, are urgently needed to ameliorate the disease burden by denv.5 ribavirin has shown activity against all flaviviruses tested in a broad array of cell types in vitro but efficacy in vivo has generally been poor, ribavirin can be toxic in vivo.6 a compound that exhibited a lower effective dose and toxicity than ribavirin while retaining its broad spectrum of activity would be particularly desirable as a candidate flavivirus therapy.5 copper and copper compounds have been used as important antiviral material.7 recently, group found that cu+ species in the related compounds is much more effective in inactivation of bacterial and viruses than copper metal and copper(ii) compounds.8 on the other hand, copper has long been used as an antibacterial material,9 and several copper compounds have been reported to exhibit viral inactivation. more recently, the inactivation of avian influenza virus by copper metal10 and divalent ions (cu2+)11 and the inactivation of human immunodeficiency virus (hiv) by copper ions12 and copper oxide have been reported.13 copper iodide nanoparticle against for feline calicivirus (fcv) was demonstrated that the antiviral behaviors of cui nanoparticles against fcv were identified to detect cu+ ions, hydroxyl radicals, and capsid protein oxidation. copper iodide nanoparticles showed high antiviral activity against fcv was attributed to cu+ ions, followed by ros (o2• or •oh) generation and subsequent capsid protein oxidation.14 the antivirus properties of the cufeo2 crystals achieved about 8 log inactivation of the phage after 4 h of contact time in the dark, cufeo2 are good chemical stability in a weak acid condition.7 copper is a bio-essential element and copper complexes have been extensively utilized in metal mediated dna cleavage for generation of activated oxygen species, was reported that teraaza macrocyclic copper coordination compounds have anti-hiv activity. macrocyclic complexes can react with dna in different binding fashions and axhibit effective nucleus activities.15 copper monodispersed nanoparticles (2-5 nm) in submicron particles of sepiolite, structure of sepiolite is mg8si12o30(oh)4(h2o)4.8h2o, have revealed as a strong bactericide so that they were able to decrease the starting microorganism concentrations of staphylococcus aureus or escherichia coli by 99.9%.16 the antibacterial stainless steels included a copper-bearing austenitic antibacterial showed excellent with antibacterial rate to e.coli over 99.99%, copper ions play the dominant role in the antibacterial effect of antibacterial stainless steels acted with e. coli.17 previous result, ribavirin exerts its toxicity through an inhibiton of intracellular energy metabolism and axidative membrane damage, leading to an accelerated extravascular hemolysis by the reticulo-endothelial. but not significant inhibiton of level, ribavirin was more to toxic to replicating cells than to stationary cell monolayers in vero cells.18 currently, there is no published data on the possible antidenv activities of copper and copper compounds. in the present study, we investigated of copper(ii)chloride dihydrate antiviral compound were futher tested for inhibitory effect on the replication of denv-2 in cell culture. material and method material chemical reagents used in this research is the copper(ii) chloride dihydrate (cucl2.2h2o) (merck 99.0%), dimethyl sulfoxide (dmso) (merck 99.98%), minimum essential medium eagle (mem media) (sigma-aldrich), denv-2 surabaya isolate, vero cell (african green monkey kidney), cell proliferation reagent wst-1 (roche applied science), and dengue virus antibody (4g2) for elisa. method antiviral activity assay confluent monolayers of vero cells were prepared in 96 wells cell culture microplate. the numbers of denv-2 were counted using a hemocytometer and the titer of virus was expressed as foci-forming-unit (ffu). seed vero cells in a 96-well plate (1x106 cells/10 ml), add serially diluted 86 indonesian journal of tropical and infectious disease, vol. 6. no. 4 january–april 2017: 84–87 6-azauridine was 1.5 μg/ml with si of wnv new york isolate. this result confirm by virus yield reduction assay when the assay when the assay was performed 2 days after initial infection in vero cells.21 copper homeostatic mechanisms play an important role in the prevention of copper toxicity, exposure to excessive levels of copper can result in a number of adverse health effects. similar to cu toxicity, cu deficiency also affects, directly or indirectly, the components of the oxidant defense system and as a result increased reactive oxygen species and oxidative damage to lipid, dna, and proteins have been observed in human cell culture models or clinical syndromes of severe copper deficiency.22 in these cases, the observed inhibition was attributed to released cupric ions which react with cysteine residues on the surface of the protease.23 maximal inhibitory concentration (ic50) of quercetin against denv-2 was 35.7 μg/ml when it was used after virus absorption to the cells and decreased to 28.9 μg/ml when the cells were treated continuously for 5 h before virus infection and up to 4 days post-infection. a weak effect for prophylactic activity of quercetin however. these findings suggest that the main anti-dengue activity of quercetin is likely due to its activity against the different stages of its replication of denv-2 instead of early stages of intracellular replication cycle such as virus attachment or entry.4 cytotoxicity of copper(ii)chloride dihydrate the cytotoxicity study was carried out for compound of copper(ii)chloride dihydrate. this extract was screened for its cytotoxicity against vero cells at different concentrations to determine the cc50 by wst-1 assay. the percentage growth cytotoxicity was found to be increasing with increasing concentration of test compound, and that show in figure 4. copper(ii)chloride dihydrate effect on vero cells (cc50) up to 5.03 μg/ml and r 2 value was 0.9174. in this work, we have examined the relationship between the concentration in the culture medium of vero cells and the cytotoxic potency of copper(ii)chloride dihydrate. test compounds to vero cells, add denv-2 solution (2x104 ffu/well) and incubate 37°c for 2 days. the percentage of inhibition concentration (ic50) compared with controls was calculated as follows: ic50 (%) = (nc-ac) x 100/ nc. where, nc is the mean of the number for negative control and ac is the number absorbance of compound. inhibition of compound to denv-2 was further verified using quantitative enzyme-linked immunosorbent assay (elisa). cytotoxicity assay cytotoxicity used wst-1 cell proliferation reagent by roche applied science, mannheim, germany.19 the dye of wst-1 reagent has a larger linear range and increased stability compared to other tetrazolium salt based assays. the wst-1 assay is suitable for use with adherent and suspension cells. the assay is very sensitive, it can detect 500 to 50,000 cells in a single well of a 96-well plate. vero cells (1x105 cells/ml) were seeded in 96-well plate at 37 °c in 5% co2 overnight. a total of 100 μl of serial delusion compound were incubated with vero cells for 24 h. a total of 10 μl of cell proliferation reagent wst-1 was added into each well, incubated for 1 hour at 37 °c. the plate was read at 450 nm (main filter) and 655 nm (reference filter) using an elisa reader (imarktm microplate absorbance reader). result and disscussion inhibitory effect of copper(ii)chloride dihydrate copper(ii)chloride dihydrate were futher studied for their inhibitory effect on replication of the denv-2 in vero cells. the ic50 (inhibitory concentration 50) was determined from the dose response curves. this compound proved to be effective as inhibitor of replication of denv-2, the ic50 value was 0.13 μg/ml and r 2 value was 0.9812 with selectivity indices (si) was 38.69. the selectivity indices of these antiviral compounds appeared to be moderately influenced by the strain of denv tested. the mechanisms of how copper(ii)chloride dihydrate exerts it is anti denv-2 effects are not known. however, the effects of other compounds against cellular rna polymerases and formation of the complex with rna have been reported suggesting that copper(ii)chloride dihydrate could also affect the similar replication enzymes. viral replication was inhibited during a simultaneous treatment assay, indicating that the entry of the virus was impeded by peptide. previous research was reported protease inhibitory activity on denv2v ns3 target, palmatine has active concentration 26.4 μm, this compound was subsequently analyzed for antiviral activity in cell-based replication assays in cell culture.20 the neutral red assay mean ec50 of ribavirin was only 106 μg/ml with si of 9.4 against west nile virus (wnv) new york isolate and ec50 of y = -4.4057x + 50.588 r² = 0.9812 0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 cu concentration (μg/ml) cu % inhibition linear (cu % inhibition) figure 1. inhibitory chart of copper(ii)chloride dihydrate for vero cells by elisa 87sucipto, et al.: antiviral activity of copper(ii)chloride dihydrate conclusion as the conclusion, the study demonstrated that the copper(ii)chloride dihydrate exhibited significant anti denv-2 replication properties. these results suggest that these copper(ii)chloride dihydrate could be further investigated ic50 was 0.13 μg/ml, cc50 was 5.03 μg/ml, and si was 38.69. acknowledgment this work was supported by the joined program of the japan initiative for global research network on infectious disease (j-grid); research grant mandat universitas airlangga (hrmua); institute of tropical disease (itd) the center of excellence (coe) program by the ministry of research and technology (ristek) indonesia; chemistry department of universitas airlangga; and chemistry department of sepuluh nopember institute of technology. references 1. halstead sb. dengue virus-mosquito interactions. annu rev entomol. 2008 jan;53(1):273–91. 2. guzman mg, harris e. dengue. lancet. 2015;385(9966):453–65. 3. sumarmo. dengue haemorrhagic fever in indonesia. southeast asian j trop med public health. 1987 sep;18(3):269–74. 4. zandi k, teoh b-t, sam s-s, wong p-f, mustafa m, abubakar s. antiviral activity of four types of bioflavonoid against dengue virus type-2. virol j. 2011;8(1):2–11. 5. sampath a, padmanabhan r. molecular targets for flavivirus drug discovery. antiviral res. 2009 jan;81(1):6–15. 6. russmann s, grattagliano i, portincasa p, palmieri vo, palasciano g. ribavirin-induced anemia: mechanisms, risk factors and related targets for future research. curr med chem. 2006;13(27):3351–7. 7. qiu x, liu m, sunada k, miyauchi m, hashimoto k. a facile onestep hydrothermal synthesis of rhombohedral cufeo2 crystals with antivirus property. chem commun. 2012;48(59):7365–7. 8. qiu x, miyauchi m, sunada k, minoshima m, liu m, lu y, et al. hybrid cu x o/tio 2 nanocomposites as risk-reduction materials in indoor environments. acs nano. 2012 feb 28;6(2):1609–18. 9. borkow g, gabbay j. copper as a biocidal tool. curr med chem. 2005;12(18):2163–75. 10. noyce jo, michels h, keevil cw. inactivation of influenza a virus on copper versus stainless steel surfaces. appl environ microbiol. 2007 apr 15;73(8):2748–50. 11. horie m, ogawa h, yoshida y, yamada k, hara a, ozawa k, et al. inactivation and morphological changes of avian influenza virus by copper ions. arch virol. 2008;153(8):1467–72. 12. sagripanti j-l, lightfoote mm. cupric and ferric ions inactivate hiv. aids res hum retroviruses. 1996 mar;12(4):333–6. 13. borkow g, lara hh, covington cy, nyamathi a, gabbay j. deactivation of human immunodeficiency virus type 1 in medium by copper oxide-containing filters. antimicrob agents chemother. 2008 feb 1;52(2):518–25. 14. shionoiri n, sato t, fujimori y, nakayama t, nemoto m, matsunaga t, et al. investigation of the antiviral properties of copper iodide nanoparticles against feline calicivirus. j biosci bioeng. 2012 may;113(5):580–6. 15. sucipto th, martak f. synthesis of metal-organic (complexes) compounds copper(ii)-imidazole for antiviral hiv candidate. indones j trop infect dis. 2016 jan 18;6(1):5–11. 16. esteban-cubillo a, pecharromán c, aguilar e, santarén j, moya js. antibacterial activity of copper monodispersed nanoparticles into sepiolite. j mater sci. 2006 aug 29;41(16):5208–12. 17. nan l, yang w, liu y, xu h, li y. antibacterial mechanism of copper-bearing antibacterial stainless steel against e. coli. j mater {…}. 2008;24(2):197–201. 18. smee df, bray m, huggins jw. antiviral activity and mode of action studies of ribavirin and mycophenolic acid against orthopoxviruses in vitro. antivir chem chemother. 2001 nov;12(6):327–35. 19. chew m-f, tham h-w, rajik m, sharifah sh. anti-dengue virus serotype 2 activity and mode of action of a novel peptide. j appl microbiol. 2015 oct;119(4):1170–80. 20. tomlinson sm, malmstrom rd, russo a, mueller n, pang y-p, watowich sj. structure-based discovery of dengue virus protease inhibitors. antiviral res. 2009 jun;82(3):110–4. 21. morrey jd, smee df, sidwell rw, tseng c. identification of active antiviral compounds against a new york isolate of west nile virus. antiviral res. 2002 jul;55(1):107–16. 22. iakovidis i, delimaris i, piperakis sm. copper and its complexes in medicine: a biochemical approach. mol biol int. 2011;2011:1– 13. 23. lebon f, boggetto n, ledecq m, durant f, benatallah z, sicsic s, et al. metal-organic compounds: a new approach for drug discovery. n1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide copper(ii) complex as an inhibitor of human immunodeficiency virus 1 protease. biochem pharmacol. 2002 may 15;63(10):1863–73. y = -14.184x + 121.4 r² = 0.9174 0 20 40 60 80 100 120 1 2 3 4 5 6 concentrati on (μg/ml) cytotoxicit y (%) linear (cytotoxicit y (%)) figure 2. cytotoxicity chart of copper(ii)chloride dihydrate for vero cells by wst-1 assay ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 2 may–august 2022 original article analysis of covid-19 surveillance system at makassar city health office 2020 fatmasari1 , fariani syahrul1* , zakiah darajat2, eva flourentina kusuma3 1epidemiology division, faculty of public health, universitas airlangga, surabaya, indonesia 2makassar city health office, makasar, indonesia 3surabaya city health office, surabaya, indonesia received: august 24th, 2021; revised: december, 17th 2021; accepted: june 21st, 2022 abstract one of the infectious diseases that emerged in indonesia in 2020 has been designated as a covid-19 pandemic since march 11, 2020, and until now, the pandemic has not been completed. surveillance has a role in providing information on targeted disease control activities; analyzed the covid-19 surveillance system based on the current system approach at the makassar city health office. methods this research is a descriptive observational study conducted in september-october 2020. data collection was carried out using in-depth interviews with people who were key informants of covid-19 surveillance activities. there are four informants in this study. in addition, secondary data was obtained from the p2p field regarding covid-19 cases. in general, the input component has not been fulfilled; hr has multiple tasks, the job desk is irregular, and several important forms are not used in the methods section. the process component has been running but has not been maximized because there are still incomplete data, no reports based on the pe form, the all-record tc-19 information system has not been used, and data analysis is still incomplete, data analysis is not equipped with data interpretation. in the output component, the success rate for public health surveillance criteria has not been evaluated, and the dissemination of information has been carried out well across sectors. the implementation of covid-19 surveillance at the makassar city health office has been carried out well, but some things are still not optimal. keywords: covid-19; input; output; proses; surveillance abstrak salah satu penyakit menular yang muncul di indonesia pada tahun 2020 telah ditetapkan sebagai pandemi covid-19, sejak tanggal 11 maret 2020 dan sampai saat ini pandemi belum selesai. surveilans memiliki peranaan untuk memberikan informasi terhadap kegiatan pemberatasan penyakit tujuan ; melakukan analisis sistem surveilans covid-19, berdasarkan pendekatan sistem yang sedang berjalan di dinas kesehatan kota makassar. metode penelitian ini merupakan penelitian deskriptif observasional yang dilakukan pada bulan september-oktober 2020. pengumpulan data dilakukan dengan cara indepth interview kepada orang yang sebagai informan kunci dari kegiatan surveilans covid-19. informan dalam penelitian ini ada 4 orang. data sekunder diperoleh dari bidang p2p tentang kasus covid-19. pada komponen input secara umum belum terpenuhi; sdm memiliki tugas rangkap, jobdesk yang tidak teratur, pada bagian metode terdapat beberapa formulir penting yang tidak digunakan. pada komponen proses sudah berjalan namun belum maksimal karena masih ada data yang kurang lengkap, tidak ada laporan berdasarkan form pe,sistem informasi allrecord tc19 belum digunakan, analisis data masih ada yang kurang lengkap, analisis data belum dilengkapi dengan interpretasi data. pada komponen output belum dievaluasinya angka keberhasilan kriteria surveilans kesehatan masyarakat dan penyebaran informasi telah dilakukan dengan baik dengan *corresponding author: fariani.s@fkm.unair.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-0890-3170 https://orcid.org/0000-0002-8100-215x https://orcid.org/0000-0001-7111-1742 84 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fatmasari, et al. analysis of covid-19 surveillance system lintas sector. dalam pelaksanaan surveilans covid-19 di dinas kesehatan kota makassar telah terlaksana dengan baik, namun masih terdapat beberapa hal yang belum maksimal. kata kunci: covid-19; input; output; proses; surveilans how to cite: fatmasari., syahrul, f., darajat, z., kusuma, e.f. analysis of covid-19 surveillance system at health department of makassar 2020. indonesian journal of tropical and infectious disease. 10(2). 83–92. aug. 2022. introduction coronavirus disease 2019 is one of the infectious diseases that coincide in indonesia and other countries.1 based on research conducted by the cdc, china found that the cause of pneumonia in this group of patients was a new species of coronavirus, namely sars cov 2.2 the coronavirus is spherical with a diameter of about 125mm as depicted in a study using cryo-electron microscopy.3 covid-19 spreads through droplets released by an infected person and is symptomatic/symptomatic through coughing or sneezing. in addition to symptomatic people, this virus can also be transmitted to asymptomatic people. cases of transmission from symptomatic hosts are generally because the host has a history of contact with positive covid-19 people.4 the cdc says that all people who have been in close contact with someone with covid-19 should be quarantined for 14 days after their last contact with that person unless they meet the requirements.5 around 80% of cases of covid-19 are mild and moderate symptoms, 13.8% are seriously ill, and 6.1% are critical cases. the percentage of subjects with no symptoms can not be known. the typical clinical symptoms of this patient are fever, dry cough, difficulty breathing, headache, and pneumonia. other symptoms that can be found are productive cough, shortness of breath, sore throat, headache, chills, nausea/vomiting, diarrhea, abdominal pain, hemoptysis, and conjunctival congestion.6 coronavirus disease 2019 (covid-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (sarscov-2). on december 31, 2019, the who china country office reported a case of pneumonia of unknown etiology in wuhan city, china. on january 7, 2020, china identified the case as a new type of coronavirus. on january 30, 2020, who designated the incident as a public health emergency of international concern (pheic). on march 11, 2020, who declared covid-19 a pandemic. indonesia reported its first case on march 2, 2020. cases continued to increase and spread rapidly throughout indonesia. as of october 16, 2020, the ministry of health reported 353,461 confirmed cases with 12,347 deaths.7 the high inflow of tourists from abroad has caused indonesia to sulawesi, particularly vulnerable to the spread of transnational diseases such as covid-19. south sulawesi is one of indonesia's provinces with a relatively high number of covid-19 cases as of october 16, 2020, with 17,286 confirmed cases with 442 deaths.8 makassar is one of the big cities in south sulawesi and has the most significant number of covid-19 cases. based on the makassar city health service report on october 17, 2020, 9,098 cumulative positive covid-19 with 282 positive cases died. makassar city has 16 sub-districts. the highest covid-19 cases occurred in the rappocini sub-district, with 1,153 cases. the distribution of positive cases of covid-19 by age group can be seen that the highest number of positive cases of covid19 occurred in the age group of 31-40 years, with 2303 cases, and the least number of positive cases of covid-19 occurred in the age group > 80 years which is 28 cases.9 maintenance of efforts prevention and control of infectious and non-infectious data support is required information through an epidemiological surveillance system disease routinely and integrated as part of the health 85 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 83–92 epidemiological surveillance system.10 surveillance is in the form of continuous and systematic observation for the sake of prevention.11 the morbidity and mortality rates due to covid-19 are increasing. therefore, prevention and control efforts are needed, one of which is epidemiological surveillance.12 surveillance is crucial to provide information on disease control activities. the concept adopted by surveillance is the principle of epidemiology in identifying diseases based on variables of person, place, and time.13 surveillance is an essential component of the health system in producing epidemiological information for health services so that diseases and risk factors can be detected early and effective and efficient responses can be made to health services. surveillance activities are an inseparable part of quarantine. during the quarantine period, surveillance is carried out to monitor changes in the condition of a person or group of people.14 surveillance also enables decision-makers to lead and manage effectively. public health surveillance provides decision-makers and managers with early warning information about health problems that need attention in a population. the performance of the health epidemiological surveillance system is measured by input, process, and output indicators. the three indicators are one unit, where the weakness of one of these indicators indicates the performance of the surveillance system is not yet adequate. evaluation is an essential tool for policymakers that help to improve the performance and productivity of health programs.15 evaluation surveillance system showed that the system was overall effective in estimating morbidity and mortality and monitoring the disease trend.16 the rationale for evaluating public health surveillance systems is to determine if the disease is being observed efficiently and effectively. every surveillance system should be evaluated periodically with recommendations to improve the surveillance system's usefulness, quality, and efficiency.17 all surveillance components such as collection, processing, analysis, and interpretation of data, follow-up, and feedback must be carried out in a systematic and organized manner.18 sensitive surveillance in detecting disease trends and being active in finding cases of covid-19 is very important in efforts to handle and monitor close contacts and people at risk.19 the covid-19 problem requires adequate and comprehensive control efforts. these efforts must be supported by providing precise and accurate data and information systematically and continuously through a good surveillance system.20 the results of surveillance activities will be used as input to reduce morbidity and mortality and improve health status.21 in general, the goal is to describe the covid-19 surveillance system based on a system approach (input, process, output) to analyze the existing health condition so that priority problems can be determined at the makassar city health office in 2020. the specific objectives to be achieved include: obtain an overview of the regional situation at the makassar city health office; get an overview of the covid-19 surveillance system at the makassar city health office; obtain an overview of the implementation of the covid-19 surveillance program at the makassar city health office based on a systems approach; studying the problems of the covid-19 surveillance program related to identifiable health problems and the quality of the data collected. determining priorities for the covid-19 surveillance program problem at the makassar city health office. and planning an alternative solution to the covid-19 surveillance program at the makassar city health office. materials and methods this research design uses a descriptive observational design that aims to describe the 86 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fatmasari, et al. analysis of covid-19 surveillance system activities of the covid-19 surveillance program at the makassar city health office through problem identification and represents the priority of the problem determined based on the method used and alternative problemsolving. the location of this research is at the makassar city health office, which will be held from 26 september to 11 october 2020. data collection was carried out using indepth interviews with the holders of the covid19 surveillance program, the head of the surveillance & immunization section, and employees involved in covid-19 activities at the makassar city health office, which was carried out on several people who were considered key informants of the covid-19 surveillance activity.22 four informants in the activity consisted of 1 coordinator of the covid-19 surveillance program, namely the head of the surveillance and immunization section and staff who joined the covid-19 surveillance program, and one student. the technique of determining the priority of the problem in this study is to use the carl method. the carl method is c is capability (availability of resources), a is accessibility (easiness), r is readiness (readiness of implementing personnel and target readiness), and l is leverage (how much influence one criterion has on another in problem-solving). this method aims to determine the problems that will be prioritized from the results of problem identification.23 the presentation of data in this activity report is in the form of tables, graphs, and images which are then analyzed using a straightforward narrative. results and discussion overview of the makassar city health office situation makassar city health office vision “healthy and comfortable makassar for all towards a world city.” and makassar city health service mission: improving quality and affordable health services based on technology. improving public health and community empowerment. ensure public health through the health insurance system. and creating a healthy environment. overview of the covid-19 surveillance system at the makassar city health office (the flow of reporting and feedback on covid-19 case management at the makassar city health office). the mechanism for reporting and feedback on the covid-19 surveillance program as shown in figure 1. figure 1. covid-19 surveillance reporting & feedback mechanism9 the importance of covid-19 surveillance the high number of covid-19 cases in makassar city has caused the local government to act to handle this case so that it does not continue to increase. however, the handling of this covid-19 case needs intervention. adequate epidemiological information is information that can provide an overview of the situation regarding covid-19 in makassar city, which includes variables of people, place, and time as well as risk factors that increase the occurrence of covid-19. therefore, the makassar city health office conducted covid-19 surveillance to obtain adequate epidemiological information. the implementation of covidmakassar city health office laboratory south sulawesi provincial health office indonesian ministry of health makassar city health office 87 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 83–92 19 surveillance at the makassar city health office is essential because it functions to evaluate programs and make recommendations so that the handling of covid19 cases can be right on target. overview of the implementation of the covid-19 surveillance system at the makassar city health office input the input components in the covid-19 surveillance system include: a. human resources (hr) human resources in covid-19 surveillance at the makassar city health office consist of 8 people from the disease prevention and control, including one program coordinator, namely the head of the surveillance and immunization section, assisted by several staff and contract workers. the latest educational background includes d3 (nursing), s1 (doctor and bachelor of public health, and master of public health). meanwhile, human resources for covid-19 surveillance activities at the health facilities level are doctors, nurses, midwives, and public health. b. funding the current source of funds for covid-19 surveillance comes from the makassar city apbd through the unexpected cost for covid-19 control and comes from the apbn. c. means and types of data 1. source and type of data the source of data on covid-19 surveillance activities comes from the results of laboratory examinations reported to the makassar city health office using form 7 (form pdp covid-19 odp covid-19 research and development center for biomedical and basic health technology, health research and development agency). the data collected included the identity of the specimen sender, patient identity, treatment history, signs and symptoms, date of onset, specimen collection, contact/exposure history, and comorbid diseases. in addition, the types of data collected are suspect case data, confirmation, close contact, death, pcr examination, serological surveillance (rapid test, rapid reactive test, rtpcr, and rtpcr +), isolation/quarantine. as well as confirmed, suspected, and probable cases. 2. office stationery, computer equipment, and internet network. facilities and infrastructure for covid-19 surveillance activities are adequate. the facilities and infrastructure for surveillance activities at the district/city level are insufficient because the availability of manual data collection forms is not yet fully complete, which is used only from form 7 (covid-19 examination application form using tcm/covid form 5). while form 3 (covid-19 case finding notification report at health facilities), form 6 (covid-19 epidemiological investigation form) is not used correctly, form 4 (covid-19 aggregate daily report) is filled in excel form but has not been consistent in filling it out. the office stationery is complete. there are several computer devices, but they also use personal laptops and printers to support the covid-19 surveillance program. d. methods the current covid-19 surveillance activities are based on the covid-19 prevention and control guidelines of the ministry of health of the republic of indonesia in july 2020.24 all policies are available at the makassar city health office starting with the revision guidelines i-v. based on interviews, the guidelines for the prevention and control of covid-19 are revised too often, so there are obstacles in the revised guideline 5 because there is no mention of swab control for those who have confirmed covid-19. at the same 88 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fatmasari, et al. analysis of covid-19 surveillance system time, many people want swab control to ensure there is no virus in the body.25 improving the quality of recording and reporting of covid-19 data must follow the form or attachment in the revision 5 guideline, but based on direct observation during field work practice at the makassar city health office still using the old form and not up to date based on the attachment of the latest form in the guide guidelines for the prevention and control of covid-19 revision 5. e. market (information dissemination) dissemination of information on the results of the covid-19 surveillance implementation at the makassar city health office is actively carried out to the provincial health office. as a result, many agencies or fields require or request the results of covid-19 surveillance, starting from the district level, bppa, bappeda, bpbd, tni/polri, and the media. the information needed or requested by the agency or field is data on patients confirmed to be covid-19. process a. data collection the covid-19 confirmed data collection activity came from the health service examination, sending specimens to the laboratory for analysis. a few days later, laboratory results from the central health laboratory and the unhas laboratory were submitted to the health office for further processing and presented to each public health center for tracing and epidemiological investigation (ei). based on interviews, data collection activities are carried out every day if there is a confirmed covid19, but it is not recorded using the attachment form 3 in accordance with the guidelines. and the ei form was also not used because the officers were lazy to fill out the form, so there was less information about the patient’s close contact, and the ei form was taken over by the provincial health office. b. data compilation data compilation is done by using a computer/ laptop. based on interviews and document studies, the data at the makassar city health office has been grouped according to the variables of the person (gender, age), place and time, and daily data on covid-19 cases. c. data analysis data analysis is used to determine the success of controlling covid-19 at the regency/city/provincial level following the indicators defined by the ministry of health. the results of the analysis at the makassar city health office as shown in figure 2. figure 2. distribution of data on the accumulation of suspected covid-19 cases9 d. interpretation based on the results of interviews, the covid-19 surveillance officers at the makassar city health service did not know how to interpret the data, the results and the presentation of the interpretation of the data (tables, graphs, diagrams) because everything was done by the expert team. however, based on the document study, the data analyzed in tables, graphs, and charts have not been equipped with data interpretation. distribution of data on the accumulation of suspected covid -19 cases 89 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 83–92 e. information dissemination data collected is disseminated by the coordinates of the covid-19 surveillance program in the form of information on the epidemiology of covid-19 in makassar city to the public, media, local government, and other cross-sectors. output the output is the result of the process of data collection, data analysis, and data interpretation. in information systems, the outcome can be in the form of information, suggestions, printed reports, etc. the covid-19 surveillance output is used as a basis for program improvement. based on interviews with program managers, the work generated from analyzing and interpreting the data is the coverage obtained from program activities. this coverage is compared with the indicators of the covid-19 surveillance program as a measure of the progress or success of the program. the indicators used at the district/city level include the epidemiological criteria, the health system criteria, and the public health surveillance criteria as many as 24. however, this evaluation focuses on the public health surveillance section, which consists of 10 indicators covering surveillance systems, case investigations, and contact tracing. then, from the data analysis results, epidemiological information about covid19 is made at the makassar city health office and then reported to the provincial health office. the output from the covid-19 surveillance data is not only used for monitoring and evaluation activities in measuring the achievement indicators of the city-scale covid-19 control program but it is also used for the preparation of makassar city health office reports such as profile of the makassar city health office; daily reports, monthly and annual reports of disease control and eradication section; makassar city health office annual report; data for covid-19 research purposes; data on cross-sectoral requests from relevant regional apparatus units; and data for ngo and community organizations. feedback feedback from covid-19 surveillance activities is used in decision-making for the program and is used as a means for program improvement. the feedback from the makassar city health office to the health facilities was not only related to the completeness, accuracy, and validity of the data but also to evaluating the achievement indicators of the covid-19 program at the health facility level. for example, in terms of the achievement of new case discovery and equipped with a notification form of new case discovery. likewise, feedback from the provincial health office to the city health office can also be through technical guidance on problems faced in covid-19 surveillance activities. identification of problems in the implementation of covid-19 surveillance activities in makassar city (based on input, process, and output components) problem identification was carried out using in-depth interviews with several sources to know the covid-19 surveillance program at the makassar city health office. the in-depth interview results are then recapitulated to determine the priority of the problem later. the input components' problem is that all those on duty in covid-19 surveillance activities have dual responsibilities, and there is an irregular job desk. and not using form attachment 3 (covid-19 case finding notification report at health facilities), form attachment 4 (covid-19 aggregate daily report) is not used consistently, and there is no attachment form 6 (covid-19 epidemiological investigation form) from fasyankes because it was taken over by the provincial health office. the problem with the process components 90 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fatmasari, et al. analysis of covid-19 surveillance system is the health facilities report only uses an excel format, but officers are also sometimes lazy to in and result in incomplete data, not reported every day, only monthly accumulation, and there is no ei report based on the ei form, and there is a the tc-19 all record information system existed in july 2020 but was not used at that time, began to be used in december 2020. data analysis is assisted by a team of experts. however, data analysis is still incomplete, and the data that has covid-19 aggregate daily report format based on attachment form 4 (aggregate daily report) but it is not consistent in filling it out, only analyzing the release of covid-19 cases; been analyzed has not been equipped with data interpretation. the problem with the output component is the success rate of public health surveillance criteria has not been evaluated yet priority determination of covid-19 surveillance problems at the makassar city health office (using the carl technique) based on the results of calculations using the carl technique, three priority problems are obtained as shown in table 1. table 1. priority problem based on carl method no problem c a r l value total rank 1 there is no ei report based on the ei form from the health facilities, and there is a covid-19 aggregate daily report format based on attachment form 4 (covid-19 aggregate daily report). still, it has not been consistent in filling it out, only analyzing case releases. 5 5 5 5 625 2371 i 5 5 5 5 625 5 3 4 4 240 4 4 4 4 256 5 5 5 5 625 2. the health facilities report only uses an excel format, but officers are also sometimes lazy to fill in, resulting in incomplete data, not reported every day, only monthly accumulation. 5 5 4 5 500 1232 ii 3 5 5 5 375 2 2 4 3 48 4 4 4 4 256 3 3 4 4 144 3 do not use form attachment 3 (covid-19 case finding notification report at health facilities), form attachment 4 (covid-19 aggregate daily report) is not used consistently, and there is no form attachment 6 (covid-19 epidemiological investigation form) from health facilities because taken over by the provincial health office. 5 5 4 5 500 1244 iii 5 3 4 5 300 3 3 4 4 144 3 3 3 4 108 4 4 3 4 192 alternative troubleshooting plan the priority is on the process component; namely, there is no epidemiology investigation (ei) report based on the ei form from the health facilities, and there is a covid-19 aggregate daily report format based on attachment form 4 (covid-19 aggregate daily report). still, it is not consistent in filling it out, only analyzing the release coronavirus case. an alternative solution to this problem is developing a brief covid-19 epidemiological investigation report through the epiinfo application so that surveillance officers are willing and consistent in reporting covid-19 epidemiological investigations. and the development of daily reports in the form of individual and aggregate data using a free application, namely google spreadsheet, to make it easier for users, in this case, the covid-19 surveillance coordinator at the makassar city health office. the individual and aggregate data obtained from the google spreadsheet application will be reported to the south sulawesi provincial health office easily, precisely, and quickly. south sulawesi to the makassar city health office. the second priority is the process component; the health service report only uses an excel format. still, officers are 91 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 83–92 sometimes lazy to fill in and result in incomplete data, not reported daily, only monthly accumulation. an alternative to solving this problem is the development of easy recording and reporting by developing the excel format based on a web application. the third priority is the input component, namely, not using form attachment 3 (covid19 case finding notification report at health facilities). form attachment 4 (covid-19 aggregate daily report) is not used consistently. there is no form attachment 6 (epidemiological investigation form covid19) from health facilities because it was taken over by the provincial health office. an alternative solution to this problem is that the holder of the surveillance program at the makassar city health office should require health facilities to fill out the notification report on the discovery of covid-19 cases in appendix 3 because this form is crucial for daily data recapitulation data. for the covid19 aggregate daily report in appendix 4, it is mandatory for one of the surveillance program officers at the makassar city health office to fill in either manually or via excel consistently because the data contained in this form is crucial for data analysis. conclusions the description of the implementation of the covid-19 surveillance system at the makassar city health office is based on a system approach: input, process, output, and feedback. the results of identifying problems in implementing covid-19 surveillance based on in-depth interviews were also based on a system approach: input, process, and output. as for the priority of covid-19 surveillance problems that were obtained 3. as well as alternative problem-solving plans, there are also three alternatives according to the problems in covid-19 surveillance at the makassar city health office. acknowledgement we would like to thank the makassar city health office. conflict of interest we declare that we have no conflict of interest. references 1. who. situation reports (internet). who. 2020 [cited 2020 april 15]. p. 12. available from: https://www.who.int/emergencies/diseases/novel -cornavirus-2019/situation-reports 2. zhu n, zhang d, wang w, li x, yang b, song j,et al. a novel coronavirus from patiens with pneumonia in china, 2019. n engl j med. 2020 feb 20, 382(8): 727-33. 3. parwanto m. corona virus (2019-ncov) causes covid-19. journal of biomedicine and health. 2020;3(1). 4. susilo a, rumende cm, pitoyo cw, santoso wd, yulianti m, sinto r, et all. coronavirus disease 2019: review of the latest literature on coronavirus disease 2019: review of current literatures j internal medicine in indonesia. 2020;7(1):45-67. 5. cdc. 2020. corona virus disease 2019 (covid 2019): situation summary (online) https://www.cdc. gov/coronavirus/2019 ncov/summary.html accessed on february 15, 2020. 6. saraswati ld, textbook of epidemiological surveillance, semarang: lp2pm undip; 2016. 204p. 7. ministry of health ri. 2020. knowing emerging infectious diseases (online) accessed from http://infectionemerging.kemkes.go.id/pengantar -elektroemerging/#.xldio2gza00 on february 20, 2020. 8. indonesian minister of health. decree of the minister of health of the republic of indonesia number 1116/menkes/sk/viii/2003. 2003; 147173. 9. makassar city health office, 2020. 10. director general of pp & pl ministry of health ri, 2003, disease epidemiological surveillance (pep), edition i ministry of health ri, jakarta. 92 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 fatmasari, et al. analysis of covid-19 surveillance system 11. j nelwan public health surveillance: an introduction. independent scholar (internet). sumatera barat; 2020. 73p. available from: https://books.google.co.id/books?id=6sefeaa aqbaj&printsec=frontcover&hl=id#v=onepag e&q&f=false 12. polak f, sumampouw o, pinontoan o.evaluation of the implementation of surveillance for corona virus disease 2019 at sam ratulangi international airport, manado in 2020. indonesian journal of public health and public medicine (internet). 2020 [cited 2020 july 18] 1(3), 55-61. available from: https://ejournal.unsrat.ac.id/index.php/ijphcm/art icle/view/29448/28589 13. riou j, althaus cl. pettern of early human-tohuman transmission of wuhan 2019 novel coronavirus (2019-ncov), desember 2019 to january 2020. vol 25, eurosurveillance. europan centre for disease prevention and contol (ecdc); 2020.p. 2000058. 14. nangi mg. basic epidemiology. special region of yogyakarta.2019: deepublish. 15. sosin dm. draft framework for evaluating syndromic surveillance systems. journal of urban health . 2003;80(1):i8-i13. 16. asif m. baig m, shah m. evaluation of the tuberculosis surveilance system in district hyderabad, province sindh-pakistan, 2012. int j trop dis heal. 2015:9 (1): 1-8, doi:10.9734/ijtdh/2015/17492. 17. german rr, lee lm, horan jm, milstein rl, pertowski ca, waller mn. updated guidelines for evaluating public health surveillance systems: recommendations from the guidelines working group. mmwr recommendations and reports: morbidity and mortality weekly report recommendations and reports/centers for disease control. 2001; (50): 35 .quiz ce1 – 7. 18. provincial health office. jatim, 2003 edition ii, guide to epidemiological surveillance of diseases-infectious diseases, food poisoning, disasters and management of extraordinary events, subdin ppdan pl, surabaya. 19. rasyidi ah. analysis of covid-19 pandemic public health indicators based on the covid-19 task force in pamekasan regency. repository unair (internet).2020. available from: https://repository.unair.ac.id/103424/ 20. naufal k. evaluation of the implementation of the covid-19 surveillance system in wonosobo regency in 2020.available from: http://eprints.undip.ac.id/82336/ 21. updated guidelines for evaluating public health surveillance systems. cdc, 2001. available from: https://www.cdc.gov/mmwr/preview/mmwrhtml /rr5013a1.htm. accessed 2020. 22. p2p filed of makassar city health office, 2020. 23. madolan a. prioritizing issues with the carl method. 2018. available from: mitrakesmas.com(internet) 24. guidelines for the control and prevention of covid-19. jakarta, indonesian ministry of health, 2020. 25. guidelines for the prevention and control of covid-19 revision 5; indonesian ministry of health, 2020. http://eprints.undip.ac.id/82336/ �� vol. 1. no. 1 january–april 2010 research report new biotype of vibrio cholerae o� from clinical isolates in surabaya garry cores de vries,1,2 emy koestanti sabdoningrum,2 and dadik rahardjo1,2 1 diarrheal study group, institute of tropical diseases, airlangga university 2 dept.of veterinary public health, faculty of veterinary medicine, airlangga university abstract a surveillance of new pathogenic variants of vibrio cholerae o1 strains was initiated to identify the emerge and spread throughout surabaya. findings from seven years (1994–2000) and from years 2008 until now by using a two-fold surveillance strategy was pursued involving 1) hospital-based case recognition, and 2) environment samples. rectal swabs and environment samples were transported to itd-unair, surabaya for culture and isolates were characterized by serotypic identification and arbitrarily primed pcr fingerprints revealed a group of strains with similar fingerprint patterns that are distinct from those of the current el tor epidemic strain. these strains have been analyzed by in vitro technique and the group has been denominated the surabaya-indonesian variant of v. cholerae o1. key words: vibrio cholerae o1, cholera toxin, ap-pcr finger print introduction cholera toxin (ct) is a major virulence determinant of vibrio cholerae. v. cholerae is indigenous to fresh and blackish water environments in worldwide especially in tropical areas primarily to developing countries with warm climates. v. cholerae causes seafood borne infection, waterborne outbreaks and epidemics in terrestrial environments (barua, d. 1974 and, bauer et al., 1966). most v. cholerae isolates from the environment do not produce cholera toxin (ct), nor do they possess the genetic potential to produce cholera toxin. v. cholerae o1 and o139 are the major seroytpes associated with illness, and some v. cholerae non-o1 and non-o139 isolates produce ct. detection of ct-producing v. cholerae using conventional culture-, biochemicaland immunologicalbased assays is time-consuming and laborious. a rapid, reliable and practical assay for the detection of ctproducing v. cholerae has been used as like as pcr assays which offer a more sophisticated approach to the identification of vibrio cholerae (brosius et al., 1981). although pcr assays provide more rapid identification of vibrio cholerae than conventional assays, they require the use of electrophoresis to detect amplified products, which is time-consuming and tedious. the worldwide epidemiological situation in cholera el tor at the beginning of this century is presented; among its characteristic features are continued extensive epidemics and outbreaks in african and asian countries with cases of import of this infection to other continents. outbreaks caused by a new variant of the infective agent of cholera, vibrio cholerae o139, are still registered at limited territories in the countries of south-east asia (oneshchenko et al., 2005). the emergence of vibrio cholerae o139 bengal during 1992–1993 was associated with large epidemics of cholera in india and bangladesh and, initially, with a total displacement of the existing v. cholerae o1 strains. however, the o1 strains reemerged in 1994 and initiated a series of disappearance and reemergence of either of the two serogroups that was associated with temporal genetic and phenotypic changes sustained by the strains. since the initial emergence of the o139 vibrios, new variants of the pathogen derived from multiple progenitors have been isolated and characterized. the clinical and epidemiological characteristics of these strains have been studied. rapid genetic reassortment in o139 strains appears to be a response to the changing epidemiology of v. cholerae o1 and also a strategy for persistence in competition with strains of the o1 serogroup. the emergence of v. cholerae o139 has provided a unique opportunity to witness ��vries et al.: new biotype of vibrio cholerae o1 genetic changes in v. cholerae that may be associated with displacement of an existing serogroup by a newly emerging one and, thus, provide new insights into the epidemiology of cholera (faruque et al., 2003). vibrio cholerae is causing a severe epidemic in east java after being absent from the region for about 10 years (tauxe et al., 1994). vibrio cholerae typically contains a prophage that carries the genes encoding the cholera toxin, which is responsible for the major clinical symptoms of the disease (safa et al., 2010). the taxonomy of this species has been the object of our interest, and we recently developed a method for distinguishing pathogenic groups by using arbitrarily primed pcr (appcr) fingerprints (coelho et al., 1993 and coelho et al., 1995) on the basis of the general methodology of ap-pcr (welsh et al., 1990 and williams et al., 1990). by this technique a single oligonucleotide with an arbitrary sequence is used in a pcr with the dna of the strain under analysis. lowstringency conditions for hybridization are used, and the oligonucleotide can find regions of pairing, leading to the amplification of various genome fragments. our study (6) involved four groups of pathogenic v. cholerae: classical, el tor, ogawa and hikojima, all of which are distinguishable with the fingerprints. when applied to strains isolated from environment the results were similar to those from clinical isolated strains. however, when this method was used to study a group of strains from patients with diarrheal disease in the north part of surabaya, a quite distinct fingerprint pattern emerged for some of these strains. in the work described here we further extended this observation by using other in vitro techniques to evaluate the degree of relatedness between this group and the other pathogenic strains. materials and methods isolation and identification of vibrio cholerae serogroups o1 and o139 enrichment in alkaline peptone water inoculated apw with liquid stool or a rectal swab. incubate the tube with the cap loosened at 37°c for 8 hours. then subculture to tcbs with two loopfuls of apw from the surface of the broth. if the broth cannot be plated after 8 hours of incubation, subculture a loopful at 18 hours to a fresh tube of apw. subculture the second tube to tcbs agar after 8 hours of incubation. inoculation of tcbs inoculate the tcbs plate after 24 hours incubation at 37° c. colonies suspicious for v. cholerae will appear on tcbs agar as yellow, shiny colonies, 2 to 4 mm in diameter. the yellow color is caused by the fermentation of sucrose in the medium. sucrose-nonfermenting organisms, such as v. parahaemolyticus, produce green to blue-green colonies. isolation of suspected v. cholerae one of each type of sucrose-fermenting colony was selected from the tcbs plate to inoculate a heart infusion agar (hia) slant. incubate the hia slants at 37° c for up to 24 hours; for serologic testing. slide serology with polyvalent o1 and o139 antisera is sufficient for a presumptive identification. screening tests for suspected v. cholerae isolates oxidase test conduct the oxidase test with fresh growth from an hia slant medium. place 2 to 3 drops of oxidase reagent (1% n,n,nn,nn-tetramethyl-p-phenylenediamine) on a piece of filter paper in a petri dish. in a positive reaction, the bacterial growth becomes dark purple immediately. oxidase-negative organisms will remain colorless or will turn purple after 10 seconds. positive and negative controls should be tested at the same time. organisms of the vibrio cholerae is oxidase positive. reactions of v. cholerae in screening tests oxidase test positive kligler iron agar (kia) alkaline/acid, no gas produced (red slant/yellow butt) triple sugar iron agar (tsi) acid/acid, no gas produced (yellow slant/yellow butt) lysine iron agar (lia) alkaline/alkaline, no gas produced (purple slant/purple butt) gram-negative curved rods serologic identification of v. cholerae o1 and o139 presumptive identification using o1 antisera slide agglutination testing with polyvalent o1 antisera, fresh growth of suspected v. cholerae from a nonselective agar medium hia should be used. using growth from tcbs agar may result in false-negative reactions. presumptive v. cholerae o1 isolates should be tested in monovalent ogawa, inaba antisera and hikojima. confirmation of v. cholerae o1 using inaba and ogawa antisera the o1 serogroup of v. cholerae has been further divided into three serotypes, inaba, ogawa, or hikojima. a positive reaction in either inaba, ogawa or both hikojima antiserum is sufficient to confirm the identification of a v. cholerae o1 isolate. isolates that agglutinate weakly or slowly with serogroup o1 antiserum but do not agglutinate with either inaba or ogawa antiserum are not considered to be serogroup o1. agglutination reactions with inaba and ogawa antisera should be examined simultaneously, and the strongest and most rapid reaction should be used to identify the serotype. strains that agglutinate very strongly and equally with both the ogawa and inaba antisera are suspected may be referred to as “possible serotype hikojima.” �0 indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 48-53 strains of v. cholerae. the strains used in the present study are listed in table 1. ap-pcr. the ap-pcr mixtures consisted of 10 mm tris-hcl (ph 8.3), 50 mm kcl, 4 mm mgcl2, 100 mm (each) deoxynucleoside triphosphate (dntp), 30 pmol of one of the oligonucleotides, and 100 ng of dna in a total volume of 25 ml. the mixture was overlaid with mineral oil, and 1.5 u of taq dna polymerase was added. the program consisted of 45 cycles, and an annealing temperature of 32o c was used (coelho et al., 1995). two sets of fingerprints were done, one of them with oligonucleotide 1 (5’-ggtgcgggaa) and the other with oligonucleotide 3 (5’-ccagatgcac) (coelho et al., 1995). analysis of the amplified fragments was done on 1.4% agarose gels (gibco-bethesda research laboratories) in tris-borate buffer (tbe) (sambrook et al., 1989) running at 100 v for 3 h. analysis of presence of virulence genes by pcr. the basic program for the pcrs for the specific genes included 1 min initial denaturation step at 94o c, followed by 35 three-step cycles at 94o c (45 sec), annealing 55o–60o c for (45 sec) and extension at 72o c (1 min). a final extension at 72° c for 5 min was included in all reactions. a total of 100 ng of dna, 20 pmol of each primer, 0.25 mm dntps, and 1.5 u of taq dna polymerase were used, with a 1.5 mm mgcl2 buffer, in a total volume of 50 ml. the oligonucleotides used for ctxa1 amplification were 5’_ cgg gca gat tct aga cct cct g _3’ (sense) and 5’_ cga tga tct tgg agc att ccc ac_ 3’ (antisense), which were designed table 1. strains of v. cholerae analyzed in the study straina placeb date serotype type vf-57 surabaya february 1995 ogawa el tor vf-58 surabaya february 1995 ogawa el tor vf-59 surabaya february 1995 ogawa el tor vf-60 surabaya october 1995 ogawa vf-61 surabaya february 1995 ogawa el tor vf-62 surabaya february 1995 ogawa el tor vf-64 surabaya march 1995 ogawa classic vf-65 surabaya january 1995 ogawa el tor vf-66 surabaya march 1995 ogawa el tor vf-67 surabaya february 1995 ogawa el tor vf68 surabaya june 1995 ogawa el tor vf 69 surabaya june 1995 ogawa classic vf70 surabaya june 1995 ogawa el tor vf-71 surabaya february 1995 ogawa el tor vf-72 surabaya february 1995 ogawa el tor vf-73 surabaya february 1995 ogawa el tor vf-74 surabaya june 1995 ogawa el tor vf-75 surabaya february 1995 ogawa el tor vf-76 surabaya february 1995 ogawa el tor vf-77 surabaya february 1995 ogawa el tor vf-80 surabaya february 1998 ogawa vf-81 surabaya february 1998 ogawa vf-82 surabaya march 1998 ogawa vf-83 surabaya march 1998 ogawa vf-84 surabaya march 1998 ogawa vf-85 surabaya march 1998 ogawa vf-86 surabaya march 1998 ogawa vf-87 surabaya march 1998 ogawa vf-88 surabaya september 1998 ogawa vf-89 surabaya september 1999 ogawa vf-90 surabaya september 1998 ogawa vf-91 surabaya september 1998 ogawa vf-92 surabaya april 1997 ogawa el tor vf-94 surabaya march 1998 ogawa vf-95 surabaya june 1998 ogawa vf-96 surabaya september 2008 ogawa vf-192 surabaya june 2009 hikojima vf-193 surabaya july 2009 ogawa vf-194 surabaya july 2009 hikojima ��vries et al.: new biotype of vibrio cholerae o1 for classical strains. the oligonucleotides used for ctxa2 amplification were 5’_ aca gag tga gta ctt tga cc_ 3’ (sense) and 5’_ ata cca tcc ata tat ttg gga g_ 3’ (antisense), which were designed for classical strains (lipp et al., 2003). biochemical identification. the biochemical characterization of surabayaindonesian strains was done by a battery of standard tests (farmer et al., 1985.) including oxidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, requirement for na+ (growth in nutrient broth with 0, 1, and 3% nacl), motility, indole, gas from glucose, susceptibility to o/129 (150-mg discs), and acid production from d-glucose, l-arabinose, cellobiose, lactose, maltose, d-mannitol, salicin, and sucrose. identification of biotypes was performed by detection of acetylmethylcarbinol (voges-proskauer test) and determination of susceptibility to polymyxin b (oxoid) by spot inoculation onto muellerhinton agar (difco) containing 15mg of polymyxin b per ml (roy et al., 1965), hemolysis of sheep erythrocytes, and hemagglutination activity for human (o group) and chicken erythrocytes. o1 somatic antigen characterization. expression of o1 antigen by ogawa-el tor strains was further evaluated by tube agglutination tests against polyvalent o1 antisera prepared by immunizing rabbits with heat-killed cells of inaba or ogawa and then absorption of the antisera with the heat-killed cells of the heterologous serotype. polyvalent o1 antiserum was obtained by mixing the two monospecific antisera. tests were performed with live cultures grown for 3 h (barua, 1974.). antimicrobial susceptibility tests. antimicrobial susceptibility testing was carried out on mueller-hinton agar (difco) by the disc diffusion method (clinical and laboratory standards institute, 2008) for levofloxacin, nalidixic acid, ampicillin, chloramphenicol, imipenem and ceftriaxone. figure 1. pathogenic v. cholerae o1 fingerprints obtained by ap-pcr with oligonucleotide ctxa1 and ctxa2. lanes 1 to 5, surabaya-indonesian variant strains (biotype ogawa-el tor vf-69, vf-59, vf-76, vf-77 and vf-92) respectively; lanes 6 to 9, biotype hikojima strains vf192, vf-200, biotype ogawa vf-193 and vf-196, respectively; lane 10 1-kb ladder size marker (gibco bethesda research laboratories). results and discussion we studied 43 v. cholerae o1 strains (table 1) isolated in 1995, 1997, 1998, 2008 and 2009 in east and south surabaya and, in particular, from the villages adjacent region north of surabaya. most of them came from patients with diarrhea, and their co-cultures did not show other enteropathogenic bacteria. a screening was done with these strains by using biochemical identification and ap-pcr fingerprints. two oligonucleotides, oligonucleotides ctxa1 and ctxa2, were used in separate reactions. each of the oligonucleotides showed that there were two markedly different groups of strains in the sample. one of these groups yielded the fingerprints found with other el tor strains (coelho et al., 1995 and lipp et al., 2003), and the other group, comprising five strains hokojima and denominated in surabayaindonesian variant in 2009–2010, produced fingerprints different from those of the other pathogenic groups studied straina placeb date serotype type vf-195 surabaya september 2009 hikojima vf-196 surabaya september 2009 ogawa vf-200 surabaya december 2009 hikojima vf-217 surabaya january 2010 ? a the 36 first strains correspond to the original group of strains analyzed. the seven el tor strains included in this group were used in the tests described in the text. the last four hikojima strains were identified later. all isolates came from patients; isolates vf-96 were obtained from environment. b all locations are in surabaya and adjacent region. �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 48-53 previously. the fingerprints of the strains within the group were identical. the results with oligonucleotide ctxa1 (fig. 1) showed a completely different pattern for the surabayaindonesian strains, in which a 1.3-kb band seemed to be the only band common to the bands for the el tor strains. in the case of oligonucleotide ctxa2, a prominent 0.55-kb el tor band was absent from the fingerprints of the surabayaindonesian strains; the other bands were the same (data not shown). these same oligonucleotides have been used against representative el tor, classical and inaba strains (coelho et al., 1995). a serotype difference is not detected with these oligonucleotides. all of the surabaya-indonesian strains tested belonged to the ogawa serotype, but other ogawa strains in the sample were normal el tor isolates, producing their characteristic ap-pcr fingerprints. the surabaya-indonesian variant strains came from the small town (villages) bordering of surabaya¸ and a few other villages 50 km adjacent surabaya. a further search of our collection revealed five other surabaya-indonesian strains among the ogawa strains from the same region. the ap-pcr fingerprints used in the present study are tuned at a less discriminative level that groups together the el tor strains (except the environment strains that form a separate group). this grouping occurs because of the choice of oligonucleotides, which were selected to distinguish between broad pathogenic groups and not strains within a group (coelho et al., 1995). a more conspicuous distinction between the surabaya-indonesian variant and the el tor strains was produced with ap-pcr. the surabaya-indonesian strains behaved in the biochemical tests as typical representatives of v. cholerae. biological markers such as o-antigen specificity and antimicrobial susceptibility were also evaluated. it was shown that all surabaya-indonesian strains but one exhibited o1 agglutination titers of 1,024 (five strains) or 2,048 (three strains). the homologous titer for the classical ogawa strain was 4,096. surabaya-indonesian strain vf-217 was autoagglutinable, preventing its testing. further testing with monospecific antiserum showed that surabaya-indonesian strains reacted only with ogawa antiserum. antimicrobial susceptibility tests showed that the surabaya-indonesian and el tor strains isolated from the same geographical area were equally susceptible to all antimicrobial agents tested. biotyping showed that these strains were vogesproskauer test positive and susceptible to polymyxin. taken together these results support the previous findings of ap-pcr analyses that surabaya-indonesian strains are a separate group distinct from the el tor (polymyxin resistant, voges proskauer test positive) and the classical (polymyxin susceptible, voges-proskauer test negative) biotypes of v. cholerae o1. the presence of ctxa gene was investigated in various ways in the surabaya-indonesian strains. pcr amplifications were done with positive results for all hikojima strain and 2 ogawa el tor strains. the restriction fragment length polymorphisms of the ctx genes were tested. a cholera toxin dna fragment of 982 bp was used as a probe. this dna fragment was produced by pcr amplification from an el tor strain, and it includes most of the ctxa1 and ctxa2 genes. this indicates that the toxin genes, if present, have a very divergent sequence. only seven strains (vf-92, vf-192, vf-193, vf-194, vf-195, vf-196 and vf-200) produced cholera toxin. the presence of other v. cholerae virulence genes was investigated by pcr. oligonucleotides specific for ctxb. all of these amplifications gave negative results. positive controls with an el tor strain were included in all of the experiments. preliminary phenetic analysis places the surabayaindonesian clone at a considerable distance from other pathogenic o1 clones (eltor, classical, and hikojima). the surabaya-indonesian variant seems to be restricted, for the time being, to a small area of the surabaya and adjacent region and is probably unable to compete with the invading el tor strains. a parallel may be traced with the early isolates of el tor from the hospitals. their epidemiological relevance is, at present, negligible, but as in the latter case, future developments of cholera in indonesia may outline its importance. references 1. barua, d. 1974. laboratory diagnosis of cholera, p. 85–126. in d. barua and w. burrows (ed.), cholera. the w. b. saunders co., philadelphia. 2. bauer, a. w., w. m. m. kirby, j. c. sherris, and m. turck. 1966. antibiotic susceptibility testing by a standardized single disk method. am. j. clin. pathol. 45:493–496. 3. coelho, a., a. vicente, h. momen, and c. salles. 1993. typing of vibrio cholerae using arbitrary primers, p. 211. volume of abstracts. seventeenth international congress of genetics, birmingham, united kingdom. the quadgraphics partnership, thatcham, united kingdom. 4. coelho, a., a. c. p. vicente, m. a. s. baptista, h. momen, f. r. w. dos santos, and c. a. salles. 1995. the distinction of pathogenic vibrio cholerae groups using arbitrarily primed pcr fingerprints. the quadgraphics partnership, thatcham, united kingdom. 5. coelho, a., o. r. joa., c. andrade., a. c. p. vicente, and c. s. a. salles. 1995. new variant of vibrio cholerae o1 from clinical isolates in amazonia. journal of clinical microbiology. 33:114– 118. 6. clinical and laboratory standards institute. 2008. performance standards for antimicrobial susceptibility testing; eighteenth informational supplement. m100-s18. 28:74–76. 7. dirita, v., c. parsot, g. jander, and j. j. mekalanos. 1991. regulatory cascade controls virulence in vibrio cholerae. proc. natl. acad. sci. usa 88: 5403–5407. 8. faruque, s.m., d.a sack, r.b sack, r.r colwell, y. takeda and g.b nair. 2003. emergence and evolution of vibrio cholerae o139. proceedings of the national academy of sciences of the united states of america. volume 100, issue 3. pp 1304–1309. 9. hayashi s, m. okura, r. osawa. 2006. soft-agar-coated filter method for early detection of viable and thermostable direct hemolysin (tdh) or tdh-related hemolysin-producing vibrio parahaemolyticus in seafood. appl environ microbiol, 72:4576–4582. 10. higgins, d. e., e. nazareno, and v. j. dirita. 1992. the virulence gene activator toxt from vibrio cholerae is a member of the arac family of transcriptional activators. j. bacteriol. 174:6974–6980. 11. iwanaga, m., and k. yamamoto. 1985. new medium for the production of cholera toxin by vibrio cholerae o1 biotype el tor. j. clin. microbiol. 22:405–408. ��vries et al.: new biotype of vibrio cholerae o1 12. jonson, g., j. holmgren, and a.-m. svennerholm. 1991. identification of a mannose-binding pilus on vibrio cholerae el tor. microb. pathog. 11:433–441. 13. jonson, g., j. sanchez, and a.-m. svennerholm. 1989. expression and detection of different biotype-associated cell-bound haemoagglutinins of vibrio cholerae o1. j. gen. microbiol. 135:111–120. 14. keasler, s. p., and r. h. hall. 1993. detecting and biotyping vibrio cholerae o1 with multiplex polymerase chain reaction. lancet 341:1661. 15. koblavi, s., f. grimont, and p. a. d. grimont. 1990. clonal diversity of vibrio cholerae o1 evidenced by rrna gene restriction patterns. res. microbiol. 141:645–657. 16. lan, r., and p. r. reeves. 2002. pandemic spread of cholera: genetic diversity and relationships within the seventh pandemic clone of vibrio cholerae determined by amplified fragment length polymorphism. jcm. 40:172-181. 17. lipp,e. k., i. n. g. rivera, a. i. gil, e. m. espeland, .n. choopun, v. r. louis, e. r. cohen, a. huq, and r. r. colwell. 2003. direct detection of vibrio cholerae and ctxa in peruvian coastal water and plankton by pcr. applied and environmental microbiology. 69: 3676–3680. 18. louis, v. r., e. r. cohen, n. choopun, i. n. g. rivera, b. gangle, s. c. jiang, a. rubin, j. a. patz, a. huq, and r. r. colwell. 2003. predictability of vibrio cholerae in chesapeake bay. applied and environmental microbiology. 69:2773–2785. 19. ogawa, a., j.-i. kato, h. watanabe, b. g. nair, and t. takeda. 1990. cloning and nucleotide sequence of a heat-stable enterotoxin gene from vibrio cholerae non-o1 isolated from a patient with traveler’s diarrhea. infect. immun. 58:3325–3329. 20. oneshchenko g. g, i. m. lomov, e. a. moskvitina, i. m. fedorov, l.s. podosinnikova, a. v. gorobets. 2005. cholera at the beginning of the xxi century. prognosis. zhurnal mikrobiologii epidemiologii i immunobiologii. pp. 44-48. 21. popovic, t., c. bopp, ø. olsvik, and k. wachsmuth. 1993. epidemiologic application of a standardized ribotype scheme for vibrio cholerae o1. j. clin. microbiol. 31:2474–2482. 22. roy, c., k. mridha, and s. mukerjee. 1965. action of polymyxin of cholera vibrios: techniques of determination of polymyxin-sensitivity. proc. soc. exp. biol. med. 119:893–896. 23. salles, c. a., h. momen, a. coelho, e. f. de oliveira, a. c. p. vicente, and g. b. nair. 1994. bengal: el tor cholera vibrio in a new robe. mem. inst. oswaldo cruz 89:115–116. 24. salles, c. a., h. momen, a. c. p. vicente, and a. coelho. 1993. vibrio cholerae in south america: polymerase chain reaction and zymovar analysis. trans. r. soc. trop. med. hyg. 87:272. 25. sambrook, j., e. f. fritsch, and t. maniatis. 1989. molecular cloning: a laboratory manual, 2nd. ed. cold spring harbor laboratory press, cold spring harbor, n.y. 26. safa a, nair g.b, kong r.y.c. 2010. trends in microbiology. 18:46–54. 27. silhavy, t. j., m. l. berman, and l. w. enquist. 1994. experiments with gene fusions, pp. 137–139. cold spring harbor laboratory press, cold spring harbor, n.y. 28. simanjuntak, c. h., w. larasati, s. arjoso, maidy putri, m. lesmana, b. a. oyofo, n. sukri, d. nurdin, r. p. kusumaningrum, n. h. punjabi, d. subekti, s. djelantik, sukarma, sriwati, muzahar, a. lubis, h. siregar, b. mas’ud, m. abdi, a. sumardiati, s. wibisana, hendarwanto, b. setiawan, w. santoso, eka putra, s. sarumpaet, h. ma’ani, c. lebron, s. a. soeparmanto, j. r. campbell, and a. l. corwin. 2001. cholera in indonesia in 1993–1999. am. j. trop. med. hyg., 65: 788–797. 29. tauxe, r., l. seminario, r. tapia, and m. libel. 1994. the latin american epidemic, p. 321–344. in i. k. wachsmuth, p. a. blake, and ø. olsvik (ed.), vibrio cholerae and cholera. american society for microbiology, washington, d.c. 30. welsh, j., and m. mcclelland. 1990. fingerprinting genomes using pcr with arbitrary primers. nucleic acids res. 18:7213–7218. 31. williams, j. g. k., a. r. kubelik, k. j. livak, j. a. rafalski, and s. v. tingey. 1990. dna polymorphisms amplified by arbitrary primers are useful as genetic markers. nucleic acids res. 18:6531–6535. 32. yamamoto, k., y. takeda, t. miwatani, and j. p. craig. 1983. purification and some properties of a non-o1 vibrio cholerae enterotoxin that is identical to cholera enterotoxin. infect. immun. 39:1128–1135. 33. yamazaki, w., k. seto, m. taguchi, m. ishibashi and k. inoue. 2008. sensitive and rapid detection of cholera toxin-producing vibrio cholerae using a loop-mediated isothermal amplification. bmc microbiology, 8:94. ijtid vol 1 no 1 jan-apr 2010.50.pdf ijtid vol 1 no 1 jan-apr 2010.51.pdf ijtid vol 1 no 1 jan-apr 2010.52.pdf ijtid vol 1 no 1 jan-apr 2010.53.pdf ijtid vol 1 no 1 jan-apr 2010.54.pdf ijtid vol 1 no 1 jan-apr 2010.55.pdf 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 124 vol. 5. no. 5 may–august 2015 the prevalence of human immunodefiency virus-1 (hiv-1) subtypes and transmission method among hiv/aids infection patient in tulungagung, east java indonesia achmad ardianto1, siti qamariyah khairunisa2, tomohiro kotaki2,3,4,adiana mutamsari witaningrum2, m. qushay2, juniastuti1,2, retno pudji rahayu,2 prihartini widiyanti,2 budi utomo1, maria inge lusida1,2, nasronudin1,2 1faculty of medicine, universitas airlangga, surabaya, indonesia. 2 indonesian-japan collaborative research center for emerging and re-emerging infectious diseases, institute of tropical disease, universitas airlangga, surabaya, indonesia. 3 center for infectious diseases, kobe university graduate school of medicine, hyogo, japan. 4 department of international health, kobe university graduate school of health sciences, hyogo, japan. abstract the rapid epidemic growth of hiv is continuing in indonesia. there are some factors which have influenced the spreading of this epidemic in indonesia, such as the poor awareness to avoid unsafe free sex attitude and the sharing of needles and syringes among intravenous drug users (idus). the sexual transmission of hiv has also apparently increased in tulungagung. commercial sex workers play a significant role in the spread of hiv in tulungagung. people in tulungagung have worked at other countries as indonesian migrants. this condition can cause the increase number of hiv-1 case and the possibility of genetic variation (subtype) hiv-1 in tulungagung. this research is aimed to analyze the subtype and to determine estimation of transmission mode on infected patient of hiv-1 and aids who came to seruni clinic dr. iskak hospital in tulungagung. 40 hiv?aidspatients were interviewed to determine the subtype and the transmission mode. the results showed that 14 of 40 plasma samples (35%) were successfully to amplified and sequenced. overallcrf01-ae wereidentified as predominant subtype among hiv/aids patients in tulungagung. based on individual information, 31 of 40 subjects (77%) were heterosexual transmission. key words: prevalence, subtype, transmission mode, hiv-1, tulungagung abstrak pertumbuhan epidemi hiv di indonesia berkembang sangat pesat. ada beberapa faktor yang mempengaruhi penyebaran epidemi di indonesia, diantaranya kesadaran masyarakat dalam melakukan hubungan seksual secara aman dan berbagi jarum suntik di kalangan pengguna narkoba suntikan (penasun). pola penyebaran hiv melalui hubungan seksual meningkat di daerah tulungagung. pekerja seks komersial memiliki peranan penting dalam penyebaran hiv di tulungagung. banyak warga di tulungagung yang bekerja di luar negeri sebagai imigran indonesia. kondisi ini dapat mempengaruhi jumlah kasus hiv/aids dan kemungkinan munculnya variasi genetik (subtipe) hiv-1 baru di tulungagung. penelitian ini bertujuan untuk menganalisis subtipe dan menentukan estimasi transmisi pada pasien yang terinfeksi hiv-1/aids yang datang ke klinik seruni rs dr iskak tulungagung. dalam penelitian ini, sebanyak40 pasien hiv/aids yang diwawancarai untuk mengetahui subtipe dan polapenyebaran hiv/aids. hasil menunjukkan bahwa dari 40 sampel plasma terdeteksi 14 (35%)sampel positif secara kualitatif. hasil analisa rip dan pohon filogentik menunjukkan bahwa 14 sampel tersebut termasuk dalam kelompok rekombinan crf01-ae. berdasarkan informasi pasien, pola penyebaran hiv/aids melalui heteroseksual sebanyak 31sampel(77%). kata kunci: prevalensi, subtipe, pola transmisi, hiv-1, tulungagung research report 125ardianto, et al.: the prevalence of human immunodefiency virus-1 (hiv-1) subtypes introduction humanimmunodeficiency virus/aquiredimmune deficiency syndrome (hiv/aids) causes a serious health problem and has a big impact on indonesian economics. in addition, rapidly growing epidemic of hiv-1 is a serious public health problem in indonesia. in indonesia, the number of people living with hiv was estimated to be 591,823 in 2012 and 735,256 in 2015. whereas newly infected with hiv were estimated to be 71,879 in 2012 and 85,523 in20151. several factors affect the rate and magnitude of growth of hiv prevalence, but two of the most important are injecting drug use and the commercial sex workers; transmission between men who have sex with men usually has a secondary role 2,3. in indonesia, crf01_ae continually dominates hiv epidemic, although hiv subtype b is responsible for a large amount of infection sexually transmitted. furthermore, circulation recombinant form (crf) can occur from one subtype to another one and nowadays it has been found 43 crf. it is stated that different hiv subtype can have different effect on its transmission, causing drug resistance and disease progressivity4. the growth and finding of hiv and aids cases in tulungagung are increasing rapidly and its transmission is getting wider. supposedly, it is triggered by hidden prostitution activities in two ex-brothels which have been closed down since 20125,6. this research is intended to identify subtype hiv-1 and to know the method of hiv1 infection transmission in dr. iskak general hospital, tulungagung. by doing so, it is very essential to get data analysis on subtype hiv-1 and its infection transmission of hiv-1 at the newest and more representative hospital, dr. iskak general hospital. methods study population and data collection a total of 40 peripheral blood samples from hiv/aids patients in dr. iskak general hospital, tulungagung were collected. a cross-sectional survey, the study population were interviewed in dr. iskak general hospital, tulungagung using a questionnaire that collected information on socio-demographic characteristics, and transmission. 5-8 ml whole blood samples werecollected from 40 hiv/aids patients by using spuit 5 cc. whole blood sample were added into a edta tube. plasma was then isolated from peripheral blood samples by centrifugation for 10 min at 2,000 rpm.plasma were analyzed at hiv/aids laboratory, institute of tropical disease (itd), universitas airlangga, surabaya. the examination of polymerase chain reaction (pcr) hiv the examination of polymerase chain reaction (pcr) hiv was done to identify cdna, as the following steps: rna hiv was extracted from plasma and synthesis of cdna hiv, amplification reaction with pcr, gel electrophoresis, and photo development using gel electrophoresis. rna virus was changed into cdna using super script iii first-stand synthesis kit (invitrogen, carlsbad, ca, usa) with reverse primer, k-envri, 5’-ccaatcagggaagaagcctgg-3’. fragment hiv-1 gene pol 33 base pair encoding fragment partial integrase was amplified with rested pcr using ex taq (takara bio, shiga, japan) and primary set as follows: for amplification of fragment gene pol virus, unipol5; 5’ –tgggtaccagcacacaaaggaataggag gaaa -3’ (nt 4152 to 4183) and unipol6; 5’ –cca cagctgatctctgccttctctgtaatsgacc-3’ (nt 4934 to 4901) is used for first pcr. unipol1; 5’ –agtggattcatagaagcagaagt-3’ (nt 4470 to 4492) and unipol2; 5’ –cccctattcctccccttctt ttaaaa-3’ (nt 4806 to 4781) is used for nested pcr. the condition of first pcr: 94ºc for 3 minutes for denaturation (one cycle), 94ºc for 1 minute for denaturation (35 cycles), 72ºc for one minute for extension (35 cycles) and 72ºc for 5 minutes for the last extension. the condition of second pcr: 94ºc for 3 minutes (1 cycle), 94ºc for 1 minute (35 cycles), 50ºc for 1 minute (35 cycles), 72ºc for 1 minute (35 cycles), 72ºc for 5 minutes.pcr products amplified at the end-point dilution of dna templates were subjected to sequencing analysisto examine the genomic fragment of the major viral population in a sample. the analysis of sequencing results sequencing analysis on fragment genomic hiv-1 which has been amplified is done by using sequencing cycle kit bigdye terminator v1.1 with abi prism310 genetic analyzer, and data is analyzed by genetyx ver10 software. hiv-1 subtyping was carried out using the recombinant identification program (rip) available on the hiv sequence database website (http://www.hiv.lanl.gov/). in addition, neighbor-joining (nj) trees with the kimura two-parameter model were constructed using mega6.2 software7,8. results demographic and behavioural characteristics we interviewed 40 hiv/aids infected patients who came to seruni clinic, dr. iskak general hospital, tulungagung on june 2014. 126 indonesian journal of tropical and infectious disease, vol. 5. no. 5 may–august 2015: 124-128 table 1. sample characteristic based on age and sex age (in years) gender total male female 21-29 7 7 13 30-39 4 9 13 >40 8 6 14 total 19 21 40 (47.5%) (52.5%) (100%) based on questionnaires, it indicates that the number of female patients is 21 (52.5%), age ranges from 30-39 while there are 19 male patients (47.5%). most of them were over 40. table 2. sample characteristic based on age and transmission methods age (in years) transmission way total h et er os ex u al h om os ex u al id u s t ra n sf u si on v er ti ca l 21-29 7 4 2 13 30-39 13 13 >40 11 1 1 1 14 total 31 5 3 1 40 (77.5%) (12.5%) (7.5%) (2.5%) (100%) there were 31 (77.5%) heterosexual patients infected by hiv-1, their ages are various, 30-39 and only 5 persons (12.5%) homosexual patients got infected by hiv-1 as well. they were 20-29 years of age. table 3. sample characteristic based on age and marital status age (in years) marital status total m ar ri ed u n m ar ri ed w id ow er w id ow 21-29 9 4 13 30-39 9 2 1 1 13 >40 12 1 1 14 total 30 7 2 1 40 (75%) (17.5%) (5%) (2.5%) (100%) the data gives us information that there were30 persons (75%), ranges of age are mostly over 40 and 7 persons (17.5%) of 20-29 are not married yet. table 4. sample characteristic based on age and the length of art usage age (in years) art duration total h av en ’t ta k en a r t < 6 m on th s 6 m on th s – 2 ye ar s < 2 y ea rs 21-29 2 7 2 2 13 30-39 3 1 3 6 13 >40 1 3 4 6 14 total 6 1 9 14 40 (15%) (27.5%) (22.5%) (35%) (100%) data obtained from art indicate that patients who have got art treatment for over 2 years are 14 (35%). they are mostly 30-39 years old, and over 40 years old. in addition, those 6 persons (15%) of 30-39 did not receive art treatment. hiv-1 subtyping hiv subtyping has been an important molecular tool for monitoring geographic changes in the worldwide aids epidemic9.of the 40 samples derived from hiv patients, 12 pol genes were succsessefully sequenced. b a s e d o n the results of rip and phylogenetic tree analyses for pol genes, overallcrf01_ae has been identified as the predominant hiv-1 subtype, similar to the epidemics in malaysia, thailand, and taiwan10,11andno other unique recombinant forms. our results were consistent with previous findings. figure 2. phylogenetic tree (dendogram) subtype hiv-1 127ardianto, et al.: the prevalence of human immunodefiency virus-1 (hiv-1) subtypes phylogenetic analysis is done to determine gene sequence pol hiv-1 with strain reference virus gen hiv1 which shows the plasma sample. those samples are: crf01_ae (01_ae). accession number gu289975 (ae-vietnam) from vietnam, kf735959 (ae-ind) from indonesia, af484509 (a1) from uganda, af069670 (a1) from somalia, af286238 (a2) from republic of congo, af286237 (a2) from cyprus, ay423387 (by) from netherlands, ay331295 (b) from usa, ay772699 (c) from south africa, af067155 (c) from india, ay371157 (d) from cameroon, ay253311 (d) from tanzania, af084936 (g) from republic of congo, af061640 (g) from finland, l39106 (02_ag) from nigeria, u54771 (01_ae) from thailand. a molecular analysis is done toward nucleotide as a dna hiv sequence result taken from patient’s plasma sample and compared with nucleotide sequences from hiv subtype which has been published before. in later analysis, from hiv-1 pol gene, the all gained sample are crf, especially one branch with hiv crf01_ae which comes from asia. those are thailand, japan, malaysia, china, and hongkong. discussion the characteristic of research sample based on age and gender is taken that women are the greatest number who suffer from hiv-1 (52.5%) age ranges from 30-39. this case may happen because the women can easily infected by hiv-1. the greatest number of spreading way is sexual activity with men who are infected. biologically, the women are easier to be infected with hiv because the flatten areas for hiv are mostly in women (vagina, cervix, and uterus) than in the men (gland penis and urethra). the women are also greater in number who roll out with ejaculate than the men (vagina liquid contains less hiv than semen). the perimenopausal and postmenopausal women who undergo increasing of mucosa genital secondary fragility toward the changing of hormones on its cycle so that it increases the spreading risk of hiv12. the characteristic of research sample based on age and assumption of transmission way is reported that heterosexual is the greatest number of transmission way (77.5%) in age between 30-39 years old. heterosexual is the transmission way which has greatest factor to infect hiv-1 in the world. it becomes worse because of risky sex behavior, for example, having sex with someone with hiv1 infected risk (a prostitute) or without using condoms. the second way of transmission is probably homosexual (12.5%) age ranges from 20-mostly 29. it shows that homosexual is risky behavior which can infect hiv-1. there is a trauma in rectum causing wound which can be an easier way of hiv to human body. rectum consists of lymphoid tissue which can make hiv-1 get into fragile lymphosit cell easily13. the characteristic of research sample based on age and marital status is reported that the biggest number is marital status (75%) age ranges from 20-29. the second biggest sample is 17.5% unmarried people with age ranging from 20-29. it has been stated that the biggest sample is married people more than 40 years old when at these age most people have been married and had children. in the previous research, 76% from hiv sufferers are married women. this fact supports the dynamics of hiv-1 infection where in many cases, the first found hiv-1 infections are men who infected their wives. the characteristic of research sample based on age and assumption of art duration is reported that the longest art duration is more than 2 years (35%) ranging from 3039 years old and more than 40 years old. most respondent state that they have been diagnosed to be infected by hiv-1 since 2 years ago and at that time, the cd4 examination was done and they continue to take arv regularly. medicine drinking obedience is always informed by the medical officer and also by odha fellows to others which take the medicine regularly in seruniclinin dr. iskak general hospital in tulungagung. the role of counselor, kpa and every element in society has helped medicine drinking obedience and routine control for odha community in tulungagung. the meeting of odha community is filled by informing from medical officers (doctors) and also giving the moral supports from odh fellows to enrich the quality of their lives. this research explains that the result of blood sample pcr of hiv-infected patients is that the percentage of positive rna hiv-1 (35%) is lesser from the percentage of negative rna hiv-1 (65%). the negative pcr hiv1 result which is quite much is possibly caused by the research subjects who have been given arv therapy. the research subjects which have not been given arv therapy are reported to have no many significant differences in the pcr result between negative rna hiv and positive rna hiv. on the negative rna hiv samples in the research, there is possibly a changing or mutation of the nucleotide sequences in the annealing primer. so that the primer cannot attach then it causes the pcr result to be negative. hiv-1 is marked by the large heterogeneity and divided into 4 groups: m (major), o (outlying), n (new or non-m, non-o) and p (pending). the viruses in group m, which are responsible for the hiv pandemic in the world, are further classified into many subtypes and circulating recombinant forms (crfs). hiv-1 subtype b is the most apparent subtype in usa, europe, and australia whereas the subtype non-b is the subtype which causes an epidemic in africa and asia. lately, new crfs, crf33_01b and cfrf34_ 01b, have been isolated in indonesia14,15. in the previous research, it has been found the genome virus fragment crf01_ae, a crf is which is dominant in southeast asia. that crf is predominantly found in hiv-positive prostitutes in surabaya, indonesia and still a dominant virus strain16. in order to know the unidentified subtype, 128 indonesian journal of tropical and infectious disease, vol. 5. no. 5 may–august 2015: 124-128 we need to examine the virus subtype in this research. the genomic fragment of virus crf01_ae which was found in this research is still the most apparent hiv-1 subtype in tulungagung, east java, indonesia. conclusion based on the research result, it can be concluded that the most found subtype in hiv-1-and-aids-infected patients in dr. iskak general hospital, tulungagung is cref01_ae. it is relevant with the subtype which develops in southeast asia. the greatest number of transmission way in hiv-1-and-aids-infected patients in dr. iskak general hospital, tulungagung is heterosexual. acknowledgments this work was supported by the program of the japan initiative for global research network on infectious diseases (j-grid); by the ministry of education, culture, sports, science and technology of japan; and the institute of tropical disease as the center of excellence (coe) program by the ministry for research and technology (ristek) of indonesia. references 1. kementrian kesehatan republik indonesia:estimasi dan proyeksi hiv/aids di indonesia: tahun 2011–2016. 2014. 2. ruxrungtham k, brown t, praphan p.: hiv/aids in asia. 2004; www.thelancet.com; vol. 364: july 3. 3. nicholas i paton: hiv in south east asia. 2005; medicine publishing company. 33: 6. 4. handajani r, nasronudin, lusida mi, lindawati, effendi f, utsumi t, 2010. ‘analisis molekuler phylogenetic human immunodeficiency virus (hiv) pada pasien di surabaya, jawa timur’. majalah kedokteran indonesia, vol. 60, pp. 172–176. 5. dinkes tulungagung, 2008. penanggulanganhiv/aids.http:// dinkestulungagung.blogspot.com/2008/01/penanggulangan-hivaids. html, diakses pada tanggal 27agustus 2014 jam 09.31. 6. dinkes tulungagung, 2013. data kumulatif hiv-aids oktober 2013. http://dinkes.tulungagung.go.id/, diakses tanggal 18 mei 2014, jam 07.30. 7. tamura k, peterson d, peterson n, et al.: mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. mol. biol. evol 2011; 28:2731–2739. 8. kimura m: a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. j mol evol 1980; 16: 111–120. 9. requejo h iz: worldwide molecular epidemiology of hiv. rev saúde pública 2006; 40: 331–45. 10. auwanit w, isarangkura-na-ayuthaya p, kasornpikul d, et al.: detection of drug resistance-associated and background mutations in human immunodeficiency virus type 1 crf01_ae protease and reverse transcriptase derived from drug treatment-naive patients residing in central thailand. aids research and human retroviruses 2009; 25: 625–631. 11. mohamada s, derisa zz, yusoffb nk, et al.: assessing subtypes and drug resistance mutations among hiv-1 infected children who failed antiretroviral therapy in kelantan, malaysia. the brazilian journal of infectious diseases 2012; 16: 284–288. 12. stone v, ojikutu b, rawlings mk, smith ky, 2009. hiv/aids in u.s. communities of color. springer science & business media, boston, pp. 86. 13. ajayi jo, 2003. the hiv-aids epidemic in nigeria: some ethical considerations. gregorian biblical bookshop, roma, pp. 24. 14. shuvra kd, nazneen z, sabrina a. molecular epidemiology of hiv in asia. polish aids research society: 2014: 1730–1270. 15. sahbandar in, takahashik, djoerbanz, firmansyahi, naganawas, motomurak, satoh, kitamurak, pohanht, satos, 2009. ‘current hiv type 1 molecular epidemiology profile and identification of unique recombinant forms in jakarta, indonesia’. u.s. national llibrary of medicine, 8600 rockville pike, bethesda md, 20894 usa. 16. kotaki t, khairunisa sq, sukartiningrum sd, arfijanto mv, utsumi t, normalina i, handajani r, widiyanti p, rusli m, rahayu rp, lusida mi, hayashi y, nasronudin, kameoka m, 2013. ‘high prevalence of hiv-1 crf01_ae viruses among female commercial sex workers residing in surabaya, indonesia’. plos one, vol. 8 (12) pp. 1–8. 68 vol. 6. no. 3 september–december 2016 case report bacterial colony growth in the ventilator circuit of the intensive observation unit at rsud dr. soetomo surabaya fajar perdhana1, arie utariani1, bambang pujo semedi1, philia setiawan1a 1 departemen anestesiologi dan reanimasi fakultas kedokteran universitas airlangga -rsud dr. soetomo a corresponding author: philstawn@yahoo.com abstract ventilator-associated pneumonia (vap) remains a problem with the highest cos, morbidity and mortalityt in the intensive care unit (icu). the correlation between mechanical ventilation and pneumonia is considered as common sense, yet scientific evidence to support this statement is still needed. this research aims to analyze the bacterial colony grows in mechanical ventilation circuit and those grew in the patient’s sputum culture. we performed an observational study. samples for bacterial culture were taken from ventilator circuit and patient sputum on day-0, day-3 and day-7.. sputum samplings are collected using double catheter tracheal aspiration technique; results are then analyzed with chi-square test. while the similarity of bacteria species in ventilator circuit to patient’s sputum is analyzed with binomial test. two samples are dropped out immediately due to the rate of bacterial growth on day-0. bacterial colony growth in ventilator circuit shows a significant difference on day-3 and day-7 at 50% and 92% respectively (p = 0.05). a comparison for the bacterial similarity of the ventilator circuit and patient’s sputum shows that the bacterial growth on day-3 is 7 out of 14 (50%) and 3 with more than 105 cfu/ml colony; while on day-7, there are 13 out of 14 positive bacterial growth, both in the circuit and the patient’s sputum. among them, 5 out of 14 (35%) of the bacterial colony which grow in the circuit have the same species as those grow in patient’s sputum. the recent study shows that there is bacteria colony growth in the ventilator circuit after day-3 and a significant increase on day-7. almost half of the colony illustrates similar species from both ventilator circuit and patient’s sputum. this suggests that the bacterial growth on day-7 in the ventilator circuit might be related to those growth in patient’s sputum. keywords: ventilator circuit, vap, bacterial colony, bacteria species abstrak ventilator-associated pneumonia (vap) masih menjadi problematik perawatan pasien di icu dan menghabiskan biaya yang besar. vap menyebabkan morbiditas dan mortalitas yang tinggi. vap spesifik untuk infeksi nosokomial yang terjadi pada pasien yang mendapat ventilasi mekanik. hubungan antara sirkuit ventilasi mekanik dengan terjadinya infeksi paru sudah dianggap sebagai suatu fakta, walaupun tanpa bukti ilmiah. penelitian ini bertujuan untuk menganalisa pertumbuhan koloni bakteri pada sirkuit ventilator yang digunakan pada pasien di ruang observasi intensif rsud dr. soetomo surabaya. penelitian ini menggunakan analisis observasional. kultur bakteri diambil dengan menggunakan swab pada bagian inspirasi dari sirkuit ventilator pada 16 pasien yang dirawat dengan ventilasi mekanik di ruang observasi intensif rsud dr. soetomo surabaya. sirkuit ventilator dilakukan swab pada hari ke-0, ke-3 dan ke-7, kemudian dilakukan perhitungan . pengambilan sampling sputum menggunakan tehnik double catheter tracheal aspiration. hasil kemudian dianalisa menggunakan uji chi square dan kesamaan spesies bakteri pada sirkuit dengan sputum pasien dianalisa dengan uji binomial. 2 sampel drop out karena terdapat pertumbuhan koloni bakteri pada hari ke-0. terdapat perbedaan yang signifikan pertumbuhan koloni bakteri pada sirkuit ventilator hari ke-3 dan ke-7, 50% dan 92% dengan nilai p=0,05. terdapat pertumbuhan koloni bakteri pada sirkuit ventilator 7 dari 14 sampel (50%) pada hari ke-3 dan 3 dengan jumlah koloni > 105 cfu/ml, sedangkan pada hari ke-7 terdapat pertumbuhan koloni 13 dari 14 sampel baik pada sirkuit ventilator maupun pada sputum pasien. dan 5 (35%) diantaranya memiliki kesamaan spesies bakteri yang tumbuh pada sirkuit ventilator maupun pada sputum pasien. penelitian ini menunjukkan ada nya pertumbuhan koloni bakteri setelah hari ke-3, dan terjadi peningkatan jumlah koloni yang signifikan pada 69perdhana, et al.,: bacterial colony growth in the ventilator circuit hari ke-7. 50% dari pertumbuhan koloni ini menunjukkan kesamaan spesien bakteri antara sirkuit ventilator dan sputum pasien. hal ini menunjukkan ada kemungkinan pertumbuhan koloni bakteri pada harike-7 berkorelasi dengan kultur sputum pasien. kata kunci: sirkuit ventilator, vap, koloni bakteri, spesies bakteri introduction ventilator-associated pneumonia (vap) is the most common nosocomial infection which occurs at the intensive care unit (icu) and plays an important role in increasing the number of mortality and morbidity. vap specifically occurs in patients who are treated with mechanical ventilation. vap which occurs in the first 48-72 hours after intubation is categorized as an early onset of vap. it might be caused by aspiration as a complication during endotracheal tube insertion. vap which occurs for more than 72 hours is identified as the late onset of vap. it is directly correlated with mechanical ventilatory support.1–4 vap as the most commonly occurs as nosocomial infection with 9%-40% incidence rate might prolong hospitalization (2-3x longer), prolongs the length of stay in icu for 5-7 days, increases health cost, and increases mortality rate to 15-45%. vap’s cost is estimated to increase about $40,000 per patient and around $1.2 billion annually. previous researches showed that pneumonia infection rate obtained in the hospital (nosocomial) was 15% of all hospital nosocomial infection and 24-27% of it occured at the icu.1,2 various researches about risk factors related to vap (such as age, sex, trauma, copd) and the use of mechanical ventilation have been done several times. understanding the pathogenesis and epidemiology of vap is important to formulate a preventive strategy to fight this infection. the most common source for vap endemic is oropharynx colonization which is caused by endogenous flora or even pathogen flora from icu environment; especially from the hand of the healthcare workers, contaminated mechanical ventilation, and the air and the water at the icu. the digestive tract is also a potential source of secondary colonization and gram-negative of nosocomial bacteria storage. microorganism aspiration from the oropharynx, gaster, and tracheal secretion around balloon of endotracheal tube towards lower respiratory tract, which are supposed to be sterile in normal condition, are the most common endemic source of vap. epidemically, vap is mostly caused by contaminated ventilation, bronchoscopy, inhalation drugs, water (eg: legionella sp.), and air (eg: aspergillus sp.). a strategy to combat microbes from the oropharynx and digestive tract is by using chlorhexidine as an oral treatment, antimicrobial prophylaxis for inhalation drugs, or sucralfate as prophylaxis stress ulcer. moreover, aspiration prevention through patient positioning and continuous suction in the subglotic area are also proven to decrease vap risks. disinfection of mechanical ventilation circuit and bronchoscopy, legionella-free hospital water, and infection control from the medical aerosol are important for vap prevention. routine inspection towards vap as an early detection is important.1,3–5 the correlation between mechanical ventilation circuit and pulmonary infection is stated as a fact, however, there is no scientific explanation about it. ting-chang hsieh did a cohort of observational study towards 96 patients who were divided into 2 groups; group 1 underwent circuit replacement every 3 days, while group 2 underwent circuit replacement every 7 days. though there was no statistically significant difference, an increasing number of vap incidence was observed. there was 13% in group 1 and around 16% in group 2; besides, there was an increase in mortality rate at 22% in group 1 and 36% in group 2. other researches showed that the main cause of pneumonia in mechanical ventilation users was the colonization of the gastrointestinal tract, followed by aspiration around balloon of the endotracheal tube, and bacteria-transmitted health products.5,6 although there is no research about vap number in indonesia, based on the worldwide database, it is stated that vap incidence is high. the correlation between the mechanical ventilation and the occurrence of vap is stated as a fact, because the mechanical ventilation circuit is proven to be contaminated by pathogen bacteria, especially from the patient’s secretion. at the intensive observation unit of rsud dr. soetomo, there is no procedure about the continual replacement of ventilation mechanic circuit, thus, it might become a primary cause of vap infection there. this study is conducted to understand the bacterial colony growth in mechanical ventilation circuit at the intensive observation unit of rsud dr. soetomo and expected to standardize the procedure of mechanical ventilation circuit replacement at the intensive observation unit of rsud dr. soetomo. method this study employs observational analysis design in time series (3x measurement) with all patients at the intensive observation unit of rsud dr. soetomo as the research subject. the inclusion criteria of this research are the patients who are using mechanical ventilation and exhibit no pneumonia sign and symptom previously based on cpis criteria. the research consents are obtained from the family members to comply with the medical research ethical regulation from rsud dr.soetomo. while the exclusion criteria are patients with underlying diseases (copd, ards, neuromuscular disease) and those who refuse to participate in this experiment. the drop out criteria are the patients with less than 7 days of extubation, died 70 indonesian journal of tropical and infectious disease, vol. 6. no. 3 september–december 2016: 68–73 counted bacterila colony in the ventilator circuit observation time day 3 (%) day 7 (%) steril 7 (50,0) 1 (7,1) < 105 cfu/ml 7 (50,0) 10(71,5) ≥ 105 cfu/ml 3(21,4) table 2. bacterial species in the ventilator circuit and sputum culture of the patient on day 3 and day 7 bacterial species ventilator circuit sputum culture day 3 (%) day 7 (%) day 3 (%) day 7 (%) steril 7(50,0) 1(7,1) 8(57,1) 1(7,1) acinobacter spp 5(35,7) 7(50,0) 5(35,7) 11(78,6) klebsiella pneumonia 1(7,1) 3(21,4) pseudomonas aeruginosa 1(7,1) 1(7,1) 1(7,1) 2(14,3) enterobacter aerogenes 2(14,3) figure 1. bacterial species in the ventilator circuit on day 3 and day 7 figure 2. patient culture on day 3 and day 7 in less than 7 days, and develop bacterial growth within 48 hours. the methods exercised in this research aim to understand the growth of bacteria using bacterial colony measurement. measuring bacterial colony can be done using pour plate method. colony counter is also used to facilitate the accurate calculation of the colony. the sample is taken in ventilator circuit on day-0 before the circuit is connected to the patient, then it is re-done on day-3 and day-7 after the ventilator application. after bacterial colony is being calculated, it will be scored. besides calculating the bacterial growth in different days, the bacterial culture is performed to study the bacterial species in patient’s sputum. all subjects are treated with usual standardized vap prevention. t-test analysis is also conducted to understand the difference between various colony growth on different days (day-0, day-3, and day-7) for the patients who undergo mechanical ventilation treatments. result and discussion from 16 samples, 2 samples are dropped out due to bacterial growth on day-0. the characteristic analysis and data distribution illustrate that male respondents are recorded in 42.9% and female in 57.1%. various colony growth is obtained from the sample. the data is categorized into 3 groups, namely a sterile group, a group with the colony number less than 105 cfu/ml, and a group with colony number more than 105 cfu/ml. table 2 explains about the observed bacterial growth on day-3 in which the number of sterile group is 7 (50%), while the less than 105 cfu/ml bacteria colony has 7 samples (50%). on day-7, the number of sterile group is 1 sample (7.1%); the less than 105 cfu/ml bacterial colony has 10 samples (71.5%); and the more than 105 cfu/ml bacterial colony has 3 samples (21.4%). table 2 and figure 1 illustrate the bacteria species growing in ventilator circuit on day-3 are derived from 5 samples of acinetobacter spp. (35.7%); 1 sample of pseudomonas aeruginosa (7.1%); 1 sample of klebsiella pneumonia (7.1%); and 7 samples with no bacteria growth (50%). on the 7th day, 7 samples (50%) of acinetobacter spp.; 3 samples (21.4%) of klebsiella pneumonia; 1 sample of pseudomonas aeruginosa (7.1%); 2 samples of enterobacter aerogenes (14.3%); and 1 sample with no bacteria colony growth. table 1. bacterial colony growth of the ventilator circuit on day 3 and day 7 furthermore, table 2 and figure 2 also displays the bacterial culture results of patient’s sputum on day-3, namely 5 samples of acinetobacter sp (35.7%); 1 sample of pseudomonas aeruginosa culture (7.1%); and 8 samples with no bacteria growth (57.1%). on the 7th day of evaluation, 11 samples of acinetobacter sp. (78.6%); 2 samples with pseudomonas aeruginosa culture (14.3%); and 1 sample with no bacterial growth (7.1%) are obtained. table 3 demonstrates data recapitulation about bacterial colony growth in ventilator circuit on day-3 and day-7. according to the data, chi-square statistic test is done to understand the significant difference in bacterial colony growth in different days. 71perdhana, et al.,: bacterial colony growth in the ventilator circuit table 3. comparison test bacterial colony growth in the ventilator circuit on day 3 and day 7 day 3 day 7 kappa harga ppositive result (%) negative result (%) positive result (%) 6 (42,9) 1 (7,1) 0,429 0,049 negative result (%) 3 (21,4) 4 (28,6) table 4. binomial test for similarity bacterial species between ventilator circuit and patient's sputum culture on day 3 ventilator circuit sputum culture kappa harga ppositive result (%) negative result (%) positive result (%) 4 (28,6) 1 (7,1) 0,689 0,010 negative result (%) 1 (7,1) 8 (57,1) table 5. binomial test for similarity bacterial species between ventilator circuit and patient's sputum culture on day 7 ventilator circuit sputum culture kappa harga ppositive result (%) negative result (%) positive result (%) 4 (28,6) 1 (7,1) 0,689 0,010 negative result (%) 1 (7,1) 8 (57,1) table 6. correlations between bacterial colony growth in the circuit ventilator with cpis >6 cpis 3 day 3 cpis 7 day 7 cpis 3 day 3 and cpis 7 day 7negative positive negative 4 7 13 positive 0 2 0.016 the statistic shows that there is a significant difference between bacterial growth in day-3 and day-7 (p = 0.049). table 4 shows that the observation on day-3 found 7 samples with no bacterial colony in ventilator circuit or in patient’s sputum (50%); 3 samples with similar bacterial species (21.4%); and 4 samples with no similar bacteria species in both ventilator circuit and patient’s sputum (28.6%). based on the statistical analysis using binomial test, there is a significant difference between both groups on day-3 (p = 0.01). table 5 shows the observation on day-7, it proves that there are 7 samples containing similar bacterial species in the ventilator circuit and patient’s sputum (50%) and 7 samples without similar bacterial species in both locations. after being statistically tested using binomial test, the bacterial species in ventilator circuit and the patient’s sputum on day-7 show no significant difference (p = 0.515). the correlation analysis of bacterial colony in ventilator circuit (cpis> 6) is also tested in this research. table 6 shows that during the day-3 observation, there are 2 patients with cpis more than 6; while during the day-7 observation, there are 12 patients with cpis more than 6. using binomial test, it is proven that there is no significant difference in the amount of bacterial colony in ventilator circuit (cpis> 6) on patients who undergo mechanical ventilation treatment. based on this research’s comparative study, it is found that there is a significant difference between bacterial colony growth on day-3 and day-7 (p = 0.049). bacterial colony growth in ventilator might be caused by many factors, such as humidifier which produces microorganism carrying water drops, thus, the water inside the humidifier might be contaminated as well. therefore, the water refill in humidifier should be done aseptically and use sterile fluid. the condenser in ventilator circuit might also contaminate the ventilator circuit, if the drainage is conducted thoroughly. around 1980, there was a high risk of infection related to the contaminated nebulizer reservoir. a drug used by nebulizer might contaminate the ventilator circuit and penetrate into patient’s respiratory tract. the contamination suffered by some health workers may also play a role in ventilator circuit contamination; therefore, all treatments (such as sterilization, ventilator circuit formation, and ventilator circuit maintenance) must be performed aseptically.1,6–8 the increase of bacterial colony significantly on day-3 to day-7 in this research still cannot predict the main role of ventilator circuit in vap contamination, because of the vap’s complex pathogenesis and various factors possibly cause vap. however, the bacterial colony growth in ventilator circuit still cannot be ignored, because the colony growth in ventilator circuit might penetrate the lung tissue through inhalation and further cause pneumonia. based long and fink’s researches, the suspension of maintenance and ventilator circuit replacement from once a day to twice a day might decrease vap occurrence. while cdc and the healthcare injection control practices advisory committee have issued some guidelines for preventing health care associated pneumonia, ventilator circuit replacement is recommended to be conducted only if the circuit is dirty or malfunctioned. other research shows that ventilator circuit replacement over 48 hours is safe and might not increase vap prevalence . 72 indonesian journal of tropical and infectious disease, vol. 6. no. 3 september–december 2016: 68–73 table 7. result of ventilator circuit and sputum culture day 0 day 3 day 7 bacterial colony growth in the circuit ventilator bacterial species in the circuit ventilator patient's sputum culture bacterial colony growth in the circuit ventilator bacterial species in the circuit ventilator patient's sputum culture bacterial colony growth in the circuit ventilator bacterial species in the circuit ventilator patient's sputum culture patient 1 steril steril > 105 cfu/ml acinetobacter sp acinetobacter sp > 105 cfu/ml acinetobacter sp acinetobacter sp patient 2 steril steril 45 cfu/ ml acinetobacter sp acinetobacter sp 105 cfu/ ml acinetobacter sp acinetobacter sp patient 3 steril steril steril steril 105 cfu/ ml klebsiella pneumonia pseudomonas aeruginosa patient 4 steril steril 75 cfu/ ml acinetobacter sp acinetobacter sp > 105 cfu/ml acinetobacter sp acinetobacter sp patient 5 steril steril 100 cfu/ ml acinetobacter sp pseudomonas aeruginosa 105 cfu/ ml acinetobacter sp acinetobacter sp patient 6 steril steril steril steril 105 cfu/ ml acinetobacter sp acinetobacter sp patient 7 steril steril steril steril 105 cfu/ ml klebsiella pneumonia acinetobacter sp patient 8 steril steril steril steril 25 cfu/ ml enterobacter aerogenes steril patient 9 steril steril steril steril steril acinetobacter sp patient 10 steril steril 105 cfu/ ml klebsiella pneumonia acinetobacter sp 12.2 × 105 cfu/ ml klebsiella pneumonia acinetobacter sp patient 11 steril steril steril steril 100 cfu/ ml acinetobacter sp acinetobacter sp patient 12 steril steril 100 cfu/ ml pseudomonas aeruginosa steril 12.5 × 105 cfu/ ml pseudomonas aeruginosa acinetobacter sp patient 13 steril steril steril steril 105 cfu/ ml acinetobacter sp pseudomonas aeruginosa patient 14 steril steril 105 cfu/ ml acinetobacter sp acinetobacter sp > 105 cfu/ml enterobacter aerogenes acinetobacter sp cfu : colony form unit table 7 is expected to inform the readers about the treatment and ventilator circuit replacement at the icu. the similarity test, which is conducted to analyze bacteria growth in ventilator circuit and patient’s sputum on day-3, indicates a significant difference (p = 0.010). it might be caused by the complex pathogenesis of vap. therefore, the bacteria growth might be caused by many factors. the bacteria in patient’s sputum might come from the digestive tract colonization of the pathogenic microorganism and increase the chance of contaminated secrete aspiration to lower respiratory tract; further, it might also colonize the oropharynx mucosal surface.1,3,4 optic fiber bronchoscopy, tracheal mucous suction, and respirator might induce pathogen bacteria contamination into the lower expiratory tract. the bacterial colony in ventilator circuit might be coming from the healthcare worker’s hand contamination, water, air, or even patient’s own secrete. bacteria test similarity shows that the bacteria growth in ventilator circuit and patient’s sputum culture on day-7 has no significant difference (p = 0.515). from the day-7 of observation, there is no proof whether the bacterial colonization of the patient’s respiratory is coming from the ventilator circuit or not. the bacterial colony growth in patient’s sputum reaches 50% of the total sample. it might be caused by the microorganism mapping at the icu itself. meanwhile, the bacteria commonly found in the ventilator circuit, humidifier, and respirometer are acinetobacter calcoaceticus, burkholderia cereus, and pseudomonas aeruginosa.1 in this research, there is no conclusion can be drawn on whether bacterial colonization in patient’s sputum is related to 73perdhana, et al.,: bacterial colony growth in the ventilator circuit the bacterial colonization in the ventilator circuit. those factors may affect this research’s results. this research also analyzes the correlation of the bacterial colony in ventilator circuit with cpis criteria more than 6 on the patient with mechanical ventilation help and diagnosed as pneumonia. this research shows no significant correlation between bacterial colony size in ventilator circuit with cpis > 6 with the help of mechanical ventilation, stated by p=0.016. on the third day of observation, there are 2 patients with cpis>16; while on the 7th day of observation, there are 13 patients with cpis> 6 out of 14 patients in this research’s sample. the early onset of vap might be observed in less than 4 days and the late onset of vap can be observed over 4 days after the patient is treated with mechanical ventilation. pathogen which causes pneumonia on the early onset of vap and late onset of vap is usually different. the early onset of vap mostly has a good prognosis. moreover, the early onset of vap might be caused by haemophilus influenzae and streptococcus pneumonia; while the late onset of vap might be caused by high-risk pathogens, such as pseudomonas aeruginosa, acinetobacter spp., and stenotrophomonas rnaltophilia. as stated by kollef, these high-risk pathogens record a high mortality rate (65%) compared to the late onset of vap which is caused by other pathogen microorganisms (31%). besides the contaminated ventilator circuit, there are various risk factors causing vap, such as surgery. post-surgery patient is exposed to higher rate of vap occurrence. based on american society of anesthesiologist, the risk factors of pneumonia post surgery include smoking, long inpatient treatment, longer surgery operation, thorax, and upper abdomen surgery.8–10 the irrational antibiotic usage also plays a role in the occurrence of nosocomial pneumonia and the infection of antibiotic resistant microbes. stress ulcer prophylaxis also influences bacterial colonization in the digestive tract. the prophylaxis stress ulcer which is unable to change gastric ph have a lower bacterial colonization rate and in turn, may lower the pneumonia nosocomial rate as well. endotracheal tube, tracheostomy, and reintubation may also lower patient’s immune system and further causes local trauma and inflammation. it can increase the probability of pathogen bacterial aspiration from the oropharynx area, specifically around the cuff. oropharyngeal secretion’s continuous suction, elevated head position, the use of nasogastric tube, and enteral feeding play a part in vap occurences.9,10 conclusion the recent study shows that there are bacterial colony growth in the ventilator circuit after day-3 and significant increase in the number of colony on day-7. almost half of the colony displays similar species in both ventilator circuit and patient’s sputum. this suggests that the growth of bactery on day-7 in the ventilator circuit might be related to the growth in patient’s sputum. references 1. safdar n, crnich cj, maki dg. the pathogenesis of ventilatorassociated pneumonia: its relevance to developing effective strategies for prevention. respir care. 2005 jun;50(6):725-3941. 2. hooser t van. ventilator-associated pneumonia ( vap ) best practice strategies for caregivers by d . theron van hooser. kimberly-clark heal care. 2002; 3. kollef mh. the prevention of ventilator-associated pneumonia. n engl j med. 1999 feb 25;340(8):627–34. 4. wiryana m. ventilator associated pneumonia. j penyakit dalam. 2007 sep;8(3):1–15. 5. branson rd. the ventilator circuit and ventilator-associated pneumonia. respir care. 2005 jun;50(6):774-87-7. 6. hess dr, kallstrom tj, mottram cd, myers tr, sorenson hm, vines dl, et al. care of the ventilator circuit and its relation to ventilator-associated pneumonia. respir care. 2003 sep;48(9):869–79. 7. craven de, kunches lm, kilinsky v, lichtenberg da, make bj, mccabe wr. risk factors for pneumonia and fatality in patients receiving continuous mechanical ventilation. am rev respir dis. 1986 may;133(5):792–6. 8. chastre j, fagon j-y. ventilator-associated pneumonia. am j respir crit care med. 2002 apr 1;165(7):867–903. 9. garibaldi ra, britt mr, coleman ml, reading jc, pace nl. risk factors for postoperative pneumonia. am j med. 1981 mar;70(3):677–80. 10. rello j, ausina v, ricart m, castella j, prats g. impact of previous antimicrobial therapy on the etiology and outcome of ventilatorassociated pneumonia. chest. 1993 oct;104(4):1230–5. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 154 vol. 6 no. 6 september–december 2017 research report prevalence of helminth eggs in cat feces contaminating public areas in surabaya nurul tri wahyudi1, lucia tri suwanti2,5a, kusnoto2, sri mumpuni2, ira sari yudaniayanti3, maslichah mafruchati4 1 veterinary medicine, universitas airlangga 2 department of parasitology of veterinary medicine, universitas airlangga 3 department of veterinary clinical of veterinary medicine, universitas airlangga 4 department of anatomy faculty of veterinary medicine, universitas airlangga 5 institute of tropical desease, universitas airlangga a corresponding author: tswanti@gmail.com abstract helminthiasis can be transmitted from animals to humans (zoonosis). helminthiasis can cause cutaneus larva migrants, visceral larva migrant, and occular larva migrants. cats are the most easily animals can found in public areas. cats have a habit of defecating in areas, such as dusty soil, gardens, sand pits, trash cans, and even children’s playgrounds. proximity of human life with a stray cats is one of the potential that can helminthiasis transmited to humans. the purpose of this study was to assess the prevalence of helminth eggs (species and number) observed in cat feces contaminating public areas in surabaya. cross-sectional study have been observations cats existense and examination laboratory of 180 cat fecal samples were collected from canteens, markets, villages, schools, and parks across 5 areas in surabaya. helminth eggs present in fecal samples were identified using direct smear, sedimentation, and flotation methodes, and quantified as fecal egg count (eggs per gram of feces) with mcmasster method. the test results positive for helminthiasis if found one or more types of helminth eggs in fecal samples. helminth eggs were present in 68 (37.8%) of the 180 cat fecal samples contaminating public areas in surabaya. results of chi-squared analysis confirmed the prevalence of helminth eggs in cat fecal samples contaminating canteen, markets, villages, schools, and parks in surabaya (p > 0.05). the species causing environmental contamination included ancylostoma sp. eggs, toxocara cati eggs, and toxascaris leonina eggs. the level of environmental contamination, as assessed using anova, was 200 eggs per gram of feces. keywords: prevalensi, helminthiasis, cats feces, helminth eggs, public areas abstrak kecacingan merupakan penyakit yang dapat menular dari hewan kepada manusia (zoonosis). penyakit kecacingan dapat mengakibatkan cutaneus larva migran, visceral larva migran, dan occular larva migran. kucing merupakan salah satu hewan yang saat mudah ditemukan di tempat umum. kucing memiliki kebiasaan buang air besar di berbagai daerah, seperti tanah berdebu, kebun, lubang pasir, tempat sampah, dan bahkan taman bermain anak-anak. kedekatan kehidupan manusia dengan kucing liar merupakan potensi yang dapat menularkan penyakit kecacingan kepada manusia. tujuan dari penelitian ini adalah untuk mengetahui prevalensi telur cacing (jenis dan menghitung jumlah telur cacing) dari pengamatan feses kucing yang mencemari lingkungan di berbagai tempat umum di surabaya. penelitian crossectional mengamati keberadaan kucing liar dan melakukan pemeriksaan laboratorium terhadap 180 feses kucing dikumpulkan dari beberapa tempat yaitu kantin, pasar, perkampungan, sekolah dan taman dari lima wilayah di surabaya. feses kucing diperiksa dengan metode natif, sedimentasi dan apung, dan dilanjutkan menghitung jumlah telur (telur per gram feses) menggunakan metode mcmasster. hasil uji dinyatakan positif helminthiasis apabila ditemukan 1 jenis atau lebih telur cacing pada sampel feses. hasil penelitian menunjukkan bahwa kandungan telur cacing pada feses kucing yang mencemari area publik di kota surabaya sebesar 37,8% atau 68 sampel positif dari 180 sampel yang diamati. hasil analisis chi square didapatkan p > 0,05 155wahyudi, et al.: prevalence of helminth eggs menunjukkan, telur cacing pada feses kucing telah mencemari lingkungan kantin, pasar, perumahan, sekolah dan taman di berbagai wilayah surabaya. pencemaran lingkungan disebabkan oleh telur ancylostoma sp., telur toxocara cati dan telur toxascaris leonina. serta hasil anova menunjukkan jumlah pencemaran lingkungan oleh telur cacing sebesar 200 telur per gram feses. kata kunci: prevalensi, kecacingan, feses kucing, telur cacing, tempat umum introduction cats often live in close proximity to humans and are common in the environment, both as pets and as stray animals. cats live in public places and have a habit of defecating in areas, such as dusty soil, gardens, sand pits, trash cans, and even children’s playgrounds. increase environmental contamination levels by feces and zoonotic eggs can caused by environmental factors and increasing cat populations.1 environmental contamination by helminth eggs can transmit helminthiasis among animals as well as from animals to humans (zoonosis). even today, helminthiasis affects many indonesians, particularly children,2 and the prevalence of helminths in indonesians is reportedly 28.12%.3 cats can transmit helminths, such as toxocara cati, toxascaris leonina, dipylidium caninum, spirometra mansoni, ancylostoma tubaeforme, ancylostoma braziliense, gnathostoma spinigerum, strongyloides sp., taenia taeniaeformis, capillaria sp., trichuris sp., and physaloptera sp., to humans, causing hemintheasis.4 helminthiasis in humans caused by transmission from cats include cutaneous larva migrans caused by ancylostoma sp. and strongyloides sp.; visceral larva migrans and ocular larva migrans caused by toxocara cati, toxascaris leonine, and gnathostoma spinigerum; and sparganosis caused by spirometra mansoni.4 according to the world health organitation,5 helminths can transmitted to humans through soil and water contaminated with high levels of helminth eggs. helminth eggs develop in the soil into infective stages that can be transmitted to humans or animals acting as reservoirs. helminth eggs can also contaminate food and water resources of human and animal consumption or can be directly ingested through contaminated soil by children on playgrounds. in addition, transdermal transmission occurs for infective helminth stages that can actively penetrate the skin after direct contact with contaminated feces or soil. thus, it is necessary to increase public awareness regarding potential diseases arising from the contamination by helminth eggs. on the basis of the abovementioned problems, this study was performed to determine the level of environmental contamination by helminth eggs present in the digestive tract of cats in various public places in surabaya. the aim of this study was to provide information on the potential transmission of diseases due to helminth eggs present in cat feces contaminating public areas in surabaya. material and method research design was cross-sectional, with two types of data collected as results of the examination of fecal samples in the laboratory and observations cats existense at the site of feces collection. samples were examined at the parasite laboratory faculty of veterinary medicine of the universitas airlangga. in total, 180 fecal samples7 in soil were collected from several public places, including markets, villages, parks, canteens, and schools, within north surabaya, east surabaya, south surabaya, west surabaya, and central surabaya. the study was conducted for 4 months, from march to june, 2017. direct smear method fecal specimens were collected using the tip of a glass stirrer and smeared onto a glass microscope slide. one to two drops of water were added and mixed well using a glass stirrer. a coverslip was placed over the mixture. the smear was then examined under a light microscope at 100× magnification.6 sedimentation method fecal samples was made into a suspension at a ratio of 1 part feces to 10 parts water and filtered through a tea strainer before placing in a conical centrifuge tube. the suspension was centrifuged at 1500 rpm for 5 minutes. the supernatant was discarded, and the sediment resuspended in water and centrifuged again for 5 min. this process was performed several times until the supernatant was clear. the final supernatant was discarded, leaving a small amount of sediment. the sediment was stirred, and a sample was removed using a pasteur pipette. the sediment was placed on a glass microscope slide and covered with a coverslip. the sample was examined under a microscope at 100× magnification.6 flotation method after the examination of fecal samples using the sedimentation method, the sediment was diluted with saturated brown sugar solution to 1 cm below the top of the conical centrifuge tube. the mixture was centrifuged at 1500 rpm for 5 min. brown sugar solution was used because it has a low viscosity and a higher specific gravity (1.20) than the organisms within the sample; thus, the helminth eggs floated to the top.10 following centrifugation, the conical tube was placed on a tube rack, and brown sugar solution was slowly added until the surface of the solution showed a convex form. a coverslip was gently placed on 156 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 154–159 the top of the tube and left for 2 minutes. the coverslip was removed and placed on a glass microscope slide, which was examined under a microscope at 100× magnification.6 mcmaster method for samples positive for helminth eggs, helminth eggs were counted using the mcmaster method. briefly, up to 2 g of feces were weighed, crushed, and 28 ml of saturated sugar solution was added. the sample was filtered into a glass scale. a pipette was used to transfer the sample, filling the chambers of the mcmaster slide. the sample was allowed to settle for 2–3 minutes until the eggs floated to the surface. the mcmaster slide was placed under a microscope at 100× magnification. the number of helminth eggs within the grid areas (0.5 ml of solution) was counted. the average number of eggs in each grid area was multiplied by 60 to obtain the number of eggs per gram of feces.6 data analysis the prevalence of parasites was calculated in terms of positive samples using the following formula: prevalence = (n positive samples/n samples examined) × 100 data were analyzed using ibm spss 24.0 followed by chi-squared analysis to assess the regional difference in eggs numbers, which possibly affect the helminth eggs in cat feces contaminating public areas in surabaya. anova was used to assess the level of environmental contamination by helminth eggs per gram of feces in various public places in surabaya. result and discussion eggs of several species of helminths, including nematodes, such as ancylostoma sp., toxocara cati, and toxascaris leonine, were detected in cat feces contaminating public areas in surabaya. the detected helminth eggs were identified by comparing the morphology and measurements of eggs with those previously reported.7,4,8 helminth eggs were measured using the optilab imageraster program. figure 1 presents eggs of ancylostoma sp. these eggs were 62.8–66.4 × 43.2–46.2 µm in size, oval-shaped, with a thin wall consisting of 2 layers, and contained 2–8 blastomers. this is consistent with the description by taylor,8 who reported eggs of ancylostoma sp. to be ovalshaped, 56–75 × 34–47 µm in size, and containing 2–8 blastomers. toxocara cati eggs were 62.4–64.5 × 73.8–74.9 µm in size, slightly rounded, with slightly mottled brownish wall, and were surrounded by thick layers of albumin. this is in accordance with the description by subekti,9 who reported that the egg diameter of toxocara cati was 65–75 µm, the egg was slightly rounded, and had a slightly mottled wall. toxascaris leonina eggs were oval-shaped, with a smooth wall, and measured 75–85 × 75 µm.9 1a 1b 2a 2b 3a 3b figure 1. egg of helminths contaminating the environment in public places in surabaya. (1). ancylostoma sp. eggs. (2). toxocara cati eggs. (3). toxascaris leonine eggs (a). 100× magnification (b). 400× magnification. ancylostoma sp. was the most common type of helminth detected in the studied public areas in surabaya. in the 180 fecal samples tested, ancylostoma sp. was the single source of contamination in 42 samples, and was present in combination with other types of helminth in 5 samples. toxocara cati was the single source of contamination in 17 samples and was present with other types of helminth in 4 samples. toxascaris leonina was the single source of contamination in 4 samples and was present with another type of helminth in 1 sample. interestingly, only helminths of the nematoda class were detected, and no cestoda class eggs were found. this may be due to the resistance of cestoda class eggs to environmental factors. moreover, cestoda helminths infecting cats may not reproduce; 1a 1b 2a 2b 3a 3b figure 1. egg of helminths contaminating the environment in public places in surabaya. (1). ancylostoma sp. eggs. (2). toxocara cati eggs. (3). toxascaris leonine eggs (a). 100× magnification (b). 400× magnification. ancylostoma sp. was the most common type of helminth detected in the studied public areas in surabaya. in the 180 fecal samples tested, ancylostoma sp. was the single source of contamination in 42 samples, and was present in combination with other types of helminth in 5 samples. toxocara cati was the single source of contamination in 17 samples and was present with other types of helminth in 4 samples. toxascaris leonina was the single source of contamination in 4 samples and was present with another type of helminth in 1 sample. interestingly, only helminths of the nematoda class were detected, and no cestoda class eggs were found. this may be due to the resistance of cestoda class eggs to environmental factors. moreover, cestoda helminths infecting cats may not reproduce; 1a 1b 2a 2b 3a 3b figure 1. egg of helminths contaminating the environment in public places in surabaya. (1). ancylostoma sp. eggs. (2). toxocara cati eggs. (3). toxascaris leonine eggs (a). 100× magnification (b). 400× magnification. ancylostoma sp. was the most common type of helminth detected in the studied public areas in surabaya. in the 180 fecal samples tested, ancylostoma sp. was the single source of contamination in 42 samples, and was present in combination with other types of helminth in 5 samples. toxocara cati was the single source of contamination in 17 samples and was present with other types of helminth in 4 samples. toxascaris leonina was the single source of contamination in 4 samples and was present with another type of helminth in 1 sample. interestingly, only helminths of the nematoda class were detected, and no cestoda class eggs were found. this may be due to the resistance of cestoda class eggs to environmental factors. moreover, cestoda helminths infecting cats may not reproduce; 1a 1b 2a 2b 3a 3b figure 1. egg of helminths contaminating the environment in public places in surabaya. (1). ancylostoma sp. eggs. (2). toxocara cati eggs. (3). toxascaris leonine eggs (a). 100× magnification (b). 400× magnification. ancylostoma sp. was the most common type of helminth detected in the studied public areas in surabaya. in the 180 fecal samples tested, ancylostoma sp. was the single source of contamination in 42 samples, and was present in combination with other types of helminth in 5 samples. toxocara cati was the single source of contamination in 17 samples and was present with other types of helminth in 4 samples. toxascaris leonina was the single source of contamination in 4 samples and was present with another type of helminth in 1 sample. interestingly, only helminths of the nematoda class were detected, and no cestoda class eggs were found. this may be due to the resistance of cestoda class eggs to environmental factors. moreover, cestoda helminths infecting cats may not reproduce; 1a 1b 2a 2b 3a 3b figure 1. egg of helminths contaminating the environment in public places in surabaya. (1). ancylostoma sp. eggs. (2). toxocara cati eggs. (3). toxascaris leonine eggs (a). 100× magnification (b). 400× magnification. ancylostoma sp. was the most common type of helminth detected in the studied public areas in surabaya. in the 180 fecal samples tested, ancylostoma sp. was the single source of contamination in 42 samples, and was present in combination with other types of helminth in 5 samples. toxocara cati was the single source of contamination in 17 samples and was present with other types of helminth in 4 samples. toxascaris leonina was the single source of contamination in 4 samples and was present with another type of helminth in 1 sample. interestingly, only helminths of the nematoda class were detected, and no cestoda class eggs were found. this may be due to the resistance of cestoda class eggs to environmental factors. moreover, cestoda helminths infecting cats may not reproduce; 1a 1b 2a 2b 3a 3b figure 1. egg of helminths contaminating the environment in public places in surabaya. (1). ancylostoma sp. eggs. (2). toxocara cati eggs. (3). toxascaris leonine eggs (a). 100× magnification (b). 400× magnification. ancylostoma sp. was the most common type of helminth detected in the studied public areas in surabaya. in the 180 fecal samples tested, ancylostoma sp. was the single source of contamination in 42 samples, and was present in combination with other types of helminth in 5 samples. toxocara cati was the single source of contamination in 17 samples and was present with other types of helminth in 4 samples. toxascaris leonina was the single source of contamination in 4 samples and was present with another type of helminth in 1 sample. interestingly, only helminths of the nematoda class were detected, and no cestoda class eggs were found. this may be due to the resistance of cestoda class eggs to environmental factors. moreover, cestoda helminths infecting cats may not reproduce; figure 1. egg of helminths contaminating the environment in public places in surabaya. (1). ancylostoma sp. eggs. (2). toxocara cati eggs. (3). toxascaris leonine eggs (a). 100× magnification (b). 400× magnification. 157wahyudi, et al.: prevalence of helminth eggs ancylostoma sp. was the most common type of helminth detected in the studied public areas in surabaya. in the 180 fecal samples tested, ancylostoma sp. was the single source of contamination in 42 samples, and was present in combination with other types of helminth in 5 samples. toxocara cati was the single source of contamination in 17 samples and was present with other types of helminth in 4 samples. toxascaris leonina was the single source of contamination in 4 samples and was present with another type of helminth in 1 sample. interestingly, only helminths of the nematoda class were detected, and no cestoda class eggs were found. this may be due to the resistance of cestoda class eggs to environmental factors. moreover, cestoda helminths infecting cats may not reproduce; therefore, cestoda eggs contained in the proglottid would not be detected in cat feces. the results of this study are similar to those reported by tun et al,10 who examined fresh cat feces contaminated with eggs of hookworms, toxocara sp., trichuris sp., spirometra, and ascaris. they also reported the presence of hookworm, ascaris sp., and toxocara sp eggs in contaminated soil samples.10 helminth infections commonly diagnosed in cats are ancylostoma sp. and toxocara sp.4 presence of helminth eggs in cat feces contaminating public areas in surabaya laboratory examination using the direct smear, sedimentation, and flotation methods of 180 cat fecal samples collected from various public places in surabaya, including markets, villages, parks, canteens, and schools, in north surabaya, east surabaya, south surabaya, west surabaya, and central surabaya from march to june 2017 as summarized in table 1 and table 2. table 1. presence of helminth eggs in cat feces contaminating public areas in surabaya result number of samples percentage (%) positive 68 37.8 negative 112 62.2 total 180 100 based on the number of fecal samples positive for helminth eggs, the level of environmental contamination was 37.8%. table 2. types of helminth eggs identified in cat feces contaminating public areas in surabaya type of helminth eggs positive samples percentage (%) ancylostoma sp. 42/180 23.3 toxocara cati 17/180 9.5 toxascaris leonina 4/180 2.2 ancylostoma sp. and toxocara cati 4/180 2.2 toxocara cati and toxascaris leonine 1/180 0.6 total 68/180 37.8 chi-squared analysis of data obtained from samples collected from various areas, including north surabaya, east surabaya, south surabaya, west surabaya, and central surabaya revealed no significant difference (p > 0.05) among the various areas of surabaya in terms of the prevalence of helminth eggs. these findings indicate that various areas across surabaya are contaminated with helminth eggs from cat feces. contamination levels and the species of helminth eggs that contaminate the environment in various areas in surabaya are summarized in table 3. the results of chi-square analysis of data obtained from samples collected from various public places (canteens, markets, villages, schools, and parks) in surabaya indicated no significant differences (p > 0.05) among the various public places in terms of environmental contamination by helminth eggs. the canteens, markets, villages, schools, and parks in surabaya had the same levels of environmental contamination by helminth eggs from cat feces. the levels of environmental contamination by helminth eggs from cat feces at different collection sites are listed in table 4. the prevalence of helminth eggs in cat feces contaminating public areas in surabaya (37.8%) is greater than the reported prevalence of helminth infections in a certain pet shops in surabaya city (30.7%).16 however, the prevalence is less than that in the feces of dogs culled for consumption and stray cats in surabaya, as reported by subekti,11 who reported a helminth infection prevalence of 63.9%. this difference may be due to the face that this table 3. prevalence of helminth eggs in cat feces contaminating public areas in surabaya area of surabaya contamination by helminth eggs type of helminth ancylostoma sp. toxocara cati toxascaris leonina north 12/36 (33.3%) + + − east 18/36 (50.0%) + + + south 14/36 (38.8%) + + + west 12/36 (33.3%) + + − central 12/36 (33.3%) + − − total 68/180 (37.8%) 158 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 154–159 study examined the prevalence of helminth infections in pet cat, who may have received antihelmintics. wastomi12 observed gastrointestinal helminths in cats, reporting the presence of eggs in stray cat feces contaminating public areas in surabaya. the helminth eggs found in cat feces in surabaya could be transmitted to humans (zoonosis) and animals. eggs of ancylostoma sp. were the most common helminth detected in our samples. infective eggs can penetrate the skin in humans and produce hives called cutaneous larva migrans.13 environmental contamination is also caused by toxocara cati and toxascaris leonina. toxocara sp. eggs develop into an infective stage, and infective eggs can be ingested by humans, causing visceral larval migrans as well as diarrhea and vomiting. ocular larval migrans may also occur due to infection of toxocara sp., and this can cause permanent eye damage in humans.14 prevalence of helminth eggs in cat feces contaminating public areas in surabaya the results of the anova analysis comparing various public areas (canteens, markets, villages, schools, and parks) in surabaya to the level of environmental contamination by helminth eggs from cat feces revealed an f-arithmetic value < f critical value, showing no real difference between the various public areas in surabaya and the level of contamination by helminth eggs. canteens, markets, villages, schools, and parks in surabaya showed the same environmental contamination levels by helminth eggs from cat feces. results of the mcmaster calculation of environmental contamination by helminth eggs from cat feces in public areas in surabaya are summarized in table 5. the prevalence may vary according to the life cycle of the genus of helminth, the presence of paratenic hosts, the number of eggs produced by females, (200–6000 eggs/day), the host immune status, and the helminth egg resistance to environmental factors.15 contamination by helminth eggs in canteens, markets, villages, schools, and parks in surabaya may be affected by various factors, such as population density, open surface area, poor environmental hygiene, waste available for cats to feed on, presence of possible paratenic hosts, species of helminths present, and the immune status of the cats against helminth infections. the areas selected for sampling in this study were places, where humans, particularly children, are in close contact with contaminated soil and water. the risk for contamination by helminth eggs from cat feces should be minimized by raising awareness regarding helminthiasis. environmental and personal hygiene should be maintained, particularly after handling animals. disposal of cat feces should be encouraged, and their population should be controlled via good cat care. healthy cat food and timely antihelmitics doses should be provided to maintain the health of cats. these measures will help in preventing the transmission of helminths in humans and animals. conclusion our findings indicate that the prevalence of helminth eggs in cat feces has contaminated canteens, markets, villages, schools, and parks in public areas of surabaya, with zoonotic helminth eggs of ancylostoma sp., toxocara cati, and toxascaris leonina, at a contamination level of 200 eggs/g of feces. table 4. presence of helminth eggs in cat feces contaminating different public areas sample of collection type of helminth number and percentage of helminth eggs in public areas canteens markets villages schools parks single contamination ancylostoma sp. 7/36 (19.5%) 8/36 (22.2%) 10/36 (27.7%) 8/36 (22.2%) 9/36 (25.0%) toxocara cati 3/36 (8.3%) 4/36 (11.1%) 3/36 (8.3%) 3/36 (8.3%) 4/36 (11.1%) toxascaris leonina 1/36 (2.7%) 1/36 (2.7%) 0/36 (0%) 2/36 (5.5 %) 0/36 (0.0%) mixed contamination ancylostoma sp. + toxocara cati 1/36 (2.7%) 1/36 (2.7%) 2/36 (5.5%) 0/36 (0.0%) 0/36 (0.0%) toxocara cati + toxascaris leonina 1/36 (2.7%) 0/36 (0.0%) 0/36 (0.0%) 0/36 (0.0%) 0/36 (0.0%) total 36.1% 38.8% 41.6% 36.1% 36.1% table 5. number of helminth eggs in cat feces contaminating public areas in surabaya public area in surabaya eggs per gram canteens 180a ± 91.6 markets 205a ± 101.8 villages 196a ± 147.7 schools 226a ± 88.8 parks 189a ± 91.1 mean 200 ± 105.9 159wahyudi, et al.: prevalence of helminth eggs acknowledgement there are no conflicts of interest to declare. i am grateful to the principal of parasitology laboratory of faculty veterinary medicine, universitas airlangga and to pt. bayer indonesia, who have provided research sponsorship. references 1. soedarto. mencegah dan mengatasi penyakit toxoplasmosis melindungi ibu dan anak. sagung seto. 2011; 2. tjahajati i, gunanti, suwarno, sutisna a, widjajanti s, raharjo e, et al. manual penyakit hewan mamalia. kesehatan d, hewan, editors. subdit pengamatan penyakit hewan direktorat kesehat hewan direktorat jenderal peternak dan kesehat hewan. 2014; 3. octama ci. angka prevalensi cacingan di indonesia mencapai 28,12 persen [internet]. (beritasatu.com. 2015 [cited 2017 feb 18]. available from: http://www.beritasatu.com/kesehatan/319918-angkaprevalensi 4. bowman dd. georgis’ parasitology for veterinarians 10th edition. saunders. 2014; 5. soil-transmitted helminth infections [internet]. world health organitation. [cited 2017 jan 16]. available from: http://www.who. int/mediacentre/factsheets/fs366/en/ 6. mumpuni s, subekti s, koesdarto s, puspitawati h, kusnoto. penuntun praktikum ilmu penyakit. 2016; 7. blagburn b. internal parasites of dogs and cats. novartis animal health us, inc. 2010. 8. taylor m, rl c, rl w. veterinary parasitology 4rd ed. blackwell publising ltd. 9. subekti s, mumpuni s, koesdarto s, puspitawati h, kusnoto. buku ajar helmintologi veteriner. airlangga univ press. 2016; 10. tun s, ithoi i, mahmud r, samsudin ni, kek heng c, ling ly. detection of helminth eggs and identification of hookworm species in stray cats, dogs and soil from klang valley, malaysia. serrano ferron e, editor. plos one. 2015 dec 15;10(12):e0142231. 11. subekti s, kusnoto, t j. prevalensi helminthiasis pada anjing yang dipotong untuk dikonsumsi dan kucing liar di surabaya. lemb penelit univ airlangga. 2005; 12. wastomi zn. prevalensi dan derajat infeksi cacing saluran pencernaan pada kucing di beberapa pet shop di kota surabaya. airlangga institutional repos. 2014; 13. kusnoto, subekti s, koesdarto s, s sm. buku teks helmintologi. zifatama. 2014; 14. e ee. toxocariasis pada hewan dan bahayanya pada manusia. wartazoa. 2005;15(3). 15. levine nd. parasitologi veteriner. ashadi wg, editor. gajah mada univ press. 1990; vol. 10 no. 1 january–april 2022 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ original article correlation between risk of febrile neutropenia based on rondinelli score with clinical outcomes in acute lymphoblastic leukemia patients dianira hanum febia alifadiningrat1, dwiyanti puspitasari2*, yetti hernaningsih3 1 medical program, faculty of medicine, universitas airlangga, surabaya, indonesia 2 department of child health, faculty of medicine, universitas airlangga, surabaya, indonesia 3 department of clinical pathology, faculty of medicine, universitas airlangga, surabaya, indonesia received: 8th january 2022; revised: 21st january 2022; accepted: 23rd march 2022 abstract febrile neutropenia (fn) is the most severe complication in patients with blood cancer and chemotherapy. acute lymphoblastic leukemia (all) is the most common type of cancer in children and the most common cause of febrile neutropenia. the low number of neutrophils in all patients due to lymphoblast cancer cells and the toxicity of chemotherapy makes patients susceptible to infection which, if not treated immediately, can lead to death. early risk assessment for infectious complications in fn patients is needed to increase clinician awareness in high-risk patients and eliminate unnecessary therapy for low-risk patients. the rondinelli scoring system is a reasonably good instrument for predicting severe infectious complications in pediatric patients with all who have febrile neutropenia. this study aims to determine the relationship between the risk category for febrile neutropenia (fn) based on the rondinelli score with clinical outcomes in fn patients with acute lymphoblastic leukemia (all) in the hematology-oncology division of the child health department of rsud dr. soetomo. this analytic observational study used secondary data fn patients with acute lymphoblastic leukemia (all) implementing a total sampling. from 30 samples of pediatric all patients with febrile neutropenia at dr. soetomo hospital for june 2018-june 2020 it was found 17 patients (56.7%) had a moderate risk score category, and 13 others were in a low-risk category (43.3 %). patients were dominated by moderate and severe severity of neutropenia respectively, 43.3%, had neutropenia for 1-7 days (50%), fever less than seven days (66.7%), had a length of stay of 8-14 days, and 15-30 days 33.3% each. conclusion from this research is that there was a signifi cant relationship between the rondinelli score category in pediatric all patients with fn with the severity of neutropenia p=0.037; r=0.383), duration of neutropenia (p=0.021; r=0.420), duration of fever (p=0.000; r=0.618), and length of stay (p-value 0.005; r=0.496). keywords: febrile neutropenia; acute leukemia lymphoblastic; cancer; rondinelli score; infection abstrak demam neutropenia (dn) merupakan komplikasi terberat pada pasien kanker darah dan kemoterapi. leukemia limfoblastik akut (lla) adalah jenis kanker yang paling umum pada anak-anak dan penyebab tersering dari demam neutropenia. rendahnya jumlah neutrofi l pada pasien lla yang diakibatkan sel kanker maupun toksisitas kemoterapi , mengakibatkan pasien mudah terinfeksi yang apabila tidak segera ditangani dapat menyebabkan kematian. penilaian risiko dini untuk komplikasi infeksi pada pasien dn diperlukan untuk meningkatkan kewaspadaan klinisi pada pasien berisiko tinggi dan mengeliminasi terapi yang tidak perlu untuk pasien berisiko rendah.. sistem penilaian rondinelli adalah instrumen yang cukup baik untuk memprediksi komplikasi infeksi berat pada pasien anak dengan lla yang mengalami demam neutropenia. penelitian ini bertujuan untuk mengetahui hubungan antara kategori risiko demam neutropenia (dn) berdasarkan skor rondinelli dengan luaran klinis pada pasien leukemia limfoblastik akut (lla) di divisi hematologi-onkologi departemen kesehatan anak rsud dr. soetomo. penelitian observasional analitik ini menggunakan data sekunder pasien dn dengan leukemia limfoblastik akut (lla) yang dilakukan secara total sampling. dari 30 sampel pasien lla anak dengan demam neutropenia di rsud dr soetomo juni 2018-juni * corresponding author: dwiyanti-p@fk.unair.ac.id ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 63dianira hanum febia alifadiningrat, et al.: correlation between risk of febrile neutropenia based on rondinelli score 2020 didapatkan 17 pasien (56.7%) memiliki kategori skor risiko sedang, dan 13 lainnya berada dalam kategori risiko rendah (43.3%). pasien didominasi oleh neutropenia derajat sedang dan berat masing-masing 43.3%, mengalami neutropenia selama 1-7 hari (50%), demam kurang dari tujuh hari (66.7%), memiliki lama rawat 8-14 hari, dan 1530 hari masing-masing 33.3%. kesimpulan dari penelitian ini ada hubungan yang bermakna antara kategori skor rondinelli pada pasien all pediatrik dengan fn dengan keparahan neutropenia p=0.037; r=0.383), lama neutropenia (p=0.021; r=0.420), lama demam (p=0.000; r=0.618), dan lama rawat inap (p=0.005; r=0.496). kata kunci: demam neutropenia; leukemia limfoblastik akut; kanker; skor rondinelli; infeksi how to cite: alifadiningrat, d. h. f., puspitasari, d., hernaningsih, y. correlation between risk of febrile neutropenia based on rondinelli score with clinical outcomes in acute lymphoblastic leukemia patients. indonesian journal of tropical and infectious disease, 10(1), p. 62–68, apr. 2022. introduction febrile neutropenia (fn) is when axillary temperature >38oc persists for more than two hours or axillary temperature >39oc on one measurement with neutrophils <1000 cells/ microliter.1 febrile neutropenia is the most severe complication that accounts for 50%-70% of deaths in patients with hematological malignancies and chemotherapy.2 acute lymphoblastic leukemia is the most common type of acute leukemia in children. at rsud dr. soetomo surabaya, acute leukemia was in the fi rst rank of malignancy patients in children within a period of 10 years (1991-2000) which is 59% of all malignancies in children.3 all is also the most common cause of fn cases in sanglah hospital, which is 33.3%.4 in leukemia patients without chemotherapy, neutrophils will actually decrease signifi cantly by themselves through the direct interaction of cancer with hematopoiesis. however, if the patient is undergoing chemotherapy, the interaction of several cancer drugs also has a myelosuppressive effect that interferes with the formation of neutrophils.2 this makes all patients very susceptible to febrile neutropenia due to systemic bacterial infection or severe sepsis, which can be life-threatening.5 the life-threatening nature of febrile neutropenia (fn) makes an initial risk assessment necessary to increase clinicians’ alertness.2 for patients with low risk, this risk stratifi cation can provide benefi ts in the form of improving quality of life, reducing the risk of exposure to nosocomial infections in hospitals, and can also reduce the burden of treatment costs.6 the rondinelli scoring system is a reasonably good instrument for predicting severe infectious complications in pediatric patients with all who have febrile neutropenia.7 fn patients will be categorized into low risk, medium risk, or high risk using several parameters: age, hemoglobin, central venous catheter access, location of focal infection, fever temperature, and the emergence of upper respiratory tract infection (urti).8 clinical outcomes in the form of the severity of neutropenia, duration of neutropenia, duration of fever, and length of stay were recorded to see clinical improvement in fn patients after empiric antibiotic therapy. this study aims to determine the relationship between the risk category for febrile neutropenia (fn) based on the rondinelli score with clinical outcomes in fn patients with acute lymphoblastic leukemia (all) in the hematology-oncology division of the child health department of rsud dr. soetomo. materials and methods materials the collected data is processed and grouped according to the rondinelli scoring criteria as shown in table 1. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 64 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 62–68 table 1. rondinelli scoring criteria clinical variables score age ≤ 5 years 1 age > 5 years 0 central venous catheter 2 without central venous catheter 0 focal infection 4,5 without focal infection 0 fever ≥ 38,5 o c 1 fever < 38,5 o c 0 hemoglobin ≤ 7 g/dl 1 hemoglobin >7 g/dl 0 without urti 0 urti 2,5 patients are categorized as low risk if the total score is <5.5, moderate risk if the score is 5.5 to 9, and more than 9 is high risk.1 methods this study is a retrospective observational analytic study using secondary data of fn patients with acute lymphoblastic leukemia (all) in the hematology-oncology division of the child health department of rsud dr. soetomo june 2018-june 2020. this research has met the requirements of ethical feasibility by the ethics committee rsud dr. soetomo, surabaya (0333/ loe/301.4.2/ii/2021). sampling is done by total sampling. the inclusion criteria for this study were medical records of pediatric patients (0-18 years) with all who were diagnosed with complications of febrile neutropenia in the complete daily records of the doctor in charge of hematology-oncology. incomplete medical record data were excluded from this study. this research analysis uses the ibm spss statistics version 24 application using the crosstabs technique and spearman’s test for non-parametric data. results and discussion there is no fn icd (the international classification of diseases) in the electronic data center, so the authors selected the inclusion data in two stages. the fi rst stage, looking for medical record numbers with icd all and icd neutropenia at the electronic medical record data center, obtained 90 medical records. in the second stage, from the 90 medical records, data were taken with the diagnosis of fn complications in printed medical records, and obtained 30 medical record data. sample characteristic fn patients with lla table 2. sample characteristic variables category n=30 percentage (%) gender male 17 56.7 female 13 43.3 age (year) >5 17 56.7 ≤5 13 43.3 hemoglobin (g/dl) >7 29 96.7 ≤7 1 3.3 central venous catheter no 30 100.0 yes 0 0.00 focal infection no 10 33.3 yes 20 66.7 temperature (°c) <38.5 21 70 ≥38.5 9 30 urti no 23 76.7 yes 7 23.3 rondinelli score category low 13 43.3 moderate 17 56.7 high 0 0 from table 2, pediatric all patients with febrile neutropenia were 17 male (56.7%) and 13 female (43.3%). this study were similar to previous studies that pediatric all patients with febrile neutropenia were predominantly male.1,7,8,9 most patients aged over fi ve years, as many as 17 people (56.7%) in line with the previous study that patients aged above 5 years more in number than under 5 years,1,8 but the proportion is almost the same.7 this study is similar to the previous studies in that patient’s hemoglobin level was predominantly above 7 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 65dianira hanum febia alifadiningrat, et al.: correlation between risk of febrile neutropenia based on rondinelli score g/dl.1,8 from table 2, similar to the previous fi ndings that none of the pediatric all patients who had fn used a central venous catheter.1,7 this study found most patients have a specifi c focus of infection same like previous studies.1,8 in this study, most patients had a subfebrile temperature that was below 38.5°c with a percentage similar to the results of rondinelli et al.8, which was 70% vs 65%. however, other studies show the opposite, the patient’s temperature is mostly above 38.5°c with a percentage of more than 60%.1 some febrile neutropenic patients whose temperature was recorded as normal in this study may be due to the author’s error in entering the data, namely choosing which fever temperature corresponds to the time when the patient was diagnosed with febrile neutropenia. another cause was that the patient had no fever when he checked his vital signs by a hematologistoncologist. from this study, patients were found to have upper respiratory tract infections (urti) only about 10%-20%.1,8 based on the calculation rules of the rondinelli score system from table 2, pediatric patients who experienced neutropenic fever at dr. soetomo hospital for the period june 2018-june 2020, 17 patients (56.7%) had a moderate risk score category, and 13 others had a low-risk category (43.3 %). this study is diff erent from previous studies because there were no high-risk patients (score > 9.0). according to the research conducted by andrieanta et al.1 at dr. cipto mangunkusumo hospital, jakarta of the 96 samples of patients with febrile neutropenia, 52% had a low category, 48% had a high category, and none had a moderate category.1 in another study conducted by hidayat et al. of 30 samples, 73.33% had low risk (score <5.5), and the rest (score 5.5) as many as 26.67% had moderate and high risk.7 previous studies have shown varied results regarding the distribution of the risk level of fn patients with rondinelli scores. so it can be concluded that there is no absolute prevalence rate related to the risk of fn patients based on the rondinelli score, depending on the situation in each hospital, and further research is needed to fi nd out this. clinical outcome fn patients with lla table 3. result of clinical outcome variables category n=30 percentage (%) severity of neutropenia low 4 13.3 moderate 13 43.3 severe 13 43.3 length of neutropenia (days) 1-7 15 50 8-14 9 30 15-25 6 20 length of fever (days) ≤7 20 66.7 >7 10 33.3 length of hospitalization (days) 1-3 1 3.3 4-7 7 23.3 8-14 10 33.3 15-30 10 33.3 >30 2 6.7 based on table 3 the fi ndings of this study were dominated by the severity of moderate and severe neutropenia, respectively 43.3%. similar to the previous study, which found that malignancy patients with fn were dominated by the severity of severe neutropenia as much as 55.55%.10 this study found that the duration of all patients with fn was in line with the previous fi nding, which was dominated by the duration of neutropenia of less than 7 days.9 the results of this study also showed that most febrile neutropenic patients had fever for less than seven days, which was 66.7%, almost the same as the previous study, which was 80.8%.11 based on table 3, patients mostly had duration of hospitalization for 8-14 days and 15-30 days, respectively 33.3%. this is in line with previous studies which found that the longest duration of hospitalization for patients was 15-30 days.9 table 4. correlation rondinelli score with clinical outcomes risk category p-value r length of hospitalization .005 .496** length of fever .000 .618** length of neutropenia .021 .420* severity of neutropenia .037 .383* bivariate analysis using spearman’s test ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 66 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 62–68 correlation rondinelli score with severity of neutropenia based on table 4, there is a moderate, positive, and signifi cant correlation between the fn risk score category and the severity of neutropenia with p=0.037 (p<0.05) and r= 0.383, which means that the higher the rondinelli score, the greater the risk of fn patients experiencing infectious disease complications, the more severe the severity of neutropenia will be. in cases of neutropenia with infectious complications, especially mucositis, and gastrointestinal infections, the patients’ anc showed a signifi cantly lower anc.12 this is in line with the fi ndings of badiei et al. that anc <100 is one of fi ve variables that have a signifi cant correlation with the incidence of life-threatening infections.13 the other four predictor variables were fever, mucositis, platelet count, and abnormal lung temperature. this variable is very similar to the component of the rondinelli score that assesses the risk of infectious complications in febrile neutropenic patients.13 furthermore, from another study, the high risk group with the mascc score that had an anc <500 showed a larger number of patients, namely 55% compared to low risk patients, which was only 45%.14 another study showed that the incidence of infection in the anc count <500 was much higher than the anc between 500-1000, which was 71.4% compared to 28.6%.15 thus, it can be concluded that there is a signifi cant relationship between the rondinelli score category and the severity of neutropenia, related to the incidence of infection. this is because neutrophils are cells that fi ght and destroy pathogens that cause infection once they are in circulation.16 correlation rondinelli score with length of neutropenia based on table 4, there is a moderate and unidirectional signifi cant relationship between the fn risk score category and the duration of neutropenia with p=0.021 (p<0.05) and r=0.420, which means that the higher the rondinelli score, the greater the risk of fn patients experiencing infectious complications, the longer the duration of neutropenia. the same result was obtained from another study in that there was a signifi cant relationship (p = 0.046) between the duration of neutropenia and the risk level of pediatric patients with fn.17 likewise from alexander et al. who said that high-risk fn patients had a signifi cantly longer duration of neutropenia than low-risk patients. this is because the longer the patient experiences neutropenia, the higher the risk of the patient experiencing infection due to the weakening of the patient’s body defenses against infectious agents.18 the guidelines of the infectious diseases working party, germany has previously classifi ed the risk of infection based on the duration of the patient experiencing neutropenia. it is said to be low risk if the duration of neutropenia is less than fi ve days, intermediate risk if the duration of neutropenia is 6-9 days, and high risk if the duration of neutropenia is at least 10, days.19 correlation pf rondinelli score with length of fever based on table 4, there is a strong, positive, and signifi cant correlation between the fn risk score category and the duration of fever with p=0.000 (p<0.0001) and r=0.618. it means that the higher the rondinelli score, the greater the risk of fn patients experiencing infectious complications, the longer the duration of the patient’s fever. the results of this study are in line with previous fi ndings which stated that the high risk group of pediatric patients with febrile neutropenia had a signifi cantly longer duration of fever (p < 0.001) than the low risk group.17 another study said that the median duration of fever in pediatric patients with fn in the group with blood stream infection was signifi cantly greater than in the group without infection.20 this is because fever is an indication of infection, which accounts for a mortality rate of 1%-3% in pediatric patients with all who are undergoing chemotherapy.21 in 20-30% of pediatric cancer patients with febrile neutropenia due to bacterial infection, other causes are viral infections, blood product transfusions, cytostatic drugs, malignancy itself, and mucositis.17 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 67dianira hanum febia alifadiningrat, et al.: correlation between risk of febrile neutropenia based on rondinelli score correlation rondinelli score with length of hospitalization based on table 4, there is a fairly strong and unidirectional signifi cant relationship between the fn risk score category and the length of stay with p=0.005 (p<0.05) and r=0.496, which means that the higher the rondinelli score, the greater the risk of fn patients experiencing infectious complications, the longer the duration of patient care. in line with another study using the clinical index of stable febrile neutropenia (cisne) score, it was said that the increase in the risk category of fn patients was directly proportional to the duration of hospital stay (p <0.001).14 likewise, the multinational association for supportive care in cancer (mascc) score system shows a longer average duration of care in the high risk group than the low risk group.22 longer duration of hospitalization in pediatric patients with high-risk fn was also found in another study.23 complications such as bacteremia/sepsis, pneumonia, bacterial or fungal infections signifi cantly increase the duration of hospitalization for pediatric patients with fn.24 another study also showed that the median duration of stay of patients with blood stream infection (bsi) was found to be longer than that of patients without an identifi ed pathogen (19 days versus 10 days).25 likewise, patients with mdi (microbiologically documented infection) were said to be 4.5 times more likely to have a prolonged duration of stay.26 the duration of hospitalization for pediatric fn patients also had a signifi cant relationship with the incidence of septicemia (p <0.0001).27 conclusions this study found that pediatric all patients who experienced febrile neutropenia in the hematology-oncology division of the child health department of rsud dr. soetomo most of them have medium risk category and the rest are low risk. there is a fairly strong and direct signifi cant relationship between the rondinelli score category and the severity of neutropenia, duration of neutropenia, and length of stay. there is also a strong and direct signifi cant relationship between the rondinelli score category and the duration of fever. thus it can be concluded that the higher the rondinelli score, the more severe the severity of the patient’s neutropenia, the longer the patient’s duration of treatment, the longer the patient’s fever, and the longer the patient’s neutropenia. acknowledgement the authors would like to thank the health research ethics committee, medical record data center offi cers, and functional medical staff of pediatrics at rsud dr soetomo. conflict of interest the authors declare that they have no confl ict of interest. references 1. adrieanta a, windiastuti e, handryastuti s. etiologi demam neutropenia pada anak dengan keganasan dan penggunaan skor klasifi kasi rondinelli. sari pediatri. 2016 nov 9;16(4):229-35. 2. rasmy a, amal a, fotih s, selwi wj. febrile neutropenia in cancer patient: epidemiology, microbiology, pathophysiology and management. j cancer prev curr res. 2016;5(3):00165. 3. widiaskara im, permono b, ugrasena id, ratwita m. luaran pengobatan fase induksi pasien leukemia limfoblastik akut pada anak di rumah sakit umum dr. soetomo surabaya. sari pediatri. 2016 nov 23;12(2):128-34. 4. nugraheni ay, rahardiani ss. evaluation of antibiotic in acute lymphoblastic leukemia patients with febrile neutropenia. jurnal farmasi sains dan praktis. 2020 apr 28;6(1):19-30. 5. nugroho s. parameter bakteremia pada anak dengan keganasan dan demam neutropenia. jurnal kedokteran brawijaya. 2013 mar 10;26(2):113-6. 6. haeusler gm, thursky ka, slavin ma, babl fe, lourenco rda, allaway z, et al. risk stratifi cation in children with cancer and febrile neutropenia: a national, prospective, multicentre validation of nine clinical decision rules. eclinicalmedicine 2020 jan 7;18:100220. 7. hidayat r, gatot d, djer mm. validasi sistem skoring rondinelli untuk mendeteksi komplikasi infeksi berat ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 68 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 62–68 pada pasien leukemia limfoblastik akut l1 dengan demam neutropenia selama kemoterapi fase induksi. sari pediatri. 2016 nov 9;15(5):325-1. 8. rondinelli pi, ribeiro kd, de camargo b. a proposed score for predicting severe infection complications in children with chemotherapy-induced febrile neutropenia. j pediatr hematol oncol. 2006 oct 1;28(10):665-70. 9. syamsuri i. hubungan karakteristik dan gambaran laboratorium terhadap luaran pada anak demam neutropenia dan leukemia limfoblastik akut (lla) di divisi hematologi–onkologi departemen ilmu kesehatan anak rsud dr. soetomo surabaya periode januari 2017–juni 2017 [thesis]. surabaya: universitas airlangga; 2017. 10. nursyirwan sr, windiastuti e. kejadian demam neutropenia pada anak dengan keganasan. sari pediatri. 2018 mar 1;19(4):220-5. 11. yu mg, villalobos re, juan-bartolome m, berba rp. predictors of outcome and severity in adult filipino patients with febrile neutropenia. advances in hematology. 2015 sep 3;2015. 12. badr m, hassan t, sakr h, karam n, rahman da, shahbah d, et al. chemotherapy-induced neutropenia among pediatric cancer patients in egypt: risks and consequences. mol clin oncol. 2016 sep 1;5(3):300-6. 13. badiei z, khalesi m, alami mh, kianifar hr, banihashem a, farhangi h, et al. risk factors associated with life-threatening infections in children with febrile neutropenia: a data mining approach. j pediatr hematol oncol. 2011 jan 1;33(1):e9-12. 14. coyne cj, le v, brennan jj, castillo em, shatsky ra, ferran k, et al. application of the mascc and cisne risk-stratifi cation scores to identify low-risk febrile neutropenic patients in the emergency department. ann emerg med. 2017 jun 1;69(6):755-64. 15. kholis fn. penilaian risiko infeksi dengan skor mascc pada penderita demam neutropenia di rumah sakit dr. kariadi dan telogorejo semarang. media medika muda. 2018 may 4;2(1). 16. hall je. guyton and hall textbook of medical physiology. 13th ed. philadelphia (pa): elsevier, inc.; 2016. 17. miedema kg, tissing wj, abbink fc, ball lm, michiels em, van vliet mj, et al. risk-adapted approach for fever and neutropenia in paediatric cancer patients–a national multicentre study. eur j cancer. 2016 jan 1;53:16-24. 18. kebudi r, kizilocak h. febrile neutropenia in children with cancer: approach to diagnosis and treatment. curr pediatr rev. 2018 aug 1;14(3):204-9. 19. link h, böhme a, cornely oa, höff ken k, kellner o, kern wv, et al. antimicrobial therapy of unexplained fever in neutropenic patients. ann hematol. 2003 oct;82(2):s105-17. 20. hazan g, ben-shimol s, fruchtman y, abu-quider a, kapelushnik j, moser a, et al. clinical and laboratory parameter dynamics as markers of bloodstream infections in pediatric oncology patients with fever and neutropenia. j pediatr hematol oncol. 2014 jul 1;36(5):e275-9. 21. reinecke j, lowas s, snowden j, neemann k. blood stream infections and antibiotic utilization in pediatric leukemia patients with febrile neutropenia. journal of pediatric hematology/oncology. 2019 may 1;41(4):251-5. 22. lathia n, mittmann n, deangelis c, knowles s, cheung m, pilotis e, et al. evaluation of direct medical costs of hospitalization for febrile neutropenia. cancer. 2010 feb 1;116(3):742-8. 23. alexander sw, wade kc, hibberd pl, parsons sk. evaluation of risk prediction criteria for episodes of febrile neutropenia in children with cancer. j pediatr hematol oncol. 2002 jan 1;24(1):38-42. 24. swati m, gita n, sujata b, farah j, preeti m. microbial etiology of febrile neutropenia. indian journal of hematology and blood transfusion. 2010 jun;26(2):49-55. 25. avilés-robles m, ojha rp, gonzález m, ojedadiezbarroso k, dorantes-acosta e, jackson be, johnson km, caniza ma. bloodstream infections and inpatient length of stay among pediatric cancer patients with febrile neutropenia in mexico city. american journal of infection control. 2014 nov 1;42(11):1235-7. 26. das a, trehan a, bansal d. risk factors for microbiologically-documented infections, mortality and prolonged hospital stay in children with febrile neutropenia. indian pediatrics. 2018 oct;55(10):85964. 27. allareddy v, rampa s, allareddy v. hospital charges and length of stay associated with septicemia among children hospitalized for leukemia treatment in the united states. world journal of pediatrics. 2012 aug;8(3):222-8. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 2 may–august 2022 original article association of il – 23 r rs 7518660 gene polymorphism with susceptibility and disease severity of pulmonary tuberculosis yenny widowati1 , yani jane sugiri1 , ngakan putu1 , nanik setijowati2 1departement of pulmonology and respiratory medicine / dr. saiful anwar hospital, malang, indonesia 2departement of public health medical faculty of universitas brawijaya, malang, indonesia received: january 25th, 2022; revised: march 21st, 2022; accepted: may 5th, 2022 abstract pulmonary tuberculosis (tb) is a global health problem. of all people infected with mycobacterium tuberculosis only a small proportion develops into tb. il 23 is the key cytokine in the pathogenesis of tb infection. this study aims to determine the association of il-23 r rs 7518660 gene polymorphism with susceptibility and disease severity of pulmonary tb. a case control study involved 105 people consisting of 31 drug sensitive pulmonary tb patients, 40 patients with drug-resistant pulmonary tb and 34 healthy subjects as a control. il-23 r rs 7518660 gene polymorphism g allele increases susceptibility to both tb drug-sensitive and drug-resistant. g and a allele, aa and ag genotypes indicates (p value >0.05) in correlation with disease severity based on lesion in chest x-ray and high load of mycobacterium tuberculosis in sputum. there was a significant relationship between allele a and susceptibility to pulmonary tb with an odds ratio of 0.231. it showed that patients with a alleles (ag and aa genotypes) were at risk of developing tb by 1/0.231 = 4.33 times lower than patients with gg genotypes. meanwhile, the relationship of the g allele with susceptibility to pulmonary tb obtained (p value <0.05) and an odds ratio value of 0.127 indicating that patients with g alleles (gg and ag genotypes) were at risk of developing tb of 1/0.127 = 7.87 times higher than in patients with the aa genotype. conclusion: we found significant correlation between il-23 r rs 7518660 gene polymorphism g allele with susceptibility to pulmonary tb, but the result was not significant with disease severity. keywords: disease severity; il-23 r rs 7518660 gene; polymorphism; pulmonary tb; susceptibility, abstrak tuberkulosis (tb) paru merupakan masalah kesehatan global. dari semua orang yang terinfeksi mycobacterium tuberculosis hanya sebagian kecil yang berkembang menjadi tb. il23 adalah sitokin kunci dalam patogenesis infeksi tb. penelitian ini bertujuan untuk mengetahui hubungan polimorfisme gen il-23 r rs 7518660 dengan kerentanan dan keparahan penyakit tb paru. studi kasus kontrol melibatkan 105 orang yang terdiri dari 31 pasien tb paru sensitif obat, 40 pasien tb paru resisten obat dan 34 subjek sehat sebagai kontrol. polimorfisme gen il-2.3 r rs 7518660 alel g meningkatkan kerentanan terhadap tb sensitif obat dan resisten obat. alel g dan a, genotipe aa dan ag menunjukkan (p value >0.05) berkorelasi dengan derajat keparahan penyakit berdasarkan lesi pada rontgen dada dan tingginya kadar mycobacterium tuberculosis dalam sputum. terdapat hubungan yang bermakna antara alel a dengan kerentanan terhadap tb paru dengan odds ratio sebesar 0,231. hal ini menunjukkan bahwa pasien dengan alel a (genotipe ag dan aa) berisiko terkena tb sebesar 1/0.231 = 4.33 kali lebih rendah dibandingkan pasien dengan genotipe gg. sedangkan uji hubungan alel g dengan kerentanan terhadap tb paru diperoleh (p<0.05) dan nilai odd ratio 0.27 yang menunjukkan bahwa pasien dengan alel g (genotipe gg dan ag) berisiko mengalami tb 1/0,127 = 7.87 kali lebih tinggi * corresponding author: yennywe83dr@gmail.com https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-7017-1855 https://orcid.org/0000-0001-9221-6463 https://orcid.org/0000-0001-7009-2180 https://orcid.org/0000-0002-4101-3727 94 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 yenny widowati, et al. association of il – 23 r rs 7518660 gene polymorphism dibandingkan pada pasien dengan genotipe aa. terdapat korelasi yang signifikan antara polimorfisme gen il23 r rs 7518660 alel g dengan kerentanan terhadap tb paru, tetapi tidak signifikan dengan keparahan penyakit. kata kunci: gen il-23 r rs 7518660; kerentanan; keparahan penyakit; polimorfisme; tb paru how to cite: widowati, y., sugiri, y. j., putu, n., setijowati, n. association of il-23 r rs 7518660 gene polymorphism with susceptibility and disease severity of pulmonary tuberculosis. indonesian journal of tropical and infectious disease. 10(2). 93–103. aug. 2022. introduction tuberculosis (tb) is an infectious disease that is currently the main cause of health problems. about a quarter of the world's population are infected with mycobacterium tuberculosis. the biggest contributors to the global increase in tb worldwide are from india and indonesia. in india, people newly diagnosed with tb increased by 1.2 million to 2.2 million between 2013 and 2019 (74%). in indonesia, the number increased from 331,703 in 2015 to 562,049 in 2019 (69%). the target of the 2030 sustainable development goals (sdgs) is to reduce tb mortality by 90% and reduce tb incidence by 80%.1 of all people infected with mycobacterium tuberculosis, only about 510% become sick; this is related to the body's immune response at the beginning of mycobacterium tuberculosis infection4. a person's susceptibility to tb is determined by genetic factors encoded in genes in deoxyribose nucleic acid (dna) molecular strand, in which the distribution is different for each population and race. the role of gene polymorphism will change the structure of the protein produced so that it will affect individual phenotypes, including susceptibility to disease.3, 5 drug-sensitive tb is a form of tb that is still sensitive to first line anti tuberculosis drugs. this condition requires fast, precise, and directed treatment and action thus tb patients do not develop to the-drug resistance stage. as many as 96% of cases of resistance to rifampicin are caused by mutations in the 'hot-spot region' by 81 bp spanning codons 507-533 in the rpob gene.6 according to the world health organization (who) in 2017, incidence of multi drug resistance (mdr) tb cases amounted to around 3.3% of all new cases, and overall patients had received anti tuberculosis drugs therapy previously (20%). data obtained from dr. saiful anwar hospital malang reached 21 new patients every month.7 several pro-inflammatory cytokine variants are associated with the possibility of pulmonary tb, one of them is the il-23 r rs 7518660 gene polymorphism. il-23 plays a role in the regulation of the immune system in the tb infection process15. based on the data above, this study analyzes the presence of the il-23 r rs 7518660 gene polymorphism which is associated with the susceptibility and severity of pulmonary tb in patients.9,11 materials and methods research design this research used a case-control study design. the aim of this study was to determine il-23 r rs 7518660 gene polymorphism with pulmonary tb by comparing the case and the control group based on their exposure status. research subjects and sample size the sample population were patients with pulmonary tb who seek treatment at the outpatient clinic of dr. saiful anwar hospital malang. all ethnic groups who seek treatment at the pulmonary clinic or being hospitalized at dr. saiful anwar hospital malang were included in the study and recorded. case group: patients with drug-sensitive and drug-resistant pulmonary tb. 95 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 93–103 control group: healthy subjects. inclusion and exclusion criteria inclusion criteria: a. patients diagnosed with drug-sensitive or drug-resistant pulmonary tb b. age between 18 – 65 years old c. willing to participate in research and sign the "informed consent" exclusion criteria: a. patients with hiv-aids b. patients with autoimmune disease c. pregnant women note: patients with diabetes mellitus, chronic kidney disease, malnutrition, and smoking were not excluded in this research, but they were still given notes for data analysis (information is listed in table 1) research variables independent variable: a. il-23 r rs 7518660 gene polymorphism dependent variables: a. susceptibility to drug-sensitive and drugresistant pulmonary tb b. the severity of pulmonary tb, based on chest x-ray lesion and the number of mycobacterium tuberculosis detected on genexpert sputum. from chest x-ray lesion, the patients with minimal and moderate lesion added categorized as mild criteria, whereas the far advanced lesion added into severe criteria. from the data of genexpert sputum, we divided into two categories regarding to the number of thresholds based on repeat cycles of mycobacterium tuberculosis dna amplification. very low and low added were categorized as mild criteria, whereas medium and high added into severe criteria. data collection samples were obtained by consecutive sampling method in patients who met the inclusion and exclusion criteria in the outpatient and hospitalized patients at dr. saiful anwar hospital malang. il-23 r rs 7518660 gene polymorphism examination procedure identification of the allele position where the polymorphism occurred was performed by incubating the polymerase chain reaction product at 94ºc for 30 seconds to denature the dna genome, followed by primer annealing at 68ºc for 20 seconds and extension at 72ºc for 20 seconds.10,14 polymerase chain reaction was performed for 35 cycles, followed by a final extension at 72ºc for three minutes. each sample is grouped according to the results of 2% agarose gel electrophoresis, while the visualization of the gel electrophoresis results were performed using a uv-transilluminator and a polaroid camera.9,10,14 data processing and analysis techniques processing and data analysis were performed with ibm spss software version 26.0. the relationship between polymorphism with susceptibility and severity of pulmonary tb was analyzed using the chi-square test using a 95% confidence level, significant if p<0.05. meanwhile, to determine the magnitude of the risk factor, it was calculated using the odds ratio (or). results and discussion sociodemographic characteristics of research subjects this research was conducted on 105 samples which were divided into three groups. the healthy control group consisted of 34 samples and the tb case group consisted of 71 samples. tb cases were composed of 31 samples of drug-sensitive tb and 40 samples of drug-resistant tb. the sociodemographic characteristics of the research subjects can be seen in table 1. 96 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 yenny widowati, et al. association of il – 23 r rs 7518660 gene polymorphism table 1. sociodemographic characteristics of research subjects characteristics healthy control (n=34) tb cases p-value drug sensitive (n=31) drug resistant (n=40) age minimum 29 19 18 0.001a maximum 58 69 69 mean ± sd 33.79 ± 4.98 40.42 ± 14.09 45.5 ± 13.06 gender male 20 (58.8%) 15 (48.4%) 24 (60%) 0.577b female 14 (41.2%) 16 (51.6%) 16 (40%) body mass index (bmi) minimum 19.3 13.8 12.1 0.000a maximum 31.1 29.9 26.1 mean ± sd 22.86 ± 2.86 18.55 ± 3.89 18.5 ± 3.04 smoking status yes 1 (2.9%) 4 (12.9%) 4 (10%) 0.329b no 33 (97.1%) 27 (87.1%) 36 (90%) diabetes mellitus (dm) yes 8 (25.8%) 13 (32.5%) 0.540b no 23 (74.2%) 27 (67.5%) chronic kidney disease (ckd) yes 2 (6.5%) 1 (2.5%) 0.412b no 29 (93.5%) 39 (97.5%) malnutrition yes 21 (67.7%) 21 (52.5%) 0.195b no 10 (32.3%) 19 (47.5%) a: kruskal wallis test (source: primary research data processed) b: chi-square test sd: standard deviation based on the characteristics of the research subjects, the average age of the healthy control group was 33.79 ± 4.98 years old, the drug-sensitive tb group was 40.42 ± 14.09 years old, and the drug-resistant tb group was 45.5 ± 13.06 years old. for the age variable, a normality test was performed using the shapiro-wilk test. the research variable was normal if the p-value > 0.05. the result of the normality test for the age variable was p-value=0.000, which showed that the normality of the data was not met for this variable. furthermore, the kruskal wallis test was performed and obtained a p-value of 0.001 (p<0.05) which proved that there was a significant difference in age characteristics in the three groups, where the healthy control group had a lower average age than the tb case group. in terms of gender characteristics, the chisquare test was performed and obtained a pvalue of 0.577 (p>0.05) which proved that there was no gender difference between the three groups. in terms of body mass index (bmi) characteristics, the average bmi of healthy control group was 22.86 ± 2.86, the tb so group was 18.55 ± 3.89 and the tb ro group was 18.5 ± 3.04. by using the kruskal-wallis test, a p-value of 0.000 (p<0.05) was obtained which proved that there was a significant difference in the characteristics of bmi in the three groups, where the tb group, both drugsensitive and drug-resistant, had a lower average bmi compared to the healthy control group. based on the smoking status and the comorbid characteristics of tb patients, such as diabetes mellitus, chronic kidney disease and malnutrition, p-value more than 0.05 (p>0.05) was obtained. from this test, it showed that there were no significant differences in smoking status and the comorbid characteristics of tb patients. 97 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 93–103 1 1.41% 12 16.90& 58 81.69% minimal moderate far advanced 5 7.04% 24 33.80% 30 42.25% 12 16.90% very low low medium high clinical characteristics of research subjects based on the imaging of the chest x-ray lesion in figure 1, it showed that the most common lesion in the tb case group, both in the drug-sensitive and drug-resistant tb groups, were more (far) advanced lesion as much as 81.69%. the minimal lesion was the least chest x-ray imaging (1.41%). the following charts describe the chest x-ray lesion in each case groups. figure 1. description of chest x-ray lesion in each case group the imaging of chest x-ray lesion in each group of tb cases is presented in table 2. based on table 2, it shows that, in the drugsensitive tb group, the most extensive or far advanced lesion description was 87.1%. likewise, in the drugresistant tb group, the most extensive lesion description was 77.5%. by using the chi-square test, a p-value of 0.474 (p>0.05) was obtained. from this test, it showed that there was no significant difference in chest x-ray images between the drug-sensitive tb and drug-resistant tb groups. table 2. the imaging of the chest x-ray lesion in the case group the imaging of chest x-ray lesion groups p-value drug sensitive tb drug resistant tb minimal 0 (0%) 1 (2.5%) 0.474 moderate 4 (12.9%) 8 (20%) far advanced 27 (87.1%) 31 (77.5%) (source: primary research data processed) based on the number of mycobacterium tuberculosis detected in sputum, figure 2 shows that the number of mycobacterium tuberculosis in the genexpert sputum was mostly in tb case group, both in the drugsensitive and drug-resistant tb groups were at a medium level of 42.25%. the very low level was a picture of the least number of genexpert sputum examination, which was 7.04%. the following chart describes the number of mycobacterium tuberculosis detected on sputum examination in each case group. figure 2. overview of the genexpert sputum of the mycobacterium tuberculosis case group based on the number of mycobacterium tuberculosis detected in sputum, figure 2 shows that the number of mycobacterium tuberculosis in the genexpert sputum was mostly in tb case group, both in the drugsensitive and drug-resistant tb groups were at a medium level of 42.25%. the very low level was a picture of the least number of genexpert sputum examination, which was 7.04%. the following chart describes the number of mycobacterium tuberculosis detected on sputum examination in each case group. the description results of the number of mycobacterium tuberculosis detected in the genexpert sputum is presented in table 3. based on table 3 shown in the drug-sensitive tb group, the number of mycobacterium tuberculosis detected in the genexpert sputum was mostly in the low category, which was 58.1%. meanwhile, in the drugresistant tb group, the genexpert sputum description was mostly in the medium category, which was 50%. by using the chisquare test, a p-value of 0.001 (p<0.05) was obtained. from this test, it showed that there were significant differences in the results of the genexpert sputum between the drugsensitive tb and drug-resistant tb groups. 98 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 yenny widowati, et al. association of il – 23 r rs 7518660 gene polymorphism table 3. overview of the number of mycobacterium tuberculosis detected on genexpert sputum in research subjects number of mycobacterium tuberculosis detected on genexpert sputum groups p-value drug-sensitive tb drug-resistant tb very low 1 (3.2%) 4 (10%) 0.001 low 18 (58.1%) 6 (15%) medium 10 (32.3%) 20 (50%) high 2 (6.5%) 10 (25%) (source: primary research data processed the distribution of patient types is described in figure 3 which shows that the most drug-resistant tb patient types were relapse and new cases, each group consisted of 14 people (35%). the lowest type of tb drug-resistant patient was k1 failure. figure 3. description of the distribution of the type of tb patients ro the allele frequencies and genotypes of the il-23 r rs 7518660 gene polymorphism in the healthy and tb control groups, on both drug-sensitive and drug-resistant tb are presented in table 4. based on table 4, it is shown that the frequency of the g allele in the control group was 17 (50%), the drug-sensitive tb group was 30 (96.8%) and the drug-resistant tb group was 33 (82.5%). the frequency of the g allele was more in the tb case group than in the control group. from the chi-square test results obtained p-values of 0.000 (k vs drug-sensitive tb) and 0.003 (k vs drugresistant tb). from this test, it was proven that there was significant difference in the frequency of the g allele between the tb group and the control group. meanwhile, the comparison of the g allele frequency between the drugsensitive tb and drug-resistant tb groups obtained a p-value of 0.059 (p>0.05), which showed that there was no significant difference in the frequency of the g allele. table 4. comparison of the allele frequencies and genotypes of il-23 r rs 7518660 gene polymorphism in tb and healthy controls variables healthy control (k) (n=34) tb cases p-value drug sensitive tb (n=31) drug resistant tb (n=40) k vs drug sensitive tb k vs drug resistant tb drugsensitive vs drugresistant tb gg 4 (11.8%) 9 (29%) 17 (42.5%) 0.082ns 0.003 0.243ns ag 13 (38.2%) 21 (67.7%) 16 (40%) 0.017 0.877ns 0.020 aa 17 (50%) 1 (3.2%) 7 (17.5%) 0.000 0.003 0.059ns alel g 17 (50%) 30 (96.8%) 33 (82.5%) 0.000 0.003 0.059ns alel a 30 (88.2%) 22 (71%) 23 (57.5%) 0.082ns 0.003 0.243ns ns: not significant (source: primary research data processed) table 5 below describes the results of analysis of the relationship between the il-23 r rs 7518660 gene polymorphism with susceptibility of pulmonary tb. gg genotype was used as a comparison. 99 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 93–103 table 5. analysis of the relationship between the il-23 r rs 7518660 gene polymorphism with the susceptibility of pulmonary tb variables healthy control drug-sensitive and drug-resistant tb p-value or 95% ci genotype gg 4 (11.8%) 26 (36.6%) (reff) ag 13 (38.2%) 37 (52.1%) 0.180ns 0.438 0.128 1.495 aa 17 (50%) 8 (11.3%) 0.000* 0.072 0.019 0.278 allele g 17 (50%) 63 (88.7%) 0.000* 0.127 0.047 0.344 allele a 30 (88.2%) 45 (63.4%) 0.008* 0.231 0.073 0.729 ns: not significant *: significant (source: primary research data processed) the relationship between the ag genotype and susceptibility to pulmonary tb obtained a p-value of 0.180 which proves that there was no significant relationship between the ag genotype and susceptibility to pulmonary tb (or = 0.438 (0.128 1.495)). while the aa genotype obtained a p-value of 0.000 which proves that there was a significant relationship between the aa genotype and susceptibility to pulmonary tb. the odds ratio of 0.072 (0.019 0.278) indicates that patients with the gg genotype are at risk of developing tb by 1/0.072 = 13.81 times higher than patients with the aa genotype. table 6. analysis of the relationship between the il-23 r rs 7518660 gene polymorphism with the severity of pulmonary tb based on chest x-ray lesion variables minimal and moderate lesion far advanced lesion p-value or 95% ci genotype gg 4 (30.8%) 22 (37.9%) (reff) ag 6 (46.2%) 31 (53.4%) 0.929 0.939 0.237 3.727 aa 3 (23.1%) 5 (8.6%) 0.176 0.303 0.051 1.805 allele g 10 (76.9%) 53 (91.4%) 0.136 0.314 0.065 1.531 allele a 9 (69.2%) 36 (62.1%) 0.628 0.727 0.200 2.647 (source: primary research data processed) table 6 describes the results of the relationship analysis between il-23r rs 7518660 gene polymorphism and the severity of pulmonary tb based on chest x-ray lesion where the gg genotype was used as a comparison. the relationship between the ag genotype and the severity of pulmonary tb obtained a p-value of 0.929, which proves that there was no significant relationship between the ag genotype and the severity of pulmonary tb (or = 0.939 (0.237 3.727). likewise, the aa genotype showed no significant relationship between the aa genotype and the severity of pulmonary tb (p>0.05). 100 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 yenny widowati, et al. association of il – 23 r rs 7518660 gene polymorphism table 7. analysis of the relationship between the il-23r rs 7518660 gene polymorphism with the severity of pulmonary tb based on the number of mycobacterium tuberculosis detected on genexpert sputum variables very low and low medium and high p-value or 95% ci genotype gg 4 (30.8%) 22 (37.9%) (reff) ag 6 (46.2%) 31 (53.4%) 0.337 1.643 0.595 4.538 aa 3 (23.1%) 5 (8.6%) 0.213 3.000 0.508 17.708 allele g 27 (93.1%) 36 (85.7%) 0.333 2.250 0.421 12.028 allele a 16 (55.2%) 29 (69%) 0.233 1.813 0.679 4.837 (source: primary research data processed) table 7 describes the results of relationship analysis between the il-23 r rs 7518660 gene polymorphism and the severity of pulmonary tb based on the number of mycobacterium tuberculosis detected in genexpert sputum where the gg genotype was used as a comparison. the relationship between the ag genotype and the severity of pulmonary tb obtained a p-value of 0.337, which proves that there was no significant relationship between the ag genotype and the severity of pulmonary tb (or = 1.643 (0.595 4.538)). likewise, the aa genotype showed no significant relationship between the aa genotype and the severity of pulmonary tb (p>0.05). in the results of relationship analysis between the g allele and the severity of pulmonary tb based on the number of mycobacterium tuberculosis detected in genexpert sputum, a p-value of 0.333 (p>0.05) was obtained, which proves that there was no significant relationship between the g allele and the severity of pulmonary tb (or = 2.250 (0.421 12.028)). likewise, the results of testing the relationship between the a allele and the severity of pulmonary tb showed that there was no significant relationship between the a allele and the severity of pulmonary tb (p>0.05). for age characteristics, a p-value of 0.001 (p<0.05) was obtained which proves that there was a significant difference in age characteristics in the healthy control group, drug-sensitive tb and drug-resistant tb, where the healthy control group has a lower average age than the tb case group.17 in this study, the average age in the drug-sensitive and drug-resistant tb groups was the productive adult age group, but the drugsensitive tb group was slightly younger than the drug-resistant tb group13. based on data from the who in 2015 and permenkes 2016, it was stated that the incidence of pulmonary tb was highest in the productive adult age group. research from dodd et al. stated that the incidence of pulmonary tb in adults was 1.5-6 times higher than in children and adolescents. this is due to the tendency of greater social interaction in adulthood. 22 the clinical characteristics of the subjects of this study used the parameters of the lesion area on the chest x-ray and the number of mycobacterium tuberculosis detected in the genexpert examination to be assessed based on the severity of pulmonary tb. the results showed that, in the drug-sensitive tb group and drug-resistant tb group, the most extensive or far advanced lesion were 87.1% and 77.5%, respectively. based on the correlation test of the x-ray lesion area parameters, it showed that there was no difference in the imaging of the chest x-ray lesion between the drug-sensitive tb and drug-resistant tb groups. icksan et al.23 stated that the most common lesion on the chest x-ray was extensive lesion consisting of cavities, consolidation, fibrosis with atelectasis, bullae, and calcifications where the degree of damage was more extensive in the drug-resistant tb group.23 based on the results of the number of mycobacterium tuberculosis detected in the genexpert sputum examination, it was 101 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 93–103 shown that there were differences in the genexpert sputum examination between the drug-sensitive tb and drug-resistant tb groups. in the drug-sensitive tb group, the most mycobacterium tuberculosis detected was in low level (58.1%), while in the drugresistant tb group, mycobacterium tuberculosis detected was in medium level (50%). genexpert examination in the drugresistant tb group showed more mycobacterium tuberculosis than the drugsensitive tb group. the number of mycobacterium tuberculosis detected on the genexpert examination was directly proportional to the viscosity of the sputum examined. our study used the genexpert sputum examination which has a more sensitive result than the acid fast bacillus (afb) examination8. the analysis of sputum samples used the polymerase chain reaction method which calculates the number of thresholds based on repeat cycles of mycobacterium tuberculosis dna amplification. the genexpert method is semi-quantitative, while the high level is stated if there is 16 mycobacterium tuberculosis ct, medium if there is 16-22 mycobacterium tuberculosis ct, low if there is 22-28 mycobacterium tuberculosis ct, and very low if there is 28-38 mycobacterium tuberculosis ct. therefore, the lower the mycobacterium tuberculosis ct number, the higher mycobacterium tuberculosis number that will be detected.23,24,26 in this study, the frequency of il-23 r rs 7518660 g allele polymorphism and the ag genotype were higher in the pulmonary tb group than in healthy controls and it was statistically significant. this was on concordance with the results of previous research on the chinese uygurs ethnic group in 2015 which stated that the allele frequencies studied showed significant differences between the case group and healthy controls, where the g allele was more dominant in the tb case group compared to healthy controls. jiang et al.’s9 study for the ag genotype with an odds ratio of 2.99 had 0.34 times less chance of developing tb than the gg genotype. based on statistical data analysis, the frequency of il-23r rs 7518660 gene polymorphism genotype aa was higher in healthy controls. this indicates the protective effect of allele a against pulmonary tb. research in tunisia in 2012 found that the frequency of the a allele and the aa genotype increased the risk 2.79 times greater for the incidence of pulmonary tb. 9,18 in this study, there was a significant relationship between allele a and susceptibility to pulmonary tb with an odds ratio of 0.231. this showed that patients with a alleles (ag and aa genotypes) were at risk of developing tb by 1/0.231 = 4.33 times lower than patients with gg genotypes. meanwhile, the relationship test of the g allele with susceptibility to pulmonary tb obtained a p-value of 0.000 (p<0.05) and an odds ratio value of 0.127, indicating that patients with g alleles (gg and ag genotypes) were at risk of developing tb of 1/0.127 = 7.87 times higher than in patients with the aa genotype. this also in concordance with the research conducted on the chinese uygur ethnic group in 2015, which stated that a person with the g allele has a higher risk of developing pulmonary tb compared to the a allele with an odds ratio of 4.83.9 colonization of mycobacterium tuberculosis caused widespread organ damage and was at risk of mutation12. the results showed that there was no significant relationship between the a, g alleles and the aa, ag genotype with the severity of pulmonary tb based on the lesion on the chest x-ray (p>0.05). previous studies on chinese uygurs also did not show significant results on the severity of lesion on chest radiographs. the same study examined the polymorphism of the il-23 r snp gene rs1884444 which showed a significant relationship to the severity of pulmonary tb based on chest x-ray lesion.19 in this study, we analyzed whether there was a relationship between the il-23 r rs 7518660 gene polymorphism and the severity 102 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 yenny widowati, et al. association of il – 23 r rs 7518660 gene polymorphism of pulmonary tb based on the number of mycobacterium tuberculosis detected in genexpert sputum. the results showed that there was no significant relationship between the a, g alleles and the aa, ag genotype with the severity of pulmonary tb based on the number of mycobacterium tuberculosis detected in genexpert sputum (p>0.05). shabbir et al. (2007) stated that one of the influencing factors was the limitation in checking the quality of sputum samples. it is known that the good sputum quality will result in a good sampling process, which that will determine the number of bacteria, which will subsequently determine the level of transmission and the severity of tb patients.20,21 the absence of a significant relationship between the il-23 r rs 7518660 gene polymorphism in terms of both alleles and genotypes on the severity of tb based on the number of mycobacterium tuberculosis detected on sputum examination was also shown by a study conducted in the uygurs of china in 2015.9,16,25 conclusions the frequency of il-23 r rs 7518660 gene polymorphism g allele and ag genotype was shown to be higher in the pulmonary tb group than in healthy control subjects. while the frequency of il-23r rs 7518660 gene polymorphism allele a was higher in healthy controls and there was a significant relationship between il-23 r rs 7518660 gene polymorphism and susceptibility to pulmonary tb, where the higher frequency of il-23 r rs 7518660 gene polymorphism allele a and g results in the higher risk of developing pulmonary tb in both drugsensitive and drug-resistant tb. the result of this study has meaningful information about the relationship between il-23 r rs 7518660 gene polymorphism with susceptibility of pulmonary tb, but it was not significant with disease severity. for researchers, the results of this study could be reference for further research using different research methods or other markers of the il23 r gene polymorphism. in future studies, risk factors should be considered in sample selection, thus the other risk factors are more homogeneous. susceptibility to pulmonary tb is polygenic, so it is necessary to examine a wider gene polymorphism with more sensitive examination methods such as restriction fragment length polymorphism (rflp) and dna sequencing; therefore, it can explain more about the influence of certain genes on pulmonary tb and even its effect on the severity of the disease caused by those genes. acknowledgement this paper and the research behind it would not have been possible without the exceptional support of my supervisor, dr. iin noor chozin, sp.p(k) was involved in supporting my academic guidance and indirectly contributed to this research. conflict of interest the authors declare that they have no conflict of interest. references 1. who. global tuberculosis reports. published online 2020. 2. boisson-dupuis s, ramirez-alejo n, li z, et al. tuberculosis and impaired il-23–dependent ifnimmunity in humans homozygous for a common tyk2 missense variant. sci immunol. 2018;3(30). 3. indonesia kkr. petunjuk teknis penanganan infeksi laten tuberkulosis (iltb). published online 2020. 4. rapolu bl, pullagurla a, ganta s, komaravalli pl, gaddam sl. immuno-genetic importance of th17 in susceptibility to tb. scand j immunol. 2021;94(2):1-7. 5. butov do, kuzhko mm, makeeva ni, butova ts, stepanenko hl, dudnyk ab. association of interleukins genes polymorphisms with multidrug resistant tuberculosis in ukrainian 103 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 93–103 population. adv respir med. 2016;84(1):168173. 6. palomino jc, martin a. drug resistance mechanisms in mycobacterium tuberculosis. antibiotics. 2014;3(3):317-340. 7. prasetya. faktor-faktor yang berpengaruh terhadap keberhasilan pengobatan paduan standar jangka pendek pasien tb resisten obat (tb ro) di rsu dr saiful anwar malang. published online 2019. 8. yani jane s, enggar fitri l, susianti h, reto prawiro s. increasing of il-17 and il-23 levels in acid-fast bacilli conversion of drug resistant tuberculosis patients after one month treatment. sysrevpharmorg. 2021;12(1):771780. 9. jiang d, wubuli a, hu x, et al. the variations of il-23r are associated with susceptibility and severe clinical forms of pulmonary tuberculosis in chinese uygurs. bmc infect dis. 2015;15(1):1-10. 10. floss dm, moll jm, scheller j. il-12 and il-23close relatives with structural homologies but distinct immunological functions. cells. 2020;9(10):1-24. 11. sary m, muhammad m, saleh i. hubungan polimorfisme gen xrcc1 arg 399 gln terhadap kejadian kanker serviks pada wanita ras melayu 2018;4(1):1-8. 12. miggiano r, rizzi m, ferraris dm. mycobacterium tuberculosis pathogenesis, infection prevention and treatment. pathogens. 2020;9(5):10-13. 13. seid g, ayele m. undernutrition and mortality among adult tuberculosis patients in addis ababa, ethiopia. adv prev med. 2020;2020:1-9. 14. singh dp, bagam p, sahoo mk, batra s. immune-related gene polymorphisms in pulmonary diseases. toxicology. 2017;383:2439. 15. floss dm, schröder j, franke m, scheller j. insights into il-23 biology: from structure to function. cytokine growth factor rev. 2015;26(5):569-578. 16. khalilullah sa, harapan h, hasan na, winardi w, ichsan i, mulyadi m. host genome polymorphisms and tuberculosis infection: what we have to say? egypt j chest dis tuberc. 2014;63(1):173-185. 17. thorson ae. gender differences in tuberculosis. int j infect dis. 2014;21:65-66. 18. urdahl kb, shafiani s, ernst jd. initiation and regulation of t-cell responses in tuberculosis. mucosal immunol. 2011;4(3):288-293. 19. khader sa, guglani l, rangel-moreno j, et al. il-23 is required for long-term control of mycobacterium tuberculosis and b cell follicle formation in the infected lung . j immunol. 2011;187(10):5402-5407. 20. hunter rl. tuberculosis as a three-act play: a new paradigm for the pathogenesis of pulmonary tuberculosis. tuberculosis. 2016;97:8-17. 21. yannam gr, gutti t, poluektova ly. il-23 in infections, inflammation, autoimmunity and cancer: possible role in hiv-1 and aids. j neuroimmune pharmacol. 2012;7(1):95-112. 22. dodd pj, sismanidis c, seddon ja. global burden of drug-resistant tuberculosis in children: a mathematical modelling study. lancet infect dis. 2016;16(10):1193-1201. 23. icksan a.g, napitupulu m.r, nawas m.a and nf. chest x-ray findings comparison between multi-drug-resistant tuberculosis and drugsensitive tuberculosis. published online 2018. 24. blakemore r, nabeta p, davidow al, et al. a multisite assessment of the quantitative capabilities of the xpert mtb/rif assay. am j respir crit care med. 2011;184(9):1076-1084. 25. ben-selma w, boukadida j. il23r(arg381gln) functional polymorphism is associated with active pulmonary tuberculosis severity. clin vaccine immunol. 2012;19(8):1188-1192. 26. governorate g. smear positive versus smear negative tuberculosis: an audit of some aspect related to management practices in mahalla al-kobra chest hospital, gharbia governorate, egypt. egypt j community med. 2015;33(3):1-18. 118 vol. 6 no. 5 may–august 2017 research report antimicrobial activity of pineapple (ananas comosus l. merr) extract against multidrug-resistant of pseudomonas aeruginosa: an in vitro study rahmat sayyid zharfan1a, priyo budi purwono1, arifa mustika1 1 faculty of medicine, universitas airlangga a corresponding author: zharfan.rs@gmail.com abstract pseudomonas aeruginosa is the main cause of nosocomial infection which is responsible for 10% of hospital-acquired infection. pseudomonas aeruginosa tends to mutate and displays potential for development of antibiotic resistance. approximately, 10% of global bacterial isolates are found as multidrug-resistant pseudomonas aeruginosa. pseudomonas aeruginosa have a quite tremendous severity index, especially on pneumonia and urinary tract infections, even sepsis, which 50% mortality rate. pineapple (ananas comosus l. merr) has antimicrobial properties. the active antimicrobial compounds in ananas comosus l. merr include saponin and bromelain. this research aims to find the potency of antimicrobial effect of pineapple (ananas comosus l. merr) extract towards multidrug-resistant pseudomonas aeruginosa. multidrug-resistant pseudomonas aeruginosa specimen is obtained from patient’s pus in orthopaedic department, dr soetomo public hospital, surabaya. multidrug-resistant pseudomonas aeruginosa specimen is resistant to all antibiotic agents except cefoperazone-sulbactam. this research is conducted by measuring the minimum inhibitory concentration (mic) through dilution test with mueller-hinton broth medium. pineapple extract (ananas comosus l. merr.) is dissolved in aquadest, then poured into test tube at varying concentrations (6 g/ml; 3 g/ml; 1.5 g/ml; 0.75 g/ml, 0.375 g/ml; and 0.1875 g/ml). after 24 hours’ incubation, samples are plated onto nutrient agar plate, to determine the minimum bactericidal concentration (mbc). the extract of pineapple (ananas comosus l. merr) has antimicrobial activities against multidrug-resistant pseudomonas aeruginosa. minimum inhibitory concentration (mic) could not be determined, because turbidity changes were not seen. the minimum bactericidal concentration (mbc) of pineapple extract (ananas comosus l. merr) to multidrug-resistant pseudomonas aeruginosa is 0.75 g/ml. further study of in vivo is needed. keywords: ananas comosus l. merr, antimicrobial, multidrug resistant pseudomonas aeruginosa, pineapple abstrak pseudomonas aeruginosa adalah penyebab utama infeksi nosokomial, dan bertanggung jawab pada sekitar 10% kasus infeksi di rumah sakit. pseudomonas aeruginosa mudah bermutasi dan memunculkan sifat resistensi antibiotik. diperkirakan 10% isolat bakteri global dinyatakan sebagai pseudomonas aeruginosa multidrug resistant (pamr). pseudomonas aeruginosa memiliki severity index yang cukup tinggi, terutama pada pneumonia dan infeksi saluran kencing, bahkan sepsis, dimana tingkat kematian mencapai 50%. buah nanas (ananas comosus l. merr) mempunyai sifat antimikroba. senyawa aktif yang terkandung dalam ananas comosus l. merr meliputi saponin dan bromelain. penelitian ini bertujuan untuk menguji potensi efek antimikroba dari ekstrak buah nanas (ananas comosus l. merr) terhadap pseudomonas aeruginosa multidrug resistant (pamr). penelitian ini menggunakan metode true experimental. specimen pseudomonas aeruginosa multidrug resistant diperoleh dari pus pasien bagian orthopedi rumah sakit umum daerah dr. soetomo surabaya. specimen pseudomonas aeruginosa multidrug resistant dinyatakan resisten pada seluruh golongan antibiotik, kecuali cefoperazone-sulbactam. penelitian dilakukan dengan mengukur konsentrasi hambat minimal (khm) melalui uji dilusi dengan media mueller-hinton broth. ekstrak buah nanas (ananas comosus l. merr) dilarutkan dalam aquades, dan dituangkan 119zharfan, et al.,: antimicrobial activity of pineapple (ananas comosus l. merr) extract ke dalam tabung uji dengan konsentrasi tertentu (6 g/ml; 3 g/ml; 1.5 g/ml; 0.75 g/ml, 0.375 g/ml; and 0.1875 g/ml). setelah diinkubasi selama 24 jam, bakteri dalam tabung uji ditanam pada nutrient agar plate, untuk menentukan konsentrasi bunuh minimal (kbm). replikasi dalam penelitian ini dilakukan empat kali. konsentrasi hambat minimal (khm) tidak dapat ditentukan, karena kekeruhan larutan yang tidak menunjukkan perubahan. konsentrasi bunuh minimal (kbm) dari ekstrak buah nanas (ananas comosus l. merr) terhadap pseudomonas aeruginosa multidrug resistant adalah 0.75 g/ml. diperlukan penelitian lebih lanjut, terutama eksperimen in vivo. kata kunci: ananas comosus l. merr, antimikroba, pseudomonas aeruginosa multidrug resistant, nanas introduction pseudomonas aeruginosa (p. aeruginosa) is a gramnegative bacterium which is categorized as an opportunistic pathogen that infects impaired immunity individuals.1 pseudomonas aeruginosa is a major cause of nosocomial infections and it is responsible for 10% of hospital-acquired infections.2 p. aeruginosa infections recurrently lead to serious and life-threatening conditions, generally difficultto-treat. multidrug resistant pseudomonas aeruginosa is pseudomonas aeruginosa which is resistant to at least one agent in three or more categories of antimicrobial, including: aminoglycoside, cephalosporin, fluoroquinolone, penicillin + β-lactamase inhibitor, monobactam, phosphonic acid, carbapenem and polymyxin.3 infections by cause of multidrug resistant pseudomonas aeruginosa have a quite tremendous severity index, especially on pneumonia and urinary tract infections, even sepsis, which 50% mortality rate.4 pseudomonas aeruginosa has intrinsic antimicrobial resistance due to the permeability of the membrane and has a wide range of efflux pumps. some strains of p. aeruginosa show mutations in the fluoroquinolone binding site, loss of porin channels, and increased production of beta lactamase as well as cephalosporinase. pseudomonas aeruginosa may acquire additional resistance mechanisms through external plasmids and has a high potential to be resistant against antimicrobials used during the treatment.5 the prevalence of p. aeruginosa that is reported to be resistant against various antimicrobial agents continues to increase, with 10% of global isolates are reported to be multidrug resistant pseudomonas aeruginosa.6 particularly in indonesia, the prevalence of multidrugresistant p. aeruginosa was reported to be 23.5%.7 a novel antimicrobial is necessary to overcome the increasing resistance of pseudomonas aeruginosa. pineapple (ananas comosus l. merr) was reported to have strong antimicrobial activity and should be taken into consideration as antimicrobials.8 the active antimicrobial compounds in ananas comosus l. merr include saponin and bromelain.9 both saponin and bromelain work as antimicrobial through membranolitic properties.10 bromelain particularly works as a proteolytic enzyme once it is bound to bacterial cell membrane, causing damage and inducing bacterial cell death.11 antimicrobial activity of the active compounds in ananas comosus l. merr had been reported to be notably potent against both gram-negative and gram-positive bacteria, including pseudomonas aeruginosa. even though pineapple extract (ananas comosus l. merr) had been reported to be a potent antimicrobial agent against pseudomonas aeruginosa, there was no research regarding its’ potential against multidrugresistant pseudomonas aeruginosa. therefore, we seek to test the antimicrobial potency of pineapple extract (ananas comosus l. merr) against multidrug resistant pseudomonas aeruginosa. material and method preparation of plant extract the pineapples were taken from pesanggrahan village, batu, east java at the age of ± 1.5 years. its surroundings on the basis of ethnopharmacological information during december 2015. the plant materials were identified and authenticated by east java department of health, indonesia. samples of 1 kg pineapple fruit were washed, cut into small parts and dried under 40°c for 72 hours. the dried plant materials were processed into subtle powder. the dried powder of any of pineapple fruit were soaked with 800 ml – 1 l of (96%) ethanol and cold distilled water, respectively for 3 x 24 hours. the pulverized materials were extracted by maceration. the filtrates were collected in a beaker and concentrated in a vacuum at a temperature below 40°c using a rotary evaporator. the resulting raw extracts obtained were stored at 4°c. preparation of bacterial specimen the specimen of multidrug-resistant pseudomonas aeruginosa was obtained from a patient in orthopaedic department, dr. soetomo public hospital surabaya. the bacterial specimen is resistant to several antibiotics agents includes: aminoglycoside, fluoroquinolone, penicillin + β-lactamase inhibitor, cephalosporin, monobactam, phosphonic acid, carbapenem, polymyxin, and only sensitive to cefoperazone-sulbactam. multidrug-resistant pseudomonas aeruginosa cultures were maintained on the sterile nutrient agar plate media and incubated at 37 ° c for 24 hours, following refrigeration storage at 4°c. 120 indonesian journal of tropical and infectious disease, vol. 6 no. 5 may–august 2017: 118–123 table 1. bacterial growth in nutrient agar plates tube pineapple fruit extract concentration (g/cc) bacterial growth of multidrug resistant pseudomonas aeruginosa 1st replication 2nd replication 3rd replication 4th replication c1 (+) 6 c2 (-) + + + + t 1 6 t 2 3 t 3 1,5 t 4 0,75 t 5 0,375 + t 6 0,1875 + + ‘+’ show bacterial growth, while ‘-‘ no bacterial growth inoculation preparation isolated colonies of the same type from a culture agar plate were selected and grabbed the surface of colony with a loop and relocated to a tube containing 4 ml of a compatible medium like nutrient broth. the suspension was incubated at 37oc and the proportion was adjusted to the 0.5 macfarland definitive turbidity, approximately 1.5 x 108 colony forming unit (cfu)/cc or 250-300 colonies in solid medium. preparation of antimicrobial test antimicrobial activity test was conducted using dilution method in mueller-hinton broth media. pineapple (ananas comosus l. merr.) extract was diluted with distilled water and divided into several tubes with particular concentrations (6 g/cc; 3 g/cc; 1.5 g/cc; 0.75 g/cc; 0.375 g/cc, and 0.1875 g/cc). bacterial specimens were diluted in isotonic solution (nacl 0,9%) to reach concentration of 0.5 mcfarland or equal to bacterial count of 1.5 x 108 colony forming unit (cfu)/cc or 250-300 colonies in solid medium. afterwards the bacterial suspension was added to each tube. after 24 hours of incubation, bacterias grown in tubes were planted into a solid medium to obtain the minimum bactericidal concentration (mbc). repetition was done for four times, according to federer formula. minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) minimum inhibitory concentration (mic) was determined as the lowest concentration that totally inhibited the growing of bacteria after 24 hours of incubation at 37oc. minimum bactericidal concentration (mbc) was determined as the lowest concentration that revealed no visible bacterial growth after sub-culturing in 37oc incubation for 24 hours, from a portion of liquid (5 µl) each tube. positive and negative cultures were also prepared. result and discussion each process used 8 tubes, including one tube for positive control group and one tube for negative control group. the pineapple extract concentrations of the treatment group are: 6 g/cc; 3 g/cc; 1.5 g/cc; 0.75 g/cc; 0.375 g/cc; 0.1875 g/cc (sorted from first tube to the sixth tube). the concentration was obtained from serial dilutions of the first tube (6 g/cc). negative control was obtained by mixing 1 cc of liquid medium (mueller-hinton broth) with 1 cc of multidrug-resistant pseudomonas aeruginosa specimen, while positive control tube was obtained by mixing 1 cc of liquid medium (mueller-hinton broth) with 1 cc of pineapple (ananas comosus l. merr.) extract at 6 g/cc concentration. observation of bacterial growth was conducted in two methods: (1) serial dilution test to determine the minimum inhibitory concentration (mic); and (2) planting the bacteria into nutrient agar plate to determine the minimum bactericidal concentration (mbc). in the serial dilution test, minimum inhibitory concentration (mic) could not be determined because the medium was discoloured by pineapple extract which made the visual observation difficult. each serial dilution tubes were planted to nutrient agar plates. the 1st, 2nd, 3rd, and 4th replication showed varying result of minimum bactericidal concentration (mbc) in table 1, which are: 0.75 g/cc; 0.1875 g/cc; 0.1875 g/cc; and 0.375 g/cc respectively. the negative control group showed bacterial growth on nutrient agar plates in all replication while the positive control group did not show bacterial growth on nutrient agar plates in all replication. the absence of bacterial growth in the positive control group indicates that there were no bacterial contaminants. antibacterial mechanism of pineapple fruit extract in the immense definition, antibacterial agents interfered with the growth and replication of bacteria. nowadays, antibacterial agents are generally defined as the agents which disinfect and eliminate adverse bacteria.12 antibacterial agents are classified into 2 groups, which are: inhibiting the growth of bacteria and killing the bacteria. mechanism of antibacterial agents confide in the structure and composition of the bacterial cell. destruction of bacterial structure led to a change in bacterial metabolism which causes cell death. antibacterial mechanism may include: cell walls destruction; disruption or injury to cell membrane; or synthesis inhibition of proteins and nucleic acids.13 121zharfan, et al.,: antimicrobial activity of pineapple (ananas comosus l. merr) extract multidrug resistant pseudomonas aeruginosa specimen in our study had categorized as resistant to several antibiotic class like aminoglycosides, beta-lactam penicillin, sulfa trimethoprime, tetracycline, chlorampenicol, macrolide, and quinolones. these bacterial specimens are sensitive only to the beta-lactam class of 3rd generation cephalosporin, such as cefoperazone sulbactam. various antibiotics had been tested against the multidrug resistant pseudomonas aeruginosa specimen which used in our experiment. we conclude that the bacteria showed resistance to diverse mechanisms of antibiotics such as: protein synthesis inhibition (aminoglycosides, tetracycline, chlorampenicol, macrolide); cell wall synthesis inhibition (beta-lactam penicillin); inhibition of folic acid pathway metabolism (sulfa trimethoprime); and nucleic acid synthesis inhibition (quinolones).14 the only mechanism that is effective against this specimen was bacterial cell wall synthesis inhibition by binding to the penicillin binding protein (cefoperazone), coupled with β-lactamase irreversible inactivation of sulbactam.15 resistance mechanism in pseudomonas aeruginosa can be classified into 2 types: intrinsic resistance and acquired resistance. intrinsic resistance is an innate mechanism which cannot be changed and unmodifiable. pseudomonas aeruginosa intrinsically possessed resistance against several classes of antibiotics such as ampicillin, amoxicillin, amoxicillin-clavulanate, as well as first and second generation of cephalosporin such as cefotaxime and ceftriaxone. intrinsic resistance is difficult to intervene; hence it is necessary to find gaps in the mechanism of acquired resistance to overcome the antibiotic resistance in pseudomonas aeruginosa. acquired resistance mechanism in pseudomonas aeruginosa includes: efflux pumps, impermeability mutation, β-lactamases, carbapenemase, and aminoglycosidemodifying enzymes.16 the phytochemical analysis of pineapple has revealed several compounds such as polyphenols, saponins, and flavonoids which are known antimicrobial agents. also, pineapple fruit contains a protease called bromelain. the exact antimicrobial mechanism of pineapple (ananas comosus l. merr) extract against gram-negative bacteria is still not clearly known. previous studies suggested the active compounds working against gram-negative bacteria are majorly bromelain and saponin, while flavonoids and polyphenols are more potent in inhibit gram-positive bacteria. flavonoids and polyphenols are phenolic compounds which have polar properties, so these compounds mostly work in the peptidoglycan layer in gram-positive bacteria, which also have polar properties, rather than the non-polar lipid layer.17 both of bromelain and saponin, act on bacterial cell walls and membranes. their antimicobial activity against multidrug-resistant pseudomonas aeruginosa in our study indicates two things. first, the active compounds in the extract of pineapple might have similar mechanism with antibiotics like cefoperazone sulbactam which is also sensitive against multidrug-resistant pseudomonas aeruginosa. second, the active compound in pineapple extracts might have antimicrobial mechanism which unrecognized by multidrug resistant pseudomonas aeruginosa bacteria. bromelain is a proteolytic enzyme. proteolytic enzymes play a role in the breakdown of proteins, as we know protein is one of the essential component in bacterial membrane. bromelain is hypothesized to induce protein breakdown in bacterial membrane, causing injury and cell death.18 the exact mechanism of how bromelain inhibits the growth of gram-negative bacteria is still not fully identified. studies suggest that bromelain operates through associated mechanism in weakening of outer membrane in gramnegative bacteria which contain proteins. bromelain are thought to disintegrate protein in surface membrane which eventually weakens the cell wall, leads to cell leakage, swells the cell, and damages the cell. the numbers of amino acids in the bacterial cell wall are thought to determine the antibacterial activity of proteolytic enzymes.11 saponins increases the permeability of the bacterial cell membrane, causing alteration of structure and function of the membrane, disrupting the surface tension of the cell wall, then allowing antibacterial substances to easily enter the cells and interfere the cell metabolism while denaturates proteins on the membrane so the cell membrane will be lysis.19 saponin selectively interacts with cholesterols on cell membrane, leaving a hole in the membrane.20 m i n i m u m b a c t e r i c i d a l c o n c e n t r a t i o n ( m b c ) determination other studies that are similar was reviewed, and taken as considerations in determining the minimum bactericidal concentration (mbc). in this study, we used g/cc in each extract concentration, namely 6 g/cc; 3 g/cc; 1.5 g/cc; 0.75 g/cc; 0.375 g/cc; and 0.1875 g/cc. respectively, which mean the concentration of 6 grams of pineapple extract in 1 cc of distilled water; 3 grams of extract in 1 cc of distilled water; 1.5 grams of extract in 1 cc of distilled water; 0.75 grams of extract in 1 cc of distilled water; 0.375 gram of extract in 1 cc of distilled water; and 0.1875 grams of extract in 1 cc of distilled water. our study was compared with earlier studies on antimicrobial activity of pineapple (ananas comosus l. merr) extract. the result of three earlier studies was compared with our research in table 2. study by ajibade et al,21 reported pineapple extract eliminate non – multidrug resistant pseudomonas aeruginosa at concentration of 0.2 g/cc. at these concentrations, pineapple extract also exhibits antibacterial activity against other bacteria, including streptococcus pneumoniae & staphylococcus aureus. in our study, bactericidal effect of pineapple fruit extract showed in ranged concentration from 0.1875 g/cc to 0.75 g/cc. compared to ajibade et al study,21 the 0.1875 g/cc is the closest concentration to 0.2 g/cc. 122 indonesian journal of tropical and infectious disease, vol. 6 no. 5 may–august 2017: 118–123 minimum bactericidal concentration (mbc) was determined differently across previous studies. this difference is highlighted especially in studies with different mbc on each replication. prihantoro et al23 mentioned that minimum bactericidal concentration (mbc) is determined when bacterial colonies growth in the media as few as <0.1% of the original inoculum. raharjoyo24 determined mbc as the smallest concentration with insignificant difference to negative control and significant difference to positive control. christiawan & perdanakusuma25 determined mbc as the smallest concentration with no bacterial growth on the growing media. other attention is the potency of bacterial resistance to antibacterial agents. pseudomonas aeruginosa is bacteria with a high mutation rate and easy to show resistant properties against antibiotic agent.5 based on this study, the mbc of pineapple fruit extract (ananas comosus l. merr.) against multidrug-resistant pseudomonas aeruginosa is starting to show at the concentration of 0.1875 g/cc. however, to guarantee the emergence of resistance not being returned, the concentration of 0.75 g/cc is considered to be a minimum bactericidal concentration (mbc). conclusion the extract of pineapple (ananas comosus l. merr) has antimicrobial activities against multidrug-resistant pseudomonas aeruginosa. the minimum bactericidal concentration (mbc) is 0.75 g/cc, which is the lowest concentration of pineapple extract (ananas comosus l. merr) that eliminate the multidrug resistant pseudomonas aeruginosa. the researcher suggests that further study of in vivo is needed. acknowledgement there is no conflict interest to declare. the authors thanks prof. dr. ni made mertaniasih, dr., ms., sp.mk from clinical microbiology department of dr soetomo general hospital and faculty of medicine universitas airlangga microbiology laboratory staff for their helpful comments, supervision and revision regarding the study and manuscript. references 1. gellatly, shaan & hancock, robert. pseudomonas aeruginosa: new insight into pathogenesis and host defenses. pathogen and disease 67, 2013; pp. 159-173 2. aloush, valerie et al. multidrug-resistant pseudomonas aeruginosa: risk factors and clinical impact. antimicrobial agents and chemotherapy. 2006; pp. 34-48 3. european center for disease prevention and control (ecdc). 2009. multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. 4. center for disease dynamics, economics, and policy (cddep). 2009. pseudomonas aeruginosa overview. [online]. accessed at: http://www.cddep.org/resistancemap/overview/pa#.u-ddqgneiyk (august 4th 2014) 5. lister, p.d., wolter, d.j., hanson, n.d. antibacterial-resistant pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. clin. microbiol. 2009; rev. 22, 582-610. 6. gales, a.c. et al. characterization of pseudomonas aeruginosa isolates: occurrence rates, antimicrobial susceptibility patterns, and molecular typing in the global sentry antimicrobial surveillance program, 1997–1999. clin infect dis.-2001-gales-s146-55 7. sulistiyaningsih. potensi daun beluntas (pluchea indica less.) sebagai inhibitor terhadap pseudomonas aeruginosa multi resistant dan methicillin resistant stapylococcus aureus. universitas padjadjaran bandung. 2009. [online]. accessed at: http://pustaka. unpad.ac.id/wpcontent/uploads/2010/11/potensi_daun_beluntas.pdf (august 9th 2014) 8. abdurrahman, farouq. uji aktivitas antimikroba ekstrak buah nanas (ananas comosus) terhadap bakteri enterotoxigenic escherichia coli secara in vitro. skripsi. 2014. surabaya. universitas airlangga 9. dalimartha s. atlas tumbuhan obat indonesia; vol 2. jakarta: trubus agriwidya. 2000; p. 1405 10. pratiwi, dewi et al. aktivitas antibakteri ekstrak etanol kelopak rosela (hibiscus sabdariffa linn) terhadap pseudomonas aeruginosa multiresisten dan shigella dysenteriae. seminar nasional. 2011 perhipba 11. eshamah, h., han, i., naas, h., rieck, j., dawson, p. bactericidal effects of natural tenderizing enzymes on escherichia coli and listeria monocytogenes. journal of food research. 2013; vol. 2, no. 1 12. alliance for the prudent use of antibiotics (apua). general background: antibiotic agents. 2014 [online]. accessed at: http:// www.tufts.edu/med/apua/about_issue/agents.shtml (december 26th 2015) 13. jawetz, melnick dan adelberg. mikrobiologi kedokteran, jilid i, edisi xxii. jakarta: salemba medika. 2003 14. antimicrobial resistance learning site (amrls). beta lactam antibiotics. 2011. [online]. accessed at: http://amrls.cvm.msu.edu/ pharmacology/antimicrobials/antibiotics-of-veterinary-importance/ beta-lactam-antibiotics (december 28th 2015) 15. mims. cefoperazone + sulbactam. 2015 [online]. accessed at: http://www.mims.com/india/drug/info/cefoperazone%20+%20 sulbactam/?type=full (december 27th 2015) 16. versalovic, j., carroll, k.c, jorgensen, j.h., funke, g., landry, m.l., warnock, d.w. manual of clinical microbiology 10th edition. washington dc: asm press. 2011; pp 683-684 17. suerni, e., alwi, m., guli, m.m. uji daya hambat ekstrak buah nanas (ananas comosus l.merr.), salak (salacca edulis reinw.) dan mangga kweni (mangifera odorata griff.) terhadap daya hambat staphylococcus aureus. jurnal biocelebes. 2013; vol. 7 no. 1 table 2. other studies of antimicrobial activity of pineapple fruit extract bacteria mc reference enterotoxigenic escherichia coli 1 g/cc 8 mdr pseudomonas aeruginosa 0,75 g/cc author’s pseudomonas aeruginosa 0,2 g/cc 21 staphylococcus aureus 0,2 g/cc 21 streptococcus pneumonia 0,2 g/cc 21 aggregatibacter actinomicetemomitans 16,6 mg/cc 22 porphyromonas gingivalis 4,15 mg/cc 22 streptococcus mutans 2 mg/cc 22 mbc: minimum bactericidal concentration green color: author’s result 123zharfan, et al.,: antimicrobial activity of pineapple (ananas comosus l. merr) extract 18. caesarita, dea p. pengaruh ekstrak buah nanas (ananas comosus) 100% terhadap bakteri staphylococcus aureus dari pioderma. skripsi. 2011. semarang. universitas diponegoro 19. karlina, muslim ibrahim, trimulyono guntur. aktivitas antibakteri ekstrak herba krokot (portulaca oleraceae l.) terhadap staphylococcus aureus dan escherichia coli. j.lentera bio. 2013; vol. ii (1): 87-93. 20. jamur mc, oliver c. permeabilization of cell membranes. methods mol biol. 2010;588:63-6 21. ajibade v. a., akinruli f. t and ilesanmi t. m. antibacterial screening of crude extract of oven-dried pawpaw and pineapple. international journal of scientific and research publications. 2015; volume 5, issue 11 22. praveen, n.c., rajesh, a., madan, m., chaurasia, v.r., hiremath, n. v., & sharma, a. m. in vitro evaluation of antibacterial efficacy of pineapple extract (bromelain) on periodontal pathogens. j int oral health. 2014;; 6(5): 96–98. 23. prihantoro, t., indra, r., sumarno. efek antibakteri ekstrak kulit buah delima (punica ganatum) terhadap shigella dysentriae secara in vitro. jurnal kedokteran brawijaya. 2006; vol. xxii, no. 3 24. raharjoyo, lanjar. profil kromatogram dan aktivitas antibakteri ekstrak etanol rimpang bengle (zingiber cassumunar roxb.) terhadap bakteri escherichia coli in vitro. skripsi. 2007. semarang. universitas diponegoro 25. christiawan, a., perdanakusuma, d. ktivitas antimikroba daun binahong terhadap pseudomonas aeruginosa dan staphylococcus aureus yang sering menjadi penyulit pada penyembuhan luka bakar. departemen/smf ilmu bedah plastik fakultas kedokteran universitas airlangga, rsud dr. soetomo surabaya 161 vol. 5. no. 6 september–december 2015 profile of hematocrit level captured by digital hematocrit test prihartini widiyanti1,2, tri arif sardjono3 1 biomedical engineering program, faculty of science and technology, airlangga university, surabaya, east java, indonesia 2 institute of tropical disease, airlangga university, surabaya, east java, indonesia 3 departement electrical engineering, faculty of industrial engineering, institut 10 nopember surabaya, east java, indonesia corresponding email: drwidiyanti@yahoo.com abstract the dengue fever is a disease caused by dengue virus which is transmitted via aedes aegypti and aedes albopictus vector. this dengue haemorrhagic fever (dhf) case in indonesia tend to rise from year to year caused by delayed detection and inadequate handling. the laboratory parameter of hematocrite had regularly been performed using invasive method by taking the blood from the patient. this method is still not been able to monitor patients with dhf by repetitive and accurate measurament. this research project aims is to perform a digital hematocrit test (dht) with non-invasive accurate sensors. digital hematocrit test (dht) is needed to presenting fast, exact, economical and accurate detection methods of hematocrit level. measureable magnitude by the instrumentation is non-absorb intensity electromagnetic waves 560 nm emitted by transmitter captured by receiver. signal captured by the receiver then converted into electrical signal. the electrical signal from receiver was the levels of hemoglobin. levels of hemoglobin then converted to hematocrit. hematokrit is three times the level of hemoglobin. technology of hematocrit monitoring is aimed to control dhf patient clinical symptoms continuously and acquisitively. key words: hematocrit (hct), hemoglobin (hb), wave 540–900 nm, non invasive, lambert beer law abstrak demam berdarah merupakan penyakit yang disebabkan oleh virus dengue yang ditransmisikanmelalui aedes aegypti dan aedes albopictus. kasus demam berdarah di indonesia cenderung meningkat dari tahun ke tahun dikarenakan terlambat dideteksi serta penanganan yang kurang memadai. parameter laboratorium dari hematokrit telah dilakukan menggunakan metode invasive denngam mengambil darah pasien. metode ini masih belum bisa memonitor pasien dengan dbd melalui pengukuran berulang dan akurat. penelitian ini bertujuan untuk menunjukkan tes hematokrit digital (thd) dengan sensor akurat yang non-invasif. tes hematokrit digital (thd) diperlukan untuk menunjukkan deteksi yang cepat, tepat, ekonomis dan akurat dari kadar hematokrit. besaran yang dapat diukur oleh instrumentasi ini adalah gelombang elektromagnetik 560 nm yang diemisikan oleh transmitter dan ditangkap oleh receiver. sinyal yang ditangkap oleh receiver dikonversi menjadi sinyal elektrik. sinyal elektrik dari receiver menggambarkan kadar hemoglobin. kadar hemoglobin kemudian dikonversi menjadi hematokrit. hematokrit merupakan tiga kali kadar hemoglobin. teknologi memonitor kadar hematokrit bertujuan untuk mengontrol pasien dengan gejala klinis dbd secera berkelanjutan. kata kunci: hematokrit (hct), hemoglobin (hb), gelombang 540–900 nm, non invasive, lambert beer law 162 indonesian journal of tropical and infectious disease, vol. 5. no. 6 september–december 2015: 161-164 introduction the dengue fever dengue hemorrhagic fever (dhf) is disease caused by dengue virus transmitted through aedes aegypti and aedes albopictus mosquito. both types of these mosquito are to be found almost in the whole parts of indonesia, with the exception of a height more than 1000 meters above the sea level. dengue fever disease often misdiagnosed by other diseases such flu or thypus. this is because an infection dengue virus that causes of dengue fever can be asimptomatik or obscure the symptoms. based on child data of cipto mangunkusumo hospital, dengue patients often showing symptoms such cough cold, vomiting, nausea and diarrhea. the problem might increase because the virus could enter at the same time with other disease such influenza or thypus. the understanding of disease infection by dengue virus, pathogenesis, and clinical observation discernment. using good and complete clinical examination supported by adequate laboratory examination then dhf diagnosis could be set up especially when symptoms are not enough. the first time dengue fever in indonesia was discovered in 1968, in surabaya it happened in 1972. since then, the disease spread across the area, until 1980 to every province in indonesia. there were, for the first time show an increase of the number of cases in the area of or infected by or in a sporadic extraordinary occurance always happening every year. the biggest extraordinary occurance of dhf happened in 1998, with incidence rate (ir) = 35,19 per 100,000 population and cfr = 2%. in 1999, a sharp declination from 10,17%, but the next year is likely to increase from 15,99 (in 2000); 21,66 (in 2001); 19,24 (in 2002); 23,87 (in 2003). the high prevalency of dhf could be caused by many factors. delayed diagnosis, incautiousness of patients’s family to monitor the physical symptoms adn inadequate laboratory examination. the dengue diagnosis according of who criteria are thrombositopenia: < 100.000 mg/dl and hemoconsentration: pack cell volume increase > 20%.1 hemoconcentration mean there was plasma leakage and it is main indicator to determine whether the patient already fall into dengue shock syndrome or not. there are several cause of inadequate detection of hematocrite by laboratory examination which could direct to false result such as first blood capillary contain interstitial liquid, sometimes blood specimen was not directly examine therefore could increase hematocrite level result, examination specimen was not mixed well until homogen, blood specimen could not contain clot.2,3 based on it above, test hematokrit digital (thd) is needed to presenting detection methods levels a hematocrit fast, exactly, economical and accurate. magnitude measured by instrumentation system is non-absorb intensity of electromagnetic waves emitted by transmitter captured by receiver as the result the remaining non-absorb waves of 560 nm. signal captured by receiver then converted into electrical signal. the electrical signal of receiver is showed levels of hemoglobin in the veins. levels of hemoglobin then converted to hematocrit level. through the technology of this hematocrit level as an indicator plasma leakage could be monitor as often as possible continuously to prevent dengue shock syndrome. material and method material spo2 oximetri nellcor, lcd graphic, arduino uno r3, shield arduino, mini-lcd probe, baterai li-po 2200 mah. method spo2 hardware accuracy spo2 data accuracy by took the normal data patient to check accuracy and calibration. by connecting hardware shield with spo2 on arduino to ensure red and ir in the right process. software process and filtering output spo2 data resulting from infra red (ir) and led red managed to find value intesity wide light absorb (r) on a finger/parts of patient bodies.4 absorbs the intensity (r) may be known by measuring value acred and dcred divided by value acir and dcir, results either absorption managed to get a saturation oxygen (sao2) by dividing value hbo2 with the result the number hbo2 + hb and multiplied 100%.value hb obtained by inserting value results saturation oxygen (sao2) by reduction constant value (110-25 x either absorption (r)) and multiplied by constants absorption hb of 13.7.value hct is value 0.33 of the value hb. result and discussion in this study, the average oxygen saturation measurement results of male samples with a dht is 97.26% and in accordance with the normal range is 95–100% spo2 levels. average hb values of all the male data is 13,328 g/dl approaching the normal range of hb values which is 13.5–18 g/dl for male.5 while the average male hct value of all male samples is 39,976% approaching the normal range of hct in male which are 40–54%.5 in this study also found the average measurement of the oxygen saturation results of female with a dht is 97.5% which is in accordance with the normal range of female spo2 levels which is 95-100%. the average value of 13.326 g hb/dl were in the normal range of hemoglobin values is 12-16 g/dl for female.5 while the average value of 40.1% of female hct in appropriate range of normal values hct 37–47% of female.5 hematocrit (hct) is an indicator of the determination of the most indicative of the symptoms of dengue fever.6 163widiyanti and sardjono: profile of hematocrit level captured by digital hematocrit test initial hematocrit levels related to the degree of clinical dhf according to who criteria. not only to assess the factual condition of the patient, but also to estimate or act as predictors the worst risk facing the patient, so it can be taken countermeasures and early prevention. as mentioned previously, plasma leakage is a causal factor that sparked the beginning of hypovolemia shock in dengue cases. and it has been proven that plasma leakage has occurred since the beginning of fever before the seizure.7,8,9 digital hematocrit test l (dht) utilizing red value at a wavelength of 540–900 nm and ir. hbo2 and hb values are the result of constituent values of saturation (sao2). to find the value of the voltage absorption wavelength figure 1. percentage spo2 of male figure 2. hb level of male figure 3. hct of male figure 4. percentage spo2 of female figure 5. hb level of female figure 6. hct of female generated by hbo2 and hb should be based on the amount of voltage that is absorbed in the spo2. the division of red voltage value with ir voltage value generating absorption voltage values (r) which were used to search hb value patient. saturation value minus the result of filtering constantas (110-25 x absorption voltage (r)). hemoglobin (hb) is obtained by multiplying the value of the saturation (sao2) to hemoglobin absorption constant value of 13.7. percentage of hct values obtained from 1/3 hemoglobin value.10 average male hct value 39,976% of all male data approaching the normal value range hct for male which are 40 to 54%. the average value of female hct 40.1% were 164 indonesian journal of tropical and infectious disease, vol. 5. no. 6 september–december 2015: 161-164 in the range of normal values female hct 37–47%.5 based on the results of measurements on male and female normal samples with 10 times showed that the measurement results are in accordance with the normal range of hematocrit values of male and female . conclusion from the research it can be concluded that, a large percentage of the value of spo2 values can be used to find the value of hb and hct. the data obtained from the normal samples showed that spo2, hb, and hct value is still in the normal range. references 1. world health organisation, regional office for south east asia, 2007. situation of dengue/dengue haemorrhagic fever in region. www.searo.who.int diakses: oktober 2008. 2. pusparini. “kadar hematokrit dan trombosit sebagai indikator diagnosis infeksi dengue primer dan sekunder”. skripsi sarjana bagian patologi klinik fakultas kedokteran universitas trisakti, 2004. 3. jaya ihsan. “hubungan kadar hematokrit awal dengan derajat klinis dbd”. skripsi sarjana fakultas kedokteran universitas muhammadiyah surakarta, 2008. 4. pallas-areny r. and webster jg. 2001. sensor and signal conditioning. 2nd edition. john wiley & sons. new york. 5. chernecky cc & berger bj, 2008. laboratory tests and diagnostic procedures 5th edition. saunders-elsevier, 2008 dan http://hnz11. wordpress.com/ 6. hardjoeno h. 2003. interpretasi hasil tes laboratorium diagnostik. hasanuddin universitas press. makassar. 7. hassan r, alatas h. (ed.), 2005. dengue, in: buku kuliah ika 2. cet. 11. jakarta: bag. ika fkui, pp. 607–16. 8. soedarmo, s.s.p., 2005. demam berdarah (dengue) pada anak. cet. 2. jakarta: penerbit universitas indonesia, pp. 26–45. 9. world health organisation, regional office for south east asia, 2007. variable endemicity for df/dhf in countries of sea region, 2007. www.searo.who.int diakses: oktober 2008. 10. enderle jd, blanchard sm, bronzino jd, 2005. introduction to biomedical engineering, 2nd ed, elsevier academic press, san diego, california. vol. 10 no. 1 january–april 2022 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ original article the longevity of aedes aegypti larvae in several water sources in surabaya antonio ayrton widiastara1, gabriel pedro mudjianto1, etik ainun rohmah2, hengki anggara putra3, martha indah widia ningtyas3, sri wijayanti sulistyawati4,5, suhintam pusarawati,4,5, fitriah5, kasiyama desi indriyani6, alpha fardah athiyyah7, sukmawati basuki4,5* 1medicine study program, faculty of medicine, universitas airlangga, surabaya, indonesia 2entomology study group/laboratory of entomology, institute of tropical disease, universitas airlangga, surabaya, indonesia 3master program of medical science, faculty of medicine, universitas airlangga, surabaya, indonesia 4department of medical parasitology, faculty of medicine, universitas airlangga, surabaya, indonesia 5malaria study group/laboratory of malaria, institute of tropical disease, universitas airlangga, surabaya, indonesia 6laboratory of medical microbiology, faculty of medicine, universitas airlangga, surabaya, indonesia 7department of child health, dr. soetomo hospital/faculty of medicine, universitas airlangga, surabaya, indonesia received: 16th december 2021; revised: 2nd march 2022; accepted: 8th march 2022 abstract aedes aegypti transmits the dengue virus that causes dengue viring the high number of dvi cases is the existing breeding places of ae. aegypti. the water sources used by the community and the surrounding environment are essential media for living ae. aegypti larvae. this recent study aimed to detect the longevity of ae. aegypti larvae in diff erent water sources in surabaya and the killing eff ect of temephos. an analytical observational and experimental study was conducted in august-september 2021. twenty-instar iii ae. aegypti larvae were put in each 100 ml beaker glass containing diff erent water sources, such as rain, well, mineral, new and used bath water, and antiseptic soapy water. fungi in water sources were examined. two groups were set with and without temephos, the fi nal temephos concentration was of 0.00001 ppm. live ae. aegypti larvae, pupae, mosquitoes were observed every 24 hours for seven days without feeding. living larvae were still found on day 7 in all water sources with and without temephos. there were more larvae live in soapy water without temephos, particularly on day 2 to day 6, compared to other water sources either without or with temephos. in contrast, many larvae died in mineral water with temephos. some larvae turned into pupae, started on day 1. pupae and mosquitoes were mostly found in rain water with temephos. ae. aegypti larvae survived better in soapy water either with or without temephos. temephos seemed to be eff ective to kill ae. aegypti larvae in mineral water, and might induce larvae in turning to pupae and mosquitoes quickly at low concentration. keywords: ae. aegypti, larvae, water sources, surabaya abstrak aedes aegypti menularkan virus dengue penyebab infeksi virus dengue. penyakit ini terjadi tertinggi di asia dan menempati urutan pertama setiap tahun, termasuk surabaya, indonesia. faktor penyebab tingginya angka kasus ivd adalah keberadaan tempat perkembangbiakan larva ae. aegypti. sumber air yang dimanfaatkan oleh masyarakat dan lingkungan sekitar merupakan media yang penting bagi kehidupan larva ae. aegypti. penelitian terbaru ini bertujuan untuk mendeteksi keberlangsungan hidup ae. aegypti di berbagai sumber air di surabaya dan efek membunuh temefos. studi observasional analitik dan eksperimental dilakukan pada bulan agustus-september 2021. dua puluh instar iii ae. larva aegypti dimasukkan ke dalam masing-masing gelas beker 100 ml yang berisi sumber air yang berbeda, seperti air hujan, sumur, mineral, air mandi baru dan bekas, dan air sabun antiseptik 0,5 ppm. jamur dalam sumber air diperiksa. dua kelompok ditetapkan dengan dan tanpa temefos, dengan konsentrasi temefos akhir 0,00001 ppm. larva ae. aegypti yang hidup, pupa, nyamuk diamati setiap 24 jam selama 7 hari tanpa diberi makan.banyak larva yang hidup * corresponding author: sukmab@fk.unair.ac.id ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 19antonio ayrton widiastara, et al.: the longevity of aedes aegypti larvae dalam air sabun tanpa temephos, terutama pada hari ke-2 hingga hari ke-6, dibandingkan dengan sumber air lain baik tanpa maupun dengan temephos. sebaliknya, banyak larva mati dalam air mineral dengan temephos. beberapa larva berubah menjadi pupa dimulai pada hari 1. pupa dan nyamuk banyak ditemukan di air hujan dengan temephos. larva ae. aegypti bertahan lebih baik dalam air sabun baik tanpa atau dengan temephos. temephos efektif untuk membunuh larva ae. aegypti dalam air mineral, dan dapat menginduksi larva berubah menjadi pupa dan nyamuk dengan cepat pada konsentrasi rendah. kata kunci: ae. aegypti, larva, sumber air, surabaya how to cite: widiastara, a. a., mudjianto, g. p., rohmah, e. a., putra, h. a., ningtyas, m. i. w., sulistyawati, s. w., pusarawati, s., fitriah., indriyani, k. d., athiyyah, a. f., basuki, s. the longevity of aedes aegypti larvae in several water sources in surabaya. indonesian journal of tropical and infectious disease, 10(1), p. 18–26, apr. 2022. introduction aedes aegypti mosquito is a global vector of human diseases, such as yellow fever, dengue, and zika through the bite of the adult female mosquito. the size and the success for being a mosquito are determined by environmental conditions during the larval growth phase to pupation.1 the geographic expansion of ae. aegypti has a signifi cant value that has been causing epidemics in diff erent countries of africa, the indian ocean, asia, pacifi c, europe, and america despite all the considerable eff orts made for their control.2 almost all tropical countries are not free from the spread of these viruses’ diseases by these mosquito carriers. especially, as a carrier of the dengue virus, ae. aegypti is the primary vector.3 in the southeast asia and western pacifi c region, about 1.8 billion people are at risk of contracting the dengue virus. dengue fever (df)/ dengue hemorrhagic fever (dhf) epidemics have been reported in bhutan, india, maldives, bangladesh, and pakistan, and due to the porous borders with india, nepal is at high risk of df/ dhf outbreaks.4 dengue virus infection (dvi) is a public health problem in indonesia with a fairly high morbidity and mortality rate, and has the potential to cause extraordinary events and can also have an impact on community economic losses.5 in 2015, cases of dvi in surabaya experienced many changes, where there was an increase and decrease in diff erent cases every month.6,7 in 2019, there were 138,127 dvi cases with an incidence rate (ir) of 51.48 per 100,000 populations. this number increased compared to 2018 of 65,602 cases. deaths due to dvi in 2019 also increased compared to 2018 from 467 to 919 deaths.8 the development of the ae. aegypti mosquito is based on its ability to adapt to the environment so that it is possible to overcome disturbances caused by natural phenomena. the ability mentioned is about surviving dry conditions and living without water for several months on the sides of the container walls or to adapt to human intervention, such as eradicating mosquito nests.9 reproduction sites of ae. aegypti are defi ned as any water retention container in which the immature stages of ae. aegypti are found. usually ae. aegypti oviposition sites are found in artifi cial containers, such as fl ower pots, stems or water storage tanks, discarded plastic or metal containers, buckets and tires.10–12 clean water used for daily needs produces domestic liquid waste, like waste water from bathrooms that contains soap (naoh and koh/ alkali).13 in a study, it showed that ae. aegypti eggs grow more quickly in water with soap than clean water. this defi nes bath soap and waste water as the most chosen and better site in the development of ae. aegypti larvae into adult.13 another study reveals that ae. aegypti larvae are able to survive in sewer water that has been remained in a single site till it is clear, which means the ae. aegypti eggs which become mosquitoes are more able to breed in clear water than dirty water.14 another previous study stated that the most preferred water reservoir properties for the reproduction of ae. aegypti mosquitoes are ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 20 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 18–26 well water sources with the complements such as dark in color, without a lid, unexposed to direct sunlight and without draining during more than a week.15 in addition, ae. aegypti larvae are able to live together alongside other microorganisms, such as fungi. fungi usually can be found growing in the same water site as ae. aegypti larvae13 and could be served as food for the larvae14. however, fungi could also be as a lethal pathogen to these larvae and they have been used to control mosquito vectors.14,15 surabaya is a dvi endemic area, and has various water sources in various circles of society. therefore, research is needed on some of these water sources in order to pay attention to their eff ect on the growth of ae. aegypti larvae. moreover, the eff ectiveness of water sources as a breeding ground for ae. aegypti larvae have not yet been fully studied. the purpose of this study was to detect the longevity of ae. aegypti larvae in several types of water sources in surabaya, as well as the eff ect of using temephos on both types of water sources. materials and methods sample collection an analytical observational and experimental study was conducted in institute of tropical disease (itd) universitas airlangga, surabaya, indonesia from august-september 2021. the sample in this study is ae. aegypti instar iii larvae that were collected from the breeding at the entomology laboratory, itd universitas airlangga. these larvae were selected using simple random sampling with a total of 20 individuals for each 100 ml beaker glass (herma, germany). type of water sources variables in this study were rain water, antiseptic soapy water (dettol16 with concentration of 0.5 ppm (mg/l)),well water, mineral water, new and used bath water. temephos preparation evaluation of the positive control in this study on the longevity of ae. aegypti instar iii larvae used temephos with a concentration of 0.00001 ppm (mg/l). the usage application of temephos was in accordance with the who recommendation using the commercial product abate® 1g (basf, indonesia).16 fungi examination fungi examination of each water source was only carried out once on the fi rst day at the laboratory of medical microbiology faculty of medicine universitas airlangga. the water sources were homogenized by vortex mixer for 30 seconds. one milliliter of each homogenized water source was put in the saboroud dextrose agar (sda) medium, and kept at room temperature for seven days. then, fungi were identifi ed from sample fi lm stained with lactophenol cotton blue under light microscope (olympus© cx22, japan) with 400 and 1000 magnifi cations. bioassay the bioassay for the longevity of ae. aegypti was performed in 14 of 100 ml beaker glasses, divided into two groups of water type. each group contained six beaker glasses each beaker glass was fi lled with each water type. first group was without temephos, second group was treated with 0.00001 ppm of temephos. each beaker glass was fi lled with 20 larvae. the other two glass beakers were used as controls, fi lled with tap water from the laboratory either with or without temephos. there were 14 beaker glasses, and the total sample was 280 ae. aegypti instar iii larvae. the variables were divided into two groups, then the fi rst group was not mixed with temephos, while the second group was mixed with temephos with a concentration of 0.00001 ppm. then these water sources were fi lled one by one in 100 ml beaker glass. these ae. aegypti larvae were observed every 24 hours for seven days without feeding until one had turned into a pupae or mosquito. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 21antonio ayrton widiastara, et al.: the longevity of aedes aegypti larvae statistical analyzes the data collected are in the form of numbers and percentages, and will be carried out in an average fi gure completed with its mean value. the data variables were also analyzed using the chi-square test with a signifi cant comparison or diff erence determined by p<0.05 value. ethical clearance this study has been approved with the license from medical research ethics commission, faculty of medicine, universitas airlangga number 242/ec/kepk/fkua/2021. results and discussion this study is the fi rst to be conducted using several diff erent water sources in surabaya. in addition, this study also used temephos which was mixed in several water sources. drastically, the average live larvae in mineral water without temephos was reduced signifi cantly on day 3 (d3) compared to day 1 (d1), 6/20 vs 16/20 (p-value = <0.00001, p<0.05, chi-square test). therefore, the average live larvae in mineral water without temephos seemed to be equal compared to used bath water without temephos since d4 until d7. interestingly, the average live larvae in soapy water without temephos were decreased little by little per day so that in soapy water without temephos many larvae could still survive (figure 1a). figure 1. live ae. aegypti larvae inside a) water sources without temephos, and b) water sources with temephos during seven days of observation. dark blue bar is mineral water, red bar is soapy water, green bar is new bath water, purple bar is used bath water, blue bar is rain water, and orange bar is well water. *means p<0.05, chi-square test y axis: percentage of live larvae ±sd in mineral water with temephos it was also decreased signifi cantly on d3 compared to d1, 5/20 vs 17/20 (p-value = <0.00001, p<0.05, chisquare test). rain water with temephos was the highest among others until d2; however, soapy water with temephos took fi rst place and remained on top until the last day (d7) of the observation (figure 1b). in control water, in which the water was taken from the laboratory, it did not demonstrate a signifi cant decrease in the number of live larvae. the results are shown in figure 2, where on d1 to d4, control water with temephos was higher than without temephos. however, on d7, the number of live larvae in control water with temephos was lower compared to that of water without temephos, and they were not signifi cantly diff erent (4/20 vs 6/20, p-value = <0. 24305, chi-square test). ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 22 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 18–26 figure 2. average living ae. aegypti larvae in control water during seven days of observation. blue bar is control water without temephos, and red bar is control water with temephos table 1 shows a calculation of signifi cant diff erences using chi-square test on the number of live larvae in six diff erent water sources with and without temephos on d1 to d7 of observation. apparently, only the number of live larvae on d6 and d7 in all water sources without temephos were insignifi cantly diff erent. thus, only the number of live larvae in all water sources with temephos on d7 was insignifi cantly diff erent. the calculation in the control waters was all insignifi cantly diff erent. apart from the larvae that managed to survive, there were also several larvae that achieved in turning to pupae and mosquitoes during the seven days of observation of this study. afterwards, the results of each water sources demonstrated, even on d1, that there were still some live pupae from mineral water, used bath water and well water without temephos. more pupae were also found in rain water, new bath water, soapy water and mineral water with temephos. during the seven days of observation, rain water with temephos resulted in thehighest number of pupae. pupae transformation to adult mosquito took at least two days at average. however, in temephos water sources, the total number of mosquitoes and pupae were unmatched due to the other two pupae that died during the study. the fact showed that some water sources, such as new bath water and rain water without temephos, as well as used bath water and well water with temephos, did not produce pupae at all (table 2). examinations of fungi on the culture of water samples used in study were also performed. however, no fungi were found in either samples of water, but only the blue color of results was seen on the surface of water culture. this was the absorption of lpcb (lactophenol cotton blue) into the water sample (figure 3). table 1. p-value of the comparison among the average of live ae. aegypti larvae inside water sources either with or without temephos during 7 days of observation ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 23antonio ayrton widiastara, et al.: the longevity of aedes aegypti larvae figure 3. no fungi were detected in the water sources; 1) mineral water, 2) rain water, 3) soapy water, 4) well water, 5) new bath water, and 6) used bath water the interesting results of this study showed that larvae were not able to survive in mineral water either with or without temephos. this might explain that the mineral water as a clean water could inhibit the process of larval development to survive and to become pupae. in fact, mineral water identifi ed as microbiologically healthy water had a guarantee of the absence of the most important contamination indicators17 and categorized its division into macronutrients like calcium, phosphorus, magnesium, sodium and potassium, and micronutrients like cobalt, iron, iodium and copper.17 in addition, the experiment was conducted in a glass container, which was not the usual breeding place of ae. aegypti. the natural breeding places of this mosquito are fl ower pots, stems or water storage tanks, discarded plastic or metal containers, buckets and tires.10–12, moreover, baharuddin and rahman22 found that ae. aegypti larvae were mostly obtained in plastic containers such as plastic barrels and used rubber tires18. it suggested that ae. aegypti larvae could not live long inside a clean glass containing mineral water. on the other hand, soapy water either with or without temephos was very prominent with a high percentage of live larvae. this means that ae. aegypti larvae could still survive better in water mixed with antiseptic soap 0.5 ppm, rather than other types of water. this might be because the soapy water contains sodium palmate, talc, sodium palm kernelate and paraffi n liquidum19 that could provide food for these larvae to survive. another study stated that soapy water with an equivalent concentration on water pollution in table 2. development of pupae and mosquito during 7 days of observation ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 24 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 18–26 nature also could become a good breeding place for ae. aegypti larva to survive; however, it only works if the ph of the soapy water is less than 12.8.20 it is suggested that temephos with a little concentration of 0.00001 ppm did not work eff ectively in soapy water. therefore, the water for bathing and water reservoir should be drained.21 in control waters, no signifi cant diff erence was found between water with or without temephos. it seemed that the control waters as media for living larvae were similar condition and the concentration of 0.00001 ppm temephos showed a low effi cacy of larvicide. there was no discovery of fungi in water sources used in this study. this happened because it was possible that the water sites where the water was taken had no prospect to grow fungi. fungi usually grow in environments that have soil debris, insect remains, or dead leaves and plants.22 a study revealed that fungi are used as food and provide nutrients for larval development. therefore, fungi-mosquitoes associations are able to form a more commensal period in the gut of mosquito with slight or no effect on host survival.23 an example is the yeast, saccharomyces cerevisiae, which is commonly used to feed the larvae during its developmental phase.23 the feeding behavior of adult mosquitoes also leads to the formation of adhesions of the fungi in the mosquitoes’ hindgut. at least there are four fungi species of the genus smittium, of the order harpelellas that can attach to and increase on various mosquito species’ hindgut without aff ecting larval development or survival.23 on the other hand, there have been studies demonstrated the potential usage of fungi as a successful and ecologically safe strategy to control mosquito vectors.24 since few studies reported that the number and diversity of fungi are greater found on the surface water than in groundwater and tap water25–27, the fungi are possibly contact with adult mosquitoes, some of which fungi are already infused together with chemical insecticides.28,29 besides chemical materials, some of the fungi itself are pathogens to mosquitoes and larvae. fungi species such as entomophthora sp. and coelomomyces sp. are known as obligate pathogens, while other fungi order such as eurotiales, hypocreales and mucorales are opportunistic pathogens that unfortunately cannot actively invade the mosquito body, but can set up an infection if ingoing through breaches in the cuticle.23 other fungal pathogens from water molds such as those in the genera lagenidium, leptolegnia and saprolegnia are identifi ed as facultative pathogens of mosquitoes, and obviously there are no commensals between these fungi and mosquitoes nor larvae.23 thus, there were no fungi in water sources in our study, which showed no fungi eff ecting into the larva life of ae. aegypti in our study. the use of temephos with concentration of 0.00001 ppm was applied in this study in order to find out its effect on the larval longevity. temephos worked very well on killing ae. aegypti larvae in mineral water, and water sources with temephos showed ae. aegypti larvae turned to pupae and adult mosquitoes rapidly. the temephos’ concentration of 0.00001 ppm seemed to eff ectively induce larva development into pupa. several studies have shown that the use of temephos could kill ae. aegypti larvae very quickly, because the toxicity of temephos is absorbed into the body of the larvae.30–32 the absorbed toxin attacks the larvae’s central nervous system, causing symptoms such as restlessness, hyperexcitability, tremors, convulsions, and paralysis.32 temephos inhibits cholinesterase enzyme, which causes a disorder in the larva nervous system due to the accumulation of acetylcholine in nerve endings, and this will lead to the larval mortality.30–33 a study in south kalimantan showed that the lowest concentration of temephos was 0.005 ppm resulted in 39% of larvae mortality. the highest concentration of 0.030 ppm resulted in 100% of larvae mortality.34 comparing to other study, the ae. aegypti larvae were continuously exposed with larvicide such as temephos, over a particular time at the larvicide would make a modifi cation in the larvae genetics and brings resistance to temephos and other larvicides.35,36 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 25antonio ayrton widiastara, et al.: the longevity of aedes aegypti larvae in this study, the use of temephos was at a concentration of 0.00001 ppm, where this concentration was very small and probably the concentration lacked the scale of larval killing when compared to the study in south kalimantan. however, if observed from the overall point of view, this very small concentration of temephos could still kill ae. aegypti larvae, particularly in mineral water, and showed the induction of larva development into pupa. regarding this point, the use of temephos for larvicide should be adequate and in appropriate dose, based on the instruction written on the package and guidelines by kemenkes ri and who.21,37 conclusions ae. aegypti larvae endured better in antiseptic soapy water with concentration of 0.5 ppm either with or without temephos compared to other water sources. temephos with concentration of 0.00001 ppm was eff ective to kill ae. aegypti larvae in mineral water, and might induce larval development into pupae and mosquitoes more quickly. acknowledgement we would like to thank the staff of institute of tropical diseases, and medical microbiologi department faculty of medicine universitas airlangga for their assistance and allowing this study to take place, and for its objectives to be achieved. our thanks also are addressed to universitas airlangga for supporting our study by a research grant with number of 2158/ un3/2019. conflict of interest there are no confl icts of interest between authors in this study. references 1. steinwascher k. competition among aedes aegypti larvae. plos one. 2018;13(11). 2. diouf b, dia i, sene nm, ndiaye eh, diallo m, diallo d. morphology and taxonomic status of aedes aegypti populations across senegal. plos one. 2020;15(11 november). 3. kinansi rr, garjito ta, prihatin mt, hidajat mc, anggraeni ym, widjajanti w. keberadaan jentik aedes sp. pada controllable sites dan dispossable sites di indonesia (studi kasus di 15 provinsi). aspirator j vector-borne dis stud. 2019;11(1). 4. thapa s, pant nd, shrestha r, gc g, shrestha b, pandey bd, et al. prevalence of dengue and diversity of cultivable bacteria in vector aedes aegypti (l.) from two dengue endemic districts, kanchanpur and parsa of nepal. j health popul nutr. 2017;36(1). 5. permenkes no. 50. peraturan menteri kesehatan republik indonesia. 2017. 6. dinas kesehatan kota surabaya. profil dinas kesehatan kota surabaya. dinas kesehat. 2017; 7. chamidah d. prevalensi dengue pada mahasiswa universitas surabaya. j ilmu kedokt wijaya kusuma. 2018;6(1). 8. kemenkes ri. profi l kesehatan indonesia tahun 2019. vol. 42, kementrian kesehatan republik indonesia. 2019. 9. cdc. help control mosquitoes that spread dengue, chikungunya, and zika viruses [internet]. 2015 [cited 2021 oct 17]. available from: www.cdc.gov/ dengue. 10. enan k, mohammed r, ahmed h, hassan sm, abdallah k, enan m. aedes aegypti in indoor and outdoor environment in kassala city. heal sci j [internet]. 2019;13:5. available from: http://www.hsj. gr/ 11. manrique-saide p. operational guide for assessing the productivity of aedes aegypti breeding sites. world heal organ. 2011;(october). 12. himatt s, osman ke, okoued si, seidahmed oe, beatty me, soghaier ma, et al. sero-prevalence of dengue infections in the kassala state in the eastern part of the sudan in 2011. j infect public health. 2015;8(5). 13. wasinpiyamongkol l, kanchanaphum p. isolating and identifying fungi to determine whether their biological properties have the potential to control the population density of mosquitoes. heliyon. 2019;5(8). 14. tawidian p, rhodes vl, michel k. mosquito-fungus interactions and antifungal immunity. insect biochem mol biol. 2019;111. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 26 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 18–26 15. accoti a, engdahl cs, dimopoulos g. discovery of novel entomopathogenic fungi for mosquito-borne disease control. front fungal biol. 2021;2. 16. martini m, triasputri y, hestiningsih r, yuliawati s, purwantisasi s. longevity and development of aedes aegypti larvae to imago in domestic sewage water. j thee med sci (berkala ilmu kedokteran). 2019;51(04). 17. jacob a, pijoh vd, wahongan gjp. ketahanan hidup dan pertumbuhan nyamuk aedes spp pada berbagai jenis air perindukan. j e-biomedik. 2014;2(3). 18. hidayah n, iskandar i, abidin z. prevention of dengue hemorrhagic fever (dhf) associated with the aedes aegypti larvae presence based on the type of water source. j trop life sci. 2017;7(2). 19. sainburys. dettol antibacterial bar soap original with moisturising agents x2 100 g [internet]. [cited 2021 dec 14]. available from: https://www. sainsburys.co.uk/gol-ui/product/soap-handwash/ dettol-antibacterial-twin-original-bar-soap 20. world health organization. temephos. 2008 [cited 2021 oct 29]; available from: https://www.who. int/pq-vector-control/prequalified-lists/temephos. pdf?ua=1 21. quattrini s, pampaloni b, brandi ml. natural mineral waters: chemical characteristics and health eff ects. vol. 13, clinical cases in mineral and bone metabolism. 2016. 22. baharuddin a, rahman r. karakteristik breeding plasces dan pertumbuhan larva aedes aegypti. heal tadulako. 2015;1(2). 23. sayono, qoniatun s, mifbakhuddin. pertumbuhan larva aedes aegypti pada air tercemar. j kesehat masy indones. 2011;7(1). 24. kemenkes ri. infodatin-situasi-demam-berdarahdengue. 2017. 25. pereira vj, basílio mc, fernandes d, domingues m, paiva jm, benoliel mj, et al. occurrence of fi lamentous fungi and yeasts in three diff erent drinking water sources. water res. 2009;43(15). 26. hageskal g, knutsen ak, gaustad p, de hoog gs, skaar i. diversity and signifi cance of mold species in norwegian drinking water. appl environ microbiol. 2006;72(12). 27. kauff mann–lacroix c, costa d, imbert c. fungi, water supply and biofi lms. in: advances in experimental medicine and biology. 2016. 28. scholte ej, ng’habi k, kihonda j, takken w, paaijmans k, abdulla s, et al. an entomopathogenic fungus for control of adult african malaria mosquitoes. science (80). 2005;308(5728). 29. farenhorst m, hilhorst a, thomas mb, knols bgj. development of fungal applications on netting substrates for malaria vector control. j med entomol. 2011;48(2). 30. world health organization. temephos evaluation june 2007. 2007 [cited 2021 nov 4]; available from: https://www.who.int/whopes/quality/temephos_eval_ june_2007_corr_aug160807.pdf 31. pradani fy. the eff ect of temephos to mortality and life level of aedes aegypti mosquitoes. insights public heal j. 2020;1(1). 32. matsumura f. toxicology of insecticides. toxicology of insecticides. springer us; 1975. 73, 141. 33. yu sj. the toxicology and biochemistry of insecticides. second edition. 2015. 34. ridha mr., nisa k. larva aedes aegypti sudah toleran terhadap temepos di kota banjarbaru, kalimantan selatan. s. vektora j vektor dan reserv penyakit. 2013;3(2 okt). 35. hendri j, jajang kusnandar a, puji astuti e, litbang pengendalian penyakit bersumber binatang lp, penelitian dan pengembangan kesehatan b, kesehatan republik indonesia k, et al. identifi kasi jenis bahan aktif dan penggunaan insektisida anti-nyamuk serta kerentanan vektor dbd terhadap organofosfat pada tiga kota endemis dbd di provinsi banten. vol. 8, aspirator. 2016. 36. shetty v, sanil d, shetty nj. inheritance pattern of temephos resistance, an organophosphate insecticide, in aedes aegypti (l.). genet res int. 2015;2015. 37. who. dengue and severe dengue [internet]. 2021 [cited 2021 dec 14]. available from: https://www. who.int/en/news-room/fact-sheets/detail/dengue-andsevere-dengue ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 2 may–august 2022 original article proportion of extrapulmonary mdr-tb confirmed by genexpert® in dr. hasan sadikin general hospital, west java, indonesia year 2012–2021 winnery dhestina1 , prayudi santoso1,2* , edhyana sahiratmadja3 1faculty of medicine, universitas padjadjaran, bandung, indonesia 2department of internal medicine, dr. hasan sadikin hospital, bandung, indonesia 3department of biomedical sciences, faculty of medicine, universitas padjadjaran, bandung, indonesia received: february 18th, 2022; revised: june 30th, 2022; accepted: july 19th, 2022 abstract as the third-highest country with tuberculosis (tb) incidence worldwide in 2020, indonesia has increasing tb cases resistant to various anti-tb therapy or multidrug-resistant (mdr)-tb, and ranked fifth for its high incidence reported in global tuberculosis report 2020. moreover, extrapulmonary tb (eptb) is rising, and data studies on eptb with mdr-tb in indonesia are scarce. this study aimed to explore the proportion of extrapulmonary mdr-tb among tb cases in dr. hasan sadikin general hospital bandung, west java, indonesia. a descriptive retrospective and cross-sectional study design were conducted, retrieving medical records from all suspect mdrtb adult patients examined by genexpert®, at dr. hasan sadikin general hospital bandung, west java period 2012–2021. those with eptb were further analyzed, and the demographic data was collected as well as clinical history, behavioral history, sites of extrapulmonary mdr-tb, and drug resistance. of a total 7,013 tb cases, 1,900 (27.1%) were mdr-tb cases, of whom 0.08% (n6) were extrapulmonary mdr-tb cases and 0.16% (n11) were combined with ptb. the main characteristics of cases with extrapulmonary mdr-tb were median age 27year-old (range 25–34), male gender (64.7%), underweight bmi (84.6%), and predominantly were primary cases (35.3%). the anti-tb drug resistance in mdr-tb were pre-xdr-tb (11.7%), xdr-tb (5.6%), mdr-tb (42%), and rr-tb (40.7%). although the proportion of extrapulmonary mdr-tb among all tb cases is small (0.2%), this disease can’t be ignored and has a great potential to be explored. most of them are rifampicin-resistant. further studies need to include a larger population to have more overview of mdr-tb with eptb. keywords: extrapulmonary; indonesia; multidrug-resistant; rifampicin; tuberculosis; abstrak sebagai negara ke-3 dengan kejadian tuberkulosis (tb) terbanyak di seluruh dunia pada tahun 2020, indonesia juga mempunyai angka kejadian tb resisten obat ganda (mdr) yang meningkat, dan diketahui sebagai negara ke-5 terbanyak untuk kejadian mdr-tb di dunia berdasarkan global tuberculosis report 2020. ditambah dengan adanya kejadian tb ekstra paru yang meningkat, namun penelitian terkait mdr-tb ekstra paru di indonesia masih sangat jarang. penelitian ini bertujuan untuk mencari proporsi kasus mdr-tb ekstra paru dibandingkan dengan keseluruhan kasus di rsup dr. hasan sadikin bandung, jawa barat. deskriptif retrospektif dan desain potong lintang, dengan pengambilan data rekam medis pasien dewasa yang merupakan suspek mdrtb dan diperiksa dengan tcm genexpert® di rsup dr. hasan sadikin bandung, jawa barat pada tahun 2012 sampai tahun 2021. proporsi mdr-tb ekstraparu kemudian dianalisis lebih lanjut, berserta dengan data demografi dan riwayat klinis, kebiasaan perilaku, lokasi infeksi mdr-tb ekstra * corresponding author: prayudi@unpad.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-3762-3784 https://orcid.org/0000-0002-7182-386x https://orcid.org/0000-0001-9092-5848 114 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 winnery dhestina, et al. proportion of extrapulmonary mdr-tb paru, dan pola resistan pada mdr-tb. dari 7,013 kasus tb, terdapat 1,900 kasus (27.1%) mdr-tb, yang terdiri dari 0.08% (n6) mdr-tb ekstra paru dan 0.16% (n11) mdr-tb kombinasi paru dan ekstra paru. proporsi karakteristik yang terbesar pada penelitian ini ditandai dengan median pada umur 27 tahun, dengan kelompok umur 25 – 34 tahun, jenis kelamin laki-laki (64.7%), imt <18.5kg/m2 (84.6%), dan kasus primer (35.3%). proporsi pola resisten obat terbanyak yang ditemukan adalah pre-xdr-tb (11.7%), xdr-tb (5.6%), mdr-tb (42%), dan rr-tb (40.7%). meskipun proporsi mdr-tb ekstra paru pada seluruh kasus tb hanya sedikit (0.2%), penyakit ini tidak bisa diabaikan dan masih mempunyai potensi yang besar untuk diteliti. proporsi terbesar merupakan resistan terhadap rifampicin. studi lebih lanjut memerlukan populasi yang lebih besar untuk mengetahui secara keseluruhan mengenai mdr-tb ekstra paru. kata kunci: ekstra paru; indonesia; resisten obat ganda; rifampicin; tuberkulosis how to cite: dhestina, w., santoso, p., sahiratmadja, e. proportion of extrapulmonary mdr-tb confirmed by genexpert® in dr. hasan sadikin general hospital, west java, indonesia year 2012–2021. indonesian journal of tropical and infectious disease. 10(2). 113–122. aug. 2022. introduction tuberculosis (tb) is well-known for its highly contagious transmission of mycobacterium tuberculosis (mtb) by airborne through coughing, sneezing, talking, or doing other things.1 although it has been identified for many years, the incidence of tb is still thriving from time to time.2 comparing the incidences between the year 2015 and 2019 in indonesia, there were rises around 69%, which caused indonesia to become the third country, coming up after india, with the highest tb incidences in the world in 2020.3 inappropriate anti-tb drugs administration would lead to failed eradication of mtb and risk leading to mtb mutation, resulting in resistance to 1st category of anti-tb drugs, which could be confirmed as multidrug-resistant tb (mdrtb) with genexpert®.4 in global tuberculosis report 2020, indonesia was the fifth country with the highest mdr-tb incidence globally.5 the mdr-tb incidence in indonesia reached 8.8 cases in 100,000 populations, with 2.4% primary cases and 13% have treated with anti-tb drugs in 2019, which unknown for its incidence in 2020 although indonesia was in the 30 high mdrtb burden countries.3,5 from inappropriate anti-tb drugs administrations, there are several risk categories defined in the national guideline for tuberculosis by the ministry of health republic of indonesia.6–9 confirmed mdr-tb patients are those whose samples have been examined and proved that there were anti-tb drug-resistant mtb, especially against isoniazid and rifampicin, can be together with or without other anti-tb drugs. mdr-tb could be grouped by its sites of infection, such as exclusively pulmonary tb (ptb), extrapulmonary tb (eptb), and eptb with ptb.10 the extrapulmonary mdr-tb proportion in china is 2.5% among all tb patients and 9.3% among mdr-tb patients in 2008 – 2017; and 5.1% among all tb patients and 12.6% among mdr-tb patients in india period 2012 – 2014.11,12 the location of extrapulmonary tb could differ in every patient, mainly manifested in lymph nodes and pleura, consecutively causing tb lymphadenitis and tb pleurisy due to their sites being the nearest from the lungs.13 apart from them, tb infections may take place in the skeletal, abdomen, pericardium, central nervous system, and other locations.11 although extrapulmonary mdr-tb cases are not rare, there have not been many studies reported, especially in indonesia. therefore, this study aimed to measure the proportion of extrapulmonary mdr-tb among tb cases and explore characteristics related to the mdr-tb cases at dr. hasan sadikin general hospital bandung, west java, indonesia in 2012-2021. 115 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 113–122 materials and methods study site and period this study used a retrospective crosssectional study design. secondary data were collected using total sampling method, retrieved from the medical record in mdr clinic dr. hasan sadikin general hospital bandung, cross-checked with data from e-tb manager, sitb (sistem informasi tuberkulosis), and lab tb 04 at dr. hasan sadikin general hospital bandung, west java, indonesia, in the period between april 2012 – august 2021. samples and data collection the inclusion criteria were all data of adult mdr-tb and tb patients (≥18 years) registered in sitb and e-tb manager as tb03 and tb06, and genexpert® lab as tb04 in dr. hasan sadikin general hospital bandung (figure 1). the incomplete data, which were completely missing from the medical record, were excluded from this study. figure 1. flowchart of mdr-tb suspect cases at dr. hasan sadikin general hospital bandung, west java the total number of cases of mdr-tb and tb sensitive were collected. the site location of mdr-tb, drugs resistances, and the extrapulmonary sites in mdr-tb was also collected and analyzed for their distribution proportion, along with the following data from the medical records, such as age, gender, body mass index, comorbid diseases (diabetes mellitus, hiv status, systemic lupus erythematosus, and others), smoking history, alcohol consumption history, and risk category for mdr-tb, were also analyzed for their proportions. the gold standard for extrapulmonary mdr-tb diagnostics in dr. hasan sadikin general hospital bandung preceded by rapid molecular test using genexpert®. the samples or tissue samples are collected from the suspected extrapulmonary tb locations. if the result appeared resistant, certified culture methods and drug susceptibility test (dst) would be performed with another collected tissue samples from the previous location. however, if the location of the samples is considered hard to reach, rapid molecular test is enough to diagnose extrapulmonary mdr-tb. data definition and analysis the risk category for mdr-tb patients according to the national guideline for tb consisted of 12 categories, which number 10 – 12 were applied since 2020:6–8 1. chronic tb patients, who are still sputum smear-positive at the end of the first line tb re-treatment (category 2); 2. tb patients with 2nd category treatment which not converted in 3 months of treatment; 3. tb patients who have a non-standard tb treatment history using quinolone and 2nd line anti-tb drug injection for at least 1 month, who received any tb treatments outside of the national program, for example, non-dots and private clinic; 4. tb patients who failed 1st category treatment; 5. tb patients with 1st category treatment who remain positive after 3 months of treatment; relapse tb patients, categories 1 and 2, whose most recent treatment outcome was ‘cured’ or ‘treatment completed’ yet return with tb symptoms; 6. returning tb patients after loss to follow-up (negligent treatment/default), who 116 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 winnery dhestina, et al. proportion of extrapulmonary mdr-tb interrupted any tb treatments for two or more consecutive months and returned with tb symptoms; 7. suspected tb patients with a history of close contact with mdr-tb patients, who live in the same household or spending many hours a day in the same indoor living space with an mdr-tb patient and show tb symptoms; 8. tb-hiv co-infection patients who do not respond to anti-tb drug administration; 9. mdr-tb patients who failed treatment; 10. relapse mdr-tb patients; 11. returning mdr-tb patients after loss to follow-up (negligent treatment/default). patients who don’t belong to these categories and tested positive for resistance to at least rifampicin and isoniazid without any history of consuming 1st line tb treatment are considered to be primary mdr-tb.9 this study still used the old definitions of pre-xdr-tb is mdr-tb with resistance to any fluoroquinolone or at least one of three second-line injectable drugs (capreomycin, kanamycin, and amikacin), and xdr-tb is tb that is resistant to any fluoroquinolone and at least one of three second-line injectable drugs (capreomycin, kanamycin, and amikacin). however, there are new definitions by who in 2021 because this study used the data from the year 2012-2021, which used the old definitions for patients grouping.14 data retrieved was further presented in the proportion of mdr-tb, characteristics, eptb sites, and anti-tb drug resistance, using a descriptive method with spss ibm® spss® ver. 22. ethical issues ethical permission for this study had been granted from the ethics committee of universitas padjadjaran with number 494/un6.kep/ec/2021 and the research permit were given from the health research ethics committee of dr. hasan sadikin general hospital bandung with number lb.02.01/x.2.2.1/16836/2021. results and discussion the data we collected was 7013 tb cases, consisting of 1900 mdr-tb cases (27.1%) and 5113 tb sensitive cases (72.9%). as we collected the data from 1900 registered mdrtb patients, we found 97 patients that came repetitively, hence re-registered into the database, of whom came five times (n1), thrice (n6), and twice (n90), thus resulting in the same name counted as different cases in this study. nevertheless, this study still included all of these data because the cases were considered different from those who lost to follow-up, relapsed, and failed conversion after treatment. of a total of 1900 adults, 1799 had pulmonary tb (ptb), 6 had extrapulmonary tb (eptb), 11 combined had eptb with ptb, and 84 with no data found, which excluded in other variables analyzed in table 1–3. table 1 shows the proportion of mdr-tb characteristics and exclusively shows the proportion of ptb mdr, eptb mdr, and their combination. most of the mdr-tb patients were in the age group 35 – 44 years old and 25 – 34 years old among eptb mdr patients. the majority of the mdr-tb patients were male (58%). underweight was found the most in mdr-tb patients (46.3%), pulmonary mdr-tb patients (61.2%), and extrapulmonary mdr-tb patients (84.6%). there were 275 pulmonary mdr-tb patients with diabetes mellitus as their comorbid disease and 80 mdr-tb patients with hypertension. interestingly, there were two mdr-tb patients with systemic lupus erythematosus (sle) and 25 mdr-tb patients with hiv co-infection, consisting of 17 pulmonary mdr-tb patients, one combined eptb with ptb mdr patient, and the rest were unknown. there were 43% of mdr-tb patients who had never smoked and 74.2% of mdr-tb patients who had never consumed alcohol. category 6 from the risk category of mdr-tb, defined as relapse tb patients (category 1 and 2), was the most frequent in this study (36.8%) and among extrapulmonary mdr-tb was primary mdr-tb (35.3%), defined as acquired mdr-tb patients who don’t belong to the 12 categories. 117 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 113–122 table 1. characteristics proportion in mdr ptb, eptb, and eptb with ptb characteristic mdr-tb total mdr-tb locations ptb eptb eptb with ptb n (%) n (%) n n 1816 1799 6 11 gender male 1058 (58.3) 1047 (58) 4 7 female 758 (41.7) 752 (42) 2 4 age (years) – median (iqr) 38 (35–44) 38 (35–44) 28 (25–34) 27 (25–34) clinical history bmi (kg/m2)* 1432 1419 6 7 <18.5 879 (46.3) 868 (61.2) 5 6 18.5-22.9 426 (22.4) 424 (29.9) 1 1 23-24.9 62 (3.3) 62 (4.4) ≥25 65 (3.4) 65 (4.6) comorbid diseases diabetes mellitus 275 (15.1) 275 (15.3) hiv/aids 25 (1.4) 18 (1) 1 hypertension 80 (4.4) 80 (4.5) sle 2 (0.1) 2 (0.1) behavioral history smoking 1734 1722 5 7 yes 101 (5.8) 99 (5.7) 1 1 ex-smoker 815 (47) 812 (47.2) 2 1 non-smoker 818 (47.1) 811 (47.1) 2 5 consuming alcohol 1733 1721 5 7 yes 8 (0.5) 8 (0.5) ex-drinker 315 (18.2) 314 (18.2) 1 non-drinker 1410 (81.4) 1399 (81.3) 4 7 risk category for mdr-tb† 1814 1797 6 11 1. chronic tb patients 184 (10.2) 183 (10.2) 1 2. tb patient with 2nd category treatment which not converted in 3 months of treatment 80 (4.3) 80 (4.5) 3. tb patient who have a non-standard tb treatment history using quinolone and 2nd line anti-tb drug injection for at least 1 month 104 (6.1) 103 (5.7) 1 4. tb patients who failed 1st category treatment 214 (11.7) 213 (11.9) 1 5. tb patients with 1st category treatment who remain positive after 3 months of treatment 76 (4.2) 74 (4.1) 2 6. relapse tb patient, categories 1 and 2 672 (36.8) 667 (37.1) 3 2 118 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 winnery dhestina, et al. proportion of extrapulmonary mdr-tb 7. returning tb patients after loss to follow-up 201 (11.3) 201 (11.2) 8. suspected tb patients with a history of close contact with mdr-tb patients 54 (2.9) 54 (3) 9. tb-hiv co-infection patients who do not respond to anti-tb drug administration 4 (0.3) 4 (0.2) 10. mdr-tb patients who failed treatment 6 (0.3) 6 (0.3) 11. relapse mdr-tb patients 13 (0.7) 12 (0.7) 1 12. returning mdr-tb patients after loss to follow-up 3 (0.2) 3 (0.2) primary mdr 208 (11) 197 (11) 3 3 note: there were samples that weren’t listed. *bmi classification based on asia-pacific guidelines; underweight: <18.5 kg/m2. normal: 18-22.9 kg/m2. overweight: 23-24.9 kg/m2. obese: >25 kg/m2 †risk category number 10 – 12 applied since the year 2020. mdr: multi drug resistant, ptb: pulmonary tb, eptb: extrapulmonary tb table 2 shows the site of infection in extrapulmonary mdr-tb, the most frequent was in the bone. the proportion of drug resistance was described in table 3, which shows the most frequent resistance from this study was the mdr-tb (41.4%) and rifampicin-resistant tb (rr-tb) in extrapulmonary mdr-tb (76.8%). table 2. distribution proportion of eptb by site in mdr-tb patients extrapulmonary mdr-tb location n (%) eptb, n (%) eptb with ptb, n (%) tb lymphadenitis 4 (23.5) 1 3 tb meningits 3 1 2 (66.7) bone or joint tb 6 (35.3) 3 (50) 3 tb spondylitis 5 (29.4) 2 (40) 3 tb colitis 3 1 (33.3) 2 (66.7) tb pleurisy 1 1 (100) total (n) 17 6 11 mdr: multidrug-resistant, ptb: pulmonary tb, eptb: extrapulmonary tb table 3. anti-tb drug resistance proportion in mdr-tb patients drug resistance n (%) n = 1816 ptb, n (%) eptb, n ptb + eptb, n pre-xdr-tb 212 (11.7) 211 (11.7) 1 xdr-tb 102 (5.6) 101 (5.6) 1 mdr-tb, n 757 (41.7) 755 (42) 1 1 rifampicinisoniazid 350 (19.3) 349 (19.4) 1 rifampicinisoniazidethambutol 141 (7.8) 140 (7.8) 1 rifampicinisoniazidstreptomycin 98 (5.4) 98 (5.4) rifampicinisoniazidethambutolstreptomycin 167 (9.2) 167 (9.3) rifampicinisoniazidethambutolstreptomycinpyrazinamide 1 (0.1) 1 (0.1) rifampicin-resistant based 745 (41) 732 (40.7) 5 8 total (n) 1816 1799 6 11 pre-xdr: pre-extensively drug resistant, xdr: extensively drug resistant, mdr: multidrug-resistant, ptb: pulmonary tb, eptb: extrapulmonary tb. 119 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 113–122 this study has explored the characteristics proportion, location, infection site in extrapulmonary and anti-tb drugs resistance of mdr-tb. with the genexpert®, mdr tb suspects have been examined since 2012. in total, 15821 adults examined, mdr tb confirmed were 1900 adults, of whom 1799 pulmonary tb (ptb), including one miliary tb case, 6 exclusively extrapulmonary tb (eptb), 11 combined eptb with ptb, and 84 unknown data locations. the total extrapulmonary mdr-tb cases were 17 cases, 0.24% of total tb cases, and 0.9% of mdr-tb cases. these results show differences with the studies in china, which were 2.5% of total tb patients and 9.3% of mdr-tb patients, and in india, were 5.1% of total tb cases and 12.6% of mdr-tb cases.11,12 however, in mdr cases, extrapulmonary tb always has shown less frequent than pulmonary tb, which suggested by raveendran et al., that could be caused by fewer number of extrapulmonary tb that has been treated before.15 in this study, the median age of mdr-tb patients was 38-year-old in the age group 35 – 44 years old and in extrapulmonary was 27year-old in the age group 25 – 34 years old, which is similar to previous studies where most patients belonged to the productive age group of 21 – 45 years old.12,16,17 most productive age people spend most of their time outside their home, meeting or passing by many people frequently, which increases the risk of tb or mdr-tb exposure, wellknown for its high incidence in indonesia.5,12 among mdr-tb and eptb mdr patients, males were found more commonly (58.3% and 64.7%), similar to previous studies in india and china, consecutively 59.5% and 69.1%.12,17 this finding is probably due to the patriarchal culture in the related country, causing females to become more passive to independently go to healthcare to check themselves up, resulting in female patients being found less than male patients.12,17,18 a different result showed from a study in peru, with a more female proportion (60.6%).19 this difference was presumptively caused by the ratio of male-tofemale that defers in each country. in 2012 – 2020, the male population outnumbered the female population in indonesia, with an average of 50.4%.20 almost half of the mdr-tb patients (46.3%) in this study had a body mass index (bmi) <18.5 kg/m2, which is categorized as underweight. ptb mdr patients (61.2%), eptb mdr patients (67%), and combined eptb with ptb patients showed a similar result. a higher underweight population in mdr-tb patients was probably resulting from a decreased immune system that existed in underweight, leading to an increased risk of getting community-acquired infection.21 this study shows 275 mdr-tb patients (15.1%)—all belonged to pulmonary mdrtb, had diabetes mellitus (dm), as the similar results found in a study in sudan (15.8%).22 this is probably related to decreasing the immune system in patients with dm, increasing the infection risk, and decreasing anti-tb drugs effectivity, resulting in delayed conversion in tb patients, leading to the risk of mtb strains resistance.23,24 unfortunately, indonesia is tb and dm high burden country, so doctors and government should pay more attention to these diseases.25 the following comorbid disease is hypertension (4.4%). its reasons are still unknown, and some studies showed no evidence between hypertension and tb.26 still, presumably, it is related to high hypertension prevalence in indonesia, resulting in its pretty high proportion in mdr-tb.28 there were also 25 patients (1.4%) with hiv co-infection, which its cause was similar with patients with dm, people living with hiv/aids (plwha) also had a decreased immune system, which could increase the risk of getting infection and malabsorption of anti-tb drugs, especially isoniazid and rifampicin, resulting in a higher risk of getting mdr-tb.28 this study showed two patients with sle, with probable mechanism, were the administration of 120 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 winnery dhestina, et al. proportion of extrapulmonary mdr-tb corticosteroid as sle therapy, which lower the patient’s immune system, resulting in an increased risk of getting any infection.29 in this study, 101 mdr-tb patients (5.8%) smoked, which had a considerable far gap than studies in india and sudan, consecutively 65.8% and 59.2%.12,22 however, in this study, there were also found 815 patients (47%) who had a smoking history but had stopped. eight patients (0.5%) also consumed alcohol, which had a considerable gap with other studies in india and sudan, with 42.1% and 47.4%, consecutively.12,22 different results in indonesia might result from their majority religion, muslim, which alcohol is considered haraam or forbidden so that most of them choose not to drink alcohol.30 according to the risk category of mdrtb, most patients came after a tb relapse, meaning that the patient had been treated with anti-tb drugs, which correspond with the study in india (56.2%).12 previous anti-tb drugs exposure might increase mdr-tb incidence globally by 18% and in indonesia 13% from an mdr-tb incidence. those numbers might differ from other studies in other countries.5,31 primary mdr-tb patients took over 11% of mdr-tb patients and were found in six out of 17 extrapulmonary mdr-tb patients, similar to the result in a study done in india (7.9%).32 yet, indonesia and india also had similar estimated percentages in primary mdr-tb incidence, consecutively 2.4% and 2.8%.5 among 17 cases of extrapulmonary infection, the most frequent sites were bone (35.3%) and lymph (23.5%), with a similar percentage compared to extrapulmonary mdr-tb and combined eptb with ptb mdr. a study in china about extrapulmonary tb also shows skeletal tb as its predominant site (41.4%), but it doesn’t exclusively show the results of extrapulmonary mdr-tb.11 meanwhile, a study in india showed that 51.3% of sites involved in extrapulmonary mdr-tb were in lymph nodes, which is reasonable due to tb pathogenesis resulting in mtb travel to lymph nodes for antigen-presenting, shuttled to other lymph nodes, or continue to travel via lymph fluid.32 this study found that the most frequent anti-tb drug resistance was rifampicin (41%), followed by rif+inhresistant (19.3%). nevertheless, more resistances towards inh+rif+emb+sm (31.1%) were found in india, which shows different results against this study.12 conclusions the retrospective method limits this study to explore the proportion of extrapulmonary mdr-tb and its characteristics proportion due to missing and incomplete data. this study was conducted in a referral hospital resulting in more frequent mdr-tb cases. the extrapulmonary mdr-tb cases are scarce, not portraying the actual proportion, unlike the mdr-tb cases. future studies are needed to include more cases in other centers to have more overview of mdr-tb with eptb. to conclude, the total extrapulmonary mdr-tb cases in dr. hasan sadikin general hospital bandung period 2012 – 2021 take over 0.24% from all of tb cases and 0.9% from all mdr-tb cases, predominated by age group 25 – 34 years old with age median 27-year-old, male gender, underweight bmi, non-smoking behavior, non-alcohol consuming behavior, and previous history of tb. most extrapulmonary mdr-tb cases are rifampicin-resistant, and the site of infection is located in bone. acknowledgement we thank all of the staff in the mdr clinic, dots clinic, and genexpert® lab in dr. hasan sadikin general hospital bandung for their approval and support in collecting the data for this study. 121 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 113–122 conflict of interest the author(s) declared no potential conflicts of interest concerning this article's research, authorship, and/or publication. references 1. churchyard g, kim p, shah ns, rustomjee r, gandhi n, mathema b, et al. what we know about tuberculosis transmission: an overview. j infect dis. 2017;216(suppl 6):s629–35. 2. barberis i, bragazzi nl, galluzzo l, martini m. the history of tuberculosis: from the first historical records to the isolation of koch’s bacillus. j prev med hyg. 2017;58(1):e9–12. 3. global tuberculosis report 2021. geneva: world health organization; 2021. 1–57 p. 4. kementerian kesehatan ri. tb mdr [internet]. 2021 [cited 2021 nov 8]. available from: https://tbindonesia.or.id/pustakatbc/informasi/teknis/tb-mdr/ 5. who. global tuberculosis report 2020 [internet]. geneva; 2020. available from: https://apps.who.int/iris/bitstream/handle/10665/ 336069/9789240013131-eng.pdf 6. kementerian kesehatan ri. peraturan menteri kesehatan republik indonesia no. 67 tahun 2016 tentang penanggulangan tuberkulosis. peraturan menteri kesehatan. [internet]. journal of chemical information and modeling indonesia; 2016. available from: https://www.kncv.or.id/publikasi/229permenkes-no-67-tahun-2016-penanggulangantuberkulosis.html 7. kementerian kesehatan ri. petunjuk teknis penatalaksanaan tuberkulosis resistan obat 2020 [internet]. jakarta; 2020. 978–979 p. available from: https://tbindonesia.or.id/wpcontent/uploads/2021/06/tbro_buku-juknistuberkulosis-2020-website.pdf 8. sahiratmadja e, mega g, andriyoko b, parwati i. performance of xpert® mtb/rif in detecting multidrug-resistance tuberculosis in west java, indonesia. mkb. 2020;52(2):99–106. 9. hamusse sd, teshome d, hussen ms, demissie m, lindtjørn b. primary and secondary antituberculosis drug resistance in hitossa district of arsi zone, oromia regional state, central ethiopia. bmc public health [internet]. 2016;16(1):1–10. available from: http://dx.doi.org/10.1186/s12889-016-3210-y 10. qian x, nguyen dt, lyu j, albers ae, bi x, graviss ea. risk factors for extrapulmonary dissemination of tuberculosis and associated mortality during treatment for extrapulmonary tuberculosis article. emerg microbes infect [internet]. 2018;7(1):1–14. available from: http://dx.doi.org/10.1038/s41426-018-0106-1 11. pang y, an j, shu w, huo f, chu n, gao m, et al. epidemiology of extrapulmonary tuberculosis among inpatients, china, 2008-2017. emerg infect dis. 2019;25(3):457–64. 12. sinha p, srivastava gn, gupta a, anupurba s. association of risk factors and drug resistance pattern in tuberculosis patients in north india. j glob infect dis. 2017;9(4):139–45. 13. rodriguez-takeuchi sy, renjifo me, medina fj. extrapulmonary tuberculosis: pathophysiology and imaging findings. radiographics. 2019;39(7):2023–37. 14. world health organization. meeting report of the who expert consultation on the definition of extensively drug-resistant tuberculosis [internet]. wold health organization. 2020. cc by-ncsa 3.0 igo. available from: https://www.who.int/publications/i/item/meeting -report-of-the-who-expert-consultation-on-thedefinition-of-extensively-drug-resistanttuberculosis 15. raveendran r, oberoi jk, wattal c. multidrugresistant pulmonary & extrapulmonary tuberculosis: a 13 years retrospective hospitalbased analysis. indian j med res. 2015;142(november):575–82. 16. kementerian kesehatan ri. profil kesehatan indonesia tahun 2019 [internet]. jakarta: sekretariat jenderal; 2019. available from: https://pusdatin.kemkes.go.id/resources/downloa d/pusdatin/profil-kesehatan-indonesia/profilkesehatan-indonesia-2019.pdf 17. lu z, jiang w, zhang j, lynn hs, chen y, zhang s, et al. drug resistance and epidemiology characteristics of multidrug-resistant tuberculosis patients in 17 provinces of china. plos one. 2019;14(11):1–14. 18. mason ph, snow k, asugeni r, massey pd, viney k. tuberculosis and gender in the asiapacific region. aust n z j public health. 2017;41(3):227–9. 19. wang s, tu j. nomogram to predict multidrugresistant tuberculosis. ann clin microbiol antimicrob [internet]. 2020;19(1):1–8. available from: https://doi.org/10.1186/s12941-02000369-9 20. staff wb. population, male (% of total population) indonesia [internet]. 2019 [cited 2021 nov 8]. available from: https://data.worldbank.org/indicator/sp.pop.to tl.ma.zs?end=2020&locations=id&start=201 2 21. dobner j, kaser s. body mass index and the risk of infection from underweight to obesity. clin 122 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 winnery dhestina, et al. proportion of extrapulmonary mdr-tb microbiol infect [internet]. 2018;24(1):24–8. available from: https://doi.org/10.1016/j.cmi.2017.02.013 22. ali mh, alrasheedy aa, a hm, kibuule d, go. predictors of multidrug-resistant tuberculosis. 2019;1–11. 23. baghaei p, tabarsi p, javanmard p, farnia p, marjani m, moniri a, et al. impact of diabetes mellitus on tuberculosis drug resistance in new cases of tuberculosis. j glob antimicrob resist [internet]. 2016;4:1–4. available from: http://dx.doi.org/10.1016/j.jgar.2015.11.006 24. saktiawati ami, subronto yw. influence of diabetes mellitus on the development of multi drug resistant-tuberculosis in yogyakarta. acta med indones. 2018;50(1):11–7. 25. restrepo bi. diabetes and tuberculosis. schlossberg d, editor. microbiol spectr [internet]. 2016 dec 23;4(6):519–22. available from: https://journals.asm.org/doi/10.1128/microbiols pec.tnmi7-0023-2016 26. seegert ab, rudolf f, wejse c, neupane d. tuberculosis and hypertension—a systematic review of the literature. int j infect dis. 2017;56:54–61. 27. peltzer k, pengpid s. the prevalence and social determinants of hypertension among adults in indonesia: a cross-sectional population-based national survey. int j hypertens. 2018;2018. 28. singh a, prasad r, balasubramanian v, gupta n. drug-resistant tuberculosis and hiv infection: current perspectives. hiv/aids res palliat care. 2020;12:9–31. 29. balbi ggm, machado-ribeiro f, marques cdl, signorellia f, levy ra. the interplay between tuberculosis and systemic lupus erythematosus. curr opin rheumatol. 2018;30(4):395–402. 30. ri kdn. visualisasi data kependudukan [internet]. 2021. available from: https://gis.dukcapil.kemendagri.go.id/peta/ 31. cheng q, xie l, wang l, lu m, li q, wu y, et al. incidence density and predictors of multidrug-resistant tuberculosis among individuals with previous tuberculosis history: a 15-year retrospective cohort study. front public heal. 2021;9(may):1–14. 32. desai u, joshi j. extrapulmonary drug-resistant tuberculosis at a drug-resistant tuberculosis center, mumbai: our experience – hope in the midst of despair! lung india [internet]. 2019;36(1):3. available from: http://www.lungindia.com/text.asp?2019/36/1/3/ 249161 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 2 may–august 2022 original article knowledge and attitudes of dengue virus infection transmission and its relationship with eradication action program in surabaya, indonesia ni njoman juliasih1* , teguh hari sucipto2 , reny mareta sari3 , zakiyathun nuha2 , soegeng soegijanto2 1school of medicine, ciputra university, surabaya, indonesia 2institute of tropical disease, universitas airlangga, surabaya, indonesia 3department of public health, institute of health sciences strada indonesia, kediri, indonesia received: june 9th, 2022; revised: june 23rd, 2022; accepted: july 4th, 2022 abstract dengue virus infection is caused by a dengue virus transmitted through mosquito bites from species aedes albopictus and aedes aegypti. the ministry of health takes action to reduce the prevalence of dhf by regulating the management of psn 3m plus. this study aimed to determine the knowledge, attitude, and compliance with the management of psn 3m plus strategies of those living in surabaya. a cross-sectional population-based google form questionnaire was conducted in january 2022 for four weeks (january 3, 2022, to january 29, 2022). based on the bivariate analysis, gender and age of respondents were no relationship between compliance with the psn 3m plus (p-value >0.05). the results also showed no relationship between education and adherence to psn 3m plus (p-value > 0.05). however, based on previous studies, people with higher education showed better compliance. public knowledge and attitude about the dengue virus and its transmission process can be increased by developing, modifying, and intervening in the people controlling dengue virus infection. most people of surabaya believe that dengue prevention is the complete responsibility of every people. based on the bivariate analysis, the characteristics of respondents had no relationship with the psn 3m plus compliance (p-value > 0.05). knowledge and attitudes of the surabaya people toward psn 3m plus are still good. however, the characteristics of the respondents did not significantly affect their knowledge and attitudes keywords: attitude; dengue virus infection; knowledge; prevention; surabaya. abstrak infeksi virus dengue merupakan penyakit yang disebabkan oleh virus dengue yang ditularkan melalui gigitan nyamuk aedes aegypti dan aedes albopictus. kementerian kesehatan melakukan tindakan untuk menurunkan prevalensi dbd dengan mengatur pengelolaan psn 3m plus. penelitian ini bertujuan untuk menganalisis korelasi antara pengetahuan dan kebiasaan terhadap kepatuhan manajemen strategi psn 3m plus pada masyarakat yang berdomisili di surabaya. kuesioner google form berbasis populasi cross-sectional dilakukan pada januari 2022, 4 minggu (3 jan 2022 hingga 29 jan 2022). berdasarkan analisis bivariat, jenis kelamin dan usia responden tidak ada hubungan antara kepatuhan mengikuti psn 3m plus (p-value >0,05). hasil analisis statistik juga menunjukkan bahwa tidak ada hubungan antara pendidikan dengan kepatuhan terhadap psn 3m plus (p-value >0,05). hasil penelitian sebelumnya menunjukkan bahwa orang-orang dengan pendidikan lebih tinggi menunjukkan kepatuhan yang lebih baik, karenan pendidikan merupakan aspek penting dari pengendalian infeksi virus dengue. pengetahuan dan sikap masyarakat tentang virus dengue dan proses penularannya dapat ditingkatkan dengan mengembangkan, memodifikasi dan mengintervensi masyarakat * corresponding author: njoman.juliasih@ciputra.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0003-2142-7882 https://orcid.org/0000-0003-0512-2990 https://orcid.org/0000-0003-3192-3302 https://orcid.org/0000-0001-8652-0469 https://orcid.org/0000-0002-9470-8223 138 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 ni njoman juliasih, et al. knowledge and attitudes of dengue virus infection yang mengendalikan infeksi virus dengue. sebagian besar masyarakat surabaya percaya bahwa pencegahan dbd adalah tanggung jawab penuh setiap orang. berdasarkan analisis bivariat, karakteristik responden tidak memiliki hubungan dengan kepatuhan masyarakat dalam menjalankan psn 3m plus (p-value > 0.05). pengetahuan dan sikap masyarakat surabaya terhadap psn 3m plus masih baik. meskipun karakteristik responden tidak berpengarus signifkan terhadap pengetahuan dan kepatuhan. kata kunci: infeksi virus dengue; pencegahan; pengetahuan; sikap; surabaya how to cite: juliasih, n. j., sucipto, t. h., sari. r. m., nuha, z., soegijanto, s. knowledge and attitudes of dengue virus infection transmission and its relationship with eradication action program in surabaya, indonesia. indonesian journal of tropical and infectious disease. 10(2). 137–143. aug. 2022. introduction dengue virus infection is a mosquitoborne infectious disease with more than 100 tropical and subtropical countries globally reported endemic.1 it was transmitted through the bites of aedes aegypti and aedes albopictus mosquitoes which have previously been infected by dengue virus from dengue sufferers. according to the world health organization, the estimated annual fatality rate was 2.5%, among individuals with severe dengue, with complications like hemorrhagic fever and fluid accumulation. dengue infection is prevalent in southeast asia because of its primary vector, aedes aegypti mosquitos.2 dengue infection was commonly reported among children and teenagers, but the prevalence among older age groups also increased in recent decades.3,4 in indonesia, dengue hemorrhagic fever (dhf) cases in 2020 with 108,303 cases.5 the incidence of dengue virus infection is caused, among others, due to high mobility, population density, environmental conditions, and the community's behaviour.6 cases of dengue virus infection in east java province in 2020 were 8,567 cases.7 surabaya city is one of the cities in east java with the highest number of dengue virus infection cases in all work areas, with 73 cases.5 the ministry of health issued a policy with the number pm.01.11/menkes/591/2016 to reduce dengue prevalence by regulating the management with eradication action program (psn 3m plus). the movement of the program consists of draining the water storage, closing the landfill, and reusing used items that have the potential for mosquito breeding, plus sprinkling larvicides, using mosquito repellent, keeping larvae eating fish in the landfill, planting mosquito repellent, and others. according to the ministry of health (2019), the one house and one larva hunter effectively prevent dengue fever.8 the other effective strategy is that the authorities need to ensure that local people have decent knowledge about vector control and follow the recommendations. the only method for controlling the dengue virus infection is vector control, as no specific treatment or vaccine is available.9–12 knowledge and behaviour greatly influence the dynamics of the aedes mosquito population, which in turn affects dengue virus transmission. therefore, vector control is critical, with knowledge and fundamental aspects of infection control and prevention of dengue virus. presently, the recommended control effort is the eradication of mosquito breeding nests. however, the local people need to have a sufficient understanding of the routes of dengue virus transmission, as their behaviour plays a vital role in limiting dengue disease transmission.13 this study aimed to determine the knowledge, attitude, and compliance with the management of psn 3m plus strategies of those living in surabaya. study findings of this research are expected to help policymakers develop strategies for more effective prevention and control of dengue virus infection and increase community participation in dengue prevention programs. 139 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 137–143 materials and methods ethics statement this study was approved by the lembaga penelitian dan pengabdian kepada masyarakat universitas airlangga with approval number 24-934/un3.14/ppd/2013. study design and study population a cross-sectional population-based google form questionnaire was conducted in january 2022 for four weeks (january 3, 2022, to january 29, 2022). the population in this study was residents with surabaya id cards concerning their age, gender, profession, monthly income, and education. this study used a random sampling method. the data processing techniques in this study were editing, coding, entry, cleaning, and saving. statistical analysis the statistical test used is chi-square using α = 0.05 with spss software. results and discussion dengue virus infection in surabaya the first dengue outbreak in surabaya was reported in surabaya in 1968.14 since then, the incidence of dengue has been increasing, with several outbreaks occurring in 1973, 1988, 1988, 2007, and 2010.15 even though dengue is a big problem in surabaya, indonesia, the incidence of dengue virus infection in surabaya has decreased over the last five years, as shown in figure 1. figure 1. dengue virus infection cases in surabaya5,7,16–19 demographic characteristics of the respondents in surabaya respondents in this study came from 5 territories of the city of surabaya (table 1). the total respondent in this study was 60, with the characteristic of respondents shown in table 2. table 1. total of respondent territory total respondent (%) east surabaya 33.3 north surabaya 13.3 central surabaya 5 south surabaya 26.7 west surabaya 21.7 table 2. the characteristic of respondent characteristic study site total (%) sex male 11 (18.3) female 49 (81.7) age (y.o) 19-32 35 (58.3) 33-45 10 (16.7) 46-60 15 (25.0) profession work 34 (56.7) doesn't 36 (43.3) education senior high school/equal 22 (36.7) bachelor/diploma 34 (56.7) master 4 (6.7) monthly income <rp. 1.000.000 19 (31.7) rp. 1.000.000 – rp. 2.500.000 10 (16.7) rp. 2.500.000 – rp. 4.000.000 15 (25.0) rp. 4.000.000 – rp. 5.000.000 7 (11.7) >rp. 5.000.000 9 (15.0) respondent's dengue virus infection history yes 3 (5.0) not yet 57 (95.0) family history of dengue virus infection yes 8 (13.3) not yet 52 (86,7) knowledge of the surabaya city people regarding dengue and its transmission the respondent's knowledge in surabaya about dengue virus infection like symptoms, vector transmission, and prevention was high (table 3). 140 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 ni njoman juliasih, et al. knowledge and attitudes of dengue virus infection table 3. respondent's knowledge characteristic category knowledge total low medium high sex male 0 0 11 49 female 0 3 46 11 total 0 3 57 60 age (y.o) < 30 0 1 26 27 30-40 0 2 11 13 40-50 0 0 9 9 >50 0 0 11 11 total 0 3 57 60 profession work 0 2 32 34 does not work 0 1 25 26 total 0 3 57 60 monthly income < rp. 1.000.000 0 0 19 19 rp. 1000.000 – rp. 2.500.000 0 3 7 10 rp. 2.500.000rp. 4.000.000 0 0 15 15 rp. 4.000.000-rp. 5.000.000 0 0 7 7 < rp. 5.000.000 0 0 9 9 total 0 3 57 60 respondent's dengue virus infection history yes 0 0 3 3 not yet 0 3 54 57 total 0 3 57 60 family history of dengue virus infection yes 0 0 8 8 not yet 0 3 49 52 total 0 3 57 60 the attitude of the surabaya city people regarding dengue virus infection the respondents' knowledge about dengue virus infection is high, and their compliance with psn 3 m plus recommendations was far from satisfactory. non-compliance can be explained by the respondent's attitude (table 4). table 4. respondents attitude characteristic category attitude total bad medium good sex male 1 4 6 11 female 5 23 21 49 total 6 27 27 60 age (y.o) < 30 4 14 9 27 30-40 2 7 4 13 40-50 0 3 6 9 >50 0 3 8 11 total 6 27 27 60 profession work 4 15 15 34 does not work 2 12 12 26 total 6 27 27 60 monthly income < rp. 1.000.000 2 8 9 19 rp. 1.000.000rp. 2.500.000 1 4 5 10 rp. 2.500.000rp. 4.000.000 2 6 7 15 rp. 4.000.000rp. 5.000.000 0 5 2 7 < rp. 5.000.000 1 4 4 9 total 6 27 27 60 respondent's dengue virus infection history yes 1 2 0 3 not yet 5 25 27 57 total 6 27 27 60 family history of dengue virus infection yes 2 2 4 8 not yet 4 25 23 52 total 6 27 27 60 knowledge high 5 26 26 57 medium 1 1 1 3 low 0 0 0 0 total 6 27 27 60 141 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 137–143 based on the bivariate analysis, gender and age of respondents were no relationship between compliance with the psn 3m plus (p-value >0.05). therefore, every male and female person should be taking action to eradicate the dengue virus infection vector to control the dengue epidemic. the characteristics of respondents had no relationship with the psn 3m plus compliance (p-value > 0.05). the results of previous studies about the psn 3m plus stated a relationship between program adherence and profession.20–22 regardless of their characteristics, people have good knowledge and attitude toward dhf prevention. many programs and information are available and can be easily acquired by people. there are several dhfrelated health promotion programs, such as psn 3m plus. some are even socialized through television. therefore psn 3m plus are popular in society. besides that, due to the number of dhf cases in indonesia, many mosquito repellent products such as insecticide spray, repellent lotion, mosquito coil, and others are easily found. public knowledge and attitude about the dengue virus and its transmission process can be increased by developing, modifying, and intervening in the people controlling dengue virus infection. prevention and control of dengue virus infection is a shared responsibility. for example, routine fogging carried out by the health service, this activity only kills adult mosquitoes, so it does not eliminate the risk of transmission of dengue virus infection, especially larvae and eggs. if fogging cannot kill the larvae in water, 3m plus prevention must still be conducted by the people, such as closing or covering water containers and reservoirs to become mosquito breeding sites potentially. most respondents showed good attitudes and habits toward preventing dengue fever outbreaks. they also took action to protect the whole family from dengue fever and mosquito bites by following the recommendation. dhf prevention practices have been implemented, and they believe that knowledge comes from both educational background and experience. the present study showed that most people surveyed had experienced dengue virus infection. mosquitoes like to breed in an environment with clean water. therefore, most people of surabaya believe that dengue prevention is the complete responsibility of every people. discipline in enforcing psn 3m plus cannot be based on the high education of an individual. highly educated individuals are usually busy with their work routines, so they cannot focus on the environment around their homes. this study found that respondents from south surabaya, sawahan district, one of the endemic areas of dhf in surabaya, have complied with the psn 3m plus policy based on the questionnaires we distributed. the high rate of dengue virus infection in this area is possible because of the poorly maintained settlements and a large number of nomadic residents. understanding the spreading pattern of dengue fever and the risk factors associated with transmission in surabaya is essential to prevent future outbreaks through targeted vector control of dengue virus infection. furthermore, disseminating appropriate behaviour change communication messages to improve household level environmental modification of destruction of breeding sites around homes, encourage adherence to personal protection, and identification of disease symptoms as successfully applied in many endemic countries.23–25 study limitations the limitation of this research study is that author unable to verify the congruence between the respondent's answers with the actions practised in everyday life. however, the strength is that respondents were recruited and information obtained based on criteria in endemic areas. therefore, the results of this research study are easily applied to the community setting. furthermore, the study's 142 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 ni njoman juliasih, et al. knowledge and attitudes of dengue virus infection eligibility criteria further strengthen the quality of the findings. conclusions respondents have a good level of knowledge and attitude towards dengue prevention efforts. however, the characteristics of the respondents did not significantly affect their knowledge and attitudes. one of the factors that influenced the results was probably the easy access to information related to dhf programs in the community.the results of this study suggest the need for future research regarding information, education and communication strategies along with current implemented intervention efforts evaluation. acknowledgement this research was supported by the centre of excellence-institute of tropical disease universitas airlangga. conflict of interest the authors declare that they have no competing interests. references 1. world health organization (who). dengue and severe dengue. who. https://www.who.int/news-room/factsheets/detail/dengue-and-severe-dengue. published 2022. accessed june 6, 2022. 2. wang e, ni h, xu r, et al. evolutionary relationships of endemic/epidemic and sylvatic dengue viruses. j virol. 2000;74(7):3227-3234. doi:10.1128/jvi.74.7.3227-3234.2000. 3. karyanti mr, uiterwaal cspm, kusriastuti r, et al. the changing incidence of dengue haemorrhagic fever in indonesia: a 45-year registry-based analysis. bmc infect dis. 2014;14(1):412. doi:10.1186/1471-2334-14-412. 4. chan eyy, lo esk, huang z, et al. sociodemographic predictors of knowledge, mosquito bite patterns and protective behaviors concerning vector borne disease: the case of dengue fever in chinese subtropical city, hong kong. beechler br, ed. plos negl trop dis. 2021;15(1):e0008993. doi:10.1371/journal.pntd.0008993. 5. kementrian kesehatan republik indonesia. profil kesehatan indonesia tahun 2020. jakarta: kementrian kesehatan republik indonesia; 2021. https://www.kemkes.go.id/downloads/resources/ download/pusdatin/profil-kesehatanindonesia/profil-kesehatan-indonesia-tahun2020.pdf. 6. ariyanto a, ibrahim e, syahribulan s, ishak h, syamsuar s, djajakusli r. density of aedes aegypti larvae based on knowledge, attitude, and action to eradicate mosquito nest in daya market of makassar city. j asian multicult res med heal sci study. 2020;1(2):84-93. doi:10.47616/jamrmhss.v1i2.52. 7. dinas kesehatan provinsi jawa timur. profil kesehatan dinas kesehatan provinsi jawa timur 2020. surabaya; 2021. https://dinkes.jatimprov.go.id/userfile/dokumen/ profil kesehatan 2020.pdf. 8. kementrian kesehatan republik indonesia. profil kesehatan indonesia tahun 2019. jakarta: kementrian kesehatan republik indonesia; 2020. https://www.kemkes.go.id/downloads/resources/ download/pusdatin/profil-kesehatanindonesia/profil-kesehatan-indonesia-2019.pdf. 9. muhammad ansori an, fadholly a, proboningrat a, et al. novel antiviral investigation of annona squamosa leaf extract against the dengue virus type-2: in vitro study. pharmacogn j. 2020;13(2):456-462. doi:10.5530/pj.2021.13.58. 10. maharani a, sucipto t, setyawati h, et al. in vitro antiviral activity of morin compound against dengue virus type 1 in vero cells. syst rev pharm. 2020;11(9):830-833. doi:10.31838/srp.2020.9.118. 11. sumarsih s, pratiwi md, ainni in, et al. the influence of metal on the performance of 2,4,5triphenylimidazole as an inhibitor of dengue virus replication. asia pacific j mol biol biotechnol. september 2020:113-121. doi:10.35118/apjmbb.2020.028.3.11. 12. sucipto th, martak f. inhibition of dengue virus serotype 2 in vero cells with [cu(2,4,5triphenyl-1h-imidazole)2(h2o)2].cl2. infect dis rep. 2020;12(11):8744. doi:10.4081/idr.2020.8744. 13. minarti m, anwar c, irfannuddin i, irsan c. community knowledge and attitudes about the transmission of dengue haemorrhagic fever and its relationship to prevention behaviour in palembang, south sumatra, indonesia. open access maced j med sci. 2021;9(e):1534-1543. doi:10.3889/oamjms.2021.7693. 143 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 indonesian journal of tropical and infectious disease, vol. 10 no. 2 may–august 2022: 137–143 14. sumarmo. dengue haemorrhagic fever in indonesia. southeast asian j trop med public health. 1987;18(3):269-274. http://www.ncbi.nlm.nih.gov/pubmed/3433157. 15. karyanti mr, uiterwaal cspm, kusriastuti r, et al. the changing incidence of dengue haemorrhagic fever in indonesia: a 45-year registry-based analysis. bmc infect dis. 2014;14(1):412. doi:10.1186/1471-2334-14-412. 16. dinas kesehatan provinsi jawa timur. profil kesehatan dinas kesehatan provinsi jawa timur 2019. surabaya; 2020. https://dinkes.jatimprov.go.id/userfile/dokumen/ profil kesehatan jatim 2019.pdf. 17. dinas kesehatan provinsi jawa timur. profil kesehatan dinas kesehatan provinsi jawa timur 2018. surabaya; 2019. https://dinkes.jatimprov.go.id/userfile/dokumen/ buku profil kesehatan jatim 2018.pdf. 18. dinas kesehatan provinsi jawa timur. profil kesehatan dinas kesehatan provinsi jawa timur 2017. surabaya; 2018. https://dinkes.jatimprov.go.id/userfile/dokumen/ buku profil kesehatan jawa timur 2017.pdf. 19. dinas kesehatan provinsi jawa timur. profil kesehatan dinas kesehatan provinsi jawa timur 2016. surabaya; 2017. https://dinkes.jatimprov.go.id/userfile/dokumen/ profil kesehatan jatim 2016.pdf. 20. wong lp, shakir smm, atefi n, abubakar s. factors affecting dengue prevention practices: nationwide survey of the malaysian public. lu s-n, ed. plos one. 2015;10(4):e0122890. doi:10.1371/journal.pone.0122890. 21. gan sj, leong yq, bin barhanuddin mfh, et al. dengue fever and insecticide resistance in aedes mosquitoes in southeast asia: a review. parasit vectors. 2021;14(1):315. doi:10.1186/s13071021-04785-4. 22. sukesi tw, satoto tbt, murhandarwati eh, padmawati rs. effects of health education based intervention on community’s perception, healthy house, and social capital of dengue in endemic area of sleman regency indonesia. open access maced j med sci. 2021;9(e):428436. doi:10.3889/oamjms.2021.6087. 23. msellemu d, gavana t, ngonyani h, mlacha yp, chaki p, moore sj. knowledge, attitudes and bite prevention practices and estimation of productivity of vector breeding sites using a habitat suitability score (hss) among households with confirmed dengue in the 2014 outbreak in dar es salaam, tanzania. hayden m, ed. plos negl trop dis. 2020;14(7):e0007278. doi:10.1371/journal.pntd.0007278. 24. guzman mg, halstead sb, artsob h, et al. dengue: a continuing global threat. nat rev microbiol. 2010;8(s12):s7-s16. doi:10.1038/nrmicro2460. 25. rakhmani an, limpanont y, kaewkungwal j, okanurak k. factors associated with dengue prevention behaviour in lowokwaru, malang, indonesia: a cross-sectional study. bmc public health. 2018;18(1):619. doi:10.1186/s12889018-5553-z. 122 vol. 7 no. 5 may-august 2019 research report microbial pattern and antibiotic susceptibility in pediatric intensive care unit dr. soetomo hospital, surabaya i wayan putra1a, arina setyaningtyas1, dwiyanti puspitasari, irwanto1, agung dwi wahyu2, ira dharmawati1, abdul latief azis1, kuntaman2 1department of child health, faculty of medicine, universitas airlangga /dr.soetomo hospital,surabaya-indonesia 2department of clinical microbiology, faculty of medicine, universitas airlangga /dr.soetomo hospital,surabaya-indonesia acorresponding author: iwayandewana@gmail.com abstract gram-negative bacterial are known as common pathogen caused infection in pediatric intensive care unit (picu). microbial pattern and antibiotic susceptibility are needed as clinical data for selected appropriate antibiotic therapy. in picu dr. soetomo hospital until now still lacking of microbial pattern and antibiotic susceptibility data. this descriptive study is to recognized microbial pattern and antibiotic susceptibility in picu patients from blood, urine, sputum, stool, cerebrospinal fluid, endotracheal tube, pus swab and pleural fluid culture specimens. patients whose admitted into picu without signs of infections were excluded from the study. the inclusion criteria are patients with sign infection as follows: fever < 36,5°c or > 37.5°c, leukocyte < 4000/mm3 or > 10000/mm3, marker infections crp >10 mg/l or pct >0,3 ng/ml, bradycardia or tachycardia, tachypnea, infiltrates on chest x-ray, turbid urine, dysuria, thrombophlebitis, abdominal pain or tenderness, and mucous or skin lesion. medical record data from 2011 to 2016, revealed 1138 patients had positive microbial culture result, wherein positive result came from blood 44.46%, urine 19.15%, sputum 11.59%, stool 8.96%, cerebrospinal fluid 7.50%, endotracheal tube 4.04%, pus swab 2.89%, and pleural fluid 1.41%. the microorganisms found in picu dr. soetomo was dominated with gram negative bacteria. commonest bacterial that recognized from blood was b. cepacea, urine was e. coli, sputum was p. aeruginosa, stool was e. coli, cerebrospinal fluid was s. cohnii, endotracheal tube was k. pneumoniae esbl, pus swab was s. aureus, and pleural fluid was s. maltophilia. both gram-negative bacteria and gram-positive bacteria isolates revealed multiple drug resistance to commonly used antibiotic, but still had good susceptibility for antibiotic such as; amikacin, cefoperazone-sulbactam, linezolid, vancomycin and carbapenem group. keywords: picu, microbial paterrn, dr. soetomo hospital, bacteria, antibiotic. abstrak bakteri gram negatif merupakan patogen tersering penyebab infeksi di ruang rawat intensif anak. pola bakteri dan kepekaan antibiotik diperlukan sebagai data klinis dalam pemilihan terapi antibiotik yang sesuai. di ruang rawat intensif anak rs.dr. soetomo hingga saat ini masih sangat kekurangan data mengenai pola bakteri dan kepekaan antibiotik. penelitian deskriptif ini bertujuan untuk membuat pola bakteri dan kepekaan antibiotika pada pasien yang dirawat di ruang rawat intensif anak dari spesimen darah, urin, sputum, feces, cairan serebrospinal,tabung endotrakeal (ett), pus luka dan cairan pleura. pasien yang masuk ke picu yang tidak menunjukkan tanda dan gejala infeksi di eklusi dari penelitian. kriteria inklusi pada penelitian ini adalah ditemukannya tanda dan gejala infeksi, antara lain ;demam < 36,5°c or > 37.5°c, kadar leukosit darah < 4000/mm3 or > 10000/mm3, marker infeksi crp >10 mg/l or pct >0,3 ng/ml, bradikardi atau takikardi, takipneu, gambaran infiltrate pada radiologi paru,urine yang keruh, nyeri berkemih, tromboplebitis, nyeri perut, dan lesi pada mukosa atau kulit. dari data rekam medis dari tahun 2011 sampai 2016 didapatkan 1138 pasien dengan hasil kultur mikrobiologi positif, dimana 44.46% dari spesimen darah, 19.15% dari urin, 11.59% dari sputum,8.96% dari feces,7.50% dari cairan cerebrospinal, 4.04% dari ett, 2.89% dari pus luka, dan 1.41% dari cairan pleura. mikroorganisme terbanyak yang ditemukan di rawat intensif anak adalah bakteri gram negatif. bakteri tersering dari spesimen darah adalah b. cepacea, e. coli pada urine, p. aeruginosa pada sputum,e. coli pada feces,s. cohnii pada cairan serebrospinal, k. 123putra, et al.: microbial pattern and antibiotic susceptibility pneumoniae esbl pada ett,s. aureus pada pus luka,s. maltophilia pada cairan pleura. isolat bakteri gram negatif maupun gram positif yang telah didapatkan menunjukan adanya resistensi berberapa golongan antibiotik yang umumnya sering digunakan tetapi beberapa jenis antibiotik lain masih menunjukan kepekaan yang baik terhadap antibiotic seperti amikasin, cefoperazone-sulbactam, linezolid, vancomycin dan grup karbapenem. kata kunci: rawat intensif anak , pola bakteri, rumah sakit dr. soetomo, bakteri, antibiotik. introduction in this two decade nosocomial infections are special health problem concerned in terms of morbidities, mortalities and economic consequences.1 especially eventful in pediatric intensive care units (picu) that have more eminent incidence rate than another ward in hospital.2 these outcome were correlated with prolonged hospital stay, severity of diseases in picu patients, excessive use of antibiotic and patients often exposed to medical intervention tools such as; peripherals intravenous or central venous lines, urinary catheterization, mechanical ventilation, etc.2-3 respiratory tract infections, and bloodstream infections are considerably occurring infection in picu.3 both gram-positive bacteria (gpb) and gram-negative bacteria (gnb) have been reported as commonly pathogen causing infection. recently, gnb have been presented more often than gpb in this setting.4 knowledge updated about prevalence of the causative agent’s infections and antimicrobial susceptibility patterns in picu are important for proper management of nosocomial infections,4-5 there were lack quantity of published studies on microbial pattern and antibiotic susceptibility in picu patients from indonesia. this study was brought to determine it, especially from picu patients in dr. soetomo hospital surabaya. this hospital provides tertiary health care as referral hospital from primary health care or secondary health care in east java and east indonesia region methods this descriptive study was carried out in dr. soetomo general hospital. the data were collected from medical record from january 2011 to january 2016. ethical clearance issued by medico-legal committee soetomo hospital. information collected include the demographic data, primary diseases diagnosis, specimen, causative agent, and antibiotic sensitivity pattern. patients admitted into picu without signs of infections were excluded from the study. the inclusion criteria are patients with sign infection as follows: fever < 36,5°c or > 37.5°c, leukocyte < 4000/mm3 or > 10000/mm3, marker infections crp >10 mg/l or pct > 0,3 ng/ml, bradycardia or tachycardia, tachypnea, infiltrates on chest x-ray, turbid urine, dysuria, thrombophlebitis, abdominal pain or tenderness, mucous or skin lesion. spss 17 version was used to process descriptive statistics data. result over period of 5 years, 4144 patients admitted in the picu were analyzed. there were 1138 (27.46%) patient with positive culture result (table 1), girls (59.92%) are dominant than boys (40.07%) with mean age 4 ± 0.8 years. primary diseases admitted patients in picu with culture positive result were dominated with respiratory tracts infection and followed by nervous system diseases (table 2). microbial culture also undertaken in patients such as; congenital heart diseases (chd), acute leukemic lymphoblastic (all), dengue hemorrhagic fever (dhf), acute diarrhea, and others, because while being treated show clinical signs or symptom suggested of infections. blood culture result were dominated with gram-negative bacteria (gnb) (14 species bacteria), followed 16 species gram-positive bacteria (gpb). the commonest gnb were b. cepacea (table 3) and gpb were s. haemolyticus (table 4). table 1. positive culture result from various specimen in picu patients specimen total sample positive result (%) blood 1345 506 37.62 urine 824 218 26.45 sputum 643 132 20.52 stool 582 102 17.52 cerebrospinal fluid 348 86 24.71 endotracheal tube 213 46 21.59 pus 102 32 31.37 pleural 87 16 18.39 table 2. primary diseases distribution of positive culture result in picu patient. primary diseases (f) (%) pneumonia 341 29.96 encephalitis 248 21.79 s. meningoencephalitis 149 13.09 bronchopneumonia 124 10.89 congenital heart diseases 97 8.52 oncologic diseases 49 4.30 renal diseases 44 3.86 post-surgery procedure 38 3.33 diarrhea 32 2.81 dengue hemorrhagic fever 25 2.19 diabetic ketoacidosis 17 1.49 biliary atresia 5 0.43 124 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 122–130 urine culture specimen were dominated with gnb (10 species) followed gpb (8 species). the commonest gnb were e. coli (table 5) and gpb were s. haemolyticus (table 6). sputum culture specimen were dominated with gnb (8 species) followed gpb (9 species). the most common gnb were p. aeruginosa (table 7) and gpb were s. epidermidis (table 8). higher rate of s. epidermidis in this study might be caused by contaminant at recruitment sampling process. stool culture specimen were also dominated with gnb (8 species) followed gpb (6 species). the commonest gnb species were e. coli (table 9) and gpb were enterococcus spp. (table 10). cerebrospinal fluid (csf) culture was dominated with gpb (8 species) followed gnb (7 species). the commonest gpb species were s. cohnii (table 11) and gnb were a. baumannii (table 12). csf culture with s. cohnii and a. baumannii in the study result, might be considered as contaminant bacteria while recruitment process because 52 patients with surgery history with device insertion. it is connected the intracerebral area with outer enviroment from external ventriculo drainage (evd) device while sampling process. endotracheal tube (ett) aspirate culture specimen was dominated with gnb (6 species) followed gpb (4 species). the commonest gnb species were k. pneumonia (esbl+) (table 13) and gpb were s. haemolyticus (table 14). pus/ wound swab culture specimen were dominated with gpb (8 species) followed gnb (5 species). the table 3. gnb species finding in blood culture bacteria species (n=334) (%) b. cepacea 57 17.06 k. pneumoniae (esbl+) 56 16.76 a. baumannii 44 13.17 k. pneumoniae 37 11.14 p. aeruginosa 33 9.88 e. coli 29 8.68 e. cloacae 21 6.28 s. marcescens 15 4.49 m. catarrhalis 12 3.59 s. typhi 9 2.69 e. coli (esbl+) 9 2.69 p. alcalifaciens 7 2.09 pasteurella spp 3 0.89 s. paratyphi 2 0.59 table 4. gpb species finding in blood culture bacteria species (n=172) (%) s. haemolyticus 55 31.97 s. hominis 35 20.35 s. epidermidis 16 9.30 s. saprophyticus 14 8.13 s. aureus 13 7.55 mrsa 12 6.97 s. intermedius 5 2.90 s. cohnii 4 2.32 e. faecalis 3 1.75 corynebacterium spp. 3 1.75 m. lylae 3 1.75 s. gallinarum 2 1.17 s. kloosii 2 1.17 s. warneri 2 1.17 s. ureolyticus 2 1.17 s. parasanguinis 1 0.58 table 5. gnb species finding in urine culture bacteria species (n=159) (%) e. coli 81 50.94 e. coli (esbl+) 33 20.75 k. pneumoniae (esbl+) 13 8.18 e. cloacae 11 6.92 b. cepacea 5 3.14 p. aeruginosa 5 3.14 a. baumannii 3 1.89 e. aerogenes 3 1.89 s. marcescens 2 1.26 p. rettgeri 1 0.63 p. mirabilis 1 0.63 aeromonas spp. 1 0.63 table 7. gnb species in sputum culture. bacteria species (n=88) (%) p. aeruginosa 42 47.72 k. pneumonia 22 25.00 e. coli (esbl+) 9 10.22 a. baumannii 6 6.82 s. maltophilia 3 3.42 e. cloacae 3 3.42 s. marcescens 2 2.27 s. fonticola 1 1.13 table 8. gpb species in sputum culture. bacteria species (n=44) (%) s. epidermidis 23 52.27 mrsa 8 18.18 s. capitis 6 13.64 s. haemolyticus 6 13.64 s. pneumonia 1 2.27 table 6. gpb species finding in urine culture bacteria species (n=59) (%) gram positive bacteria : s. haemolyticus 11 18.64 s. epidermidis 10 16.94 s. cohnii 9 15.26 e. faecalis 9 15.26 mrsa 8 13.56 e. faecium 5 8.47 s. warneri 3 5.09 s. ureolyticus 2 3.39 s. parasanguinis 2 3.39 125putra, et al.: microbial pattern and antibiotic susceptibility pleural fluid culture specimen was dominated with gnb (5 species bacteria) followed gpb (4 species bacteria). the commonest gnb were s. maltophilia (table 17) and gpb were s. epidermidis (table 18). antibiotic sensitivity pattern of gnb (table 19) are showed that almost all of the isolate are resistant to; penicillin cephalosporin, tetracycline, chloramphenicol, sulfa and quinolones groups. among gnb isolate, cefo-sulbactam has the highest susceptibility rate (87.71%) for b. cephacea in blood, nitrofurantoin (97.53%) for e. coli in urine, cefo-sulbactam (88.09%) for p. aeruginosa in sputum, both of amikacin table 9. gnb species in stool culture bacteria species (n=65) (%) e. coli 34 52.30 e. cloacae 21 32.32 e. coli (esbl+) 2 3.07 e. aerogenes 2 3.07 k. pneumoniae (esbl+) 2 3.07 c. youngae 2 3.07 c. jejuni 1 1.55 c. testosteroni 1 1.55 table 10. gpb species in stool culture bacteria species (n=37) (%) e. cloacae 19 51.35 s. aureus 10 27.03 s. epidermidis 3 8.11 mrsa 3 8.11 s. paratyphi 1 2.70 c. difficile 1 2.70 table 11. gpb species in csf culture bacteria species (n=56) (%) s. cohnii 11 19.64 s. epidermidis 9 16.07 s. haemolyticus 8 14.28 s. aureus 7 12.50 e. faecium 7 12.50 e. faecalis 7 12.50 a. viridans 4 7.15 mrsa 3 5.36 table 12. gnb species in csf culture bacteria species (n=30) (%) a. baumannii 10 33.33 e. cloacae 7 23.34 p. aeruginosa 6 20.00 e. coli (esbl+) 3 10.00 b. diminuta 2 6.67 p. stutzeri 1 3.33 b. cepacea 1 3.33 table 13. gnb species in ett aspirate bacteria species (n=37) (%) k. pneumonia (esbl+) 16 43.24 p. aeruginosa 11 29.72 a. baumannii 6 16.22 e. coli (esbl+) 2 5.42 s. marcescens 1 2.70 b. cepacea 1 2.70 table 14. gpb species in ett aspirate bacteria species (n=9) (%) s. haemolyticus 6 66.67 mrsa 1 11.11 s. epidermidis 1 11.11 s. capitis 1 11.11 table 15. gpb species in pus wound swab bacteria species (n=23) (%) s. aureus 9 39.13 s. epidermidis 7 30.43 s. haemolyticus 2 8.69 s. constellatus 1 4.35 s. acidominus 1 4.35 e. faecalis 1 4.35 mrsa 1 4.35 s. capitis 1 4.35 table 16 gnb species in pus wound swab bacteria species (n=9) (%) p. aeruginosa 5 55.56 k. pneumonia (esbl+) 1 11.11 p. mirabilis 1 11.11 c. testosteroni 1 11.11 c. striatum 1 11.11 table 17. gnb species in pleural fluid. bacteria species (n=10) (%) s. maltophilia 4 40.00 p. putida 3 30.00 l. adecarboxylata 1 10.00 c. farmeri 1 10.00 k. pneumonia (esbl+) 1 10.00 table 18. gpb species in pleural fluid. bacteria species (n=6) (%) s. epidermidis 3 50.00 s. haemolyticus 1 16.66 s. capitis 1 16.66 mrsa 1 16.66 commonest gpb were s. aureus (table 15) and gnb were p. aeruginosa (table 16). over 32 wound positive culture isolate in our study were undertaken from 37 pediatric patients with history surgical site infection. 126 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 122–130 t ab el .1 9. a nt ib io ti cs s en si ti vi ty p at te rn o f gr am n eg at iv e ba ct er ia i so la te f ro m d if fe re nt s am pl es a n ti b io ti cs t h e m os t gr am n eg at iv e b ac te ri a (g n b ) sp ec ie s in i so la te s am p le s b lo od u ri n e s p u tu m s to ol c s f f lu id e t t p u s/ w ou n d p le u ra l f lu id g n b s en si ti vi ty b . c ep ac h ea n = 57 e . c ol i n = 81 p . a er u gi n os a n = 42 e . c ol i n = 34 a . b au m an ii n = 10 k . p n eu m on ia n = 16 p .a er u gi n os a n = 5 s .m al to p h il ia n = 4 a m ik ac in 50 ( 87 .7 1% ) 79 ( 97 .5 3% ) 33 ( 78 .5 7% ) 33 ( 97 .0 5% ) 7 (7 0. 00 % ) 15 ( 93 .7 5% ) 4 (8 0. 00 % ) 3 (7 5. 00 % ) 84 .9 5% % t ob ra m yc in 39 ( 68 .4 2% ) 49 ( 60 .4 9% ) 23 ( 54 .7 6% ) 22 ( 64 .7 0% ) 5 (5 0. 00 % ) 11 ( 68 .7 5% ) 2 (4 0. 00 % ) 2 (5 0. 00 % ) 57 .1 4% g en ta m yc in 37 ( 64 .9 1% ) 47 ( 58 .0 2% ) 32 ( 76 .1 9% ) 20 ( 58 .8 2% ) 5 (5 0. 00 % ) 13 ( 81 .2 5% ) 3 (6 0. 00 % ) 3 (7 5. 00 % ) 65 .5 2% a st re on am 20 ( 35 .0 8% ) 32 ( 39 .5 0% ) 27 ( 64 .2 8% ) 14 ( 41 .1 7% ) 0 (0 % ) 8 (5 0. 00 % ) 3 (6 0. 00 % ) 2 (5 0. 00 % ) 42 .5 0% a m ox ic il li n c la vu la ni c 18 ( 31 .5 7% ) 38 ( 46 .9 1% ) 0 (0 % ) 15 ( 44 .1 1% ) 0 (0 % ) 8 (5 0. 00 % ) 0 (0 % ) 0 (0 % ) 21 .5 7% a m pi ci ll in 4 (7 .1 7% ) 13 ( 16 .0 4% ) 0 (0 % ) 5 (1 4. 70 % ) 0 (0 % ) 0 (0 % ) 0 (0 % ) 0 (0 % ) 4. 74 % a m pi ci ll in s ul ba ct am 25 ( 43 .8 5% ) 32 ( 39 .5 0% ) 5 (1 1. 90 % ) 13 ( 38 .2 3% ) 7 (7 .0 0% ) 7 (4 3. 75 % ) 1 (2 0. 00 % ) 1 (2 5. 00 % ) 28 .6 5% p ip pe t az ob ac ta m 35 ( 61 .4 0% ) 59 ( 72 .8 3% ) 27 ( 64 .2 8% ) 25 ( 73 .5 2% ) 4 (4 0. 00 % ) 10 ( 62 .5 0% ) 3 (6 0. 00 % ) 3 (7 5. 00 % ) 63 .6 9% c ef az ol in 13 ( 22 .8 0% ) 23 ( 28 .3 9% ) 0 (0 % ) 10 ( 29 .4 1% ) 0 (0 % ) 8 (5 0. 00 % ) 0 (0 % ) 0 (0 % ) 16 .3 3% c ef ta zi di m e 21 ( 36 .8 4% ) 38 ( 46 .9 1% ) 36 ( 85 .7 1% ) 16 ( 47 .0 5% ) 4 (4 0. 00 % ) 9 (5 6. 25 % ) 4 (8 0. 00 % ) 4 (1 00 % ) 61 .6 0% c ef ot ax im e 22 ( 38 .5 9% ) 34 ( 41 .9 7% ) 0 (0 % ) 14 ( 41 .1 7% ) 4 (4 0. 00 % ) 7 (4 3. 75 % ) 0 (0 % ) 0 (0 % ) 25 .6 9% c ef tr ia xo ne 11 ( 19 .2 9% ) 9 (1 1. 11 % ) 12 ( 28 .5 7% ) 4 (1 1. 76 % ) 2 (2 0. 00 % ) 2 (1 2. 50 % ) 1 (2 0. 00 % ) 1 (2 5. 00 % ) 18 .5 3% c ef o – s ul ba ct am 54 ( 94 .7 3% ) 76 ( 93 .8 2% ) 37 ( 88 .0 9% ) 32 ( 94 .1 1% ) 8 (8 6% ) 15 ( 93 .7 5% ) 4 (8 0. 00 % ) 4 (1 00 % ) 91 .3 1% c ef ep im e 27 ( 47 .3 6% ) 35 ( 43 .2 0% ) 26 ( 61 .9 0% ) 15 ( 44 .1 1% ) 4 (4 0. 00 % ) 7 (4 3. 75 % ) 3 (6 0. 00 % ) 3 (7 5. 00 % ) 51 .9 2% c ot ri m ox az ol e 32 ( 56 .1 4% ) 29 ( 35 .8 0% ) 7 (1 6. 66 % ) 12 ( 35 .2 9% ) 8 (8 0. 00 % ) 12 ( 75 .0 0% ) 1 (2 0. 00 % ) 1 (2 5. 00 % ) 42 .9 9% t et ra cy cl in e 21 ( 36 .8 4% ) 22 ( 27 .1 6% ) 0 (0 % ) 10 ( 29 .4 1% ) 4 (4 0. 00 % ) 8 (5 0. 00 % ) 0 (0 % ) 0 (0 % ) 22 .9 3% c hl or am ph en ic ol 30 ( 52 .6 3% ) 58 ( 71 .6 0% ) 12 ( 28 .5 7% ) 25 ( 73 .5 2% ) 0 (0 % ) 12 ( 75 .0 0% ) 1 (2 0. 00 % ) 1 (2 5. 00 % ) 43 .2 9% c ip ro fl ox ac in 31 ( 54 .3 8% ) 32 ( 39 .5 0% ) 27 ( 64 .2 8% ) 14 ( 41 .1 7% ) 5 (5 0. 00 % ) 9 (5 6. 25 % ) 3 (6 0. 00 % ) 2 (5 0. 00 % ) 51 .9 5% l ev of lo xa ci n 33 ( 57 .8 9% ) 35 ( 43 .2 0% ) 30 ( 71 .4 2% ) 15 ( 44 .1 1% ) 6 (6 0. 00 % ) 9 (5 6. 25 % ) 3 (6 0. 00 % ) 3 (7 5. 00 % ) 58 .4 8% f os fo m yc in 37 ( 64 .9 1% ) 79 ( 97 .5 3% ) 20 ( 47 .6 1% ) 32 ( 94 .1 1% ) 1 (1 0. 00 % ) 7 (4 3. 75 % ) 2 (4 0. 00 % ) 2 (5 0. 00 % ) 55 .9 9% n it ro fu ra nt oi n 80 ( 98 .7 6% ) 0 (0 % ) 33 ( 97 .0 5% ) 0 (0 % ) 12 ( 75 .0 0% ) 0 (0 % ) 0 (0 % ) 38 .6 9% im ip en em 46 ( 80 .7 0% ) 69 ( 85 .1 8% ) 32 ( 76 .1 9% ) 29 ( 85 .2 9% ) 7 (7 0. 00 % ) 15 ( 93 .7 5% ) 4 (8 0. 00 % ) 3 (7 5. 00 % ) 80 .7 6% m er op en em 45 ( 78 .9 4% ) 73 ( 90 .1 2% ) 32 ( 76 .1 9% ) 31 ( 91 .1 7% ) 7 (7 0. 00 % ) 14 ( 87 .5 0% ) 4 (8 0. 00 % ) 3 (7 5. 00 % ) 81 .1 2% 127putra, et al.: microbial pattern and antibiotic susceptibility t ab el .2 0. a nt ib io ti cs s en si ti vi ty p at te rn o f gr am p os it iv e ba ct er ia i so la te f ro m d if fe re nt s am pl es a n ti b io ti cs t h e m os t gr am p os it iv e b ac te ri a (g p b ) sp ec ie s in i so la te s am p le s b lo od u ri n e s p u tu m s to ol c s f f lu id e t t p u s/ w ou n d p le u ra l f lu id g p b s en si ti vi ty s . b -h ae m ol yt ic u s n = 5 5 s . b -h ae m ol yt ic u s n = 1 1 s . c oa gu la se n eg at if n = 2 3 e . c lo ac ae n = 1 9 s . c oh n ii n = 1 1 s . b -h ae m ol yt ic u s n = 6 s . a u re u s n = 9 s . h ae m ol yt ic u s n = 4 g en ta m ic in 16 ( 29 .0 9% ) 2 (1 8. 18 % ) 12 ( 52 .1 7% ) 8 (4 2. 10 % ) 6 (5 4. 54 % ) 2 (3 3. 33 % ) 8 (8 8. 89 % ) 3 (7 5. 00 % ) (4 9. 16 % ) a m pi ci ll in 1 (1 .8 1% ) 1 (5 .5 6% ) 8 (3 4. 78 % ) 4 (2 1. 05 % ) 1 (5 .5 6% ) 0 (0 % ) 1 (1 .1 1% ) 1 (2 5. 00 % ) (1 1. 86 % ) a m pi ci ll in -s ul ba ct am 9 (1 6. 36 % ) 2 (1 8. 18 % ) 6 (2 6. 08 % ) 3 (1 5. 78 % ) 1 (5 .5 6% ) 1 (1 6. 66 % ) 7 (7 7. 78 % ) 2 (5 0. 00 % ) (2 8. 30 % ) p en ic il li n 2 (3 .3 6% ) 1 (5 .5 6% ) 4 (1 7. 39 % ) 4 (2 1. 05 % ) 2 (1 8. 18 % ) 1 (1 6. 66 % ) 1 (1 .1 1% ) 1 (2 5. 00 % ) (1 3. 54 % ) o xa ci ll in 12 ( 21 .8 1% ) 3 (2 7. 27 % ) 10 ( 43 .4 7% ) 8 (4 2. 10 % ) 6 (5 4. 54 % ) 1 (1 6. 66 % ) 6 (6 6. 67 % ) 3 (7 5. 00 % ) (4 3. 44 % ) c ot ri m ox az ol e 4 (7 .7 2% ) 2 (1 8. 18 % ) 11 ( 47 .8 2% ) 8 (4 2. 10 % ) 5 (4 5. 45 % ) 1 (1 6. 66 % ) 8 (8 8. 89 % ) 1 (2 5. 00 % ) (3 6. 48 % ) t et ra cy cl in e 29 ( 52 .7 2% ) 6 (5 4. 54 % ) 6 (2 0. 08 % ) 7 (3 6. 84 % ) 3 (2 7. 27 % ) 0 (0 % ) 0 (0 .0 0% ) 1 (2 5. 00 % ) (2 7. 06 % ) c hl or am ph en ic ol 25 ( 45 .4 5% ) 5 (4 5. 45 % ) 8 (3 4. 78 % ) 9 (4 7. 36 % ) 3 (2 7. 27 % ) 3 (5 0. 00 % ) 6 (6 6. 67 % ) 2 (5 0. 00 % ) (4 5. 87 % ) e ry th ro m yc in 18 ( 32 .7 2% ) 3 (2 7. 27 % ) 13 ( 56 .5 2% ) 11 ( 57 .8 9% ) 6 (5 4. 54 % ) 3 (5 0. 00 % ) 6 (6 6. 67 % ) 2 (5 0. 00 % ) (4 9. 45 % ) c li nd am yc in 21 ( 38 .1 8% ) 5 (4 5. 45 % ) 16 ( 69 .5 6% ) 12 ( 63 .1 5% ) 4 (3 6. 36 % ) 2 (3 3. 33 % ) 6 (6 6. 67 % ) 2 (5 0. 00 % ) (5 0. 34 % ) c ip ro fl ox ac in 38 ( 69 .0 9% ) 7 (6 3. 63 % ) 0 (0 .0 0% ) 12 ( 63 .1 5% ) 6 (5 4. 54 % ) 3 (5 0. 00 % ) 0 (0 .0 0% ) 2 (5 0. 00 % ) (4 3. 80 % ) l ev of lo xa ci n 31 ( 56 .3 6% ) 9 (8 1. 81 % ) 14 ( 60 .8 6% ) 13 ( 68 .4 2% ) 7 (6 3. 63 % ) 4 (6 6. 67 % ) 3 (3 3. 33 % ) 2 (5 0. 00 % ) (6 0. 14 % ) m ox if lo xa ci n 49 ( 89 .0 9% ) 9 (8 1. 81 % ) 16 ( 69 .5 6% ) 12 ( 63 .1 5% ) 6 (5 4. 54 % ) 4 (6 6. 67 % ) 3 (3 3. 33 % ) 3 (7 5. 00 % ) (6 6. 64 % ) f os fo m yc in 4 8 (8 7. 27 % ) 10 ( 90 .9 0% ) 19 ( 82 .6 0% ) 11 ( 57 .8 9% ) 9 (8 1. 81 % ) 3 (5 0. 00 % ) 8 (8 8. 89 % ) 3 (7 5. 00 % ) (7 6. 80 % ) n it ro fu ra nt oi n 46 ( 83 .6 3% ) 9 (8 1. 81 % ) 20 ( 96 .9 5% ) 14 ( 73 .6 8% ) 6 (5 4. 54 % ) 3 (5 0. 00 % ) 8 (8 8. 89 % ) 3 (7 5. 00 % ) (7 5. 56 % ) m er op en em 8 (1 4. 54 % ) 2 (1 8. 18 % ) 18 ( 78 .2 6% ) 10 ( 52 .6 3% ) 5 (4 5. 45 % ) 2 (1 8. 18 % ) 8 (8 8. 89 % ) 2 (5 0. 00 % ) (4 5. 77 % ) v an co m yc in 52 ( 94 .5 4% ) 11 ( 10 0. 00 % ) 22 ( 95 .6 5% ) 16 ( 84 .2 1% ) 9 (8 1. 81 % ) 5 (8 3. 33 % ) 9 (1 00 .0 0% ) 4 (1 00 .0 0% ) (9 2. 44 % ) l in ez ol id 53 ( 96 .3 6% ) 11 ( 10 0. 00 % ) 20 ( 86 .9 5% ) 17 ( 89 .4 7% ) 11 ( 10 0. 00 % ) 5 (8 3. 33 % ) 8 (8 8. 89 % ) 4 (1 00 .0 0% ) (9 3. 13 % ) d ap to m yc in 52 ( 94 .5 4% ) 10 ( 90 .9 1% ) 21 ( 91 .3 0% ) 16 ( 84 .2 1% ) 9 (8 1. 81 % ) 4 (6 6. 67 % ) 8 (8 8. 89 % ) 4 (1 00 .0 0% ) (8 7. 29 % ) 128 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 122–130 and nitrofurantoin (97.05%) for e. coli in stool isolate. amikacin, cefo-sulbactam and imipenem (93.75%) had highest sensitivity for k. pneumonia in ett isolate and at the last cefo-sulbactam (100%) also had highest sensitive for s. malthopnilia in pleural isolates. gpb antibiotic sensitivity pattern (table 20), are showed that almost all of isolate resistant for aminoglycoside, penicillin, macrolide, tetracycline, and carbapenem antibiotic groups. in gpb isolate, linezolid (100%) has highest susceptibility rate for s. cohnii in csf fluid and vancomycin (100%) high sensitive for p. aeruginosa in pus/wound swab isolated. discussion in this study totally 4144 picu patients were followed the study and only 27.46% had positive culture result, female higher than male in distribution gender, with mean age 4 ± 0.8 years. primary diseases distribution was dominated with respiratory tracts infection. previous study by camilla et.al, positive culture result in picu patient dominant respiratory tract infection as primary diseases (33.26%), female more often than male, with higher age incidence at less than five years.5 other study in picu mohammad hoesin palembang stated that commonest respiratory tract infection diagnosis was broncho-pneumonia (33.3%).6 in our present study, the frequency gram-negative bacteria (gnb) isolates was slightly higher than that gran-positive bacteria (gpb) isolates. gnb constituted the majority of bacterial pathogens associated with the 6 major specimen, blood (66.01%), urine (72.69%), sputum (66.67%), stool (63.73%), ett aspirate (80.43%) and pleural fluid (62.50%). the predominance of gnb is a relevant reminder that these pathogens were once the most common human pathogens.3,7 for approximately the past 2 decades, gnb have been the pathogens most frequently associated with respiratory system diseases and urinary tracts infections(utis).4,7 blood culture result of our study demonstrated gnb (66.01%) were the most common organisms causing blood stream infection, various literatures from the world are showed these phenomena such as; gupta et.al, haeusler et.al, and kirsty et.al, showed gnb as predominant pathogen for blood stream infection.7,8,9 our study is gained commonest gnb species was b. cepacea (11.26%). b. cepacea has emerged as a serious human pathogen in the last two decades, causing fatal necrotizing pneumonia and bacteremia. b. cepacea has been associated with out breaks involving infections of the bloodstream, respiratory tract, and urinary tract in intensive care unit setting.7,8,10 antony et.al. stated that the intensive care unit bloodstream infections in tertiary hospital often caused by b. cepacea infections.10 urine culture demonstrated positivity rate for 26.45%, clinically with urinary tract infection cases. several study are showed vary positivity of the urine culture e.g. salar et.al 17%. ‘kaur et.al 15.7% are showed an occurrence of urinary tract infection among picu patients11,12 this difference could possibly due to various antibiotic prescribing practices, variations in sample collection, culture technique and susceptibility testing practices in our hospital than others. our study was also shown that gnb, e. coli (50.94%) was the most common organisms in urinary tract infection. this finding similar with microbial pattern in adult patients in same hospital, that e. coli is the most common cause utis.13 sputum culture revealed positivity rate 20.52% of respiratory tract infection cases. its majority caused by gnb with dominant p. aeruginosa (47.72%). piyush et.al is stated 34.23% patient had p. aeruginosa etiology from sputum sample in respiratory tract infection.14 p. aeruginosa is a gram-negative aerobic rod. it became considered as most challenging pathogen bacteria globally because of its high rate of resistance to antimicrobial agent.3,15 it was also reported that p. aeruginosa is one of the most common nosocomial pathogen and a leading cause of nosocomial respiratory tract infection.5,15 stool culture is obtain positivity rate was 17.52% majority caused by gnb (63.73%) were dominant e. coli (52.30%). e. coli has been reported as the most frequently identified pathogen in other study throughout the world like china.16 some country reported different bacteria as the leading entero-pathogen, such a salmonella spp in south korea,17 and aeromonas spp. in singapore. 18 some of these regional differences may be related to study population or stool culture techniques. the endotracheal tube aspirated is performed 21.59% positivity rate which were dominated with gnb dominantly k. pneumonia (esbl+) (43.24%). in contrast to our study rehman et.al are reported 93.65% culture positive in ett tips, they also revealed that k. pneumoniae (41.93%) was the most common isolate.19 kalanuria et.al are stated that pseudomonas spp was common isolate from ett tips.20 this differences result may be most of these microorganism acquired from environment and their concentrations varying depend on hospital geographical distribution and their ability to survive in particular conditions. the lumen of ett in patients using mechanical ventilation usually became colonized with gnb which commonly appeared to survive within a biofilm.21 while it appears that colonization of the ett may begin from as early as 12 hours, it is most abundant at 96 hours.20,21 pleural fluid culture is attained positivity rate for 18.39% and were dominated with gnb (62.50%) with majority species s. maltophilia (40.00%). jones et.al. in their study are got positivity culture of pleural fluid rate was 11.50% with s. maltophilia (59.16%) are commonest bacterial.22 chawla et.al are stated that s. maltophilia often cause pneumonia infection.23 in our study these microbes may affect pleural fluid after infected lower respiratory tract such as pneumonia by organ lesion caused diseases progression. at present, the incidence of nosocomial infections cause by s. maltophilia is increasing; in 129putra, et al.: microbial pattern and antibiotic susceptibility particular, intensive care units are leading areas with high risk of these infections.23,24 these organisms also resistance to many broad-spectrum antibiotics including carbapenem causes an increase in the mortality and morbidity rates in the intensive care units.22,24 cerebrospinal fluid culture had gram positive bacteria (65.12%) as the common microorganism with majority species s. cohnii (52.30%). previous study conducted by jiang et.al. are showed (50.8%) acute bacterial meningitis in pediatric caused gpb infections.25 zhu et.al. are found gpb predominant pathogen in pediatric patients caused purulent meningitis were e. coli and staphylococcus spp.26 in our finding has similar perform with the other literature, its suggest that the development of nosocomial staphylococcal meningitis may subsequent to central nervous system conditions and neurosurgery interventions, which include ventriculo-peritoneal shunts, or other embedded devices. in this study over 52 patients also known had surgery history for inserting neurosurgery device. generally, as is common in other surgical practice, the risk factor of inserting device infection, the venue of procedure and the surgical technique are know by surgeon’s experience.27 wound culture were dominated with gpb (71.87%), with isolate was s. aureus (39.13%) and followed gnb p. aeruginosa (55.56%). negi et.al are found 96,4% surgical site infection yielding bacteria growth with s. aureus (54.4%), p. aeruginosa (21.7%) and e. coli.28 these infections are usually caused by exogenous or endogenous microorganisms that enter the operative wound during the course of the surgery.29 in our study over 32 of 37 patients with history surgical site infection had positive result culture. these wound infections may have occurred at hospital and recognized to be associated with an infection before-after or during surgery, extended length of hospital stays and prolonged or permanent disability. antibiotics susceptibility pattern of gnb isolates (blood, urine, sputum, stool, ett and pleural fluid) in our study finding were resistant to three or more groups antimicrobial agents and therefore consider multidrug resistant (mdr), almost all of the isolate are resistant to; penicillin, cephalosporin, tetracycline, chloramphenicol, sulfa and quinolones groups, the development of antibiotic resistant in our hospital might be caused by unnecessary, inappropriate, or suboptimal prescribed antibiotic therapy from community before, previous health care and our hospital itself. previous study similar that, find very high level of resistance penicillin derivate, approximately one half isolate in infants and young children.30 other study in africa 75% isolate are mdr to ampicillin, chloramphenicol and cotrimoxazole.31 who in 2014 report that five out of the six who regions had more than 50% resistant to third generation of cephalosporin and fluoroquinolones in hospital setting.32 in gpb isolates (csf and wound swab) also found multidrug resistance, over two third of antibiotic testing had resistance. only vancomycin, linezolid and daptomycin had highest susceptibility for all gpb isolates. sarangi et.al and singh et.al. were also found that vancomycin and linezolid had highest antibiotic susceptibility nicu setting.33,34 highest prevalence isolates with multiple drug resistance that observed in our study may cause our hospital is a tertiary care center with large range health service not only in east java but also in east region indonesia, patient adjoining provinces are admitted for treatment that before attending the hospital, most of the patient get different antibiotic from low level heath care centers or due to over the counter sell of antibiotics often in improper dose. limited population in some specimen and obtain of some pathogen or contaminant bacteria were all limitation in our study, multicenter prospective studies are needed to validated our finding. conclusion our study revealed gnb isolates as the predominant pathogen in all picu isolates sampling, with most microorganism found were b. cepacea in blood, p. aeruginosa in sputum, e. coli in urine and stool, s. cohnii in csf fluid, k. pneumoniae esbl in ett aspirate, s. aureus in pus, and s. maltophilia in pleural fluid culture. both gnb and gpb isolates showed multiple drug resistance to commonly used antibiotic but still had good susceptibility for amikacin, cefoperazone-sulbactam, linezolid, vancomycin and carbapenem group. reference 1. eyal z, daniel h, orly t et.al. health –care associated infection, a meta-analysis of cost and financial impact on the us health-care system. jama intern med,2013:173(22) 2039-96. 2. stephen wp, allison tk, ken k, robert j, louise v, charlene g, et.al. health-care associated infection among critically ill children in the us. aap news and journals, 2014:3:120-36. 3. navaeifar m, and rezai fm. device associated nosocomial infection in children. j.pediatr.rev, 2013;3:25-41. 4. berezin en, and solorzano f. gram negative infection in pediatric and neonatal intensive care unit of latin america. j.infect dev.ctries,2014 (8):942-53. 5. camilla s, carlos a, lital m, sulim a, eduardo j. the epidemiological profile of pediatric intensive care center at hospital israelita albert einstein. einstein, 2012, vol 10; 6-21. 6. suryadi t. pola kuman dan resistensi antibiotik di picu rs. dr. mohammad hoesin palembang tahun 2013. jurnal kedokteran & kesehatan, 2015: 2: 91-97. 7. gupta s, kashyap b. bacteriological profile and antibiogram of blood culture isolates from a tertiary care hospital of north india. trop j med res, 2016:19:94-9. 8. haeusler gm, mechinaud f, daley aj, starr m, shann f, connel tg, bryant pa, donath s, et.al. antibiotic resistant gram negative bacteremia in pediatric oncology patients-risk factor and outcomes. the pediatric infectious diseases journal,2013:32:723-26. 9. kirsty ld, julia b, paul th, and mike s. systematic review of antibiotic resistance rates among gram-negative bacteria in children with sepsis in resource-limited countries. journal of the pediatric infectious diseases society, 2015:4:11-20. 10. antony b, cherian ev, boloor r, shenoy kv. a sporadic outbreak of bulkhoderia cepacea complex bacteremia in pediatric intensive care unit of a tertiary hospital in coastal karnataka, south india. indian j pathol microbial, 2016;59:197-9. 130 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 122–130 11. behzadnia s, davoudi a, and ahangarkani f. nosocomial infection in pediatric population and antibiotic resistance of the causative organism in north of iran. iranian red cresc med j, 2014:2:1-17. 12. kaur n, sharma s, and hans c. urinary tract infection: etiology and antimicrobial resistance pattern in infant from a tertiary care hospital in northern india. j clin diagn res,2014:8:1-13. 13. yuanita v, kuntaman, dan hadi u, pola kepekaan antibiotika pada bakteri resisten karbapenem di rsud dr sutomo surabaya. 2018: 39-44. 14. piyush t, gopa b, shivani s, pramod w. antibiotic resistance pattern of pseudomonas aeruginosa isolated from patient of lower respiratory tract infection. african journal of microbiology research, 2011; 2: 9-11. 15. juhas m, pseudomonas aeruginosa essential: an updated on investigation of essential genes. microbiology, 2015 :161:20532060. 16. wang x, wang j, sun h, xia s, duan r, liang j, hiao y et.al. etiology of childhood infectious diarrhea in a developed region of china: compared to childhood diarrhea in a developing region and adult diarrhea in developing region. plos one,2015;3:1-14. 17. young lj, young cs, hae sh, young jr, jongjin l,do is, jin bk, et.al. diagnostic yield of stool culture and predictive factors for positive culture in patients with diarrhea illness. medicine, 2017:96:p e7641. 18. chau ml, hartantyo shp, yap m, et.al. diarrhea genic pathogens in adults attending a hospital in singapore. bmc infect dis, 2016:16: 32-38. 19. rehman s, zafar a, qureshi ah, haq ui. frequency of endotracheal tube inhabiting klebsiella pneumonia and their antibiogram isolated from children’s hospital lahore pakistan. online journal of bioscience and informatics, 2014:3:589-9. 20. kalanuria aa, zai w, mirski m. ventilator-associated pneumonia in the icu. critical care, 2014 :18:208-11. 21. ferreira ot, koto yr, leite cf, klautau bg, nigro s, silva bc, souza fi, et.al. microbial investigation of biofilm recovered from endotracheal tubes using sonication in intensive care unit pediatric patients. braz j infect dis, 2016 (20) 1678-84. 22. jones r. microbial etiologies of hospital-acquired bacterial pneumonia and ventilator-associated pneumonia. clin. infect. dis, 2010;51: 81-87. 23. chawla k, viswanath s, gupta a. stenothropomonas maltophilia in lower respiratory tract. journal of clinical and diagnosis research, 2014:8:12-19. 24. adegoke aa, stenstorm at, okoh ia, stenotrophomonas malthophilia as an emerging ubiquitous pathogen: looking beyond contemporary antibiotic therapy. front microbial, 2017. 25. jiang h, su m, kai l, huang h, qiu l, mu j, du t et.al. prevalence and antibiotic resistance profile of cerebrospinal fluid pathogen in children with acute bacterial meningitis in yunnan province china 2012-2015. plos one, 2017 (6):c0180161 26. zhu m, hu q, mai j, and lin z. analysis of pathogenic bacteria and drug resistance in neonatal purulent meningitis. pubmed, 2015(1):51-6. 27. hussein k, bitterman r, shoffy b, paul m, neuberger a. management of post-neuro surgical meningitis, narrative review. clinical microbiology and infection, 2017(23): 621-28. 28. negi v, pal s, juyul d, sharma km, sharma s. bacteriological profile of surgical site infection and their antibiogram: a study from resource constructed rural setting of uttarakand india. j clin diag res,2015;10: 17-20. 29. bhattacharya s, pal k, jain s, chatterjee ss, konar j. surgical site infection by methicillin resistant staphylococcus aureus on decline ?. j clin diagn res, 2016;9: 32-36. 30. downie l,armiento r, subi r, et.al. community acquired neonatal and infants sepsis in developing countries: efficacy of who currently recommended antibiotic-systematic review and meta-analysis, arch dis child,2013:83; 140-5. 31. phoba mf,de boeck h,ifeka bb, et.al. epidemic increase in salmonella blood stream infection in children bawanda, republic democratic congo, eur j clin microbial infect dis, 2014:33: 79-87. 32. founou rc, founou ll, essack sy. clinical and economic impact of antibiotic resistance in developing countries: a systematic review and meta-analysis. plos one, 2017:12; e0189621. 33. sarangi kk, pattnaik d,mishra sn,nayak mk, jena j. bacteriological profile an anti biogram of blood culture isolates done by automated cultured and sensitivity method in a nicu in a tertiary care hospital in odisha india. int j adv med.2015;2:387-92. 34. singh hk, sharja p,onkar k. bacterial profile of neonatal sepsis in nicu in a tertiary care hospital: prevalent bugs and their susceptibility pattern. eur j pharm med res, 2016;3:241-45. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid vol 6 no 4 jan-maret 2017.indd 92 vol. 6. no. 4 january–april 2017 research report plasma leakage profiles of dengue hemorrhagic fever patients in rsud dr. soetomo, surabaya, east java, indonesia january – june 2014 ferdian rizaliansyah1a, aryati1,2, musofa rusli1,3 1 faculty of medicine – universitas airlangga, surabaya 2 department of clinical pathology, faculty of medicine – rsud dr. soetomo, surabaya 3 department of internal medicine, faculty of medicine – rsud dr. soetomo, surabaya a corresponding author: ferdian.rizaliansyah@gmail.com abstract plasma leakage is one crucial point of dengue hemorrhagic fever (dhf) that differentiates it from dengue fever (df). dhf has to meet 4 criteria which are 2 – 7 days of acute fever, hemorrhagic manifestation, thrombocytopenia (≤100.000 cells/mm3) and evidence of plasma leakage. plasma leakage consists of increasing hematocrit ≥20%, hypoalbuminemia or evidence of pleural effusion or ascites. often doctors only base their dhf diagnosis on the presence of thrombocytopenia. this study analyzed the presence of plasma leakage between adult and pediatric patients with a dhf diagnosis in rsud dr. soetomo in order to make the diagnosis and healthcare services better in the future. this was a retrospective study which used medical records of dhf patients admitted from january to june 2014. 78 cases were included, 24 adult patients (31%) and 54 pediatric patients (69%). 29/78 (37%) patients had no evidence of plasma leakage. no adult patients had ascites whereas 11/54 (20%) pediatric patients presented with ascites. no adult patients had pleural effusion whereas 25/54 (53%) pediatric patients did. most adult patients that had serum albumin checked had normal albumin levels (12/14 [86%]) while only 14/28 (52%) pediatric patients had normal albumin level. 5/22 (23%) adult patients versus 32/53 (60%) pediatric patients showed hematocrit increments ≥20%. patients admitted with dengue virus infection may currently be often misclassified as dhf because there are no plasma leakage manifestation in some patients.. there are significant differences in plasma leakage manifestations between adult and pediatric patients which poses a theory that pediatric patients are more susceptible to have plasma leakage manifestations than adult patients. keywords: plasma leakage, dengue hemorrhagic fever, dengue fever, pediatric patients, adult patients abstrak kebocoran plasma adalah salah satu gejala penting demam berdarah dengue (dbd) yang membedakan dengan demam dengue (dd). ada 4 kriteria dalam penegakan diagnosis dbd yaitu demam tinggi mendadak 2 – 7 hari, manifestasi perdarahan, trombositopenia (≤100.000 sel/mm3) dan bukti dari kebocoran plasma. kebocoran plasma terdiri dari peningkatan hematokrit ≥20%, hipoalbuminemia, bukti dari efusi pleura atau asites. masih banyak dokter yang hanya berpatokan pada kriteria trombositopenia saja pada penegakan diagnosis dbd. penelitian ini bertujuan untuk menganalisis profil dari kebocoran plasma dari pasien dbd dewasa dan anak di rsud dr. soetomo pada periode januari – juni 2014 sehingga bisa didapatkan diagnosis dan pelayanan kesehatan yang lebih baik ke depannya. penelitian ini adalah penelitian studi retrospektif dengan menggunakan rekam medik pasien dbd. 78 rekam medik pasien dbd ditemukan, terdiri dari 24 pasien dewasa (31%) dan 54 pasien pediatri (69%). 29/78 (37%) pasien ditemukan tanpa adanya manifestasi kebocoran plasma. sama sekali tidak ditemukan pasien dewasa dengan asites tetapi ditemukan 11/54 (20%) pasien pediatric dengan asites. tidak ada pasien dewasa dengan manifestasi efusi pleura sama sekali sedangkan pada pasien pediatri ditemukan 25/54 (53%) pasien dengan efusi pleura. mayoritas pasien dewasa yang telah dicek serum albumin memiliki kadar albumin normal (12/14 [86%]), di sisi lain hanya 14/28 pasien pediatri yang telah dicek serum albumin memiliki kadar albumin normal. 5/22 (23%) pasien dewasa berbanding 32/53 (60%) pasien anak menunjukkan kenaikan hematokrit ≥20%. dalam penelitian ini dapat disimpulkan bahwa masih cukup banyak terjadi misdiagnosis dari dbd karena dapat ditemukan beberapa pasien yang tidak memiliki manifestasi kebocoran 93rizaliansyah, et al.,: plasma leakage profiles of dengue hemorrhagic fever patients plasma. pasien anak dan pasien dewasa memiliki perbedaan yang signifikan sehingga dapat memunculkan teori bahwa pasien anak lebih rentan untuk memiliki manifestasi kebocoran plasma. kata kunci: kebocoran plasma, demam berdarah dengue, demam dengue, pasien pediatri, pasien dewasa introduction who classifies symptomatic dengue virus infection into four groups which are undifferentiated fever, dengue fever (df), dengue hemorrhagic fever (dhf), and expanded dengue syndrome. dengue hemorrhagic fever (dhf) remains one of the tropical diseases with an enormous worldwide caseload. around 50 million dengue virus infections happen annually of which 500,000 people are diagnosed with dhf and need hospitalization. approximately 90% of dhf patients are children aged less than five years and 2.5% die.1 indonesia in 2014 had 100,347 cases of dhf of which 907 (0.9%) were lethal.2 df and dhf mostly have similar symptoms. the requirement for dhf diagnosis is acute onset of fever lasting 2 – 7 days, hemorrhagic manifestation, a platelet count ≤100,000 platelets/mm3 and evidence of plasma leakage. plasma leakage is the main hallmarks of dhf and it differentiates dhf from df. dhf and df nearly have a similar sign & symptom (like thrombocytopenia, hemorrhagic manifestation, fever etc.). plasma leakage manifestation is essential for the diagnosis making of dhf because any signs and symptoms of dengue viral infections without plasma leakage manifestations is considered to be df. plasma leakage is considered to have occurred in case of a suddenly rising hematocrit to ≥20% from baseline or decrease in convalescence, the presence of ascites, a new pleural effusion on lateral decubitus chest x-ray (cxr), or low serum albumin or protein for age and sex.1 many doctors base their diagnosis of dhf only on the presence of thrombocytopenia.3 who changed the classification of dengue in 2011 which refers back to the classification from 1997 with some changes. many doctors were confused about the 2009 who dengue classification since the who 2009 classification created about twice the workload for health care personnel and required a dengue confirmatory test.4 the changes made in 2009 created confusion among healthcare personnel and may have had unwanted consequences for healthcare services. by the latest 2011 dengue classification, most patients did not fulfill the dhf case definition: evidence of plasma leakage in the presence of thrombocytopenia.4 in the early phase of mild cases of dhf one might not find evidence of plasma leakage by physical examination.1 this phenomenon makes it easier misclassify patients with dengue virus infection. this goal of study was to analyze the presence of plasma leakage between adult and pediatric patients with a dhf diagnosis in rsud dr. soetomo surabaya in the period of january – june 2014. material and method this study was a retrospective study using medical record of patients that had had a dhf diagnosis in rsud dr. soetomo, surabaya between january – june 2014. dhf patients which had unusual manifestations or complications that were not correlated with dengue virus infection were excluded. these exclusions were based on the ‘comprehensive guidelines for prevention and control of dengue and dengue hemorrhagic fever’ published by who in 2011. these exclusions would minimalize plasma leakage manifestations that caused by non dengue virus infection. variables recorded in this study consisted data regarding plasma leakage, i.e. increasing hematocrit ≥20%, hypoalbuminemia, and evidence of pleural effusion and ascites. there were no data available regarding the baseline levels of hematocrit in indonesia, instead we therefore subtract the lowest hematocrit level from the highest hematocrit level, then dividing that value by the lowest hematocrit level and multiplying by 100.5 as normal albumin levels in this study we used 3.4 – 5 g/dl. pleural effusion were checked by chest x-ray (cxr) and the presence of ascites was ascertained by physical examination only. table 1. grading of disease severity among patients admitted to rsud dr. soetomo academic hospital with a diagnosis of dengue hemorrhagic fever. diagnosis adult patients pediatric patients frequency percentage (%) frequency percentage (%) dhf grade i 13 54.2 16 29.6 dhf grade ii 11 45.8 19 35.2 dhf grade iii 0 0 16 29.6 dhf grade iv 0 0 3 5.6 total 24 100 54 100 p value calculated by mann – whitney test = 0.002 94 indonesian journal of tropical and infectious disease, vol. 6. no. 4 january–april 2017: 92-96 adult data are the data of patients treated in internal medicine department and pediatric data are from cases treated in pediatric medicine department. result and discussion 48 patients were excluded based on exclusion criteria. there were 78 dhf patients who fulfilled this study’s criteria in rsud dr. soetomo between january – june 2014 that consisted of 24 adult patients (31%) and 54 pediatric patients (69%). 76% patients enter the hospital in critical phase while 6% and 18% enter the hospital in febrile and recovery phase. no dengue shock syndrome (dss) happened in adult patients while in pediatric patients there were 19 dss patients. the clinical presentation of adult and pediatric with dhf differed significantly (table 1). overall, 29/78 patients (37%) did not have any evidence of plasma leakage. these 29 patients consisted of 17 adult patients and 12 pediatric patients (table 2). none of the adult patients had ascites on physical examination while 11 pediatric patients did have ascites. among the 11 pediatric patients with ascites, 9 patients had developed ascites before entering the hospital and 2 patients developed ascites during their hospital stay. none of the adult patients had radiological evidence of pleural effusion while pleural effusions were detected in 25/34 (74%) pediatric patients that had cxr taken. 11 adult patients and 20 pediatric patients did not have a cxr taken during their hospital stay. from the albumin level data presented in table 3 it is become evident that there were more patients which had normal albumin level than patients that had hypoalbuminemia. 12/14 (86%) adult patients had normal albumin levels while only 14/27 (52%) pediatric patients table 2. distribution of clinical and laboratory manifestations of plasma leakage in patients admitted with a dengue hemorrhagic fever diagnosis. manifestation frequency percentage (%) percentage with no manifestations (%) no evidence of plasma leakage 29 37.2 ascites only 1 1.3 2.0 pleural effusion only 4 5.1 8.2 hypoalbuminemia only 2 2.6 4.1 hematocrit increase ≥20% only 19 24.4 38.8 pleural effusion + hypoalbuminemia 1 1.3 2.0 pleural effusion + hematocrit increase ≥20% 7 9.0 14.3 hypoalbuminemia + hematocrit increase ≥20% 1 1.3 2.0 ascites + hematocrit increase ≥20% 1 1.3 2.0 ascites + pleural effusion + hematocrit increase ≥20% 3 3.8 6.1 pleural effusion + hypoalbuminemia + hematocrit increase ≥20% 4 5.1 8.2 ascites + pleural effusion + hypoalbuminemia 3 3.8 6.1 ascites + pleural effusion + hypoalbuminemia + hematocrit increase ≥20% 3 3.8 6.1 total 78 100 100 table 3. albumin level distribution of dhf patients albumin adult pediatric frequency total percentage (%) frequency total percentage (%) normal 12 14 85.7 14 27 51.9 hypoalbuminemia 2 14.3 13 48.1 no data 10 27 total 24 100 54 100 p value calculated by chi – square test = 0.03 table 4. changes in hematocrit in adult and pediatric patients with a dengue hemorrhagic fever diagnosis. hematocrit adult pediatric frequency total percentage (%) frequency total percentage (%) increased ≥20 % 5 22 22.7 32 53 60.4 negative 17 77.3 21 39.6 only tested once 2 1 total 24 100 54 100 p value calculated by chi – square test = 0.003 95rizaliansyah, et al.,: plasma leakage profiles of dengue hemorrhagic fever patients had normal albumin level (table 3, p = 0.03). however, in a large proportion of patients suspected of dhf, their serum albumin level was not determined. 37/75 (49%) patients had hematocrit increments ≥20 %. more so among pediatric patients (32/53 [60%]) than among adult patients (only 5 out of 22 patients [22.7%]), a highly significant difference (table 4, p = 0.003. in three patients their increments could not be determined because they had their hematocrit level determined only once during their hospital stay. discussion plasma leakage is a crucial point that differentiates dhf from df. the possibility to misdiagnose df into dhf is high because plasma leakage manifestations are difficult to observe and ascertain. evidence of plasma leakage may not be detectable by physical examination alone, especially in the early phase of plasma leakage or in mild cases of dhf. 29 out of 78 patients (37.2%) had no evidence of plasma leakage. however, many of those did not receive a full clinical and laboratory work-up. this poses the question how did doctors make the dhf diagnosis in these cases if there was no plasma leakage evidence? these 29 patients included 17 adult patients and 12 pediatric patients. for the first manifestation of plasma leakage, ascites, there were no cases among the adult patients while there were 11 cases among the pediatric patients, nine had already developed ascites before entering the hospital. patients with ascites in rsud dr. soetomo, surabaya received a full physical examination. other studies showed varied results. srikiatkhachorn et al.6 (2011) in his research in pediatric patients with dhf found that 34% had ascites, while navarrete-espinosa et al.7 (2005) shows that there were only 4 ascites cases among a cohort of 898 dhf patients. another study by balasubramanian et al.8 (2005) showed that by using ultrasonography, 91% of their dhf patients had ascites. this big gap between physical examination and usg results showed that in many, possibly in the majority of them, ascites cannot be detected by physical examination alone. ascites developed during the patient’s stay in the hospital may not be the direct consequence of the virus infection but rather should be considered as an early symptom of fluid therapy overload.1 routine usage of ultrasonography is thus recommended as the method of choice in detecting cases with ascites which couldn’t be detected by physical examination alone. presence of fluid leakage by ultrasound might differentiate cases with borderline hematocrit levels (10 – 20%).9 classification of patient’s data based on the course of dengue illness in febrile phase, critical phase and recovery phase based on who 2009 guidelines.10 mostly, patients enter the hospital in the critical phase. the most important things to do in the critical phase is give fluid therapy as fast as possible. it is better to use ultrasound but it is overrated because we could use other sign and examination which cheaper and easier in critical phase like temperature, potential clinical issues, and laboratory changes to make a dengue viral infection suspect. because of fluid therapy importance, probably there were patients with possible over fluid therapy. the other plasma leakage manifestation, pleural effusions did not occur in adult patients with dhf. in contrast, a large majority, approximately 74% of pediatric patients had radiological evidence of pleural effusions. in the study by navarrete-espinosa et al.7 (2005), 3/898 dhf patients had pleural effusions whereas srikiatkhachorn et al.6 (2011) reported that 78% of their cases had pleural effusions. the study by balasubramanian et al.8 (2005) in 89% of dhf patients checked by ultrasonography had pleural effusions. there were no significant difference in the presence of pleural effusions when examination by cxr and ultrasonography were compared. however, 31 patients in our retrospective were not checked by cxr. transudates pleural effusion could be caused by hypoalbuminemia.11 minimum volume of pleural effusion that become visible in cxr is 50 ml.12 mild pleural effusion/less than 50 ml couldn’t be analyzed by cxr. this number is probably relative based on thoracic volume. 74% of pediatric patients had radiological evidence of pleural effusion while none of adult patients had radiological evidence of pleural effusion. smaller thoracic volume in pediatric patients make the ratio between fluid volume and thoracic volume bigger than the same fluid volume in adult thoracic volume. hypoalbuminemia was found in only a minority of the cases enrolled in this study, especially among adult patients whereas in pediatric patients, hypoalbuminemia was detected in close to half of the patients tested. however, not all of the patients got their albumin level tested (37/78 patients had not been tested). this study is somewhat contradictive with a study by villar-centeno et al.13 (2008) reported that 57% of their dhf patients presented with hypoalbuminemia. albumin levels less than 4 g/dl may be an early indicator of vascular permeability alteration.13 lower albumin levels could be caused by liver involvement in denv infections which makes severe liver damage that leading to decreased production of albumin.14 mostly dhf patients in this study have normal albumin level. the final plasma leakage manifestation was the hematocrit. nearly half number patients had increases of their hematocrit ≥20%. only few adult patients have developed increasing hematocrit ≥20% while 60% of pediatric patients have increasing hematocrits of ≥20%. this finding corresponds with srikiatkhachorn et al.6 (2011) and guilarde et al.15 (2008) who showed that among pediatric patients, increasing hematocrit ≥20% is the rule, whereas other studies found that among adult patients with hematocrits increasing ≥20% constituted a minority, albeit a sizable minority of 49%. 96 indonesian journal of tropical and infectious disease, vol. 6. no. 4 january–april 2017: 92-96 increasing hematocrit levels is a consequence of plasma leakage. extravasations of plasma without rbc makes the ratio between rbcs and total volume of blood is higher. a rising hematocrit, e.g. 10% to 15% above baseline, is the earliest evidence of plasma leakage1 but dhf would be misdiagnosed if only based on hematocrit as a diagnostic criterion.9 highly significant difference of hematocrit level and albumin level between adult and pediatric patients poses a theory which pediatric patients are more susceptible to have a plasma leakage than adult patients. conclusion plasma leakage is the most important signs / symptoms for the diagnosis making of dhf. signs and symptoms of dengue viral infection without any of plasma leakage manifestations is not classified as dhf but is classified as df. this study found that patients admitted with dengue virus infection may currently be often misclassified as dhf because there are no evidences of plasma leakage manifestations. there are significant differences in plasma leakage manifestations between adult and pediatric patients which poses a theory that pediatric patients are more susceptible to have plasma leakage manifestations than adult patients. acknowledgement writer gratefully and sincerely thanks for patients of dr. soetomo general hospital which always become the first consideration, dean of faculty of medicine of airlangga university, director of dr. soetomo general hospital for motivating, inspiring and spending their precious time to guide and direct this experiment until this study is done. references 1. who, editor. comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. 1st ed. world health organization. new delhi-india: world health organization regional office for south-east asia; 2011. 2. indonesia kkr. profil kesehatan indonesia tahun 2014. jakarta: kementerian kesehatan ri; 2015. 1-382 p. 3. aryati. buku ajar demam berdarah dengue (tinjauan laboratoris). surabaya: global persada press; 2011. 4. kalayanarooj s. dengue classification: current who vs. the newly suggested classification for better clinical application? j med assoc thai. 2011 aug;94 suppl 3:s74-84. 5. suwarto s, nainggolan l, sinto r, effendi b, ibrahim e, suryamin m, et al. dengue score: a proposed diagnostic predictor for pleural effusion and/or ascites in adults with dengue infection. bmc infect dis. 2016 dec 8;16(1):322. 6. srikiatkhachorn a, gibbons r v, green s, libraty dh, thomas sj, endy tp, et al. dengue hemorrhagic fever: the sensitivity and specificity of the world health organization definition for identification of severe cases of dengue in thailand, 1994-2005. clin infect dis. 2010 apr 15;50(8):1135–43. 7. navarrete-espinosa j, gómez-dantés h, germán celis-quintal j, vázquez-martínez jl. clinical profile of dengue hemorrhagic fever cases in mexico. salud publica mex. 2005 jun;47(3). 8. balasubramanian s, janakiraman l, kumar ss, muralinath s, shivbalan s. a reappraisal of the criteria to diagnose plasma leakage in dengue hemorrhagic fever. indian pediatr. 2006 apr;43(4):334–9. 9. srikiatkhachorn a, krautrachue a, ratanaprakarn w, wongtapradit l, nithipanya n, kalayanarooj s, et al. natural history of plasma leakage in dengue hemorrhagic fever: a serial ultrasonographic study. pediatr infect dis j. 2007 apr;26(4):283-90-2. 10. world health organization. dengue: guidelines for diagnosis, treatment, prevention, and control. spec program res train trop dis. 2009;x, 147. 11. hooper c, lee ycg, maskell n. investigation of a unilateral pleural effusion in adults: british thoracic society pleural disease guideline 2010. thorax. 2010 aug 1;65(suppl 2):ii4-ii17. 12. craig blackmore c, black wc, dallas r v., crow hc. pleural fluid volume estimation: a chest radiograph prediction rule. acad radiol. 1996 feb;3(2):103–9. 13. villar-centeno la, díaz-quijano fa, martínez-vega ra. biochemical alterations as markers of dengue hemorrhagic fever. am j trop med hyg. 2008 mar;78(3):370–4. 14. martina bee, koraka p, osterhaus adme. dengue virus pathogenesis: an integrated view. clin microbiol rev. 2009 oct;22(4):564–81. 15. guilarde ao, turchi md, jr. jbs, feres vcr, rocha b, levi je, et al. dengue and dengue hemorrhagic fever among adults: clinical outcomes related to viremia, serotypes, and antibody response. j infect dis. 2008 mar 15;197(6):817–24. �0 vol. 1. no. 1 january–april 2010 research report cognitive behavior change for the improvement of health care, cognitive function and school achievement in helminth infected children hastaning sakti faculty of psychology, diponegoro university abstract overall helminth infection prevalence in central java in 2002 was high (20–100%). unfortunately, the knowledge, attitude and practice (kap) of children and their parent’s about worm infection were in general very low. objective of this study was to determine the improvement of parents’ health care behavior on helminth infected children by improving their kap, in order to decrease re-infection rate of helminth infestation and improving school achievement. design study is quasi experiment with pre and post test control group design on community participation toward school aged children in sragen sub-district in central java. a sample of one hundred and thirty one of children deriving from a systematic random sampling was used in the study and divided into 66 children for intervention population and 65 children for control population. during the study, the children in selected area were given with 400mg chewable albendazole, and after a month, their stools were examined to find out the negatives. parent’s participation was needed in improving the kap on re-infestation that was performed by an intervention using a module. data were analysed using chi-square test to determine the relationship between variables among groups. stools were examined for the presence of geohelminth’s egg using kato-katz technique. three subject matters (mathematics, indonesian language and natural science) were obtained by a special tool. digit span, coding and stroop test were used to determine the level of cognitive function. monitoring and the ladder-snake game were use to observe the improvement of the behavior. it revealed that re-infestation of ascaris lumbricoides, trichuris trichhiura and oxyuris vermicularis occurs 2 times greater in control group, even there was no significant different. in both groups, there was no hookworm re-infestation. a good knowledge and attitude can be predicted to be a better practice to prevent the re-infestation. oxyuris vermicularic re-infection could have adverse effect on study achievement (mathematics, indonesian language and natural science) and the cognitive function, such as memory, attention, visual-motoric co-ordination. the moshly affected cognitive function was memory-based and specifically represented the components of the working memory. from the monitoring of health care behavior, this study found that the more convinced the mothers in changing their degree of family’s health were, the higher the level of children’s health was. key words: worm re-infestation, school achievement, cognitive function. introduction school performance can be affected by family care pattern and health status. in most agricultural areas, where the sanitation program and city planning are not carefully prepared, this situation can increase the prevalence of helminth infestation in children, especially in school-age children. worm infection can bring about the decrease of nutritional status. as a consequence, this makes someone easy to get other infected diseases. many studies showed that there was a strong relationship between helminth infection and malnutrition, health status, absenteeism, sanitation, and school achievement. in 1996, a biomedical survey on school-age children in 5 countries, including indonesia, confirmed that 2396% were infected by parasitic infection (pcd, 1997). the overall helminth infection prevalence in central java indonesia in 1996 was 20–100% (satoto, hertanto and hendratno, 1997). of all helminthes, hookworm causes the most severe anemia because of the iron deficiency due to chronic blood loss. it is also well known that iron deficiency is the most prevalent nutritional disorder in the world, affecting some 50 million people. nutritional anemia caused by iron deficiency is common throughout indonesia, being most prevalent in pregnant women, under five-year-old ��sakti: cognitive behavior change for the improvement of health care children and school-aged children, especially in lower socio-economic groups. the result of the study in karanganyar (central java), in 1996, as the previous study, showed that helminth infection especially hookworm, can influence the concentration and motivation, particularly of 11–13 years rather than 8–9 years old children. in further analysis, it was found that there was a strong correlation between long term memory skill and school performance in mathematics (p=0.004) and indonesian language (p<0.0001). the results suggest that hookworm infestation can have a significant adverse effect on children’s working memory which may have consequences for a child’s reasoning ability and reading comprehension. on the other hand, parents’ education and the willingness of parents to closely assist their children while they learn, can influence the school achievement. unfortunately, the study about health care education related to reducing re-infection were rarely done. based on the description above, the prevention for helminth infection would yield better results if it is conducted in the early age. the most important one is the parent’s participation in medication management, so that the parents will be able to make their own decision and have a sense of belongings and responsibility to do their decision. another study done by andre and rachel, in 1995 found that the program derived from educational diagnosis requires the participation of representative advisory boards and multiple actors, including parents. school health research, whose work has demonstrated positive outcomes, are unanimous in their conclusion that development of relevant cognitive skills, resistance to peer pressure, and social competence skills, in some combinations, facilitates change or resistance to change. concerning to this result, the parents at home, who deal with the children every day, are the right persons to maintain health care and act as a model of healthy behavior to minimize the effect of health problem and to improve school achievement. rousham (1994) argued that health education is considered to have an important role in the prevention and control of helminths, but the dissemination and implementation of health message is rarely evaluated. without information on these broader aspects of helminth treatment and prevention, the design of appropriate deworming programs is hindered. so far, the studies about the effect or benefits of parent participation on reducing re-infection rate are rarely done. a study on effectiveness of albendazole toward hookworm infection showed that it 85.8% affective for the cure rate, but there was 14.2% who got re-infected. helminth infection can adversely affect the cognitive function of children, specifically on attention ability and working memory. it can provide a negative affect on intelligence, concentration and learning ability, that in turn it can affect the school achievement (sakti et al., 1997). the qualitative study in evaluating the knowledge, attitude and practice of school aged-children and their parents about worm infection and related issues appeared very low in general. parents’ knowledge about transmission and prevention of helminth infection much lower than their children, and it is assumed that it can stimulate the re-infection. even the knowledge attitude and practice about helminth infection among the parents in karanganyar, central java, was very low, but the willingness of the parents to participate in the prevention program was a great. the module has been arranged with the title: “modul pelatihan peningkatan perilaku sehat anak dan ibu terhadap kecacingan (peningkatan perisai kecacingan)”. the module used affective approach containing: (1) who we are, (2) children and us, (3) what should we do, (4) talk about health and helminth infection and (5) our responsibility. working memory refers to a brain system that provides temporary storage and manipulation of the information necessary for more complex cognitive tasks such as language comprehension, learning and reasoning (waters, caplan, 1996). within the system, there is attentional controller / central executive which is supplemented by two subsidiary systems called the phonological loop and the visual-spatial sketchpad. the phonological loop is assumed to be responsible for maintaining speech-based information. the digit span test provides a good example of this role and was significantly adversely affected by hookworm and t.trichiuria infection (sakti et al., 1997). whatever the mechanism, if working memory is affected, it could have broader implication for the educational achievement and development of a child, not least because of the underlying association of working memory with complex real-world tasks such as reasoning ability and reading comprehension (water, caplan, 1996). cognitive factors also determine whether or not individuals practice health behaviours. such cognitive factors include the belief that a particular health practice is beneficial, the belief that it can help stave off a particular illness a sense of vulnerability to illness and feeling of self-efficacy (bandura, 1977). participatory research method can be used not only to enable local people to seek their own solutions according to their priorities, but also to secure funding, to co-operate with local people into the agendas of others or to justify short cut research within a top-down process (rifkin, 1994). this study revealed that re-infestation of ascaris lumbricoides, trichuris trichiura and oxyuris vermicularis occurred (two) times greater on control group even there was no significant different. in both groups, there was no hookworm re-infestation. a good knowledge and attitude can predict a better practice to prevent the reinfestation. oxyuris vermicularis re-infection could provide adverse effect on the school achievement (mathematics, indonesian language and natural science) and the cognitive functions, such as memory, attention, visualmotoric co-ordination. the cognitive function which was mostly affected were memory-based and specifically represented the components of the working memory. from �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 10-16 the monitoring of health care behaviour, this study found that the more confinced the mothers in changing their degree of family’s health were, the higher the level of the children’s health. materials and methods study sites-selection of schools of children this study was held in sragen regency in central java indonesia, which is an agricultural area with high prevalence on hookworm (74%) in 1996 (mitra, bulletin, 1996). the decision to use these areas for intervention and control group had been discussed and consulted with regional head department of central java province in sragen, in collaboration with department of health and department of education. two areas had been selected in accordance with the similarities in terms of physical and environmental factors related to health. schools were also selected by soil sample test. the similar geographical condition and the school condition was the pre-requirement for this experimental study. the target population on school-age children in high prevalence of hookworm infection, sitting in grade 3, 4, and 5 in primary school. they have been tested with school achievement test and cognitive function test. they were also suggested to collect their stools. a day after, they were dewormed by giving albendazole skb 400mg single dose (chewable). a month later, the stools were collected again to see the negative infection to determine the selected children on each intervention and non intervention group. intervention to their parent conducted through the health counseling and education course and followed by giving post test of stools on the sixth month. the selected parents were interviewed to collect the information about their perception on worm infection and the willingness to participate for the prevention and other related things. question on knowledge, attitude and practice of the parents were administered by fgd and in-depth interview for gathering the data. participative observation using home visit was performed to observe the environmental condition. the observation included the portable water resource, the latrine, the place to wash hands, etc. furthermore, the study also collected social economy status of the family, such as parent’s job, parent’s final education. experimental design a quasi experiment with pre test and post test design. in a quasi-experimental design, a group of intervened parents was compared to those who were not intervened. this two groups were separated by distance. the inclusion criteria for students were: (a) students of grades 3, 4, and 5 (age 9–11) in elementary school, (b) students who suffered from hookworm infestation. the exclusion criteria were: (i) students who had mental and physical defect (hands and legs), (ii) students who were colored-blind, and (iii) the ones who had not been trained as the little doctor. some characteristic for environments were: (1) area with high prevalence more than 60% in worm infection, (2) developing agricultural and industrial area which have plantation and manpower who worked there, (3) the soil around schools had been tested and recommended not to have worm eggs or was minimally the same between the two areas, (4) criteria determination for the two sub-districts was as follows: (a) each of the sub-districts had the same distance from the capital of the district, (b) each of the subdistricts had the same distance from puskesmas and had the same geographical condition, i.e., near from reservoir, (c) each of the sub-districts had the same socio-economical condition (non idt), (d) each of the sub-districts had the same condition of playing place in school (unpaved yard), (e) each of the sub-districts had the same school’s condition/ status, i.e./ government school and had school health unit (unit kesehatan sekolah). material and administration test parasite infection pots for faeces were distribute to all subjects one day before examination in selected school. an illustrated direction sheet had been provided to all children on method to collect faeces using the pots correctly. on the following day, the other team collected post filled with fresh faeces and were brought to the puskesmas’ laboratory in sragen, maximum 24 hours after taking. microscopic examination of worm eggs in the faeces was administered using kato katz technique (who, 1982). the result of the examination included eggs of ascaris lumbricoides, trichuris trichiura, hookworm and oxyuris vermicularis recorded in a form in accordance with the total number of each type. knowledge, attitude and practice for parent and children the objective of the baseline data collection on knowledge, attitude, and practice of school children and their parent was to assess the health behavior on worm infection, personal hygiene and health care behavior. this test administered in class for the children, and for parents by home visit and environmental condition that had been observed during visit, including potable water resources, the latrine, the place to wash hands, etc. school achievement test this study used a set of school achievement test instrument that was developed by indonesian partnership of child development, 1997. this test consists of 3 subjects: mathematics, indonesian language and natural science for grade 3, 4, and 5, in which each subject consists of 40–50 questions. it was developed for the first quarter. their test was conducted in natural setting class by teachers and performed as a common test on the 3 subjects. the inter-ratter reliability test for mathematics was 1.0 and indonesian language was 0.99. the result of interratter split-half for mathematics was 0.88 and indonesian language 0.78. test tools for mathematics and indonesian ��sakti: cognitive behavior change for the improvement of health care language had been developed beforehand. for natural science test used uub (quartile evaluation test). cognitive function test this study used 3 battery tests to measure cognitive function, which were: stroop test, digit span and coding. low cognitive phase was attention, was measured by stroop test. medium phase was memory which were measured by digit span test. visual motor test were measured by coding test. the duration of cognitive test per child was about 30 minutes (wisc-r manual, 1974). 1. stroop test stoop test had been adapted into indonesian language and is thus a culture-fair test. the stroop test consist of two tasks: the colour task which consists of a card with the names of four colour (tan, red, blue, and green). the procedure was started by asking children to read the word written and ignoring the colour in which the word was print. the score was the total number of correct answer given in 2 minutes. the correlation of the stroop test from the first and second administration was 0.90, suggesting a high degree of temporal stability. validity of the stroop test was indicated from discriminate analysis which were performed by using the presence or absence of brain damage as dependent variables and colour-word score as the predictor. the correlation was 0.70 (max r, 1989). 2. digit span test digit span test is part of the weschler intelligence scale for children (wisc-r, 1974) used for measuring the phonological loop which includes memory skill. increasingly longer string of number were read with the speed of one number per second, and performed soon after each trial. the child was required to recall or repeat the string of numbers either forward or backwards. the score was the total number of correct trials. for digit span forward the reliability test was 0.74 and backward was 0.72 (sakti et al., 1997) 3. coding it is also part of wisc test, consisting of 93 blocks with numbers that had to be joined with the symbols. the score was the total number of correct trials in 2 minutes. the reliability was 0.78. the sequence of test device administration was based on the child psychology. it means that the test aimed at being a game so that the child did not feel tense. the tests given were started with the easy questions and going through to the difficult ones. therefore, the 2 testers had been trained beforehand and received the test-retest reliability 0.88. school characteristic the objective of survey on school characteristic was to find out the condition of soil surrounding school. it was expected to be clean of worm eggs or minimally the same condition between the two schools. results a total of 131 children participated in the course of this study in sragen, central java. the children were geographically divided into two areas: (1) sixty-six children were in the south area intervention group and (2) sixty-five children were in the north area as the control group, who did not receive any intervention for their parents. re-infestation analysis table 1. shows that there was a re-infestation on trichuris trichiura (3%) and oxyuris vermicularis (1.5%) on intervention group, even it was not statistically significant. re-infestation mostly occurred on control goup (10.6%) rather than intervention group (4.5%). three types of worm that re-infested positively in control group were ascaris lumbricoides, trichuris trichiura and oxyuris vermicularis. the greater re-infestation worm was on oxyuris vermicularis (6.1%) rather than trichuris trichiura (3%) and ascaris lumbricoides (1.5%). only two types of worm that was re-infesting the intervention group, they were trichuris trichiura (3%) and oxyuris vermicularis (1.5%). both on intervention and control group, there was no positive re-infestation of hookworm. there was no hookworm re-infestation, both in intervention and control group. the intervention group was only re-infested, whereas in control group which were no intervention, there was re-infestation on ascaris lumbricoides, trichuris trichiura and oxyuris vermicularis. re-infestation of oxyuris vermicularis can induce adverse effect to indonesian language and natural science. there was a strong correlation between the re-infestation on oxyuris vermicularis toward the decreasing grade of indonesian language (p<0.001) and natural science (p<0.001). in control group, the re-infestation of ascaris lumbricoides had strong correlation with the decreasing grade on mathematics (p<0.001). it means that the re-infestation of ascaris lumbricoides could provide adverse effect for the school achievement especially on mathematics. this study also found that re-infestation of oxyuris vermicularis statistically correlated with mathematics in control group (p=0.037). the re-infestation of oxyuris vermicularis could provide adverse effect on the decreasing grade of mathematics. another result conducted by sadjimin and rini (2000) found that infestation of worms could decrease children’s verbal ability, comprehension, reasoning, and cognitive ability. �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 10-16 table 1. prevalence of re-infestation type parent participation non parent participation preinfest.(-) n (%) reinfest.(+) n (%) reinfest.(-) n (%) reinfest.(+) n (%) ascaris 66 (100%) 65(98.5%) 1 (1.5%) 1 trychuris 66 (100%) 2 (3%) 64 (97%) 2 (3%) 0.496 hookworm 66 (100%) 66 (100%) oxyuris 65 (98.5%) 1 (1.5%) 62 (93.4%) 4 (6.1%) 0.365 total 4.5% (10.6%) table 2. correlation between ∆ egg worm and ∆ subject matters for intervention and control group group subject matters ∆ egg worm (reinfestation) ascaris trichuris hookworm oxyuris intervention ∆ mathematics 0.452 ∆ ind. language <0.001 ∆ natural science <0.001 control ∆ mathematics <0.001 0.841 0.037 ∆ ind. language 0.992 ∆ natural science 0.980 table 3. correlation between ∆ cognitive function, ∆ re-investation and ∆ school achievement ∆ cognitive function test ∆ subject matters of school achievement ∆ mathematics ∆ ind. language ∆ natural science as tr hk ox as tr hk ox as tr hk ox digit span fw 0.050 0.014 digit span bw coding 0.051 0.046 0.031 stroop c 0.023 stroop cw 0.046 0.014 0.046 cognitive function analysis the cognitive function test used in this study were: coding test, digit span test and stoop test. the functions which were most affected were memory-based and specifically represented the components of working memory model (baddeley, 1992). those three tests measured the concentration and short term memory. low cognitive phase was attention, which in this study was measured by stroop test. medium phase was memory which was measured by digit span test. visual motor test was measured by coding test. this result found that there was no significant difference in both pre test and post test. re-infestation of ascaris lumbricoides can influence the school achievement on indonesian language and also the cognitive function on visual motor and attention. this study shows that the re-infestation of oxyuris vermicularis can effect mathematics, indonesian language and natural science and it was shown in the correlation with the cognitive function tests. the cognitive functions which were most affected were memory-based and specifically represented the components of the working memory model (baddeley, 1992). this drove an emerging hypothesis that helminth infection adversely affects working memory (nokes & bundy., 1994) and especiallythe result of digit-span forwards test (boivin et al., 1993). working memory refers to a brain system that provides temporary storage and manipulation of the information necessary for more complicated cognitive tasks such as language comprehension, learning, and reasoning (waters & caplan, 1996). within memory system, there is an attentional controller central executive which is supplemented by two subsidiary systems called the phonological loop and the visual-spatial sketchpad. the phonological loop is presumed to be responsible for maintaining speech-based information. the stroop test provides a good example of this role and was significantly adversely affected by hookworm. the central executive in the cerebral function was considered to be a type of attentional controller (baddeley, 1996). the 4 tasks, which could be considered measures of the central executive and neuropsychologycal factor, were adversely affected by all types of worm infestation after controlling the confounding variables. the reason for why working memory, as oppose to other cognitive domains, could be affected by helminths ��sakti: cognitive behavior change for the improvement of health care infection was still unclear. central executive tasks in particular involve the frontal lobe, a part of the brain which seems to continue developing through to adolescence and is thus still developing during the period when children are most likely to he infected with helminths. whatever the mechanism, if working memory is affected, it could have broader implications for the educational achievement and development of a child, not least because of the underlying association of working memory with complex real-world tasks such as reasoning ability and reading comprehension (waters, caplan, 1996). health behavior analysis the previous result conducted by sakti and nokes (1997) demonstrated that hookworm infestation could deteriorate children’s memory and had an effect on reasoning ability and reading comprehension. a further result also suggested that infestation of hookworms was primarily found in older children (age between 11 and 12 years old) rather than younger children (age between 8 and 9 years old). based on those results, a conclusion could drawn was that the longer the infestation of hookworms occurred, the worse the anemie condition appeared. this condition give an effect in children’s cognitive ability. an interesting result which had been found at the beginning of information-providing was that all mothers considered that wormness was not a dangerous disease if compared to diarrhea, blood fever, tuberculosis and malaria. after monitoring for four times, it was found that mothers, belief on the increase in children’s health had an extremely significant correlation with mothers’ perception on the level of children’s health. the different amount of wormeggs between pre-test and post test had a strong correlation between level of mother’s feeling was accompanied with the increase in children’s level of health (p=0.12). the same results was also shown in the significant correlation between level of children’s health and level of mother’s belief to their degree of family health (p=0.003). from the result of monitoring health behavior both in children and in parents, it was well-demonstrated that there had been an improvement in the practice of selfcleanliness, healthy behavior, environmental cleanliness, mother’s attention, and children’s perception on their mother’s attention. this result represents an indication that the more frequent the behavior appeared, the more possible a significant behavior modification occurred. it was also found that monitoring result, mothers attention on their children’s health did not correlate significantly with modification in children’s health behavior, but mother’s belief in modifying children’s health degree was more influential on children’s behavior. the assumption which could be put forward was that level of mother’s belief of family’s health was accompanied with the changes in children’s health. in other words, the more convinced mothers in changing their degree of family’s health were, the higher level of children’s health was. this also meant that mothers or parents could become an effective medium for the modification of children’s health behavior, especially when coping with the danger of wormness. in respondent’s evaluation about the ever-received information-providing before this research and the present information-providing, it was found that there was a significant change, especially in the change of environmental health (p=0.047). this was shown by the change in environmental cleanliness which could be regarded as a manifestation of an attention to family’s health and mother’s self-convince to improve their family’s degree of health this result was in line with the result shown by sadjimin (2000). acknowledgements i would like to thank all the children and their parents who participated in the study for their warmness and enthusiasm. i also extremely grateful to the teachers who made us so welcome in the schools and facilitated the field work. i also indebted for the excellent work to the cognitive tester, lintang, fitri and kartika, and the health analysts nani and agam. thanks are also given to the field observers tina, cecilia, astri, and the nice caricaturist illustrator game nurjanah. i would like to express my gratitude to certc gadjah mada university in term of chance to participate as a fellow and also for inclen and rockefeller foundation for a providing financial support for this study. very extremely grateful for my advisers prof. dr. mohammad hakimi, spog, phd, dra. supra wimbarti, msc. phd, and drs. zulaela dipl.med.stats, msi for their guidance and encouragement in finishing this study. references 1. andrea c., rachel j., 1995, what is participatory research. social science medicine, 41, 12, 1667–1676. 2. beddeley a., exploring the central executive, quarterly journal of experimental psychology 49a. 5–28. 3. beddeley a., exploring the central executive, quarterly journal of experimental psychology 49a. 5–28. 4. bandura, a., 1977, self efficacy: “toward a unifying theory of behaviour change”. psychological review,. 84, 191–215. 5. bulletin mitra, 1997, mitra, bulletin kemitraan indonesia untuk perkembangan anak . april, 2, 5. 6. kato-katz technique, who, 1982, programme on intestinal parasitic infection, division of communicable disease. 7. max r., 1989, stroop, neuropsychological screening test manual, psychological assessment resources, inc. 8. nokes c., bundy d., 1994, does helminth infection affect mental processing and educational achievement ? parasitology today, 10, 4–8. �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 10-16 9. pcd, 1997, partnership for child development, report v: programme monitoring and evaluation, collaboration between partnership for child development and ministry of health indonesia, semarang, indonesia, 1997. 10. rifkin s., 1994, participatory research and health. proceeding of the international symposium on participatory research in health promotion. liverpool school of hygiene and tropical medicine. 11. rousham e.k., 1994, perception and treatment of intestinal worms in rural bangladesh, local differences in knowledge and behaviour, social science medicine, 39, 8, 1063–1068. 12. sakti h., nokes c., hertanto w., sri h., andrew h., donald b., atoto, 1997, evidence for baseline association between helminth infection on cognition and motor function and educational achievement in indonesia school-age children, journal of tropical medicine, 1999, may, 22, 223–229. 13. sadjimin t., rini j., 2000, hubunganantara infestasi cacing dan prestasi belajar anak sekolah dasar di kecamatan ampama kota kabupaten poso sulawesi tengah, jurnal epidemiologi indonesia, 4: 1: 17–26. 14. satoto, hertanto w.s., sri h., 1996, mitra, partnership for child development”. collaboration between partnership for child development and ministry of health indonesia, semarang, indonesia, 1997. 15. waters g.s., caplan., 1996, the measurement of verbal working memory capacity and its relation to reading comprehension. quarterly journal of experimental psychology 49 a, 51–65. ijtid vol 1 no 1 jan-apr 2010.12.pdf ijtid vol 1 no 1 jan-apr 2010.13.pdf ijtid vol 1 no 1 jan-apr 2010.14.pdf ijtid vol 1 no 1 jan-apr 2010.15.pdf ijtid vol 1 no 1 jan-apr 2010.16.pdf ijtid vol 1 no 1 jan-apr 2010.17.pdf ijtid vol 1 no 1 jan-apr 2010.18.pdf ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 11 no. 1 january–april 2023 case report severe leptospirosis (weil’s disease) with multiple organ failure in urban setting: a case report samuel halim1,2* , bryan arista hartono3 1internal medicine department, hermina hospitals kemayoran, jakarta, indonesia 2department of internal medicine, faculty of medicine, universitas tarumanegara, jakarta, indonesia 3hermina hospitals kemayoran, jakarta, indonesia received: september 29th, 2022; revised: january 1st, 2023; accepted: march 1st, 2023 abstract leptospirosis is a rare disease that could cause multiple organ failures and death if left untreated. the correct treatment will determine the recovery of patients. a 28-years old male came to the emergency department with profuse diarrhea. no prior medical history; worked as a private employee recently assigned to collect rat traps one week before. laboratories show severe thrombocytopenia, acute liver failure, and acute renal failure support by imaging with the conclusion of hepatomegaly with normal kidney size. during observation in the emergency room, the patient worsens into septic shock. the patient was treated in intensive care, diagnosed with weil's disease, and treated given antibiotics with aggressive fluid therapy; dialysis was postponed, and close monitoring of the patient's symptoms and organ function. after five days of care, clinical symptoms and organ function improved, and the patient was discharged well. diagnosis of leptospirosis is challenging with a combination of signs and symptoms that are not commonly found. therefore, primary treatment is antibiotic and supportive care such as renal replacement therapy is not routinely needed as long there are improvements in close monitoring. this objective is to increase awareness and treatment option for further severe leptospirosis cases keywords: dialysis; fluid therapy; leptospirosis, multi organ failure; weil’s disease highlights: . novelty in this case is weil’s disease could manifest as severe acute kidney injury without prominent icteric whilst hepatomegaly with increase liver function occur will be reversable with appropriate conservative management. it benefits as reference to postpone dialysis with proper conservative management. how to cite: halim, s., hartono, b. a. severe leptospirosis (weil’s disease) with multiple organ failure in urban setting: a case report. indonesian journal of tropical and infectious disease. 11(1). 12–17. apr. 2023. doi: 10.20473/ijtid.v11i1.39466 * corresponding author: samuelhalim2000@yahoo.com https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0009-0002-7305-5777 https://orcid.org/0009-0006-7492-446x 13 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 12–17 introduction leptospirosis is a zoonotic infection that affects both humans and animals.1according to who, in 2019, there were 920 cases reported in indonesia, with 122 deaths. however, this reported case number is a severe underestimate of leptospirosis occurrence in indonesia, given that the annual morbidity of leptospirosis in the population was recently estimated at 39.2 per 100,000 people.2clinical symptoms are undistinguishable from other infectious diseases such as hepatitis, dengue, and typhoid. severe cases, rather known as weil’s syndrome, are the triad of haemorrhage, jaundice, and acute kidney injury.3the primary treatment for leptospirosis is antibiotic such as penicillin and supportive care. hemodialysis as the early supportive therapy for kidney injury did not associate with the mortality rate in a critically ill patient.4 the objective of this report is to increase awareness and as the reference consideration to treat severe leptospirosis in further cases. case report a 28th-year-old man from kemayoran, central jakarta, was admitted to the emergency department with profuse diarrhoea, nausea, yellowish-red sclera, malaise, and muscle pain, especially below the knee. the stool is brown-yellow with soft consistency without blood; meanwhile, the urine is dark. symptoms occur around two days before admission with a fever that has never been felt before. there is no prior medical history or high-risk lifestyles such as needle injection and promiscuity. patient work as a private employee. one week before admission, he was assigned to collect rat traps around the corner of a warehouse. he did not catch a single rat and managed to clear up the trap. no evidence of rat bite or prior flood was recorded during that time. physical examination on admission shows vital signs of low blood pressure (109/75 mmhg), regular pulse, breath and no fever (36.5c). conjunctival suffusion, normal breath and heart sound, abdominal pain at the epigastric, and no swelling nor jaundice on the extremities. the patient was suspected of having a hepatitis a infection. laboratories and imaging did the further investigation. laboratory finding shows anaemia, leucocytosis, thrombocytopenia, hyponatremia, increased bilirubin level, slight hypo albumin, normal blood coagulation test, liver injury and renal failure—serologic tests of anti-hav, hbsag, anti-hcv, and anti-hiv show negative results (table 1). chest x-ray (figure 1) was clear, and abdominal ultrasonography (figure 2) shows nonspecific hepatomegaly without other organs abnormality. by the time examination was done, blood pressure had dropped to 85/34 mmhg, pulse rate 102 times per minute, respiratory rate 25 times per minute, with a normal temperature of 38.2c, fall into the diagnosis of septic shock then given norepinephrine 0,1 mcg per bodyweight per minute. transfusion of one unit thrombocyte concentrate followed by hydration of nacl 3% 500 ml with crystalloid 2000 ml over 24 hours, antibiotic, proton-pump inhibitor (ppi) and attapulgite was given as initial therapy. the patient was admitted to the icu for further monitoring. figure 1. chest x-ray, shows no abnormality in lungs and heart 14 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license samuel halim, et al. severe leptospirosis (weil’s disease) figure 2. abdominal ultrasonography, shows a non-specific hepatomegaly with normal kidney structure during intensive care, the patient clinical was improved with normal vital signs, decreasing icteric, and no other symptoms. follow-up laboratory findings were done with anaemic, improving leucocytes, thrombocytes, and liver and renal function. norepinephrine support was tapered down, and the patient planned to move to the general wards. additional test igm anti-leptospirosis was done and shows a positive result. treatment of antibiotics, rehydration, and ppi was continued. the patient was hospitalized for another three days. on the last day, the patient clinically improves, and symptoms are all gone but icteric slightly remains. stool and urine are within normal colours. the patient then discharges with antibiotics and ppi as home medicine. table 1. laboratory examination examination result 22/8 23/8 24/8 25/8 26/8 hemoglobin (g/dl) 10.3 9.5 9.8 10.1 10.2 hematocrit (%) 27.8 25.3 27.0 27.8 28.1 leucocyte (/ul) 20,400 27,680 12,600 6,080 5,950 thrombocyte (/ul) 36,000 71,000 91,000 119000 163,000 natrium (mmol/l) 126.0 kalium (mmol/l) 3.67 chloride (mmol/l) 95.5 alt (g/dl) 79.7 ast (g/dl) 170.4 albumin (mg/dl) 2.8 creatine (mg/dl) 11.45 8.76 2.29 urea (mg/dl) 236.1 283.1 145.7 egfr ( ml/min/1.73m2) 6 8 39 anti hav non-reactive hbsag non-reactive anti hcv non-reactive anti hiv non-reactive total bilirubin (mg/dl) 8.0 5.05 conjugated bilirubin (mg/dl) 6.45 3.93 unconjugated bilirubin (mg/dl) 1.55 1.12 pt (second) 17.8 control pt 14 aptt (second) 31.4 control aptt 31.3 igm anti-leptospira reactive abbreviations: g = grams; dl = deciliter; ul = microliter; mm = millimeter; u = unit; l = liter; meq = milliequivalent, min = minutes 15 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 12–17 discussion in this report, we have described a case of severe leptospirosis or known as weil’s disease.1,3 on admission, the patient presented with fever, conjunctiva suffusion, dark urine, and myalgia with leucocytosis, thrombocytopenia, aki, liver failure, and hyperbilirubinemia. patients experience septic shock in the er and are given norepinephrine as support. treatment given was antibiotics and aggressive hydration. dialysis was postponed while watchful waiting for the improvement of kidney functions by fluid therapy. strict monitoring of kidney function and haematology was done. symptoms and kidney function then recover with the treatment given. leptospira is a zoonotic disease that is an emerging global public health problem. indonesia, with a high incidence of flooding and subsequent presence of stagnant water and poor sanitation conditions in some housing areas, is at high risk for leptospirosis. the transmission from infected animals through their urine (rodents, dogs, livestock, pigs, horses, wildlife) can survive for weeks to months in water and soil. a human can be infected through direct contact with the urine, urine-contaminated water, and wet soil, or ingestion of urine-contaminated food or water.1,5,6 in the present case, there is no contact with water or soil, but our patient does risk contact with a rat trap which could be contaminated with rodent urine. high-risk infection activities include wading, swimming, boating, and activities that could lead to skin abrasion and water or soil exposure. leptospirosis symptoms are usually a flulike illness of sudden onset, fever, headache, nausea, vomiting, abdominal pain, conjunctival suffusion, and myalgia, typically on the calves and lower back. severe cases have a classic presentation known as weil’s syndrome consists of the triad of haemorrhage, jaundice, and aki.1,3,5 incidence of severe leptopirosis estimated 5% to 15% of patients.7symptoms that occur in our patients fulfil the severe symptoms. thrombocytopenia is common in leptospirosis, which suggested a mechanism caused by peripheral platelet consumption due to widespread haemorrhages, immunemediated platelet destruction caused by antiplatelet antibodies, and inhibited platelet production by bone marrow.8 it aggravates hemorrhagic manifestation, as does installation access for dialysis if needed. therefore, transfusion of thrombocyte concentrate was given as a preventative strategy.9septic shock occurs because of severe infection from leptospira which causes vasculitis and systemic inflammatory response syndrome.10 it could develop into an immunosuppressive state as it evolves until the death of the host.11 early administration of the vasoactive drug norepinephrine is beneficial in restoring organ perfusion in septic shock patients.12 treatment of leptospirosis consists of antibiotics and supportive therapy. antibiotics chosen are penicillin group or cephalosporin such as ceftriaxone that was given to our patient. leptospira are highly susceptible to a broad range of antibiotics. a jarischherxheimer reaction may occur as a response to the clearance of spirochetes from the circulation. it is an acute inflammatory response characterized by fever, rigors, and hypotension with a 21% incidence according to guerrier et al which is not found in this report.13,14 supportive therapies are based on clinical manifestation with renal replacement therapy, ventilatory support, and blood products. a study in brazil shows that leptospirosis patients with complications of acute respiratory distress syndrome and aki benefit from daily hemodialysis to lower the mortality rate.15 while the starrt-aki (standard versus accelerated initiation of renal-replacement therapy in acute kidney injury) investigation concluded that among critically ill patients with aki, an accelerated renal-replacement strategy within 12 hours was not associated with a lower risk of death than the standard strategy.4 this study supports the present case in which dialysis, as renal replacement therapy, was not given to the patient and, as a result of clinical laboratories, does improve with aggressive 16 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license samuel halim, et al. severe leptospirosis (weil’s disease) fluid therapy alone. the choice made was risky yet convenient and promising as for the patient condition and psychology that he did not need dialysis. strengths and limitations the strength of this study were the detail information given from patient history prior medication to condition and treatment given until discharge. the limitation of this study were treatment decision are based on physician experience and patient profile therefore not always applicable in every cases. conclusions overall, diagnosis and treatment of leptospirosis are challenging. typical infection symptoms of fever, when followed by icteric, hemorrhagic, and aki, should be asked for further anamnesis of contact with rodent or other leptospirosis risks to ensure the diagnosis. treatment given for leptospirosis is mainly antibiotics and close monitoring. in contrast, other supportive care, such as renal replacement therapy, is not routinely needed because renal failure will recover itself as the infection diminishes. acknowledgement we are grateful to all medical workers and nurses in hermina hospital kemayoran for their support and facilities in managing the patient. we would like to thank the director hermina hospital kemayoran for allowing us to publish our findings. funding this study did not receive any funding. conflict of interest the authors declare that they have no conflict of interest in this report. author contribution conceptualization and supervision: sh. data curation, writing-original draft, review, and editing: bah. references 1. wang s, stobart gallagher ma, dunn n. leptospirosis. in: statpearls [internet]. treasure island (fl): statpearls publishing; 2022 [cited 2022 sep 7]. available from: http://www.ncbi.nlm.nih.gov/books/nbk441858/ 2. leptospirosis prevention and control in indonesia [internet]. [cited 2022 sep 9]. available from: https://www.who.int/indonesia/news/detail/2408-2020-leptospirosis-prevention-and-controlin-indonesia 3. jameson, fauci, kasper, hauser, longo, loscalzo. harrison’s priciples of internal medicine. 20th edition. 1290–1295 p. 4. timing of initiation of renal-replacement therapy in acute kidney injury. new england journal of medicine. 2020 jul 16;383(3):240–51. 5. leptospirosis | cdc [internet]. 2019 [cited 2022 sep 21]. available from: https://www.cdc.gov/leptospirosis/index.html 6. gasem mh, hadi u, alisjahbana b, tjitra e, hapsari mmdeah, lestari es, et al. leptospirosis in indonesia: diagnostic challenges associated with atypical clinical manifestations and limited laboratory capacity. bmc infectious diseases. 2020 feb 27;20(1):179. 7. duarte-neto an, croda j, pagliari c, soriano fg, nicodemo ac, duarte mis. severe leptospirosis features in the spleen indicate cellular immunosuppression similar to that found in septic shock. frontiers in immunology [internet]. 2019 [cited 2022 sep 24];10. available from: https://www.frontiersin.org/articles/10.3389/fim mu.2019.00920 8. lippi g, favaloro ej, buoro s. platelet transfusion thresholds: how low can we go in respect to platelet counting? semin thromb hemost. 2020 apr;46(3):238–44. 9. lie kc, lau cy, van vinh chau n, west te, limmathurotsakul d, sudarmono p, et al. utility of sofa score, management and outcomes of sepsis in southeast asia: a multinational multicenter prospective observational study. j intensive care. 2018 feb 14;6(1):9. 10. yilmaz h, turhan v, yasar kk, hatipoglu m, sunbul m, leblebicioglu h. characteristics of leptospirosis with systemic inflammatory response syndrome: a multicenter study. ann clin microbiol antimicrob. 2015 dec 21;14:54. 17 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 12–17 11. el hasbani g, farooqui sr, kofahi a, saeed y, tayeh o, abu-hishmeh m, et al. unusual presentation of urban leptospirosis complicated by a septic shock. idcases. 2019 jun 13;17:e00574. 12. hamzaoui o, scheeren twl, teboul jl. norepinephrine in septic shock: when and how much? current opinion in critical care. 2017 aug;23(4):342–7. 13. guerrier g, lefèvre p, chouvin c, d’ortenzio e. jarisch-herxheimer reaction among patients with leptospirosis: incidence and risk factors. am j trop med hyg. 2017 apr;96(4):791–4. 14. guerrier g, d’ortenzio e. the jarischherxheimer reaction in leptospirosis: a systematic review. plos one. 2013;8(3):e59266. 15. goarant c. leptospirosis: risk factors and management challenges in developing countries. research and reports in tropical medicine. 2016 dec 31;7:49–62. 152 vol. 5. no. 6 september–december 2015 an appropriate diagnosis of dengue virus infection in some cases who had and were being treated in soerya hospital sepanjang – indonesia soegeng soegijanto1, desiana ws, dyah wikanesthi, eva chilvia2, oedojo soedirham3 1 head of dengue team institute tropical disease, airlangga university surabaya 2 medical residents of soerya hospital 3 senior lecturer of faculty of public health, airlangga university abstract since january 2014, soerya hospital has found many cases with positive result of ns1 or igm and igg dengue. the clinical manifestations mostly were high fever with headache, vomiting and also malaise convulsion and unconsciousness. aim of the study is to find out an appropriate diagnosis of dengue virus infection. observasional study had been done since january–april 2014 with 50 cases of dengue virus infection. the diagnostic procedure was made based on the who 2011 criteria. result many cases had come with fever within couple days, some of them showed convulsions. therefore, it should be made a differential diagnosis with other disease, such as acute tonsilopharingitis, etc. the patient also had to be tested with ns1 if the patient come in the first, second and third day of fever and followed by igm/igg dengue on the fourth, fifth or sixth days of fever. the diagnosis of dengue virus infection was made based on the who criteria 2011. this study showed that not all cases showed positive result of ns1 or igm/igg dengue on the first or second test. for the negative result, we should not think that the case is not a case of dengue virus infection, especially if it happens at aedes aegypti breeding season, the patient should be observed and performed the test again to get a proper diagnosis for dengue virus infection. monitoring clinical manifestation should always be done, to predict the appropriate diagnosis of dengue virus infection. key words: dengue virus, diagnosis dengue virus ns1, igm test, igg test, who criteria abstrak sejak januari 2014, rumah sakit soerya telah menemukan banyak kasus dengan hasil positif dari ns1 atau igm dan igg dengue. manifestasi klinis sebagian besar adalah demam tinggi dengan sakit kepala, muntah dan juga kejang, lemas. tujuan penelitian untuk mengetahui diagnosis yang tepat dengue virus infeksi. materi dan metode penelitian observasional telah dilakukan sejak januari– april 2014 dengan 50 kasus infeksi virus dengue. prosedur diagnostik dibuat berdasarkan kriteria who 2011. hasil yang ditemukan diantaranya demam dalam beberapa hari, beberapa dari mereka menunjukkan kejang. oleh karena itu, harus dibuat diagnosis diferensial dengan penyakit lain, seperti tonsilopharingitis akut, dll. pasien juga harus diuji dengan ns1 jika pasien datang pertama, kedua dan ketiga hari demam dan diikuti oleh igm/igg dengue pada hari keempat, kelima atau keenam demam. diagnosis virus dengue infeksi dibuat berdasarkan kriteria who 2011. studi ini menunjukkan bahwa tidak semua kasus menunjukkan hasil positif dari test ns1 atau igm/igg dengue pada pertama atau kedua menunjukkan hasil negatif, kita tidak harus berpikir bahwa kasus ini bukan kasus dengue virus infeksi, terutama jika hal itu terjadi pada musim nyamuk aedes aegypti, pasien harus diamati dan dilakukan tes lagi untuk mendapatkan diagnosa yang tepat untuk dengue infection. kesimpulannya pemantauan virus berdasarkan manifestasi klinis harus selalu dilakukan, untuk memprediksi diagnosis yang tepat dengue virus infeksi. kata kunci: dengue virus, diagnosis virus dengue ns1, tes igm, tes igg, kriteria who research report 153soegijanto, et al.: an appropriate diagnosis of dengue virus infection in some cases introduction dengue fever and severe dengue infection an important causes of morbidity in tropical and sub tropical region. most half world population live in area at risk infection.1,2 one step dengue ns1 antigen test is a highly conserved glycoprotein that seems to be essential for virus viability, but has no established biological activity, this ns1 antigen is present, at high concentration in the sera of dengue virus infection patients during the early clinical phase of the disease so it could be used as a suitable marker of dengue virus infection.3,4,5 since january 2014, soerya hospital has found many cases with positive result of ns1 or igm and igg dengue. the clinical manifestations mostly were high fever with headache, vomiting and also malaise convulsion and unconsciousness. pathogeneis of dhf and dss is still controversial. two theories, which are not mutual exlusive-were frequently invited to explain the pathogenetic changes; secondary infection or immune enchacement hypothesis, viral virulence theory. both theory is supported by epidemiologic and laboratory evidence, are most probably valid. risk factor reported for dhf; virus strain, pre-existing anti-dengue antibody: previous infection, maternal antibodies in infant, host genetics, age, higher risk in secondary infections, higher risk in locations with two or more serotypes circulating simultaneously at high levels (hyperendemic transmission). diagnosis dengue nsi ag as rapid test is an in vitro immunochromatographic, one step assay designed to detect dengue virus ns1 antigen human serum, plasma or whole blood.11,12,13 diagnosis early acute dengue infection to detect nsi antigen. dengue nsi ag can be detected from day 1 after on set of fever.14 sensitivity-92.8%, spesificity98.4%. the speciment: serum, plasma an wholeblood (100 μl).9,10 test result: 15–20 minutes. the introduction of few device model: fully covered device. diagnosis dengue igg/igm test is a solid phase in vitro immunochromatographic test for the qualitative and differential detection of igg and igm antibodies to dengue virus serotype den-1,2,3 and 4. differential detection of igg and igm antibodies. serum, plasma, and whole blood. test result: 3-lines (igg, igm, control). highest accuracy in low titer specimen.15 presumptive differentiation between primary and secondary dengue infection have good correlation with haemagglutination-inhibition (hi) test.11 material and methods to make a diagnosis a cases, the doctor showed ask to family who brought the patient to the hospital for getting history why the patient suffer for illness. what is the reason? based on his answer or her history: the doctor in charge should make a plan the laboratory examination with can support the diagnosis. what kind laboratory should be done? laboratary examination was done based on clinical manifestation that had been found. for doing laboratory examination the doctor in charge should know the clinical manifestation of cases based on answer of the question. therefore the doctor incharge had found a sign an symptoms of dengue virus infection (dvi). observasional study had been done since january–april 2014. there were 50 cases of dengue virus infection had been studied. the diagnostic procedure was made based on the who 2011 criteria. sample collection and diagnosis of dengue. the patient came early had to be tested with ns1 if the patient come in the first, second and third day of fever and followed by igm/igg dengue on the fourth, fifth or sixth days of fever. the diagnosis of dengue virus infection was made based on the who 2011 criteria. the patient came late (4, 5, 6 dengue of fiver) should be test igm igg dengue. result and discussion 10 cases who came early (1, 2, 3 dengue of fiver) showed positip ns1 test and the other 7 cases also came early but showed negatif ns1 test. see table 1. and then 7 cases who had a negatif result ns1, were followed igm igg test on the fourth until sixth day fever. table 1. ns1 test as early diagnosis in suspected dvi who had come earlier in soerya hospital sepanjang day of fever ns1 test examination total cases + – first day 1 1 second day 5 3 8 third day 5 3 8 total cases 10 7 17 table 2. igm/igg/igm & igg rapid test followed negatif test ns1 that had been done in earlier cases dengue virus infection day of fever dengue test examination total cases igm (+) igm & igg(+) fourth day 1 2 3 fifth day 3 3 sixth day 1 1 total cases 5 2 7 154 indonesian journal of tropical and infectious disease, vol. 5. no. 6 september–december 2015: 152-155 the result there were 5 cases showed an igm (+), 2 cases showed igm igg positip. see table 2. there were 33 cases suspected dengue virus infection came late: all of them had been identified igm igg dengue test. the result 9 cases should igm (+), 2 cases igg (+), 12 cases igm igg dengue (+). see table 3. buy doing faster test of igm igg an all suspected dengue cases with day of fever 4, 5, 6,7 could be identified as a true cases of dengue virus infection. dengue virus ns1 antigen was detected in 199 of 213 acute serum samples from patients with laboratory confirmation of acute dengue virus infection. the dengue ns1 antigen – capture elisa sensitivity of 93.4%, specificity of 100%. the sensitivity was significant higher in acute primary dengue (97.3%) than in acute secondary dengue (70.0%). the positive predictive value the dengue ns1 antigen –capture elisa 100%. negative predictive value was 97.3%. virus isolation gave on overall positive isolation rate 68.1% with a. positive isolation rate 73.9 for acute primary dengue and 31.0% acute secondary dengue. molecular detection of dengue rna by rt-pcr gave on overall positive detection rate 66.7%. detection rate of 65.2 for acute primary dengue and 75.9% for acute secondary dengue.14 w e h a v e f o u n d t h a t n s 1 s e r o t y p e s p e c i f i c immunoglobulin g (igg) enzyme linked immunosorbent assay (elisa) can be used differentiate primary and secondary dengue virus infection.4,6 this is due to the fact that the ns1 specific igg antibody cannot be detected before day 6 of illness for primary infection. so the ns1specific igg antibody measure in acute phase serum some of them as previous infection. comparison of ns1 specific igg elisa with envelope-and membrane-specific capture igm and igg elisa in the differentiation of primary and secondary dengue virus infection showed correlation (95,90% agreement). most important we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identifitied when convalescent-phase or postinfection sera were analyzed by ns1 serotype-specific igg elisa. these findings suggested the ns1 serotype-specific igg elisa could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection.7,8,15 50 cases of suspected dengue virus infection who had been admitted in soerya hospital sepanjang sidoarjo and had been collected since january 1–april 30, 2014 had been studied. they had come with clinical manifestation of fever, vomitting, convulsion, head ache and gastric pain. and than two groups of cases suspected dengue virus infection had been made, as: 1. first) who had come on the first, second and third of fever and 2. second) who had come on the fourth, fifth and sixth of fever. ns1 test had been done in the first group cases of dengue virus infection and the result showed on table 1. there were five cases who had shown a clinical manifestation on the second days of fever and had been identified as a positive result of ns1 test. these event were also found on the following five cases that had a clinical manifestation on the third days of fever. the result showed that there were 10 cases who came early had shown as a positive result of ns 1 test but the others 7 cases who came early had shown as a negative result of ns 1 test. it mean that all cases of suspected dengue virus infection who had early come in hospital had been test by ns 1, not always shown totally had a positive result but only 58,8% showed positive result. these negative result cases should be observed and followed on the next day for getting igm, igg and igm & igg test, the result had been showed on table 2. all of them were positive. this experience give an idea that: if we found some cases which have been identified as the true a suspected clinical manifestation of dengue virus infection, we should try to follow the clinical manifestation and try to do the other test dengue related with occurring antibody. for some cases who came late more than 3 days of fever we should test igm & igg dengue. the result were showed on table 3. there were 19 cases positive only igm, it mean that all cases has been suffered from primary infection of dengue virus. all of them showed a mild clinical manifestation and didn’t show plasma leakage and shock. 2 cases showed a positive igg and 12 cases showed a positive igm & igg; it mean that all cases had been suffered from secondary infection dengue; these cases showed severity of clinical manifestation of dengue hemorrhage fever. it was due to enhancement ag ab reaction that promoting increasing plasma leakage and shock. in some cases this event occurred and showed a clinical manifestation of plasma leakage and promoting shock and need a special treatment. conclusion monitoring clinical manifestation should always be done, to predict the appropriate diagnosis of dengue virus infection for making a good management of df or dhf and dss. table 3. igm/igg/igm & igg rapid test for identification dengue virus infection in who had came late to the hospital day of fever dengue test examination total igm igg igm & igg fourth 8 2 6 16 fifth 6 3 9 sixth 2 2 4 seven 3 1 4 total cases 9 2 12 33 155soegijanto, et al.: an appropriate diagnosis of dengue virus infection in some cases literature 1. world health organization. geneva, switzerland: who; 2010. working to overcome the global impact of neglected tropical diseases. first who report on neglected tropical diseases. 2. bandyopadhyay s, lum lc, kroeger a. classifying dengue: a review of the difficulties in using the who case classification for dengue haemorrhagic fever. trop med int health. 2006; 11: 1238–55. [pubmed]. 3. world health organization. dengue and dengue haemorrhagic fever. fact sheet 117, 2009 [cited 28 november 2011] available from: www.who.int/mediacentre/factsheets/fs117/en/ 4. world health organization. geneva, switzerland: who. 2009. dengue: guidelines for diagnosis, treatment, prevention and control. 5. avirutnan p, punyadee n, noisakran s, komoltri c, thiemmeca s, auethavornanan c, 2006. vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein ns1 and complement. j. of infect. dis. 193: 1078–1088. 6. darmowandowo w, faizi m, 2001. identifikasi jenis infeksi primer dan sekunder melalui penetapan rasio igm & igg pada penderita demam berdarah dengue 2001. tdc unair, h. 12–26. 7. departemen kesehatan republik indonesia, 2003. pencegahan dan penanggulangan penyakit demam dengue dan demam berdarah dengue. terjemahan dari who regional publication searo no. 29: prevention and control of dengue haemorrhagic fever. kerja sama who dan dep.kes. ri, jakarta. 8. dins kesehatan propinsi jawa timur 2007. situasi penyakit dbd propinsi jatim & kebijakan program p2 dbd. dinas kesehatan propinsi jawa timur, surabaya. 9. dussart p, labeau b, lagathu g, louis p, nunes mrt, rodriques sg, storck-hermann c, cesaire r, morvan j, flamand m, baril l, 2006. evaluation of an enzyme immunoassay for detection of dengue virus ns1 antigen in human serum. clinical and vaccine immunology, 13(11): 1185–1189. 10. dussart p, petit l, labeau b, bremand l, leduc a, moua d, matheus s, baril l, 2008. evaluation of two new commercial tests for the diagnosis of acute dengue virus infection using ns1 antigen detection in human serum. plos negl. trop. dis. 2(8): 57–61. 11. faizi m, 1998. validitas ratio igm/igg sebagai pembeda infeksi primer dan sekunder pada penderita demam berdarah dengue. penelitian karya ilmiah akhir untuk dokter spesialis 1 ilmu kesehatan masyarakat fakultas kedokteran universitas airlangga/ rsud. dr. soetomo, surabaya. 12. flamand m, megret f, mathieu m, lepault j, rey f, deudel v, 1999. dengue virus type 1 nonstructural glycoprotein ns1 is secreted form mammalian cells as a solube hexamer in glycosylationdependent fashion. j of virol, 73(7): 6104–6110. 13. flammand m, alcon – lepoder, drouet mt, sivard p, 2005. detection of ns1 from dengue virus: basis for early diagnosis and aprognostic marker of disease progression (2). virologi departement, pasteur institute, paris, france, p. 43. 14. v.kumarasamy a.h, abdul wahaps.k,chua,z.hassan. 2007. evaluation of a commercial dengue ns1 antigen-capture elisa for laboratory diagnosis of acute dengue virus infection. journal of virology method, volume 140, issue 1-2, march 2007, pages 75–79. 15. pei yun shu, cheng-li kuang, chang fen shi.2003.comparison of capture immunoglobulin m (igm) and igg enzyme-linked immunosorbent assay (elisa) and nonstructural protein ns1 serotype – spesific igg elisa for differentiation of primary and secondary dengue virus infections. received 13 january 200., returned for modification 17 march 2003/accepted 1 april 2003. vol. 10 no. 1 january–april 2022 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ * corresponding author: ingelusida@itd.unair.ac.id review article human norovirus molecular analysis and development of norovirus vaccine adinda juwita syakila elizafanti1, maria inge lusida2,3*, muhammad miftahussurur3,4, alpha fardah athiyyah5 1faculty of medicine universitas airlangga, surabaya, indonesia 2department of medical microbiology, faculty of medicine universitas airlangga, surabaya, indonesia 3institute of tropical disease, universitas airlangga, surabaya, indonesia 4gastroenterology and hepatology division, department of internal medicine, faculty of medicine dr. soetomo teaching hospital, universitas airlangga, surabaya, indonesia 5department of pediatry, faculty of medicine universitas airlangga/dr. soetomo general academic hospital surabaya, indonesia received: 5th december 2021; revised: 16th december 2021; accepted: 4th january 2022 abstract the most common organism of acute viral gastroenteritis is norovirus, which accounts for roughly 20% of all occurrences of acute gastroenteritis globally. the virus kills over 200,000 children each year and is the leading cause of childhood diarrhea in the rotavirus-vaccinated population. this study aims to review available studies regarding the information on the genogroup norovirus in humans, development of norovirus vaccines, and effectiveness of norovirus vaccines. a systematic review using science direct, pubmed, and scopus databases to identify eligible case studies. the search was conducted in september-october 2021. the quality of the included literature used checklists from the critical appraisal skills program (casp). all of the six selected studies with populations given rt-pcr intervention showed positive for norovirus infection. the most predominant genogroups in humans are gi and gii. as for the research results of the two selected studies on norovirus vaccine, namely the human phase 2 trial containing two virus-like particles (vlp) genotypes, one study showed efficacy at 18-49 one study at ≥ 60 years of age. this study analysis uses takeda bivalent vaccine. the vaccine includes norovirus antigens of the gi and gii genogroups, intending to expand its protective immune potential. gi, gii, and giv genogroups are prevalent in humans. vlp that contains gi.i and consensus gii.4c have been created as the nov vaccine, providing signifi cant efficacy. very likely because they contain gi dan gii antigens, which are the genogroups that infect humans the most. patients given a placebo developed acute gastroenteritis due to norovirus gii.2, indicating a genotype cross-reactivity. keywords: norovirus, genogroups, vaccine, human, systematic abstrak norovirus adalah agen etiologi paling umum dari gastroenteritis akut yang menyebabkan sekitar 20% dari seluruh kasus gastroenteritis akut yang terjadi secara global. virus ini menyebabkan sekitar 200.000 kematian anak setiap tahun, dan sekarang menjadi penyebab paling umum dari diare anak pada populasi yang divaksinasi rotavirus. tujuan penelitian ini adalah untuk meninjau studi yang tersedia mengenai informasi tentang genogroup norovirus pada manusia, pengembangan vaksin norovirus, dan efektivitas vaksin norovirus. systematic review menggunakan database science direct, pubmmd dan scopus untuk mengidentifi kasi studi kasus yang memenuhi kriteria. pencarian dilakukan pada bulan september-oktober 2021. kualitas literatur yang disertakan dinilai menggunakan checklist dari critical appraisal skills program (casp). keenam studi terpilih dengan populasi yang diberikan intervensi rt-pcr menunjukkan positif terinfeksi norovirus. genogroup yang paling dominan pada manusia adalah gi dan gii. adapun hasil penelitian dari dua studi terpilih pada vaksin norovirus, yaitu uji coba fase 2 pada manusia yang mengandung dua genotipe virus-like particles (vlp), satu studi menunjukkan efi kasi pada usia 18-49 tahun dan satu studi pada usia ≥ 60 tahun. analisis studi ini menggunakan takeda bivalent vaccine. vaksin ini mengandung antigen ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 9adinda juwita syakila elizafanti, et al.: human norovirus molecular analysis and development norovirus genogroup gi dan gii, yang bertujuan untuk memperluas potensi imun protektifnya. genogroup gi, gii, dan giv banyak terdapat pada manusia. vlp yang mengandung gi.i dan konsensus gii.4c telah dibuat sebagai vaksin nov dan memberikan efi kasi yang signifi kan. hal ini sangat memungkinkan karena mengandung antigen gi dan gii yang merupakan genogroup yang paling banyak menginfeksi manusia. pasien yang diberi plasebo mengalami gastroenteritis akut akibat norovirus gii.2, yang menunjukkan adanya reaktivitas silang antar genotype. kata kunci: norovirus, genogroup, vaksin, manusia, systematic how to cite: elizafanti, a. j. s., lusida, m. i., miftahussurur, m., athiyyah, a. f. human norovirus molecular analysis and development of norovirus vaccine. indonesian journal of tropical and infectious disease, 10(1), p. 8–17, apr. 2022. asymptomatic norovirus is estimated to be 7%, with africa, mesoamerica, and south america having a high incidence (11-15%). however, europe and north america are still having a low prevalence.7 people of all ages can be infected with norovirus, with the elderly and youngsters being most vulnerable.8 norovirus is the second most frequent source of death by diarrhea for children under the age of five across the region of the world health organization.9 norovirus outbreaks are prevalent in nursing homes, daycare facilities, and hospitals.10 norovirus is highly contagious and pervasive.11 various factors that could aff ect the increase of norovirus transmission are the minimal amount of inoculum required to cause infection (100 particles of the viral agents), prolonged discharge of the viral agent, and its survivability in the environment.12 this virus plays a significant role in the food-borne epidemic.13 efforts are currently underway to develop an effective norovirus vaccine using virus-like particles (vlp), which is regarded as a promising approach to administering cases of norovirus infection.10 identification from the study of vlp bivalent vaccine usage on animals and human suggest that multivalent vaccination may be an eff ective strategy for inducing a broadly neutralizing antibody protective against challenge with the latest and heterologous norovirus strain.14 an efficient norovirus vaccine can lower direct influence on gastroenteritis and have indirect socioeconomic costs. the relationship between protective immunity and norovirus infection must be further explored to permit more acceptability and efficacy of norovirus vaccine candidates in humans.8 therefore, this study was introduction norovirus is a ribonucleic acid virus (rna) discovered in 1970 with 27 nm diameter, single strand, and no veil. they belong to the family caliciviridae.1 norovirus is a virus that may cause outbreaks of severe gastroenteritis with the main symptom of diarrhea, which appeared after the rotavirus vaccine was discovered. norovirus was found positive in 64 (19%) samples of 340 stools of children with a mean age of 11–12 months (11.75 months). it was generally found in patients suffering from diarrhea under 24 months (95%), with 64% of sufferers male in a study in indonesia. infection from norovirus is most common in november, followed by may and april in 2020 because, in that month, it is a rainy season in indonesia. important factors that play a role in transmitting norovirus are raining. rainwater changes the norovirus viral load, and thus, norovirus transmission is easier.2 clinical attributes of norovirus infection consist of asymptomatic and symptomatic infections.3 clinical symptoms of infection are fever (72%), bloating (59%), vomiting (66%), abdominal colic (34%), anal fistula (27%), seizures (8%), and abdominal distension (16%).2 natural infection increases in children during the first two years of life.4 noroviruses are varied in genetic and antigenic 6 6 kim performed a study and found that of the 230 genotyped norovirus strains, gii.4 (77.3%) was the most prevalent capsid genotypes, followed properties.5 one of the epidemics caused by norovirus was the outbreak in korea in 2013. by gii.3 (6.1%) and gii.13 (3.9%). according to a meta-analysis study, the global prevalence of ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 10 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 8–17 conducted to obtain information related to the molecular analysis of norovirus in humans and the development of the norovirus vaccine. methods the method used in this study is a systematic review, which focuses on searching comprehensive and detailed data on several relevant works of literature. it aims to reduce biased information by identifying, assessing, and synthesizing all studies relevant to related topics.15 the following criteria are used to consider the study for this review: literature that discusses genogroup of norovirus and that of norovirus vaccine, published in 2017-2021, types of literature research articles, and randomized controlled trial (rct) with full text in english. while the exclusion criteria in this review are works of literature on norovirus genogroup other than in humans and those on norovirus vaccines tested besides humans. finding strategy literature was carried out using the boolean operator in or for literature search with alternative keywords. thus, broader literature coverage and and for a complete literature search keyword. the keywords used are entered together in the database using the advanced search. the literature search strategy is carried out with the following two keywords of (norovirus or nov or norwalk like viruses) and gastroenteritis and human and (molecular or rt-pcr) and (norovirus or nov or norwalk like viruses) and gastroenteritis and vaccine and (efficacy or immunogenicity). study selection articles considered potentially eligible for inclusion criteria are selected based on the abstract with pico criteria. it consists of norovirus patients as a population, molecular development in detection with rt-pcr and the provision of norovirus vaccines as interventions, norovirus identification with analysis (sequencing, phylogenetic analysis, rna extraction) and placebo administration as a comparison, norovirus genogroup and vaccine effectiveness norovirus as an outcome. then assessed for quality, literature quality was assessed using the critical appraisal skills program (casp). the collected data is then managed by applying the preferred reporting items for systematic review and meta-analysis (prisma). fundamental literature research obtained will be carried out for identification, screening, eligibility, and include. data extraction this data extraction is done by transferring important information from the literature figures selected into the data collection form. the data are modified from cochrane (the cochrane library). the cochrane data collection form contains the identity, characteristics, methods, and research results to make it easier for researchers to analyze the writing reviewed for further presentation in a summary table, thereby making it easier for writers and readers to understand the results. results and discussion norovirus in humans the search results acquired 3,114 articles from science direct, 662 from pubmed, and 3,237 from scopus. the strategy used to filter research articles is with boolean logic with the keyword. furthermore, it was screened using an advanced filter, and the results of the obtained screening of 1,256 works of literature with 17 duplicated literature excluded so that 1,239 works of literature are obtained. the literature was screened by reading titles and abstracts to find 1,228 pieces of literature that do not meet the pico and sample criteria. after screening, 11 appropriate pieces of literature were obtained to be studied in this systematic review. after further study throughout the literature, five studies were excluded because: not only focusing on norovirus and discussing other enteric viruses (n=3), discussing other norovirus strains besides gi, gii, and giv (n=2). so that the results of the literature screening are six pieces of literature that meet the criteria to be studied in this systematic review (figure 1). ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 11adinda juwita syakila elizafanti, et al.: human norovirus molecular analysis and development , , , finding on sciences direct (n=3.114) finding on pubmed (n=662) finding scopus (n=3.237) total number 7.013 screening criteria : year of publication 2017-2021 full text type of research articles and rct screening= 1.256 same literature (n=17) not in english(n=14) the literature type is review article (n=283) literature which not discuss norovirus in medical term (n=246) the population is not norovirus infected patients (n=315) not intervened detection with rt-pcr, only stated in the literature (n=164) not discuss norovirus (n=206) full-text literature by identified topic (n=11) not only focus on norovirus and discuss other enteric viruses (n=3) discuss other norovirus strains that are not on humans (n=2) literature included in this study (n=6) figure 1. diagram of preferred reporting items for systematic review and meta-analysis (prisma) molecular norovirus table 1. analysis of molecular norovirus no. author title background methods time participants inclusion criteria exclusion criteria variables variables bound research findings 1 toren et al., 202116 risk factors for norovirus infection in healthcare workers during nosocomial outbreaks: a crosssectional study, gothenburg, sweden norovirus detection by gii specifi c rt-pcr with a cross-sectional study januaryapril 2012 308 participants health workers in the ward infected with norovirus at sahlgrenska university hospital cleaners are not employed by the hospital fecal samples strains of norovirus a total of 26 out of 129 patients were positive for gii norovirus 2 john et al., 202117 high proportion of norovirus infection and predominance of gii.3[p12] genotype among the children younger than 5 in sabah, malaysian borneo sabah, malaysia rt-pcr assay with amplifi cation of c of the capsid region january 2018– march 2019 299 participants pediatric patient under 5 years old with acute gastroenteritis admitted to hospital sabah, malaysia children aged >5 years old, not suff ered diarrhea or fever fecal samples strains of norovirus a total of 17.7% of patients were positive for norovirus infection, the majority were caused by the genotypes gii (71,7%), then gi (24,5%), and the combination of gii and gi (3,8%) 3 cao et al., 202118 epidemiology of norovirus gastroenteritis in hospitalized children under fi ve years old in western china, 20152019 chengdu, china rt-pcr assay with nucleic acid norovirus kit using applied biosystems 7500 2015-2019 1181 participants pediatric patients under 5 years old with acute gastroenteritis who are hospitalized at chengdu hospital, china children aged >5 years old, did not experience acute gastroenteritis fecal samples strains of norovirus 20% of patients were infected with norovirus. most of the samples came from genotypes gii, id est gii.4 sydney 2012 and gii.3 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 12 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 8–17 no. author title background methods time participants inclusion criteria exclusion criteria variables variables bound research findings 4 utsumi et al., 202120 molecular epidemiology and genetic diversity of norovirus infection in children hospitalized with acute gastroenteritis in east java, indonesia in 2015-2019 east java, indonesia conventional taqman rt-pcr based uses applied biosystems 7300 june 2015-july 2019 966 participants pediatric patients aged 1-191 months with acute gastroenteritis that hospitalized in the east java hospital region, indonesia children aged>191 months, no age fecal samples strains of norovirus a total of 12.3% of samples were detected as positive for norovirus. the predominant genotypes in each year were gii.13 in 2015, gii.4 sydney in 2016, gii.3 in 2017, and gii.4 sydney in 2018 5 shen et al., 202021 molecular epidemiology of norovirus associated with acute gastroenteritis in taizhou, china: a retrospective study taizhou, china multiplex rt-pcr with the applied biosystems 7500 using agpathid one step rt-pcr kit january 2016december 2017 1464 participants patient of acute gastroenteric, episodic diarrhea and vomiting is in emergency hospital that handle diarrhea no age fecal samples strains of norovirus 9.49% of samples of patients with acute gastroenteritis were positive for norovirus. gii was the main genotype. a total of 12 genotypes and seven recombinant strains were found in the study. 6 bonura et al., 202119 recombinant gii.p16 genotype challenges rt-pcr based typing in region a of norovirus genome palermo, italy qiaamp viral rna extraction kit and rt-pcr assay using two sets of modifi ed gii primers (deg 1 and deg 2) january 2016– december 2019 2194 participants children under 5 years old being treated for acute gastroenteritis at the children hospital in palermo, italy no age children aged>5 years old rna extracted from fecal samples strains of norovirus norovirus gii identifi ed the most between 2016-2019 were gii.2 and gii.4 sydney the study characteristics of molecular norovirus studies used in this literature review came from five different regions, each from sweden, malaysia, china, indonesia, and italy. as shown in table 1, the population used in the study involved health workers in hospitals in one study16, pediatric patients aged under five years with acute gastroenteritis in three studies17–19, children were suffering from severe gastroenteritis in private and government hospitals in surabaya, east java in one study20, acute gastroenteritis patients in southeast china in one study.21 the intervention used in six references is rectal samples detected for norovirus by specific rt-pcr16, rt-pcr assay with amplification of region c17, rt-pcr assay with norovirus nucleic acid kit using applied biosystems 750018, rt-pcr assay with qiaamp viral rna kit19, taqman test based on conventional rt-pcr using the applied biosystems 7300 system20, multiplex rt-pcr assay with applied biosystems 7500 using agpath-id one step kit.21 two studies stated that norovirus-positive patients had gii norovirus, two studies with gii and gi genotypes, and two studies with gii, gi, combination gii, and gi genotypes as shown in table 1. individual contact is strongly associated with norovirus infection. health workers in large hospitals, especially psychiatric wards, are highly susceptible to norovirus infection. the main symptom of health workers infected with norovirus is experiencing acute gastroenteritis, and the most common norovirus strain is gii.4.16 the risk of norovirus is higher in children under five years old. it is primarily due to the ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 13adinda juwita syakila elizafanti, et al.: human norovirus molecular analysis and development strain when the capsid and rdrp genotypes are combined. the highest prevalence of the nov strain was gii.3[p12], followed by gii.6[p7] and gii.17[p17] in sabah, malaysia, from 2018 to 2019.17 norovirus of strains gii.17 and gii.2, is one of the most prevalent causes of viral gastroenteritis in children under five in china.18 children under two years of age tend to be more susceptible to norovirus infection. in indonesia, gii norovirus is more connected with disorders that require medical care, whereas gi norovirus causes moderate symptoms and does n o t necessitate hospitalization. indonesia’s norovirus genotypes are genetically varied and similar to those seen in asia and europe.19 the norovirus genotypes were dominated by gii, and a small proportion is gi and the combination of gi and gii. the most common strains found in taizhou, china, are gii.p17/ gii.17, gii.pe/gii.4, and gii.p16/gii.2. the percentage of norovirus infection was 9.49% among all acute gastroenteritis patients of all age groups in taizhou. the incidence of norovirus infection in various age groups is connected to population immunity, public health, and the usage of over-the-counter medications.20 norovirus gii was the most dominant genotype infecting acute gastroenteritis patients in taizhou. the three main norovirus strains that infect patients in the region are gii.p17/gii.17, gii.pe/gii.4 and gii.p16/gii.2.20 the molecular examination is quintessential because the identification of norovirus is generally made by identifying the rna. most norovirus outbreaks globally are caused by strains gii.4, including a new variety of gii.4 that appears every 2 to 3 years. the plasticity of the norovirus genome requires periodic renewal of the primers used for rt-pcr amplification21 (table 1). norovirus vaccines based on the search results, there are 2,717 articles with potential from science direct, 662 from pubmed, and 3,237 from scopus. furthermore, it was screened using an advanced. the results from 135 works of literature were obtained. then, the researchers excluded six duplications of them to obtain 129 works of literature. the literature was re-screened through reading the title and abstract so that two works of literature were obtained that met the criteria to be studied in this systematic review (figure 2). finding in scopus (n=1.327) finding in sciences direct (n=1.205) finding in pubmed (n=365) total number 2.717 screening criteria : year of publication 2017-2021 full text type of research articles and rct screening= 134 literature exclusion (n=132): same literature (n=6) not in english (n=7) the literature type is review article (n=39) the population of not norovirus infected patients (n=43) no placebo control (n=33) tested on animals (n=4) literature included in this study (n=2) figure 2. diagram of preferred reporting items for systematic review and meta-analysis (prisma) norovirus ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 14 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 8–17 study characteristics of norovirus vaccines the studies used in these two literature reviews are from the united states. the populations used in the literature review are one study using a population of naval soldiers aged 18-49 years22, and a study using a population of adult subjects aged ≥ 60 years.23 the two norovirus takeda vaccine (tak-214) studies contained 15 μg gi.1 and 50 μg gii.4c vlps, 0.5 mg al(oh)3].22,23 one study showed vaccine effectiveness, indicated by antibodies that can block genotype specific histo-blood group antigen (hbga), 80% for gi.i/gii.4 homogenotypes, and 61.8% for any genotypes.22 there was no safety concern and similar effectiveness of vaccinees over 60 years of age as those of 18-48 years of age23 (table 2). levels of antibodies that block gi.1 and gii.4c increased in the vaccinated population aged 18-49 years. at the same time, some given a placebo developed acute gastroenteritis due to the gii.2, which means cross-reactivity among genotypes.22 the adult population over 60 years also showed no worrying conditions when given the vaccine, and the response was similar to the vaccine given to the younger population23 (table 2). table 2. analysis of the norovirus vaccine study no author title backg round methods time participants inclusion criteria exclusion criteria variables variables bound research findings 1 sherwo od et al., 202022 effi cacy of an intramuscular bivalent norovirus gi.1/gii.4 virus-like particle vaccine candidate in the healthy us adults illinois, united states randomized controlled phase 2b trial june 2016 – june 2018 4,712 participants: 2,355 vaccinated and 2,357 saline placebo soldier navy united states in illinois aged 18-49 years, healthy have comorbid disease causative genotypes eff ectiveness and immunoge nicity to vaccine level of gi.1 and gii.4c hbgablocking antibodies increased on vaccines and in some placebo age cases infected with gii.2. it shows that there is a genotype crossreactivity 2 treanor et al., 202023 a phase 2 study of the bivalent vlp norovirus vaccine candidate in older adults; impact of mpl adjuvant or a second dose 10 trial centers in the united states randomized controlled phase 2 trial february 2016 – october 2017 294 participants adult subject healthy one same age as or older than 60 years with bmi <35 kg/m2 hypersensitivity to vaccine, showing the presence of fever or infection, clinical and mental disorders in immunosup pressive condition age strata 60-74, 75-84, ≥ 85 years of age; second vaccination; mpl (monophos phoryl lipid a, an adjuvant) immunoge nicity to vaccine adults over 60 years old do not present worrisome conditions when vaccinated and respond similarly to that of younger vaccine, and not aff ected by second vaccination and mpl discussion molecular norovirus in humans norovirus is a positive-sense, single-stranded, non-enveloped rna virus. the norovirus genomes evolve rapidly, resulting in a wide range of genotypes. noroviruses are now divided into ten genogroups (gi-gx) based on the variations in orf2, which encodes the vp1 protein. human infections are caused by the genogroups gi, gii, and giv. this genogroup was further subdivided into nine gi, 27 gii, and two giv genotypes. since recombination in the norovirus genome frequently occurs at the orf1-orf2 junction, each genotype consists of those of capsid and polymerase gene, h currently of a polymerase (rdrp) and a capsid (vp1). based on the rdrp ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 15adinda juwita syakila elizafanti, et al.: human norovirus molecular analysis and development gene sequence, gi and gii viruses are presently divided into 14 and 37 p-genotypes, respectively. the norovirus capsid consists of 90 capsid protein dimers, forming a shell of 90 protruding arch-like dimers.19 different types of cells in the human gut are involved in norovirus infection. the human gut’s predominant cell is a single layer of intestinal epithelial cells (enterocytes), including many immune cells. according to multiple studies, norovirus infects and replicates immune cells such as dendritic cells, b cells, and macrophages. the mechanism is that it enters the host through m cells, which lack microvilli and do not secrete mucous, allowing norovirus to enter the host, attack immune cells, and cause inflammation. the median duration between viral inoculation and clinical manifestations is 1-2 days, and norovirus symptoms usually clear up within 1-3 days. in some cases, symptoms may disappear, and the virus can be excreted in the stool for a prolonged period of up to 60 days.24 nov, specifically genotypes gii.4, evolves quickly through the mutation and recombination events, resulting in the recurrent emergence of new antigenic variants impacted in the global outbreak of acute gastroenteritis.16 according to bonura et al.19, the majority of norovirus outbreaks and sporadic cases worldwide are caused by norovirus gii.4, with new gii.4 variants appearing every 2 to 3 years. the gii.4 variant results from antigenic drift and recombination mechanisms, as evidenced by gii.4 strains epidemics in 2009 and 2012. additional recombinant norovirus strains have emerged in recent years, particularly at the orf1-orf2 junction, including two different gii.4 sydney 2012 recombination viruses, gii.4 sydney 2012[p4 new orleans] and gii.4 sydney 2012[p16], as well as strains gii.2[p16], gii.3[p12], and gii.3[p16]. phylogenetic analysis revealed that the taizhou strains gii.p16/gii.2 shared an evolutionary pattern with the strains discovered in the united states and japan in 2016.20 the gii.p16/gii.2 recombination genotypes, previously reported infrequently in china, were first observed in wuhan province in 2010. this uncommon variant of gii.p16/gii.2 caused a rapid increment in sporadic age patient populations in europe and asia during the winter of 2016–2017. it can emerge as a widespread strain that can trigger an epidemic or pandemic. norovirus vaccine the development of norovirus vaccines is a priority for both public health and economic benefits. however, there are difficulties in vaccine development due to the complex nature of norovirus, human immune response, viral culture, and limited animal models for vaccine testing. several vaccines are presently in pre-clinical progress, one of which has finished phase ii elderly clinical study. because of viral evolution, research has focused on multivalent vaccines similar to influenza vaccines, namely virus like particles (vlp).23 vlp nov is a structure that resembles the original virus organization and conformation but lacks the viral genome, potentially resulting in safer and less expensive vaccine candidates.25 takeda vaccine has developed a bivalent intramuscular norovirus vaccine candidate containing two vlp genotypes, gi.1 and consensus genotypes gii.4c synthesized from three variants, gii.4-2006a (yerseke), 2006b (den haag), and 2002. (houston), intended to provide broad cross-reactivity against various gii.4 strains. clinical trials have shown that the tak-214 candidate formulation is safe, well tolerated, and immunogenic in healthy adults. the ideal adult tak-214 regimen is a single intramuscular dosage based on the development of inhibitory antibodies against histo-blood group antigen (hbga), which are expected to correlate with norovirus illness prevention.22 effective norovirus vaccinations for all age ranges are required to lower the worldwide illness burden of the extremely contagious age norovirus. sherwood et al.22 found that of the 48 cases of moderate or severe age norovirus, 29 cases receiving placebo and 19 cases receiving the vaccine, the causative genotypes are gi.1 (n = 1), g1.7a (n = 1), gii.2 (n = 39) and gii.4 (n = 7). due any to norovirus genotypes, 26 placebo and 10 vaccination groups showed 61.8% ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 16 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 8–17 (95.01 % ci, 20.8 to 81.6; p = 0.0097) vaccine effectiveness. while in vaccine genotypes age cases, the placebo (five cases) vs. vaccination (one case) group yielded a primary endpoint vaccine effectiveness of 80% (99.99 % ci, 1318.1 to 99.7; p = 0.142).22 while treanor et al.23 showed that this vaccine, with or without mpl, with or without second dose, had similar immune responses. it is expected that the development of the norovirus vaccine will continue to reduce the rate of acute gastroenteritis that has spread throughout the world. conclusions the conclusion drawn from the literature review is that the genogroups that infect humans are gi, gii, and giv, of which gi and gii are the most. the bivalent vaccine being developed, tak-214, showed significant efficacy to gi.i and gii.4c vaccine antigens and another genotype (gii.2) indicating a genotype cross-reactivity immunity. our systematic review is based on many studies and includes several countries worldwide, not only indonesia. this study provided a more probable estimate of norovirus vaccines worldwide and methods of diagnosing norovirus infection. this study also provided the details of g.i and g.ii strains infection in humans. the limitation of this study is the lack of articles about the norovirus vaccine in indonesia. we could not assess several studies, and their exclusion may have biased our results. acknowledgement the authors would like to thank sciencedirect website, pubmed website, and scopus website, for the data and resource. conflict of interest we declare that we have no conflict of interest. references 1. weinberg ga. outbreak epidemiology: one of many new frontiers of norovirus biology. j infect dis. 2019;219(9):1349–1352. 2. a t h i y y a h af, wa r d h a n i s, d a r m a a, r a n u h rg, raharjo d, shirakawa t, et al. the clinical epidemiology of norovirus infection in children with diarrhea at regional public hospital dr. soetomo. j berk epidemiol. 2020;8(3):200–7. 3. utsumi t, lusida mi, dinana z, wahyuni rm, yamani ln, juniastuti, et al. occurrence of norovirus infection in an asymptomatic population in indonesia. infect genet evol. 2017;55:1–7. 4. cannon jl, lopman ba, payne dc, vinjé j. birth cohort studies assessing norovirus infection and immunity in young children: a review. clin infect dis. 2019;69(2):357–65. 5. vinjé j. advances in laboratory methods f o r detection and typing of norovirus. j clin microbiol. 2014;53:373–381. 6. kim js, kim hs, hyun j, kim hs, song w. molecular epidemiology of human norovirus in korea in 2013. biomed res int. 2015;2015. 7. qi r, huang yt, liu jw, sun y, sun xf, han hj, et al. global prevalence of asymptomatic norovirus infection: a meta-analysis. eclinicalmedicine. 2018;2–3:50–8. 8. cates j, vinjé j, parashar u, hall a. recent advances in human norovirus research and implications for candidate vaccines. expert rev vaccines. 2020;19(6):539–48. 9. world health organization. introduksi keamanan vaksin [internet]. in.vaccine-safety-training.org. 2021 [cited 2021 dec 20]. available from: https:// in.vaccine-safety-training.org/pre-licensure-vaccine safety.html 10. nordgren j, svensson l. genetic susceptibility to human norovirus infection: an update. viruses. 2019;11(3):226. 11. wulandari ps, juniastuti, wahyuni rm, amin m, yamani ln, qushai m, et al. predominance of norovirus gi.4 from children with acute gastroenteritis in jambi, indonesia, 2019. j med virol. 2020;92(12):3165– 3172. 12. robilotti e, deresinski s, pinsky ba. norovirus. clin microbiol rev. 2015;28(1):134–64. 13. lopman ba, steele d, kirkwood cd, parashar ud. the vast and varied global burden of norovirus: prospects for prevention and control. plos med. 2016;13(4):e1001999. 14. cortes-penfield nw, ramani s, estes mk, atmar rl. prospects and challenges in the development of a norovirus vaccine. clin ther. 2017;39(8):1537– 1549. 15. uman l. systematic reviews and meta-analyses’, j . c a n . a c a d . c h i l d a d o l e s c . p s y c h i a t r y. 2011;20:57–9. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 17adinda juwita syakila elizafanti, et al.: human norovirus molecular analysis and development 16. torén k, schiöler l, nenonen n, hannoun c, roth a, andersson lm, et al. risk factors for norovirus infection in healthcare workers during nosocomial outbreaks: a cross-sectional study. antimicrob resist infect control. 2021;10(1):1–9. 17. john jl, mori d, amit ln, mosiun ak, chin az, ahmed k. high proportion of norovirus infection and predominance of gii. 3 [p12] genotype among the children younger than 5 in sabah, malaysian borneo. j clin virol. 2021;143, 104968. 18. cao r, ma x, li w, wang b, yang y, wang h, et al. epidemiology of norovirus gastroenteritis in hospitalized children under five years old in western china’, 2015–2019,. j microbiol immunol infect. 2021. 19. bonura f, urone n, bonura c, mangiaracina l, filizzolo c, sciortino g, et al. recombinant gii. p16 genotype challenges rt-pcr-based typing in region a of norovirus genome’,. j infect. 2021. 20. utsumi t, lusida m, dinana z, wahyuni r, soegijanto s, soetjipto. molecular epidemiology and genetic diversity of norovirus infection in children hospitalized with acute gastroenteritis in east java, indonesia in 2015–2019. infect genet evol. 2021;88:104703. 21. shen w, sheng y, weng j, li g, wang d, qiu d, et al. molecular epidemiology of norovirus associated with acute gastroenteritis in taizhou, china: a retrospective study. j infect public health. 2020;13(1):34–9. 22. sherwood j, mendelman p, lloyd e, liu m, boslego j, borkowski a, et al. efficacy of an intramuscular bivalent norovirus gi. 1/gii. 4 virus-like particle vaccine candidate in healthy us adults. vaccine. 2020;38(41):6442–9. 23. treanor j, sherwood j, cramer j, le cam bouveret n, lin s, baehner f, et al. a phase 2 study of the bivalent vlp norovirus vaccine candidate in older adults; impact of mpl adjuvant or a second dose. vaccine. 2020;38(36):5842–5850. 24. capece g, gignac e. norovirus. treasure island (fl), editor. statpearls: statpearls publishing; 2021. 25. esposito s, principi n. norovirus vaccine: priorities for future research and development. front immunol. 2020;11:1383. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 141 vol. 6 no. 6 september–december 2017 inhibitory activity of cobalt(ii)–morin complex against the replication of dengue virus type 2 teguh hari sucipto1a, siti churrotin1, harsasi setyawati2, kris cahyo mulyatno3, ilham harlan amarullah1, shuhai ueda4, tomohiro kotaki4, sri sumarsih2, puspa wardhani1, sri subekti bendryman3, aryati1, soegeng soegijanto1, masanori kameoka4 1 dengue study group, institute of tropical disease, universitas airlangga, indonesia 2 department of chemistry, faculty of science and technology, universitas airlangga, indonesia 3 entomology study group, institute of tropical disease, universitas airlangga, indonesia 4 center of infectious disease, kobe university graduate school of medicine, japan a corresponding author: teguhharisucipto@staf.unair.ac.id abstract dengue virus (denv) is a significant pathogen emerging worldwide as a cause of infectious disease. antidengue treatments are urgently required to control the emergence of dengue. denv is a mosquito-borne disease responsible for acute systemic diseases and serious health conditions. denvs were distributed in the tropical and sub-tropical areas and transmitted to humans by aedes agypty and aedes albopictus. dengue vaccine or antiviral has not yet been clinically approved for humans, even though there have been great efforts toward this end. antiviral activity against denv is an important alternative for the characterization and development of drugs. metal–organic compounds were reported to exhibit fungicidal, bactericidal, and antiviral activities its inhibitory activity was not significant, at high concentration it was more toxic to replicating cells than to stationary cell monolayers of vero cells. the aim of this study is to investigate the antiviral effects of cobalt(ii)–morin complex. this compound was further investigated for its inhibitory effect on the replication of denv-2 in vero cells. the replication of denv was measured by enzyme-linked immunosorbent assay and the value of selectivity index (si). si was determined as the ratio of the 50% cytotoxic concentration (cc50) to the 50% inhibitory concentration (ic50). the ic50 value of the cobalt(ii)–morin complex for denv-2 was 3.08 µg/ml, and the cc50 value of the complex for vero cells was 3.36 µg/ml; thus, the si value was 1.09. the results of this study demonstrate the antidengue serotype 2 inhibitory activity of cobalt(ii)–morin complex and its high toxicity in vero cells. further studies are not required before co(ii)–morin can be applied in the treatment of denv-2 infections. keywords: cobalt(ii), morin, complex compound, inhibitory activity, denv-2 abstrak virus dengue (denv) adalah patogen yang muncul secara global pada penyakit menular. pengobatan anti-demam diperlukan untuk mengendalikan demam berdarah. virus dengue (denv) adalah penyakit yang ditularkan melalui nyamuk atas penyakit sistemik akut dan kondisi kesehatan yang memilukan. denvs didistribusikan di daerah tropis dan sub-tropis dan ditransmisikan ke manusia oleh agregat aedes dan aedes albopictus. kini, vaksin dengue atau antivirus untuk manusia tidak disetujui secara klinis, meski telah ada upaya besar untuk mencapai tujuan ini. aktivitas antiviral melawan denv merupakan alternatif penting untuk karakterisasi dan pengembangan obat-obatan. senyawa organik-logam dilaporkan menunjukkan aktivitas fungisida, bakterisida, dan antivirus, aktivitas penghambatannya tidak signifikan, dengan konsentrasi tinggi, lebih beracun untuk mereplikasi sel daripada monolay sel steroid sel vero. tujuan dalam proyek ini adalah investigasi senyawa antiviral kompleks kobalt (ii) -morin diuji lebih lanjut untuk efek penghambatan pada replikasi denv-2 pada sel vero. replikasi virus dengue dilakukan dengan metode enzyme-immunosorbent assay (elisa) dan nilai indek selektifitas (si), si ditentukan sebagai rasio konsentrasi sitotoksik 50 (cc50) terhadap konsentrasi hambat 50 (ic50) untuk senyawa. nilai ic50 cobalt (ii) -morin untuk virus dengue tipe 2 adalah 3,08 µg / ml, dan nilai cc50 cobalt (ii) -morin research report 142 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 141–144 untuk sel vero adalah 3,36 µg / ml; demikian nilai si untuk cobalt (ii)-morin adalah 1.09. hasil penelitian ini menunjukkan bahwa cobalt (ii) -morin menunjukkan aktivitas penghambatan serotipe 2 anti-dengue dan memiliki sifat beracun pada sel vero. studi lebih lanjut tidak diperlukan sebelum co (ii)-morin dapat diterapkan dalam pengobatan infeksi denv-2. kata kunci: kobalt(ii), morin, senyawa kompleks, aktivitas penghambatan, denv-2 introduction dengue virus (denv) serotypes denv-1–denv-4 are enveloped viruses that belong to the genus flavivirus of the flaviviridae; it is widespread in the tropical and subtropical areas globally. the world health organization reported that the incidence of dengue increased 30-fold in the last five decades, and it is estimated that about 390 million people are infected with denv worldwide.1 many efforts have been made to prevent and treat denv infection, and clinical trials of a number of vaccines are currently underway.2 antiviral activity against denv is an important alternative for the characterization and development of drugs. complementary to vaccine, inhibitors of any natural step of the virus’s replicative cycle have the potential for the treatment of denv infection and indeed compounds such as inhibitors of rna replication are already tested as such.3 however, there is no drug commercially available yet with antiviral activity for denv.4 m o r i n o r 2 ( 2 , 4 d i h y d r o x y p h e n y l ) 3 , 5 , 7 trihydroxychromen-4-one is a flavonoid that exhibits various biological activities, such as anti-bacillus cereus and anti-salmonella enteritidis,5 antioxidant,6 antiinflammatory,7 and antiviral for equid herpesvirus 1.8 however, the antiviral activity for denv has not been reported yet. metals have been used in the treatment and prevention of diseases of humans since ancient times. already in 2500 bc in china elemental gold was in use as therapy for certain diseases, so-called chrysotherapy.9 gold and the more recently developed nano-gold have already had a large impact on medicine, especially in hiv therapy and cancer treatment.10 metal–organic compounds were reported to exhibit fungicidal,11 bactericidal,11–14 and antiviral13,15 activities. in a previous study, ribavirin was shown to exert its toxicity by inhibiting the intracellular energy metabolism and oxidative membrane damage, leading to accelerated extravascular hemolysis by the reticuloendothelial system. although its inhibitory activity was not significant, at high concentration it was more toxic to replicating cells than to stationary cell monolayers of vero cells.16 currently, there are no published data on the possible anti-denv activities of cobalt compounds. in the present study, the inhibitory activity of cobalt(ii)–morin complex against the replication of denv-2 in cell culture was investigated. material and method chemicals and media the chemical reagents used in this research were the cobalt(ii)–morin complex compound, dimethyl sulfoxide (merck 99.98%, germany), minimum essential eagle medium (sigma-aldrich, germany), dengue virus serotype 2 surabaya isolate (kt012513), vero cell (african green monkey kidney), celltiter96® non-radioactive proliferation reagent (promega, usa), and denv antibody (4g2) for enzyme-linked immunosorbent assay (elisa). antiviral activity assay confluent monolayers of vero cells were prepared on a 96-well plate (1 × 106 cells/10 ml) and counted using a hemocytometer, and the titer of denv-2 (2 × 104 ffu/well) was expressed in foci-forming units (ffu) after incubating at 37°c for 2 days. the 50% inhibitory concentration (ic50) was calculated as follows: ic50 = (nc − ac) × 100/nc, where nc is the mean of the number of negative controls and ac is the absorbance of the compound tested. the inhibition of denv-2 replication by each compound was further investigated by using quantitative elisa. cytotoxicity assay a cytotoxicity assay was performed using celltiter96® non-radioactive proliferation reagent. the celltiter96® assay is a modification of the mtt assay method described by mosmann.17 the assay is very sensitive: it can detect 1,000 cells/well of a 96-well plate reader. vero cells (1 × 105 cells/ml), 500 µl of serial dilution compound, and a total of 100 µl of cell proliferation reagent was added to each well of a 96-well plate and incubated under 5% co2 at 37°c for 1–4 hours. the plate was read at 570 nm using an imarktm microplate absorbance reader. results and discussion antiviral activity of cobalt(ii)–morin a significant inhibitory activity to that of the complex cobalt(ii)–morin was displayed against the tested pathogenic denv-2 virus in vero cells. in the inhibitory activity test, we studied the ability of the compound to produce a direct virus-inactivating effect. the ic50 value was determined from the concentration–response curve (figure 1); the ic50 value was 3.08 µg/ml, r 2 was 0.9404, 143sucipto, et al.: inhibitory activity of cobalt(ii)–morin and the value of the selectivity index (si) was 1.09. the si of the antiviral compound appeared to be moderately influenced by the strain of denv tested.18 the emergence of arboviruses worldwide raises the necessity of developing a new strategy for the treatment of the diseases they cause. several approaches have been demonstrated for the treatment of denv-2 in which cobalt(ii)–morin at concentrations of micrograms per milliliter promote inactivation. the antiviral activity of natural and synthetic morin has been described for distinct viruses.8,19 in members of the flaviviridae family, the activity of morin has been described for the replication system of canine distemper virus). here, the ic50 value of morin is 40.52 ± 1.69 µg/ml for a 1-hour incubation. investigation of morin’s structure showed that the compound is able to inhibit the adsorption and penetration stages.19 in hepg2 cells, the protection conferred by heme and co(ii)–protoporphyrin ix seems to be the result of decreased denv-2 replication upon treatment. the ic50 value was reported to be 3.912 ± 1.4 µmol/l. treatment of thp-1 cells with co(ii)–protoporphyrin ix, after infection at a low multiplicity of infection showed similar results regarding cell viability and denv replication after 72 hours post inoculation. this in vitro studies indicate a potential therapeutic use porphyrins in the treatment of flavivirus infection.4 figure 2 shows the structure of morin. morin has hydroxyl groups at c-2′ and c-4′ (meta position). the activity of morin might be related to the position of the hydroxyl groups at c-2′, which might prevent its biological effects on virus. however, the specific mechanism of co(ii)–morin is still unclear. antiviral activity assay confluent monolayers of vero cells were prepared on a 96-well plate (1 × 106 cells/10 ml) and counted using a hemocytometer, and the titer of denv-2 (2 × 104 ffu/well) was expressed in foci-forming units (ffu) after incubating at 37°c for 2 days. the 50% inhibitory concentration (ic50) was calculated as follows: ic50 = (nc − ac) × 100/nc, where nc is the mean of the number of negative controls and ac is the absorbance of the compound tested. the inhibition of denv-2 replication by each compound was further investigated by using quantitative elisa. cytotoxicity assay a cytotoxicity assay was performed using celltiter96® non-radioactive proliferation reagent. the celltiter96® assay is a modification of the mtt assay method described by mosmann.17 the assay is very sensitive: it can detect 1,000 cells/well of a 96-well plate reader. vero cells (1 × 105 cells/ml), 500 µl of serial dilution compound, and a total of 100 µl of cell proliferation reagent was added to each well of a 96-well plate and incubated under 5% co2 at 37°c for 1–4 hours. the plate was read at 570 nm using an imarktm microplate absorbance reader. results and discussion antiviral activity of cobalt(ii)–morin a significant inhibitory activity to that of the complex cobalt(ii)–morin was displayed against the tested pathogenic denv-2 virus in vero cells. in the inhibitory activity test, we studied the ability of the compound to produce a direct virus-inactivating effect. the ic50 value was determined from the concentration–response curve (figure 1); the ic50 value was 3.08 µg/ml, r2 was 0.9404, and the value of the selectivity index (si) was 1.09. the si of the antiviral compound appeared to be moderately influenced by the strain of denv tested.18 y = -6.2655x + 69.307 r² = 0.9404 0 10 20 30 40 50 60 70 co-morin concentration (µg/ml) co-morin % inhibition linear (co-morin % inhibition) 50 25 12.5 6.25 3.13 1.56 0.78 0.39 concentrations of compound (µg/ml) v al ue n um be r figure 1. inactivation of denv-2, a member of the flavivirus genus, at several concentrations of cobalt(ii)–morin figure 2. structure of morin figure 3. cytotoxicity of cobalt(ii)–morin for vero cells at several concentrations cobalt(ii) is stable in water by coordinating to ligands or chelators and is more stable than co(iii).13 when compared with a previous study, it has been revealed that co(ii) is more toxic than cu(ii) with a cc50 value of 5.03 µg/ml. 18 copper(ii) was found to be nontoxic to human erythrocyte cells even at a concentration of 500 µg/ml.20 conclusion further studies are not required before co(ii)–morin can be applied in the treatment of denv-2 infections. this study did not show the potential of the co(ii)–morin complex as a candidate for antiviral agent against denv-2 because it was shown to be toxic to vero cells. acknowledgement this research was supported by the joint program of the japan initiative for global research network on infectious disease (j-grid); a research grant from mandat universitas airlangga (hrmua); the institute of tropical disease (itd); the center of excellence (coe) program by the ministry of research and technology (ristek) indonesia; and the chemistry department of universitas airlangga. references 1. bhatt s, gething pw, brady oj, messina jp, farlow aw, moyes cl, et al. the global distribution and burden of dengue. nature. 2013 apr 7;496(7446):504–7. 2. woodland dl. vaccines against dengue virus. viral immunol. 2015 mar;28(2):75–75. 3. lim sp, noble cg, shi p-y. the dengue virus ns5 protein as a target for drug discovery. antiviral res. 2015 jul;119:57–67. 4. assunção-miranda i, cruz-oliveira c, neris rls, figueiredo cm, pereira lps, rodrigues d, et al. inactivation of dengue and yellow fever viruses by heme, cobaltprotoporphyrin ix and tin-protoporphyrin ix. j appl microbiol. 2016 mar;120(3):790– 804. y = 13.965x 1.2521 r² = 0.9493 0 10 20 30 40 50 60 70 80 concentration (µg/ml) % viability linear (% viability) 50 25 12.5 6.25 3.125 concentrations of compound (µg/ml) v al ue n um be r figure 3. cytotoxicity of cobalt(ii)–morin for vero cells at several concentrations 144 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 141–144 cytotoxicity of co(ii)–morin to vero cells the cytotoxic activity of co(ii)–morin was investigated. this compound was tested against vero cells at different concentrations to determine the cc50 value using celltiter96® non-radioactive proliferation reagent. the cc50 value was found to increase with an increasing concentration of the test compound, as shown in figure 3. the cc50 of cobalt(ii)–morin for vero cells was 3.36 µg/ml, with an r2 value of 0.9493. in this study, we have examined the relationship between the concentration of vero cells in the culture medium and the cytotoxic potency of cobalt(ii)–morin. cobalt(ii) is stable in water by coordinating to ligands or chelators and is more stable than co(iii).13 when compared with a previous study, it has been revealed that co(ii) is more toxic than cu(ii) with a cc50 value of 5.03 µg/ml. 18 copper(ii) was found to be nontoxic to human erythrocyte cells even at a concentration of 500 µg/ml.20 conclusion further studies are not required before co(ii)–morin can be applied in the treatment of denv-2 infections. this study did not show the potential of the co(ii)–morin complex as a candidate for antiviral agent against denv-2 because it was shown to be toxic to vero cells. acknowledgement this research was supported by the joint program of the japan initiative for global research network on infectious disease (j-grid); a research grant from mandat universitas airlangga (hrmua); the institute of tropical disease (itd); the center of excellence (coe) program by the ministry of research and technology (ristek) indonesia; and the chemistry department of universitas airlangga. references 1. bhatt s, gething pw, brady oj, messina jp, farlow aw, moyes cl, et al. the global distribution and burden of dengue. nature. 2013 apr 7;496(7446):504–7. 2. woodland dl. vaccines against dengue virus. viral immunol. 2015 mar;28(2):75–75. 3. lim sp, noble cg, shi p-y. the dengue virus ns5 protein as a target for drug discovery. antiviral res. 2015 jul;119:57–67. 4. assunção-miranda i, cruz-oliveira c, neris rls, figueiredo cm, pereira lps, rodrigues d, et al. inactivation of dengue and yellow fever viruses by heme, cobalt-protoporphyrin ix and tinprotoporphyrin ix. j appl microbiol. 2016 mar;120(3):790–804. 5. arima h, ashida h, danno g. rutin-enhanced antibacterial activities of flavonoids against bacillus cereus and salmonella enteritidis. biosci biotechnol biochem. 2002 jan 22;66(5): 1009–14. 6. subash s, subramanian p. morin a flavonoid exerts antioxidant potential in chronic hyperammonemic rats: a biochemical and histopathological study. mol cell biochem. 2009;327(1–2): 153–61. 7. fang s-h, hou y-c, chang w-c, hsiu s-l, lee chao p-d, chiang b-l. morin sulfates/glucuronides exert anti-inflammatory activity on activated macrophages and decreased the incidence of septic shock. life sci. 2003 dec;74(6):743–56. 8. gravina hd, tafuri nf, silva júnior a, fietto jlr, oliveira tt, diaz man, et al. in vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1. res vet sci. 2011 dec;91(3):e158–62. 9. merchant b. gold, the noble metal and the paradoxes of its toxicology. biologicals. 1998 mar;26(1):49–59. 10. nagender reddy panyala, eladia maría peña-méndez, josef havel. gold and nano-gold in medicine: overview, toxicology and perspectives. j appl biomed. 2009;7(may):75–91. 11. mao w, bao k, feng y, wang q, li j, fan z. synthesis, crystal structure, and fungicidal activity of triorganotin(iv) 1-methyl1h-imidazole-4-carboxylates. main gr met chem. 2015 jan 1;38(1–2). 12. lu x, ye j, zhang d, xie r, bogale rf, sun y, et al. silver carboxylate metal–organic frameworks with highly antibacterial activity and biocompatibility. j inorg biochem. 2014 sep;138: 114–21. 13. chang el, simmers c, knight da. cobalt complexes as antiviral and antibacterial agents. pharmaceuticals. 2010 may 26;3(6): 1711–28. 14. aguado s, quirós j, canivet j, farrusseng d, boltes k, rosal r. antimicrobial activity of cobalt imidazolate metal–organic frameworks. chemosphere. 2014 oct;113:188–92. 15. sucipto th, martak f. synthesis of metal-organic (complexes) compounds copper(ii)-imidazole for antiviral hiv candidate. indones j trop infect dis. 2016 jan 18;6(1):5. 16. smee df, bray m, huggins jw. antiviral activity and mode of action studies of ribavirin and mycophenolic acid against orthopoxviruses in vitro. antivir chem chemother. 2001;12(6):327–35. 17. mosmann t. rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. j immunol methods. 1983 dec;65(1–2):55–63. 18. sucipto th, churrotin s, setyawati h, kotaki t, martak f, soegijanto s. antiviral activity of copper(ii)chloride dihydrate against dengue virus type-2 in vero cell. indones j trop infect dis. 2017 aug 22;6(4):84. 19. carvalho ov, botelho cv, ferreira cgt, ferreira hcc, santos mr, diaz man, et al. in vitro inhibition of canine distemper virus by flavonoids and phenolic acids: implications of structural differences for antiviral design. res vet sci. 2013 oct;95(2):717–24. 20. lv j, liu t, cai s, wang x, liu l, wang y. synthesis, structure and biological activity of cobalt(ii) and copper(ii) complexes of valinederived schiff bases. j inorg biochem. 2006 nov;100(11):1888–96. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 3 september–december 2022 original article long-term consequences, chances of re-infection, and outcomes among cases recovered with severe covid-19 at a tertiary care centre in central india talha saad1 , satyendra mishra1, hindeshwari rai2, sumit kumar rawat3* 1department of chest and tb, bundelkhand medical college, sagar, mp, india 2department of medicine, bundelkhand medical college, sagar, mp, india 3department of microbiology, bundelkhand medical college, sagar, india received: june 14th, 2022; revised: july 14th, 2022; accepted: november 23rd, 2022 abstract covid-19 has a wide disease spectrum. different presentations may be seen in different people, with uncertain long-term fate. the amount and longevity of immunity provided among the infected also vary from person to person which might in turn affect the chances of re-infection. current study tries to uncover the incidence, disease severity and outcomes amongst those who have been previously hospitalized for covid-19. a prospective cohort study where all patients admitted to intensive care facility at the tertiary care center were followed up for any occurrences of re-infection for more than one year. all cases were followed up telephonically and at scheduled visits to the hospital by trained personnel. a total of 410 cases with a mean age of 59.8 years, including 310 (75.6%) males and 100 (24.4%) females. among these 410 patients 287 remained alive till the end of study period. re-infection rates among recovered icu admitted seriously ill patients were 1.4% whereas the rate of icu readmission due to covid-19 re-infection was only 0.7%. re-infection among female was 1.1% whereas in male was 1.5%. icu readmission rate among female was 1.1% while in male was 0.5% only. the chances of re-infection in female were seen less than that in males, but the severity of re-infection in females was found to be higher. covid-19 re-infection in previously severely infected covid-19 patient is not so common. the chances of a severe disease among such cases are even rarer. keywords: covid-19; icu patients; re-infection; sars-cov-2 abstrak covid-19 memiliki spektrum penyakit yang luas. presentasi yang berbeda dapat dilihat pada orang yang berbeda, dengan keadaan jangka panjang yang tidak pasti. jumlah dan masa kekebalan yang diberikan di antara orang yang terinfeksi juga bervariasi dari orang ke orang yang pada gilirannya dapat mempengaruhi kemungkinan infeksi ulang. studi saat ini mencoba mengungkap kejadian, tingkat keparahan penyakit, dan hasil di antara mereka yang sebelumnya dirawat di rumah sakit karena covid-19. sebuah studi kohort prospektif di mana semua pasien yang dirawat di fasilitas perawatan intensif di pusat perawatan tersier ditindaklanjuti untuk setiap kejadian infeksi ulang selama lebih dari satu tahun. semua kasus ditindaklanjuti melalui telepon dan pada kunjungan terjadwal ke rumah sakit oleh personel terlatih. sebanyak 410 kasus dengan usia rata-rata 59,8 tahun, termasuk 310 (75,6%) laki-laki dan 100 (24,4%) perempuan. di antara 410 pasien 287 tetap hidup sampai akhir masa studi. tingkat infeksi ulang di antara pasien yang dirawat di icu yang pulih dan sakit parah adalah 1,4% sedangkan tingkat masuk kembali ke icu karena infeksi ulang covid-19 hanya 0,7%. infeksi ulang pada wanita adalah 1,1% sedangkan pada pria adalah 1,5%. tingkat penerimaan kembali icu pada wanita adalah 1,1% sedangkan pada pria hanya 0,5%. kemungkinan infeksi * corresponding author: rawat5000@gmail.com https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-2661-7688 https://orcid.org/0000-0002-8222-5486 145 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license talha saad, et al. long-term consequences chances of re-infection, and outcomes ulang pada wanita terlihat lebih kecil dibandingkan pria, tetapi tingkat keparahan infeksi ulang pada wanita ditemukan lebih tinggi. infeksi ulang covid-19 pada pasien covid-19 yang sebelumnya terinfeksi parah tidak begitu umum. kemungkinan penyakit parah di antara kasus-kasus seperti itu bahkan lebih jarang. kata kunci: covid-19; infeksi ulang; pasien icu; sars-cov-2 how to cite: saad, t., mishra, s., rai, h., rawat, s. k. long-term consequences, chances of re-infection, and outcomes among cases recovered with severe covid-19 at a tertiary care centre in central india. indonesian journal of tropical and infectious disease. 10(3). 144–149. dec. 2022. introduction covid-19 often presents with an extensive clinical spectrum varying from asymptomatic infection to severe lifethreatening viral pneumonia often requiring admission to intensive care, and sometimes even leading to death.1 persisting symptoms, and unforeseen organ dysfunction has been observed subsequently to sars-cov-2 infection in an escalating quantum of those who have recovered, as was observed in the past during sars outbreak.2 however, since covid-19 is not a classical disease, we need to keep vigil about gaining new insights in it and an uncertainty prevails concerning its long-term health sequelae amongst those who have recovered from it. this is of immediate relevance and warrants attention since patients presenting with grave disease including those requiring mechanical ventilation during their initial medical admission, for whom long-term complications, persisting symptoms and sometimes who might lack a complete recovery on discharge.3 this is an initial general concept that the patients who have recovered from covid-19 natural infection generate a robust immune response which help in clearing the virus. however, currently it is not very clear whether such primary exposure or disease confers a shielding immunity to successive infections with this virus. recent studies suggest that antibodies generated after a recent covid-19 infection might help in providing some protection against reinfection in most patients but despite this, reinfection or break-through infection is possible.4 from previous research, it is clear that despite the presence of antibodies reinfection is common with other human corona viruses.5 according to a recent report the working epidemiological case definition for re-infection after initial infection of covid-19 was suggested as two positive tests at an interval of at least 102 days with one interim negative pcr test report.6 few case series show that recurring covid-19 infections might be worse in approximately 20% of patients and even severe complications may occur among the higher those with advanced age and immunecompromised patients.7 re-infection with covid-19 is not limited to any particular stain, there are multiple variants with a differing genetic sequence, thus causing reinfection.8 subsequently, to the emergence of the newer mutants and variants of concern of covid-19 from the uk, india and south africa; it becomes indispensable to see whether these newer mutants cause any infection to patients who were affected with this disease during the ‘first wave’ prior to the appearance of these variants.7 it is thought that as there is priming of adaptive immune response by the previous infection, reinfection is usually associated with milder symptoms, protection from severe disease but the robust response has also been reported.9,10 there might be numerous sars-cov-2 reinfection cases than have been currently reported.11,12 it is very difficult to estimate the true prevalence of these re-infections as the genome sequencing data are not available in most covid-19 cases and many of the asymptomatic and mildly symptomatic patients were not seeking medical advice. for 146 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 144–149 identification of true prevalence of covid-19 re-infection population-based studies are more useful. it might often be challenging to differentiate between covid-19 relapse, reinfection and rt-pcr re-positivity in a few cases. recently, yahav et al had suggested that re-infection is considered in those case who become negative after infection and again became pcr positive after more than 90 days.13 two of the meta-analyses performed during early phase of the pandemic reported that re-infection or repositivity were rare but such case reports and studies were performed without considering genome sequencing data.14 few studies have shown that subsequent infection is possible in those persons already having a previous exposure to covid-19. therefore, practicing social distancing and wearing mask at all public places, irrespective of history of prior infection or vaccination is very essential to prevent the spread of further waves of the current pandemic. without which, it’s likely that the sars-cov-2 virus may continue to transmit and circulate in various populations despite the achievement of herd immunity by vaccination or natural infection.15 this study is concerned with the detection of re-infection if any, the associated disease severity and the outcome of such re-infection during the subsequent waves of covid-19 infections among icu hospitalized patients during the earliest wave of this disease. materials and methods research design this study consisted of cohorts enrolled prospectively at a tertiary care center, in central india, madhya pradesh. study was started after due approval from institutional human ethical committee reference number, iecbmc/2021/32. inclusion and exclusion criteria inclusion criteria: 1. we included and followed up all critically ill icu patients with laboratoryconfirmed sars-cov-2 pcr positivity via recommended throat swabs or nasopharyngeal swabs, 2. those who were discharged from the institute between august 2020 to november 2020 during the first wave of covid-19. exclusion criteria: 1. those who refused to participate, 2. those who died before the follow-up visit, 3. those who could not be contacted. all discharged patients met uniform discharge criteria according to the government of india imcr guidelines for covid-19.16 patient follow-ups phone calls were used to schedule followup visits and done by trained medical staff. post-discharge such patients were contacted in the order of their symptom onset date as per initial admission record. if the follow-up appointment was missed, 2 more chances on further dates were provided. follow-up consultations were done with face-to-face interviews and examinations performed by trained medical personnel. data analysis data were abstracted and fed into computer on excel sheets, percentages and proportions were calculated using the same software. 147 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license talha saad, et al. long-term consequences chances of re-infection, and outcomes results and discussion during the study 410 cases in total between the ages of 1-month old to 95 years, and population having mean age 59.8 years. more than half i.e. 209 (51%) patients were between 51 to 70 years of age. the sex distribution of study cases was observed to have 310 (75.6%) males versus 100 (24.4%) females. among 410 patients, 287 remained alive after first wave was over (shown in table 1). between first wave and second wave during the study, 5 persons died with the reasons behind their death remaining unclear and not directly related to covid-19. a single dose of vaccine was received by 196 (partially vaccinated) while 78 were vaccinated by both the doses of vaccination before second wave of covid-19. among these 287 individual only 4 were infected in the second wave of and only 2 were admitted in icu. the two patients those who were readmitted in icu were partially vaccinated. former being a 48 years old female and later one a 50-year-old male. the other two who were admitted in general ward were 32 years old male, he was fully vaccinated and other one was youngest patient 10 months old nonvaccinated male child, all four were discharged successfully. the mean age of those re-infected was 33±19 years and this study population comprising of 25% females and 75% males as shown in figure 1. only female patient was having hypertension as a co-morbidity. figure 1. sex wise distribution of re-infected patient re-infection rate among recovered icu admitted seriously ill patients was 1.4% whereas the rate of icu re-admission due to covid re-infection was only 0.7%. reinfection among female was 1.1% whereas in male was 1.5%. icu readmission rate among female was 1.1% while in male was 0.5% only. the chances of re-infection in female were seen less than that in males, but the severity of re-infection in females is more was found to be higher. table 1. age-wise distribution of study cohorts no age group (year) patients deaths male female total male female total 1 ≤ 30 18 7 25 2 1 3 2 31–40 34 8 42 6 2 8 3 41–50 58 16 74 8 3 11 4 51–60 70 33 103 23 6 29 5 61–70 86 20 106 32 9 41 6 > 70 44 16 60 22 9 31 in a meta-analysis study by ghorbani et al, the overall estimation of reinfection, was 3% (95% ci: 0.8–5), recurrence was 133 (95% ci: 105–160), with readmissions being 75 (95% ci: 54–96) per 1000 patients17, but in our study rate of re-infection leading to hospitalization was only 0.7%. this is close to the study done by arafkas et al. where the prevalence of re-infection was reported as zero.18 other study done by of ren et al. reported a re-positivity of 12%, while piri et al. concluded in their systematic review a recurrence rate between 2.3% to 21.4%.19,20 in addition their review indicates that the recurrence was 47.7% in male and 53.3% in female which is in contrast to our study in 148 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 144–149 which recurrence was most common in male than female. in our study males were three times more affected than females.20 in some of the studies, re-infected, recurrent, and readmitted cases were either asymptomatic or had mild to moderate symptoms17, but in our study all patients were symptomatic, some of them even had rare symptoms and complications like hepatitis which were rarely seen earlier.21 this may be attributed to the reason that asymptomatic or patients with mild symptoms were not reported to near medical facilities and therefore they did not get tested for covid 19. few patients even had severe symptoms in the second phase of infections of the disease, implying that the severity of its subsequent infection may vary according to the demographics, health status of the patients, and immune system status.22,23 in the study incidence density per 100,000 person days was 1.0 (95%, ci 0.5–1.5) among persons having previous history of infection and 15.1 (95% ci, 14.5–15.7) for persons lacking such infection in the past.24 our findings are in agreement to the to those of harvey and colleagues, who found that persons with a positive diagnostic rt-pcr test for sars-cov-2 and for antibodies to it were much less likely to develop sars-cov-2 infection within initial 3 months than those with absence of antibodies.25 conclusions covid-19 re-infection in previously severely infected covid-19 patient is not so common. the chance of having a severe disease in these patients upon re-infection is even rarer. however, large scale population based elaborate case control study may be required in this field in order to provide further insights. acknowledgement we the authors, acknowledge the dean and the authorities for the support provided during the study and in providing the timely approvals for conduction of the research. we are also grateful to dr. shraddha mishra for helping us with the statistical analysis part of the research. conflict of interest the authors declare that they have no conflict of interest. references 1. zhou f, yu t, du r, fan g, liu y, liu z, et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet. 2020 mar 28;395(10229):1054–62. 2. zhang p, li j, liu h, han n, ju j, kou y, et al. long-term bone and lung consequences associated with hospital-acquired severe acute respiratory syndrome: a 15-year follow-up from a prospective cohort study. bone res. 2020 feb 14;8(1):1–8. 3. cortinovis m, perico n, remuzzi g. long-term follow-up of recovered patients with covid-19. lancet. 2021;397(10270):173–5. 4. hall v, foulkes s, charlett a, atti a, monk ej, simmons r, et al. do antibody positive healthcare workers have lower sars-cov-2 infection rates than antibody negative healthcare workers? large multi-centre prospective cohort study (the siren study), england: june to november 2020. medrxiv. 2021. 5. galanti m, shaman j. direct observation of repeated infections with endemic coronaviruses. j infect dis. 2020;jiaa392. 6. mukherjee a, anand t, agarwal a, singh h, chatterjee p, narayan j, et al. sars-cov-2 reinfection: development of an epidemiological definition from india. epidemiology & infection. 2021;149. 149 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license talha saad, et al. long-term consequences chances of re-infection, and outcomes 7. karthik k, senthilkumar tma, udhayavel s, raj gd. role of antibody-dependent enhancement (ade) in the virulence of sars-cov-2 and its mitigation strategies for the development of vaccines and immunotherapies to counter covid-19. hum vaccin immunother. :1–6. 8. jain vk, iyengar k, garg r, vaishya r. elucidating reasons of covid-19 re-infection and its management strategies. diabetes & metabolic syndrome: clinical research & reviews. 2021 may 1;15(3):1001-6. 9. perez g, banon t, gazit s, moshe sb, wortsman j, grupel d, peretz a, tov ab, chodick g, mizrahi-reuveni m, patalon t. a 1 to 1000 sars-cov-2 reinfection proportion in members of a large healthcare provider in israel: a preliminary report. medrxiv. 2021. 10. harnath at, payne ba, duncan cj. prior sarscov-2 infection is associated with protection against symptomatic reinfection. journal of infection. 2021 apr 1;82(4):e29-30. 11. shastri j, parikh s, agrawal s, chatterjee n, pathak m, chaudhary s, sharma c, kanakan a, srinivasa vasudevan j, maurya r, fatihi s. clinical, serological, whole genome sequence analyses to confirm sars-cov-2 reinfection in patients from mumbai, india. frontiers in medicine. 2021:215. 12. singh pp, tamang r, shukla m, pathak a, srivastava a, gupta p, bhatt a, shrivastava ak, upadhyay sk, singh a, maurya s. estimation of real-infection and immunity against sars-cov2 in indian populations. medrxiv. 2021 jan 1. 13. yahav d, yelin d, eckerle i, eberhardt cs, wang j, cao b, et al. definitions for coronavirus disease 2019 reinfection, relapse and pcr repositivity. clinical microbiology and infection. 2021 mar 1;27(3):315–8. 14. azam m, sulistiana r, ratnawati m, fibriana ai, bahrudin u, widyaningrum d, et al. recurrent sars-cov-2 rna positivity after covid-19: a systematic review and meta-analysis. sci rep. 2020 nov 26;10(1):20692. 15. to kk-w, hung if-n, ip jd, chu aw-h, chan w-m, tam ar, et al. coronavirus disease 2019 (covid-19) re-infection by a phylogenetically distinct severe acute respiratory syndrome coronavirus 2 strain confirmed by whole genome sequencing. clinical infectious diseases [internet]. 2020 aug 25 [cited 2021 jul 31];(ciaa1275). available from: https://doi.org/10.1093/cid/ciaa1275 16. updated clinical management protocol for covid 19 dated 03072020.pdf [internet]. [cited 2022 july 13]. available from: https://www.mohfw.gov.in/pdf/updatedclinical managementprotocolforcovid19dated030720 20.pdf. 17. sotoodeh ghorbani s, taherpour n, bayat s, ghajari h, mohseni p, hashemi nazari ss. epidemiologic characteristics of cases with reinfection, recurrence, and hospital readmission due to covid-19: a systematic review and meta-analysis. journal of medical virology. 2022;94(1):44–53. 18. arafkas m, khosrawipour t, kocbach p, et al. current meta-analysis does not support the possibility of covid-19 re-infections. j med virol. 2021; 93(3): 15991604. 19. ren x, ren x, lou j, et al. a systematic review and meta-analysis of discharged covid-19 patients retesting positive for rt-pcr. eclinicalmedicine. 2021; 34: 100839. 20. piri sm, edalatfar m, shool s, jalalian mn, tavakolpour s. a systematic review on the recurrence of sars-cov-2 virus: frequency, risk factors, and possible explanations. infect dis. 2021; 53(5): 315324. 21. rawat sk, asati aa, jain a, mishra n, ratho rk covid-19 associated hepatitis in children (cah-c) during the second wave of sars-cov2 infections in central india: is it a complication or transientphenomenon?medrxiv2022. https://www.medrxiv.org/content/10.1101/2021. 07.23.21260716v7 22. azam m, sulistiana r, ratnawati m, et al. recurrent sars-cov-2 rna positivity after covid-19: a systematic review and metaanalysis. sci rep. 2020; 10(1):20692. 23. chakravarty d, nair ss, hammouda n, et al. sex differences in sars-cov-2 infection rates and the potential link to prostate cancer. commun biol. 2020; 3(1): 12. 24. assessment of sars-cov-2 reinfection 1 year after primary infection in a population in lombardy, italy | infectious diseases | jama internal medicine | jama network [internet]. [cited 2021 nov 15]. available from: https://jamanetwork.com/journals/jamainternalm edicine/fullarticle/2780557 25. hall vj, foulkes s, charlett a, et al; siren study group. sars-cov-2 infection rates of antibody-positive compared with antibodynegative health-care workers in england: a large, multicentre, prospective cohort study (siren). lancet. 2021;397(10283):1459-1469. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 11 no. 1 january–april 2023 case report a young female with acute acalculous cholecystitis associated with hepatitis a viral infection: a case report bonfilio neltio ariobimo 1* , nurun nujum2, daniel ponco harto saputro3 1faculty of medicine, universitas airlangga, surabaya, indonesia 2department of surgery, st. vincentius a paulo catholic hospital, surabaya, indonesia received: october 3rd, 2022; revised: march 13th, 2023; accepted: april 3rd, 2023 abstract most hepatitis a infections are acute, self-limiting, and asymptomatic. in rare instances, extra hepatic complication, such as acute cholecystitis, may emerge. acute cholecystitis is inflammation of the gallbladder wall and is classified into calculus and acalculus. about 90–95% of cases are brought on by bile duct stones. acute acalculous cholecystitis can be brought on by structural and functional abnormalities in the gallbladder brought on by viral hepatitis infection. here we present a 20 years old female patient with acute acalculous cholecystitis associated with hepatitis a infection. gallbladder distention, thickening of the gallbladder wall, absence of acoustic shadow or biliary sludge, perivesical liquid buildup, and absence of dilatation of the intraand extrahepatic bile ducts are among the ultrasonographic criteria for diagnosing acute acalculous cholecystitis. the viral hepatitis serology revealed acute hepatitis a infection with positive anti-hav igm. hepatitis a testing should be considered in patients suspected with acalculous cholecystitis of undefined etiology in markedly deranged liver function test adult patients. keywords: acalculous cholecystitis; acute cholecystitis; gallbladder inflammation; hepatitis a infection; viral cholecystitis highlights: a rare entity of extrahepatic complication from hepatitis a viral infection in the form of acalculous cholecystitis. recognized and treated the acalculous cholecystitis could prevent the morbidy and mortality. how to cite: ariobimo, b. n., nujum, n., saputro, a young female with acute acalculous cholecystitis associated with hepatitis a viral infection: a case report. indonesian journal of tropical and infectious disease. 11(1). 66–72. apr. 2023. doi: 10.20473/ijtid.v11i1.39532 * corresponding author: neltioario@gmail.com https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0003-2534-5358 67 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: x–xx introduction the hepatitis a virus, which causes hepatitis a infection, is spread by the ingestion of contaminated food and drink. transmission through sharing needles and sexual activity is also possible, however this is unusual.1–4 acute cholecystitis is inflammation of the gallbladder wall and is classified into calculus and acalculus.5 as many as 90–95% of cases are brought on by bile duct stones.6,7 acute acalculous cholecystitis can be brought on by structural and functional abnormalities in the gallbladder brought on by viral hepatitis infection, including gallbladder wall thickening, perivascular edema, and poor bile filling.8,9 there are extremely few cases of acute acalculous cholecystitis as a result of hepatitis infection. only few previous studies have ever reported it.1–3 even while this condition normally resolves on its own, it can occasionally proceed to gangrene, perforation, and even death.10 in acute acalculous cholecystitis instances that do not exhibit the traditional signs of acute cholecystitis, delayed identification and treatment can result in serious consequences and high death rates.11 here we reported a case of young female who was came with acalculous cholecystitis associated with hepatitis a virus infection which a rare entity that needs to be recognized and treated to prevent morbidity and mortality. case report this is a 20 years old single female patient, who came to emergency department with 4day history of epigastric pain, nausea, anorexia, and generalized fatigue. the pain increased over the last 2 days with the radiation to right hypochondriac area along with feeling of rising in body temperature. the patient also complain vomiting five times before she came. she denied any pale stool and dark colored urine. past medical illnesses were only reported tonsillectomy procedure two years ago. same complains in her family was denied. upon physical examination, vital sign was in normal state with maximum temperature was 37°c, her sclera is anicteric, abdominal examination revealed epigastric and right hypochondriac area tenderness. blood investigations showed normal complete blood count, normal electrolytes, normal renal function test, normal prothrombin time (pt) and partial thromboplastin time (ptt), normal urinalysis, and negative qualitative pregnancy test. there was significant raise in liver function test with alanine aminotransferase (alt) 1716 u/l (normal 0– 31u/l) and aspartate aminotransferase (ast) 1564 u/l (normal 0– 30 u/l), total bilirubin 3.98 mg/dl (normal 0.1-1 mg/dl) and direct bilirubin 2.07 mgl/dl (normal <0.2 mg/dl). her wbc is 5100, hb is 13.8 gm/dl, platelets are 188,000, mild elevation in esr 25 mm/hour (normal 0-20 mm/hour). abdominal ultrasound (figure 1) indicated a collapsed gallbladder and ±12 cc of free fluid in the pelvic cavity. no stones or sludge were seen inside the gallbladder, and neither the intranor extrahepatic bile duct was dilated. abdominal ct (figure 2) demonstrated diffuse thickening of gallblader wall (8 mm) and pericholecystic fluid without any calculus found. the serology for viral hepatitis suggested acute hepatitis a infection with positive antihav igm and was negative for other viral hepatitis causes, where hbsag (-) and antihcv (-). thus, the diagnosis was acute acalculous cholecystitis due to viral hepatitis. patient was treated with supportive therapy of intravenous (i.v.) fluid, antinausea and vomiting, analgetic, hepatoprotector, and low fat diet. the abdominal pain is gradually diminishing but her scleral found to be mild icteric since the second day of hospitalization. repeated liver function test in day 4th showed resolution of alt 815 u/l and ast 68 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license bonfiloio neltio ariobimo, et al. a young female with acute acalculous cholecystitis 463 u/l. her symptoms of abdominal pain and nausea improved gradually and settled completely by day 5th. evaluation of liver function test in day 7th showed the following: alt 355 u/l, ast 110 u/l, total bilirubin 1.93 mg/dl, and direct billirubin 0.97 mg/dl. the patient was then discharged with good general condition and a medical clinic appointment for follow up was given. during the follow up at the 16th day, she had no complaint and the liver function test showed resolution alt 31.5 u/l, ast 27.9 u/l, direct billirubin 0.6 mg/dl, and total bilirubin 1.03 mg/dl. figure 1. abdominal ultrasound indicated a collapsed gallbladder and free fluid in the pelvic cavity. (rll : right liver lobe; vu: vesica urinary; gb: gallbladder; ff: free fluid) figure 2. abdominal ct demonstrated diffuse thickening of gallblader wall (8 mm) and pericholecystic fluid without any calculus found (white arrow). discussion in this report, we have described a case of severe leptospirosis or known as weil’s disease.1,3on admission, the patient presented with fever, conjunctiva suffusion, dark urine, and myalgia with leucocytosis, thrombocytopenia, aki, liver failure, and hyperbilirubinemia. patients experience septic shock in the er and are given norepinephrine as support. treatment given was antibiotics and aggressive hydration. dialysis was postponed while watchful waiting for the improvement of kidney functions by fluid therapy. strict monitoring of kidney function and haematology was done. symptoms and kidney function then recover with the treatment given. most hepatitis a infections are acute and self-limiting. infection is usually asymptomatic, but in rare instances, fulminant hepatitis, which can be fatal, or extra hepatic symptoms, such as acute acalculous cholecystitis, may emerge.4,12 acute cholecystitis is an emergency that often occurs in the emergency department.3,13 an inflammation of the gallbladder without the presence of stones or sludge is known as acute acalculous cholecystitis.10,14 acute acalculous cholecystitis caused by viral hepatitis has a significant morbidity and death rate due to changes in gallbladder conditions such as gangrene, perforation, and empyema, even though the precise pathophysiology of the disorder is yet unknown.6 up to 95% of cases of acute cholecystitis are caused by stones that block the bile duct or gallbladder neck, with the remaining cases being inflammation without the presence of stones or sludge. about 5–15% of cases of acute cholecystitis are acalculous, and 47% of these cases follow surgery, extended immobility, prolonged starvation, such as long-term intravenous feeding, elderly patients, patients receiving care in the intensive care unit, and septic conditions.7,10,14,15 hepatitis a infections linked to pancreatitis and cholecystitis happen in 5% of instances as a result of the 69 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: x–xx virus's direct invasion.16 acute acalculous cholecystitis is a very rare condition and has not been widely reported.6,12,17,18 acute acalculous cholecystitis is an uncommon gallbladder infection without gallstones, yet it progresses rapidly. infection in acute acalculous cholecystitis cases progresses faster than in acute calculous cholecystitis cases, and 10% of patients also have consequences including gangrene or perforations. while acute acalculous cholecystitis instances are generally encountered in men and older adults in their sixth decade of life, acute calculous cholecystitis cases are typically seen in women in their fourth and fifth decades of life.11 uncertainty surrounds the precise pathophysiology of acute acalculous cholecystitis. the influence of bile chemicals on the epithelium, ischemia in the gallbladder epithelium, formation of bile wall immune complexes leading to bile fluid stasis, and bacterial invasion are some of the claims made as the reason.9,10,17 hepatitis a infections can lead to the virus to invade the gallblader and bile duct epithelium because of high viral antigen levels followed by an immune complex-mediated immune response.9,10,12,17 the existence of hypoalbumin conditions, the localized spread of the inflammatory process, and increased portal venous pressure, which results in edema of the gallbladder wall, sludge development, and reduced volume during fasting, are structural and functional alterations in the gallbladder.6,10,18 some of the mechanisms thought to be the pathogenesis of acute acalculous cholecystitis include direct injury to the mucous and muscular layers due to direct invasion of the hepatitis a virus in the gallbladder, impaired production and excretion of bile substances due to damage to hepatocytes, and spread of inflammatory mediators from surrounding organs due to hepatocyte cell necrosis.8,10,19 the majority of hepatitis a infections are asymptomatic, however common symptoms often include a mix of gastrointestinal, cholestasis, and flu-like illness.4 while adult hav infection rates are thought to be more than 70%, around 70% of hav infections in children are asymptomatic.1 hepatitis a infection is frequently accompanied by diarrhea, stomach discomfort, fever, anorexia, nausea, vomiting, fever, malaise, dark urine, pale stool, and jaundice.1,17 hepatitis a infection symptoms are quite similar to those of acute cholecystitis patients in terms of their complaints. acute renal failure, autoimmune hemolytic anemia, pleural or pericardial effusion, acute pancreatitis, encephalopathy, ascites, and cholecystitis are only a few of the extremely uncommon extrahepatic problems that might develop.1,3,18,19 the clearance of the hepatitis infection typically results in an improvement of the symptoms caused by these consequences.18 although it is mentioned that the etiology of ascites in hepatitis a infection is still unknown, it is believed to be caused by an increase in portal venous pressure because of damage to the structure and cells of the liver organ.19 this patient had mild ascites. it is difficult to separate hepatitis a from other viral hepatitis types solely on the basis of clinical characteristics. serologic testing, which recognizes the presence of immunoglobulin m (igm) anti-hav in the acute phase of infection and immunoglobulin g (igg) anti-hav in the convalescent phase of infection, is necessary for a definitive diagnosis of hepatitis a.2 although not specific, laboratory testing of cases with acute acalculous cholecystitis as a complication from hepatitis a infection revealed an increase in white blood cells (wbc), c-reactive protein (crp), alanine aminotransferase (alt), aspartate aminotransferase (ast), and anti-hav igm.1,11 70 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license bonfiloio neltio ariobimo, et al. a young female with acute acalculous cholecystitis when acute gallbladder inflammation is unrelated to gallstones or sludge, the diagnosis of acalculous cholecystitis is established.6 due to its low cost, ease of accessibility, quick examination time, and absence of ionizing radiation, ultrasonography (us) continues to be the recommended first imaging modality for the assessment of suspected acute cholecystitis. high sensitivity and specificity in identifying gallstones, as well as the ability to elicit "murphy's sign" using the ultrasonic transducer, are key benefits of us over other imaging modalities.15 gallbladder distention, thickening of the gallbladder wall (>3.5 mm), absence of acoustic shadow or biliary sludge, perivesical liquid buildup, and absence of dilatation of the intraand extrahepatic bile ducts are among the ultrasonographic criteria for diagnosing acute acalculous cholecystitis.4,12,20 although the usual gallbladder wall is thin-hairline or undetectable, a modestly thicker wall was not included.20 the specificity and accuracy of ultrasonography for the identification of acute acalculous cholecystitis are 97.8 and 96.1%, respectively, while the sensitivity is 88.9%.12 computed tomography (ct) scans must be conducted in the event of non-contributory ultrasound imaging or clinical warning signals in order to prevent erroneous diagnoses and identify gangrenous forms necessitating a change in therapeutic approaches.3 overextended gallbladder, mural thickness, mural enhancement with intramural gas buildup, pericholecystic fat stranding, pericholecystic fluid, and enhanced hyperenhancement of the neighboring liver are all characteristics of acute acalculous cholecystitis on ct.14,15,20 the discovery of vha antigen during immunohistochemical analysis in the gallbladder wall of a patient exhibiting anc while vha confirms the causal relationship between vha and anc.3 hepatitis a individuals with noticeably abnormal liver function tests should be tested if they are suspected of having acalculous cholecystitis of unknown cause.4,6,12 depending on the clinical appearance, several acute acalculous cholecystitis related with acute viral hepatitis treatments are available.12 the majority of cases are selflimited, and with therapy for the underlying systemic condition, the gallbladder may spontaneously decompress in about two weeks.10 surgery may be indicated by related problems such gallbladder perforation and worsening of abdominal symptoms.10,12 when the viremia drops within a few days and gallbladder wall thickness recovers to normal under conservative care. these cases don't need to have surgery.18 hepatoprotective maintenance therapy is sufficient for the majority of mild and selflimiting cases.8 if medical treatment alone often is ineffective in treating acute acalculous cholecystitis, early cholecystectomy or percutaneous cholecystostomy application is advised as a treatment; nonetheless, cholecystectomy may be a difficult procedure for the surgeon.11 the recommended course of treatment for complex acute cholecystitis is an early cholecystectomy performed within 7 days of the beginning of symptoms.13 acute acalculous cholecystitis is an extremely rare complication of acute viral hepatitis, and the mortality from acute acalculous cholecystitis with viral hepatitis is extremely low in comparison to acute acalculous cholecystitis of other origin that needs urgent surgical intervention.10 some of these are self-limiting and heal spontaneously, while a limited number of cases progress to a gangrenous state, gallbladder perforation, and even to death.14 mortality rates range from 10%–50% due to the severity of underlying illness.9 strength and limitation the strength of this study was reporting rare case which need to be recognized as soon as possible to prevent the morbidity and mortality. the limitation of this study was no 71 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: x–xx follow-up results were done in the form of ultrasound or abdominal ct scan. conclusions acute acalculus cholecystitis is a rare condition and although it was extremely rare it needs to be considered as a complication in cases with viral hepatitis infection. on the other hand, hepatitis a testing should be considered in patients suspected with acalculous cholecystitis of undefined etiology in markedly deranged liver function test adult patients. early diagnosis and treatment in acute acalculous cholecystitis cases that do not show classical acute cholecystitits symptoms could prevent the case progressed to emergence of severe complications and high rates of mortality. acknowledgement we are grateful to all medical workers and nurses in hermina hospital kemayoran for their support and facilities in managing the patient. we would like to thank the director hermina hospital kemayoran for allowing us to publish our findings. funding this study did not receive funding. conflict of interest the authors have no conflicts of interest to declare. all co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report author contribution data curation, writing–review and editing, validation: bna. data curation, writing– review, and editing: nn. data curation, supervision, validation: dhp. references 1. wang m, feng z. mechanisms of hepatocellular injury in hepatitis a. viruses. 2021;13(5):1–9. 2. guzman-holst a, luna-casas g, garcia ab, madrid-marina v, cervantes-apolinar my, andani a, et al. burden of disease and associated complications of hepatitis a in children and adults in mexico: a retrospective database study. plos one. 2022;17(5 may):1–19. 3. mejri a, arfaoui k, hospital jr, hospital jr. case report acute cholecystitis that must not be operated. 2020;2797:1–5. 4 .bura m, michalak m, chojnicki mk, kowalapiaskowska a, mozer-lisewska i. viral hepatitis a in 108 adult patients during an eight-year observation at a single center in poland. adv clin exp med. 2015;24(5):829–36. 5. albu s, voidăzan s, popa d. gallbladder hydrops associated with an episode of acute liver toxicity in the adult: may it be considered a surgical emergency or not? j interdiscip med. 2016;1(2):180–2. 6. omar a, osman m, bonnet g, ghamri n. acute acalculous cholecystitis caused by hepatitis c: a rare case report. int j surg case rep [internet]. 2016;19:78–81. available from: http://dx.doi.org/10.1016/j.ijscr.2015.12.020 7. radunović m, terzić d, mugoša b, terzić z, andrić b, ratković m, et al. cholecystitis as a cause of abdominal pain in patients with acute viral hepatitis a and b. acta medica median. 2012;(november):20–3. 8. velev v, popov m, tomov l, golemanov b. involvement of the gallbladder in the course of viral hepatitis a in childhood. trop doct. 2019;49(4):271–3. 9. wright wf, palisoc k, pinto cn, lease ja, baghli s. hepatitis c virus-associated acalculous cholecystitis and review of the literature. clin med res. 2020;18(1):33–6. 10. mohammed ra, ghadban w, mohammed o. acute acalculous cholecystitis induced by acute hepatitis b virus infection. case reports hepatol. 2012;2012:1–4. 11. oztas m, peker ys. new diagnostic and predisposing parameters for acute acalculous cholecystitis. indian j pharm sci. 2020;82(5):16– 21. 12. kaya s, eskazan ae, ay n, baysal b, bahadir mv, onur a, et al. acute acalculous cholecystitis due to viral hepatitis a. case rep infect dis. 2013;2013:1–4. 13. shahramian i, parooie f, salarzaei m. acute cholecystitis management during the covid19 pandemic – a systematic review and metaanalysis. polish j surg. 2022;94(4):1–8. 72 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license bonfiloio neltio ariobimo, et al. a young female with acute acalculous cholecystitis 14. unal h, korkmaz m, kirbas i, selcuk h, yilmaz u. acute acalculous cholecystitis associated with acute hepatitis b virus infection. int j infect dis. 2009;13(5):310–2. 15. chawla a, bosco ji, lim tc, srinivasan s, teh hs, shenoy jn. imaging of acute cholecystitis and cholecystitis-associated complications in the emergency setting. singapore med j. 2015;56(8):438–44. 16. albert a, valencia r, smereck j. acute hepatitis b with pancreatitis and cholecystitis leading to acute liver failure and death. clin pract cases emerg med. 2018;2(4):304–8. 17. tarawneh a, alkhatib am, al-namorah ba. acute acalculous cholecystitis in a child with hepatitis a infection. j pediatr surg case reports [internet]. 2021;66:101778. available from: https://doi.org/10.1016/j.epsc.2020.101778 18. nazan;i̇nce d. acute viral acalculous cholecystitis due to viral hepatitis a hepatita’ya bağlı akut taşsız kolesistit. ankara üniversitesi tıp fakültesi mecmuası. 2005;58(2):1. 19. israel s, fruchtman h, hakimian d, ackerman z. ascites and gallbladder abnormalities are frequent findings in adults with hepatitis a virus infection. isr med assoc j. 2019;21(1):24–8. 20. yeo dm, jung se. differentiation of acute cholecystitis from chronic cholecystitis. medicine (baltimore). 2018;97(33):e11851. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 11 no. 1 january–april 2023 original article epidemiological and clinical features of critical and non-critical elderly covid-19 patients in udayana university academic hospital: a retrospective study cokorda agung wahyu purnamasidhi1* , i ketut agus somia1 , darren junior2 , richard christian suteja2 , i komang hotra adiputra2 , giovanca verentzia purnama2 , i gede purna weisnawa2 , jerry2 , putu kintan wulandari2 , dewa ayu fony prema shanti2 , i gusti ngurah ariestha satya diksha2 1tropical and infectious diseases division, internal medicine department, universitas udayana, bali, indonesia 2faculty of medicine, universitas udayana, bali, indonesia received: february 1st, 2023; revised: april 4th, 2023; accepted: april 5th, 2023 abstract elderly covid-19 patients have been associated with worse outcomes and have been presented with the highest mortality rate. however, studies on the clinical features and the differences between critical and non-critical elderly covid-19 patients in indonesia and even other countries are still lacking and rare. in this retrospective study, the epidemiological and clinical features of critical and non-critical elderly covid-19 patients admitted to udayana university academic hospital between april 2020 and march 2021 were analyzed and then compared. of the 280 medical records analyzed, 60.7% were male and the median age was 65.0 years old. based on the medical records, 18.2% of elderly patients met our criteria of critical patients. the most common symptoms presented in both category upon admission included fever and coughing. the most common comorbidity found in critical patients was heart disease and hypertension in non-critical patients. laboratory results differences included leukocytes, neutrophils, lymphocytes, neutrophil-to-lymphocyte ratio, platelets, sgot, sgpt, and urea. only 9.9% of critical patients and 6.1% of non-critical patients were given antiviral therapy. in contrast, 68.6% of critical patients and 76% of non-critical patients were given antibiotics. the mortality rate in critical patients was 70.6% and 0.4% in non-critical patients. based on the results, a multimodal approach in the treatment of elderly covid-19 patients is very essential. the higher mortality rate in elderly patients should be able to be reduced by giving early and timely antiviral therapy with the addition of effective choice of drugs. keywords: covid-19; epidemiology; elderly patients; geriatric; sars-cov-2 highlights: the novelty of this study is that it is the first study focusing on the clinical profile of elderly covid-19 patients in bali. the benefit of this study through its description and comparison is for clinicians to be able to provide a more structured and comprehensive approach towards elderly covid-19 patients. how to cite: purnamasidhi, c. a. w., somia, i. k. a., junior, d., suteja, r. c., adiputra i. k. h., purnama, g. v., et al. epidemiological and clinical features of critical and non-critical elderly covid-19 patients in udayana university academic hospital: a retrospective study. indonesian journal of tropical and infectious disease. 11(1). 27–34. apr. 2023. doi: 10.20473/ijtid.v11i1.43097 * corresponding author: purnamasidhi@unud.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-1646-3793 https://orcid.org/0000-0003-4168-9572 https://orcid.org/0000-0002-7324-9910 https://orcid.org/0000-0001-7649-0913 https://orcid.org/0000-0002-5922-4127 https://orcid.org/0000-0002-5470-6098 https://orcid.org/0000-0001-5895-8340 https://orcid.org/0000-0002-3822-0119 https://orcid.org/0000-0002-8172-3061 https://orcid.org/0000-0002-3632-6892 https://orcid.org/0000-0002-1837-7282 28 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license cokorda agung wahyu purnamasidhi, et al. epidemiological and clinical features introduction novel coronavirus disease 2019 (covid19) has been a worldwide phenomenon since it was initially found in china in december 2019. it was first declared as a worldwide pandemic by the world health organization (who) in march 2020.1 indonesia reported its first cases in early march 2020 and by late may 2020 the number of cases had undergone significant increase, reaching a total of 21430 cases with 1326 deaths. a study on the epidemiology of covid-19 in indonesia, which observed a total of 8211 cases between march 2nd and april 24th 2020, showed that 16% of the patients were from the age group of above 60 years old. patients from this age group were also presented with the highest mortality rate of 43.6%.2 elderly patients have been associated with a higher susceptibility to covid-19 and worse outcomes due to several reasons such as immunosenescence and comorbidities which usually worsen with aging. deterioration of pathophysiological functions in the body systems of elderly patients might also contribute to higher mortality rates.3 however, studies on the clinical features of elderly covid -19 patients and the differences between critical and noncritical elderly covid-19 patients in indonesia and even other countries are still lacking and rare. through this descriptive study, we aimed to summarize the clinical features and to provide a comparison between critical and non-critical elderly covid-19 patients who were admitted to udayana university academic hospital in hope that this could act as a reference for physicians to have a better understanding and to provide better treatment for elderly covid-19 patients in the future. materials and methods study design and setting this study was designed as an observational cross-sectional study conducted at udayana university academic hospital which serves as a health facility in jimbaran, bali and was the province’s main referral site for covid-19 patients. data collection secondary data of all elderly covid-19 patients admitted to udayana university academic hospital between april 2020 and march 2021 were collected through medical records using the method of total sampling. the inclusion criteria for our study were all elderly covid-19 patients who were aged 60 to 90 years old. although there are different ways to classify elderly age range, several studies such as the study by alterovitz and mendelsohn4 in journal of aging studies had used 60 years old as the baseline age for elderly people according to the us census bureau age divisions as well as to previous researches on physical and cognitive aspects of aging. in addition, three other studies which discussed about clinical characteristics of elderly covid-19 patients in jakarta, hunan, and hainan had also used 60 years old as the baseline age for their population of elderly patients.5–7 the patients were then divided into two categories, including critical and non-critical elderly covid-19 patients. critical patients were patients who met any of the following criteria, including patients with acute respiratory distress syndrome (ards), shock, and/or sepsis; patients who were admitted to the icu; and patients who died in a short duration upon admission, whereas non-critical patients consisted of the rest. patients were excluded if they were not aged 60 to 90 years old and/or were not diagnosed with covid-19. the collected data only consisted of epidemiological features, clinical manifestations, comorbidities, laboratory results, treatment, and patients’ outcome, whereas any data regarding patients’ identity were not disclosed due to patients’ right to privacy. patients who decided to opt out of the study were also given the opportunity to contact the authors 29 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 27–34 and all data were only accessible to the authors. this study had been approved by the ethical commission of udayana university (no. 1010/un14.2.2.vii.14/lt/2020). data analysis all statistical analyses were performed using ibm spss statistics 20. all data, except sex and prognosis, were tested using kolmogorov-smirnov z test and continued by either independent-samples t test if the asymptotic significance 2-tailed was > 0.05 or mann-whitney u test if the asymptotic significance 2-tailed was < 0.05. sex and prognosis variables were tested using chisquare test. continuous variables are described in median (interquartile range, iqr) and categorical variables are presented as n (%). results and discussion a total of 280 elderly covid-19 patients were included in this study and all patients were admitted to udayana university academic hospital, bali. the median age of all elderly patients were 65.0 years old. this finding correlates with a study in china where covid-19 had the epidemiological characteristics which commonly affect age group ranging from 30 to 79 years old.8 other specific studies on elderly covid-19 patients also showed similar results where a study in jakarta by azwar et al.5 found that the majority of its patients were aged 60 to 69 years old whereas another study in hunan by guo et al.6 found that the median age of all its elderly patients was 67 years old. more than half of the patients in this study were constituted by male patients in both critical and non-critical criterion. the data samples in several other studies from different countries were also predominantly constituted by male patients. a study in a tertiary hospital in north india by soni et al.9 reported that 57.8% of its patients were males and an even higher percentage were reported from a study in al ain hospital of united arab emirates by ismail et al.10 where 84.6% of its critical patients were also males. aside from the prevalence of covid-19 cases among males, it was also found that there was a difference in terms of fatality rate where males showed higher results.11 there are several mechanisms which may contribute to the possible correlation between male sex and a higher incidence of covid-19 cases among them. the sars-cov-2 is known to infect humans by binding to angiotensinconverting enzyme 2 (ace2) receptor. after binding to the ace2 receptor, the virus is then able to enter human tissue through cell surface fusion mediated by transmembrane protease serine 2 (tmprss2).12 in molecular perspective, ace2 gene is found to be located on the x chromosome which means there should be alleles that regulates resistance towards covid-19.11 in addition, ace2 was also found to be a constitutive product of leydig cells which is a factor affecting testosterone secretion in males. to further support this incidence, it was also found that androgen receptor activity had been considered as a factor in the transcription of tmprss2 gene, thus implying that the expression of tmprss2 modulated by testosterone might contribute to male predominance in covid-19 cases.13 despite several supporting factors which may explain male predominance in covid-19 cases, there are also several studies which had higher cases among females rather than males.6,14 30 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license cokorda agung wahyu purnamasidhi, et al. epidemiological and clinical features table 1. demographics and clinical characteristics of elderly covid-19 patients all patients (n = 280) critical (n = 51) non-critical (n = 229) p value age, years 65.0 (62.0-72.0) 68.0 (63.0-74.0) 65.0 (62.0-71.0) 0.061 sex 0.059 male 170 (60.7) 37 (72.5) 133 (58.1) female 110 (39.3) 14 (27.5) 96 (41.9) symptoms fever 276 (98.6) 51 (100) 225 (98.3) 0.344 cough 252 (90) 46 (90.2) 206 (90) 0.959 cold 46 (16.4) 2 (3.9) 44 (19.2) 0.000 sore throat 116 (41.4) 17 (33.3) 99 (43.2) 0.187 cephalgia 78 (27.9) 8 (15.7) 70 (30.6) 0.015 myalgia 90 (32.1) 10 (19.6) 80 (34.9) 0.020 diarrhea 15 (5.4) 5 (9.8) 10 (4.4) 0.223 comorbidities hypertension 132 (47.1) 17 (33.3) 115 (50.2) 0.026 heart disease 68 (24.3) 23 (45.1) 45 (19.7) 0.000 diabetes 72 (25.7) 13 (25.5) 59 (25.8) 0.968 kidney disease 12 (4.3) 2 (3.9) 10 (4.4) 0.888 malignancy 5 (1.8) 1 (2) 4 (1.7) 0.917 hiv 0 (0) 0 (0) 0 (0) autoimmune 0 (0) 0 (0) 0 (0) data are presented in median (iqr) or n (%) the study of critical patients by ismail et al.10, showed that the most common symptoms presented in its patients were coughing followed by fever. another study from china by liu et al.7 which compared the clinical features manifested in elderly patients with young patients also showed that the most common symptoms in both age groups were fever and cough. these clinical features are in accordance with our findings where both non-critical and critical patients were most commonly presented with symptoms of fever followed by coughing. while ace2 acts as an entry point for covid19, it also has a crucial anti-inflammatory role by converting angiotensin ii, which is a perpetrator of inflammation, to angiotensin 17 in the renin-angiotensin signaling system. the expression of ace2 declines with aging and in patients with cardiovascular diseases. when sars-cov-2 binds to ace2 receptors in patients, it further reduces the ace2 cell surface expression. hence, elderly covid19 patients with cardiovascular comorbidities are suspected to have a significantly low level of ace2 which contributes to the predisposition of a more severe outcome.15 a study from central sulawesi by faustine16 which focused on the severity profile of covid-19 patients with hypertension concluded that there was no significant correlation between high blood pressure and the severity and mortality of covid-19 patients. on the other hand, it was stated that cardiovascular comorbidities other than hypertension were associated with the severity of covid-19.16 another study stated that the presence of heart lesion in covid19 patients was associated with poor prognosis where these patients were five to ten times more at risk. the cardiac manifestations found were predominated by acute myocardial damage.17 these findings may provide an explanation to the difference found in terms of the most common comorbidity suffered by the elderly patients in our study where hypertension was more prevalent in non-critical elderly patients whereas heart disease was more prevalent in critical elderly patients. all data on demographics and clinical characteristics are presented in table 1. 31 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 27–34 table 2. laboratory results of elderly covid-19 patients all patients (n = 280) critical (n = 51) non-critical (n = 229) p value hemoglobin, d/dl 13.2 (12.1-14.1) 13.7 (11.9-14.3) 13.2 (12.1-14.0) 0.967 hematocrit, % 39.0 (36.1-41.5) 39.1 (35.5-42.4) 38.9 (36.2-41.5) 0.718 leukocyte count, x103µl 6.77 (5.1-8.8) 8.1 (6.1-12.4) 6.3 (4.9-8.3) 0.000 neutrophil count, x103µl 4.5 (3.2-6.6) 6.9 (4.4-10.5) 4.2 (3.1-5.9) 0.000 lymphocyte count, x103µl 1.2 (0.8-1.7) 0.7 (0.5-1.2) 1.35 (0.9-1.9) 0.000 neutrophil-to-lymphocyte ratio 3.4 (2.0-5.7) 10.0 (4.7-16.1) 3.1 (1.9-4.6) 0.000 platelet count, x103µl 210.5 (165.0-272.0) 187.0 (163.0-234.0) 219.0 (165.5-276.0) 0.037 liver function sgot, u/l 33.0 (26.0-50.0) 52.0 (34.3-70.0) 31.0 (25.0-45.0) 0.000 sgpt, u/l 29.0 (21.5-47.0) 44.5 (28.0-67.5) 28.0 (20.0-40.5) 0.000 kidney function blood urea nitrogen, mg/dl 15.0 (11.0-21.0) 20.0 (15.0-30.5) 14.0 (11.0-19.0) 0.000 creatinine, mg/dl 0.8 (0.6-1.1) 0.9 (0.7-1.2) 0.8 (0.6-1.0) 0.122 random blood sugar, mg/dl 114.5 (97.0-152.0) 121.0 (107.0-152.0) 114.0 (96.0-152.0) 0.959 data are presented in median (iqr) or n (%) similar to our study, the study by faustine et al.16 found that most patients’ laboratory test showed an increase in neutrophils level but a decrease in lymphocytes level. according to several other studies, majority of the covid-19 cases also displayed low lymphocytes level, especially in critical patients. the sars-cov-2 virus is known to induce the manifestation of cytokine storm during infection which causes an excessive inflammatory reaction. the persistent stimulation in this phenomenon may lead to a reduction in lymphocytes.18 the higher value of neutrophil and lower value of lymphocyte in critical patients when compared to the lower value of neutrophil and higher value of lymphocyte resulted in a disparity of nlr value where the median nlr in critical patients was significantly higher. the value of nlr was found to be constantly higher in severe covid-19 patients in several other studies. a study which focused on the predictive values of nlr found that nlr has good specificity and sensitivity, thus makingit a good predictive value on the severity and mortality of covid-19 patients.19 the median platelet level of critical patients was found to be approximately 32 x 103/µl lower than non-critical patients in this study. mild thrombocytopenia had been reported as one of the laboratory findings in 58-95% of severe covid-19 cases. viral infections are able to cause thrombocytopenia through various causes. the development of thrombocytopenia in response to viral infections is generally mediated via enhanced platelet clearance. viruses are also known to interact with megakaryocytes and reduce platelet synthesis.20 the correlation between elevated levels of sgot and sgpt in liver function test and covid-19 is still a subject of debate and needs further investigation.21 direct cytopathic effect may not be the main mechanism for sars-cov-2 to induce liver damage since ace2 receptors are found to be more abundant in cholangiocytes than in hepatocytes.22 however, other factors such as covid-19-induced cytokine storm, sepsis, or drug-induced liver injury should be considered as possible mechanisms of covid19-related liver injury. in addition, covid19 may also worsen underlying chronic liver disease which contributes to a higher mortality outcome.23 the disparities of kidney function test in median urea between critical and non-critical patients may be due 32 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license cokorda agung wahyu purnamasidhi, et al. epidemiological and clinical features to the role of covid-19 in causing kidney damage. the infection of sars-cov-2 may contribute to the impairment of kidney through multiple mechanisms. the viral load of sars-cov-2 was found to be able to directly induce cytotoxicity of renal resident cell. symptoms manifested in covid-19 patients such as fever, vomiting, diarrhea, and shock could also cause kidney hypoperfusion. in addition, the cytokine storm induced by sars-cov-2 should also be considered.24 all data on laboratory results are presented in table 2. table 3. treatment and outcomes of elderly covid-19 patients all patients (n = 280) critical (n = 51) non-critical (n = 229) p value treatment antiviral therapy 19 (6.8) 5 (9.8) 14 (6.1) 0.353 antibiotics 209 (74.6) 35 (68.6) 174 (76) 0.035 vitamin c 262 (93.6) 43 (84.3) 219 (95.6) 0.038 anticoagulants 48 (17.1) 14 (27.5) 34 (14.8) 0.066 prognosis 0.000 discharge 243 (86.8) 15 (29.4) 228 (99.6) death 37 (13.2) 36 (70.6) 1 (0.4) data are presented in median (iqr) or n (%) a significantly higher overall mortality rate in elderly covid-19 patients was found in this study and the study conducted by azwar, et al.5 in jakarta when compared to the study by guo et al.6 in hunan, china. the mortality rate in our study and the one in jakarta was 13.2% and 23% respectively in contrast to the only 2.9% in hunan.5,6 there are several factors that we supposed may have contributed to higher mortality rate in indonesian studies when compared to china. these factors included the lower usage of antiviral therapy and choice of antibiotics administered to patients. only 6.8% from all the elderly patients in our study were given antiviral therapy with the choice of either aluvia (lopinavir-ritonavir), favipiravir, or remdesivir. the administration of antibiotics was much higher with the choice of either azithromycin, levofloxacin, or combination of both. in comparison, 93.3% of the elderly patients in hunan were given antiviral therapy and the most common antibiotic administered was moxifloxacin.6 a study by wu et al.25 found that patients experiencing mild symptoms received earlier initiation of antiviral therapy, thus indicating that early and timely administration of antiviral therapy may contribute to the slowing of covid-19 progression into a more severe state and may improve the prognosis of patients under care. in a randomized controlled trial, which assessed the clinical efficacy and safety of moxifloxacin compared to levofloxacin plus metronidazole in treating community-acquire pneumonia (cap), it was found that moxifloxacin monotherapy was more effective in treating cap with a clinical cure rate of 76.7% compared to 51.5% in the levofloxacin plus metronidazole group. the administration of moxifloxacin also showed lower incidence of adverse events with a more convenient dosing regimen.26 all data on the treatments and outcomes of patients are presented in table 3. strengths and limitations the strengths of this study were being the first few studies to focus on the clinical profile of elderly covid-19 patients and to further compare the characteristics of critical and non-critical patients in indonesia and even southeast asia. the limitations of this study were not including the data of the second wave of covid-19 in indonesia, which was predicted to have higher 33 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 27–34 mortalities, and not further classifying the elderly age group into youngest-old (65-74 years), middle-old (75-84 years), and oldestold (≥ 85 years) for a more in-depth comparison. conclusions in conclusion, elderly patients are more susceptible to develop a severe outcome with covid-19. there is a diverse number of possible factors which affect bodily functions in elderly patients and contribute to the progression of the disease. hence, a multimodal approach in the treatment of elderly covid-19 patients is very essential. the higher mortality rate in elderly patients should be able to be reduced by giving early and timely antiviral therapy with the addition of effective choice of drugs. acknowledgement the authors would like to acknowledge the director of udayana university academic hospital dr. dr. i dewa made sukrama, m.si, sp.mk (k) and the dean of faculty of medicine udayana university dr. dr. komang januartha putra pinatih, m.kes. ethical clearance the research protocol was approved by the ethical commission of udayana university (no. 1010/un14.2.2.vii.14/lt/2020). funding this paper did not receive nor use any sorts of funding before, during, and after its making. conflict of interest the authors declared that there was no conflict of interests that might bias or fabricate the information and work stated within the paper. author contribution study design and data collection: cawp. clinical advice and data collection: ikas. data analysis and report writing: dj and rcs. report writing and manuscript review: ikha, gvp, igwp, j, and pkw. manuscript review and revision: dafps and ignasd. references 1. who. who director-general’s opening remarks at the mission briefing on covid-19 12 march 2020 [internet]. world health organization. 2020 [cited 2022 jul 16]. available from: https://www.who.int/directorgeneral/speeches/detail/who-director-general-sopening-remarks-at-the-mission-briefing-oncovid-19---12-march-2020 2. hikmawati i, setiyabudi r. epidemiology of covid-19 in indonesia: common source and propagated source as a cause for outbreaks. j infect dev ctries. 2021;15(5):646–52. 3. perrotta f, corbi g, mazzeo g, boccia m, aronne l, d’agnano v, et al. covid-19 and the elderly: insights into pathogenesis and clinical decision-making. aging clin exp res [internet]. 2020;32(8):1599–608. available from: https://doi.org/10.1007/s40520-020-01631-y 4. alterovitz ssr, mendelsohn ga. relationship goals of middle-aged , young-old , and old-old internet daters : an analysis of online personal ads. j aging stud [internet]. 2013;27(2):159–65. available from: http://dx.doi.org/10.1016/j.jaging.2012.12.006 5. azwar mk, setiati s, rizka a, fitriana i, saldi srf, safitri ed. clinical profile of elderly patients with covid-19 hospitalised in indonesia’s national general hospital. acta med indones. 2020;52(3):199–205. 6. guo t, shen q, guo w, he w, li j, zhang y, et al. clinical characteristics of elderly patients with covid-19 in hunan province, china: a multicenter, retrospective study. gerontology. 2020;66(5):467–75. 7. liu k, chen y, lin r, han k. clinical features of covid-19 in elderly patients: a comparison with young and middle-aged patients. j infect [internet]. 2020;80(6):e14–8. available from: https://doi.org/10.1016/j.jinf.2020.03.005 8. pericàs jm, hernandez-meneses m, sheahan tp, quintana e, ambrosioni j, sandoval e, et al. 34 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license cokorda agung wahyu purnamasidhi, et al. epidemiological and clinical features covid-19: from epidemiology to treatment. eur heart j. 2020;41(22):2092–108. 9. soni sl, kajal k, yaddanapudi ln, malhotra p, puri gd, bhalla a, et al. demographic & clinical profile of patients with covid-19 at a tertiary care hospital in north india. indian j med res [internet]. 2021;115–25. available from: http://www.ncbi.nlm.nih.gov/pubmed/23144490 10. ismail k, bensasi h, taha a, nazir a, abdelkhalek m, mohamed w, et al. characteristics and outcome of critically ill patients with coronavirus disease-2019 (covid19) pneumonia admitted to a tertiary care center in the united arab emirates during the first wave of the sars-cov-2 pandemic. a retrospective analysis. plos one [internet]. 2021;16(10 october):5–17. available from: http://dx.doi.org/10.1371/journal.pone.0251687 11. tay mz, poh cm, rénia l, macary pa, ng lfp. the trinity of covid-19: immunity, inflammation and intervention. nat rev immunol [internet]. 2020;20(6):363–74. available from: http://dx.doi.org/10.1038/s41577-020-0311-8 12. jackson cb, farzan m, chen b, choe h. mechanisms of sars-cov-2 entry into cells. nat rev mol cell biol. 2022;23(1):3–20. 13. pozzilli p, lenzi a. commentary : testosterone , a key hormone in the context of covid-19 pandemic. 2020;(january):19–21. 14. fauzar f, kurniati r, abdullah f, kam a. the profile of covid-19 patients in semen padang hospital indonesia. j kesehat andalas. 2021;10(1):45. 15. alghatrif m, cingolani o, lakatta eg. the dilemma of coronavirus disease 2019, aging, and cardiovascular disease insights from cardiovascular aging science. jama cardiol. 2020;5(7):747–8. 16. faustine i, malik a, andrajati r, wanandi si. clinical characteristics and severity profile of covid-19 patient with hypertension in palu, central sulawesi. indones j pharm. 2021;32(4):563–72. 17. zakhama l, yahia f, ben abdelaziz a, maghrebin prp r, faten yahia c. covid-19 and cardiovascular diseases. scoping review study. tunis med [internet]. 2020;98(04):283– 94. available from: https://www.researchgate.net/publication/34135 5098 18. zhang hj, qi gq, gu x, zhang xy, fang yf, jiang h, et al. lymphocyte blood levels that remain low can predict the death of patients with covid-19. med (united states). 2021;100(28). 19. li x, liu c, mao z, xiao m, wang l, qi s, et al. predictive values of neutrophil-to-lymphocyte ratio on disease severity and mortality in covid-19 patients: a systematic review and meta-analysis. crit care [internet]. 2020;24(1):1–10. available from: https://doi.org/10.1186/s13054-020-03374-8 20. wool gd, miller jl. the impact of covid-19 disease on platelets and coagulation. pathobiology. 2021;88(1):15–27. 21. moon am, barritt as. elevated liver enzymes in patients with covid-19: look, but not too hard. dig dis sci [internet]. 2021;66(6):1767–9. available from: https://doi.org/10.1007/s10620020-06585-9 22. xu w, huang c, fei l, li q, chen l. dynamic changes in liver function tests and their correlation with illness severity and mortality in patients with covid-19: a retrospective cohort study. clin interv aging. 2021;16:675–85. 23. jothimani d, venugopal r, abedin mf, kaliamoorthy i, rela m. covid-19 and the liver. j hepatol. 2020;73(5):1231–40. 24. liu yf, zhang z, pan xl, xing gl, zhang y, liu zs, et al. the chronic kidney disease and acute kidney injury involvement in covid-19 pandemic: a systematic review and metaanalysis. plos one [internet]. 2021;16(1 january):1–13. available from: http://dx.doi.org/10.1371/journal.pone.0244779 25. wu j, li w, shi x, chen z, jiang b, liu j, et al. early antiviral treatment contributes to alleviate the severity and improve the prognosis of patients with novel coronavirus disease (covid-19). j intern med. 2020;288(1):128–38. 26. sun ty, sun l, wang rm, ren xp, sui dj, pu c, et al. clinical efficacy and safety of moxifoxacin versus levofoxacin plus metronidazole for community-acquired pneumonia with aspiration factors. chin med j (engl). 2014;127(7):1201–5. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 �� vol. 1. no. 1 january–april 2010 research report ttc repeats variation of mycobacterium leprae isolates for analysis of leprosy transmission in leprosy endemic area in east java, indonesia dinar adriaty, ratna wahyuni, iswahyudi, indropo agusni, and shinzo izumi leprosy study group, institute of tropical disease, airlangga university abstract east java province still has some pocket of leprosy endemic areas. in order to solve the problem, molecular typing will make it feasible to study the transmission pattern of mycobacterium leprae in leprosy endemic area. the present study is to analyze the presence of m.leprae dna in the environment and to study variation number of ttc repeats and their distribution. poteran island is located in madura, east java and was chosen because this island has a high prevalence of leprosy and remains stable for the last five years. all samples were analyzed by pcr and the numbers of ttc repeats were confirmed by direct sequencing. of all collected samples, 26.4% isolates of water resources (24); 61.9% nasal swabs (26); and 35.3% skin tissues (24) are positives. no statistically difference in the pattern distribution of ttc repeats between skin tissues of patients and nasal swab of households contact (p=0.594); also distribution of ttc repeats between skin tissues of leprosy patients and those of water resources (p=0.441); and distribution of ttc repeats between nasal swab of households contact with water resources (p=0.906). it means that the transmission of m.leprae in leprosy endemic area has closely related in 3 aspects: agent, host & environment. key words: ttc repeats, mycobacterium leprae , leprosy transmission, endemic area, east java introduction leprosy is a chronic infectious disease caused by mycobacterium leprae and is still a major health problem in the developing countries of asia, latin america and africa. indonesia is one of asian countries that also have problems with leprosy elimination. it is commonly believed that the humans (multibacillary patients) are the host and reservoir of m.leprae and global efforts to control leprosy by distributing a multidrug therapy (mdt) regimen should eliminate leprosy. however, after following the leprosy elimination program, the prevalence rates in indonesia has reduced into 0.84 per 10.000 inhabitants, but in the contrary new cases of leprosy, has remained unchanged over the last 10 years.1 there are many hyper endemic areas distributing in several provinces especially in the eastern part of indonesia, which called pocket area. east java province is the one of many provinces that has some pockets areas of leprosy. until in the middle of 2004, prevalence rate in east java province still 1,39 per 10.000 inhabitants distribute to 38 districts with 4298 registered cases. from whole districts in east java province, sampang has the highest prevalence rate (6,41), followed by sumenep (6,29), pamekasan (4,01), lamongan (3,94) and tuban (3,54 per 10.000 inhabitants).2 sumenep is one of endemic areas which have many hyper endemic areas especially in the islands region that still isolated from outsider. one of them is poteran island that has 39.479 inhabitants living in 8 villages with prevalence of leprosy is 24,1 per 10.000 inhabitants. poteran island was chosen because this island has a high prevalence of leprosy and remains stable for last five years.3 it has been difficult to identify sources of infection of leprosy because of the protracted incubation period preceding clinical disease made natural history of the disease unclear although port of entry and port of exit of the bacilli via the nasal passages have been proposed. this condition was aggravated by the fact that m.leprae remains uncultivable on artificial media.4 these factors have slowed our understanding about the route of transmission of m.leprae which could inhibit our ability to target drug therapy campaigns and to improved control strategies. humans are considered to be the principal reservoir. the disease is thought to be spread most effectively through ��adriaty, et al.: ttc repeats variation of mycobacterium leprae long-term, intimate contact with an infected individual, but the majority of new cases presenting in some area in indonesia are unable to relate any close association with another person who had leprosy.5 in order to understand the problems, molecular typing would be a great value to study the transmission pattern and geographical distributions of m.leprae for epidemiological investigation. the aim of this study is to detect the presence of m.leprae in environment of endemic leprosy area and to analyze the variation number of ttc repeats and their distribution in leprosy endemic area especially in east java province. it has been possible to recognize potential polymorphic sites from the genome sequence of m.leprae. as is the case in several eukaryotic and prokaryotic genomes that have been sequenced, short stretches of dna that occur in tandem repeats are also found in m.leprae.6 matsuoka7 first reported that 6-bp sequence (gacatc) was found as two alleles in the rpot gene of m.leprae. this was followed by the recognition of variable number tandem repeats (vntrs) of the ttc triplet in a noncoding region of the m.leprae cosmid mlcb2407 (genbank accession no. al023596).8 according to the report from shin,9 the gene location of the ttc repeats were not found in mycobacterium tuberculosis, mycobacterium avium, mycobacterium marinum, or human tissues, which indicated their specificity to m.leprae. truman10 also report the stability of the ttc (vntr) by testing this gene from m.leprae that obtained from armadillo and nude mice tissues and investigated for 121 months, so this gene is reliable as a marker of strain differentiation for epidemiological investigations of leprosy. materials and methods mycobacterium leprae isolates and preparation of genomic dna a total of 201 m.leprae isolates were collected and divide into 3 groups: 91 water sources, 42 nasal swabs of household contact and 68 slit-skin tissues of leprosy patients. water samples. water samples were collected from one village in poteran island, sumenep, madura, east java. all water came from natural sources. no piped water supply is available in this village. well water was used for drinking and bathing in this area. one well usually used for several houses surround it. samples that were collected were being kept cool until preparing the templates. for preparation, 10 ml sample centrifuge at 4000g for 10 min. discard supernatant and take filtrate to sterile 1.5 ml tube and again centrifuge at 10.000g for 20 min in 4ºc and pellet were collected.11 slit-skin specimens. samples were obtained from leprosy patients in the village. we collected all patients who are villagers of that area categorized as multibacillary (mb), pausibacillary (pb) cases based on a criteria of who (world health organization). slit-skin smear specimens were collected from the skin lesion of patients in the same manner as the routine slit-skin smear test for bacterial index examination. the samples on the disposable surgical blade was soaked into 70% ethanol and kept in a refrigerator until use. the bacilli were removed from the blade and collected as a pellet by centrifugation at 10.000g for 20 min in 4º c until serving and then washed with phosphate-buffered saline when doing isolation.12 nasal swabs specimens. nasal swabs were collected from healthy villagers that lived in that area including household contact (persons that live in the same house with the patients). nasal swabs were taken by using a sterilized cotton-tipped and made it wet by phosphate-buffered saline and swabs were kept in freezer until use. the bacilli were removed by gently rubbing the swab into 1.5 ml sterile tube which contained sterile distilled water (0.6 ml) and centrifuge at 10.000g for 20 min in 4ºc. all isolates were prepared for dna extraction by treatment qiagen qiaprep spin miniprep kit cat. no. 27106 as mentioned in qiagen protocol.13 mycobacterium leprae detection detection of m.leprae dna. to identify m.leprae, the 18 kda antigen m.leprae in regio rlep3 repetitive element (x17153) (14) was chosen to amplify by nested pcr. amplification will produce about 129 bp for external (outer) and 99 bp for internal (inner) product. pcr was carried out using a premix g mixture of failsafe pcr system cat.no.fsp995g (epicentre, madison, wi, usa) in a 20 µl volume of reaction mixture containing at least 0.1 pg of genomic dna in 2µl of template dna solution and 2 µl of 5 µm primers using failsafe pcr enzyme taq mix 250 u@2.5 u/µl cat. no. fs99250. primers lp1 5’ tgcatgtcatggccttgagg 3’ and lp2 5’ caccgataccagcggcagaa 3’ were produced by takara (japan) and the amplification was done in a thermal cycler machine (biorad i-cycler) under the conditions of 2 min at 98º c for preheating, 20 sec at 98º c for denaturation, 30 sec at 56º c for annealing and 30 sec at 72º c for elongation/extension repeated for 35 cycles followed by prolong extension of 5 min at 72º c then inactivation at 4º c. amplicon was then being nested with primers lp3 5’ tgaggtgtcggcgtggtc 3’ and lp4 5’ cagaaatggtgcaaggga 3’ under the conditions of 2 min at 98º c for preheating, 20 sec at 98º c for denaturation, 30 sec at 56ºc for annealing and 30 sec at 72º c for elongation/extension repeated for 30 cycles followed by prolong extension of 5 min at 72 ºc then inactivation at 4º c. the full length of this amplicon were separated by electrophoresis in 3% hs agarose gel code no. 312-01431 (cambrex bioscience, rockland, me, usa) using tbe (tris/boric/edta, ph 8.0) buffer at 100 v. all the positives samples were continued to genotyping analysis. �0 indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 38-43 genotyping of ttc repeats pcr amplification. pcr was carried out as described before and make it in a 50 µl volume of reaction mixture containing at least 0.1 pg of genomic dna in 5µl of template dna solution and 2 µl of 5 µm primers. primers ttc-a (5’ggacctaaaccatcccgttt3’) and ttc-b (5’ctacagggggcacttagctc3’) were used for amplification and pcr will produce about 200bp amplification product. the amplification was done in a thermal cycler machine (biorad i-cycler) under the conditions of 2 min at 98º c for preheating, 20 sec at 98º c, 30 sec at 58º c, 30 sec at 72º c for 40 cycles followed by prolong extention of 5 min at 72º c then inactivation at 4º c. the full length of this amplicon were separated by electrophoresis in 3% nusieve gtg agarose gel cat no. 50080 (cambrex bioscience, rockland, me, usa) using tae (tris/acetate/edta, ph 8.0) buffer at 100 v. sample preparation for sequence analysis. the numbers of ttc repeats were confirmed by direct sequencing. dna samples for sequencing were recovered by gfxtm pcr, dna and gel band purification kits (amersham biosciences/ ge healthcare) with product code: 27-9602-01 according to the manufacture’s manual (amersham, 2002). before sequencing reaction, the quantity and quality of purified dna was examine by uv spectrophotometer. dual cydyetm terminator sequencing kits cat. no 258226-01 (amersham biosciences/ ge healthcare) was used in the preparation of sequencing reaction. the mixture for cycle sequencing (labeling) was performed according to the manufacture’s manual. the sequencing reaction was also done in a thermal cycler machine (biorad i-cycler) under the following condition : 20 sec at 95° c, 15 sec at (tm of sense primer + 3) °c , 1 min at 70° c and repeat for 35 cycles. the sequencing product was then purified by ethanol precipitation and dried followed by dissolving in 2 µl of loading dye and was loaded into prepared acrylamide gel in long-read towertm system version 3.01. sequence analysis was done using long-read towertm system15 with the temperature was set on 60°c as described as in the protocol. results and discussions detection of m.leprae dna out of 24 isolates of water resources (26.4%); also 26 nasal swabs (61.9%); and 24 skin tissues (35.3%) showed the 99 bp pcr products of the 18 kda antigen m.leprae and indicated that samples contained m.leprae (fig.1). the pcr positives results are mostly from nasal swabs taken from healthy villagers. nasal mucosa is reported as a port of entry for m.leprae surrounds environment. it means the leprosy transmission in a highly endemic area is very active. the fact that water and nasal mucosa samples give many positive results indicates that environmental sources play an important role in m.leprae infection and transmission of the disease other than patients.11 figure1. pcr products of m.leprae detection. lane 1 : the dna size marker of 20bp ladder ; lane 2-15 : isolates from poteran island; lane 16 : nc, negative control; lane 17 : pc, positive control (m.leprae strain thai53) ttc repeats distribution of m.leprae in poteran island according to the report from shin,9 the gene location of the ttc repeats were not found in mycobacterium tuberculosis, mycobacterium avium, mycobacterium marinum, or human tissues, which indicated their specificity to m.leprae. truman10 also report the stability of the ttc (vntr) by testing this gene from m.leprae that obtained from armadillo and nude mice tissues and investigated for 121 months, so this gene is reliable as a marker of strain differentiation for epidemiological investigations of leprosy. all the positives samples which have positive pcr according to the 18kda detection were then continued to be analyzed by ttc repeats genotyping. all samples were amplified by primers that recommended by matsuoka.16 amplicon has sizes about 200bp and m.leprae strain thai53 was used as positive control. other samples are varying as seen in fig.2. figure 2. pcr product of ttc repeats. samples were: lane 1, the dna size marker of 100bp ladder; lane 2,3,4,5, isolates from poteran island; nc, negative control; pc, positive control (m.leprae strain thai-53) ��adriaty, et al.: ttc repeats variation of mycobacterium leprae figure 3. ttc repeat amplified product after sequencing repeat region 80...121 (ttc-14 copy belong to m.leprae strain thai-53) table 1. frequency (%) of ttc genotypes in each isolates no. of repeats slit-skin specimens nasal swabs water sources ttc-9 0 0 4 ttc-10 20 10 8 ttc-11 32 42 36 ttc-12 4 4 0 ttc-13c-13 8 4 12 ttc-14 16 28 32 ttc-15 0 0 4 ttc-16 4 0 0 ttc-17 0 4 0 ttc-24 4 0 0 ttc-28 4 0 0 ttc-40 4 0 0 ttc-44 0 8 0 ttc-49 0 0 4 ttc-60 4 0 0 total 100% (24 cases) 100% (26 cases) 100% (24 cases) the copy number of ttc repeats in poteran island varied from 9 to 60 copies (table 1). the 11-copy ttc genotype was the most frequent in all samples. our report and mostly from south east asian region such as the m.leprae strain thai-53 from thailand (ttc-14 copy) and from philippines (mostly ttc-14, followed by ttc-24 and ttc-25 copies) that has been reported by shin et al.,9 all isolates have a short tandem repeats of ttc and this was the same with the isolates from latin america countries that commonly have ttc-10 copy.8 it is different than the isolates that found in the africa and india which are have longer repeated (m.leprae strain tamil nadu india has ttc-21 copy; m.leprae strain ethiopia has ttc-29 copy). based on these molecular typing, it could be related with the origin of leprosy that came from indian subcontinent and from india, leprosy is thought to have spread to china, japan reaching pacific islands until america as described by monot.17 after collected the data from poteran island and analyzed by non-parametric (kolmogorov-smirnov) test, we concluded that no statistically difference in the pattern distribution of ttc repeats between skin tissues of patients and nasal swab of households contact (p=0.594); also there is no statistically difference in distribution of ttc repeats between skin tissues of leprosy patients and water resources (p=0.441); and no statistically difference in distribution of ttc repeats between nasal swab of households contact with water resources (p=0.906). it could be concluded that the existence of mycobacterium leprae in the leprosy endemic area has closely related in 3 aspects: agent, host and environment and this mode of transmission might be the problems of leprosy elimination in a highly endemic leprosy area. table 2. example of ttc genotypes diversity in a multicase family in poteran island location family member relationship ttc repeat nasal swab slit skin spec. water resources house 1 : well no.1 mb patient husband 10 10 household contact wife 11 household contact daughter a suspect leprosy son 11 household contact mother 10 household contact sister in law 11 household contact mother in law 10 household contact father in law 11 house 2 : (neighbourhood) mb patient son 11 11 household contact mother 11 household contact father 10 household contact son household contact son 11 house 3 : mb patient daughter 11 11 (neighbourhood) household contact aunt 11 household contact mother 14 well no. 2 14 a _ , absence of data as a result of insufficient material or failure to amplify a pcr product. �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 38-43 targeted analysis of multicases family demonstrated (table 2) that the microsatellite profile was conserved in the context of a presumed transmission link, and the pattern observed for the overall patient population suggests that the continuing incidence of leprosy in this community was the result of a complex series of transmission events. further studies of genetic diversity in samples with known epidemiological links will be important in establishing the extent to which microsatellite mapping can be used as a reliable marker for longer transmission chains. in addition to further exploration of microsatellite diversity, it will be important to search for other forms of genetic variation suitable for strain typing; a systematic screening for single nucleotide polymorphisms may be useful, for example. important goals will be to identify typing systems capable of providing reliable information about the m.leprae transmission and to use these to assist in the search for interventions that will reduce the number of new cases of leprosy.21 founding of m.leprae dna in water resources is also interesting we can see acid fast bacilli in some water samples and several of them are pcr positives with primers specific of m.leprae genome ( fig. 4). figure 4. poteran water source with acid fast bacilli positive by ziehl nielsen staining (100×× magnification) the results seems strongly suggest water as a probable source of infection in leprosy endemic area other than patients. water borne infection or presence of m.avium, m.ulcerans, m.marinum, m.kansasii, m.intrcellulare, m.scrofulaceum, m.chelonae and m.fortuitum in water has been reported.11 detection of m.leprae from water at the place where leprosy was previously endemic has also been reported.18 because of these findings, water was assumed to be the most likely reservoir of the bacilli. in this study, pcr technique was applied for the detection of m.leprae dna and detection of dna itself does not necessarily mean the existence of live bacilli. since m.leprae has known as an obligate intracellular, the existence of live bacilli could be found as saprophytes, commensalisms and symbionts in the environment with any other microorganism. it has been reported that m.avium enters and replicates in the amoebae,19 also m.leprae has been found in acanthamoeba.20 therefore, the significance of the presence of m,leprae in the water (environment) as another source of infection will be observed in the future study. conclusions we found the presence of m.leprae dna from environment as well as the households contact. it means that environment has influence to become a competent reservoir of the leprosy transmission. in the m.leprae strains of all isolates that has collected from poteran island, east java, m.leprae with ttc-11 copies was most common among all strains and from the statistic analysis we conclude that environment has a great influence to a leprosy transmission besides humans. all the information shows that although the results seems strongly suggest water as a probable source of infection, more investigation still need to explain the existence of live bacilli and how can they infect to human. however, this information has benefit in order to show that the leprosy eradication program which is still ongoing, such as early detection of m.leprae, prevention, promotion among the inhabitants in endemic leprosy area and it must be continuously conducted by public health official. acknowledgements this work was financially supported by jica and leprosy ngo from japan. thank you for all the attentions. we are grateful to all people and paramedics in poteran island for all kindness, cooperation and support in this observation. references 1. world health organization. 2006. who expert committee on leprosy. 12th ed. who technical report series . no. 874. 2. dinas kesehatan jawa timur. 2006. laporan kusta tahun 2005. dinkes jatim. surabaya. 3. dinas kesehatan kabupaten sumenep. 2006. laporan kusta tahun 2005. dinkes kabupaten sumenep. 4. rees, rjw; young, db. 1994. the microbiology of leprosy. in: hastings,rc. leprosy. churchillivingstone. edinburg. p 47–98. 5. fine, p.e.m.1982. leprosy:the epidemiology of a slow bacterium. epidemiol. rev. 4:161–187. 6. young, sk; taylor, gm; jain, s; suneetha, lm; sunetha, s; lockwood, dnj and young, db. 2004. microsatellite mapping mycobacterium leprae population in infected humans. j. clin. microbiol. 42: 4931–4936. 7. matsuoka, m; zhang, l; budiawan, t; saeki, k and izumi, s. 2004. genotyping of mycobacterium leprae on the basis of the polymorphism of ttc repeats for analysis of transmission. j. clin. microbiol. 42(2): 741–745. 8. matsuoka, m; zhang, l; morris, mf; legua, p and wiens, c. 2005. polymorphism in the rpot gene in mycobacterium leprae isolates obtained from latin american countries and its possible ��adriaty, et al.: ttc repeats variation of mycobacterium leprae correlation with the spread of leprosy. fems. microbiol. letters. 243: 311–315. 9. shin, yc; lee, h; walsh, gp; kim, jd. and cho, sn. 2000. variable of ttc repeats in mycobacterium leprae dna from leprosy patients and use in strain differentiation. j. clin. microbiol. 38 (12):38 (12): 537–544. 10. truman, r; fontes, ab; miranda, ab; suffys, p. and gillis, t. 2004. genotypic variation and stability of four variable-numbergenotypic variation and stability of four variable-number tandem repeats and their suitability for discriminating strain of mycobacterium leprae. j. clin. microbiol. 42: 2558–2565. 11. matsuoka, m; izumi, s; budiawan t; nakata, n. and saeki, k. 1999, mycobacterium leprae dna ini daily using as a possible source of leprosy infection. indian journal of leprosy. 71 (1) 61–67. 12. izumi, i; budiawan, t; saeki, k; matsuoka, m; kawatsu k.1999. an epidemiological study on mycobacterium leprae infection and prevalence of leprosy in endemic villages by molecular biologila technique. indian j lepr. vol.71(1) 37–43. 13. qiagen. 2005. qiaprep ® miniprep handbook. usa. 14. donoghue h.d.; holton j. and spigelman m. 2002. pcr primers that can detect low levels of mycobacterium leprae dna. j. med. microbiol. vol 50. p.177–182. 15. amersham biosciences. 2002. long read tower dna sequencing ver.3.01 (manual). usa 16. matsuoka, m; maeda, s; kai, m; nakata, n; chae, gt, gillis, tp; kobayashi, k; izumi, s; kashiwabara, y. 2000. mycobacterium leprae typing by genomic diversity and global distribution of genotypes. int. j. lepr. 68(2): 122–128. 17. monot, h; honore, n; garnier, t; araoz, r; coppee, jy; lacroix, c; sow, s; spencer, js; truman, rw; williams, dl; gelber, r; virmond, m; flageul, b; cho, sn; ji, b; mondolfi, ap; convit, j; young, s; fine, pe; rasolofo, v; brennan, pj. and cole, st. 2005. on the origin of leprosy. science. 308: 1040–1042. 18. kazda, j. 1981. occurance of non-cultivable acid-fast bacilli in the environment and their relationship to mycobacterium leprae. lepr. rev. suppl 1: 85–91. 19. cirillo, jd; falkow, s; tomkins, ls and bermudez, le. 1997. interaction of mycobacterium avium with environmental amoebae enhance virulence. infect. immun. 65: 3759–3767. 20. jadin, j.b. 1975. amoebes limax: vecteurs possibles de mycobacteriesamoebes limax: vecteurs possibles de mycobacteries et de m. leprae. acta leprol. 59: 57–69. 21. groathouse, na; rivoire, b; kim, h; lee, h; cho, sn; brennan, pj. and vissa, vd. 2004. multiple polymorphic loci for molecular typing of strains of mycobacterium leprae. j. clin. microbiol. 42: 1666–1667. ijtid vol 1 no 1 jan-apr 2010.40.pdf ijtid vol 1 no 1 jan-apr 2010.41.pdf ijtid vol 1 no 1 jan-apr 2010.42.pdf ijtid vol 1 no 1 jan-apr 2010.43.pdf ijtid vol 1 no 1 jan-apr 2010.44.pdf ijtid vol 1 no 1 jan-apr 2010.45.pdf 59 vol. 6. no. 3 september–december 2016 case report s e z a r y s y n d r o m e m i m i c k i n g g e n e r a l i z e d p s o r i a s i s vulgaris eko rianova lynoora1a, rahmadewi2 1 dept. of dermato-venereology, medical faculty, universitas airlangga 2 dr. soetomo general hospital – surabaya a corresponding author: lynoora@gmail.com abstract sezary syndrome is the leukemic variant of cutaneous t cell lymphoma. this disease is characterized by some reddish patches or plaques all over the skin which extends to the whole body into erythroderma, lymphadenopathy. it is also indicated by the presence of atypical lymphocytes called sezary cells. this case report is aimed to know clinical manifestation, examination and management of sezary syndrome which clinically resembles generalized psoriasis. a 60 years old man came with scaly reddish brown plaques almost all over his body. it was accompanied by lymphadenopathy on the supraclavicular lymph node right and left as well as intense itchy. other clinical features were alopecia, palmoplantar hyperkeratosis, onychodysthropy, facies leonine without anesthesia on the lesion and enlargement of peripheral nerve. from a laboratory test, an increase in the number of leukocytes and, sezary cells were found in peripheral blood smear examination; while the histopathology showed focal athrophy and acanthosis of the epidermis and dense infiltration of lymphocytes in the dermo-epidermal junction and superficial dermis. patient received 3 x 5 mg (1 cycle) of methotrexate (mtx) with 0,1% cream mometasone furoate and 3x1 tablet of ctm for adjunctive therapy. methotrexate was discontinued because there was a disturbance in liver function and deterioration of patient’s condition. after 25 days of treatment, the patient got sepsis and then passed away. early onset of sezary syndrome in this case is difficult to know because the clinical manifestation is similar with psoriasis vulgaris. supporting examination such as laboratory test, blood smears and histopathology examination could help the diagnosis. the presence of lymphadenopathy, and atypical lymphocytes in the peripheral blood and the extensive skin involvement reflect the poor prognosis. the most common cause of death was sepsis. keywords: sezary syndrome, cutaneous t cell lymphoma, psoriasis vulgaris, erythroderma, sezary cells abstrak sindrom sezary merupakan salah satu jenis limfoma pada sel t kutaneus. penyakit ini ditandai dengan bercak atau plak kemerahan pada kulit yang meluas ke seluruh tubuh menjadi eritroderma, limfadenopati dan terdapatnya limfosit atipikal yang disebut dengan sel sezary. tujuan dari laporan kasus ini adalah untuk mengetahui manifestasi klinis, pemeriksaan dan penatalaksanaan sindroma sezary yang secara klinis menyerupai generalized psoriasis. seorang pria berusia 60 tahun datang dengan plak coklat kemerahan dengan skuama tebal pada hampir seluruh tubuh disertai limfadenopati kelenjar getah bening supraklavikular kanan dan kiri yang disertai rasa gatal pada lesi. gambaran klinis lainnya adalah alopesia, hiperkeratosis palmoplantar, onikodistrofi, facies leonina tanpa anestesi pada lesi dan tanpa pembesaran saraf tepi. pada pemeriksaan laboratorium terdapat peningkatan jumlah leukosit, pada pemeriksaan hapusan darah tepi ditemukan sel sezary dan gambaran histopatologi menunjukkan atrofi dan akantosis fokal pada epidermis dan infiltrasi padat limfosit pada dermo-epidermal junction dan dermis superfisial. pasien mendapatkan pengobatan methotrexate (mtx) 3 x 5 mg (1 siklus) dengan terapi tambahan krim mometason furoate 0,1% dan ctm 3 x 1 tablet. pemberian methotrexate tidak dilanjutkan karena terjadi gangguan pada fungsi hepar pasien dan terjadi pemburukan kondisi pasien. setelah 25 hari perawatan, pasien mengalami sepsis dan kemudian pasien meninggal dunia. onset awal dari sindrom sezary pada kasus ini sulit untuk diketahui karena gambaran klinisnya yang mirip dengan psoriasis vulgaris. pemeriksaan penunjang berupa pemeriksaan 60 indonesian journal of tropical and infectious disease, vol. 6. no. 3 september–december 2016: 59-62 3 to the admission to the dermatology ward, the patient was hospitalized in the internal department for granulomatous lymphadenitis and treated with 3 series of methotrexate (mtx) course. some improvements were observed and the patient was discharged from the hospital. in clinical examination the patient looked weak though all vital signs were normal, except for the body temperature (390c). the enlargements of both supraclavicular lymph nodes were palpable. thick brown plaques with some erythematous skin areas were found all over the body, while hyperkeratosis of the skin was observed with some fissures on his palms and soles. erythematous nodules were found all over the face and alopecia of the scalp was also present. however, there was no skin anesthesia, madarosis, or thickening of the ear lobes. there was no peripheral nerve enlargements on palpation either (figure 1-4). figure 1. erythematous nodules on face and madarosis figure 2. alopecia figure 3. psoriasiform plaques and infiltrated plaques with hyperkeratosis and scaling figure 4. plaques with superficial erosion and fissuring on the soles figure 1. erythematous nodules on face and madarosis. laboratorium, hapusan darah tepi dan pemeriksaan histopatologi membantu menegakkan diagnosis. adanya limfadenopati dan ditemukannya sel limfosit atipikal pada darah tepi dan keterlibatan kulit yang luas menggambarkan prognosis yang buruk pada kasus ini. penyebab kematian tersering adalah sepsis. kata kunci: sindrom sezary, limfoma sel t kutaneus, psoriasis vulgaris, eritroderma, sel sezary introduction lymphoma is the name for various types of cancer which emerge in the lymphocytes (immune cells). there are three types of lymphocytes: b-lymphocyte (b-cell), t-cell and the natural killer lymphocyte (nk-cell). in general the b-cell lymphoma is more prevalent, while t-cell lymphoma is specifically common for the skin.1 the non-hodgkin cutaneous t-cell lymphoma is manifested on the skin. it has a variety of skin manifestations like skin patch or plaque, tumor, or erythrodermic lesions.1,2 the proportion of t-cell lymphoma was accounted for approximately 4% of the whole non-hodgkin lymphoma.1 among many types of cutaneous-t cell lymphoma, mycosis fungoides is the slow growth clinical type while the sezary syndrome (ss) is an aggressive, leukemic cutaneous t-cell lymphoma variant which manifests as erythroderma. sezary syndrome is the most aggressive type of lymphoma due to its influence to skin and blood.1,2 the incidence of cutaneous lymphoma has been increasing since the last decades and mostly occurred among black people at the age of 50–60 years old.1,3. changes in the amount of tumor suppressor and gen that control apoptosis were found in cutaneous t cell lymphoma. however, how these changes affect the t cell activities are still not clear.3 genetic disorder of nav3 expression (the tumor suppressor) was found in 50-85% patient. mutations of p53, p15, p16, junb and pten gen were observed in advance cases. the clinical manifestation of mf and ss may be similar with some mild dermatoses like dermatitis, psoriasis vulgaris, pityriasis lichenoides chronic, and pityriasis lichenoides et varioliformis acuta.2,4 there are trias in clinical symptom of ss which consist of erythroderma, lymphadenopathy and the typical sezary cell.3,5 itchy skin lesions and thick squamae especially over the palmar and plantar areas, often occurred along with alopecia. histopathologically, the ss shows non-specific inflammatory dermatosis, but in immuno-histochemistry staining some atypical cd4 t-cells are found with the increase ratio of cd4 / cd8 recorded at more than 10.3,6,7 the prognosis of ss is not suitable with low remission and slow response to the few available treatment modalities.3,8,9 case a 60 years old man was hospitalized in the dermatology ward of dr. soetomo hospital, complaining about itchy and thick patches all over the body in the last 3 days before admission. the erythematous plaques started in the back for a year previously and spread to the other parts of the body. he was treated at the health center and got some tablets and injections without any improvement; then he was referred to the hospital. one month prior to the admission to the dermatology ward, the patient was hospitalized in the internal department for granulomatous lymphadenitis and treated with 3 series of methotrexate (mtx) course. some improvements were observed and the patient was discharged from the hospital. in clinical examination the patient looked weak though all vital signs were normal, except for the body temperature (390c). the enlargements of both supraclavicular lymph nodes were palpable. thick brown plaques with some erythematous skin areas were found all over the body, while hyperkeratosis of the skin was observed with some fissures on his palms and soles. erythematous nodules were found all over the face and alopecia of the scalp was also present. however, there was no skin anesthesia, madarosis, or thickening of the ear lobes. there was no peripheral nerve enlargements on palpation either (figure 1-4). laboratory results of peripheral blood revealed a leukocytosis (17.9x103/μl) with the increasing neutrophil. hemoglobin of 11.2 g/dl and low albumin level (2.4 g/l) were also observed. the liver and renal function tests were in normal limits. peripheral blood smear examination showed a typical lymphoid cell, and the sezary cell was found (figure 5). 61lynoora and rahmadewi: sezary syndrome mimicking generalized psoriasis vulgaris figure 4. plaques with superficial erosion and fissuring on the soles figure 5. sezary cell 4 laboratory results of peripheral blood revealed a leukocytosis (17.9x103/μl) with the increasing neutrophil. hemoglobin of 11.2 g/dl and low albumin level (2.4 g/l) were also observed. the liver and renal function tests were in normal limits. peripheral blood smear examination showed a typical lymphoid cell, and the sezary cell was found (figure 5). figure 5. sezary cell in histopathological examination of the skin plaque biopsy, a hyperkeratotic corneum layer and epidermic atrophy with inflammatory process in the dermis were identified. it was indicated by the dense lymphocyte infiltrating at the dermisepidermis junction and some focal acanthosis. there was no acid fast bacilli (afb) found in the fite faraco staining (figure 6). figure 6. focal acanthosis and dense lymphocytes (400x ampl) the diagnosis of sezary syndrome was established, then the treatment using methotrexate (mtx) 3x5 mg every 12 hours (one cycle) combined with ciprofloxacin 2x500 mg daily and chlorpheniramine maleat (ctm) 3x4 mg daily as the antipruritic drug was started. physiologic saline with 10% dextrose and 20% albumin was administered through infusion. emollients was also applied all over his body as a topical therapy. after one cycle of mtx therapy (3 times 5 mg, every 12 hour), the general condition of the patient got worse. blood culture showed a positive staphylococcus aureus growth, the liver function test was abnormal and the patient passed away after 25 days being hospitalized due to sepsis. discussion sezary syndrome (ss) can mimic several mild dermatoses like dermatitis, psoriasis vulgaris, pityriasis lichenoides chronica, figure 6. focal acanthosis and dense lymphocytes (400x ampl) in histopathological examination of the skin plaque biopsy, a hyperkeratotic corneum layer and epidermic atrophy with inflammatory process in the dermis were identified. it was indicated by the dense lymphocyte infiltrating at the dermis-epidermis junction and some focal acanthosis. there was no acid fast bacilli (afb) found in the fite faraco staining (figure 6). the diagnosis of sezary syndrome was established, then the treatment using methotrexate (mtx) 3x5 mg every 12 hours (one cycle) combined with ciprofloxacin 2x500 mg daily and chlorpheniramine maleat (ctm) 3x4 mg daily as the antipruritic drug was started. physiologic saline with 10% dextrose and 20% albumin was administered through infusion. emollients was also applied all over his body as a topical therapy. after one cycle of mtx therapy (3 times 5 mg, every 12 hour), the general condition of the patient got worse. blood culture showed a positive staphylococcus aureus growth, the liver function test was abnormal and the patient passed away after 25 days being hospitalized due to sepsis. figure 3. psoriasiform plaques and infiltrated plaques with hyperkeratosis and scaling figure 2. alopecia 62 indonesian journal of tropical and infectious disease, vol. 6. no. 3 september–december 2016: 59-62 discussion sezary syndrome (ss) can mimic several mild dermatoses like dermatitis, psoriasis vulgaris, pityriasis lichenoides chronica, pityriasis lichenoides et varioliformis acuta, and actinic reticuloid.2,4,10 these mild dermatoses can progress to erythrodermic skin lesions due to the extension of the plaques; thus it is spread all over the body. the ss patients were mostly 50-70 years old. the trias of ss are pruritic erythroderma, lymph nodes enlargement and sezary cells in the blood circulation.7,11,12 sometimes it is difficult to differentiate ss from erythroderma which is caused by other agents, especially in a case of severe skin desquamation. however, there is an exception in some a case where the infiltration is more dominant than the desquamation.13 this patient fulfilled those three criteria of ss with severe skin manifestations and many other systemic symptoms. the first differential diagnosis is psoriasis vulgaris due to the thick scales; however, based on the history of early lesions sites, it does not occur in the traumatic area like elbow, knee and shoulder. clinically, the typical scales of psoriasis were not present and the auspitz’ sign was also negative. the second differential diagnosis is leprosy reaction, such as enl reaction, but this can be excluded if there is by no cardinal signs of leprosy identified. both differential diagnosis could be excluded by histopathological examination because psoriasis vulgaris and leprosy have some specific histopathological patterns. the results of the histopathological examination for the thick scale revealed focal atrophy and acanthosis of the epidermis, as well as dense infiltration of lymphocytes in the dermo-epidermal junction and superficial dermis. the result of peripheral blood smear showed the presence of sezary cells which ensures a definite diagnosis of sezary syndrome on this patient. the staging system of mycosis fungoides is usually applicable for ss and the clinical manifestation of this case was classified as grade 3. the malignancy related to this condition was identified prior to or after the diagnosis of mf and ss.14 only a few data or reports on ss cases are available. likewise, there are limited amount of treatment available in the clinical setting due to rarity of the case. some topical treatments could be given for ss cases, such as nitrogen mustard, corticosteroids or bexarotene. the ultra violet treatment has been used to control the plaques; while the systemic treatment is used when topical therapy shows no progress.3,8,9 methotrexate (mtx) and other antimitotic drugs are the other available choices to manage ss.14 in this case, mtx was only applied for one cycle and had to be discontinued due to the clinically worsening condition of the patient and the disturbance of his liver function. the low immune level of the patient was the main factor in the fatal sepsis caused by staphylococcus aureus infection. this kind of situation was also reported by mirvish et al who worked on ss cases.15 in general, the prognosis of ss is not suitable with low remission and slow response to therapy, especially for the pruritus of the skin which influences the patient’s quality of life. a guide of ss management has been proposed by us cutaneous lymphoma consortium (usclc) and modified by the national comprehensive cancer network (nccn).11 conclusion sezary syndrome (ss) is rare and relatively difficult to diagnose due to the similarities of the clinical manifestation with any skin diseases. the presence of lymphadenopathy, and atypical lymphocytes in the peripheral blood and the extensive skin involvement reflect the poor prognosis. references 1. h ky. cutaneous t-cell lymphoma facts. leukemia and lymphoma society; 2011. 1-9 p. 2. jawed si, myskowski pl, horwitz s, moskowitz a, querfeld c. primary cutaneous t-cell lymphoma (mycosis fungoides and sézary syndrome): part i. diagnosis: clinical and histopathologic features and new molecular and biologic markers. j am acad dermatol. 2014 feb;70(2):205.e1-16; quiz 221-2. 3. hwang st, janik je, jaffe es, wilson wh. mycosis fungoides and sézary syndrome. lancet. 2008 mar;371(9616):945–57. 4. doukaki s, aricò m, bongiorno mr. a rare presentation of mycosis fungoides mimicking psoriasis vulgaris. case rep dermatol. 2009;1(1):60–5. 5. cyriac mj, kurian a. sézary cell. indian j dermatol venereol leprol. 2004;70(5):321–4. 6. demierre m-f. mycosis fungoides and sézary syndrome: the burden of pruritus. community oncol. 2010 sep;7(9):399–404. 7. shaikha sa, mansoor k, homsi ha, riad h. sézary syndrome. gulf j dermatology venerol. 2005 apr;12(1):50–3. 8. olsen ea, rook ah, zic j, kim y, porcu p, querfeld c, et al. sézary syndrome: immunopathogenesis, literature review of therapeutic options, and recommendations for therapy by the united states cutaneous lymphoma consortium (usclc). j am acad dermatol. 2011 feb;64(2):352–404. 9. whittaker sj, marsden jr, spittle m, russell jones r, british association of dermatologists, u.k. cutaneous lymphoma group. joint british association of dermatologists and u.k. cutaneous lymphoma group guidelines for the management of primary cutaneous t-cell lymphomas. br j dermatol. 2003 dec;149(6):1095–107. 10. pacheco d, fraga a, travassos ar, antunes j, freitas j, soares de almeida l, et al. actinic reticuloid imitating sézary syndrome. acta dermatovenerologica alpina, pannonica, adriat. 2012 sep;21(3):55–7. 11. yamashita t, abbade lpf, marques mea, marques sa. mycosis fungoides and sézary syndrome: clinical, histopathological and immunohistochemical review and update. an bras dermatol. 2012 dec;87(6):817–30. 12. desar ime, stevens wbc, verweij pe, velden wjfm van der. a sezary cryptogram. netherlands j med. 2011;69(1). 13. zackheim hs, epstein eh. low-dose methotrexate for the sézary syndrome. j am acad dermatol. 1989 oct;21(4 pt 1):757–62. 14. jawed si, myskowski pl, horwitz s, moskowitz a, querfeld c. primary cutaneous t-cell lymphoma (mycosis fungoides and sézary syndrome): part ii. prognosis, management, and future directions. j am acad dermatol. 2014 feb;70(2):223.e1-17; quiz 240-2. 15. mirvish ed, pomerantz rg, geskin lj. infectious agents in cutaneous t-cell lymphoma. j am acad dermatol. 2011 feb;64(2):423–31. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 10 no. 3 september–december 2022 original article epidemiology of escherichia coli as a critical pathogen of bloodstream infection patients in dr. soetomo general hospital, surabaya, indonesia pepy dwi endraswari1,2,4* , firman setiawan1,2,4 , ayu lidya paramita3 , ni made mertaniasih1,2,4 1department of medical microbiology, faculty of medicine, universitas airlangga, surabaya, indonesia 2dr. soetomo academic hospital, surabaya, indonesia 3study program of clinical microbiology, faculty of medicine, universitas airlangga, surabaya, indonesia 4unit of clinical microbiology, universitas airlangga hospital, surabaya, indonesia received: september 30th, 2022; revised: november 14th, 2022; accepted: november 18th, 2022 abstract bloodstream infections (bsi), caused primarily by multidrug-resistant escherichia coli, are a significant cause of morbidity and mortality worldwide. this study aims to evaluate the epidemiology of e. coli as a critical pathogen in patients with bloodstream infections in a tertiary referral hospital. this is a retrospective study using a descriptive observational research design. this study used a medical record instrument for bloodstream patients in dr. soetomo hospital's inpatient ward with gram-negative bacteria results of blood cultures in the clinical microbiology laboratory from april 2021 to september 2021. the observed variables include; antimicrobial sensitivity, patient clinical characteristics, demographic data, clinical diagnosis, and clinical outcome. in 6 months, 276 gram-negative bloodstream infection patients were treated at dr. soetomo hospital. the proportion of e. coli was 17 %. the main characteristics of patients were over 60 years old (28%), and 54% were female. 63% of e. coli were esbl, and 9% were carbapenem-resistant microorganisms. high antimicrobial resistance was found in quinolones (100%), ampicillin (93%), piperacillin (74%), tetracycline (72%), ceftriaxone (66%), cefotaxime (65%), ceftazidime (60%), cefazolin (65%), and trimethoprim-sulfamethoxazole (65%). the most common potential determinant profile discovered was linked to immunocompromised status due to malignancy. the high number of antimicrobial-resistant bacteria showed the importance of strict infection control and updated epidemiology data as a guide for empirical antimicrobial therapy. keywords: bloodstream infection; e.coli; epidemiology; esbl; resistance abstrak infeksi aliran darah (iad), yang terutama disebabkan oleh escherichia coli yang bersifat multi-drug resistance microorganisms (mdro), merupakan penyebab signifikan morbiditas dan mortalitas di seluruh dunia. penelitian ini bertujuan untuk mengevaluasi epidemiologi e. coli sebagai patogen pada pasien infeksi aliran darah di rumah sakit rujukan tersier. penelitian ini merupakan penelitian deskriptif dengan desain penelitian observasional menggunakan alat rekam medis aliran darah pasien di ruang rawat inap rsud dr. soetomo dengan bakteri gram negatif hasil kultur darah di laboratorium mikrobiologi klinik pada bulan april 2021 sampai september 2021. variabel yang diamati meliputi; sensitivitas antimikroba, karakteristik klinis pasien, data demografis, diagnosis klinis, dan hasil klinis. dalam 6 bulan, didapatkan 276 pasien infeksi aliran darah gram-negatif dirawat di rs dr. soetomo. proporsi e. coli adalah 17%. karakteristik utama pasien berusia di atas 60 tahun (28%), dan 54% berjenis kelamin perempuan. 63% e. coli adalah esbl, dan 9% adalah mikroorganisme yang resisten terhadap karbapenem. resistensi antimikroba yang tinggi ditemukan pada kuinolon (100%), ampisilin (93%), piperacillin (74%), tetrasiklin * corresponding author: pepy.dr@fk.unair.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-0271-8505 https://orcid.org/0000-0002-5478-6673 https://orcid.org/0000-0002-8917-0406 https://orcid.org/0000-0002-0594-2385 206 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 205–213 (72%), ceftriaxone (66%), cefotaxime (65%), ceftazidime (60%), cefazolin (65%), dan trimethoprim-sulfamethoxazole (65%). profil penentu potensial yang paling umum ditemukan terkait dengan status immunocompromised karena keganasan. tingginya jumlah bakteri resisten antimikroba menunjukkan pentingnya pengendalian infeksi yang ketat dan data epidemiologi terkini sebagai panduan terapi antimikroba empiris. kata kunci: e. coli; epidemiologi; esbl; infeksi aliran darah; resistensi how to cite: endraswari, p. d., setiawan, f., paramita, a. l., mertaniasih, n. m. epidemiology of escherichia coli as a critical pathogen of bloodstream infection patients in dr. soetomo general hospital, surabaya, indonesia. indonesian journal of tropical and infectious disease. 10(3). 205–213. dec. 2022. introduction bloodstream infection (bsi) is a big challenge of infectious diseases. it represents 40% of community-acquired (ca) cases, hospital-acquired (ha) sepsis and septic shock, and approximately 20% of icuacquired cases.1 it is invariably associated with poor outcomes significantly when adequate antimicrobial therapy and source control are delayed.2 the pathogens causing bloodstream infection majority caused by gram-negative bacteria, including e. coli.3, 4 e. coli is a bacteria that often has resistant mechanisms to multiple antibiotics. these bacteria have built-in resistance mechanisms and can pass on genetic material that allows other bacteria to become drug-resistant. because of this, e. coli was covered as a critical pathogen by the who in 2017.5,6 bsi caused by multi-drug resistant (mdr) organism make the management difficult because the antibiotic therapy is limited and unsuitable empirical antibiotic treatment is given. bsi by mdr e. coli was associated with poorer outcomes and a higher overall mortality rate.7 e. coli, including extended-spectrum beta-lactamase (esbl) producing and carbapenem-resistant, can cause severe and frequently lethal infections, especially bloodstream infections (bsis).4,8,9 esblsproducing bacteria can hydrolyze broadspectrum cephalosporins, monobactams, and penicillins, while carbapenem-resistant is an e. coli isolate resistant to ertapenem, imipenem, meropenem, or any carbapenem antimicrobial.10 bloodstream infection caused by those organisms represents a challenge due to the limitation of antimicrobials as a drug of choice; furthermore, it can cause significant morbidity and mortality. the critical pathogens in bloodstream infection are majority caused by gramnegative bacteria, the most frequent pathogen was e. coli3,4 which could be characteristically different profiles in various hospitals or patient care units. in a hospital setting, it is crucial to evaluate and monitor the updated epidemiology of causative agents of infection due to the prevention and infection control program and the updated empirical antibiotics in the hospital. therefore, epidemiological studies on microorganism infection must be updated periodically. this research focuses on local epidemiology data of e. coli as a pathogen detected in bloodstream infection, including the resistance pattern and the determinants factor related to invasive devices and immunocompromised conditions. materials and methods materials we used data from the records of blood culture results from the clinical microbiology unit and medical records of patients with gram-negative bloodstream infection in inpatient wards of dr. soetomo hospital from april 2021 until september 2021. ethical clearance from the ethics committee has been obtained by number 0660/loe/301.4.2/ x/2021. methods this research is descriptive research. all medical records containing escherichia coli 207 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license pepy dwi endraswari, et al. epidemiology of escherichia coli as a critical pathogen detected, antimicrobial sensitivity, and other determinant factors, i.e., patient clinical characteristics, demographic data, clinical diagnosis, history of invasive devices, antibiotic use, and clinical outcomes. in addition, species identification, antimicrobial susceptibility testing, and determination of resistant patterns, including esbl-producing strains and carbapenem-resistant strains, using bd bactec™ blood culture system and bd phoenixtm system. antimicrobial sensitivity interpreted based on clinical laboratory and standards institute (clsi) guideline 2021. statistical analysis data were analyzed with microsoft excel and presented in a frequency table with the percentage of each variable which was then converted into a descriptive form. results and discussion there were 276 gram-negative bacteria of a total of 973 (28.4%) positive blood cultures of hospitalized patients in dr. soetomo hospital surabaya within 6 months. e. coli was found in 48 patients of 276 gramnegative bacteria (17%). it can be seen that e. coli was the third rank of gram-negative bacteria causing bloodstream infection (table 1). forty-three of the 48 patients with e. coli bloodstream infection with the complete medical record were analyzed. table 1. distribution of gram-negative bacteria detected of bloodstream infection in dr. soetomo hospital, surabaya, from april 2021 – september 2021 gram-negative bacteria n (%) acinetobacter baumannii/calcoaceticus complex 67 (24) klebsiella pneumoniae 63 (23) escherichia coli 48 (17) pseudomonas aeruginosa 22 (8) enterobacter cloacae 19 (7) other gram-negative bacteria 57 (21) total 276 (100) this study's results align with the surveillance study about the trend of bloodstream infection in the usa reported that the most prevalent gram-negative bacteria causing bacteremia from 2005 until 2016 was e. coli, with an incidence range of 20-24%.3 in comparison, e. coli also was found to be the most prevalent pathogen (32.8% of cases), followed by staphylococcus aureus (20.6%), klebsiella pneumoniae (16.1%), and pseudomonas aeruginosa (11.6%), in a study of bloodstream infections at a major teaching hospital in rome within 9 years period, according to angelis et al.4 another data of 382 bsi cases in a tertiary teaching hospital icu revealed the most frequently isolated microorganisms to be klebsiella pneumoniae (11.52%), followed by escherichia coli (9.95%).11 furthermore study about the profile of blood culture of sepsis patients in the intensive care unit (icu) – dr. soetomo hospital surabaya revealed that gram-negative bacteria were 25% of the total positive culture. of the gram-negative bacteria, enterobacteriaceae showed a proportion of 59%, followed by acinetobacter baumannii at 29%.12 the 43 e. coli strains isolated from the blood culture of bsi comprised 20 male patients and 23 females. this data aligns with a systematic literature review report that women were more likely than men to develop e. coli bacteremia overall. according to age group stratification, this connection was only present in young and middle-aged individuals; in adults over 60, the incidence rates for men and women were comparable.13 the age of the patients showed in table 2, where the subjects were between 0.01 years (neonates) to 81 years, with the most age distribution being over 60 years, namely 12 patients. this data is in accordance with a systematic literature review report that the incidence rate considerably rose with age. with estimated rates of 110, 154, and 319 episodes per 100,000 person-years among those aged 60 to 69, 70 to 79, and 80 years and older, respectively, older individuals' incidence rates were higher than the population norm.13 https://www.bd.com/en-us/products-and-solutions/products/product-families/bd-bactec-fx-blood-culture-system 208 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 205–213 table 2. age distribution of patients with bloodstream infection caused by e. coli age group (year) n (%) 0–10 6 (14) 11–20 5 (12) 21–30 1 (2) 31–40 3 (7) 41–50 7 (16) 51–60 9 (21) >60 12 (28) out of 43 patients, 16 (37%) were referred from other hospitals (table. 3); this could be associated with the role of dr. soetomo hospital as the tertiary referral hospital. the primary diagnoses of patients in this study were grouped into several criteria, namely malignancy, coronavirus infection, primary infection other than bsis, bile duct atresia, and other diagnoses composed of the small proportion of diagnoses listed in the footnotes of the table. the primary disease diagnosis was malignancy in 12 patients (28%), coronavirus infection in 11 patients (26%), primary infection other than bsis in 8 patients (19%), bile duct atresia in 3 patients (7%), and others in 9 patients (21%). the most significant proportion of patients with primary diagnoses were malignancy and coronavirus infections. table 3. characteristics of the patients in the study characteristics (n=43) patients (n/%) age (year; means, min-max) 43; 0.01–81 gender (m/f) 20 (46)/23 (54) referral patients 16 (37) diagnosis for hospitalization malignancy* 12 (28) coronavirus infection 11 (26) infection ** 8 (19) bile duct atresia 3 (7) other *** 9 (21) nasogastric tube 27 (63) ventilator/intubation 19 (44) surgery 14 (33) using a central venous catheter (cvc) 14 (33) immunosuppressant therapy in 30 days 13 (30) total of patients 43 (100) *acute myeloid leukemia, acute lymphocytic leukemia, anaplastic anemia, non-hodgkin's lymphoma, malignant neoplasm of the placenta, malignant neoplasm of the ovary, malignant neoplasm of the cervix, malignant neoplasm of uteri, malignant neoplasm of the bile duct, malignant neoplasm of the pancreas **septicemia, abscess of the liver, acute pancreatitis, pneumonia, cholecystitis, acute peritonitis, intestines tuberculosis, congenital pneumonia ***myelodysplastic syndrome, myasthenia gravis, morbidly adherent placenta, other and unspecified ovarian cysts, congenital hydronephrosis, acute renal failure, communicating hydrocephalus, burn multi regions several determinants were recorded, including invasive devices and others that may be associated with bacteremia (table 3). the data showed the use of invasive devices was nasogastric tubes (63%), ventilators/ intubation (44%), and the central venous catheter (cvc) (33%). furthermore, we found surgery cases (33%), immunosuppressant therapy (30%), and neutropenia (16%). a systematic literature review concluded that central and peripheral venous catheters increased the risk of e. coli bacteremia: by 10-fold and 7.5-fold, respectively. in contrast, suprapubic and urethral urinary catheters increased the risk by 6-fold and 3-fold, respectively.13 the proportion of patients with malignancy was relatively high, namely 12 patients (12%), consisting of 6 patients with leukemia and 6 with solid organ malignancy. this result supports the available epidemiological data that the percentage of bacteremia patients infected by e. coli is associated with particular underlying clinical conditions. a study by bonten et al. mentioned that the highest rate of patients with bacteremia resulting from e. coli was lymphocytic leukemia and multiple myeloma (12–13%). the neoplastic disease has a relative risk (rr) of developing e. coli bacteremia 14.9 fold compared with the general population.13 the proportion of bacteremia cases with a primary diagnosis of covid-19 was relatively high, namely 11 patients (26%). bhatt et al14 report that the bloodstream infections observed in patients with covid-19 may have contributed to the more severe presentation and clinical course. furthermore, it reflects other underlying physiological and immunological complications of covid-19. alternatively, a complicated hospital course may have contributed to more risk factors for developing bloodstream infections.14 in this 209 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license pepy dwi endraswari, et al. epidemiology of escherichia coli as a critical pathogen research, 26% of covid patients with coinfection by bloodstream infection due to e. coli need attention to the importance of surveillance and prevention of the possibility of a healthcare-associated infection bsis. no review of the source of the bloodstream infection was carried out in this study. however, several studies have reported that central line is the most common presumed source of bloodstream infections.14 antimicrobial resistance profile the microorganism was classified based on antimicrobial resistance profiles. of 43 isolates, 15 (35%) were non-mdro, 28 (63%) were esbl-producing microorganisms, 4 (9%) were carbapenemresistant. in addition, three esbl-producing microorganisms were carbapenem-resistant microorganisms (table 4). table 4. types of organisms based on antimicrobial resistance profile types of the organism n (%) non-mdro 15 (35) esbl-producing strain 24 (56) carbapenem-resistant strain 1(2) esbl-producing strain and carbapenem-resistant strain 3 (7) total 43 (100) the esbl-producing bacteria were higher than the non-mdro bacteria, 63% and 35%, respectively. this number was very high. the study showed that the prevalence of esblproducing bacteria is increased in the latest period. the clinical relevance of infections caused by esbl-producing organisms has been outlined in several studies.15,16 in a retrospective analysis of patients with e. coli bsi over four years in a teaching hospital, 58.9% developed esbl-producing e. coli.17 no risk factor analysis for esbl infection in this study, but several studies report that esbl-producing e. coli bacteremia is associated with prior urinary tract infections,17 previous cephalosporin exposure17, central venous cathether15, and history of admission to a long-term care hospital.18 bloodstream infections, particularly bsis due to mdr e. coli, can be caused by hospitalacquired or community-acquired infections. it has been widely reported that infections caused by mdr bacteria are associated with hospital/healthcare-associated infections. several studies supported communityacquired bsis by mdr bacteria, which reported the presence of carriers of esblproducing e. coli bacteria in communities with varying prevalence between different populations. 19,20,21 globally, an 8-fold growth in the bowel carriage rate of esbl e. coli in the community during the last decade. the pooled incidence confirmed an upward trend of e. coli carriage in the community, growing from 2.6% in 2003–2005 to 21.1% in 2015–2018. over the entire period, the highest carriage rate happened in south-east asia (27%), while the lowest happened in europe (6.0%).22 in addition, the carrier of esbl-producing e. coli bacteria was reported to develop bloodstream infection.19 bsi caused by emerging multidrugresistant e. coli strains is more challenging to treat and confers a higher risk of death. although it cannot be concluded that the cause of death was purely due to e. coli bsi, 68% of patients died in this study. a study reported that in e. coli bsi, 50% of the patients died, and the mortality analysis showed that 33.3% of the deaths were associated with bsi.23 https://milissehatyop.org/carbapenem-resistant-enterobacteriaceae-cre/ https://milissehatyop.org/carbapenem-resistant-enterobacteriaceae-cre/ 210 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 205–213 table 5. antibiotics resistant pattern of e. coli isolated from bacteremia hospitalized patients antibiotics tested number resistance n (%) ciprofloxacin 27 27 (100) levofloxacin 27 27 (100) ampicillin 42 39 (93) piperacillin 43 32 (74) tetracyclin 43 31 (72) cefotaxime 41 27 (66) cefazolin 43 28 (65) ceftriaxone 43 28 (65) trimetoprimsulfametoxazole 43 28 (65) aztreonam 43 27 (63) ceftazidime 43 26 (60) moxifloxacin 42 25 (58) cefepime 37 17 (46) gentamicin 42 15 (36) ampicillin sulbactam 43 10 (23) chloramphenicol 43 8 (19) amoxicillinclavulanate 42 7 (17) cefoxitin 37 5 (14) fosfomycin 42 5 (12) imipenem 41 4 (10) meropenem 42 4 (10) tigecycline 30 2 (7) cefoperazonesulbactam 43 2 (5) amikacin 43 1 (2) piperacillin tazobactam 40 0 (0) the antimicrobial resistance pattern of tested antimicrobials against e. coli from bloodstream infection patients in table 5 revealed a high proportion of strains of e. coli were resistant to ampicillin (93%), piperacillin (74%), and tetracycline (72%). the resistance of third-generation cephalosporin ceftriaxone, cefotaxime, and ceftazidime was 66%, 65%, and 60%, respectively, while the fourth-generation cephalosporin cefepime was lower (46%). the resistance to trimethoprim-sulfamethoxazole was 65%. carbapenem as a drug of choice for multi-drug resistance e coli showed resistance was 10%. a low proportion of strains of e. coli were resistant to tigecycline (7%), cefoperazone-sulbactam (5%), and amikacin (2%). no resistance to piperacillin tazobactam was found. quinolone antibiotics (levofloxacin and ciprofloxacin) were tested only in 27 isolated, and the result was 100% resistance. the resistance to several drugs, including carbapenem antibiotics (imipenem and meropenem), was meagre. carbapenems are β lactam antibiotics, as are penicillins and cephalosporins, but differ from these other classes in their exact chemical structure. the bactericidal activity of carbapenem results from the inhibition of cell wall synthesis. carbapenem penetrates the cell wall of most gram-positive and gram-negative bacteria to bind penicillinbinding-protein (pbp) targets.24 esbls are enzymes that inactivate most penicillins, cephalosporins, and aztreonam. esbl producing bacteria generally remain susceptible to carbapenems. therefore, it is relevant to the current data that carbapenem is still an effective drug for treating infections caused by esbl producers.25 high resistance (>60%) to the antibiotics ciprofloxacin, levofloxacin, ampicillin, piperacillin, tetracycline, cefotaxime, cefazolin, ceftriaxone, trimethoprimsulfamethoxazole, aztreonam, and ceftazidime was shown. this result supports another study that antimicrobial resistance among e. coli causing bloodstream infection was common; 36% of e. coli blood isolates were nonsusceptible to ciprofloxacin, and 23% were non-susceptible to third generation cephalosporins.26 esbls do not inactivate non–β-lactam agents (eg, ciprofloxacin, trimethoprimsulfamethoxazole, gentamicin). however, organisms that carry esbl genes often carry additional genes or mutations in genes that mediate resistance to a broad range of antibiotics. the number of esbl-producing e. coli is relatively high (63%). esbls producing enterobacteriaceae, including e. coli, can hydrolyze broad-spectrum cephalosporins, monobactams, and penicillins. enzymes of class a β-lactamases, like tem-1, tem-2, and shv-1, are responsible for the resistance to ampicillin, amoxicillin, and early generation cephalosporins. resistance to third-generation cephalosporins arises when mutation of genes encoding tem-1, tem-2, https://www.sciencedirect.com/topics/medicine-and-dentistry/ciprofloxacin https://www.sciencedirect.com/topics/medicine-and-dentistry/cephalosporin 211 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license pepy dwi endraswari, et al. epidemiology of escherichia coli as a critical pathogen or shv-1 gives rise to new β-lactamases that can hydrolyze them.10 this study's resistance rate to fluoroquinolones in esbl-producing e. coli is high (100%). this result supports another study that extended-spectrum β-lactamase (esbl) constitutes the most common antibiotic resistance mechanism often found on the same resistance plasmids. 27 these epidemiological data provide good information on the resistance profile of e. coli causing bsi in the tertiary referral hospital. the high prevalence of bloodstream infections caused by mdro e. coli necessitates strict infection control in order to reduce the number of mdro e. coli infections in tertiary hospitals. high levels of antimicrobial resistance encourage clinicians to carry out culture and antibiotic susceptibility testing as soon as possible after the appearance of signs and symptoms of infection to provide definitive and appropriate treatment immediately. while waiting for the definitive antibiotics, the local antibiotic sensitivity pattern in the hospital needs to be taken into account to choose the right empirical antibiotics. therefore, the role of updated epidemiology data as the guide for empirical antimicrobial therapy is essential. conclusions according to epidemiology statistics, 17% of gram-negative bacteria identified from bloodstream infections were the pathogen e. coli. quinolones, ampicillin, piperacillin, tetracycline, beta-lactam antibiotics, and trimethoprim-sulfamethoxazole were all linked to high levels of antimicrobial resistance. strict infection control is required due to the high occurrence of bloodstream infections caused by mdro e. coli. acknowledgement the author expressed gratitude to dr. soetomo hospital, who provided the data, also inna fairuuza firdaus and ega rischella, who collected the data for this publication. conflict of interest the authors declare that they have no conflict of interest. references 1. timsit jf, ruppé e, barbier f, tabah a, bassetti m. bloodstream infections in critically ill patients: an expert statement. intensive care med [internet]. 2020;46(2):266–84. available from: https://doi.org/10.1007/s00134-020-05950-6 2. adrie c, garrouste-orgeas m, ibn essaied w, schwebel c, darmon m, mourvillier b, et al. attributable mortality of icu-acquired bloodstream infections: impact of the source, causative micro-organism, resistance profile and antimicrobial therapy. j infect [internet]. 2017;74(2):131–41. available from: http://dx.doi.org/10.1016/j.jinf.2016.11.001 3. diekema dj, hsueh pr, mendes re, pfaller ma, rolston k v., sader hs, et al. the microbiology of bloodstream infection: 20-year trends from the sentry antimicrobial surveillance program. antimicrob agents chemother. 2019;63(7). 4. de angelis g, fiori b, menchinelli g, d’inzeo t, liotti fm, morandotti ga, et al. incidence and antimicrobial resistance trends in bloodstream infections caused by eskape and escherichia coli at a large teaching hospital in rome, a 9-year analysis (2007–2015). eur j clin microbiol infect dis. 2018;37(9):1627–36. 5. who global priority pathogens list of antibioticresistant bacteria combat amr [internet]. [cited 2022 oct 31]. available from: https://www.combatamr.org.au/newsevents/who-global-priority-pathogens-list-ofantibiotic-resistant-bacteria 6. mancuso g, midiri a, gerace e, biondo c. bacterial antibiotic resistance: the most critical pathogens. pathogens. 2021;10(10):1–14. 212 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 10 no. 3 september–december 2022: 205–213 7. eun y, kang c in, kyeong m, yeon s, mi y, ryeon d, et al. international journal of antimicrobial agents epidemiology and clinical outcomes of bloodstream infections caused by extended-spectrum-lactamase-producing escherichia coli in patients with cancer. 2013;42:403–9. 8. gladstone ra, mcnally a, pöntinen ak, tonkin-hill g, lees ja, skytén k, et al. emergence and dissemination of antimicrobial resistance in escherichia coli causing bloodstream infections in norway in 2002–17: a nationwide, longitudinal, microbial population genomic study. the lancet microbe. 2021;2(7):e331–41. 9. de lastours v, laouénan c, royer g, carbonnelle e, lepeule r, esposito-farèse m, et al. mortality in escherichia coli bloodstream infections: antibiotic resistance still does not make it. j antimicrob chemother. 2020;75(8):2334–43. 10. jubeh b, breijyeh z, karaman r. resistance of gram-positive bacteria to current antibacterial agents and overcoming approaches. molecules. 2020;25(12). 11. wu hn, yuan ey, li w bin, peng m, zhang qy, xie kl. microbiological and clinical characteristics of bloodstream infections in general intensive care unit: a retrospective study. front med. 2022;9(april):1–14. 12. octora m, mertaniasih nm, semedi bp, koendhori eb. predictive score model of clinical outcomes sepsis in intensive care unit tertier referral hospital of eastern indonesia. open access maced j med sci. 2021;9(apache ii):1710–6. 13. bonten m, johnson jr, van den biggelaar ahj, georgalis l, geurtsen j, de palacios pi, et al. epidemiology of escherichia coli bacteremia: a systematic literature review. clin infect dis. 2021;72(7):1211–9. 14. bhatt pj, shiau s, brunetti l, xie y, solanki k, khalid s, et al. risk factors and outcomes of hospitalized patients with severe coronavirus disease 2019 (covid-19) and secondary bloodstream infections: a multicenter casecontrol study. clin infect dis. 2021;72(12):e995–1003. 15. liang t, xu c, cheng q, tang y, zeng h, li x. epidemiology, risk factors, and clinical outcomes of bloodstream infection due to extended-spectrum beta-lactamase-producing escherichia coli and klebsiella pneumoniae in hematologic malignancy: a retrospective study from central south china. microb drug resist. 2021;27(6):800–8. 16. lovayová v, čurová k, hrabovský v, nagyová m, siegfried l, toporová a, et al. antibiotic resistance in the invasive bacteria escherichia coli. cent eur j public health [internet]. 2022;30(88):s75–80. available from: https://doi.org/10.21101/cejph.a7384 17. xiao t, wu z, shi q, zhang x, zhou y, yu x, et al. a retrospective analysis of risk factors and outcomes in patients with extended-spectrum beta-lactamase-producing escherichia coli bloodstream infections. j glob antimicrob resist [internet]. 2019;17:147–56. available from: https://doi.org/10.1016/j.jgar.2018.12.014 18. baek yj, kim ya, kim d, shin jh, uh y, shin ks, et al. risk factors for extended-spectrumβ -lactamaseproducing escherichia coli in community-onset bloodstream infection : impact on long-term care hospitals in korea. 2021;455–62. 19. malande oo, nuttall j, pillay v, bamford c, eley b. a ten-year review of esbl and nonesbl escherichia coli bloodstream infections among children at a tertiary referral hospital in south africa. plos one. 2019;14(9):1–16. 20. martinez ae, widmer a, frei r, pargger h, tuchscherer d, marsch s, et al. esblcolonization at icu admission: impact on subsequent infection, carbapenem-consumption, and outcome. infect control hosp epidemiol. 2019;40(4):408–13. 21. kawamura k, nagano n, suzuki m, wachino j ichi, kimura k, arakawa y. esbl-producing escherichia coli and its rapid rise among healthy people. food saf. 2017;5(4):122–50. 22. bezabih ym, sabiiti w, alamneh e, bezabih a, peterson gm, bezabhe wm, et al. the global prevalence and trend of human intestinal carriage of esbl-producing escherichia coli in the community. j antimicrob chemother. 2021;76(1):22–9. 23. daga ap, koga vl, soncini jgm, de matos cm, perugini mre, pelisson m, et al. escherichia coli bloodstream infections in patients at a university hospital: virulence factors and clinical characteristics. front cell infect microbiol. 2019;9(jun). 24. hawkey pm, livermore dm. carbapenem antibiotics for serious infections. bmj. 2012;344(7863):1–7. 25. tamma pd, aitken sl, bonomo ra, mathers aj, van duin d, clancy cj. infectious diseases society of america 2022 guidance on the treatment of extended-spectrum β-lactamase producing enterobacterales (esbl-e), carbapenem-resistant enterobacterales (cre), and pseudomonas aeruginosa with difficult-totreat resistance (dtrp. clin infect dis. 2022;75(2):187–212. 26. blandy o, honeyford k, gharbi m, thomas a, ramzan f, ellington mj, et al. factors that 213 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license pepy dwi endraswari, et al. epidemiology of escherichia coli as a critical pathogen impact on the burden of escherichia coli bacteraemia: multivariable regression analysis of 2011–2015 data from west london. j hosp infect [internet]. 2019;101(2):120–8. available from: https://doi.org/10.1016/j.jhin.2018.10.024 27. salah, fortune djimabi, soubeiga st, ouattara ak, sadji ay, metuor-dabire a, obiri-yeboah d, banla-kere a, et al. distribution of quinolone resistance gene (qnr) in esbl-producing escherichia coli and klebsiella spp. in lomé , togo. antimicrob resist infect control. 2019;8:1–8 �� vol. 1. no. 1 january–april 2010 a case report histoid leprosy umi rinasari,1 sawitri,1 m. yulianto listiawan,1 cita rosita s prakoeswa,1 indropo agusni,1,3 rachmat santoso,2 and shinzo izumi3 1 dept. of dermatology, school of medicine, airlangga university 2 dept. of pathology, school of medicine, airlangga university 3 institute of tropical disease, airlangga university surabaya, indonesia abstract histoid leprosy is a variant of lepromatous leprosy with characteristic clinical and histopathological features. usually it is occured in lepromatous patients who relaps after dapsone monotherapy, in those with dapsone resistance , sometimes even after multidrug treatment, or at times, de novo with characteristic clinical and histopathological features. a 36 years old male, originated from papua, visited to the skin outpatient clinic with translucent shiny nodules on the left elbow and thumb for the last 18 months. the nodules were multiple, painless and firm. there were nasal congestion, tickening of ear lobes and loss of eye brows. patient did not have any history of previous antileprotic treatment. routine blood examination was normal. bacteriological examination of slit skin smear revealed acid-fast bacilli of bacterial index 4+ and morfologic index 10%. histopathology of skin suggested lepromatous leprosy of histoid type with characteristic interlacing bundles of spindle shaped cells. anti-pgl1 antibody (elisa) revealed high titer of igm (>5.300 u/ml) and also igg anti pgl-1 (>5.300 u/ml). polymerase chain reaction examination test to detect m.leprae was positive and direct sequencing of m.leprae isolate shows no mutation, which means no resistancy to mdt treatment. treatment with mdtwho regiment give clinical improvements and the histoid lesions disappered after 3 months treatment.the histoid form of leprosy in this case developed without any prior treatment of anti leprotic drugs ( de novo ). some theoretical aspects of the patho-mechanism of histoid leprosy are discussed. key words: histoid leprosy, lepromatous leprosy, de novo background histoid leprosy is an uncommon variant of lepromatous leprosy with characteristic clinical, histopathological and bacteriological findings.1,2,9 clinically it is characterized by multiple discrete shiny, smooth, painless, succulent, globular, protuberant, firm, skin colored to yellow brown nodules and papules on normal appearing skin.5,12 slit skin smear from histoid lesions shows abundant acid fast bacilli appear long when compared to ordinary lepra bacilli.15 the mean baseline bacteriological index was 4–6 (range 3–6). the mean morfological index was 4% (range 0–10%).9 histopathologically the epidermis shows grenz zone and the dermis shows sheets of round to spindle-shaped histiocytes.6,16 wade described this pattern in 1960 and 1963 in patients from the phillipines. almost 50 years after its description by wade, it remains an interesting enigmatic form of leprosy mostly reported from india. histoid leprosy occurs in lepromatous patients who relapse after dapsone monotherapy, in those with dapsone resistance, sometimes even after multidrug treatment, or at times, de novo.7,8,9 the pathogenesis of this rare and unusual variant of leprosy still remains unresolved. the interplay of genetic factors, immune response and treatment received in a given patient seems to influence the manifestations of histoid leprosy.9 treatment of histoid leprosy includes not only antimycobacterial chemotherapy, but also patient education about the disease, treatment of reactions, monitoring for and care of nerve damage, care of any disability, social support, physical and occupational therapy, and rehabilitation.8 although, there is no clear recommendation regarding the treatment regimens for histoid leprosy, it has been treated on the lines of multibacillary leprosy9 and its managed by initially giving rom therapy with rifampicin 600 mg, ofloxacin 400 mg, minocycline 200 mg once, which is followed by multidrug regiment therapy.7,12 �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 27-31 case report a 36 years man, army soldier, originated from papua, visited to the skin clinic with transluscent shiny nodules on the left elbow and thumb for the last 18 months. these nodules were multiple, firm, and painless. this complaint was accompanied with nasal congestion. after 12 months, he noticed tickening of the ear lobes and loss of his eye brows. there was no history of any other painful eruptions or constitutional symptoms. no complaint of epistaxis or eyes involvement, as well as enlargement on his genital or pain on joints. there were no history of decrease of sweating on his body, nor dryness of the skin. no complaint about ulcer on his feet and any deformity of hands and feet. family history of the same disease was denied. he never got any medication or taking any antileprosy drugs. two years ago, he was operated for a single of nodule on his face at army hospital but no further information about the diagnosis. physical examination of general state showed an alert male with the blood pressure was 120/90 mmhg, the pulse rates was 80 times per minute, the respiration rates was 20 times per minute and the body temperature was 36.5° c. from head and neck, there were no anemic, cyanotic, ictreus and respiratory distress. there were madarosis on his eye brows and tickening of his both ear lobes, but no lagophtalmus or nose deformity. there were tickening of n. auricularis magnus dextra and sinistra. heart and lungs were normal; liver and spleen were not palpable. on his upper extremities, there were no edema and warm on palpation. bilateral medianus nerves were tickened but no glove anaesthetic pattern of the skin. there were nodules on his left elbow and thumb. on his lower extremities, there were tickening of n. peroneus lateralis dextra and sinistra, without stocking pattern of anaesthesia of the skin. no deformities of extremities fingers. dermatological examination on the left elbow and thumb, the nodules were 2-4 cm in size, multiple, mobile with hard in consistency. regular in contour with translucent shiny and sharply marginated. these lesions arising from apparently normal-looking skin. crusting were positive (figure 1 a,b,c). on his ears, multiple, skin colored plaques varying in size from 0.2-0.5 cm on normal appearing skin (figure 2 a,b). there were madarosis on his bilateral eye brows (figure 2c). differential diagnosis of this patient were histoid leprosy, dermatofibroma, neurofibroma, histiocytoma and eritema nodosum leprosum.laboratory examination revealed hemoglobin 12.0 g/dl, white blood cell count 11.500 k/ul. there was no abnormality from urinalysis figure 1. (a and b) the localization of lesions found in the patient. translucent nodules located over an apparently normal skin on the left elbow. (c) single nodule on the left thumb. figure 2. (a,b) multiple, shiny skin nodules varying in size on the ear lobes. note intervening normal looking skin. (c) madarosis ��agusni, et al.: histoid leprosy and urine sedimentation. bacterial examination from the ear lobe and shiny nodule smear for acid-fast bacilli showed a bacterial index (bi) of 4+ and a morfologic index (mi) of 10% (figure 3 ). figure 3. slit skin smear from ear lobe and nodule showed bacilli in globi,numerous and appear long with tappering ends (ziehl neelsen staining). (magnification 1000×) biopsy specimen of the nodules showed a wellcircumscribed area of the dermis packed with many acidfast organisms and foamy macrophages, consistent with histoid leprosy. fite-faraco stain demonstrated cells packed with lepra bacilli with a characteristic interlacing bundles of spindle-shaped histiocytes (figure 4 a,b,c). elisa examination of anti pgl-1 examination revealed igm > 5300 u/ml and igg > 5300 u/ml with cut off for igm: 605 u/ml and igg: 630 u/ml. pcr examination from nasal and skin swabs were positive and no mutation.polymerase chain reaction to detect m.leprae using the lpf-r/lp 1–2 nested primers were positive from skin smear, nasal swab and blood specimens. the results of drug resistance study using direct sequencing method for rpob, folp and gyra areas of m.leprae revealed no mutation, which means that the bacilli still sensitive to rifampycin, dapsone and quinolone treatment. the patient was treated with rifampicin 600 mg and ofloxacin 400 mg daily for ten days therapy initially, then followed by who-mdt therapy for multibacillary leprosy. improvement of clinical symtomps have been achieved after 3 months therapy and nodule lesions were disappeared. the treatment is continuing and the patient is still under monitoring. discussion histoid leprosy is uncommon variant of lepromatous leprosy.7 the term ‘histoid leprosy’ was first coined by wade in 1960 as a histological concept of bacillary rich leproma composed of spindle-shaped cells along with an absence of globus formation. since then few case series have been published, mostly from india 9 it constitutes 1.2–3.6 % of all leprosy cases, however, studies regarding this form of disease are rare.9,11 this variant of leprosy is considered as a well-recognized expression of multibacillary leprosy characterized by typical clinical, histopathological, immunological and bacteriological findings.3 based on the history, physical examination, and supported by laboratory result as well as histopathology, bacteriology and serology examination we diagnosed this patient as histoid leprosy. the histoid lesions commonly appear as smooth, shinny, hemispherical, dome-shaped, nontender soft to firm nodules which may be superficial, subcutaneus or fixed deeply under the skin and plaques or pads appearing on otherwise normal-looking skin.4,6,8 in this case, nodules/subcutaneus translucent shinny nodules were the morphological pattern and a significant proportion of this patient, arising from apparently normal skin. there are three type lesions of histoid leprosy; subcutaneus nodule, cutaneus nodule, and cutaneus plaque.4 in this case, the patient showed all of the typically condition features of histoid leprosy, like cutaneus nodules on his elbow, subcutaneus nodules on his left thumb and he also had skin colored plaques over the ear lobes. the nodules of histoid lesions which occur over the extensor surface of the extremities, back, buttock and face. they may be localized to bony prominences, especially around the elbows and knees.6 in this case, bony prominences around the elbow and thumb were the sites of involvement. leprosy bacilli in slit skin smear of a patient figure 4. skin biopsy showing (a) epidermal atrophy with grenz zone. (b) domination of histiocytes (magnification 1000x). (c) spindle shaped histiocytes, foamy macrophages. special fite-faracomethod �0 indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 27-31 with histoid leprosy are numerous.9 slit skin smear from histoid lesions shows abundant acid-fast bacilli occuring in clusters, singly or tightly. the histiocytes and macrophages are packed with lepra bacilli characteristically longer than ordinary lepra bacilli and with tappering ends.4,6,9 bacteriological index may be 3+ to 6+ and morphological index may be 0 to 10% or very high.9 slit skin smear from our patient were taken by standard methods from the skin and nodule, its revealed abundant organisms occuring in globi, appearing long with tappering ends as compared with lepra bacilli in patients with other types of leprosy. the bacteriological index and morphological index from our patient revealed 4+ and 10%. the microscopic pathology of histoid leprosy evolves with time and is altered by therapy. it is imperative to take biopsy of the entire lesion early in its evolution and before the administration of multidrug therapy.6 in this case, we did an excisional biopsy to taken the entire lesion. histopathology finding is unique in histoid leprosy.6 histopathological features in haematoxylin eosin and ziehl-neelsen stained skin biopsy specimens were studied to confirm the diagnosis of histoid leprosy, as suggested by sehgal and srivastava and wade.9 classical histopathology findings include epidermal atrophy as a result of dermal expansion by the underlying leproma and an acellular band (unna band) located immediately below the epidermis. this dermal expansion of histiocytes pushes aside the dermal collagen resulting in the formation of pseudo capsule. the leproma consists of fusiform histiocytes arranged in a whorled, criss-cross or storiform pattern. these histiocytes resemble fibroblast and it is suggested than these fibroblast-like macrophages may have arisen from tissue histiocytes rather than from blood monocytes. usually there is epidermal atrophy with a subepidermal grenz zone and a well-circumscribed dermal area of closely packed spindle-shaped histiocytes foaming interlacing bands and whorls surrounded by a pseudocapsule. foamy macrophages may be found. however, epidermal atrophy is the rule of thumb in histoid leprosy. 6 histopathological findings in this case included a free subepidermal zone (grenz) and intertwining of strands of spindle-shaped histiocytes, and also epidermal atrophy as a striking features of histoid leprosy. rapid molecular-type assays have been developed for detection of m. leprae directly from patient specimens using available genetic data. these assyas have been based primarily on the amplification of m. leprae-specific sequences using polymerace chain reaction (pcr) and identification of the m. leprae dna fragment. this technique has been applied not only to skin biopsy samples but also to several different types of specimens.4,15 we performed this examination to identify of acid-fast organisms and to detect any mutation. pcr examination of our patient taken from nasal and skin swab showed positive result and no mutation. pcr is indicated to identification of acid-fast organisms when bacilli are numerous but tissue site, clinical history, or their circumstances are questionable. pcr has thus generated new approaches to the detection and identification of m. leprae and, coupled with mutation detection analyses, has the ability to provide rapid drug susceptibility results from specimens taken directly from the patient. pcr can provide an excellent adjunct to clinical and histopathological diagnosis of leprosy.5,8 histoid leprosy has been reported generally to manifest in patients after long-term dapsone monotherapy, irregular or inadequate therapy, developing as relaps after successful treatment or even appearing de novo without a prior history of any antileprosy treatment6 like in this case. de novo, means, without any previous lesions or treatment, thus without the possibility of being relapsing cases.10,13 the pathogenesis of histoid leprosy still remains unresolved.4 the interplay of genetic factors, immune response and treatment received in a given patient seems to influence the manifestations of histoid leprosy. despite the presence of adequate numbers of macrophages, it has been claimed that they lack the functional property to kill bacilli that exist in high numbers in histoid lesions. it is possible that under the influence of m. leprae antigens they lose their bacteriolytic property or produce ‘suppressor’ cytokines, such as interleukin-10, that adversely inhibit t cell-mediated responses to m. leprae.4,9 when histoid leprosy occurs in the appropriate clinical setting, that is, in patient of lepromatous leprosy on antileprosy therapy, the diagnosis is rarely a problem. problems in diagnosis may occur when patients, particularly travelers, are present in nonendemic areas where the level of suspicion and familiarity with leprosy is low or when the preceeding ll leprosy is missed or is not evident. lepromatous nodules, erythema nodosum leprosum, von reklinghausen disease and histiocytoma are the conditions that need to be evaluated for the differential diagnosis of histoid leprosy. however, there are certain distinguishing features to be looked upon in cutaneous histoids. unlike the resilient von reklinghausen nodules, they are firm to palpation and also lack umbilication so typical of molluscum contagiosum.6,14 erythema nodosum lesions are red, hot, tender nodules; are associated with systemic manifestations; and tend to disappear and reappear.6 dermatofibroma is a common benign fibrous skin lesion, is also called a fibrous histiocytoma. it is due to a non-cancerous growth of dermal dendritic histicyte cells. in some cases it arises at the site of a minor injury, especially an insect bite or thorn prick. dermatofibroma most often occur on the legs and arms. once developed, they usually persist for years. they appears as firm-feeling nodules, often yellow-brown in colour, sometimes pink or quite dark. lepromatous leprosy nodules arise from infiltrated skin. in contrast, lesions of histoid leprosy arise from apparently normal skin. classical lepromatous leprosy presents with generalized symmetric lesions, while histoid lesions presents with localized asymetric lesions.11there is no clear recommendation regarding the treatment regimens for histoid leprosy. as some workers have considered it to be a variant of ll disease, it has been treated on the lines of multibacillary leprosy. between 1982 and 1994, multibacillary patients were treated with 2 years mdt multibacillary regimen ��agusni, et al.: histoid leprosy (mbr) or until bacillary negativity, whichever was later. between 1994 and 1998 and subsequently from 1999 onwards, multibacillary patients were treated with 2 years or 1 year fixed duration mdt mbr.9 with the advent of the new era of more effective treatment (mdt) modalities available for multibacillary leprosy, the same regimens can be applied to the patients with histoid disease. histoid leprosy being a highly bacillary form of leprosy and concern regarding the efficacy of fixed dose (12 months, after 1998). researchers have used ofloxacin in combination with standard mdt mbr or pefloxacin alone in treatment. in this case, we have also used ofloxacin that may rapidly reduce the bacillary load. as we have known that ofloxcacin have a strong bactericidal effect like other drugs; minocycline, clarithromycin, and levofloxin.8,11 it is the general belief that histoid disease requires longer for bacteriological clearance than does ll.11 according to report of two cases in india, histoid leprosy was managed by initially giving rom therapy with rifampicin 600 mg, ofloxacin 400 mg, minocycline 200 mg once, which is followed by mdt therapy.12 the disease responded satisfactorily. us national hansen disease program (nhdp) treatment recommends minocycline 100 mg daily, which can be used as a substitution for dapsone in individuals who do not tolerate this drug. it can also be used instead of clofazimine, although evidence of the efficacy of its anti-inflammatory activity against type 2 reactions is not as substantial as the evidence for clofazimine. clarithromycin, 500 mg daily, is also effective against m. leprae and can be used as substitution for any of the other drugs in a multiple drug regimen.8 refferences 1. brycesen a, pfalzgraff re. leprosy. 3th ed. edinburg: churchill livingstone; 1990. p. 80–1. 2. jopling wh, mcdougall ac. handbook of leprosy. 5th ed. india: cbs publis & dist; 1996. p. 35–6. 3. amirudin m. ilmu penyakit kusta. makasar: penerbit hasanuddin university press; 2003. h. 115–123. 4. scollard dm, adams lb, gillis tp, krahenbuhl jl, truman rw, williams dl. the continuing challenges of leprosy. clinical microbiology review. 2006; 19(2): 338–381. 5. sehgal vn, srivastava g, singh n. histoid leprosy: histopathological connotations’ relevance in contemporary context. am j dermapathol. vol 31. no. 3. 2009. p. 268–271. 6. manoharan r, madhu r, srinivasan ms. histoid hansen-a case report. e-journal of the indian society of teledermatology,2008;vol2, no. 2. p. 12–16. 7. worobec sm. treatment of leprosy/hansen’s disease in the early 21st century. dermatologic therapy,vol. 22, 2009. p. 518–37. 8. kaur i, dogra s, de d, saikia un. histoid leprosy: a retrospective study of 40 cases from india. british j of dermatology 2009 160, pp. 305–310. 9. bopp c, bakos l. the histoid variety of lepromatous leprosy. arch.derm.forsch.1975. 252, 1–10. 10. nair sp, moorthy kp, suprakasan s, jayapalan s, mini g. histoid leprosy-unsual presentation. international j of dermatology 2006, 45, 433–434. 11. annigeri sr, metgud sc, patil jr. lepromatous leprosy of histoid type. indian j med microbiol 2007; 25: 70–1. 12. sehgal vn, aggarwal a, srivastava g, sharma n, sharma s. evolution of histoid leprosy (de novo) in lepromatosa (multibacillary) leprosy. international journal of dermatology 2005; 44(7). p. 576–78. 13. nayar a. narayanan js, job k charles. histopathology study of early skin lession in leprosy . archieves of pathol 1992; 94: 199–204. 14. nkinda sj, reddy nbb. skin smears for leprosy. nilson t, sparel g. eds.2nd ed. wurzburg:germany leprosy relief association 1990. p. 67–71. 15. sharma ka. histopathology of histoid leprosy. international journal of leprosy and other mycobacterial diseases, mar 1997. ijtid vol 1 no 1 jan-apr 2010.29.pdf ijtid vol 1 no 1 jan-apr 2010.30.pdf ijtid vol 1 no 1 jan-apr 2010.31.pdf ijtid vol 1 no 1 jan-apr 2010.32.pdf ijtid vol 1 no 1 jan-apr 2010.33.pdf 11 vol. 7 no. 1 january–april 2018 cryptococcal antigenemia in hiv/aids patients using lateral flow immunoassay detection at dr. soetomo general hospital surabaya sajuni widjaja1,2a, erwin astha triyono3, arthur pohan kawilarang2, abu rohiman2 1 clinical microbiology study program,faculty of medicine universitas airlangga, dr. soetomo hospital, surabaya, indonesia. 2 departement of clinical microbiology, faculty of medicine universitas airlangga, dr. soetomo hospital, surabaya, indonesia. 3 departement of internal medicine, faculty of medicine universitas airlangga, dr. soetomo hospital, surabaya, indonesia. a corresponding author: sajuni.widjaja-2014@fk.unair.ac.id abstract cryptococcus infection in hiv / aids patients results in cryptococcal meningitis, a major cause of subacute meningitis with 100% mortality if not receiving appropriate antifungal therapy. an examination of cryptococcal antigen will provide risk information for patients who will experience cryptococcal meningitis. better diagnosis in asymptomatic and symptomatic phases of cryptococcosis are key components to reduce morbidity and mortality. this study aims to determine the proportion of cryptococcal antigenemia in hiv / aids patients treated at intermediate treatment-infectious diseases unit of dr. soetomo general hospital surabaya. cryptococcal antigenemia was examined in hiv / aids patients with suspected cryptococcus infection and cd4+ t cell lymphocyte count <200 cell /μl. the examination used a lateral flow assay diagnostic tool, a simple fda(food and drug administration)-approved immunochromatographic test system for detection of capsular polysccharide antigens of cryptococcus species complex (cryptococcus neoformans and cryptococcus gattii) in blood. this test meets all of the world health organization assured criteria (affordable, sensitive, specific, user friendly, rapid/robust, equipment-free, and delivered). sensitivity and specifiticy of this method from serum are both 100%. there were 3 positive cryptococcal antigenemia from 41 serum hiv / aids patients with suspected cryptococcus infection at intermediate treatmentinfectious diseases unit of dr. soetomo general hospital surabaya. all of these patients were male aged over 36 years, had cd4+ t cell lymphocytes <100 cell /μl and had never received antiretroviral therapy before. the proportion of cryptococcal antigenemia in hiv / aids patients with suspected cryptococcus infection at intermediate treatment-infectious diseases unit of dr. soetomo general hospital surabaya was 7.32%. keywords: cryptococcal antigenemia, aids, hiv, dr. soetomo hospital, surabaya abstrak infeksi jamur cryptococcus pada pasien hiv/aids mengakibatkan cryptococcal meningitis, yang merupakan penyebab utama meningitis subakut dengan mortalitas sebesar 100% bila tidak mendapatkan terapi antijamur yang tepat. pemeriksaan antigen cryptococcal akan memberikan informasi risiko pasien yang akan mengalami cryptococcal meningitis. semakin baik dan cepat diagnosis cryptococcus antigenemia baik dilakukan pada fase simptomatik maupun asimptomatik cryptococcosis merupakan kunci penting dalam mengurangi morbiditas dan mortalitas. penelitian ini bertujuan mengetahui proporsi cryptococcal antigenemia pada pasien hiv/aids yang dirawat di unit perawatan intermediate penyakit infeksi rumah sakit dr. soetomo surabaya. dilakukan pemeriksaan cryptococcal antigenemia pada pasien hiv/aids dengan kecurigaan infeksi cryptococcus dan hitung limfosit t cd4+<200sel/μl. pemeriksaan menggunakan alat diagnostik lateral flow assay yang merupakan teknik imunokromatografi yang telah disetujui oleh fda (food and drug administration) dan dapat mendeteksi antigen kapsul polisakarida dari kompleks spesies cryptococcus (cryptococcus neoformans dan cryptococcus gattii) dari darah. pemeriksaan ini memenuhi kriteria dari who (world health organization) antara lain mudah dijangkau, sensitif, spesifik, mudah digunakan, cepat, tidak memerlukan peralatan yang sulit didapat, dan mudah dibawa. sensitifitas dan spesifisitas pemeriksaan ini dari serum adalah sebesar 100%. didapatkan hasil penelitian yaitu 3 positif cryptococcal antigenemia dari 41 serum pasien hiv/aids dengan kecurigaan infeksi cryptococcus di unit perawatan intermediate penyakit infeksi rumah sakit research report 12 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 11–14 dr. soetomo surabaya. semua pasien tersebut adalah laki-laki, berusia diatas 36 tahun , memiliki hitung limfosit t cd4+<100 sel/μl dan belum pernah mendapat terapi arv sebelumnya. proporsi cryptococcal antigenemia pada pasien hiv/aids dengan kecurigaan infeksi cryptococcus di unit perawatan intermediate penyakit infeksi rumah sakit dr. soetomo surabaya adalah sebesar 7.32%. kata kunci: cryptococcal antigenemia, aids, hiv, rsud dr. soetomo, surabaya introduction hiv/aids has been a cause of death for mostly humans in their productive times. death is mainly due to opportunistic infection. opportunistic infection is an immune system will not cause illness but is fatal in people with decreased immunity, as in hiv/aids patients.1 cryptococcal meningitis is a major cause of subacute meningitis and death in patients with advanced hiv infection, affecting an estimated one million people each year, especially in sub saharan africa.2,3 cryptococcal antigenemia testing will provide risk information for patients who will experience cryptococcal meningitis and death. therefore, appropriate antifungal administration in patients with positive cryptococcal antigenemia (crag) is highly recommended. rapid diagnostic testing using lfa (lateral flow immunoassay) method recommended by who is also recommended as a screening diagnostic method especially in hiv/aids patients with mild symptoms (no neurological disorders) or asympomatic. lfa examination is inexpensive and easy to perform, giving a sensitivity and specificity of more than 95% within 10 minutes.4 more than 80% positive crag was obtained in hiv/ aids patients with cd4+ t cell lymphocyte count <100 cells/µl. in other african and american studies, it was also found in hiv/aids patients with cd4+ t cell lymphocyte count between 100-200 cells/µl.5,6 this study aim to determine the proportion of cryptococcal antigenemia in hiv/aids patients treated at dr. soetomo hospital surabaya. material and method study design and patients this was an observational study with cross sectional study design to study the proportion of cryptococcal antigenemia. hiv/aids patients ≥21 years old confirmed by three methods of hiv examination, cd4+ count <200 cells/µl with suspected cryptococcal antigenemia (had fever with or without headache) were enrolled. study participants were not required to be art naive. patients treated for cryptococcal infection in the last three months or currently taking an antifungal agent were excluded from study participation. ethics statement the study was conducted according to the principles of the declaration of helsinki. written informed consent was required from all study participants and the study was approved by ethical committee of dr. soetomo hospital, surabaya, indonesia (no. 382/panke.kke/v/2017). procedures a blood sample was obtained from each study participant to perform cryptococcal antigen lateral flow assay (immy, usa), a simple fda-approved immunochromatographic test system for detection of capsular polysaccharide antigens of cryptococcus species complex (cryptococcus neoformans and cryptococcus gattii) in blood. serum was separated from the blood sample. according to immy manufacturer’s recommendation one drop of lateral flow specimen was added to microtube, and then 40µl of serum was added to microtube. after that, lateral strip was immersed to the mixture and stayed for 10 minutes. the test is positive if there were 2 line on strip, negative test if only 1 line on strip. step by step of immy crag lfa can be seen in figure 1. data analysis figure 1. step by step of immy crag lfa.7 all analyses were performed using spss 16. chi square was used to determine factors associated with positive cryptococcal antigenemia. a p value ≤0.05 was considered significant. result and discussion the diagnostic use for detection of cryptococcal capsular polysacchride antigen (crag) in serum by latex agglutination test (crag-latex) or enzym-linked immunoassay (eia) has been available for over decades. better diagnostics in asymptomatic and symptomatic phases of cryptococcosis are key components to reduce mortality.8 cryptococcal antigen lateral flow assay (crag lfa) was included in the armamentarium for diagnosis. unlike the other tests, the crag lfa is a dipstick immunochromatographic assay, in a format similar to the home pregnancy test, and requires little or no lab infrastructure. this test meets all of the world health organization assured criteria (affordable, sensitive, 13widjaja, et al.: cryptococcal antigenemia in hiv/aids patients specific, user friendly, rapid/robust, equipment-free, and delivered).8 crag lfa has better analytical sensitivity for cryptococcus gattii than crag-latex or eia. sensitivity and specificity of crag lfa from serum are both 100%.7 a total of 41 hiv/aids patients were enrolled into the study. the mean age of those enrolled was 36.37 years and 90.24% were male. the prevalence of male against female can be explained by the higher incidence of hiv in men. the prevalence of cryptococcal antigenemia in soetomo hospital is 7.32%. the mean global prevalence of cryptococcal antigenemia is 6%.4 positive crag lfa were 3 males from 41 patient. a 3% prevalence of cryptococcal antigenemia is the point at which the cost of treating cryptococcal meningitis with amphotericin b is greater than the cost of screening for crag examination. in uganda, screening to prevent a case of cryptococcal meningitis requires a cost of $28, whereas to prevent a single death due to cryptococcal meningitis costs $40.9 the gold standard treatment for cryptococcal meningitis is to use amphotericin b which are known to be expensive and difficult to obtain especially by low-income countries, so more often fluconazole is used.10 other analyses reported that crag screening in areas with prevalence of <1% remained “cost-effective” due to lower lfa screening costs when compared with crag latex.9,11 the host gender also plays a role in the pathogenesis of cryptococcal, which estrogen can inhibit the growth of cryptococcus in vitro. male immune responses are less efficient in controlling cryptococcus infections, because male macrophages tend to be killed by cryptococcus rather than phagocytosis of cryptococcus.12,13 in this study there is no correlation between male and female with positivity of cryptococcal antigenemia (p:1.0). there is also no correlation between male and female with positivity of cryptococcal antigenemia in another studies. all of the positive patients are >36 years old, but there is no correlation between age with positivity of cryptococcal antigenemia (p:0.091) in this study. it is the same with another studies.7,11 one patient is 42 years old, another 54 years old and 71 years old. old patient (71 years old) was died, but the others were alive. the prognosis of cryptococcosis occuring in patients >60 years is generally worse.14 baseline characteristics of patients according to cryptococcal antigenemia status can be observed in table 1. the positive test of cryptococcal antigen lateral flow assay can be seen in figure 2. there were 15 patients (36.6%) that already take arv, 96% from that have cd4+ t cells lymphocyte count <100 cells/µl. in this study, all of crag positive patients were naive arv, but there is no significant correlation (p:0.287), the same as studies that have already done by vidal and manga.6,12 there is no correlation within symptoms and crag positive antigenemia in this study (p:0.143 for fever, and p:0.539 for headache). in manga’s study, fever present in 82.5% of cases, was more frequent in patients with positive table 1. baseline characteristics of patients according to cryptococcal antigenemia status. parameter cragpositive cragnegative p age (years) <36 >36 0 3 22 16 0.091 sex male female 3 0 34 4 1.000 fever yes no 2 1 37 1 0.143 headache yes no 3 0 25 13 0.539 cd4+ cells <100 cells/µl >100 cells/µl 3 0 36 2 1.000 arv yes no 0 3 15 23 0.287 figure 2. positive test of cryptococcal antigen lateral flow assay antigenemia, but also have no significant corellation,7 but presenting headaches has significantly associated with positive cryptococcal antigenemia (p:0.000008).12 from total patients enrolled in this study, 95% have cd4+ t cell lymphocyte count <100 cells/µl, mean cd4+ t cell lymphocyte count is 31.5 cels/µl. crag positive patients in this study have cd4+ t cell lymphocyte count <100 cells/µl. but there is no significant correlation between cd4+ t cell lymphocyte count with positivity of crag lfa. research conducted by manga, alemu and ganiem also get the value of p>0.05.5,12,13 more opportunistic infections will occur in hiv/aids patients that have cd4+ lymphocyte count <100 cells/µl. the prevalence of opportunistic infections varies between regions.15 opportunistic infections encountered in this study include pulmonary tuberculosis, cerebral toxoplasmosis, oropharyngeal candidiasis, pcp. the most common types of opportunistic infections in this study are pulmonary tuberculosis. research conducted by andama16 and jarvis17states that tuberculosis is an infection that is often encountered in conjunction with cryptococcus antigenemia. 14 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 11–14 indonesia, china and india are known to be a country with high tuberculosis burden.18 several studies suggest that tuberculosis alone may result in a decrease in cellular immune function (a decrease in cd4+ t cell lymphocyte count), and that concurrent tuberculosis infection with hiv will result in significantly lower cd4+ t cell lymphocyte count compared to hiv monoinfection or tuberculosis monoinfection.19–21 our study is subject to several limitations. these include absence of lumbar punctures, which would help define the proportion of patients with cryptococcal meningitis among those with a positive cryptococcal antigen test, and no long-term clinical follow up of our patient cohort, also the high difference between cd4+<100 cells/µl and cd4+ 100-200 cells/µl. conclusion the prevalence of cryptococcal antigenemia in soetomo hospital’s hiv/aids patients is 7.32%. it means that crag screening in soetomo hospital will be “costeffective”. future studies should be conducted to optimize screening and pre-emptive treatment of cryptococcosis. references 1. denning dw. minimizing fungal disease deaths will allow the unaids target of reducing annual aids deaths below 500 000 by 2020 to be realized. philos trans r soc b biol sci. 2016;371(1709):20150468. 2. park bj, wannemuehler ka, marston bj, govender n, pappas pg, chiller tm. estimation of the current global burden of cryptococcal meningitis among persons living with hiv/aids. aids. 2009 feb 20;23(4):525–30. 3. jarvis jn, meintjes g, williams a, brown y, crede t, harrison ts. adult meningitis in a setting of high hiv and tb prevalence: findings from 4961 suspected cases. bmc infect dis. 2010 dec 15;10(1):67. 4. rajasingham r, smith rm, park bj, jarvis jn, govender np, chiller tm, et al. global burden of disease of hiv-associated cryptococcal meningitis: an updated analysis. lancet infect dis. 2017 aug;17(8):873–81. 5. alemu as, kempker rr, tenna a, smitson c, berhe n, fekade d, et al. high prevalence of cryptococcal antigenemia among hivinfected patients receiving antiretroviral therapy in ethiopia. plos one. 2013;8(3). 6. vidal je, toniolo c, paulino a, colombo a, dos anjos martins m, da silva meira c, et al. asymptomatic cryptococcal antigen prevalence detected by lateral flow assay in hospitalised hiv-infected patients in são paulo, brazil. trop med int health. 2016;21(12):1539–44. 7. use i. cryptococcal antigen lateral flow assay performance summary. culture. (figure 1). 8. vidal je, boulware dr. lateral flow assay for cryptococcal antigen: an important advance to improve the continuum of hiv care and reduce cryptococcal meningitis-related mortality. rev inst med trop sao paulo. 2015 sep;57 suppl 1:38–45. 9. meya db, manabe yc, castelnuovo b, cook ba, elbireer am, kambugu a, et al. cost-effectiveness of serum cryptococcal antigen screening to prevent deaths among hiv-infected persons with a cd4+ cell count < or = 100 cells/microl who start hiv therapy in resourcelimited settings. clin infect dis. 2010 aug 15;51(4):448–55. 10. parkes-ratanshi r, wakeham k, levin j, namusoke d, whitworth j, coutinho a, et al. primary prophylaxis of cryptococcal disease with fluconazole in hiv-positive ugandan adults: a double-blind, randomised, placebo-controlled trial. lancet infect dis. 2011 dec;11(12):933–41. 11. rajasingham r, meya db, boulware dr. integrating cryptococcal antigen screening and pre-emptive treatment into routine hiv care. jaids j acquir immune defic syndr. 2012 apr;59(5):85– 91. 12. manga nm, cisse-diallo vmp, dia-badiane nm, diop-nyafouna sa, er d, yengo n, et al. prevalence and factors associated with positive cryptococcal antigenemia among hiv infected adult hospitalized in senegal epidemiological aspects. 2016;1–6. 13. ganiem ar, indrati ar, wisaksana r, meijerink h, van der ven a, alisjahbana b, et al. asymptomatic cryptococcal antigenemia is associated with mortality among hiv-positive patients in indonesia. j int aids soc. 2014;17:1–7. 14. kauffman ca. fungal infections in older adults. clin infect dis. 2001;33(4):550–5. 15. gangcuangco lma, sawada i, tsuchiya n, do cd, pham ttt, rojanawiwat a, et al. regional differences in the prevalence of major opportunistic infections among antiretroviral-naïve human immunodeficiency virus patients in japan, northern thailand, northern vietnam, and the philippines. am j trop med hyg. 2017 jul;97(1):49–56. 16. andama ao, den boon s, meya d, cattamanchi a, worodria w, davis jl, et al. prevalence and outcomes of cryptococcal antigenemia in hiv-seropositive patients hospitalized for suspected tuberculosis in uganda. jaids j acquir immune defic syndr. 2013 jun;63(2):189–94. 17. jarvis jn, harrison ts. hiv-associated cryptococcal meningitis. aids. 2007 oct;21(16):2119–29. 18. world health organization. global tuberculosis report. 2016. 19. rajian m, gill ps, chaudhary u. effect of tuberculosis co infection on virological failure in hiv patients on first line of highly active antiretroviral therapy. intjcurrmicrobiolappsci. 2017; 6(1):78–81. 20. davoudi s, rasoolinegad m, younesian m, hajiabdolbaghi m, soudbakhsh a, jafari s, et al. cd4+ cell counts in patients with different clinical manifestations of tuberculosis. brazilian j infect dis. 2008 dec;12(6):483–6. 21. pawlowski a, jansson m, sköld m, rottenberg me, källenius g. tuberculosis and hiv co-infection. hobman tc, editor. plos pathog. 2012 feb 16;8(2):e1002464. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 27 vol. 7 no. 2 may–august 2018 soil-transmitted helminth infection and eosinophil level among waste collectors in banda aceh teuku romi imansyah putra1,2a, ricke loesnihari3, merina panggabean4 1 tropical medicine program, faculty of medicine, university of north sumatera, dr t mansur st, padang bulan, medan, sumatera utara, indonesia. 2 department of parasitology faculty of medicine, syiah kuala university, tgk tanoh abee st, darussalam, banda aceh, indonesia. 3 department of clinical pathology haji adam malik general hospital, bunga lau st, medan tuntungan, medan, north sumatra, indonesia. 4 department of parasitology faculty of medicine, university of north sumatra, dr t mansur st, padang bulan, medan, north sumatera, indonesia. a coresponding author: teukuromiimansyahputra@unsyiah.ac.id abstract soil-transmitted helminth (sth) has infected more than one billion people worldwide. in indonesia, the prevalence of worms is still relatively high in 2006, which amounted to 32.6% and in 2007 reached 65% mainly on the economically disadvantaged group. primary infections are usually occured in children and can persist into adulthood through exposure to recurrent infections to stoolcontaminated environments and may be chronic to residents living in endemic areas. waste collectors are one of the groups associated with land at risk for sth infection. the work environment of waste collectors which has many seeds of disease, causing pollution and other negative effects. waste collectors are also often contact with the ground directly. the research was conducted to find out the association of sth infection with eosinophil level in waste collectors from sanitation department in banda aceh city. the design of this research is an observational study using cross sectional method. statistical analysis using chi square test with significance p < 0.05. stool and blood samples were collected from 60 workers who were willing to participate (with informed consent). the katokatz method was used to determine sth infection and absolute eosinophil count was performed on blood preparations seeing in the count chamber to find out the number of eosinophils in the waste collectors. the prevalence of sth infection is 23.3% (14/60) consisted of t.trichiura infection (21.7%) and a mixed infection of 1.6% (both of a. lumbricoides and t.trichiura) there was no single infection of a. lumbricoides and hookworm infection. the prevalence of eosinophilia is 21.7% (13/60). there was no significant association between sth infection and blood eosinophil level (p value = 1.00). this study does not recomemend the use of eosinophilia as a marker for sth infection. keywords: eosinophilia, kato katz, sth infection, waste collectors, banda aceh abstrak soil-transmitted helminth (sth) menginfeksi lebih dari satu milyar orang di seluruh dunia. di indonesia, prevalensi kecacingan masih relatif tinggi pada tahun 2006, yaitu sebesar 32,6% dan pada tahun 2007 mencapai 65% terutama pada golongan penduduk kurang mampu dari sisi ekonomi. infeksi primer biasanya terjadi pada anak-anak dan bisa menetap sampai dewasa melalui paparan infeksi berulang terhadap lingkungan yang terkontaminasi tinja dan dapat bersifat kronis pada penduduk yang tinggal di daerah endemis. petugas sampah merupakan salah satu kelompok yang berhubungan dengan tanah yang beresiko terinfeksi sth. lingkungan kerja petugas sampah banyak mengandung bibit penyakit, menimbulkan polusi dan berbagai efek negatif lainnya. petugas pengangkut sampah juga sering bersentuhan langsung dengan tanah. penelitian ini dilaksanakan untuk mengetahui hubungan infeksi sth dengan kadar eosinofil pada petugas pengangkut sampah dinas kebersihan dan keindahan kota banda aceh. desain studi observasional menggunakan metode cross sectional. analisis statistik menggunakan uji chi square dengan kemaknaan p < 0,05. sampel tinja dan darah dikoleksi dari 60 petugas yang bersedia berpartisipasi (dengan informed consent). metode kato-katz digunakan untuk mengetahui adanya infeksi sth dan hitung eosinofil absolut dilakukan pada sediaan darah yang dilihat pada kamar hitung untuk mengetahui jumlah eosinofil pada petugas pengangkut sampah. prevalensi infeksi sth sebesar 23,3% (14/60) terdiri dari infeksi t.trichiura 21,7% dan infeksi campuran 1,6% (a. lumbricoides dan t. trichiura). tidak ditemukan infeksi tunggal a.lumbricoides dan infeksi hookworm. research report 28 indonesian journal of tropical and infectious disease, vol. 7 no. 2 may–august 2018: 27–34 prevalensi eosinofilia adalah 21,7% (13/60). tidak terdapat hubungan yang signifikan antara intensitas infeksi sth dengan kadar eosinofil darah (p value = 1.00). penelitian ini tidak menganjurkan eosinofilia sebagai marker adanya infeksi sth. kata kunci: eosinofilia, kato-katz, infeksi sth, petugas sampah, banda aceh introduction soil-transmitted helminth is a parasitic disease caused by a nematode worm transmitted to humans through faecally contaminated soil.1 more than one billion people have been infected by at least one species of soiltransmitted helminth (sth) group. nematodes, also called geohelminth, includes the roundworm (ascaris lumbricoides), whipworm (trichuris trichiura), hookworm (necator americanus and ancylostoma duodenale) and strongyloides stercoralis.2,3 waste collectors are at risk of being infected by sth due to the frequent contact with soil and waste.1,4 a study conducted in pekanbaru showed that 77.78% of park workers were positively infected by ascarislumbricoides and trichuris trichiura.5 another study was conducted among waste collectors in pematang siantar found that 11.1% of waste collectors were infected byworm.6 a study in yogyakarta was revealed that waste collectors have yet to make adequate efforts in protecting themselves against waste-related diseases.7 the human immune response to worm infection is associated with increased level of ige, eosinophils, and mastocytosis that stimulate th2 production, namely interleukin 4 (il-4) and interleukin 5 (il-5). one of the main functions of eosinophils is to protect against infections and fully responsible for worm infestation inflammatory pathology.8 study in northern mindanao, the philippines is showed the association between the increased number of eosinophils (eosinophilia) and sth parasite infections.9 eosinophilia, as a th2 cell response marker, can be used to assess worm infections10 but the effect of sth infection on eosinophilia may vary depending on the distribution, maturation, and chronicity and type of parasites.8 the researcher was also observed that the waste collectors did not use or wear the standard personal protective equipment (ppe) such as gloves or shoes while working, thus exposed to garbage-related diseases, including the sth. data on the sth infection among waste collector is not available and there is no existing research on the association between sth infection and eosinophil level among waste collectors in banda aceh. therefore, it is necessary to conduct such a research to find out the prevalence and the association between sth infection and eosinophil level among waste collectors in the city of banda aceh. material and method this cross-sectional study was conducted to determine the prevalence and the association of sth infection and eosinophil level by performing faecal and blood eosinophil examination. the research was also wanted to know the association between the hand-washing habit and sth infection, and the association between the use of standard ppe and sth infection. the research subjects were 60 waste collectors in banda aceh. they were selected using the simple random sampling method. they clearly informed about the objective of the study. waste collectors are involved in this study were provided a written informed consent for examining their faecal and blood. for their handwashing habit and standard ppe, authors were interviewed by person and gave questionnaire to be filled. medical research ethics commission of faculty of medicine university of north sumatera (usu) approved the study. (no:780/tgl/kepk fk usu-rsup ham/2016). the study was conducted in march 2017 at the sanitation department of banda aceh. the examination of stool samples using the kato-katz technique was performed at the parasitology laboratory, faculty of medicine, syiah kuala university in banda aceh. absoluted eosinophil count examination was conducted at the health clinic laboratory of prodia in banda aceh. chi square test was used to estimate the association between sth infection and eosinophil level. if it does not meet the test requirements then fisher’s exact test were conducted through a computer software with significance value of<0.05. kato katz method study participants were instructed how to collect stool samples and provided with labeled clean plastic container, toilet tissue paper and applicator sticks. the team was labeled empty containers with identification (id) numbers, distributed these to the waste collectors and collected filled containers that had been distributed the day before. each day, approximatel 5-6 workers were enrolled and lime-sized early morning stool samples were collected. the kato-katz method is a recommended examination with a thick quantitative smelling technique. this method is most recommended and widely used for epidemiologic surveys, as it is both easy and cost-prohibitive.11,12 the materials used were prepared in accordance with standard 29putra, et al.: soil-transmitted helminth infection and eosinophil laboratory in-house procedures. thus, the glycerinmalachite green solution was mixed with 1 ml of 3% malachite green, 100 ml of 6% phenol and 100 ml of pureglycerin. the cellophane strips, each 22x40 mm, were soaked in this solution for at least 24 hours before use.13 immediately after the stool sample arrived at syiah kuala parasitology laboratory (skpl), kato-katz thick smears from each stool sample were prepared by 2 members of skpl, using 41.7 mg templates. the cellophane is then placed directly on the stool, so that the eggs are more easily visible and stored for long periods in slide. after a clearing time of 20-40 min, each kato-katz thick smear was examined quantitatively for geohelminth eggs by one of two experienced microscopists. in the afternoon, 3-6 hour after slide preparation, the thick smears were re-examined by the other one of two experienced microscopists, who counted eggs of a. lumbricoides, t. trichiura and hookworm then recorded them separately. additionally, in order to eliminate fibers or seed, the technique was modified by pressing a 105-mesh stainless steel grid onto the sample which was then filtered, transferred to slides covered by the cellophane soaked cover slips and allowed to stand for 30 minutes. all preparations were initially screenedwith a low-power (10x) objective lens. suspected parasitic objects were subsequently examined under a high-power (40x) objective.13,14 the stool samples were preserved in 10% formalin for later confirmation, if needed. absolute eosinophil count the health clinic laboratory of prodia in banda aceh uses the manual method. the equipment consists of improved neubauer counting chamber, white blood count (wbc) pipette and diluting dunger’s fluid. the technique are gently mix the blood in the edta vial, so that the cells mix well with plasma. draw the blood in the wbc pipette up to mark 1. wipe off the excess blood from sides of the tip of the pipette. dip the tip of the pipette in the dunger’s fluid and draw the fluid up to mark 11. the dilution is 1 in 10. holding the pipette horizontally in its long axis, rotate it slowly to ensure thorough mixing of blood and diluent. this is facilitated by the white bead in the bulb. place the cover slip on the cleaned ruled area of the counting chamber. we were discarded the first 2 to 3 drops (since the fluid has not mixed with blood) of wbc fluid from the pipette and charged the chamber by placing the tip of the pipette just under the cover slip and fluid flows under it by capillary action. then we were allowed untill allow till the counting chamber is just filled. we were waited for 5 minutes for the eosinophils to settle in the chamber and counted the number of eosinophils in the 4 corner squares under the microscope using a low power objective. eosinophils are identified because of their bright red granules and count should be done within 30 minutes. absolute eosinophil count (aec) = total number of eosinophils in 4 squares × 25. eosinophils constitute 1 to 6% of circulating wbcs. the range of absolute eosinophil count is 40–450 cells/ µl.15 result and discussion table 1 was showed the demography and clinical features of the respondents. from the 60 subjects, half of them were aged between 31–40 years (50%) and the majority (83.3%) did not wear personal protection equipment (ppe) while working. the prevalence of sth infection was 23.3%, where 21.7% were infected by t. trichiura, and 1.6% by mixed infections. based on the frequency distribution of absolute eosinophil level, it was found that there was 21.7% of subjects with an increase of absolute eosinophil and 78.3% of them with normal level. the majority of the waste collectors (96.7%) is washed their hands after working and the other 3.3% did not. meanwhile only 16.7% of them use or wear the ppe while working and the other 83.3% did not maintain that habit. table 1. d e m o g r a p h y a n d c l i n i c a l f e a t u r e s o f s t u d y respondents no category frequency % 1 2 3 4 5 6 age (year) 20-30 31-40 41-50 >50 positive kato-katz t. trichiura a. lumbricoides hookworm mixed infection (a.lumbricoides+t.trichiura) negative kato-katz absolute eosinophil level normal elevated hand-washing habit after work yes no personal protective equipment yes no 20 30 8 2 13 0 0 1 46 47 13 58 2 10 50 33,3 50 13,3 3,3 21,7 0 0 1,6 76,7 78,3 21,7 96,7 3,3 16,7 83,3 the results of this study were showed that of the 60 sth-infected subjects, t. trichiura infection was the most prevalent worm infection which was suffered by the subjects (21.7%) and 1.6% of subjects suffered from one mixed infection subject (t. trichiura and a. lumbricoides) and no one suffered from a. lumbricoides single infection as we see in table 2. this is probably because the subject’s work environment is located in a humid area which facilitates the growth of t. trichiura worm eggs. t. trichiura eggs will be able to grow optimally at 30°c. in addition, the fewer number of sth-infected subjects may also be caused by the administration of worm infestation medication albendazol 400 mg (single dose) in the office. as many as 600 tablets of medication supply were obtained from banda aceh city health office but there was 30 indonesian journal of tropical and infectious disease, vol. 7 no. 2 may–august 2018: 27–34 no accurate record of the waste collectors who received it or who directly took it. in populations receiving treatment of worm infection, the natural pattern of infection is altered because single dose albendazole is effective for the treatment of ascaris lumbricoides infection compared to the other two types of worm.16 based on a meta-analysis study, single dose administration of albendazole was not adequate for the treatment of t. trichiura. the cure rate increases after the administration of albendazole for three consecutive days.17 table 2 was showed the quantitative data of each study subjects obtained from the examination. subjects infected with sth and also experiencing eosinophilia are given an arrow and highlight marker. there were 13 people who experienced an increase in eosinophil count and among 13 people only 3 people who were detected had sth infection. table 2 also was showed the intensity of worms based on the value of eggs per gram (epg) from each subject infected sth as mild intensity category. table 3 was shows the association between handwashing habits and sth infection. from 60 samples, the subjects who did not wash their hands all had sth infection (100%), subjects who washed their hands and got infected with sth were as many as 20.7%, subjects who washed their hands and were not infected with sth were 9.3%. prevalence ratio (pr) of 0.2 (95% ci = 0.12-0.34). however, the statistical test revealed p-value of 0.051. therefore, the study is found that there is no significant association between hand-washing habits and sth infection. hygiene (in this case hand washing treatment) in waste collector is very necessary because they always contact with garbage which can lead to susceptibility to some garbage diseases such as infection of worms.18 this study is revealed that from the sample of 60 subjects examined by kato-katz method, there were 23.3% of the subjects who were positively infected with sth. the fact that there were fewer subjects infected with sth may result from their maintaining good personal hygiene (hand-washing habits) although they are still at risk of being infected with sth due to constant exposure to waste and soil. another possible explanation is that because the subjects routinely take anti-helmintic medication for prevention which are available in their office as part of worm medication program. however, there is no available data regarding the number of waste collectors who have taken such a medication because it is distributed through the team foreman. the association between hand-washing habit after work and sth infection incidence is showed that of 58 subjects who washed hands, there were 46 sth-uninfected subjects (79.3%) and 12 sth-infected subjects (20.7%) as well as 2 subjects (100%) who did not wash their hands and got infected with sth. the finding that subjects who wash their hands but got infected with sth was probably due to their washing their hand without using soap or antiseptics. this study is found that there is no association between hand-washing habits and sth infection (p-value = 0.051). table 2. absolute eosinofil count value and eggs per gram value from each waste collector (quantitative data) no. id absolute eosinofil count (x 10³/µl) helminth eggs by kato katz examination epg 1 88 0,070 tt = 2 48 2 313 0,080 n 3 29 0,462 ↑ n 4 515 0,110 n 5 118 0,144 tt = 1 24 6 392 0,188 n 7 288 0,270 n 8 87 0,070 n 9 301 0,144 n 10 374 0,490 ↑ tt = 8 192 11 123 0,165 n 12 400 0,929 ↑ n 13 25 0,120 n 14 207 0,122 n 15 508 0,160 tt = 4 96 16 240 0,350 n 17 226 0,090 tt = 2 48 18 103 0,070 n 19 502 0,130 n 20 227 0,621 ↑ n 21 375 0,187 n 22 455 0,270 n 23 298 0,110 n 24 214 0,440 tt = 4 96 25 315 0,150 n 26 133 0,187 n 27 94 0,121 n 28 264 0,133 tt = 3 72 29 403 0,110 n 30 348 0,649 ↑ n 31 31 0,550 ↑ n 32 95 0,160 n 33 193 0,210 n 34 439 0,122 n 35 449 0,070 al = 32 tt = 20 al=768. tt=480 36 73 0,831 ↑ n 37 507 2,480 ↑ n 38 251 0,310 n 39 46 0,357 tt = 6 144 40 99 0,132 n 41 286 0,080 n 42 121 0,110 n 43 128 0,324 n 44 209 0,460 ↑ n 45 228 0,070 n 46 506 0,570 ↑ tt = 2 48 47 349 0,050 n 48 444 0,180 n 49 336 0,200 n 50 407 0,080 tt = 5 120 51 291 0,120 tt = 13 312 52 199 0,050 n 53 248 0,230 n 54 257 0,570 ↑ tt = 2 48 55 202 0,198 n 56 389 0,467 ↑ n 57 144 1,800 ↑ n 58 377 0,150 tt = 3 72 59 371 0,120 n 60 360 0,060 n note: tt = t. trichiura al = a. lumbricoides n = normal epg = eggs per gram 31putra, et al.: soil-transmitted helminth infection and eosinophil this finding was similar to a study wich was conducted by butarbutar et al who found no association between hygiene and sth infection among collectors in pematang siantar city.6 however, the finding was differ to a study in pekanbaru, where siregar found a significant association between hand-washing with antiseptic soap and worm infestation incidence (p-value 0,024).5 a research conducted in tanzania was showed that sth infection can spread in the case of hand-washing without soap and clean water so the risk of sth infections is lowered when washing hand.19 the personal hygiene habits among waste collectors include changing clothes after work, washing work clothes, washing hands and feet after work (in contact with waste) and using soap during shower after getting in contact with garbage or waste..18 the present study is found that hand washing is a common practice among waste collectors in banda aceh, where 96.7% washed their hands after work. this indicates that the subjects have made a conscious effort to maintain their personal hygiene, one of which is by washing their hands. however, it was not clear if the subjects washed their hand using soap, because according to the researcher’s observation in the waste collectors’ workplace, only tap water for hand washing was available, without soaps or antiseptics. the finding of this study was significantly different from a research conducted by maywati, where she found that waste collectors in tasikmalaya did not maintain personal hygiene.20 table 4 was showed the association between the complete use of ppe and sth infection. from 60 subjects, there were 74.0% of subjects with incomplete use of ppe and were not infected with sth, 26.0% of subjects with incomplete use of ppe and got infected with sth, 90% of subjects who completely used ppe and were not infected with sth and 10.0% of subjects who completely used ppe and got infected with sth. the prevalence ratio of 0.3 (95% ci = 0,5–2,6) indicates that complete use of ppe is not necessarily a factor of protection against sth infection among waste collectors. statistical test revealed the p-value of 0.427, which suggests there is no significant association between the use of ppe and sth infection. ppe which is a compulsory must be used when working as needed to maintain workers’ safety and health such as gloves or shoes.4 ppe is the completeness that must be wornwhen working as needed to maintain safety. a small number of waste collectors who used ppe (16.7%) demonstrates their lack of awareness on the safety at work. irregular availability of ppe in each work unit also contributes to the few workers who wear ppe. the ministry of manpower regulation of 2010 was mentioned that the use of ppe is aimed to protect a person and isolate some or all of the body from potential hazards in the workplace that can cause illness or work accident. the use of ppe at work can include wearing closed shoes, gloves, masks and hats.18 the study is also found that the proportion of the subjects who did not completely use ppe and were not infected with sth was higher than the subjects who did not completely use ppe and been infected by sth (74%: 26%), while the subjects who completely used ppe and were not infected with sth were also higher than those subjects with complete use of ppe and infected with sth (46%: 14%). based on the incidence of the subjects positively suffering from worm infestation, the proportion of sth-infected subjects and incomplete use of ppe was higher than that of sth-infected subjects and complete use of ppe. this suggests that complete use of ppe at the workplace reduces the risk of sth infection. this is similar to a research conducted by islami et al in wakatobi which was showed that sth infection was higher in subjects who did not completely use ppe (60%), whereas in those subjects with complete use of ppe, there were 28.6% of subjects who got infected with sth.4 this was indicates that sth infection does not originate from the workplace but may come from elsewhere. the results of this study was found no association between the use of ppe and the incidence of sth infection (p-value = 0.427). the finding table 3. the association between hand-washing habits and sth infection hand-washing sth infection based on kato-katz total p-value prpositive negative n % n % n % yes 12 20.7 46 79.3 58 100 0.051 0.2 no 2 100 0 0 2 100 total 14 23.3 46 76.7 60 100 table 4. association between complete use of ppe and sth infection complete use of ppe sth infection based on kato-katz total p-value prpositive negative n % n % n % yes 1 10.0 9 90.0 10 100.0 0.427 0.3 no 13 26.0 37 74.0 50 100.0 total 14 23.3 46 76.7 60 100.0 32 indonesian journal of tropical and infectious disease, vol. 7 no. 2 may–august 2018: 27–34 was similar to a study in yogyakarta and pematang siantar where they found that the association between the use of ppe and worm infection among collectors was poor (p-value > 0.05).6,18 this finding however was differed from a study conducted by islami et al which shows a significant association between the use of ppe and the incidence of sth infection (p-value = 0.04).4 according maywati, ppe cannot completely eliminate the work-related hazards or diseases. workers who wear ppe do not come into direct contact with the source of the disease or danger.20 table 5 was showed the association between sth infection and eosinophil level. in regard to sth-infected subjects, there were 78.6% of sth-infected subjects with normal eosinophil count and 21.4% of sth-infected subjects with eosinophilia. while among sth-uninfected subjects, there were 78.3% of them with normal eosinophil level and 21.7% of them with elevated eosinophil level. the prevalence ratio of 0.98 (95% ci = 0.2 4.2) is indicates that sth infection may not be considered a factor of protection against the increase of eosinophil level among waste collectors. the statistical test is revealed the p-value of 1.00, which suggests no significant association between sth infection and eosinophil level. the present study is also showed that of the 60 subjects were examined, there were 21.7% who suffered from eosinophilia. because eosinophilia not only occurs in sth infection but can occurs due to allergies, malignancy and vasculitis but there was no anamnesis in this study nor any physical examination nor a specific allergy test for atopy. the results of this study are similar to the findings in surakarta,21 where they were found that of 96 samples of residents living around a landfill, there was 27.1% of them who suffered from eosinophilia. however, our findings were differed from a study in puerto rico in densely populated and low-income areas, where they were found that eosinophilia was present in 15 out of 16 people infected with worms (94%).22 this study is also indicates that there is a mild intensity of sth infection among waste collectors. the nature of the worm, which continues to lay eggs, will cause the daily accumulation of worm eggs in the host’s body so that the length and severity of the worm infection may affect the intensity of sth infection. the current study also found that the sth-infected subjects within normal eosinophil level are more prevalent than sth-infected subjects with eosinophilia (78.6%: 21.4%). this is probably due to the mild intensity of worm eggs in the feces of sth infected patients and there is a possibility that the subjects suffered from chronic sth infection. this is consistent with the research that has been conducted by darmadi et al among elementary school students which is revealed that the more number of eggs found in per gram of stool, the higher the eosinophils level, with the elevation of eosinophils level of> 9%.23 the results of this study were differed from that of bestari et al in surakarta which suggests that sth-infected subjects with normal eosinophils level were fewer than sthinfected subjects with eosinophilia (43%: 57%).21 chronic eosinophilia are associated with chronic inflammation that may contribute to the absence of eosinophils in peripheral blood.24 in the case of worm infestation, normal eosinophil level can be found because eosinophil maturation and age are heavily dependent on the level of il-5 which cause eosinophils to be more responsive to granulocyte and macrophage colony-stimulating factor (gm-csf).25 ascaris lumbricoides are tends to cause chronic infections that may interfere with th2 responses. the association between sth infection and allergies can occur due to several factors namely first-time infection, infection intensity, and genetics.26 furthermore, symptoms of invisible worm infection in patients indicating eosinophilia are common in rural areas.9 the findings of this study is based on the absence of sth infections and the increase in the eosinophils level (eosinophilia) at 21.7%. in addition, 78.3% of the subjects were not infected with sth and had normal eosinophil level. this is likely due to eosinophilia not only occuring in sth infection but can also occur in patients with allergies, cancer, and vasculitis. the results of this study were similar to the research conducted by bestari et al which showed that the sth-uninfected subjects with normal eosinophils level are 75.3% and the sth-uninfected subjects with eosinophilia of 24.7%.21 the results of a research which was conducted by heukelbach et al in brazil was showed that there were 14% of samples with eosinophilia without sth infection.22 the finding of poor statistical association between sth infection and eosinophil level (p-value = 1.00) may be due to eosinophils level being higher in the early invasive phase than in the chronic phase. the eosinophil level depends on the host factor so it depends not only on previous exposures but also in this study using the stool samples examination showed infected sth results in mild intensity. table 5. the association between sth fection and eosinophil level sth infection eosinophil level total p-value princreased normal n % n % n % positive 3 21.4 11 78.6 14 100.0 1.00 0.98 negative 10 21.7 36 78.3 46 100.0 total 13 21.7 47 78.3 60 100.0 33putra, et al.: soil-transmitted helminth infection and eosinophil lastly, not every sth infection is followed by eosinophilia. some sth infection is induced eosinophilia only during the stage of tissue invasion in the development of the worm. in addition, the highest eosinophilia is usually present in acute worms infection.21 this is common in developing countries where eosinophilia is caused by parasite invasion, one of which is worms. sth infection has a strong association with eosinophilia, especially in the initial stages of infection, when migratory larvae occur.27 schulte et al was stated that eosinophilia is only one of the diagnostic support tools (biomarker) for people affected by worms.28 eosinopilia as a th2 cell response marker can be used to assess worm infections.28 however, according to gabrie et al there is no agreement among experts whether eosinophils could be as a biomarker for worm infection or not. some researchers still consider eosinophilia as a predictor of worm infection that is still used temporarily in people living in tropical and sub-tropical regions.27 figures of eosinophils associated with parasitic infection are determined by the development, migration and distribution of the parasite and the host immune response 24,29 which is characterized by product parasite’s interaction with effector cells of the immune system in the tissue, especially in the migration phase.29 the research which was conducted by cabada et al in peru was showed that eosinophilia was found in 21.2% of subjects. one out of five children with eosinophilia (> 500 eosinophils / ml) increased with the discovery of the parasite in the tissue.30 eosinophilia is not seen in intraluminal parasites such as adult tapeworms or cysts (e.g hydatid cysts) unless there is an integrity disruption in the cyst wall which enables it to escape the cyst.29 conclusion the study among waste collectors in banda aceh was found no association between sth infection and eosinophil level (p = 1.00), between hand-washing habit and sth infection (p = 0.051), nor between the use of ppe and sth infection (p = 0.427). therefore, elevated eosinophil level (eosinophilia) can not be used as sth infection marker. acknowledgement i sincerely thanks to faculty of medicine university of north sumatera and faculty of medicine syiah kuala university for providing me an opportunity to do this research and for their guidance in carrying out this project work. i also wish to express my grattitude to the officials and other staff members of sanitation departement of banda aceh and the health clinic laboratory of prodia banda aceh who rendered their help during the period of my research. references 1. world health organization. soil-transmitted helminthiases: eliminating soil-transmitted helmnthiases as a public health problem in children. prog rep. 2012;1–90. 2. mascarini-serra l. prevention of soil-transmitted helminth infection. j glob infect dis. 2011 apr;3(2):175–82. 3. andiarsa d, hairani b, meliyanie g, fakhrizal d. helminth infection, immunity and allergy. j buski. 2012;4(1):47–52. 4. islami ln, ode w, asfiah s. perbedaan kejadian infeksi cacing antara petugas pengangkut sampah yang menggunaan alat pelindung diri dengan petugas pengangkut sampah yang tidak menggunakan alat pelindung diri. medula. 2014;2(1):108–11. 5. anita s, siregar i, zulkarnain z. hubungan personal higiene dengan penyakit cacing (soil transmitted helminth) pada pekerja tanaman kota pekanbaru. pus penelit lingkung hidup univ riau 102. 2013;93–102. 6. butarbutar m, ashar t, santi d. hubungan hygiene perorangan dan pemakaian alat pelindung diri (apd) dengan keluhan gangguan kulit dan kecacingan pada petugas pengangkut sampah kota pematang siantar. 2012;3(2):1–7. 7. adnani h. perilaku petugas pengumpul sampah untuk melindungi dirinya dari penyakit bawaan sampah di wilayah patangpuluhan yogyakarta tahun 2009. kesmas. 2010;4(3):144–52. 8. silalahi rhb, wistiani, dharmana e. jumlah eosinofil pada anak dengan soil transmitted helminthiasis yang berusia 6-10 tahun. sari pediatr. 2014;16(2):79–85. 9. sumagaysay jb, emverda fm. eosinophilia and incidence of soiltransmitted helminthic infections of secondary students of an indigenous school. asian j heal. 2011 jan 25;1(1). 10. janeway c. immunobiology : the immune system in health and disease. new york. 2005. 29-30, 48-49, 68, 79, 517-517, 542-547 p. 11. speich b, knopp s, mohammed ka, khamis is, rinaldi l, cringoli g, et al. comparative cost assessment of the kato-katz and flotac techniques for soil-transmitted helminth diagnosis in epidemiological surveys. parasit vectors. 2010;3(1):71. 12. taye s. comparison of kato-katz and formol-ether concentration methods for the diagnosis of intestinal helminthic infections among school children of wonji shoa town, eastern ethiopia: a school based cross-sectional study. am j heal res. 2014;2(5):271. 13. tungtrongchitr a, chiworaporn c, praewanich r, radomyos p, boitano jj. the potential usefulness of the modified kato thick smear technique in the detection of intestinal sarcocystosis during field surveys. southeast asian j trop med public health. 2007 mar;38(2):232–8. 14. ri k. 5. peraturan menteri kesehatan republik indonesia, no.15 tahun 2017 tentang. penanggulangan cacingan. 2017;1–78. 15. nayak r, rai s, gupta a. essentials in hematology and clinical pathology. fiirst edi. new delhi: jaypee brothers medical publishers; 2012. 368-369 p. 16. sanchez a, mahoney d, gabrie j. interleukin-10 and soil-transmitted helminth infections in honduran children. bmc res notes. 2015;8(1):55. 17. keiser j, utzinger j. efficacy of current drugs against soiltransmitted helminth infections. jama. 2008 apr 23;299(16). 18. mulasari sa, maani d. hubungan antara kebiasaan penggunaan alat pelindung diri dan personal hygiene dengan kejadian infeksi kecacingan pada petugas sampah di kota yogyakarta. j ekol kesehat. 2013;12(2):161–70. 19. kumar d, kumari r, james j, sekharan b. soil-transmitted helminth infections and the associated risk factors in pre-primary school children, kiwangwa rural ward, bagamoyo district, tanzania. tanzania asian j med pharm res. 2016;6(3):24–31. 20. maywati s. kontribusi penggunaan alat pelindung diri terhadap kejadian infeksi nematoda usus (studi pada petugas pengangkut sampah di kota tasikmalaya). j kesehat komunitas indones. 2013;9(1):1–10. 34 indonesian journal of tropical and infectious disease, vol. 7 no. 2 may–august 2018: 27–34 21. bestari rs, supargiyono, sumarni, suyoko. derajad eosinofilia pada penderita infeksi soil-transmitted helminth (sth). biomedika. 2015;7(2):27–34. 22. heukelbach j, poggensee g, winter b, wilcke t, kerr-pontes lrs, feldmeier h. leukocytosis and blood eosinophilia in a polyparasitised population in north-eastern brazil. trans r soc trop med hyg. 2006;100(1):32–40. 23. darmadi d, irawati n, nasrul e. perbandingan kadar il-5 dan jumlah eosinofil antara anak dan orang dewasa yang terinfeksi ascaris lumbricoides. j kesehat andalas. 2015;4(3):756–64. 24. kremyanskaya m, ackerman s, butterfield j, mascarenhashas j, hoffmann r. eosinophilia, eosinophil-associated diseases, chronic eosinophil leukemia, and the hypereosinophilic syndromes. in: hematology: basic principles and practice. 6th ed. philadelphia, pa: elsevier; 2013. p. 1082–3. 25. medeiros d, silva ar, rizzo ja, motta me, oliveira fhb de, sarinho esc. total ige level in respiratory allergy: study of patients at high risk for helminthic infection. j pediatr (rio j). 2006 aug 9;82(4):255–9. 26. sarinho es, medeiros d, silva a, rizzo jâ. specific ige anti-ascaris in brazilian children and adolescents. world allergy organ j. 2010 mar;3(3):53–6. 27. gabrie ja, rueda mm, rodríguez ca, canales m, sanchez al. immune profile of honduran schoolchildren with intestinal parasites: the skewed response against geohelminths. j parasitol res. 2016;2016:1–13. 28. schulte c, krebs b, jelinek t, nothdurft hd, von sonnenburg f, loscher t. diagnostic significance of blood eosinophilia in returning travelers. clin infect dis. 2002 feb 1;34(3):407–11. 29. khanna v, tilak k, ghosh a, mukhopadhayay c. significance of high eosinophilic count in non-helminthic parasitic infections. int j microbiol parasitol. 2015;1(1):1–4. 30. cabada mm, goodrich mr, graham b, villanueva-meyer pg, deichsel el, lopez m, et al. prevalence of intestinal helminths, anemia, and malnutrition in paucartambo, peru. rev panam salud publica. 2015;37(2):69–75. 143 vol. 5. no. 6 september–december 2015 research report evaluation of salmonella sp contamination and its antibiotics resistance patterns isolated from broiler meat sold at wet market in center of surabaya risky aprillian1,a, dadik rahardjo2, setiawan koesdarto3 1 bachelor of veterinary, the faculty of veterinary medicine, airlangga university surabaya 2 veterinary public health departement, the faculty of veterinary medicine, airlangga university surabaya 3 parasitology departement, the faculty of veterinary medicine, airlangga university surabaya a email of corresponding author: riskyaprillian@gmail.com abstract antibiotic resistance now days become a main issue to the medical researches as found many positive result of antibiotic resistance test. one of the causes of antibiotic resistance is using antibiotic as a feed additive to animal. bacteria that are resistant to antibiotics can be a danger to humans, in this case the resistant bacteria as a result of treatment errors animals, especially chickens that uses low-dose antibiotics as growth promoters. this study aimed to determine the contamination of salmonella sp and its antibiotics resistance patterns of salmonella sp isolated from broiler meat sold at wet market in the center of surabaya: (a) pasar kembang, (b) pasar kupang, (c) pasar dukuh kupang, (d) pasar kedungsari, (e) pasar kedungdoro and (f) pasar keputran. the method that used in this study was bacteriological isolation and identification method. the method started with pre-enrichment using buffered pepton water, selective enrichment using tetrathionate broth and selenite cysteine broth, selective media using salmonella-shigella agar, biochemical test using triple sugar iron agar, simon citrate, methyl red – voges proskauer, and sulfide indol motility, and followed with susceptibility test according to kirby-bauer method using mueller-hinton agar. the antibiotics that used in susceptibility test were: (a) meropenem, (b) ampicillin sulbactam, (c) amikacin, (d) ofloxacin and (e) nalidixic acid. the results of this study were found 90% or 27 of 30 samples positive contaminated with salmonella sp. the results of antibiotics resistance from 27 isolates 0% were resistant to meropenem, 0% were resistant to amikacin; 3.7% were resistant to ampicillin-sulbactam; 11.1% were resistant to ofloxacin and 44.4% were resistant to nalidixic acid. key words: salmonella sp, wet market, broiler meat, antibiotic resistance, center of surabaya abstrak resistansi antibiotik sekarang menjadi isu utama pada penelitian medis seiring ditemukannya banyak hasil positif pada uji resistansi antibiotik. satu dari penyebab resistansi antibiotik adalah penggunaan antibiotik sebagai makanan aditif pada hewan. bakteri yang resistan terhadap antibiotik dapat membahayakan manusia, pada kasus ini resistansi bacteria merupakan hasil dari kesalahan perlakuan pada hewan, terutama pada ayam yang menggunakan antibiotik dosis rendah sebagai pemicu pertumbuhan. penelitian ini membahas mengenai cemaran dan resistensi terhadap antibiotika dari bakteri salmonella sp yang diisolasi dari daging ayam broiler di pasar tradisional surabaya pusat (pasar kembang, pasar kupang, pasar dukuh kupang, pasar kedungsari, pasar kedungdoro dan pasar keputran). penelitian ini menggunakan metode isolasi dan identifikasi bakteri yang dilanjutkan dengan uji sensitivitas antibiotika menggunakan metode difusi dari kirby bauer. antibiotika uji yang digunakan pada uji sensitivitas adalah: (a) meropenem, (b) ampicillin sulbactam, (c) amikacin, (d) ofloxacin dan (e) nalidixic acid. hasil dari penelitian ini adalah ditemukan 144 indonesian journal of tropical and infectious disease, vol. 5. no. 6 september–december 2015: 143-146 27 dari 30 sampel positif terkontaminasi bakteri salmonella sp. hasil uji sensitivitas terhadap antibiotika, 0% resisten terhadap antibiotik meropenem dan amikacin; 3,7% resisten terhadap antibiotika ampicillin sulbactam; 11,1% resisten terhadap ofloxacin dan 44,4% resisten terhadap nalidixic acid. kata kunci: salmonella sp, pasar basah, daging ayam broiler, resistensi antibiotika, surabaya pusat introduction the poultry product consumption especially broiler meat is predicted will climb up as increaseas the number of indonesian population, lifestyle changes and the high awareness of the importance of protein consumed. on 2008, broiler meat consumption got up to 3,8 kg/capita/year. the total of broiler meat consumption reached at 84.07% from total consumption of the other livestock.1 broiler meat is a product that easy contaminates with pathogenic or nonpathogenic microorganism.2 one of the microorganisms that often contaminate broiler meat is salmonella sp, a bacteria caused salmonellosis and recorded as the main cause of food borne disease.3 there are 21.6 million cases of salmonellosis in the world with 216.000 victim dies, and more than 90% happened in asia.4 directorate general of medical services, indonesian department of health in 2008 reported typhoid fever was on second rank of the top ten main diseases of inpatients in indonesia’s hospitals with 81.116 cases (proportion 3,15%), the first rank was occupied by diarrhea with the amount of 193.856 cases (proportion 7,52%).5 as the high level of demand for broiler meat, many farmers choose a shortcut way to increase the chicken’s perform with giving feed additive, such as antibiotic to fast the growth of the chicken. monitoring and surveillance in 2004 at padang and palembang reported that there were chicken, meat, and egg contained antibiotic residues. in padang, from 98 specimens were found 3% contained tetracycline residues and 2% contained aminoglycoside residues. in pekanbaru, from 22 specimens were found 4,8% contained penicillin residues.6 317 salmonella sp isolated from immanuel hospital in bandung were testedthe resistance of antibiotic and found that resistant to trimetoprim-sulfametisazol (7,89%), trimetoprim (6,95%), ciprofloxacin (4,11%), chloramphenicol (0,95%), and amoxicillin (0,62%).7 seeing the potential incidence of salmonellosis and broiler meat as the media vulnerable to contamination by bacteria and the phenomenon of antibiotics as a feed additive for maintenance broilers, the researchers wanted to know the existence of contamination of salmonella sp in broiler chicken meat sold in wet markets in the center of surabaya and its antibiotic resistance against salmonella sp. material and methods a total of 30 specimens (musc. pectoralis) were collected randomly from 7 wet markets at center of surabaya between november–december, 2014. the list of wet market presented in table 1. bacterial test including isolation, identification and susceptibility test were done at gastroenteritis and salmonellosis laboratory, institute of tropical disease, airlangga university. the bacteriological test started with pre-enrichment, 25 gram specimen put into an erlenmeyer with 225 ml buffered peptone water sterile (oxoid®) and incubates for 24 hours at 37oc.8 the next day, inoculate 1ml isolate from pre-enrichment media to selective enrichment media using 10 ml tetrathionate broth (bd) and 10 ml selenite cystine broth (bd), incubate for 24 hours at 37oc.8 the culture from each selective enrichment media were inoculated on selective media: salmonella shigella agar (oxoid®) sterile with streaking using sterile loop on the surface of the plate, incubate all media for 24 hours at 37oc.8 biochemical tests were started with colony selection. colonies that showed suspect of salmonella sp were the colonies with black spot. take five colonies and inoculate each to biochemical media: triple sugar iron agar (oxoid®), simons citrate agar (oxoid®), sulfide indol motility (bd), and methyl-red voges-proskauer (oxoid®), incubate for 24-48 hours at 37oc, then confirmation the positive salmonella sp isolates. purify the positive salmonella sp using nutrient agar (merck®). table 1. list of wet market in center of surabaya and total specimens no wet market spesimens 1 pasar kembang 5 specimens 2 pasar keputran 5 specimens 3 pasar dukuh kupang 4 specimens 4 pasar kupang 4 specimens 5 pasar pandegiling 4 specimens 6 pasar kedungsari 4 specimens 7 pasar kedungdoro 4 specimens total 30 specimens 145aprillian, et al.: evaluation of salmonella sp contamination and its antibiotics resistance patterns isolated each of purified positive salmonella sp from nutrient agar were sub-cultured to pz sterile and the turbidity of the isolates equivalent to 0.5 mcfarland. susceptibility of isolates to selected antibiotics was carried out using the kirby bauer’s disk diffusion method on muellerhinton agar (bd).9 susceptibility to the following antibiotics was determined: ampicillin-sulbactam 10 μg (oxoid®), amikacin 30 μg (oxoid®), meropenem 10 μg (oxoid®), ofloxacin 1 μg (oxoid®), nalidixic acid 30 μg (oxoid®). result and discussion any colonies that grow on salmonella shigella order taken five colonies were selected for the best. then conducted to biochemical tests on triple sugar iron agar, simon citrate, indol motility sulfide and methyl redvoges proskauer. out of a total of 30 specimens examined, 27 (90%) were positive for salmonella sp (table 2). contamination could happen when processing on poultry slaughter house until the meats were consumed. the contaminants are soil contamination, dirt, water, processing equipment, air, human.10 samples with positive results and then tested again to see the level of sensitivity to antibiotics. this sensitivity test using kirby-bauer method and the antibiotics that used are a class of β-lactam antibiotics (ampicillin sulbactam), a sub-class of carbapenems (meropenem), aminoglycosides (amikacin), fluoroquinolones (ofloxacin), and quinolones (nalidixic acid). high percentages of theisolates were susceptible to meropenem and amikacin (100%), ampicillin sulbactamand ofloxacin (88.9%). however, 44,4% isolates were resistant to nalidixic acid. mechanism of antibiotic resistance could be transferred via plasmid (r factor), a genetic mutation of bacteria that could change the location of binding sites of antibiotics, bacterial metabolic change so that is not affected by antibiotics, or the change of bacteria cell membrane permeability and its difficult to be penetrated by antibiotics.11,12 meropenem has a good result, 27 samples was susceptible to salmonella sp. meropenem is antibiotic that could be the final choice for treating the gram-negative bacteria infection. from center of disease control and prevention (cdc) report on antibiotics resistance threat in the united states 2013, antibiotic resistance of carbapenems sub-category could be found on gramnegative bacteria, included pseudomonas and acinetobacter spp. after the bacteria became resistant to carbapenems, the bacteria normally resistant to all β-laktam antibiotics. amikacin resistance occured due to the expression of the gene encoding β-lactamase. this gene encodes the enzyme β-lactamase that inactivates β-lactam ring of amikacin, therefore becoming resistant to amikacin.13 amikacin is a good antibiotic for salmonella sp, 100% samples were positive sensitive to this antibiotic. amikacin is one of semi synthetic aminoglycoside antibiotic that is highly resistant to enzymes modification. resistance may occur because of three things, the decline retrieval; the absence of oxygen-dependent transport system for aminoglycosides, lack of receptor; 30s ribosomal sub-unit has a low affinity for aminoglycosides, modification of the enzyme; plasmids that carry r.factor which encodes an enzyme formation (example: acetyl transferase, nucleotidyl transferase and phosphotransferase) change and inactivation of aminoglycosides antibiotics.14 antibiotic ampicillin-sulbactam has only one positive isolates resistant. two samples including intermediates to antibiotics and the rest of 88.9% is still sensitive samples. the occurrence of resistance to ampicillinsulbactam due to the expression of the gene, i.e. the gene encoding β-lactamase is located on gram-negative bacteria chromosome. this gene encodes the enzyme β-lactamase that inactivates β-lactam ring of ampicillin by means of hydrolyzing β-lactam ring, thereby becoming resistant to ampicillin.15 antibiotic sensitivity of ofloxacin is 88.9% of isolates of salmonella sp still sensitive to these antibiotics. three isolates were resistant or 11.1%, so it can be said that table 2. salmonella sp contamination on broiler meat at wet market in center of surabaya no wet market number of specimen positive salmonella sp total proportion 1 ps. kembang 5 3 60% 2 ps. kupang 4 3 75% 3 ps. dukuh kupang 4 4 100% 4 ps.pandegiling 4 4 100% 5 ps. kedungsari 4 4 100% 6 ps. kedungdoro 4 4 100% 7 ps. keputran 5 5 100% total 30 27 90% table 3. antibiotic susceptibility pattern of salmonella isolated from broiler meat at wet market in center of surabaya no antibiotics sa % ib % rc % 1 ampicillin sulbactam 24 88,9% 2 7,4% 1 3,7% 2 meropenem 27 100% 0 0% 0 0% 3 amikacin 27 100% 0 0% 0 0% 4 ofloxacin 24 88,9% 0 0% 3 11,1% 5 nalidixic acid 13 48,2% 2 7,4% 12 44,4% a sensitive, b intermediate, c resistant 146 indonesian journal of tropical and infectious disease, vol. 5. no. 6 september–december 2015: 143-146 salmonella sp begin resistant to ofloxacin. ofloxacin is an antibiotic that belongs to the class of fluoroquinolones. the mechanism of resistance to fluoroquinolones is this antibiotic bound to the β subunit of the bacterial enzyme dna gyrase and block the activity of enzymes that are essential in maintaining dna supercoiling and important in the process of dna replication. mutations in encoding gene of the dna gyrase could produce active enzyme but could not be bound by fluoroquinolones.14 bacteria salmonella sp has the highest resistance to the antibiotic level nalidixic acid as many as 12 samples or 44.4% resistant and two samples or 7.4% intermediates. nalidixic acid is active against gram-negative bacteria coliform. these antibiotics work by inhibiting the enzyme activity of bacterial dna gyrase that disrupts dna supercoiling.16 nalidixic acid resistance to antibiotics is not transferred via plasmids (r factor), but by other mechanisms. the mechanism is a genetic mutation of bacteria that can change the location of the protein and binding sites of antibiotics, bacterial metabolic change so it is not affected by antibiotics, or bacteria alter the permeability of the cell membrane so difficult to be penetrated by antibiotics. this resistance has led to clinical problems, bacteria normally resistant to nalidixic acid is pseudomonas spp.11, 12 conclusion from the result of this study, broiler meat sold in wet markets surabaya center 90% positive contaminated with salmonella sp (27 of 30 samples). salmonella sp isolated from broiler meat sold in wet markets surabaya center were 0% resistant to meropenem and amikacin, ampicillin sulbactam (3.7%), ofloxacin (11.1%), and nalidixic acid (44.4%). references 1. direktorat jenderal peternakan, departemen pertanian (ditjennak). 2008. statistik peternakan. jakarta: direktorat jenderal peternakan. 2. lawrie ra, 2003. ilmu daging. edisi kelima. universitas indonesia press, jakarta. p. 132–157. 3. world health organization (who). 2014. salmonella. http://www. who.int/topics/salmonella/en/. [16 september 2014]. 4. crump ja, sp. luby, and ed. mintz. 2004. the global burden of typhoid fever. bull world health organ 82: 346–353. 5. departemen kesehatan ri (depkes ri). 2009. profil kesehatan indonesia tahun 2008. jakarta: depkes ri. 6. fitria y, rh. nugroho, hb. sosiawan, noviarti, dan nurhayati. 2004. hasil monitoring dan surveilanse cemaran mikroba dan residu antibiotika di kota padang, pekanbaru dan jambi. tahun 2004. informasi kesehatan hewan. 7. mulyana, yanti. 2007. sensitivitas salmonella sp. penyebab demam tifoid terhadap beberapa antibiotik di rumah sakit immanuel bandung. bandung: fakultas kedokteran universitas padjajaran. 8. bell c, and a. kyriakides. 2002. salmonella a practical approach to the organism and its control in foods. uk: blackwell science ltd. 9. reynolds j. 2012. kirby-bauer (antibiotic sensitivity). dallas, usa: richland college. 10. soeparno. 2005. ilmu dan teknologi daging. yogyakarta: gadjah mada university press. p. 113–114. 11. kalalo lp, aryati, dan b. subagjo. 2004. pola bakteri dan tes kepekaan antibiotika wanita hamil dengan bakteriuria asimtomatis. surabaya: universitas airlangga. 12. suyatna f. dan h. toni. 1995. farmakologi dan terapi: edisi keempat. jakarta: penerbit bagian farmakologi fakultas kedokteran universitas indonesia. p. 595. 13. center of disease control and prevention (cdc). 2013. antibiotics resistance threat in the united states. usa: cdc. p. 23. 14. pratiwi st. 2008. mikrobiologi farmasi. penerbit erlangga: jakarta. p. 136; 149–160; 165–171. 15. russell ad and i. chopra. 1990. understanding antibacterial action and resistance. new york: ellis horwood series in pharmaceutical technology. 16. rang hp and mm. dale. 1991. pharmacology. uk: churchill livingstone. p. 824–825. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 21 vol. 7 no. 1 january–april 2018 fever as indicator to secondary infection in dengue viral infection soegeng soegijanto1,3, sufiandika nuryandari2, siti churrotin3, teguh hari sucipto3a 1 faculty of medicine, universitas airlangga, indonesia 2 mother and child hospital of soerya, indonesia 3 dengue study group, institute of tropical disease, universitas airlangga, indonesia a corresponding author: teguhharisucipto@staf.unair.ac.id abstract dengue virus infections are distributed in tropical and sub-tropical regions and transmitted by the mosquitoes such as aedes aegypti and aedes albopictus. dengue virus can cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome or dengue and severe dengue classified by world health organization. beside it concurrent infection virus salmonella had been found some cases who showed fever more than 7 days. concurrent infection with two agents can result in an illness having overlapping symptoms creating a diagnostic dilemma for treating physician, such as dengue fever with typhoid fever. the aim of this research is detection of dengue virus and secondary infection with salmonella typhi in patients suspected dengue virus infection. detection of dengue virus and salmonella typhi using immunochromatography test such as ns1, igg/igm for dengue virus infection, and igm/igg salmonella and blood culture. the fifty children with dengue virus infection came to soerya hospital and 17 cases suspected dengue virus infection, five cases showed a positive ns1 on the second day of fever and one case concurrent with clinical manifestation of convulsi on the third days of fever there were five cases only showed positive. it was showed in this study that on the fourth to six day of fever in dengue virus infection accompanied by antibody igm & igg dengue. there were 12 cases showed the clinical manifestation of concurrent dengue viral infection and salmonella, all of them showed a mild clinical manifestation and did not show plasma leakage and shock. in this study we found the length of stay of concurrent dengue virus infection and salmonella infection is more than 10 days. these patients were also more likely to have co-existing haemodynamic disturbances and bacterial septicaemia which would have required treatment with inotropes and antibiotics. this idea is very important to make update dengue viral management to decrease mortality in outbreak try to gain new prevention method before the occurrence of outbreak. keywords: dengue viral, salmonella typhi, co-infection, secondary infection, laboratory test abstrak infeksi virus dengue didistribusikan di daerah tropis dan sub-tropis dan ditularkan oleh nyamuk seperti aedes aegypti dan aedes albopictus. virus dengue dapat menyebabkan demam berdarah, demam berdarah dengue dan sindrom syok dengue atau demam berdarah dan demam berdarah parah yang dikelompokkan oleh organisasi kesehatan dunia. disamping itu infeksi sekunder oleh salmonella telah ditemukan beberapa kasus yang menunjukkan demam lebih dari 7 hari. infeksi bersama dengan dua agen dapat menyebabkan penyakit yang memiliki gejala tumpang tindih yang menciptakan dilema diagnostik untuk pengobatan, seperti demam berdarah dengan demam tifoid. tujuan dari penelitian ini adalah deteksi virus dengue dan infeksi sekunder dengan salmonella typhi pada pasien yang diduga terinfeksi virus dengue. deteksi virus dengue dan salmonella typhi menggunakan uji imunokromatografi seperti ns1, igg/igm untuk infeksi virus dengue, dan igm/igg salmonella dan kultur darah. lima puluh anak dengan infeksi virus dengue datang ke rumah sakit soerya dan 17 kasus yang diduga terinfeksi virus dengue, lima kasus menunjukkan ns1 positif pada hari kedua demam dan satu kasus bersamaan dengan manifestasi klinis konvulsi pada hari ketiga demam terjadi lima kasus. hal ini ditunjukkan dalam penelitian ini bahwa pada demam dengue keempat sampai enam hari disertai dengan antibodi igm dan igg. ada 12 kasus yang menunjukkan manifestasi klinis infeksi virus dengue bersamaan dan salmonella, semuanya menunjukkan manifestasi klinis ringan dan tidak menunjukkan kebocoran dan kejutan plasma. dalam penelitian ini kami menemukan lamanya tinggal bersamaan infeksi dengue virus dan infeksi salmonella lebih dari 10 hari. pasien-pasien ini juga cenderung memiliki gangguan hemodinamik yang research report 22 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 21–25 menyertai dan bakteri septicemia yang memerlukan pengobatan dengan inotrop dan antibiotik. ide ini sangat penting untuk membuat update manajemen virus dengue untuk menurunkan angka kematian saat wabah dan mencoba mendapatkan metode pencegahan baru sebelum terjadinya wabah. kata kunci: virus dengue, salmonella typhi, koinfeksi, infeksi sekunder, uji laboratorium introduction dengue virus infections are distributed in tropical and sub-tropical regions and transmitted by the mosquitoes such as aedes aegypti and aedes albopictus.1–3 dengue virus can cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome or dengue and severe dengue classified by world health organization.4 the patient came to the doctor in first, second, third days of fever or more days fever than following by other clinical manifestation such as salmonella infection some other bacterial infection and virus. based on clinical manifestation some laboratory test that should be done such as (1); for dengue viral infection: ns1, igm & igg5 (2); for salmonella infection: igm igg salmonella6 (3); culture of blood for looking other bacterial infection. based on these information the doctor should be aware to care and prevent to dengue virus infection. the pathogenic mechanism of dhf has not been fully elucidated, but it is generally accepted that disease severity is related to viremia level. antibody-dependent enhancement (ade) of infection is a hypothetical mechanism that possibly leads to increased viremia levels. a major factor in ade is the cross-reactive non-neutralizing antibodies induced by a former (primary) infection. these can enhance denv infection in monocytes/macrophages in an fc gamma receptor (fcyr)medicated manner upon secondary exposure to a heterotypic infection, thus they are called ‘enhancing anti bodies’.7 in contrast, neutralizing antibodies can decrease viremia levels and are considered to play a protective role against infection. under in vivo conditions, the enhancing and neutralizing antibody species are present in the circulation as polyclonals. therefore, the total activity, namely the balance of these two opposing activities, is a potentially critical factor determining viremia level and disease outcomes.8 our present sero-epidemiological study is aimed to investigate dengue antibody status in endemic children. we used serum samples collected from healthy philippine children in 1993 and from a population of indonesia children between 1999 and 2000. we are measured the balance of enhancing and neutralizing activities against search denv type at varying serum dilutions in the presence or absence of complement. a variety of dose (serum dilution)-dependent antibody activity patterns were observed among the children. in the present study is revealed that most infected children possessed enhancing antibodies against one or more the denv types at a serum dilution of 1:10.8 importantly, enhancing activity was not reduced by the addition of complement in our in vitro assay condition. concurrent infection with two agents can result in an illness having overlapping symptoms creating a diagnostic dilemma for treating physician, such as dengue fever with typhoid fever. the similarity in symptoms and differential diagnoses of these diseases often mimic those of dengue and thereby makes accurate clinical diagnosis and treatment difficult without laboratory confirmation. some laboratory test was taken more of ns1, igm dengue to identify for viral infection.9,10 igm/igg salmonella and blood culture to identify for salmonella infection.11,12 this idea is very important to make update dengue viral management to decrease mortality in outbreak try to gain new prevention method before the occurrence of outbreak. material and method since 1st january until 30th april 2017, there were 50 children with dengue virus infection came to soerya mother and child hospital and 17 cases suspected dengue virus infection who came late. all of them were coming to doctor due illness feeling and showed the clinical manifestation such as fever, headache, vomiting, and tired and following by diarrhea, colic abdomen, and cough. to complete the data, the laboratory test such as ns1 kit by sd bioline (us), igm & igg dengue for dengue virus infection identification by sd bioline (us), igm/igg salmonella by sd bioline (us) and blood culture for salmonella infection identification. result and discussion there were 17 cases came early to the hospital due to the clinical manifestation of high fever and one until three cases suffer from convulsion. all of them were examined physically and were done ns1, igm and igg dengue test to identify dengue viral infection. one case is showed a positive ns1. it was a case with clinical manifestation of fever more the 39°c with convulsion on the first day. five cases are showed a positive ns1 on the second day of fever and one case concurrent with clinical manifestation of convulsion on the third days of fever. there were five cases which only showed positive, the study is showed table 1 fever with test of positive ns1. clinical manifestation in patient of suspected dengue virus infection with ns1 positive such as fever, nausea, vomiting, abdominal pain, diarrhea, plasma leakage and epistaxis. two groups of cases are suspected dengue virus infection had been made, as: 1) who had come on the 23soegijanto, et al.: fever as indicator to secondary infection first, second, and third of fever as shown in table 1 and 2) who had come on the fourth, fifth, and sixth of fever as shown in table 2. ns1 test had been done in the first group cases of dengue virus infection and the result showed on table 1. seven cases who had a negative result ns1 had been followed by of igm test on the four until six day of fever. the result see table 2. it is showed in this study that on the fourth to six day of fever in dengue virus infection accompanied by antibody igm & igg dengue as shown in table 2 and 3. there were 5 cases who had shown a clinical manifestation on the fourth, fifth, and sixth days of fever and had been identified as a positive result of igm test. these event were also found of the following 5 cases that had a clinical manifestation on the third days of fever. clinical manifestation in patient of suspected dengue virus infection with igm positive such as fever, nausea, vomiting, and abdominal pain. seven cases who had a negative result ns1 had been be followed by of igg & igm test on the four until six day of fever. the results as shown in table 2. it was showed in this study that on the fourth to six day of fever in dengue virus infection usually accompanied by antibody to viral of dengue as shown in table 2 and 3. on the table 4 there were 12 cases showed the clinical manifestation of concurrent dengue viral infection and salmonella. there were 12 cases positive only igm, it mean that all cases has been suffered from primary infection of dengue virus. all of them are showed a mild clinical manifestation and did not show plasma leakage and shock. two were cases showed a positive igg and 12 cases were table 1. ns1 test result in suspected dengue virus infection before three days in soerya hospital sepanjang day of fever ns1 test examination total cases + first day 0 1 1 second day 5 3 8 third day 5 3 8 total cases 10 7 17 table 2. igm, igg, igm & igg rapid test followed negative test ns1 day of fever ns1 test examination t o t a l casesigm (+) igg (-) i g m & igg (+) fourth 1 0 2 3 fifth 3 0 0 3 sixth 1 0 0 1 total 5 0 2 7 table 3. patient’s igm, igg, igm & igg rapid test for identification dengue virus infection come in day 4th until day 7th day of fever ns1 test examination total casesigm igg igm & igg fourth 8 2 6 16 fifth 6 0 3 9 sixth 2 0 2 4 seven 3 0 1 4 total 19 2 12 33 table 4. the clinical manifestation of concurrent dengue virus infection and salmonella symptoms dengue virus infection dengue virus infection+salmonella fever 100% 30 100% 12 nausea 62% 18 83% 10 vomiting 40% 12 60% 7 diarrhea 6% 2 20% 2 abdominal pain 34% 10 71% 8 plasma leakage 2% 1 10% 1 epistaxis 2% 1 showed a positive igm and igg. it mean that all cases had been suffered from secondary infection dengue. these cases are showed severity of clinical manifestation of dengue haemorrhagic fever. it was due to enhancement ag ab reaction that promoting the inclination of plasma leakage and shock. dengue fever manifests as spectrum of illness ranging from in apparent or mild febrile illness to serve and fatal hemorrhagic disease. in a typical case of dengue fever, the patient experiences high fever lasting for 5 to 7 days. concomitantly, a severe frontal and retro orbital headache, myalgias, especially lower back, arm, and leg pains, malaise, arthralgia and anorexia. in typhoid fever, a dull continuous frontal headache begins during the first two days of fever, mild arthralgia involving multiple joints and vague, poorly localized back pain may occur.13 in the previous study, risk factors for bacterial co-infection in children with dengue have not been well characterized. in a study done in adult patients, prolonged fever (> 5 days) was an independent risk factor for co-infection. the reasons for bacterial co-infections in some patients with dengue virus can cause a diminished t cell proliferation in response to mitogens in vitro. however, the in vivo effects of these observations have not been studied.14 on 2017 there were 24 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 21–25 found 12 cases of concurrent dengue virus infection and salmonella. in this study we are found the length of stay of concurrent dengue virus infection and salmonella infection is more than 10 days. these patients were also more likely to have co-existing haemodynamic disturbances and bacterial septicaemia which would have required treatment with inotropes and antibiotics. furthermore, older patients would have other co-morbidities and have rehabilitation issues which may complicate admissions and extend hospital stay.15 in november 2008 the predominant serotype from denv type 2 (denv-2) to denv type 1 (denv-1) had been found,16–18 july 2013 the predominant of denv-1 to denv-2 and the predominance of denv-2 continued in 2014. all denv-2 which isolated in surabaya were classified into the cosmopolitan genotype, and further divided into 6 clusters defined as the “surabaya lineage”.19 in previous study was reported that genome sequence of denv-1, which is phylogenetic close to the japanese outbreak strain of 2014. this finding is indicated that the southeast asian region was the source of the dengue outbreak in japan 2014. this abstract based on the reviewed of some studies that had been done in 2008-2014.20,21 based on this result the monitoring of emergence imported or mutated strain of dengue virus in human being and mosquito emergence should be done. therefore continuous surveillance of circulating viruses is required to predict the risk of dhf and df. this idea is very important to make update dengue viral management to decrease mortality in in outbreak try to gain new prevention method before the occurrence of outbreak. conclusion monitoring clinical manifestation should always be done, to predict the appropriate diagnosis of dengue virus infection. and sero-epidemiology study should be continued doing in many the capital city of island in indonesia. acknowledgement this work is supported by the joined program of the japan initiative for global research network on infectious disease (j-grid); research grant mandat universitas airlangga (hrmua); and institute of tropical disease (itd) as the center of excellence (coe) program by the ministry of research and technology (ristek) indonesia. references 1. carrington lb, simmons cp. human to mosquito transmission of dengue viruses. front immunol. 2014 jun 17;5:1–8. 2. lambrechts l, scott tw, gubler dj. consequences of the expanding global distribution of aedes albopictus for dengue virus transmission. halstead sb, editor. plos negl trop dis. 2010 may 25;4(5):e646. 3. watts dm, burke ds, harrison ba, whitmire re, nisalak a. effect of temperature on the vector efficiency of aedes aegypti for dengue 2 virus. am j trop med hyg. 1987 jan;36(1):143–52. 4. jayaratne s, atukorale v, gomes l, chang t, wijesinghe t, fernando s, et al. evaluation of the who revised criteria for classification of clinical disease severity in acute adult dengue infection. bmc res notes. 2012;5(1):645. 5. wattal c, goel n. infectious disease emergencies in returning travelers: special reference to malaria, dengue fever, and chikungunya. med clin north am. 2012 nov;96(6):1225–55. 6. kuhn kg, falkenhorst g, ceper th, dalby t, ethelberg s, molbak k, et al. detecting non-typhoid salmonella in humans by elisas: a literature review. j med microbiol. 2012 jan 1;61(1):1–7. 7. yamanaka a, kosugi s, konishi e. infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type 2 and 4 viruses are controlled by complement levels. j virol. 2008 jan 15;82(2):927–37. 8. yamanaka a, tabuchi y, mulyatno kc, susilowati h, hendrianto e, soegijanto s, et al. dengue virus infection-enhancing and neutralizing antibody balance in children of the philippines and indonesia. microbes infect. 2012 nov;14(13):1152–9. 9. aryati a, trimarsanto h, yohan b, wardhani p, fahri s, sasmono rt. performance of commercial dengue ns1 elisa and molecular analysis of ns1 gene of dengue viruses obtained during surveillance in indonesia. bmc infect dis. 2013 dec 29;13(1):611. 10. shu p-y, chen l-k, chang s-f, yueh y-y, chow l, chien l-j, et al. comparison of capture immunoglobulin m (igm) and igg enzymelinked immunosorbent assay (elisa) and nonstructural protein ns1 serotype-specific igg elisa for differentiation of primary and secondary dengue virus infections. clin diagn lab immunol. 2003 jul;10(4):622–30. 11. jesudason m, esther e, mathai e. typhidot test to detect igg & igm antibodies in typhoid fever. indian j med res. 2002 aug;116:70–2. 12. bhatti ab, ali f, satti sa. cross-reactivity of rapid salmonella typhi igm immunoassay in dengue fever without co-existing infection. cureus. 2015 dec 4;7(12):e396. 13. sharma y, arya v, jain s, kumar m, deka l, mathur a. dengue and typhoid co-infectionstudy from a government hospital in north delhi. j clin diagn res. 2014 dec;8(12):09–11. 14. srinivasaraghavan r, narayanan p, kanimozhi t. culture proven salmonella typhi co-infection in a child with dengue fever: a case report. j infect dev ctries. 2015 sep 27;9(9):1033. 15. khalil mam, tan j, khalil mau, awan s, rangasami m. predictors of hospital stay and mortality in dengue virus infection-experience from aga khan university hospital pakistan. bmc res notes. 2014;7(1):1–7. 16. sucipto th, ahwanah nlf, churrotin s, matake n, kotaki t, soegijanto s. immunofluorescence assay method to detect dengue virus in paniai-papua. in 2016. p. 40001. 17. yamanaka a, mulyatno kc, susilowati h, hendrianto e, ginting ap, sary dd, et al. displacement of the predominant dengue virus from type 2 to type 1 with a subsequent genotype shift from iv to i in surabaya, indonesia 2008–2010. coffey ll, editor. plos one. 2011 nov 7;6(11):e27322. 18. kotaki t, yamanaka a, mulyatno kc, churrotin s, labiqah a, sucipto th, et al. continuous dengue type 1 virus genotype shifts followed by co-circulation, clade shifts and subsequent disappearance in surabaya, indonesia, 2008–2013. infect genet evol. 2014 dec;28:48–54. 25soegijanto, et al.: fever as indicator to secondary infection 19. kotaki t, yamanaka a, mulyatno kc, churrotin s, sucipto th, labiqah a, et al. divergence of the dengue virus type 2 cosmopolitan genotype associated with two predominant serotype shifts between 1 and 2 in surabaya, indonesia, 2008-2014. infect genet evol. 2016 jan;37:88–93. 20. churrotin s, kotaki t, sucipto th, ahwanah nlf, deka pt, mulyatno kc, et al. dengue virus type 1 strain isolated in indonesia shows a close phylogenetic relation with the strains that caused the autochthonous dengue outbreak in japan in 2014. jpn j infect dis. 2016 sep 21;69(5):442–4. 21. kotaki t, yamanaka a, mulyatno kc, labiqah a, sucipto th, churrotin s, et al. phylogenetic analysis of dengue virus type 3 strains primarily isolated in 2013 from surabaya, indonesia. jpn j infect dis. 2014;67(3):227–9. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 80 vol. 7 no. 3 september–december 2018 literature review potency of luteolin with solid lipid nanoparticle (sln)-polyethylene glycol (peg) modification for artemisinin-resistant plasmodium falciparum infection william kamarullah1a, erika indrajaya1, janice emmanuella1 1 faculty of medicine, atma jaya catholic university of indonesia, jakarta, indonesia a corresponding author: williamkamarullah@hotmail.com abstract falciparum malaria is still considered as one of the important global health problems and its causal agent (plasmodium falciparum) is reported to be the third most common factor for contributing the number of deaths in the world. as we all know, artemisinins are the most rapidly acting of currently available antimalarial drugs. along with artesunate, these two combining drugs, the so-called artemisinin-based combination therapies (acts) has become the foundation of modern falciparum malaria treatment globally. nowadays, however, there have been reports about intricate cases of resistance against artemisinin in various southeast asian countries and it is predicted to spread over several other countries, including indonesia. therefore, adjuvant therapy is required along with first-line therapy administration to help eradicate both artemisinin-sensitive and resistant p. falciparum. luteolin in vitro has a prospective inhibitory activity (ic50<50 μg) in inhibiting the development of parasite’s life cycle. nonetheless, its poor bioavailability and pharmacokinetics restrict clinical application. the low bioavailability of luteolin requires encapsulation using solid lipid nanoparticle (sln) and polyethylene glycol (peg). sln is useful for improving the bioavailability of luteolin in the body, whereas peg is needed in order to prevent the destruction of luteolin-sln substance by the reticuloendothelial system. here in this literature review, we’re trying to demonstrate the benefits, potential, way of constructions, pharmacokinetics, and pharmacodynamics of luteolin encapsulated with sln with peg modification. thus, it is hoped that the results of this literature study may encourage further research in assisting the development of adjuvant therapy for cases of artemisinin-resistant p. falciparum infection. keywords: luteolin, plasmodium falciparum, artemisinin resistance, sln, peg abstrak malaria falsiparum masih menjadi salah satu dari masalah kesehatan global penting dan agen kausalnya (plasmodium falciparum) dilaporkan menempati posisi ketiga tersering dalam mengontribusi jumlah kematian di dunia. seperti yang telah kita ketahui, artemisinin adalah obat antimalaria yang memiliki onset kerja paling cepat yang dapat digunakan saat ini. seiring dengan artesunate, dua kombinasi obat ini, atau yang biasa disebut sebagai terapi kombinasi berbasis artemisinin (acts) telah menjadi dasar pengobatan malaria falsiparum modern secara global. namun, saat ini, hal ini diperparah dengan adanya laporan kasus resistensi terhadap artemisinin di berbagai negara asia tenggara, serta diprediksi akan menyebar ke beberapa negara lainnya, termasuk indonesia. untuk itu, diperlukan terapi adjuvan selain terapi lini pertama untuk membantu eradikasi plasmodium falciparum baik yang sensitif maupun resisten. luteolin secara in vitro memiliki aktivitas inhibisi yang cukup prospektif (ic50<50 μg) dalam menghambat perkembangan hidup parasit. meskipun demikian, bioavailabilitas yang buruk serta proses farmakokinetik yang kurang memuaskan membatasi penerapan klinis dari substansi ini. rendahnya bioavailabilitas luteolin membutuhkan enkapsulasi menggunakan nanopartikel lipid padat (sln) dan polietilen glikol (peg). sln bermanfaat untuk meningkatkan bioavailabilitas luteolin di dalam tubuh, sedangkan peg bermanfaat untuk mencegah destruksi substansi luteolin-sln oleh sistem retikuloendotelial. di sini, dalam tinjauan literatur ini, kami mencoba untuk memaparkan manfaat, potensi, cara konstruksi, farmakokinetik, dan farmakodinamik luteolin yang dienkapsulasi 81kamarullah, et al.: potency of luteolin with solid lipid nanoparticle (sln)-polyethylene glycol (peg) introduction malaria falciparum, one of the life-threatening diseases caused by plasmodium falciparum is still one of the top priority health problems in indonesia as well as around the world. in fact, malaria falciparum is one of the most common cases of infection in tropical countries such as indonesia with a prevalence of 86.4%.1 currently, the management of p. falciparum infection is a combination of drugs that one of its components is artemisinin or any of its derivatives (artesunate, artemether, dihydroartemisinin). the combination of these drugs is also commonly referred as artemisinin-based combination therapy (act). however, several studies in 2010 state that in some populations, artemisinin substances that enter into the ring stages of parasites do not directly kill the parasites intracellularly. instead, they only stay at their dormancy stage.2,3 in addition, a study called tracking resistance to artemisinin collaboration (trac) in 7 asian countries show that there is a decrease in the clearance of plasmodium parasite with artemisinin therapy in several southeast asian countries, such as cambodia, myanmar, thailand as well as vietnam, and is predicted to spread to other countries including ours, indonesia.4 therefore, it is necessary to develop additional therapies to improve the effectiveness of primary therapy in eradicating the p. falciparum parasite. one of the bioactive components that can be used as an adjuvant therapy in accordance with eradicating artemisinin-resistant p. falciparum is luteolin. luteolin is a polyphenol-containing flavonoid that is widely found in a variety of plants, especially on scallion (allium fistulosum). from that information, we can deduce that luteolin can easily be utilized to fathom malaria problems in indonesia.5 luteolin has a role in inhibiting the process of fatty acid synthesis in parasites, knowing that this whole synthesis process is very important in order to form new organelles and biomembranes. thus, this can result in termination of the parasite’s life cycle.6 in vitro studies also show that luteolin is able to inhibit the development of young trophozoite (ring stage) so that the intraerythrocytic parasite cycle can be cut off.7 unfortunately, just like the other natural active compounds, luteolin has low stability and bioavailability in human body so that its delivery method must be carefully considered. solid lipid nanoparticle (sln), a drug delivery method with submicron particle size is a solution to overcome these weaknesses. sln as a luteolin encapsulation will increase the biocompatibility and bioavailability of the compound. however, due to the hydrophobic nature of sln, this encapsulation will easily be phagocytosed by the reticuloendothelial system as a result of being recognized as foreign body by macrophages.8 therefore, further modification is required to eliminate the opsonization properties. according to some studies, the use of poly ethylene glycol (peg) modification agent was found to be successful in altering the hydrophobic part of the sln particle surface. thus, it is desirable that the bonding process by opsonin factor causing destruction by the reticuloendothelial system can be inhibited.9 based on these information, the authors offer the idea of luteolin utilization and optimized by peg modified sln delivery method as the idea of developing additional therapies in improving the effectiveness of primary therapy to eliminate the p. falciparum parasite. falling artemisinin susceptibility in several southeast asian countries based on several reports of published studies, poor therapeutic responses to artemisinins have occurred for the last few years in southeast asia. the studies of teuscher et al10 and witkowski et al11 in 2010 state that some populations, the substance of artemisinin that enters the ring stage do not directly kill intracellular parasites, but only in the dormancy stage. therefore, parasites can grow sustainably. this is one of the hypothesises that is thought to be one mechanism of artemisinin resistance against plasmodium falciparum parasite. it is also suspected to be the cause of recrudescence in falciparum malaria. hien et al12 and kyaw et al13 ‘s study of tracking resistance to artemisinin collaboration (trac) is showed that there is a decrease in the clearance of plasmodium parasite with artemisinin therapy in some southeast asian countries, especially in cambodia and in myanmar. the decrease in the parasitic clearance rate is associated with the interaction of many kinds of genotypes. one of the most widely accepted theory is that there’s an area on the 13th chromosome of p. falciparum which is strongly associated with decreased in vivo parasite clearance. the mutated genes, now commonly known as k13, encode proteins that contain the “kelch” motive, a figure that resembles six-bladed structure. mutations occurring in the k13 gene will cause impaired function of ubiquitin pathway so that the apoptotic process does not occur and the parasite will escape the artemisinin’s mechanism of actions.14,15 dengan sln dengan modifikasi peg. dengan demikian, diharapkan hasil studi literatur ini dapat mendorong penelitian lebih lanjut dalam membantu pengembangan terapi adjuvan untuk kasus infeksi p. falciparum. kata kunci: luteolin, plasmodium falciparum, resistensi artemisinin, sln, peg 82 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 80–86 luteolin: chemical structure and its potential sources figure 1. the chemical structure of luteolin. as a flavonoid compound, luteolin has a basic structure comprising two phenyl groups and is interconnected with a triple carbon group.16 l u t e o l i n o r s o m e t i m e s c a l l e d a s 3 ’ , 4 ’ , 5 , 7 tetrahydroxyflavone, is a flavonoid of plants that are often utilized for its pharmacological activities as herbal medicine ingredients (as seen in figure 1).16 in addition, luteolin is often used as an anticarcinogenic, antithrombotic, antiallergic, antidiabetic, antiobesitic, immune-enhancer, and also for its antimalarial effect.16 one of the potential plants that contain luteolin is scallion (allium fistulosum). this is because the luteolin contained in scallion is considered highest when compared with other tropical plants, reaching 391 mg/kg.5 mechanism of luteolin as an antimalarial agent as mentioned earlier, the presence of resistance from artemisinin drug requires an adjuvant therapy agent with different working mechanisms to help eradicate the p. falciparum parasite. the results showed that luteolin works by targeting apicoplast in plasmodium falciparum, an organelle analogous to plastids which function in a wide range of intracellular activities including fatty acid synthesis. the synthesis of fatty acids is very important for plasmodium falciparum parasites. this is due to the role of essential fatty acids in the parasitic life cycle, which is an important component in the process of synthesizing new organelles. the process of synthesizing the new organelles in question takes place in one of its life cycle’s process, which is when the parasites are turning the ring stage into a schizon (a collection of merozoites) or into a gametocyte. fatty acids are also known to be important components in the biomembranes synthesis of plasmodium falciparum.6,7 a study from the last five years conducted by qidwai et al17 showed that luteolin has a different way of eradication mechanism from previous malaria medicines. luteolin works specifically on the regulation of fatty acid synthesis of plasmodium falciparum primarily in the inhibition of three of the four key enzymes, namely fabg, fabz, and fabi (fab=fatty acid biosynthesis). fab is one of the enzymes that play an important role in the process of fatty acid synthesis of plasmodium falciparum especially in the process of fatty acid elongation into a functional structure for the parasite.17 based on these findings, it is hoped that luteolin can be the latest breakthrough in initiating the development of adjuvant therapy of artemisinin-resistant plasmodium falciparum. as seen in figure 2, giemsa-stained smears were prepared at 0, 19, 26, and 48 hours after the addition of luteolin (20 µm) dissolved in dmso (dimethyl sulfoxide) or an equal volume of dmso (control; 0.025% dmso) to infected erythrocytes. while control parasites developed into mature trophozoites (26 hours) that subsequently gave rise to daughter parasites (48 hours), the growth of luteolintreated parasites was arrested at the young trophozoite stage. luteolin-treated parasites did not give rise to daughter parasites, resulting in a reduced parasitemia compared to the control at the 48 hours.7 giemsa-stained smears were prepared at 0, 19, 26 and 48 h after the addition of luteolin (20 µm) dissolved in dmso or an equal volume of dmso (control; 0.025% dmso) to 7g8-infected erythrocytes (1% parasitemia, 2% hematocrit). while control parasites developed into mature trophozoites (26 h) that subsequently gave rise to daughter parasites (48 h), the growth of luteolin-treated parasites was arrested at the young trophozoite stage. luteolin-treated parasites did not give rise to daughter parasites, resulting in a reduced parasitemia compared to the control at the 48 h time point.7 pharmacokinetic of luteolin when luteolin is administered orally, it will be recognized by the body as a substrate for the conjugation and hydrolysis process by various enzymes that present in the small intestine. the conjugation process will convert luteolin into glucuronide and sulphate derivatives, thereby facilitating its absorption and excretion through the gallbladder and bile secretion. the absorption process of luteolin in the form of glucuronide will undergo further process by the microbiota of the small intestine, thereby altering the chemical properties of glucoronide to the aglycone compound. aglycone will experience several figure 2. the effect of luteolin on the intraerythrocytic growth of p. falciparum. 83kamarullah, et al.: potency of luteolin with solid lipid nanoparticle (sln)-polyethylene glycol (peg) catabolism phases into a low molecular weight compound so that it can be readily absorbed by the small intestinal villi. meanwhile, a group of components that has not been absorbed by the small intestine will lead to the large intestine and undergo further modification by the normal flora of the colon.18 unfortunately, those several metabolic processes cause incomplete absorption of luteolin by the human digestive system. thus, this results in the low bioavailability of luteolin in the human body.18 t h e e n c a p s u l a t i o n o f s o l i d l i p i d nanoparticle (sln) with polyethylene glycol (peg) modification although bioflavonoids like luteolin have a wide variety range of health benefits and potentials in vitro, studies have shown that the role of bioflavonoids in vivo is somewhat ineffective, even has no benefit at all. this is due to the poor lipid solubility properties of luteolin and the unsuitable molecular size of the absorption, leading to poor absorption and bioavailability levels of bioflavonoids. nevertheless, the demand for bioflavonoid use as an herbal treatment is reported to have increased worldwide due to the lack of its side effects and better therapeutic effects in compare with modern medicine. thus, a drug delivery medium may be required to improve the bioavailability of the herbs.19 one of the lipid-based drug delivery media that has been recognized for its effectiveness in improving drug bioavailability is solid lipid nanoparticle (sln). sln was introduced in 1991 and is a lipid-based medium of a colloidal system used as an alternative in drug delivery processes. sln is a submicron particle, having a size from 50 nm to 1000 nm and is made from modified lipid materials so that it has a solid form at the room temperature. compared to other lipid-based drug delivery media, sln has several advantages, among others, such as sln-based materials have good biocompatibility with the human body, easily measured and sterilized, drug release can be controlled with components bioactive in sln, has a large drug capacity, has proven to be the best lipid-based drug delivery substance in enhancing the bioavailability of the encapsulated drug, and has a high degree of stability in the human body.20 in general, the process of preparing encapsulation with the sln method is divided into two, namely the heat homogenization technique and the cold homogenization technique. the cold homogenization technique is judged to have less weakness when compared with the heat homogenization technique. whereas, the technique of heat homogenization can cause various kinds of damage to the medical substance, such as degradation of drugs due to high temperature.20 the heat homogenization technique is carried out at temperatures above the melting point of the lipid (± 30°c to 48°c), so it can be said that the homogenization process is a process for the emulsion. first, the substance of the drug to be encapsulated is mixed with the melted lipids. subsequently, there will be a dispersion process from the lipid phase to a hot mixture called a liquid-surfactant. at this stage, a fast but regular stirring process is required to produce a mixture called pre-emulsion. then, once pre-emulsion formation has created, a high pressure homogenization process is performed. this homogenization process aims to make the mixture blended in very well for the purpose of producing two phase heterogeneous substances called colloids. the colloids are further cooled at room temperature (20°c to 25°c) in order to make the solidification process into a drug that has been encapsulated by sln.20 in the cold homogenization technique, the initial process of cold homogenization techniques is not much different from the high-temperature techniques. the process starts with mixing the desired substance with melted lipids. after that, a process of reducing the size of the substance is done to break down the fat globules into some smaller fat particles. thereafter, the mixture was suspended with water and followed by high pressure homogenization. when the process is complete, a drug product is encapsulated by sln.20 although sln has good biocompatibility, some studies show that sln is often recognized as a foreign antigen in the human body. this is caused by hydrophobic molecules that act as opsonin factors of macrophages so that sln tends to be phagocytosed by various components present in the reticuloendothelial system. to prevent the occurrence of this phagocytosis and to keep the level of the drug in blood plasma remained high, the surface of the drug needs to be modified furthermore using polyethylene glycol (peg). peg works by decreasing the immunological properties of the sln’s hydrophobic molecule like bovine serum albumin in order to avoid immunological reactions between protein-breaking enzymes, such as superoxide dismutase, arginase, and asparginase.21 peg also has dual molecule properties (hydrophilic and hydrophobic surfaces) that can bind to sln hydrophobic part (figure 3). the outer part of peg (hydrophilic) serves to conceal the sln hydrophobic molecules that are figure 3. representation scheme of sln modified with peg and the molecular structure of peg.9 84 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 80–86 believed to be the opsonin factor of the macrophages so that in this case, peg acts indirectly to protect sln from the phagocytic process.21 biopharmaceutical characterization information the construction process of this product the sln-encapsulated luteolin construction process with further peg modification is divided into various stages. the first stage is to extract luteolin from allium fistulosum. some studies recommend that if you want to extract compounds that have limited solubility in a solvent, such as bioflavonoids, you need to use a distillation-based extraction method called soxhlet extraction. the process of luteolin extraction by this method lasted for 8 hours and resulted in a concentrated solution containing 174.76 µg/ ml luteolin (2 grams of dried allium fistulosum equals to 174.76 µg/ml luteolin).22,23 furthermore, luteolin encapsulation with sln was prepared using a cold homogenization method. the liquid lipid phase was prepared using glyceryl monostearate (tween 80) and soy lecithin aqueous chloroform solution in 1:1 ratio. the extracted luteolin solution was mixed with the lipid matrix and then reduced in size (micronization) using nitrogen liquid and high pressure homogenization. the solution phase is then combined using peg-400 (polyethylene glycol) in water and heated until it reaches 80°c. finally, the two phases were combined under mechanical agitation (for 6 minutes at an amplitude of 60%) and the solvent was evaporated by heating the mixture at a temperature of 80°c. the emulsion obtained after evaporation was then cooled to 0°c for 1 hour until it crystallized completely.24,25 pharmacokinetic of this product sln can be administered by mouth, parenteral, and topical. however, luteolin as a flavonoid compound derivative exhibits better efficacy if administered orally or topically.27 based on these findings, the preferred administration method for this product is oral administration. pharmacokinetic reviews include the subject of absorption, distribution, metabolism, and excretion. slnlike lipid properties result in a similar process of digesting lipid-containing materials. the sln digestion process begins with the breakdown of fat through the process of hydrolysis into fatty acids and glycerol using lipase enzymes that are secreted by the chief cells in the stomach. after that, the breakdown product is carried to the duodenum. furthermore, the fatty acid induces the pancreas to secrete the cholecystokinin enzyme to release the colipase enzyme to break the triglycerides into simpler form.26 micelles, a colloidal substance that has a function to make the fat particles can be readily absorbed by the intestinal villi, will be absorbed by several mechanisms such as the passive diffusion transport method, facilitated diffusion, and active transport through the enterocyte membrane. once absorbed into cytosol, the carrier protein will bring the fatty acid into the smooth endoplasmic reticulum. in the smooth endoplasmic reticulum, fatty acids and glycerol will be brought to the golgi apparatus and released by exocytosis to the extracellular space in the form of vesicles. another important step is the absorption of the medicinal active ingredient (luteolin) through an intestine’s intermediate lipoprotein called chylomicron. during the absorption phase, the drug molecule is usually metabolized with the enzyme cytochrome p450-3a4 present at the end of the small intestinal villi. studies are showed that the presence of these enzymes increases the bioavailability of the drug if administered with lipids. this causes the lipophilic drugs tend to have a higher half-life when compared to hydrophilic drugs (figure 4).26 the majority of drugs that administered orally have access to systemic circulation via portal circulatory system. however, highly lipophilic drugs such as peg modified sln will have access to systemic circulation through the lymphatic circulatory system. thus, lipophilic drugs tend to have higher bioavailability in the human body because they do not pass through the first-pass metabolism presented in the liver. in contrast, the addition of peg to the product reduced the drug clearance by the reticuloendothelial system so that the half-life of the drug would be increased.26 this was supported by a study conducted by das et al28 figure 4. various mechanisms of enhancement of drug bioavailability in the presence of lipid substance (sln) within the drugs: (a) solubilization of drug in the intestinal fluid by formation of colloidal substances, vesicles, mixed micelles and micelles; (b) interference with enterocyte-based transport and metabolic processes, thereby potentially changing drug uptake, efflux, disposition, and the formation of metabolites (m) within the enterocyte; (c) by selective lymphatic uptake which reduces first-pass drug metabolism as intestinal lymph travels directly to the systemic circulation.26 85kamarullah, et al.: potency of luteolin with solid lipid nanoparticle (sln)-polyethylene glycol (peg) showing that the half-life of sln encapsulated luteolin with peg modification can reach seven days in circulation. the last pharmacokinetic aspect, excretion, shows that luteolin will be effectively eliminated in the kidneys due to its highly biodegradable nature.26 evidence-based studies regarding improvement of drug’s bioavailability, pharmacokinetics by solid lipid nanoparticle (sln) (in vivo studies) the only study on the efficacy of solid lipid nanoparticles in improving bioavailability and pharmacokinetics of a drug derived from a study that was conducted by dang et al.8 the drug luteolin was detected by a high-performance liquid chromatograph (hplc) and the uv detector was operated at the 350 nm wavelength. also, they used male sprague–dawley rats, weighing from 180 to 220 g and were kept in environmentally controlled room (temperature 25±2°c, humidity 60±5 %, 12/12h dark/light cycle) for 1 week prior to the experiments. they were given daily fresh diet with free access to water.8 luteolin-sln (lu–sln) were prepared by slightly modification in hot-microemulsion ultrasonic technique. briefly, soybean lecithin, tween 80, and water were placed together in a beaker and heated to the lipid melting temperature. glyceryl monostearate with luteolin was also melted at 75±2°c separately. the hot aqueous emulsifier mix was injected into the lipid melt containing luteolin drop by drop, under magnetic stirring to obtain a microemulsion. the obtained pre-emulsion was sonicated by an ultrasonic cell pulverizer for 20 minutes and cool it with ice bath immediately.8 the rats were fasted overnight before experiments with free access to water and randomly divided into two groups (n=6). the free luteolin suspension and lu–sln were administrated to rats by oral gavage at a dose of 20 mg/kg. 0.3-0.5 ml of blood was collected into heparinized tubes at 0, 0.167, 0.333, 0.5, 0.667, 1, 2, 4, 6, 9, 12, 15, and 24 hours after administration. then, plasma was separated immediately by centrifugation at 10,000 rpm for 10 minutes and stored at -20°c for analysis.8 luteolin was extracted from the plasma by liquid–liquid extraction method for lc–ms/ms (liquid chromatography– mass spectrometry) analysis. briefly, plasma sample (100 ll) was spiked with 500 ll mtbe (methyl tert-butyl ether) and 100 ll internal standard solution (diosmetin, 500 lg/l in methanol) by vortex mixing for 30 ss. after centrifugation at 10,000 rpm for 10 minutes at 4°c, the supernatant was transferred to a fresh tube, and evaporated to dryness under a nitrogen gas. the residue was dissolved in 100 ll methanol. the reconstituted extraction was thoroughly mixed and then centrifuged again at 5,000 rpm for 2 min at 4°c. finally, the supernatant solution was injected into the tandem liquid chromatography-mass spectrometry system (figure 5).8 orally administered of lu–sln was rapidly absorbed, as evidenced with less tmax for lu–sln than the pure suspension. the relative bioavailability of luteolin was improved (more than 4.89-fold) when incorporated into the slns. at the same time, distribution and clearance of luteolin with slns were decreased. these data make a clue for supporting slns are a promising delivery system for the enhancement of oral administration of a poorly water-soluble drug.8 human effect matrix the human effect matrix discusses the toxicology as well as the side effects that a drug can cause on the human body. however, no studies have been done to test the toxicity of luteolin-sln-peg against humans. nevertheless, based on a review study conducted by chen et al29 regarding toxicology reviews from luteolin substance, it was mentioned that high doses of luteolin had no toxic effect. therefore, luteolin is expressed as a gras (generally recognized as safe) substance. summary luteolin encapsulated by sln with further peg modification is highly potential to be used as an innovative adjuvant therapy for either artemisinin-resistant or artemisinin-sensitive p. falciparum parasite. however, preclinical studies in the pharmacology field need to be done to confirm the pharmacokinetics and the potential dose of this product. references 1. kementrian kesehatan republik indonesia. riskesdas 2013. 2013. 2. teuscher f, chen n, kyle de, gatton ml, cheng q. phenotypic changes in artemisinin-resistant plasmodium falciparum lines in vitro: evidence for decreased sensitivity to dormancy and growth inhibition. antimicrob agents chemother. 2012;56(1):428–31. 3. witkowski b, amaratunga c, khim n, sreng s, chim p, kim s, et al. novel phenotypic assays for the detection of artemisinin-resistant plasmodium falciparum malaria in cambodia: in-vitro and ex-vivo drug-response studies. lancet infect dis. 2013 dec;13(12):1043–9. 4. ashley ea, dhorda m, fairhurst rm, amaratunga c, lim p, suon s, et al. spread of artemisinin resistance in plasmodium figure 5. mean plasma concentration–time profile of luteolin with and without sln after single dose oral administration of pure luteolin suspension to rats (20 mg/kg).8 86 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 80–86 falciparum malaria. n engl j med. 2014;371(5):411–23. 5. kh m, s m. flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants. j agric food chem. 2001;49(3):106–3112. 6. tasdemir d, lack g, brun r, rüedi p, scapozza l, perozzo r. inhibition of plasmodium f alciparum fatty acid biosynthesis: evaluation of fabg, fabz, and fabi as drug targets for flavonoids. j med chem. 2006 jun;49(11):3345–53. 7. am l, kj s. common dietary flavonoids inhibit the growth of the intraerythrocytic malaria parasite. bmc res notes. 2015;1(26). 8. dang h, meng mhw, zhao h, iqbal j, dai r, deng y, et al. luteolinloaded solid lipid nanoparticles synthesis, characterization, &amp; improvement of bioavailability, pharmacokinetics in vitro and vivo studies. j nanoparticle res. 2014 apr 16;16(4):2347. 9. uner m, yener g. importance of solid lipid nanoparticles (sln) in various administration routes and future perspectives. int j nanomedicine. 2007;2(3):289–300. 10. teuscher f, gatton ml, chen n, peters j, kyle de, cheng q. artemisinin‐induced dormancy in plasmodium falciparum : duration, recovery rates, and implications in treatment failure. j infect dis. 2010 nov;202(9):1362–8. 11. witkowski b, khim n, chim p, kim s, ke s, kloeung n, et al. reduced artemisinin susceptibility of plasmodium falciparum ring stages in western cambodia. antimicrob agents chemother. 2013;57(2):914–23. 12. hien t, thuy-nhien n, phu n, boni mf, thanh n, nha-ca n, et al. in vivo susceptibility of plasmodium falciparum to artesunate in binh phuoc province, vietnam. malar j. 2012;11(1):355. 13. kyaw mp, nyunt mh, chit k, aye mm, aye kh, aye mm, et al. reduced susceptibility of plasmodium falciparum to artesunate in southern myanmar. borrmann s, editor. plos one. 2013 mar 8;8(3):e57689. 14. mita t, tachibana s-i, hashimoto m, hirai m. plasmodium falciparum kelch 13 : a potential molecular marker for tackling artemisinin-resistant malaria parasites. expert rev anti infect ther. 2016 jan 2;14(1):125–35. 15. woodrow cj, white nj. the clinical impact of artemisinin resistance in southeast asia and the potential for future spread. van ooij c, editor. fems microbiol rev. 2017 jan;41(1):34–48. 16. shimoi k, saka n, nozawa r, sato m, amano i, nakayama t, et al. deglucuronidation of a flavonoid, luteolin monoglucuronide, during inflammation. drug metab dispos. 2001 dec;29(12):1521–4. 17. qidwai t, khan f. antimalarial drugs and drug targets specific to fatty acid metabolic pathway of plasmodium falciparum. chem biol drug des. 2012 aug;80(2):155–72. 18. thilakarathna sh, rupasinghe hpv. flavonoid bioavailability and attempts for bioavailability enhancement. nutrients. 2013 aug 28;5(9):3367–87. 19. kesarwani k, gupta r, mukerjee a. bioavailability enhancers of herbal origin: an overview. asian pac j trop biomed. 2013 apr;3(4):253–66. 20. ekambaram p, sathali a ah, priyanka k. solid lipid nanoparticles : a review. sci rev chem commun. 2012;2(1):80–102. 21. c b, o c, r c, l g, a m, mr g. phagocytic uptake of fluorescent stealth and non-stealth solid lipid nanoparticles. int j pharm. 2001;175:185–193. 22. journal ai. an indian journal. biochemistry. 2011;5(2):7–10. 23. wb j. the origin of the soxhlet extractor. j chem educ. 2007;84:1913. 24. singh h. nanotechnology applications in functional foods; opportunities and challenges. prev nutr food sci. 2016 mar 31;21(1):1–8. 25. vijayakumar a, baskaran r, jang ys, oh sh, yoo bk. quercetin-loaded solid lipid nanoparticle dispersion with improved physicochemical properties and cellular uptake. aaps pharmscitech. 2017 apr 1;18(3):875–83. 26. jawahar n, meyyanathan sn, reddy g, sood s. solid lipid nanoparticles for oral delivery of poorly soluble drugs. j pharm sci res. 2012;4(7):1848–55. 27. mukherjee s, ray s, thakur r. solid lipid nanoparticles: a modern formulation approach in drug delivery system. indian j pharm sci. 2009;71(4):349. 28. das s, chaudhury a. recent advances in lipid nanoparticle formulations with solid matrix for oral drug delivery. aaps pharmscitech. 2011 mar 21;12(1):62–76. 29. chen z, kong s, song f, li l, jiang h. pharmacokinetic study of luteolin, apigenin, chrysoeriol and diosmetin after oral administration of flos chrysanthemi extract in rats. fitoterapia. 2012 dec; 83(8):1616–22. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 11 no. 1 january–april 2023 review article impact of hypertension and cardiovascular diseases to immune response in covid-19 vaccination: a systematic review karin dhia fahmita1,2 , gatot soegiarto2,3* , laksmi wulandari2,4 , dewajani purnomosari5 1department of internal medicine, faculty of medicine, universitas airlangga, surabaya, indonesia 2dr. soetomo general academic hospital, surabaya, indonesia 3division of allergy and clinical immunology, department of internal medicine, faculty of medicine, universitas airlangga, surabaya, indonesia 4department of pulmonology and respiratory medicine, faculty of medicine, universitas airlangga, surabaya, indonesia 5department of histology and cell biology, faculty of medicine public health and nursing, universitas gadjah mada, yogyakarta, indonesia received: november 2nd, 2022; revised: march 6th, 2023; accepted: march 13th, 2022 abstract to determine impact of hypertension and cardiovascular diseases towards effectivity and safety of covid-19 vaccination. systematic review based on prisma statement was done. searching was conducted in pubmed, sciencedirect, scopus, and proquest and resulting in 6 studies involving 4,053 participants which deemed on good quality according to joanna briggs institute tools for critical appraisal. after thorough analysis, we found that two out of four studies assessing mrna-based vaccine found out that hypertension lower antibody response significantly. two out of two studies assessing inactivated virus vaccine shown that hypertensive patients tend to have lower antibody titers compared to control. one of studies mentioned above found that antibody titer was not different between populations with cardiovascular diseases and control. hypertension lessened response to covid-19 vaccination regardless of vaccine type used. however, lack of studies on cardiovascular disease suggested that more studies should be conducted, along with hypertension, in-order to make meta-analysis possible to provide better evidence. keywords: antibody; cardiovascular disease; covid-19; efficacy; hypertension highlights: the discovery of the phenomenon of hypertensive patients having lower antibody titers when vaccinated against covid-19 how to cite: fahmita, k. d., sugiarto, g., wulandari, l., purnomosari, d. impact of hypertension and cardiovascular diseases to immune response in covid-19 vaccination: a systematic review. indonesian journal of tropical and infectious disease. 11(1). 44–51. apr. 2023. doi: 10.20473/ijtid.v11i1.40266 * corresponding author: gatot_soegiarto@fk.unair.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-9575-8142 https://orcid.org/0000-0002-9197-3873 https://orcid.org/0000-0002-5000-0151 https://orcid.org/0000-0002-0594-2385 45 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 44–51 introduction coronavirus disease 2019 (covid-19) is a global pandemic resurging from wuhan, china. it involve mild to severe respiratory symptoms which could be left fatal at various cases. in order to end its pandemic status, world health organization (who) has mandated vaccine to be developed and applied. covid-19 vaccine was first introduced in late 2020 and early 2021, with implementation vaccine begun ever since. in general, covid-19 vaccine consists of either mrna or inactivated virus as its base. recent meta-analysis shown that mrna-based and inactivated virus covid-19 vaccines provided efficacy of 94.6% (95% ci 93.6–95. 4) and 80.2% (95% ci 98.0–98.4) respectively.1 it was also proven safe in pregnancy. a meta-analysis studying mrna vaccines shown that efficacy rate was 89.5% (95% ci 69.0–96.4) along with low risk of stillbirth and no addition to risk of miscarriage, earlier gestation at birth, pulmonary embolism, placental abruption, and maternal death.2 emergence of newer variants, which known as variants of concern also did not damper its effectivity, with another study shown that fully vaccinated patients shown efficacy of 88.0%, 73.0%, 63.0%, 77.8%, and 55.9% to alpha, beta, gamma, delta, and omicron variants respectively. boosted patients were more immune to delta and omicron variants with effectivity of 95.5% and 80.8% respectively.3 systematic review by mohammed shown that covid-19 vaccines deemed to suppress infection rate among population and severity, hospitalization rate, and mortality among covid-19 patients. 4 a study by gram found that covid-19 vaccines successfully reduced hospitalization rates for 14–30 days by 98.1%, 98.1%, and 95.5% for alpha, delta, and omicron variant respectively. 5 even though several reports have shown that covid-19 vaccine effectiveness wanes as weeks pass, covid-19 has been proven to protect population from severity and mortality because of covid-19 and to improve health and well-being. 5,6 response to covid-19 vaccination was not the same for every recipient, there were several factors playing part. a study in japan showed that age which older than 60 years, hypertension, high hba1c (>6.5%), and sedentary lifestyle were significant for inhibiting immune response in covid-19 vaccination.7 other studies mention age, sex, nutritional status, obesity, gut microbiota, polymorphisms, and immune system as determinants.8 there were several limitations for populations with high blood pressure and cardiovascular disease to take covid-19 vaccines, even though the limitations have been leniently loosened.9,10 however, impact of hypertension and cardiovascular diseases to immune response to covid-19 vaccination is not fully known. therefore, we conducted a systematic review to determine its relationship to provide better knowledge on covid-19 vaccination. materials and methods materials we conducted systematic review based on the preferred reporting items of systematic review and meta-analysis (prisma) statement.11 searching was conducted on pubmed, scopus, proquest, and sciencedirect published in 2022 using specific keywords and medical subheading (mesh). methods searching was conducted on pubmed, scopus, proquest, and sciencedirect using specific keywords and medical subheading (mesh) terms (table 1). we applied following inclusion criteria: (1) clinical studies; (2) studying population of people with hypertension and/or cardiovascular disease; (3) studying all sort of covid-19 vaccine as intervention; (4) studying effectivity as outcome. in addition, we applied following exclusion criteria: (1) co46 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license karin dhian fahmita, et al. impact of hypertension and cardiovascular diseases existence of other comorbidities; (2) language other than english. selected studies were appraised using the joanna briggs critical appraisal tools.12 studies were extracted for characteristics and result. qualitative analysis was conducted to determine the relationship between variables. table 1. keywords being used for searching. database keywords filters pubmed ("covid-19 vaccines"[mesh]) and (("cardiovascular diseases"[mesh]) or "hypertension"[mesh]) scopus ("covid-19 vaccine") and (("hypertension") or ("cardiovascular disease")) proquest ("covid-19 vaccine") and (("hypertension") or ("cardiovascular disease")) “scholarly journals”, “covid-19 vaccines” sciencedirect ("covid-19 vaccine") and (("hypertension") or ("cardiovascular disease")) “research articles” results and discussion we found total six studies after application of searching strategies and criteria (figure 1).13–18 there were three studies across asia, two across europe, and one american study involving total 4,053 subjects. there were two studies studying coronavac, which is an inactivated virus, and four studies studying bnt162b2 vaccine which is based on mrna. all studies were eligible to be included in this study after appraisal using joanna briggs institute critical appraisal tools (table 2). studies characteristics could be seen in table 3. all four studies studying mrna vaccines shown that hypertensive patients tend to have lower antibodies level compared to control, but only two deemed significant.13–15,17 on the other hand, hypertensive patients which underwent inactivated virus covid-19 vaccination shown significantly lower antibody level compared to control based on both two studies.16,18 one of the studies stating that cardiovascular diseases yet to contribute on antibody level. 16 all results could be seen on table 4. table 2. critical appraisal results of selected studies.12 studies aspect overall 1 2 3 4 5 6 7 8 9 10 11 watanabe et al, 2022 y y y y y y y y y na y include ebinger et al, 2022 y y y y y y y y y y y include delgado et al, 2022 y y y y y y y y y y y include soegiarto et al, 2022 y y y y y y y y y y y include parthymou et al, 2022 y y y y y y y y y y y include rifai et al, 2022 y y y y y y y y y y y include 47 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 44–51 figure 1. schematic workflow of studies’ finding.11 table 3. characteristics of selected studies author year location sample size ab vaccine dose measurement (weeks after dose 2) age (years) male (%) bmi (kg/m 2 ) hypertension (%) diabetes (%) smokers (%) watanabe et al13 2021 japan 68 igs bnt162b2 2 1-4 29.0 (17.0) 39.5 22.4 (5.5) 15.3 2.4 31.7 ebinger et al14 2022 usa 843 igs bnt162b2 2 1, 2, 8, 16, 24, 32, 40 45.0 (13.0) 30.0 15.2 delgado et al15 2022 spain 2174 igs bnt162b2 2 12 45.9 19.9 24.1 8.1 22.2 soegiarto et al16 2022 indonesia 101 igg coronavac 2 4, 12, 20 47.7 (18.9) 59.5 23.7 17.8 10.9 parthymo u et al17 2022 greece 712 igs bnt162b2 2 3, 12 50.8 (11.4) 37.6 26.7 (4.9) 16.2 7.0 34.4 rifai et al18 2022 indonesia 155 igg coronavac 2 8, 24 39.0 (9.2) 48.3 27.9 (7.3) 18.7 table 4. results of selected studies author vaccine results watanabe et al13 mrna hypertensive patients presented lower antibody response compared to normotensive (650 ± 1192 vs 1911 ± 1364, p = 0.001). hypertensive patiens shown significant beta coefficient on univariate and multivariate analysis with -1033.16 (p = 0.005) and 973.27 (p = 0.036) respectively. ebinger et al14 mrna hypertensive patients shown significant beta coefficient on multivariate analysis with -0.17 and se of 0.08 (p = 0.041). delgado et al15 mrna hypertensive patients shown insignificant fold changes with -1.02 (p = 0.8584). soegiarto et al16 inactivated hypertensive patients shown significant beta coefficient on multivariate analysis with -11.208 (p = 0.038). patients with history of cardiovascular diseases shown nonsignificant beta coefficient on multivariate analysis with -10.040 (p = 0.969) parthymou et al17 mrna hypertensive patients shown insignificant beta coefficient on multivariate analysis with -0.0454 (p = 0.3276). rifai et al18 inactivated patients with high systolic blood pressure and high diastolic blood pressure shown significant correlation with lower antibody response with r coefficient of -0.172 (p = 0.016) and -0.139 (p = 0.043) respectively second months after vaccination, and r coefficient of -0.284 (p = 0.046) and -0.475 (p = 0.006) respectively six months after vaccination. 48 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license karin dhian fahmita, et al. impact of hypertension and cardiovascular diseases hypertension accounted for lower antibody response in covid-19 vaccination which was stated in all adjuvant vaccine studies and in most of mrna vaccine studies. however, some studies showed that there were reports of non-significant differences between groups. study by delgado et al involving mrna vaccines reported there were positively increased anti-s protein antibody level after vaccination in patients with older age, more bmi, and arterial hypertension, but exclusive to infected subjects which explained the non-significant of result.15 however, another mrna vaccines study by parthymou et al reported that non-significant difference of immune response between hypertensive and nonhypertensive groups was due to confounding factors and differences in size, age, and selfreporting of the populations.17 it is known that vaccine response was based on cascades of immune system responses. it depends on the role of t helper 2 (th2) and b cells to provide a connection to produce long-lived plasma cells which secrete antibodies with high affinity.19 however, there is differences between mrna vaccine and adjuvant vaccine in terms of immune response, whereas mrna vaccine is stimulating cellular immune response and adjuvant vaccine stimulates humoral immune response. hypertension played role in impairing both of mechanisms. hypertensive patients had lower th2 and interleukin 4 (il4) levels significantly, thus immune response was impaired.20 in addition, hypertensive patients developed proinflammatory t cells as a result of high blood pressure which could produce cytokines relating to th1 and th17 such as interferon-gamma and interleukin 17a (il-17a).21 another piece of evidence found that angiotensin ii, which was overactivated on hypertension, was accounted for the increase in th1 production and th2 suppression.22 th1 will inhibit humoral immune response, thus inhibiting antibody production.23 many other evidences have stated similar hypertension’s role in modulating t cell immune metabolism.24–26 in addition, another study stated that chronic inflammatory due to hypertension will release cytokines due to endothelial dysfunction which included reactive oxidative species (ros) and interleukins such as il-1-beta, il-6, il-8, il-17, il-23, and tnf-alpha. all of these cytokines were responsible for dysfunction of angiotensin ii which worsen blood pressure. these cytokines also could alter immune response in hypertension.27 besides applying a damper effect to the immune response of covid-19 vaccination, hypertension accounted for more severe covid-19 outcomes.28–29 hypertension was found to be the most common comorbidity observed in covid-19 infection and alongside cardiovascular disease accounted for 2.36 folds higher chance of mortality compared to control.30 not only as comorbid, hypertension also played its role as an adverse event towards covid-19 vaccination. a meta-analysis showed that 3.20% of patients who underwent covid-19 vaccination showed an abnormal increase in blood pressure, with 0.6% of patients developed hypertensive urgencies and emergencies.31 this was further confirmed by other studies which stated similar findings.31– 36 therefore, hypertension provided difficult challenges for healthcare workers who administered covid-19 vaccine. not only being impactful to lessen antibody response, but it also accounted for more severe covid-19 outcomes and more risk towards adverse events. therefore, hypertension in populations who were prospective for covid-19 vaccine administration should be taken cautiously and seriously in order to prevent adverse events or severe outcomes. vaccine developers should be able to make sure that covid-19 vaccine provided the expected antibody response when given to hypertensive populations in a safe fashion. relation between cardiovascular diseases and antibody response is still yet to be known with unclear mechanisms. however, it is 49 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 44–51 suspected to accounted towards blood circulation and component. therefore, more studies should be conducted further to determine relation and mechanism of cardiovascular disease impact towards covid-19 vaccination. this was a systematic review which provided information on the impact of hypertension and cardiovascular diseases and hypertension towards covid-19 vaccine response. however, there were limited studies available. in addition, studies included in this review is limited to widescope of hypertension which is yet to be graded or classified. this make reviewer could not determine stage which is more responsible for impairment of immune response after vaccination. therefore, it was recommended that more high-quality studies which involved graded hypertension should be done to make meta-analysis possible to provide a better understanding and knowledge of this field. strength and limitation the strength of this study was a comprehensive literature search and a bias study was carried out. the limitation of this study is that the amount of literature found is very small. conclusions hypertension was linked with lesser antibody response to covid-19 vaccination in both mrna-based and inactivated virustype vaccines. however, cardiovascular diseases are yet to be linked to covid-19 vaccination response. due to the few studies which have been retrieved, more studies should be conducted to make a meta-analysis with higher and stronger evidence to be conducted to provide better knowledge on this field. funding this study did not receive any funding. conflict of interest the authors confirm that they have no conflict of interest. author contribution writer, literature searcher, collecting data from literature: kdf. conceptor and supervision: gs. review and supervision: lw and dp. references 1. pormohammad a, zarei m, ghorbani s, mohammadi m, razizadeh mh, turner dl, et al. efficacy and safety of covid-19 vaccines: a systematic review and meta-analysis of randomized clinical trials. vaccines (basel). 202; 9(5): 467 2. prasad s, kalafat e, blakeway h, towsend r, o’brien p, morris e, et al. systematic review and meta-analysis of the effectiveness and perinatal outcomes of covid-19 vaccination in pregnancy. nature communications. 2022; 13: 2414 3. zeng b, gao l, zhou q, yu k, sun f. effectiveness of covid-19 vaccines against sars-cov-2 variants of concern: a systematic review and meta-analysis. bmd med. 2022; 20(1): 200 4. mohammed i, nauman a, paul p, ganesan s, chen k, jalil s, et al. the efficacy and effectiveness of the covid-19 vaccines in reducing infection, severity, hospitalization, and mortality: a systematic review. hum vaccin immunother. 2022; 18(1): 2027160 5. gram ma, embord h, schelde ab, friis n, nielsen kf, mousten-helms i, et al. vaccine effectiveness against sars-cov-2 infection or covid-19 hospitalization with the alpha, delta, or omicron sars-cov-2 variant: a nationwide danish cohort study. 2022; 19(9): e1003992 6. andrews n, stowe j, kirsebom f, toffa s. covid-19 vaccine effectiveness against the omicron (b.1.1.529) variant. n engl j med. 2022; 386: 1532–46 50 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license karin dhian fahmita, et al. impact of hypertension and cardiovascular diseases 7. mitsunaga t, ohtaki y, seki y, yoshioka m, mori h, suzuka m, et al. the evaluation of factors affecting antibody response after administration of the bnt162b2 vaccine: a prospective study in japan. peerj. 2021; 9: e12316 8. falahi s, kenarkoohi a. host factors and vaccine efficacy: implications for covid-19 vaccines. j med virol. 2022; 94(4): 1330–5 9. block jp, boehmer tk, forrest cb, carton tw, lee gm, ajani ua, et al. cardiac complications after sars-cov-2 infection and mrna covid-19 vaccination – pcornet, united states, january 2021–january 2022 [internet}. cdc. 2022. available from: https://www.cdc.gov/mmwr/volumes/71/wr/mm 7114e1.htm 10. antopolis s. covid vaccines are safe for patients with cardiovascular disease [internet]. esc. 2022. available from: https://www.escardio.org/the-esc/pressoffice/press-releases/covid-vaccines-are-safefor-patients-with-cardiovascular-disease 11. page mj, mckenzie je, bossuyt pm, boutron i, hoffmann tc, mulrow cd, et al. the prisma 2020 statement: an updated guideline for reporting systematic reviews. systematic reviews. 2021; 10: 89. 12. checklist for cohort studies [internet]. the joanna briggs institute. available from: https://jbi.global/sites/default/files/201905/jbi_critical_appraisalchecklist_for_cohort_studies2017_0.pdf 13. watanabe m, balena a, tuccinardi d, tozzi r, risi r, masi d, et al. central obesity, smoking habit, and hypertension are associated with lower antibody titres in response to covid-19 mrna vaccine. diabetes metab res rev. 2022; 38(1): e3465 14. ebinger je, joung s, liu y, wu m, weber b, claggett b, et al. demographic and clinical characteristics associated with variations in antibody response to bnt162b2 covid-19 vaccination among healthcare workers at an academic medical centre: a longitudinal cohort analysis. bmj open. 2022; 12(5): e0599994 15. delgado jf, berenguer-llergo a, julia g, navarro g, espasa m, rodriguez s, et al. antibody response induced by bnt162b2 and mrna-1273 vaccines against the sars-cov-2 in a cohort of healthcare workers. viruses. 2022; 14: 1235 16. soegiarto g, wulandari l, purnomosari d, fahmita kd, gautama hi, hadmoko st, et al. hypertension is associated with antibody response and breakthrough infection in healthcare workers following vaccination with inactivated sars-cov-2. vaccine. 2022; 40: 4046–56 17. parthymou a, habeos ee, habeos gi, deligakis a, livieratos e, marangos m, et al. factors associated with anti-sars-cov-2 antibody titres 3 months post-vaccination with the second dose of bnt162b2 vaccine: a longitudinal observational cohort study in western greece. bmj open. 2022; 12: e057084 18. rifai a, pratama mz, wahono cs, kalim h. association between the effectiveness and immunogenicity of inactivated sars-cov-2 vaccine (coronavac) with the presence of hypertension among healthcare workers. clin exp hypertens. 2022. 19. inoue t, moran i, shinnakasu r, phan tg, kurosaki t. generation of memory b cells and their reactivation. immunol rev. 2018;283:138– 49 20. ji q, cheng g, ma n, huang y, lin y, zhou q, que b, dong j, zhou y, nie s, et al. circulating th1, th2, and th17 levels in hypertensive patients. dis markers. 2017;2017:7146290 21. asadikaram g, ram m, izadi a, sheikh fathollahi m, nematollahi mh, najafipour h, shahoozehi b, mirhoseini m, masoumi m, shahrokhi n, et al. the study of the serum level of il-4, tgf-β, ifn-γ, and il-6 in overweight patients with and without diabetes mellitus and hypertension. j cell biochem. 2019;120(3):4147–57 22. mikolajczyk tp, guzik tj. adaptive immunity in hypertension. curr hypertens rep. 2019;21:68 23. shao j, nangaku m, miyata t, inagi r, yamada k, kurokawa k, fujita t. imbalance of t-cell subsets in angiotensin ii-infused hypertensive rats with kidney injury. hypertension. 2003 jul;42:31–38 24. mattson dl, abais-battad jm. t cell immunometabolism and redox signaling in hypertension. curr hypertens rep 2021;23(12):45 25. moshfegh cm, case aj. the redox-metabolic couple of t lymphocytes: potential consequences for hypertension. antioxid redox signal 2021;34 (12):915–35 26. rai a, narisawa m, li p, piao l, li y, yang g, et al. adaptive immune disorders in hypertension and heart failure: focusing on t-cell subset activation and clinical implications. j hypertens 2020;38(10):1878–89 27. tanase md, gosav me, radu s, ouatu a, rezus c, ciocoiu m, et al. arterial hypertension and interleukins: potential therapeutic target or future diagnostic marker?. int j hypertens. 2019; 2019. 1–17. 51 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 44–51 28. maddaloni e, d'onofrio l, alessandri f, et al. cardiometabolic multimorbidity is associated with a worse covid‐19 prognosis than individual cardiometabolic risk factors: a multicentre retrospective study (covidiab ii). cardiovasc diabetol. 2020; 19(1):164 29. maddaloni e, d'onofrio l, alessandri f, et al. clinical features of patients with type 2 diabetes with and without covid‐19: a case control study (covidiab i). diabetes res clin pract. 2020;169: 108454 30. yang j, zheng y, gou x. prevalence of comorbidities and its effects in coronavirus disease 2019 patients: a systematic review and meta-analysis. int j infect dis. 2020; 94: 91–5 31. angeli f, reboldi g, trapasso m, santilli g, zappa m, verdecchia p. blood pressure increase following covid-19 vaccination: a systematic overview and meta-analysis. j cardiovasc dev dis. 2022; 9(5): 150 32. khani e, entezari-maleki t. hypertensive crisis following covid-19 vaccination. j clin pharmacol. 2022; 62: 1047–8 33. soegiarto g, purnomosari d, wulandari l, et al. incidence of sars-cov-2 infection in hospital workers before and after vaccination programme in east java, indonesia-a retrospective cohort study. lancet reg health southeast asia. 2023 mar;10:100130.doi:10.1016/j.lansea.2022.1001 30. epub 2022 dec 12. pmid: 36531927; pmcid: pmc9742226. 34. modin d, claggett b, jørgensen me, et al. flu vaccine and mortality in hypertension: a nationwide cohort study. j am heart assoc. 2022 mar 15;11(6):e021715. doi: 10.1161/jaha.121.021715. epub 2022 feb 8. pmid: 35132866; pmcid: pmc90752 35. ying cq, lin xq, lv l, et al. intentions of patients with hypertension to receive a booster dose of the covid-19 vaccine: a crosssectional survey in taizhou, china. vaccines (basel). 2022 sep 29;10(10):1635. doi: 10.3390/vaccines10101635. pmid: 36298500; pmcid: pmc9608070. 36. zhang y, chen h, lv j, et al. evaluation of immunogenicity and safety of vero cellderived inactivated covid-19 vaccine in older patients with hypertension and diabetes mellitus. vaccines (basel). 2022 jun 25;10(7):1020. doi: 10.3390/vaccines10071020. pmid: 35891184; pmcid: pmc9315836. 57 vol. 7 no. 3 september–december 2018 blastocystis and other intestinal parasites infections in elementary school children in dukuh village, karangasem district, bali ni luh putu eka diarthini1,2, i kadek swastika2, luh ariwati2, rahmadany isyaputri3, moh. yasin fitri n3, sri hidajati3, sukmawati basuki3,4a 1 basic medical science program, faculty of medicine, universitas airlangga, surabaya 2 department of parasitology, faculty of medicine, udayana university, denpasar, bali 3 department of parasitology, faculty of medicine, universitas airlangga, surabaya 4 malaria study group, institute of tropical disease, universitas airlangga, surabaya a corresponding author: sukmabas@fk.unair.ac.id abstract blastocystis spp. is the most common enteric parasitic infection found in several community surveys from developing countries. blastocystis infections may cause gastrointestinal symptoms, but also cause extraintestinal symptoms such as urticaria and joint pain. blastocystis infection can also be asymptomatic or a carrier. however, the prevalence of blastocystis infection in children has not yet been fully investigated in indonesia, particularly in bali province. this study aimed to determine the prevalence of blastocystis and other intestinal parasites in elementary school children stools in dukuh village, karangasem regency. a cross sectional study was conducted in september 2016. a total of 103 school children stools were collected by informed consent and parasites were examined by microscopy with wet mounts method using lugol’s iodine solution. thirty-five school children were infected with blastocystis spp. (35/103, 34%) that consisted of a single infection (29/35, 82.9%) and mix infection with other parasites (6/35, 17.1%). the mix infections were blastocystis spp. and hookworm infection (1/6, 16.7%), blastocystis spp. and entamoeba coli (1/6, 16.7%), blastocystis spp. and giardia lamblia (2/6, 33.3%), blastocystis spp. and entamoeba histolytica/ entamoeba dispar (1/6, 16.7%) and blastocystis spp. and entamoeba histolytica/ entamoeba dispar and giardia lamblia (1/6, 16.7%). the vacuolar forms of blastocystis were dominantly found, in which was non-infectious form, whereas the infectious form is the cyst form and blastocystis density was observed less than 5 cells per field of view at 400 magnification in all cases. this study concluded that the high prevalence of blastocystis infection in elementary school children in dukuh village, karangasem district, bali that were dominantly single infections and several mix infections with other intestinal parasites. the high prevalence of blastocystis infection in elementary school children suggested that it needs proper prevention measures for the children in this study area. keywords: blastocystis spp., intestinal parasites, prevalence, children, bali abstrak blastocystis spp. adalah infeksi parasit usus yang paling umum ditemukan di beberapa survei masyarakat dari negara-negara berkembang. infeksi blastocysts dapat menyebabkan gejala gastrointestinal, namun dapat juga menyebabkan gejala ekstraintestinal seperti urtikaria dan nyeri sendi. infeksi blastocystis dapat juga asimptomatik atau sebagai carrier. namun, prevalensi infeksi blastocystis pada anak belum sepenuhnya diketahui di indonesia, khususnya di provinsi bali. penelitian ini bertujuan untuk mengetahui prevalensi blastocystis dan parasit usus lainnya pada anak-anak sekolah dasar di desa dukuh, kabupaten karangasem. penelitian cross sectional dilakukan pada bulan september 2016. sebanyak 103 tinja anak sekolah dikumpulkan dan parasit diperiksa secara mikroskopis dengan metode preparat basah menggunakan larutan yodium lugol. tiga puluh lima anak sekolah terinfeksi blastocystis spp. (35/103, 34%) yang terdiri dari satu infeksi tunggal (29/35, 82.9%) dan infeksi campuran dengan parasit lain (6/35, 17.1%). infeksi campuran infeksi blastocystis spp. dengan cacing tambang (1/6, 16.7%), blastocystis spp. dengan entamoeba coli (1/6, 16.7%), blastocystis spp. dengan giardia lamblia (2/6, 33.3%), dan blastocystis spp. dengan entamoeba histolytica/entamoeba dispar (1/6, research report 58 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 57–61 16.7%) dan blastocystis spp. dengan entamoeba histolytica/ entamoeba dispar dan giardia lamblia (1/6, 16.7%). bentuk vakuolar merupakan bentuk blastocystis yang dominan ditemukan, yang mana bentuk ini adalah tidak infeksius, namun bentuk kista adalah bentuk infeksius, serta kepadatan blastocystis diamati kurang dari 5 sel per lapang pandang pada pembesaran 400 yang ditemukan pada semua kasus. penelitian ini menyimpulkan bahwa tingginya prevalensi infeksi blastocystis pada anak sekolah dasar di desa dukuh, kabupaten karangasem, bali yang mana infeksi tunggal adalah dominan ditemukan dan beberapa infeksi campuran dengan parasit usus lainnya. prevalensi infeksi blastocystis yang tinggi pada anak sekolah menunjukkan bahwa diperlukan tindakan pencegahan yang tepat untuk anak-anak di wilayah studi ini. kata kunci: blastocystis spp, parasit usus, prevalensi, anak-anak, bali indonesia in september 2016. the karangasem district is the eastern part of bali island (figure 1). this study area is selected as a research area due to associate with various risk factors of blastocystis infection that are found in this village such as poor individual hygiene, lack of family latrines for defecation, lack of proper sanitation, poverty, close contact with animals, consuming contaminated food and water, dry natural conditions without any wellsprings and lack of clean water supply for daily living. large rainy water reservoirs named ‘cubang’ are built at several points that serve to accommodate water during the rainy season.13 residents in the dukuh village there are those who keep animals without cages so that life together and close contact with animals (figure 2), such as pigs, dogs, chickens, cows, rats and cockroaches. people share water resources with various animals. introduction diarrhea is still major cause of childhood morbidity and mortality in developing countries.1,2 one of the most common intestinal protozoa found in several community survey is blastocystis spp.3,4 that able to cause diarrhea. blastocystis spp. is an intestinal protozoan and most commonly reported human intestinal protozoan in children and adults in developing country, has a world-wide distribution with prevalence of 30% to 60 % in developing country and 1,5% to 20% in developed country.5–7 the prevalence of blastocystis infection is reported in japan 0.5-1%, in singapore 3.3%, but in developing countries the prevalence is much higher, as in argentina 27.2%, in egypt 33.3%, in cuba 38.5%, in brazil 40.9%, and in indonesia 60%.6 pegelow8 found that the incidence of blastocystis infections in elementary school children in sukaraja, west java was 60%3,8 and in elementary school children in hygiene poor areas in sumba island found a prevalence of blastocystis infections was 29.9%.9 the high prevalence of blastocystis infection in children is attributed to many factors, particularly environmental and personal hygiene. this infection is mainly through fecal-oral route and belongs to water borne disease. the pathogenic potential is controversial because the infection can be asymtomatic.6 blastocystis infections may cause gastrointestinal (gi) symptoms, but may also cause extraintestinal symptoms such as urticaria and joint pain.7,10,11 blastocystis can cause irritable bowel syndrome (ibs) so that it can affect the occurrence of malnutrition. in addition, immunocompromised hosts are more susceptible to these organisms than immunocompetent hosts, which is common in hiv/aids patients and cancer patients.11,12 bali island is a tropical region, where has some areas with poor hygiene, such as in dukuh village, karangasem district, blastocystis infection prevalence has not been investigated. this study aimed to determine the prevalence of blastocystis infection in this area with poor hygiene as one of the risk factors for parasitic infections, especially blastocystis. material and method this cross-sectional study was conducted at elementary school in dukuh village, karangasem district, bali province, figure 2. people keep pigs, chickens and cows without cages figure 1. map of bali island. the study was conducted in karangasem area (hatched) 59diarthini, et al.: blastocystis and other intestinal parasites infections stools were collected from 103 students (6-13 years old) at sd 2 dukuh, an elementary school, by informed consent. the procedure for stool collection was thoroughly explained and clean plastic containers were distributed to each student on the day before specimen collection. all specimens were collected into wide-mouth, screw-capped containers and were immediately preserved in 10% formalin. blastocystis spp. and other intestinal parasites in stools were examined and identified by wet mounts method using lugol iodine solution as described by world health organization (who) manual of basic technique for a health laboratory.14 microscopic examinations of stool samples, which are based on morphology, are commonly used for identification of parasites. however, these methods are unable to differentiate subtype of blastocystis. microscopic examination for the blastocystis was viewed by magnification of 400 and 1000 times under a light microscope. blastocystis is a polymorphic intestinal parasite that is common in humans. the four major morphologic forms: vacuolar (most common), cyst, granular, and amoeboid can be seen in stools.5 to determine the density of blastocystis was performed by counting the number of blastocystis per field of view in 400 magnification. this study was approved by the ethical committees, faculty of medicine, universitas airlangga. result and discussion this study was conducted to determine the prevalence of blastocystis infections in elementary school children located in dukuh village, karangasem district, bali province in september 2016. stools were collected from 103 students, age from 6 to 13 years, and examined microscopically to determine blastocystis infection by wet mount method using lugol iodine solution. samples examined found that (35/103, 34%) student stools infected with blastocystis, asymptomatic infection. a single infection of blastocystis (29/35, 82.9%) dominantly detected than mix infections, blastocystis and other parasites (6/35, 17.1%). among mix infections, blastocystis and giardia lamblia were commonly observed (2/6, 33.3%) therefore, the mix infection prevalence occurred less 10 % (6/103, 5.8%) (see on table 1). in the microscopic examination we found that the blastocystis density image in the sample examined in figure 3. and blastocystis density was found to be less than 5 cells per field at 400 magnification in all cases (table 2). the dominant forms were vacuolar forms on microscopic examination of infected samples of blastocystis (in figure. 4). the prevalence of blastocystis infection in elementary school children at dukuh village, karangasem district was 34% that was similar with blastocystis infection prevalence in sumba island in 2016 (29.9%). those prevalence of blastocystis infection were commonly found in developing countries, 30-60% compared with industrialized countries5–7, and indonesia is a developing country. it has been reported by pegelow et al8 that the incidence of blastocystis infections reached to 60% in elementary school children in sukaraja, west java in 1997.3,8 high prevalence of blastocystis infection in elementary school children at dukuh village, karangasem district might due to the risk factors of blastocystis infection, particularly table 1. blastocystis and other intestinal parasite infections in elementary school student stools in dukuh village parasites stools (n=103) number of infection (n, %) single infection blastocystis spp. 29 (28.1) mix infections blastocystis spp., hookworm blastocystis spp., e. histolytica/ e.dispar blastocystis spp, e.coli blastocystis spp, g.lamblia blastocystis spp, e. histolytica/ e.dispar, g.lamblia 6 ( 5.9) 1 ( 1.0) 1 ( 1.0) 1 ( 1.0) 2 ( 1.9) 1 ( 1.0) total 35 (34.0) table 2. blastocystis density was calculated by the viewer with magnification of 400 number of blastocystis per field (40x objective) no. infected samples (n,%) 1/field 23 (65.7) 2/field 6 (17.1) 1/3 field 1 (2.9) 1/5 field 4 (11.4) 1/10 field 1 (2.9) total 35 (100.0) figure 3. blastocystic density is examined under a light microscope at 400 magnification (blastocystis is shown with arrows) 60 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 57–61 the poor hygiene and sanitation in this study area such as sharing water resources (cubang) between human and animals, lack of family latrines for defecation, lack of proper sanitation, and close contact with animals (fig.3). it is known that blastocystis infection belongs to water borne diseases15,16, so the above factors are involving in the transmission of blastocystis infection which is thoroughly the fecal-oral route by water contamination. thus, it needs to study further the transmission of blastocystis infection in this area by the examination of blastocystis in animal stools and water resources (cubang). in many epidemiologic surveys, blastocystis is the most frequently isolated parasite, with a higher prevalence in underdeveloping countries, where poor hygiene, exposure to animals, and consumption of contaminated food or water are observed. it is more common than other protozoan parasites, giardia lamblia and dientamoeba fragilis.5 in this study showed that hookworm, entamoeba coli, giardia lamblia and e. histolytica/ entamoeba dispar were also detected together with blastocystis, however, the prevalence of them were low (1%, 1%, 3% and 1% respectively). even though, their prevalence was low, hookworm disease belongs to the neglected tropical disease17 that causes anemia, which affect to the children growth and development18. so, it needs to be concerned for disease control and eradication, for example use the toilet for defecation, use the shoes or slipper during playing in the ground. giardia lamblia was more common obtained with blastocystis than other intestinal protozoan and the cyst form, the infective form, of giardia lamblia was dominantly found in student stools. the students seemed to be carrier hosts who can transmit the infective form through water contamination19,20. health education needs to be conducted at elementary school in order to improve student behavior on use the proper toilet for defecation. mix infection between blastocystis and entamoeba in school children stools were found. both protozoan parasites cause water borne disease 21. it means that the water at this study area is poor quality or contaminated water, thus the treated water for either cooking or drinking is required. this study revealed that predominant intestinal parasite infection in the school children was blastocystis infection without intestinal symptoms and density of blastocystis observed by 400x magnifications was less than 5 parasites per field (see on table 2). previous studies suggested that more than 5 parasites per highpower field (40x objective) for wet mount or by oil immersion (100x objective) were associated with the presence of gastrointestinal disease6,22,23 and treated by metronidazole.22,23 in addition, the blastocystis parasite seems to be more common in healthy individuals than in patients with inflammatory bowel disease and is associated with certain gut microbiota and health indicators. although the parasite may elicit disease under certain conditions, the focus on blastocystis infection may be shifting from a clinical to a public health viewpoint.24 a vacuolar form of blastocystis spp. a polymorphic intestinal parasite, was dominantly detected in school children stools in dukuh village. this form is one of blastocystis forms in its life cycle that are, cyst, granular, and amoeboid in which can be seen in stool and axenic cultures.25,26 some studies proposed that morphologic features of blastocystis have a role as predictors of clinical symptoms.25,26 unfortunately, this study did not investigate the subtype of blastocystis, whereas the variability of intrasubtype may result in the production of effectors that contributes to blastocystis pathogenicity in correlation with the presence of clinical symptoms.26 diverse morphologic features (vacuolar transiting to amoeboid), probably reflecting the progression from an asymptomatic to a symptomatic state, were observed in an asymptomatic subtype 3 carriers who later had symptoms. searching for amoeboid forms might be helpful to presumptively screen symptomatic patients with subtype 3 or to follow up an asymptomatic subtype 3 carrier in case symptoms become evident before antiprotozoal treatment was attempted.26 diverse forms of blastocystis have been reported by various researchers: vacuolar forms are observed in feces and in cultures, granular forms are considered degenerative products, and amoeboid stages of blastocystis are detected whenever symptoms are reported.27,28 beside vacuolar forms, we also found a few of granular forms but not amoeboid and cyst forms in school children stools. both forms might be correlated to asymptomatic infection as the previous study reported that vacuolar forms and granular forms are found in people who have asymptomatic conditions, and when found in amoeboid form the possibility of progression from an asymptomatic to a symptomatic state.26 our study revealed the first report of the high prevalence of blastocystis spp in an elementary school children stools, dukuh village, karangasem district, bali, with less density of blastocystis and dominant vacuolar forms. it suggested that the water quality for drinking or cooking a 3,37 μm c b 4,41 μm d figure 4. blastocystis vacuolar form was found on microscopic examination using wet mount method with 1000 magnification (blastocystis is shown with arrows) 61diarthini, et al.: blastocystis and other intestinal parasites infections needs to be improved, and also hygiene and sanitation be refined in this area. blastocytis transmission, and the roles of morphologic features and variation within blastocystis sub-types as predictor of symptomatic infection need to be elaborated further. conclusion the prevalence of blastocystis infection was high in elementary school children in dukuh village, karangasem district, bali that were dominantly single infections and several mix infections with other intestinal parasites. it suggested that needs proper prevention measures for the children in this area, particularly improving water quality. the result of this study could be used for other studies on the prevalence of blastocystis infection in elementary school children, and for establishing policy in control and prevention programs of blastocystis infection. acknowledgment the authors would like to thank all the children for their participation in this study. we thank ministry of education, headmasters and teachers of the respective schools at which specimens were collected, for their kind help and cooperation. this study was supported by department of parasitology, faculty of medicine, universitas airlangga and department of parasitology, faculty of medicine, udayana university as a part fulfillment of a master degree. references 1. farthing m, salem m, lindberg g, dite p, khalif i, salazer-lindo e, et al. acute diarrhea in adults and children: a global perspective. world gastroenterol organ. 2012;(february): 1–24. 2. subagyo b, santoso n. buku ajar gastro-enterologi-hepatologi. 1st ed. jakarta: idai; 2012. 87-102 p. 3. nofita e, harminarti n, rusjdi sr. identifikasi blastocystis hominis secara mikroskopis dan pcr pada sampel feses di laboratorium rsup. dr. m. djamil padang. maj kedokt andalas. 2014; 37(1): 26–31. 4. rebolla mf, silva em, gomes jf, falcão ax. high prevalence of blastocystis spp. infection in children and staff members attending public urban schools in sao paulo state, brazil. rev inst med trop sao paulo. 2016;(2). 5. wawrzyniak i, poirier p, texier c, delbac f, viscogliosi e, dionigia m, et al. blastocystis, an unrecognized parasite: an overview of pathogenesis and diagnosis. ther adv infect dis. 2013;1(5): 167–78. 6. tan ksw. new insights on classification, identification, and clinical relevance of blastocystis spp. 2008; 21(4): 639–65. 7. abdulsalam am, ithoi i, al-mekhlafi hm, al-mekhlafi am, ahmed a, surin j. subtype distribution of blastocystis isolates in sebha, libya. plos one. 2013; 8(12): 1–11. 8. stensvold c, arendrup m, jespersgaard c, molbak k, nielsen h. detecting blastocystis using parasitologic and dna-based methods: a comparative study. diagnostic microbiol infect disesase. 2007; 59: 303–7. 9. yoshikawa h, tokoro m, nagamoto t, arayama s, asih pbs, rozi ie, et al. molecular survey of blastocystis sp. from humans and associated animals in an indonesian community with poor hygiene. parasitol int. 2016; 65(6): 780–4. 10. dogruman-al f, kustimur s, yoshikawa h, tuncer c, simsek z. blastocystis subtypes in irritable bowel syndrome and inflammatory bowel disease in ankara, turkey. mem inst oswaldo cruz, rio jaeniro. 2009;104(august): 724–7. 11. popruk s, pintong a, radomyos p. diversity of blastocystis subtypes in humans. j trop med parasitol. 2013; 36(2): 88–97. 12. eassa sm, ali hs, masry sa el, el-fattah aha. imedpub journals blastocystis hominis among immunocompromised and immunocompetent children in alexandria, egypt abstract. 2016; 1–7. 13. daya pertiwi foundation. selayang pandang proyek 2014/2015: pulau nusa penida &amp; kubu kecamatan karangasem, bali. 2015. 14. who. manual of basic technique for a health laboratory. 2003. 15. leelayoova s, rangsin r, taamasri p, naaglor t, thathaisong u, mungthin m. evidence of waterborne transmission of blastocystis hominis. am j trop med hyg. 2004; 70(6): 658–62. 16. leelayoova s, siripattanapipong s, thathaisong u, naaglor t, taamasri p, piyaraj p, et al. drinking water: a possible source of blastocystis spp. subtype 1 infection in schoolchildren of a rural community in central thailand. am j trop med hyg. 2008; 79(3): 401–6. 17. kline k, mccarthy js, pearson m, loukas a, hotez pj. neglected tropical diseases of oceania: review of their prevalence, distribution, and opportunities for control. plos negl trop dis. 2013; 7(1): 1–9. 18. hotez pj, broker s, bethony jm, bottazi me, loukas a, xiao s. hookworm infection. n engl j med. 2004; 2: 799–807. 19. gardner tb, hill dr. treatment of giardiasis. clin microbiol rev. 2001; 14(1): 114–28. 20. bedell da, garrett re. giardidasis. seventh. rakel re, editor. elsevier; 2007. 477–479 p. 21. mh fh, karamkhani a, r fh. waterborne parasites : a recent status of occurrence, source and human intestinal parasites in sources and tap. 2016;1(61): 17–21. 22. kaya s, çeti̇n es, aridoğan bci�, arikan s, demi̇rci̇ m. pathogenicity of blastocystis hominis, a clinical reevaluation. turkiye parazitoloji derg. 2007; 31(3): 184–7. 23. moghaddam d, ghadirian e, azami m. blastocystis hominis and the evaluation of efficacy of metronidazole and trimethoprim/ sulfamethoxazole. parasito res. 2005;96 (4): 273–5. 24. andersen lob. blastocystis in health and disease are we moving from a clinical to a public health perspective ? pubmed commons. j clin microbiol. 2016; 54(3): 524/8. 25. coyle cm, varughese j, weiss lm, tanowitz hb. blastocystis : to treat or not to treat. 2012; 54: 105–10. 26. vassalos cm, spanakos g, e v, c p, n v. differences in clinical significance and morphologic features of blastocystis sp subtype 3. 2010; 251–8. 27. tan tc, suresh kg, smith h v. phenotypic and genotypic characterisation of blastocystis hominis isolates implicates subtype 3 as a subtype with pathogenic potential. parasitol res. 2008; 104(1): 85–93. 28. vdovenko aa. blastocystis hominis: origin and significance of vacuolar and granular forms. parasitol res. 2000;86(1): 8–10. 109 vol. 6 no. 5 may–august 2017 research report relationship between growth rate and different types of infection in children under 5 years on west papua fransiskus aryo pratomo1a 1 faculty of medicine, universitas airlangga a corresponding author: fransiskus_aryo@yahoo.com abstract malnutrition is still a significant problem in the world and in indonesia. among the factors underlying it, the role of growth faltering is often underestimated. considering infection as a factor that affects growth and that indonesia is endemic to various different infectious diseases, to understand its role, a study on infants is conducted using using longitudinal study design in the sumuri district, bintuni bay regency, west papua province. a total of 138 children aged 6 months to 5 years is followed for 6 months in february to august 2014. weight gain data and frequency of infection is collected, with the infections divided into four category of disease: upper respiratory tract infection, skin infection, gastroenteritis, and malaria. these data are gathered by puskesmas daily and monthly records followed by home visit. this study found that the prevalence of malnutrition for the area covered by puskesmas tanah merah is 15.9% for moderate malnutrition and 2.9% for severe malnutrition, with the mean sd value in the beginning of the study -1.15 and at the end of study -1.12, with the difference of sd value calculated as weight gain. total incidence of infections and mean duration of each infection is then compiled and calculated with weight gain data using linear regression method statistical test to understand the difference of role of each infection to weight gain. the result of the study shows that gastroenteritis has a significant negative effect to weight gain and upper respiratory tract infection has a negative effect to weight gain on children in the villages handled by puskesmas tanah merah west papua. keywords: gastroenteritis, upper respiratory tract infection, growth rate, weight gain, malnutrition abstrak malnutrisi masih menjadi masalah yang signifikan di dunia dan di indonesia. di antara faktor-faktor yang mendasarinya, gagal tumbuh sebagai sebuah faktor seringkali masih diremehkan. meperetimbangkan infeksi sebagai faktor yang mempengaruhi pertumbuhan dan bahwa indonesia endemik terhadap berbagai penyakit menular, untuk memahami peran faktor tersebut, penelitian pada anak dilakukan dengan menggunakan desain penelitian longitudinal di distrik sumuri, kabupaten teluk bintuni, provinsi papua barat. sebanyak 138 anak usia 6 bulan sampai 5 tahun diikuti selama 6 bulan pada februari sampai agustus 2014. data penambahan berat badan dan frekuensi infeksi dikumpulkan, dengan infeksi dibagi menjadi empat kategori: infeksi saluran pernafasan bagian atas, infeksi kulit, gastroenteritis, dan malaria. data ini dikumpulkan dengan catatan harian dan bulanan puskesmas yang diikuti dengan kunjungan ke rumah. studi ini menemukan bahwa prevalensi malnutrisi untuk area yang dicakup oleh puskesmas tanah merah adalah 15,9% untuk malnutrisi sedang dan 2,9% untuk malnutrisi berat, dengan nilai sd rata-rata pada awal penelitian -1,15 dan pada akhir penelitian -1,12, dengan selisih nilai sd dihitung sebagai penambahan berat badan. jumlah kejadian infeksi dan durasi rata-rata setiap infeksi kemudian dikompilasi dan dihitung dengan data penambahan berat badan dengan menggunakan metode statistik regresi linier untuk mengetahui perbedaan peran masing-masing infeksi terhadap penambahan berat badan. hasil penelitian menunjukkan bahwa gastroenteritis memiliki efek negatif yang signifikan terhadap kenaikan berat badan dan infeksi saluran pernapasan bagian atas memiliki efek negatif terhadap penambahan berat badan pada anak-anak di desa-desa yang ditangani oleh puskesmas tanah merah papua barat. kata kunci: gastroenteritis, infeksi saluran pernapasan atas, pertumbuhan, penambahan berat badan, malnutrisi 110 indonesian journal of tropical and infectious disease, vol. 6 no. 5 may–august 2017: 109–112 introduction infection is one of the key factors regarding the problem of nutrition.1,2 when an infection happens, the immune system needs various nutrients to maintain immunity against pathogens. various hypothesis has said that frequent infections may make nutritional interventions may not as effective.3 based on riskesdas 2010 data the prevalence of malnutrition in infants in indonesia is still very high at 17,9%.4 due to its place in the tropical region, indonesia is endemic to various different infectious diseases. considering also various factors such as the lower per capita income and low education rates of rural areas of indonesia, this contributes a lot to the persistence of malnutrition in indonesia. malnutrition is closely linked with risks of infection and infection is the leading cause of child mortality. from worldwide data regarding estimated death in children younger than 5 years in 2008, infectious diseases is the leading cause with 68%, with the leading infectious disease being pneumonia (18%) and diarrhea (15%).5 in indonesia, data from riskesdas 2010 tabulates a total of 276 deaths of children aged 1 month to 4 year with the leading disease being diarrhea (25,2%) and pneumonia (15,5%).4 among the key factors identified underlying the higher prevalence of malnutrition in low-income countries is growth faltering.6 association between malnutrition, immunodeficiency, incidence of infections and morbidity in children has been shown by various studies.7-10 however, few studies have tried to link the problem of growth rate itself with the prevalence of different types of infection beside gastroenteritis.11-13 a province in the eastern indonesia, west papua, hold the distinction of having the lowest population density in indonesia.14 the aforementioned low population density makes it replete with problems such as inadequate infrastructure and low economic prowess. in sumuri district, bintuni bay regency of west papua, there are three villages in areas handled by puskesmas tanah merah: tanah merah baru, saengga, and onar village. these villages are relatively isolated, with no land route and a distance of ± 35 kilometers by sea from the regency capital. there are no reliable mass transportation method, a small merchant boat comes around every approximately two weeks carrying with a small boat carrying groceries and other necessities becomes the primary mean of villagers without their own boat for transportation. this makes problems such as inadequacy of food, sanitary, and health needs persistent. by studying the children in the aforementioned villages covered by puskesmas tanah merah, this study attempts to find how much the role of different types of infection affects the growth rate of children in rural papua. the purpose of this study is not to examine the direct effect of infection on malnutrition, since it is a broader topic and of which the relationship has been more established, but to determine whether how much the specific factor of prevalence of infection affects growth rate. between other factors such as the high rate of poverty, low education and inadequate infrastructure, the result of this study may be able to help the policymakers to decide a more effective approach to reducing malnutrition in such regions. material and method subjects and collection methods puskesmas tanah merah is located southwest of the regency capital in a small rural peninsular. it can be considered isolated since there are no land routes and no regular transportation to that area. the sample is all children between 6 months to 5 years in the villages of tanah merah baru, saengga, and onar. the datas are collected between february to august 2014. due to the relatively high growth rate, children under 6 months of age were not included in this study. data of frequency of infection is gathered by puskesmas daily records, combined with home visit and inquiries pertaining to duration of infection. data of nutrition is gathered by posyandu monthly records or home visit records. due to the technical difficulty of calibration of measuring tools, height data is not considered in this study. disease categories diseases are divided into four categories of most common type of infection to aid analysis. the four categories are: 1. upper respiratory tract infection. this covers throat, nose, and ear infections; 2. gastroenteritis, defined by a history of diarrhea without or with vomiting; 3. skin infections, regardless of viral or bacterial cause; 4. malaria, an endemic infection to the region, diagnosed by a positive value on rapid test and confirmed by blood smear examination. statistical analysis from a period of 6 months, the weight data is gathered and the z score of each children during the beginning and the end of the survey is determined. it is then compared by who standard and the difference in z score is considered as weight gain. frequency of infection data in the same interval is then decided by the relative period of disease, gauged by days of sickness divided by total interval duration. the relative effect of each disease is then measured by linear regression analysis of the difference in z score and the frequency of infection of each disease with 95% confidence interval. a less than 0.05 p values is considered significant. data is gathered in libre office calc for windows and analysed using spss 21.0 for windows 111pratomo: relationship between growth rate and different types of infection result and discussion total number of children data gathered is 138 children, with dropout rate of 3.5% due to relocation to other geographic areas. prevalence of malnutrition in the beginning of the study, february 2014, the prevalence of moderate malnutrition, defined by a score of between -2 sd and -3 sd in comparison to standard of who is 20 children (14.5%). the prevalence of severe malnutrition, defined by a score of under -3 sd compared to standard of who is 6 children (4.3%). the mean sd value of all children at the beginning of the survey is -1.15. in the end of the study (august 2014), the prevalence of moderate malnutrition is 22 children (15.9%). the prevalence of severe malnutrition is 4 children (2.9%). the mean sd mean value in the end of the study is -1.12 frequency of infection from the results of the data collection, total incidence and mean duration of each infections were calculated as listed in table 1. relationship between weight gain and infection linear regression analysis is done to calculate the relationship between each type of infection and weight gain, to determine the difference in value of each type to growth rate. the results from table 2 established that gastroenteritis has a significant negative effect (p<0.001) to weight gain and upper respiratory tract infection has a negative effect (p<0.05) to weight gain. this shows that gastroenteritis is a very significant factor concerning the growth rate of children. the most universally used method to determine nutritional status is anthropomometry.15 yet in a limited public health settings it has many limitations. growth rate isn’t well calculated in those settings in service of nutritional interventions. in addition, other key factors such as infection typically are not prioritized for helath interventions because it may not have immediate effects. based on riskesdas 2010,4 the prevalence of malnutrition according to weight for age in indonesia is 13% for moderate malnutrition and 4.9% for severe malnutrition. in west papua province, the prevalence of malnutrition is 17.4% for moderate malnutrition and 6.3% for severe malnutrition. this study founds that the prevalence of puskesmas tanah merah area is 15.9% for moderate malnutrition and 2.9% for severe malnutrition. this shows that the number for moderate malnutrition is higher than national average but lower than provincial average, and the number for severe malnutrition is lower than both provincial and national average. the active role of a local non-govermental organization yayasan sosial agustinus possibly have a big role in this fact, since they are active in giving nutritional interventions for children with severe malnutrition. however, children with less than severe malnutrition doesn’t get as much consideration. based on riskesdas 2010,4 no data is collected about frequency of infection on specific age ranges nor about the average duration of infections. however, from international data and studies, a very strong relationship in both incidence and duration between gastroenteritis and malnutrition has been established.8,10,16,17 in both incidence and the rate of development into more serious infections, a relation has also been established between upper respiratory tract infections and malnutrition.7,9,18 for the specific factor of growth rate, some studies have shown a relationship between growth faltering and gastroenteritis.11-13 a combined analysis of data from nine studies in five countries (bangladesh, brazil, ghana, guinea-bissau and peru) shows that 25% of stunting at 24 months of age was associated with five or more episodes of gastroeneteritis in the first 2 years of life.12 less clear is the relationship between growth rate and respiratory infections. some studies have associated growth faltering and febrile respiratory infections.19,20 no studies were found about these relationship and associations in regard of rural indonesia. the result of this study shows that infections such as upper respiratory tract infections and gastroenteritis especially have a negative effect on weight gain in rural papua. this is in line with previous studies. the resulf of this study doesn’t show that malaria and skin infection to have an effect to weight gain. a possible explanation is because skin infections do not alter dietary intake as much as respiratory infections or gastroenteritis. other studies have demonstrated a relationship between malaria and nutrtion,21,22 but the low number of children in this study table 1. total incidence and mean duration (in days) of each infections category of infection total incidence mean duration (in days) upper respiratory tract infection 188 7.9 gastroenteritis 29 7.2 skin infection 54 12.9 malaria 6 5.7 table 2. regression coefficients of weight gain from linear regression analysis with four category of infections and their p value category of infection regression coefficients of weight gain (-sd difference) p value upper respiratory tract infection -0.184 0.01 gastroenteritis -0.553 0.000 skin infection -0.053 0.448 malaria -0.082 0.205 112 indonesian journal of tropical and infectious disease, vol. 6 no. 5 may–august 2017: 109–112 that is diagnosed with malaria, with a total of 5 makes it hard to show a significant effect. from the result of this study, regarding nutritional status in children, the focus should not be solely on improving the anthropometry status of children, but to also consider the overall health condition, because upper respiratory tract infections and gastroenteritis is caused by many different factors. nutritional intervention programs like dietary supplementation should therefore give more attention to children who have long and/or frequent respiratory infections or gastroenteritis. other than nutritional interventions, methods to control and reduce infection such as education on hygiene, sanitation, water quality, food preparation should not be less prioritized than nutritional intervention to develop an integrated cost-effective and efficient programs to combine all those priorities, it is recommended to do further research on the efficacy and effectiveness of methods that combine strategies of infection control and prevention with nutritional intervention. this should be considered a high priority to solve the problem of malnutrition in indonesia. conclusion gastroenteritis has a significant negative effect to weight gain and upper respiratory tract infection has a negative effect to weight gain on children in the villages handled by puskesmas tanah merah west papua. references 1. keusch gt. the history of nutrition: malnutrition, infection and immunity. the journal of nutrition. 2003 jan 1;133(1):336s-40s. 2. schaible ue, stefan he. malnutrition and infection: complex mechanisms and global impacts. plos medicine. 2007 may 1;4(5):e115. 3. dewey kg, mayers dr. early child growth: how do nutrition and infection interact?. maternal & child nutrition. 2011 oct 1;7(s3):12942. 4. bppk kemenkes ri. riskesdas 2010 dalam angka dan buku. 2010 5. black re, cousens s, johnson hl, lawn je, rudan i, bassani dg, jha p, campbell h, walker cf, cibulskis r, eisele t. global, regional, and national causes of child mortality in 2008: a systematic analysis. the lancet. 2010 jun 11;375(9730):1969-87. 6. lundeen ea, behrman jr, crookston bt, dearden ka, engle p, georgiadis a, penny me, stein ad. growth faltering and recovery in children aged 1–8 years in four low-and middle-income countries: young lives. public health nutrition. 2014 sep;17(9):2131-7. 7. schaible ue, stefan he. malnutrition and infection: complex mechanisms and global impacts. plos medicine. 2007 may 1;4(5):e115. 8. caulfield le, de onis m, blössner m, black re. undernutrition as an underlying cause of child deaths associated with diarrhea, pneumonia, malaria, and measles. the american journal of clinical nutrition. 2004 jul 1;80(1):193-8. 9. cunha al. relationship between acute respiratory infection and malnutrition in children under 5 years of age. acta paediatrica. 2000 may 1;89(5):608-9. 10. caulfield le, de onis m, blössner m, black re. undernutrition as an underlying cause of child deaths associated with diarrhea, pneumonia, malaria, and measles. the american journal of clinical nutrition. 2004 jul 1;80(1):193-8. 11. moore sr, lima aa, conaway mr, schorling jb, soares am, guerrant rl. early childhood diarrhoea and helminthiases associate with long-term linear growth faltering. international journal of epidemiology. 2001 dec 1;30(6):1457-64. 12. checkley w, buckley g, gilman rh, assis am, guerrant rl, morris ss, mølbak k, valentiner-branth p, lanata cf, black re, childhood malnutrition and infection network. multi-country analysis of the effects of diarrhoea on childhood stunting. international journal of epidemiology. 2008 jun 20;37(4):816-30. 13. checkley w, epstein ld, gilman rh, cabrera l, black re. effects of acute diarrhea on linear growth in peruvian children. american journal of epidemiology. 2003 jan 15;157(2):166-75. 14. badan pusat statistik, 2005b. pedoman pengawas dan pemeriksa; survei penduduk antar sensus (supas) 2005. jakarta: bps. 15. world health organization. physical status: the use of and interpretation of anthropometry, report of a who expert committee. 16. wierzba tf, el-yazeed ra, savarino sj, mourad as, rao m, baddour m, el-deen an, naficy ab, clemens jd. the interrelationship of malnutrition and diarrhea in a periurban area outside alexandria, egypt. journal of pediatric gastroenterology and nutrition. 2001 feb 1;32(2):189-96. 17. moore sr, lima nl, soares am, oriá rb, pinkerton rc, barrett lj, guerrant rl, lima aa. prolonged episodes of acute diarrhea reduce growth and increase risk of persistent diarrhea in children. gastroenterology. 2010 oct 31;139(4):1156-64. 18. broor s, pandey rm, ghosh m, maitreyi rs, lodha r, singhal t, kabra sk. risk factors for severe acute lower respiratory tract infection in under-five children. indian pediatrics. 2001 dec 1;38(12):1361-9. 19. weisz a, meuli g, thakwalakwa c, trehan i, maleta k, manary m. the duration of diarrhea and fever is associated with growth faltering in rural malawian children aged 6-18 months. nutrition journal. 2011 mar 20;10(1):25. 20. moffat t. diarrhea, respiratory infections, protozoan gastrointestinal parasites, and child growth in kathmandu, nepal. american journal of physical anthropology. 2003 sep 1;122(1):85-97. 21. ehrhardt s, burchard gd, mantel c, cramer jp, kaiser s, kubo m, otchwemah rn, bienzle u, mockenhaupt fp. malaria, anemia, and malnutrition in african children—defining intervention priorities. the journal of infectious diseases. 2006 jul 1;194(1):108-14. 22. caulfield le, richard sa, black re. undernutrition as an underlying cause of malaria morbidity and mortality in children less than five years old. the american journal of tropical medicine and hygiene. 2004 aug 1;71(2_suppl):55-63. 147 vol. 5. no. 6 september–december 2015 differences of universal and multiplex primer for detection of dengue virus from patients suspected dengue hemorrhagic fever (dhf) in surabaya arif nur muhammad ansori1, teguh hari sucipto2, pemta tia deka2, nur laila fitriati ahwanah2, siti churrotin2, tomohiro kotaki3,soegeng soegijanto2 1 biology department, faculty of science and technology, universitas airlangga 2 dengue study group, institute of tropical disease, universitas airlangga 3 center for infectious disease, kobe university graduate school of medicine, japan abstract dengue hemorrhagic fever (dhf) is a global health problem in tropical and subtropical regions, as well as endemic in110 countries around the world. indonesia is one of the largest countries in the region of endemic dengue. in indonesia, dengue virus infection has become a contagious disease that was very important and was reported in 1968. many molecular epidemiological approaches have been developed to look for factor that has been assumed as the cause of the increase of prevalence dengue virus infection in the world. the aim of this study is for the detection and determination of serotype of dengue virus in surabaya. the method used was the technique of reverse transcription-polymerase chain reaction (rt-pcr) and polymerase chain reaction (pcr) with specific primers for dengue virus. samples suspected dhf patients were obtained from various health center and hospital in surabaya. results of this research detected negative result for dengue virus in all samples of patients suspected dhf. negative results caused by dengue virus titers in serum samples of patients who had been dropped due to long storage time and taken after the third day of fever in early period. key words: dengue hemorrhagic fever, pcr, rt-pcr, dengue virus abstrak demam berdarah dengue (dbd) merupakan permasalahan kesehatan global di daerah tropis dan subtropis, serta penyakit endemik di 110 negara di dunia. indonesia adalah salah satu negara terbesar di wilayah endemik dbd. di indonesia, infeksi virus dengue telah menjadi penyakit menular yang sangat penting dan dilaporkan pada tahun 1968. banyak pendekatan epidemiologi molekuler telah dikembangkan untuk mencari faktor-faktor yang telah dianggap sebagai penyebab meningkatnya infeksi virus dengue prevalensi di dunia. penelitian ini bertujuan untuk deteksi dan penentuan serotipe virus dengue di kota surabaya. metode yang digunakan adalah teknik reverse transcription polymerase chain reaction (rt-pcr) dan polymerase chain reaction (pcr) dengan primer spesifik untuk virus dengue. sampel pasien diduga dhf didapatkan dari berbagai puskesmas dan rumah sakit di kota surabaya. hasil penelitian terdeteksi negatif virus dengue pada semua sampel pasien yang diduga dhf. hasil negatif yang disebabkan oleh titer virus dengue dalam sampel serum dari pasien yang telah turun karena waktu penyimpanan yang lama dan diambil setelah hari ketiga demam pada periode awal. kata kunci: dengue hemorrhagic fever, pcr, rt-pcr, surabaya, virus dengue research report introduction dengue hemorrhagic fever (dhf) is a global health problem in tropical and subtropical regions, also endemic diseases in 110 countries around the world.1, 2, 3, 4 indonesia is one of the largest country in dengue endemic regions, with a population reaching 251 million people.5 dhf is caused by the dengue virus and transmitted through 148 indonesian journal of tropical and infectious disease, vol. 5. no. 6 september–december 2015: 147-151 mosquito vector aedes aegypti and aedes albopictus.6 in 2010, indonesia became the first ranked country in asean by the highest number of cases in dhf and hyperendemic predicate.7 t h e d e n g u e v i r u s i s t h e f l a v i v i r i d a e f a m i l y members and genus flavivirus. consists of four dengue virus serotypes (denv-1, denv-2, denv-3 and denv4).8,9,10 dengue virus serotype denv-1 was first discovered in hawaii in 1944 and denv-2 in papua new guinea in the same year. dengue virus serotype denv-3 and denv4 was found at philippines in 1956.11 dengue virus is transmitted to humans by the mosquito aedes aegypti and aedes albopticus.12 in indonesia, dengue virus infection has become a contagious disease that was very important and was reported in 1968.13,14 many molecular epidemiological approach have been developed to look for factors that has been assumed as the cause of the increase of prevalence dengue virus infection in the world. that ranges of strains in serotypes classified in different groups can genetically sequencing revealed the dengue virus. differences of nucleotide caused biological diversity in nature and their antigenicity.15,16 the aim of this study is for detection and determination of serotype of dengue virus in surabaya. dengue virus serotypes that have been known can be compared with previous studies, so the movement of dengue virus serotypes could be discovered. material and methods samples serum samplesof patients suspected of dengue put in eppendorf tubes and storedina refrigerator at a temperature -80ºc in dengue laboratory, institute of tropical disease airlangga univesity. extraction of dengue virus ribonucleic acid(rna) ribonucleic acid or rna of dengue virus extracted from serum samples with extraction device qiaamp viral rna mini kit (qiagen), following a step works: put 560 μl of buffer avl and carrier rna in a 1.5 ml eppendorf tube, put1 40 μl of serum samples and vortex for 15 seconds, incubation at room temperature for 10 minutes and then centrifuge, added 560 μl of ethanol 96-100% then vortex for 15seconds and centrifuged, put 630μl of solution to the qiaamp mini coloumn, centrifuged at 8000 rpm for 1 minute, the remaining solution was included to qiaamp mini coloumn, centrifuged at 8000 rpm for 1 minute, put 500 μl of buffer aw1, centrifuged at 8000 rpm for 1 minute, put 500 μl of buffer aw2, centrifuged at 8000 rpm for 1min, transferred to a 1.5 ml microtube, added 60 μl of buffer ave, incubated at room temperature for 1 minute, centrifuged at 8000 rpm for 1 minute and showed rna. reverse transcription-polymerase chain reaction (rt-pcr) and polymerase chain reaction (pcr) for the detection and determination of dengue virus serotype molecular examination using rt-pcr and pcr for detecting nucleic acid of dengue virus sample and also for testing dengue virus serotypes is known as serotyping. the function of rt-pcr is to transcript the rna into cdna, then thecdnawas amplified bypcr. primers used for serotyping is d1 (forward), ts1, ts2, ts3 and ts4 (reverse). in the process of rt-pcr was performed with 3 stages: the first stage component of reagents consists of 1 μl of primer, 5 μl of rna, 1 μl of dntp, and 7 μl of water; the second stage components of reagents consists of 4μlof 5x fs buffer, 1 μl of dtt, 0.5 μl of water, 0.5 μlof rnase out, and 0.5 μlof superscript; the third stage was added 0.5 μl rnaseh. the first stage of the rt-pcr reaction carried out at a temperature of 65 °c for 5 minutes, the second stage carried out at a temperature of 55 °c for 50 minutes and 85 °c for 5 minutes, and the third stageincubated at a temperature of 37 °c for 20 minutes. in the process of pcr, component of reagents consists of 5 μlof cdna, 2 μl of 10x pcr buffer, 2 μlof dntp, 2 μl of primer, 0.1 μl of rtaq, and 9 μl of water. pcr reactions were performed as many as 30 to 40 cycles of pcr, the temperature was 94 °c for 4 minutes (pre-denaturation), 94 °c for 1 minute (denaturation), 50 °c for 1 minute (annealing), 72 °c for 12 minutes (extension) and 4 ºc. temperature 94 °c for 5 minutes in a pcr reaction aiming to denature doublestranded dna, so dna into single strands. pcr products were analyzed by electrophoresis on a 1.5% agarose gel and staining is done with ethidium bromide. marker used is a 100 bp ladder. on the implementation of the detection table 1. primer oligonucleotides used for amplification and determination dengue virus serotypes primer sequences d1 5’-tcaatatgctgaaacgcgcgagaaaccg-3’ ts1 5’-cgtctcagtgatccggggg-3’ ts2 5’-cgccacaagggccatgaacag-3’ ts3 5’-taacatcatcatgagacagagc-3’ ts4 5’-ctctgttgtcttaaacaagaga-3’ according lanciotti et al. (1992) [17] and harris et al. (1998) [9], dengue virus serotype determination was based on the size of dna band formed after visualization on agarose gel electrophoresis, 482 bp for denv-1, 119 bp for denv-2, 290 bp for denv-3, and 389 bp for denv-4. 149ansori, et al.: differences of universal and multiplex primer for detection of dengue virus reaction and serotyping, always included a positive comparison (positive control dengue virus). additionally, in this studyuseda specific primer for flavi virus, which cfd2, mamd, and fs778. the process of rt-pcr with specific primers for flavi virus was done in 3 stages: the first stage component of reagent sconsists of 1 μl of cfd2 primer, 5 μl of rna, 1 μl of dntp, and 7 μl ofwater; the second stage components of reagent sconsists of 4 μl of 5 xfs buffer, 1 μl of dtt, 0.5 μl of water, 0.5 μl of rnase out, and 0.5 μl of superscript; the third stage was added 0.5 μl of rnaseh. the first stage of the rt-pcr reaction carried out at a temperature of 65°c for 5 minutes, the second stage carried out ata temperature of 55°c for 50 minutes and 85°c for 5 minutes, and the third stage incubated at a temperature of 37°c for 20 minutes. in the process of pcr, component of reagents consists of 5 μl of cdna, 2 μl of 10x pcr buffer, 2 μl of dntp, 2 μl of primer (1 μl of cfd2 and 1 μlof mamd), 0.1 μl rtaq, and 9 μl of water. pcr reactions were performed by 25 cycles of pcr, the temperature was 94°c for 4 minutes (pre-denaturation), 94 °c for 1 minute (denaturation), 54°c for 1 minute (annealing), 72°c for 1 minute (extension), and 72ºc for 10 minutes (extension). next process was heminested-pcr, component of reagents consists of 5 μl of dna,2 μl pcr buffer 10x, 2 μl of dntp, 2 μl primer (1 μl of cfd2 and 1 μl of fs778), 0.1 μlof rtaq, and 9 μl of water. heminested-pcr was performed by 25 cycles of pcr, 94°c for 2 minutes (pre-denaturation), 94°c for 30 seconds (denaturation), 55°c for 30 seconds (annealing), 60°c for 4 minutes (extension), and 60°c for 10 minutes (extension). heminested-pcr products were analyzed by electrophoresis method on a 1.5% agarose gel and staining is done with ethidium bromide. marker used is a 100 bp ladder. result and discussion reverse transcription-polymerase chain reaction or rtpcr and polymerase chain reaction or pcr for detection of dengue virus performed on serum samples of patients suspected of dhf were taken from medokan ayu health center, manukan kulon health center, pacar keling health center, tenggilis health center, krembangan selatan health center,and soerya hospital child and maternity. this method, obtained negative results for all samples. negative results caused by dengue virus titers in serum samples of patients who had been dropped due to long storage time and taken after the third day of feverin early period. in figure 1 was a serum samples obtained from soerya hospital child and maternity. the results obtained through out the sample is negative, there is no dengue virus dna bands appearing. however, the positive control dengue virus dna bands appear. positive control was used the dengue virus serotype denv-2 (119 bp). in figure 2 was a serum sample obtained from medokan ayu health center, manukan kulon health center, pacar keling health center, tenggilis health center, and krembangan selatan health center. the negative results of the samples based on the condition that there is no dna table 2. primer oligonucleotides used for detection of flavivirus group primer sequences cfd2 5’-gtgtcccagccggcggtgtcatcagc-3’ mamd 5’-aacatgatgggraaragrgaraa-3’ fs778 5’-aargghagymcdgchathtggt-3’ figure 1.results of electrophoresis of pcr samples obtained from soerya hospital child and maternity with primer d1, ts1, ts2, ts3, ts4. m: marker; 1: positive control denv-2; 2-6: samples. figure 2.results of electrophoresis of pcr samples with primer cfd2, mamd, and fs778.m: marker; 1: medokan ayu health center; 2: manukan health center; 3: pacar keling health center; 4: tenggilis health center; 5: krembangan selatan health center; 6: manukan health center; 7: positive control; 8: positive control. discussion in addition, dengue virus consists of four serotypes (denv-1, denv-2, denv-3 and denv-4) [8, 9, 10]. in indonesia gained dominance serotype denv-2, followed denv-3 in 2003 to 2005 [18]. in indonesia, the four serotypes of dengue virus have been discovered but denv-3 is often associated with severe dengue cases [19, 20]. in surabaya in 2005 was dominated serotype denv-2, followed by denv-3 and denv1. in surabaya in 2008-2009 was also dominated by denv-2 [21]. according aryati et. al. (2012) [22], in surabaya was predominant by denv-1, followed by denv-2, denv-4, and denv-3. detection of dengue virus serotypes is very important because secondary infection with a different serotype may impact more severe. likewise, an infection caused by two serotypes or more in a single individual (double infection) can contribute to the severity of the infection. the serotyping is very important in the management of patients with dengue virus infection [23, 24]. virus serotype can be demonstrated by molecular techniques such as pcrandrtpcr. this is very important because it changes serotypes causing an indication of the threat of dengue fever in this population [25]. unavailability of vaccines or antiviral drugs for the prevention of dengue virus infection is the cause of the development of research based surveillance systemics needed inearly warning (early warning) dbd. such information can be used as a preventive measure and alert in preparation foran outbreak of dengue. early warning is given each year prior to the extraordinary incident in dengue. societies can play an active role in efforts to combat the vector which is an important factor for breaking the chain of transmission and prevention of dengue disease that reappeared in the future. conclusion 119 bp 200 bp bp figure 1. results of electrophoresis of pcr samples obtained from soerya hospital child and maternity with primer d1, ts1, ts2, ts3, ts4. m: marker; 1: positive control denv-2; 2-6: samples. figure 1.results of electrophoresis of pcr samples obtained from soerya hospital child and maternity with primer d1, ts1, ts2, ts3, ts4. m: marker; 1: positive control denv-2; 2-6: samples. figure 2.results of electrophoresis of pcr samples with primer cfd2, mamd, and fs778.m: marker; 1: medokan ayu health center; 2: manukan health center; 3: pacar keling health center; 4: tenggilis health center; 5: krembangan selatan health center; 6: manukan health center; 7: positive control; 8: positive control. discussion in addition, dengue virus consists of four serotypes (denv-1, denv-2, denv-3 and denv-4) [8, 9, 10]. in indonesia gained dominance serotype denv-2, followed denv-3 in 2003 to 2005 [18]. in indonesia, the four serotypes of dengue virus have been discovered but denv-3 is often associated with severe dengue cases [19, 20]. in surabaya in 2005 was dominated serotype denv-2, followed by denv-3 and denv1. in surabaya in 2008-2009 was also dominated by denv-2 [21]. according aryati et. al. (2012) [22], in surabaya was predominant by denv-1, followed by denv-2, denv-4, and denv-3. detection of dengue virus serotypes is very important because secondary infection with a different serotype may impact more severe. likewise, an infection caused by two serotypes or more in a single individual (double infection) can contribute to the severity of the infection. the serotyping is very important in the management of patients with dengue virus infection [23, 24]. virus serotype can be demonstrated by molecular techniques such as pcrandrtpcr. this is very important because it changes serotypes causing an indication of the threat of dengue fever in this population [25]. unavailability of vaccines or antiviral drugs for the prevention of dengue virus infection is the cause of the development of research based surveillance systemics needed inearly warning (early warning) dbd. such information can be used as a preventive measure and alert in preparation foran outbreak of dengue. early warning is given each year prior to the extraordinary incident in dengue. societies can play an active role in efforts to combat the vector which is an important factor for breaking the chain of transmission and prevention of dengue disease that reappeared in the future. conclusion 119 bp 200 bp bp figure 2. results of electrophoresis of pcr samples with primer cfd2, mamd, and fs778. m: marker; 1: medokan ayu health center; 2: manukan health center; 3: pacar keling health center; 4: tenggilis health center; 5: krembangan selatan health center; 6: manukan health center; 7: positive control; 8: positive control. 150 indonesian journal of tropical and infectious disease, vol. 5. no. 6 september–december 2015: 147-151 band of dengue virus appearing. negative results caused by dengue virus titers in serum samples of patients who had been dropped due to long storage time and taken after the third day of fever in early period. however, the positive control of dengue virus dna bands appear at about 200 bp position. primers used were primer for flavi virus, which are cfd2, mamd, and fs778. primer of flavi virus used to detect a group of viruses belonging to the genus flavivirus. in addition, dengue virus consists of four serotypes (denv-1, denv-2, denv-3 and denv-4).8,9,10 in indonesia gained dominance serotype denv-2, followed denv-3 in 2003 to 2005.18 in indonesia, the four serotypes of dengue virus have been discovered but denv-3 is often associated with severe dengue cases.19,20 in surabaya in 2005 was dominated serotype denv-2, followed by denv-3 and denv-1. in surabaya in 2008-2009 was also dominated by denv-2.21 according aryati et. al. (2012),22 in surabaya dominated by denv-1, followed by denv-2, denv-4, and denv-3. detection of dengue virus serotypes is very important because secondary infection with a different serotype may impact more severe. likewise, an infection caused by two serotypes or more in a single individual (double infection) can contribute to the severity of the infection. then serotyping very important in the management of patients with dengue virus infection.23,24 virus serotype can be demonstrated by molecular techniques such as pcrandrt-pcr. this is very important because it changes serotypes causing an indication of the threat of dengue fever in this population.25 unavailability of vaccines or antiviral drugs for the prevention of dengue virus infection is the cause of the development of research based surveillance system is needed in early warning (early warning) dbd. such information can be used as apreventive measure and alert in preparation for an outbreak of dengue. early warning is given each year prior to the extraordinary incident in dengue. societies can play an active role in efforts to combat the vector which is an important factor for breaking the chain of transmission and prevention of dengue disease that reappeared in the future. conclusion in this study, all samples of patients suspected of dengue hemorrhagic fever (dhf) which is obtained from various health center and hospital in surabaya detected negative and dengue virus serotype can’t be known. acknowledgements thanks to institute of tropical disease airlangga univesity for research internship opportunity in the dengue laboratory, institute of tropical disease airlangga university. references 1. pinheiro fp, corber sj. 1997. global situation of dengue and dengue haemorrhagic fever and its emergence in the americas. world health stat. quart, 50, 161–169. 2. gubler dj. 1998. dengue and dengue hemorrhagic fever. clin. microbiol. rev. 11, 480–496. 3. lindegren g, vene s, lundkvist a, falk kl. 2005. optimized diagnosis of acute dengue fever in swedish travelers by a combination of reverse transcription-pcr and immunoglobulin m detection. j. clin. microbiol. 43, 2850–2855. 4. ranjit s, kissoon n. 2011. dengue hemorrhagic fever and shock syndromes. pediatr. crit. care med. 2011 jan; 12(1): 90–100. 5. karyanti mr, uiterwaal c.s.p.m, kusriastuti r, hadinegoro sr, rovers mm, heesterbeek h, hoes aw, bruijning-verhagen p. 2014. the changing incidence of dengue haemorrhagic fever in indonesia: a 45-year registry-based analysis. bmc inf. dis., 14: 412. 6. rahayu df, ustiawan a. 2013. identifikasi aedes aegypti dan aedes albopictus. balaba, j. lit. peng. peny. ber. bin. banjarnegara 9(1), juni 2013, 7–10. 7. rehatta nm, hasan h, setyoningrum ra, andajani s, ida r, umijati s, mertaniasih nm, retnowati e, yotopranoto. 2013. pedoman survei penyakit tropis. surabaya: airlangga university press. 8. trent dw, manske cl, fox ge, chu mc, kliks sc., monath tp. 1990. the molecular epidemiology of dengue viruses. appl. virol. res. 2, 293–315. 9. harris e, roberts tg, smith l, selle j, kramer ld, valle s, sandoval e, balmaseda a. 1998. typing of dengue viruses in clinical specimens and mosquitoes by single-tube multiplex reverse transcriptase pcr. j. clin. microbiol.; 36(9): 2634–9. 10. yong yk, thayan r, chong ht, tan c.t, sekaran sd. 2007. rapid detection and serotyping of dengue virus by multiplex rt-pcr and real-time syber green rt-pcr. sing. med. j. 48, 662. 11. ananthanarayan r, paniker ckj. 2000. textbook of microbiology. 6th ed. pp. 487–491. orient longman, hyderabad. 12. kristina, isminah, wulandari l. 2004. kajian kesehatan demam berdarah dengue. jakarta: badan penelitian dan pengembangan kesehatan departemen kesehatan. 13. soedarmo sp. 1995. demam berdarah dengue. medika. 10, 798–808. 14. hadinegoro srh, soegijanto s, wuryadi s, dan suroso t. 2006. tata laksana demam berdarah dengue di indonesia. departemen kesehatan republik indonesia, direktorat jenderal pengendalian penyakit dan penyehatan lingkungan, jakarta. 15. salda ltd, parquet mdc, matias rr, natividad ff, kobayashi n, morita k. 2005. molecular epidemiology of dengue 2 viruses in the philippines: genotype shift and local evolution. am. j. trop. med. hyg. 2005; 73(4): 796–802. 16. a n o o p m , i s s a c a , m a t h e w t , p h i l l i p s , k a r e e m n a , unnikrishnan r, sreekumar e. 2010. genetic characterization of dengue virus serotypes causing concurrent infection in an outbreak in ernakulam, kerala, south india. indian j. exp. biol. 48: 849–57. 17. lanciotti rs, calisher ch, gubler dj, chang gj, vorndam av. 1992. rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase polymerase chain reaction. j. clin. microbiol; 30(3): 545–51. 18. aryati, soetjipto, hariadhi s, rantam fa, soegijanto s. 2006. profile serotype virus dengue di indonesia tahun 2003–2005. maj. ked. trop. ind. (mkti). 17(1): 72–80. 19. rice cm, lenches em, eddy sr, shin sj, sheet rl, strauss jh. 1985. nucleotide sequence of yellowever virus: implication for flavivirus gene expression and evolution. science 229: 726–733. 20. juniarti f, irawan d, subintoro, wahyunita k, rudiyanto a, malik c, narita v. 2012. optimasi produksi protein non struktural 1 (ns1) virus dengue serotipe 3 (denv-3). prosiding insinas 2012: 5–12. 21. aryati, wardhani p. 2010. profil virus dengue di surabaya tahun 2008–2009. ind. j. of clin. path. and med. lab. (ijcp & ml). 17(1): 21–24. 151ansori, et al.: differences of universal and multiplex primer for detection of dengue virus 22. aryati, wardhani p, yohan b, aksono eb, sasmono rt. 2012. distribusi serotipe dengue di surabaya tahun 2012. ind. j. of clin. path. and med. lab. (ijcp & ml) 2012; 19(1): 41–44. 23. saxena p, dash pk, santhosh sr, shrivastava a, parida m, rao p.v.l. 2008. development and evaluation of one step single tube multiplex rt-pcr for rapid detection and typing of dengue viruses. j. of virol. 5(20). 24. sarkar a, taraphdar d, chatterjee s. 2012. molecular typing of dengue virus circulating in kolkata, india in 2010. j. trop med; 2012: 960329. 25. chung yk, fung yp. 2002. dengue virus infection rate in field of population of female aedes aegypti and aedes albopictus in singapore. trop. med. & int. heal., 7: 32. 6 vol. 7 no. 1 january–april 2018 the difference of map1lc3 level as macrophage autophagy marker between resistant and sensitive tuberculosis patients on rifampicin dian novita w1a, jusak nugraha2, soedarsono3 1 master of immunology, faculty of pasca sarjana, universitas airlangga 2 clinical patology, dr. soetomo teaching hospital 3 pulmonology & respiratory, dr. soetomo teaching hospital a corresponding author: diannovii13@gmail.com abstract mycobacterium tuberculosis (mtb) is an intracelular bacteria that live in the host macrophage cells. several organs can be affected by tuberculosis but most major illnesses are lung diseases. immediately after infection, mtb will be phagocytosed by the alveolar macrophage cells and can survive in the phagosome. the macrophage plays a role in innate immunity towards an infection using autophagy by removing the microbe directly via phagocytosis. when bacteria phagocytosized, vacuole membrane formed double membranes called autophagosome, and followed by degradation by lysosome, which known as autolysosome. induction of autophagy can be observed on the formation of microtubule-associated proteins 1b lightchain 3b (map1lc3b/lc3). map1lc3b is protein that have role at autophagic way for selection autophagy substrate and biogenesis. in this study we are used serum from patients tb with rifampicin resistant and rifampicin sensitive as control. samples were divided using gene expert to differentiate between resistant and sensitive rifampicin.this research aims to compare map1lc3b levels in resistant and sensitive rifampicin to study macrophages respond in autophagic way in tuberculosis patients, and give information for define therapy plan to improve therapy for mdr-tb patients. type of this research is a case control study design with cross sectional research with each groups sample is 19 from age 18-65 years old. result, map1lc3b serum levels on the rifampicin resistant group are lower compared to rifampicin sensitive group. this occur because mtb is able to hide and evade innate immune defense mechanisms. mtb can maintain intracellular growth inside the phagosome by inhibiting phagolysosome formation in autophagy process especially inhibit map1lc3b formation by pdim. keywords: mycobacterium tuberculosis, drug resistance, rifampicin, autophagy, map1lc3b abstrak mycobacterium tuberculosis (mtb) adalah bakteri intraseluler yang hidup dalam makrofag pada sel inang. beberapa organ dapat dipengaruhi oleh tuberkulosis tetapi yang paling utama adalah penyakit paru. segera setelah terjadi infeksi, kuman tb akan difagositosis oleh sel makrofag alveolar dan tetap bertahan hidup dalam fagosom. makrofag mempunyai peranan penting dalam respon imun bawaan terhadap infeksi melalui autofagi dengan mengeliminasi bakteri secara langsung dengan cara fagositosis. ketika bakteri di fagositosis membran vakuola membentuk dua lapisan membran yang disebut dengan autofagosom dan didegradasi oleh lisosom, yang biasa dikenal dengan autolisosom. induksi autofagi dapat dipantau pada pembentukan formasi microtubule-associated protein 1b light chain 3b (map1lc3b/lc3). map1lc3b adalah protein yang mempunyai peranan pada jalur autofagi untuk seleksi subrat dan biogenesis. penelitian ini menggunakan serum darah pasien tb yang resisten dan sensitif rifampisin sebagai kontrol. sampel resisten dan sensitive dibedakan menggunakan tes gen expert. penelitian ini bertujuan untuk membandingkan kadar map1lc3b pada resisten dan sensitif rifampisin untuk mempelajari autofagi makrofag pada pasien tuberkulosis dan memberikan informasi untuk meningkatkan terapi pada pasien mdr-tb. jenis penelitian ini adalah case control study dengan rancangan penelitian cross sectional dengan besar sampel tiap kelompok sebesar 19 dengan rentang umur 18-65 tahun. hasilnya, kadar map1lc3b pada kelompok resisten rifampisin research report 7novita, et al.: the difference of map1lc3 level as macrophage autophagy marker memiliki kadar lebih rendah dibandingkan dengan kelompok sensitif.hal ini disebabkan karena mtb dapat menghindari sistem pertahanan respon imun bawaan. mtb dapat mempertahankan pertumbuhan intraseluler di dalam fagosom dengan menginhibisi formasi fagolisosom pada proses autofagi terutama menghambat pembentukan map1lc3b oleh pdim. kata kunci: mycobacterium tuberculosis, resisten obat, rifampisin, autofagi, map1lc3b introduction mycobacterium tuberculosis (mtb) can cause a dangerous disease called tuberculosis (tb). this microbacteria can attack various organs, mostly the lungs. the tb infection can spread from coughing or sneezing which allows mtb to enter the body along with dusts or droplets.1 there are 6 countries with the world’s largest tb disease spread: south africa, nigeria, china, pakistan, india and indonesia. mtb can evolve its resistance against antimicrobial drugs. there is a type of tb called multidrug-resistant tb (mdr-tb) which cannot be treated by at least with two of the potent first line anti-tb drugs like isoniazid and rifampicin. to improve detection of the case and treatment for mdr-tb, any further research is needed. there are 300,000 cases of mdr-tb patients that were estimated in 2013. around 45% cases from them were detected among all pulmonary tb in the world while around 5% of cases of mdr-tb that are not detected or not managed outside the national tb programs were not reported.2 comparative genomic analyses drug resistance on mtb can be caused by 3 things, they are chromosomal mutations that required for the action of antibiotics, gene that encodes the protein targets of drugs applied, or enzymes that are required to activate pro-drug. the target of antibiotics is important to cell function. resistant mutations encode gene target will affect pathogenesis.3 in every 106 to 108 replications, wild strains of mtb will undergo spontaneous mutations that confer resistance to a single drug, mutations variety to antibiotic shown at table 1. tb therapy with fast onset needs rifampicin (rif) as critical component of first-line therapy.4 almost 90% of rif resistant strains are also resist to isoniazid. rif resistant is used as subtitution marker for detecting mdr tb.5 rif resistant is caused by mutation of a single nucleotidesubstitution on rpob region. in this mutation process, table 1. mutations in antibiotic1 drug average mutation rate isoniazid 2.56 x 10-6 rifampicin 2.25x 10-10 ethambutol 1 x 10-7 streptomycin 2.95 x 10-8 pyrazinamide 1 x 10-3 the gene encodes the β-subunit of rna polymerase into dna-dependent (rnap).6 transcription of the rnap from the mutations of rpob in the gene has some effects toward physiology of the mtb. mutations in this site can cause secondary mutations which lead resistance to another antibiotic.7 autophagy is a complex process involving multiple protein that consist of complex formation and initiation of double membrane development phagophore as nucleation, elongation of the membrane and completion of autophagosome vesicles surround the cargo, and then they will fuse with lysosome (figure 1). lysosome is contained hydrolase that can degrade and dispose component.8 mtb persist and multiply within infected macrophage, where it resides in host-derived phagosome which fails to fuse with lysosom.9 autophagy is caused by metabolic and immune signals consists of recognition of pathogen and stimulation by pro-inflammatory cytokines. autophagy trigger microtubule-associated proteins 1b light chain 3b (map1lc3b/lc3), a protein encoded by the gene map1lc3b in humans.10 lc3 was first identified as a protein co-purified with microtubule-associated protein 1a and 1b from rat brains. this protein is derived from 28% of amino acids with apg8/aut7p who plays a role in autophagy in yeast, undergoes complex c-terminal proteolitic and lipid (phosphathydil ethanolamine) modifications, which is translocate from cytosol to the autophagosomal membrane.11 map1lc3b functions are for biogenesis, autophagy and substrate selection autophagosome.10 if mtb resistance to rifampicin, it physiology change, map1lc3b could not form autophagosome vesicles so elimination of bacteria with autophagy process not formed, result mtb survive inside body. this research is conducted to analyze the differences between the map3lc1b level in tuberculosis patient with sensitive and resistant rifampicin where this protein used as autophagy marker from macrophage. figure 1. autophagy process 8 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 6–10 material and method a retrospective cross-sectional study was conducted from may 2017 to september 2017 at the dr. soetomo general hospital. samples are used are serum from tuberculosis patients who visited dr. soetomo general hospital during study period. when patients coming they have blood tested and fill information for medical record. patients are divided into sensitive and resistant rifampicin using gene expert test, and sample used were patients that meet the inclusion and exclusion criteria based on medical record. based on the who (2013) the proportion value of tb with mdr was 4% of new tb cases, so the number of samples obtained were 19 sensitive (as control) and 19 resistant. normal groups were used as map1lc3b baseline. after all samples collected, samples are processed by elisa. these were diluted and decontaminated, and map1lc3b kit performed according to the manual of manufacturer. result were analyzed using one-way annova p<0.05, and comparisons between groups using tukey. result and discussion results are obtained as concentration levels of map1lc3b (ng/ml) which showed at table 2 and analyzed with a value of p<0.05 (table 3). table 2. map1lc3b concentration (ng/ml) normal sensitive resistance 1.061 2.291 0.136 1.418 0.866 0.475 1.537 0.983 0.321 1.753 3.268 3.473 1.978 0.482 1.067 2.435 2.268 0.435 2.45 0.684 0.402 2.504 1.072 0.776 2.538 3.012 0.796 3.812 0.526 0.949 0.345 0.512 1.432 1.637 0.381 1.828 1.584 1.435 2.35 0.954 4.137 0.529 2.033 1.505 1.973 0.59 2.156 0 table 3. comparison between groups (ng/ml) groups mean significant p val. normal vs. sensitive 0.4727 no 0.3908 normal vs. resistant 1.211 yes 0.0042 sensitive vs. resistant 0.7381 yes 0.0434 based on the graphic (figure 2), anti-tb resistant group have map1lc3b level lower than sensitive group. the highest mean value from highest to the lowest are the normal group (2.1486), sensitive group (1.6759), and resistant groups (0.9378). no rm al se ns itiv e res ist an t 0 1 2 3 4 5 groups m a p 1l c 3b le ve ls ( ng /m l) figure 2. map1lc3b levels comparison for group (n normal=10; n sensitive=19; and n resistant=19) each macrophages are important fundamental for host defense system with phagocytic cells i.e neutrophil and monocyte which recognize and eradicate pathogenic bacteria. pathogen are destroyed by macrophages directly or indirectly through the innate and adaptive immune system.12 macrophages are target for bacterial pathogens that also can give an advantage for bacteria to evade the immune system.2 phagocytosis is an ingestion of antigens that are large into membrane vacuole commonly known as the phagosome.13 autophagy is isolated cargo into the membrane with double structure commonly referred by autophagosome.14 induction of autophagy can be monitored by map1lc3b (lc3) formation.14 to survive inside macrophages, intracellular bacteria develop a variety of strategies to avoid or fight the host defense system.12 in this case, mtb has the ability to hold phagosome maturation.15 autophagy can act as a tumor suppressor in normal cells based on the efficiency of non-apoptotic cell death from malignant cells and dna damage by inhibiting ros formation.8 antimicrobial activity and apoptosis of human macrophages can be triggered by cytosolic phospholipase 9novita, et al.: the difference of map1lc3 level as macrophage autophagy marker activity through mtb which catalyze the release of arachidonic acid. arachidonic acid is product of a second messenger of tnf which induce apoptosis and oxygen radicals, which are produced during arachidonic acid lipoxygenation, thus inducing the production of reactive oxidative species and are involved in cell death.16 bacteria that are resistant to drugs is a threat to human health. resistant to antibiotics can be against two things: bacterial survival ability and the ability to reproduce in the presence of macrophages. when bacteria enter the macrophages, they will experience environmental stress such as nutritional restriction induced by the host, acidification, toxic peptides, osmotic stress, and reactive oxygen species (ros), is later became the biggest cause the death of the bacteria.17 to survive inside macrophages, mtb developed a variety of strategies to avoid or fight the host defense system.12 one of the mechanisms of mtb to survive is manipulating the host cell death pathways in infected cells. one of the virulence factors are surface glycolipid pdim (phthiocerol dimycocerosates).18 lipid is not directly genetically encoded and therefore is not amenable to traditional tagging methods, also cell wall lipids have multiple overlapping functions.17 multiple role functions from pdim on pathogenesis has been investigated before, including the invasion of macrophages, masking of pathogen-associated molecular pattern (pamps), resistance to death with nitric oxide, and the prevention of the recruitment of active macrophages to infected area.1 pdim suppress recruitment of microbicidal, inos positive macrophages by inhibiting tlr signaling (figure 3).19 interactions between host and bacterial cell wall are likely to be bidirectional and change when infection.2 pdim in vivo18 abundance depend on expression of bisynthetic enzymes which decrease upon macrophage infection, shift metabolic flux which occur during host lipid catabolism9 and insertion of molecule into host membranes20. there is variable amount of pdim on mtb surface is at different time points after infection.19 mtb initiated human infections in distal lung, and reside in upper respiratory tract. tlr signalling stimulated by pamps from lung overrides pdim and pgl-mediated immune evasion.2 there is site named resistancedetermining region (rrdr)18 that caused by mutations in mtb strains at 81-bp region of rpob. this mutations result is high levels of resistance to rifampicin. according to comas21 all laboratory-generated mutans of mtb with rifampicin resistant mutations in the rrdr reduced fitness compared to their respond for drug ancestors when without rifampicin13, mtb with rif resistant caused by mutations in the rpob gene, where the majority is on codon 531 and 52611. according to kawamura mutation in codon 526 related to oxidative stress sensitivity.22 in addition, some reports say that just one gene mutations in the rpob encodes in sub-unit of rna polymerase β can cause interaction between the rna polymerase and some promoter also transcription regulation that trigger changes in phenotype.17 the mechanism of the rpob gene mutation is caused by resistant rifampicin indicates that specific lead to mutations in the rpob changes aspects of transcription. these transcription factors causing changes in gene expression which encodes the protein secretion, and proteomic changes produce some enzymes and lipid biosynthetic of intermediate in the path of phthiocerol dymycocerosate (pdim). to prove pdim plays role in induction of autophagy and necrosis on mtb, quigley observed conversion of cytosolic lc3i to autophagosome-bound lc3ii, using the expression of green fluorescent proteinlc3 (gfp) and flow cytometry.18 as a result, autophagy was decreased in cells infected by mtb. pdim plays role in induction of autophagy with decreasing autophagy on infected cells by mtb.18 resistance to rifampicin caused by mutations in rpob gene related with physiological and metabolic changes in bacterial systems.3 these rif resistant might be under dual selection in mtb, combined benefit and physiological advantage of rpob gene can fix rpob mutants to infect in mtb populations. conclusion levels of map1lc3b on groups rifampicin resistant groups lower than on sensitive groups, that indicate no autophagy process or only few at macrophage on resistant groups than sensitive groups. this process occures because mtb successfully evade host defense by innate immune mechanisms. mtb can maintain intracellular growth inside the phagosome by inhibiting phagolysosome formation especially inhibiting map1lc3b formation by pdim. figure 3. mtb cell wall lipids modulate macrophage composition at sites of infection19 10 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 6–10 references 1. patel dm, patel sd, jaiswal ps, brahmbhatt kj. drug resistant mycobacterium tuberculosis and new drug development. int j drug dev res. 2012;4(2):76–91. 2. lory s, delong ef, thompson f, stackebrandt e, lory s, delong ef, et al. the prokaryotes-human microbiology. fourth edi. new york: springer heidelberg; 2013. 3. koch a, mizrahi v, warner df. the impact of drug resistance on mycobacterium tuberculosis physiology: what can we learn from rifampicin? emerg microbes infect. 2014 mar 12;3(3):e17–e17. 4. boehme cc, nabeta p, hillemann d, nicol mp, shenai s, krapp f, et al. rapid molecular detection of tuberculosis and rifampin resistance. n engl j med. 2010 sep 9;363(11):1005–15. 5. aziz ma, wright a, laszlo a, de muynck a, portaels f, van deun a, et al. epidemiology of antituberculosis drug resistance (the global project on anti-tuberculosis drug resistance surveillance): an updated analysis. lancet (london, england). 2006 dec 16;368(9553):2142–54. 6. du preez i, loots dt. altered fatty acid metabolism due to rifampicinresistance conferring mutations in the rpob gene of mycobacterium tuberculosis: mapping the potential of pharmaco-metabolomics for global health and personalized medicine. omics. 2012 nov;16(11):596–603. 7. gagneux s, long cd, small pm, van t, schoolnik gk, bohannan bjm. the competitive cost of antibiotic resistance in mycobacterium tuberculosis. science. 2006 jun 30;312(5782):1944–6. 8. patel as, morse d, choi amk. regulation and functional significance of autophagy in respiratory cell biology and disease. am j respir cell mol biol. 2013 jan;48(1):1–9. 9. goletti d, petruccioli e, romagnoli a, piacentini m, fimia gm. autophagy in mycobacterium tuberculosis infection: a passepartout to flush the intruder out? cytokine growth factor rev. 2013 aug;24(4):335–43. 10. rosenberger cm, finlay bb. phagocyte sabotage: disruption of macrophage signalling by bacterial pathogens. nat rev mol cell biol. 2003 may;4(5):385–96. 11. kabeya y, mizushima n, ueno t, yamamoto a, kirisako t, noda t, et al. lc3, a mammalian homologue of yeast apg8p, is localized in autophagosome membranes after processing. embo j. 2000 nov 1;19(21):5720–8. 12. gong l, devenish rj, prescott m. autophagy as a macrophage response to bacterial infection. iubmb life. 2012 sep;64(9):740–7. 13. flannagan rs, heit b, heinrichs de. antimicrobial mechanisms of macrophages and the immune evasion strategies of staphylococcus aureus. pathog (basel, switzerland). 2015 nov 27;4(4):826–68. 14. bento cf, empadinhas n, mendes v. autophagy in the fight against tuberculosis. dna cell biol. 2015 apr;34(4):228–42. 15. podinovskaia m, lee w, caldwell s, russell dg. infection of macrophages with mycobacterium tuberculosis induces global modifications to phagosomal function. cell microbiol. 2013 jun;15(6):843–59. 16. behar sm, divangahi m, remold hg. evasion of innate immunity by mycobacterium tuberculosis: is death an exit strategy? nat rev microbiol. 2010;8(9):668–74. 17. miskinyte m, gordo i. increased survival of antibiotic-resistant escherichia coli inside macrophages. antimicrob agents chemother. 2013 jan;57(1):189–95. 18. quigley j, hughitt vk, velikovsky ca, mariuzza ra, el-sayed nm, briken v. the cell wall lipid pdim contributes to phagosomal escape and host cell exit of mycobacterium tuberculosis. mbio. 2017 mar 7;8(2). 19. siegrist ms, bertozzi cr. mycobacterial lipid logic. cell host microbe. 2014 jan 15;15(1):1–2. 20. astarie-dequeker c, le guyader l, malaga w, seaphanh f-k, chalut c, lopez a, et al. phthiocerol dimycocerosates of m. tuberculosis participate in macrophage invasion by inducing changes in the organization of plasma membrane lipids. flynn jl, editor. plos pathog. 2009 feb 6;5(2):e1000289. 21. comas i, borrell s, roetzer a, rose g, malla b, kato-maeda m, et al. whole-genome sequencing of rifampicin-resistant mycobacterium tuberculosis strains identifies compensatory mutations in rna polymerase genes. nat genet. 2011 dec 18;44(1):106–10. 22. kawamura n, kurokawa k, ito t, hamamoto h, koyama h, kaito c, et al. participation of rho-dependent transcription termination in oxidative stress sensitivity caused by an rpob mutation. genes cells. 2005 may;10(5):477–87. 35 vol. 7 no. 2 may–august 2018 research report effectiveness of meniran (phyllanthus niruri linn) as antibacterial for antibiotics resistance enterotoxigenic escherichia coli sri hidanah1, emy koestanti sabdoningrum1, retno sri wahyuni2, arini rahmi dewi3, erma safitri3a 1 departmen of animal husbandry, faculty of veterinary medicine, universitas airlangga 2 department of basic medicine veterinary, faculty of veterinary medicine, universitas airlangga 3 student of faculty of veterinary medicine, universitas airlangga 4 departemen of reproduction veteriner, faculty of veterinary medicine, universitas airlangga a corresponding author: erma-s@fkh.unair.ac.id abstract escherichia coli (e. coli) can be isolated from the environment both inside and outside the hospes body. there were 89 serotypes in which showed 21% resistance to various antibiotics, such as enterotoxigenic e.coli. alternative efforts are needed to overcome this, one of them through the use of medicinal plants, such as meniran (phyllanthus niruri linn). meniran plant is an immunomodulator that serves to repair the immune system of the body. the aim of research is the research was done through several stages: isolation and identification of enterotoxigenic e. coli from several broiler farms in east java using the polymerase chain reaction (pcr) method, e. coli resistance test against some antibiotics, making meniran extract and activation test against enterotoxigenic e. coli n the study was divided into five treatments: t0+ (group of chickens were infected by enterotoxigenic e. coli t0(control group, not infected), t1 (infected by enteroxigenic e. coli + 20% meniran extract), t2 (infected by enterotoxigenic e. coli + 25% extract meniran), t3 (infected by enterotoxigenic e. coli + 30% extract meniran). data were analyzed by anova (analysis of variance). the results show that all of e. coli dna isolates which tested by the pcr method show positive reactions at 600 bp. the next stage, the enterotoxigenic e. coli are resistant to some antibiotics, such as amoxicillin, ampicillin, erythromycin, cephalosporins, tetracycline, cloxacillin and gentamicin. furthermore, the 30% phyllanthus niruri linn extract is effective as an antibacterial for antibiotic resistance enterotoxigenic e. coli. next necessary to write that: the 30% meniran extract is potential for kill of enterotoxigenic e. coli keywords: phyllanthus niruri linn, enterotoxigenic e. coli, antibiotic resistance, medicinal plants, immunomodulator abstrak escherichia coli (e. coli) dapat diisolasi dari lingkungan baik di dalam maupun di luar tubuh inang. ditemukan 89 serotipe dimana 21% menunjukkan resistensi terhadap berbagai antibiotik, seperti e. coli enterotoxigenic. diperlukan upaya alternative sebagai pengganti antibiotik, salah satunya melalui pemanfaatan tanaman obat, seperti meniran (phyllanthus niruri linn). tanaman meniran merupakan imunomodulator yang berfungsi memperbaiki sistem imun tubuh. penelitian ini melalui beberapa tahap yaitu: isolasi dan identifikasi e. coli enterotoxigenic dari beberapa peternakan ayam pedaging di jawa timur menggunakan metode polymerase chain reaction (pcr), uji resistensi e. coli terhadap beberapa antibiotik, pembuatan ekstrak meniran dan uji aktivasi meniran terhadap e. coli enterotoxigenic. penelitian ini dibagi menjadi lima perlakuan: t0+ (kelompok ayam yang terinfeksi oleh e. coli enterotoxigenic), t0(kelompok kontrol, ayam tidak terinfeksi enterotoksigenic e. coli, t1 (kelompok ayam yang terinfeksi oleh e. coli enterotoxigenic + ekstrak meniran dosis 20%), t2 (ayam terinfeksi oleh e. coli enterotoxigenic + ekstrak meniran dosis 25%), t3 (ayam terinfeksi e. coli enterotoxigenic + ekstrak meniran dosis 30%). data dianalisis dengan anova (analisis varians). hasil penelitian menunjukan semua isolat dna e. coli yang diuji melalui metode pcr menunjukkan reaksi positif berada pada posisi 600 bp. pada tahap penelitian berikutnya ditemukan bahwa e coli enterotoksigenic resisten terhadap beberapa antibiotik, seperti: amoxicillin, ampicillin, eritromisin, cephalosporins, tetracycline, cloxacilin dan gentamicin. selanjutnya 30% ekstrak meniran (phyllanthus niruri linn) 30% efektif sebagai 36 indonesian journal of tropical and infectious disease, vol. 7 no. 2 may–august 2018: 35–39 antibakteri untuk pencegahan resisten antibiotik dari e. coli enterotoxigenic. kesimpulan dari penelitian ini, 30% meniran extract efektif unuk membunuh e. coli enterotoksigenic kata kunci: phyllanthus niruri linn, e. coli enterotoxigenic, resisten antibiotik, tanaman obat, imunomodulator introduction the poultry, especially chickens are farm animals which particularly vulnerable to diseases. this condition leads to decrease in productivity and causes major losses if the treatment is not successful.1 one of the diseases that are common and detrimental to farmers is a bacterial infection such as e. coli.2 e. coli especially due to enterotoxigenic from e. coli was commonly cause diarrheal disease in chickens, is called colibacillosis.3 the losses due to diseases such as colibacillosis is high chicken mortality which can reach 30%. colibacillosis disease attacks young chickens until harvesting on the age around of 25-35 days old broiler and 40-50 days old layer.4 incorrect diagnosis, treatment and control of e. coli infections often cause resistance to antibiotics.5 there are 89 serotypes e. coli can be isolated from the host and 21% exhibit resistance to various antibiotics, because e. coli contains plasmids.6 use of antibiotics should be re-evaluated, as well as against the enterotoxigenic and virulence factors of e. coli. the meniran (phyllanthus niruri linn) is a plant that can be used as an alternative prevention and treatment of diseases which caused by e. coli.7 the chemicals contain in meniran are flavonoids and tannins.8 the flavonoids action of phyllanthus niruri linn is an imunnomodulator whose role is to boost the immune system and improve dysfunctional of the immune system.9 immune dysfunctional is caused a decrease in number of immune cells such as t-cd5+,cd4+, cd8a+,cd8b+ in the lamina proria and intraepithelial villi intestine.10 the immune dysfunctional, which can lead to infection by bacterial or parasites with symptoms of chronic diarrhea and that, will increase mortality. from previous studies, the animal laboratory were conditioned immune dysfunctional in addition to obtain high expression of hsp70, low expression of pros taglandin e2, expression of immunoglobulin a, histologically visible damage of intestinal mucosa epithelium and is showed villus atrophy, which will lead to decrease in the absorption function. the impact is as reduced nutritional intake and organs dam age, including liver damage.11 this will reduce the reproductive rate of both male12 and female,13,14 which in turn decreases productivity for livestock, especially poultry. tannins efficacious as an antibacterial (prevents bacterial growth) and hemostatic (bleeding stoped).15 phenolic compounds may be bactericidal or bacteriostatic depending on the concentration used. antibacterial substances have various ways in inhibiting bacterial growth.16 damage to one of the structures of the bacterial cell can cause changes in the structure and action of bacteria. this can lead become stunted bacterial growth and even lead to cell death.17 cytoplasmic membrane is the outer part of the cytoplasm which is located under the cell wall, composed of proteins, lipids and carbohydrates. this membrane plays a role to regulate the incoming of matter such as water and mineral salts needed by the cell. the parts of the cell in the cytoplasm are ribosomes, nuclei, granules and mesosomes. ribosomes are small follicles composed of proteins and ribonucleic acid (rna), which their function were act in protein synthesis. the nucleus contains dioxyribonucleic acid (dna) as a carrier of genetic information. granules are chemical substances that can functionate as food reserves for cells. the mesosome is the fold of the cytoplasmic membrane into the cytoplasm. in connection with this, the damage to cell membranes by antibacterial substances can lead stunted cell growth and even result in the death of bacterial cells.16 the meniran plant as the composition of feed as well as a single oramixture of food ingredients, processed or unprocessed, which is given for animal’s survival, production and breed.18 the research purposes is isolation and identification of enterotoxigenic e. coli was antibioticresistant as the causative agent of loss and death of broilers. furthermore, applying meniran (phyllanthus niruri linn) as antibacterial for the prevention enterotoxigenic e. coli of antibiotic-resistant in broiler. material and method the research was done through several stages: first stage. isolation and identification of enterotoxigenic e. coli from several broiler farms in east java (mojokerto and tuban). the identification using the polymerase chain reaction (pcr) method19 with primer: forward (5’tagagaaattatcaagttagttcc-3’) reverse: (5’ atagttatgaacatcttgtttagc-3’) (17). the second stage. e. coli resistance test against some antibiotics were used dilution method, namely minimum inhibitory concentration (mic)20 and minimum bacteriocide concentration (mbc). the test were done through several antibiotics: amoxicillin, amphicillin, erythromycin, cephalosporins, tetracycline, cloxacilin and gentamicin. the third stage. the synthesis of meniran extract with ethanol solven. ried meniran plants have been milled to obtain powder. pollen meniran 1 kg extracted using maceration method by immersion in a solution of ethanol 96% as much as five liters for 3 x 24 hours. stirring is 37hidanah, et al.: effectiveness of meniran (phyllanthus niruri linn) done twice, morning and afternoon. maceration process is performed three times. the marinade filtrate is then filtered to evaporate using a rotary evaporator which will yield a concentrated plant extracts meniran21 and then, meniran extract was already for application to broiler chicken. the fourth stage. activation test of the meniran extract against enterotoxigenic e. coli were applicated to 25 broiler chicken aged 19 days, with treatment as follow: e. coli infected t0+: broiler chickens infected with e. coli at 28 days of age with a concentration of 106 cfu/ ml/chicken orally without meniran plant extract (control positive); t0-: broiler chickens at 28 days without any treatment (control negative); e. coli infected t1: broiler chickens 28 days of age with a concentration of 106 cfu/ml/chicken orally and then given the extract of meniran plants with a dose of concentration of 20% /ml/chicken orally; e. coli infected t2: broiler chickens 28 days of age with a concentration of 106 cfu/ml/chicken orally and then given the extract of meniran plants with a dose of concentration of 25%/ml/ chicken orally; e. coli infected t3: broiler chickens at 28 days of age with a concentration of 106 cfu/ml/ chicken orally and they had been given the extract of meniran plants with a dose of concentration of 30%/ml/chicken orally. sampling for the calculation of the number of e. coli bacteria was done by killing the broiler chickens in each treatment to take 1 g of broiler chicken liver sample and inserted into a venoject tube containing 9 ml of physiological nacl solution and labeled treatment for each sample. all samples that have been taken are saved into the coolbox. data on the number of e. coli with most probable number (mpn) were analyzed by anova (analysis of variance). the data was analysed using spss version 20. p value at the level of < 0,05 was refer to significant. result and discusion the isolation and identification of escherichia coli bacteria results on broiler chicken liver samples from broiler farms in tuban and mojokerto, where showed of e. coli infected typical symptoms such as diarrhea and sepsis. the body was bluish, e. coli isolated bacteria was identified on hepar. 29 samples including 17 samples from tuban and 12 samples from mojokerto, were showed positive for e.coli in 20 samples consist of 12 samples from tuban and 8 samples from mojokerto. the results of e.coli isolation on emba media were showed metallic green colonies (figure 1). biochemical test results are as follows tsia (+), indole (+), urea agar (-) and sca (-). result and discusion the isolation and identification of escherichia coli bacteria results on broiler chicken liver samples from broiler farms in tuban and mojokerto, where showed of e. coli infected typical symptoms such as diarrhea and sepsis. the body was bluish, e. coli isolated bacteria was identified on hepar. 29 samples including 17 samples from tuban and 12 samples from mojokerto, were showed positive for e.coli in 20 samples consist of 12 samples from tuban and 8 samples from mojokerto. the results of e.coli isolation on emba media were showed metallic green colonies (figure 1). biochemical test results are as follows tsia (+), indole (+), urea agar (-) and sca (-). figure 1. e. coli isolation in emba media the identification of enterotoxigenic e. coli dna from several broiler farms in east java (mojokerto and tuban) using the polymerase chain reaction (pcr) method was showed a positive reaction for all isolates tested the pcr fragments which was located position at 600 bp (figure 2). figure 2. gene analysis coding enterotoxigenic e. coli dna with pcr electrophoresis was visualized on 2% agarose gel ethidium bromide staining. the results show a 600 bp cdna string on all samples from chicken farms in mojokerto and tuban figure 1. e. coli isolation in emba media. the identification of enterotoxigenic e. coli dna from several broiler farms in east java (mojokerto and tuban) using the polymerase chain reaction (pcr) method was showed a positive reaction for all isolates tested the pcr fragments which was located position at 600 bp (figure 2). result and discusion the isolation and identification of escherichia coli bacteria results on broiler chicken liver samples from broiler farms in tuban and mojokerto, where showed of e. coli infected typical symptoms such as diarrhea and sepsis. the body was bluish, e. coli isolated bacteria was identified on hepar. 29 samples including 17 samples from tuban and 12 samples from mojokerto, were showed positive for e.coli in 20 samples consist of 12 samples from tuban and 8 samples from mojokerto. the results of e.coli isolation on emba media were showed metallic green colonies (figure 1). biochemical test results are as follows tsia (+), indole (+), urea agar (-) and sca (-). figure 1. e. coli isolation in emba media the identification of enterotoxigenic e. coli dna from several broiler farms in east java (mojokerto and tuban) using the polymerase chain reaction (pcr) method was showed a positive reaction for all isolates tested the pcr fragments which was located position at 600 bp (figure 2). figure 2. gene analysis coding enterotoxigenic e. coli dna with pcr electrophoresis was visualized on 2% agarose gel ethidium bromide staining. the results show a 600 bp cdna string on all samples from chicken farms in mojokerto and tuban figure 2. gene analysis coding enterotoxigenic e. coli dna with pcr electrophoresis was visualized on 2% agarose gel ethidium bromide staining. the results show a 600 bp cdna string on all samples from chicken farms in mojokerto and tuban. enterotoxigenic e. coli dna detection by pcr was showed a positive reaction for all isolates tested the pcr fragments which was located at position 600 bp. the used of primer has a great prospect when used in the early detection of enterotoxigenic e. coli strain enterohemorrhagic for local (indonesian) because it has a fairly high specificity.2 furthermore, enterotoxigenic e. coli resistance to some antibiotics such as amoxicillin, ampicillin, erythromycin, cephalosporins, tetracycline, cloxacilin and gentamicin. the using of antibiotics is widely used in the livestock 38 indonesian journal of tropical and infectious disease, vol. 7 no. 2 may–august 2018: 35–39 industry to prevent infection of e. coli.22 because e. coli is a commensal bacterium that has a live strain not only in the gastrointestinal tract but also in various internal organs.23 bacteria become resistant to antibiotic agents due to mutations, transformations, transduction or conjugation.24 the mechanism of resistance can be through various means, among others: drug activation, altering the structure of enzymes or bacterial membranes, decreasing the accumulation of drugs by cells, the presence of variations in metabolic pathways as well as increased metabolic concentration.25 enterotoxigenic e. coli resistance to some antibiotics is showed resistant to amoxicillin, amphicillin, erythromycin, cephalosporins, tetracycline, cloxacilin and gentamicin. the ability of meniran (phyllanthus niruri linn) extract in inhibiting the growth of bacteria due to the chemicals contains found in plant meniran (phyllanthus niruri linn) extracts namely flavonoids, tannins and saponins. plant phenolic compounds and phenol compounds in general is a class of materials that has the ability to kill and inhibit the growth of bacteria. damage to one of the constituent structures of bacterial cells can cause changes in the structure and working of bacteria.22 after meniran extract already for application to broiler chicken, further observations were made on the activation test from the meniran extract against enterotoxigenic. the meniran extract potential for enterotoxigenic e. coli was showed the power to kill the enterotoxigenic e. coli at concentrations of 30% (table 1). table 1. mean and standard deviation (sd) number of e. coli (mpn) in broiler chickens with various treatment treatments mean ± sd t0 (control group, not infected) 0.7593a ± 0,32578 t3 (infected by enterotoxigenic e. coli + 30% extract meniran) 1.1799a ± 0,.81489 t2 (infected by enterotoxigenic e. coli + 25% extract meniran) 1.4210ab ± 0,32792 t1 (infected by enterotoxigenic e. coli + 20% meniran extract) 2.3181bc ±0, 83574 t0 + (group of chickens were infected by enterotoxigenic e. coli) 2.7732 c ± 0.53645 a, b superscript different in the same column indicate significant differences (p < 0.05) the analysis of data that has been done using anova showed significant result between t0 + and t0-. while t0 + and t1 showed insignificant results, significant results were also seen between t0 + and t2. t0 + and t3 were significantly different, significant results were also seen between t0and t1, and t1 and t3. between t0and t2 are not significant. while in t0and t3 the results are significant. the concentration of 30% is a concentration that can kill the bacteria e. coli according to the result. the cytoplasmic membrane is the outermost part cytoplasm that lies beneath the cell wall, composed of protein compounds, lipids and carbohydrates. this membrane acts to regulate the materials out of cells such as water and mineral salts needed cells. parts of the cell in the cytoplasm include the ribosomes, nuclei, granules and mesosom. ribosomes shaped small particles consisting of protein and ribonucleic acid (rna), which functions as a protein synthesis. the tannin compound which is a component of meniran has a working mechanism that inhibits and kills the growth of bacteria by reacting with membrane cells as well as the destruction or inactivation of the function of the genetic material.16 tanin is also toxic and the nature of astrigensia works against bacterial cell membranes, by inhibiting certain enzymes.24,25 other antibacterial compounds are saponins. saponins can increase the permeability of bacterial cell membranes in order to alter membrane structure and function, causing membrane protein denaturation so that cell membranes will be damaged and lysis.16 saponins can increase the permeability of the intestinal wall, improve nutrient absorption and also inhibit the activity of the urease enzyme.17 meniran plants also contain alkaloids that are toxic to microbes, so effectively kill gram-negative and gram-positive bacteria. alkaloids act as antibacterial by destroying the peptidoglycan component in bacterial cells, so that the cell wall layer is not fully formed and causes the cell death.26 conclusion enterotoxigenic e. coli dna was showed a positive reaction for all isolates tested the pcr fragments which was is located at position 600 bp. the enterotoxigenic e. coli are resistance to some antibiotics, such as amoxicillin, ampicillin, erythromycin, cephalosporins, tetracycline, cloxacillin and gentamicin. 30% concentration phyllanthus niruri linn extract effective as an antibacterial for the prevention of antibiotic resistance from enterotoxigenic e. coli of broilers. acknowledgement we thank to directorate general of higher education for funding through scheme program penelitian unggulan perguruan tinggi (pupt) universitas airlangga, 2016. references 1. review m. salmonellosis a potential threat to poultry: j cell tissue res. 2015;15(3):5209–13. 2. he x, qi w, quiñones b, mcmahon s, cooley m, and mandrell re. comparison of the specificities and efficacies of primers for aromatic dioxygenase gene analysis of environmental samples. appl env microbiol. 2011;77(11):3551–3557. 3. bergeron cr, prussing c, boerlin p, daignault d, dutil l, reidsmith rj, et al. chicken as reservoir for extraintestinal pathogenic escherichia coli in humans, canada. 2012;18(3):415–21. 39hidanah, et al.: effectiveness of meniran (phyllanthus niruri linn) 4. matin ma, islam ma, khatun mm. prevalence of colibacillosis in chickens in greater mymensingh district of bangladesh. vet world. 2017 jan;10(1):29–33. 5. beckson d. b bbl prepared tubed medium for detection of fecal coliform bacteria. 2011;8806881. 6. trivedi rn, akhtar p, meade j, bartlow p, ataai mm, khan sa, et al. high-level production of plasmid dna by escherichia coli dh5α ω sacb by introducing inc mutations. parales re, editor. appl environ microbiol. 2014 dec 1;80(23):7154–60. 7. yuniati y, rollando r. isolation of antibacterial compounds from endophyte fungal of fusarium isolation of antibacterial compounds from endophyte fungal of fusarium sp. in phyllanthus niruri linn. leaves. 2018;(march). 8. permata da, murtius ws, august j. research journal of pharmaceutical, biological and chemical sciences alpha amylase inhibition and antioxidant activity of phyllanthus niruri powder drink. 7(656):656–62. 9. aldi y, rasyadi y, handayani d. immunomodulatory activity of meniran extracts (phyllanthus niruri linn.) on broiler chickens. j sains farm klin. 2014;1(1):20–6. 10. prasetyo rh, safitri e. effects of honey to mobilize endogenous stem cells in efforts intestinal and ovarian tissue regeneration in rats with protein energy malnutrition. asian pacific j reprod. 2016 may;5(3):198–203. 11. prasetyo rh, hestianah ep. honey can repairing damage of liver tissue due to protein energy malnutrition through induction of endogenous stem cells. vet world. 2017 jun;10(6):711–5. 12. safitri e, utama s, widiyatno tv, sandhika w, prasetyo rh. autoregeneration of mice testicle seminiferous tubules due to malnutrition based on stem cells mobilization using honey. asian pacific j reprod. 2016 mar;5(1):31–5. 13. safitri e, widiyatno tv., prasetyo rh. honeybee product therapeutic as stem cells homing for ovary failure. vet world. 2016 nov;9(11):1324–30. 14. samik a, safitri e. mycotoxin binders potential on histological of ovary mice exposed by zearalenone. vet world. 2017 mar;10(3):353–7. 15. dandjesso c, klotoé jr, dougnon tv, sègbo j, atègbo jm, gbaguidi f, et al. phytochemistry and hemostatic properties of some medicinal plants sold as anti-hemorrhagic in cotonou markets (benin). indian j sci technol. 2012;5(8):3105–9. 16. maddox ce, laur lm, tian l. antibacterial activity of phenolic compounds against the phytopathogen xylella fastidiosa. curr microbiol. 2010 jan 8;60(1):53–8. 17. jawetz, melnick and a. medical microbiology. 6th ed. new york: a langa medial book; 2013. 18. samik a, safitri e. potency of mycotoxin binders on mda level, expressions of caspase 9 and caspase 3 in the uterus of mice exposed to zearalenone. iraqi j vet sci. 2017;31(1):29–33. 19. lluque a, mercado e, riveros m, alvarado l, carlos e., colichón a, salazar e, ochoa t. comparison of enteropathogenic escherichia coli (epec) diagnosis by serology and by polymerase chain reaction (pcr). rev gastoenterol peru. 2010;30(2):121–5. 20. lara vm, carregaro ab, santurio df, sá mf de, santurio jm, alves sh. antimicrobial susceptibility of escherichia coli strains isolated from alouatta spp. feces to essential oils. evidence-based complement altern med. 2016;2016:1–4. 21. rumagit bi, banne y, dilampudi y. preparation of ulcer ointment from meniran (phyllanthus niruri l.) herb extract. j stifa makassar. 2015;1(1):15–9. 22. landers tf. a review of antibiotic use in food animals: perspective, policy, and potential. 127(february 2012). 23. katouli m. population structure of gut escherichia coli and its role in development of extra-intestinal infections. 2010;2(2):59–72. 24. willey jm, sherwood lm, woolverten cj. prescott’s principle of microbiology. new york: mcgraw-hill higher education; 2009. 532–567 p. 25. munita jm, arias ca, unit ar, santiago a de. hhs public access. 2016;4(2):1–37. 26 cushnie tpt, cushnie b, lamb aj. international journal of antimicrobial agents alkaloids: an overview of their antibacterial, antibiotic-enhancing and antivirulence activities. int j antimicrob agents. 2014;44(5):377–86. 79 vol. 7 no. 4 january-april 2019 research report prevalence and risk factors of diabetes mellitus in tuberculosis patient at patrang district indonesia hasri yulia sasmita1a, irma prasetyowati2, pudjo wahjudi2 1 student of epidemiology and biostatistics population, faculty of public health, jember university, indonesia 2 department of epidemiology and biostatistics population, jember university, jember, indonesia a corresponding author: hasriyuliasasmita@gmail.com abstract tuberculosis (tb) is one of cause of death in infectious disease domain. the control of tb is complicated because the inclination of case numbers people with diabetes mellitus. diabetes mellitus (dm) is an important risk factor for tb development, with prove that more than ten percent of tb patient is dm patient. people with dm have risk three times more likely to suffer from tb than people without dm. the results of tb treatment with comorbid dm will be easier to be failed. puskesmas patrang have the highest bacteriologically confirmed bta tb cases and dm cases in jember during 2014 until 2016. the aim of this research is to know the dm prevalence in tb patients and to analyze the correlation between dm risk factors in tb patient to tb-dm incidence at puskesmas patrang jember in 2017. the research uses observasional analytic with cross sectional approach. the sampling technique uses simple random sampling with 47 samples. the independent variables include respondent characteristics (age, sex, type of tb, medication category, and family history of dm), central obesity and smoking behavior. while the dependent variable is the dm status. the result shows that the prevelance of dm in tb patients at puskesmas patrang jember regency is 23.4%. factors associated with tb-dm are age (p-value = 0.012), family history of dm (p-value = 0.003), and smoking status (p-value = 0.035). factors that do not associated with tb-dm are sex (p-value = 0.731), type of tb (p-value = 0.170), treatment category of tb (p-value = 0.560), central obesity (p-value = 0.435), the number of cigarette (p-value = 1.000) and smoking duration (p-value = 1.000). the most important factor of tb-dm is family history of dm that 10,850 times higher of getting tb-dm than patients without family history of dm. keywords: tb, dm, risk factors of dm abstrak tuberkulosis (tb) adalah salah satu penyebab kematian dalam kelompok penyakit menular. pengendalian tb dipersulit karena kecenderungan kasus penderita diabetes melitus. diabetes melitus (dm) merupakan faktor risiko penting untuk perkembangan tb, dengan bukti bahwa lebih dari sepuluh persen penderita tb merupakan penderita dm. penderita dm berisiko 3 kali lebih tinggi untuk menderita tb dibandingkan dengan orang tanpa dm. hasil pengobatan tb dengan komorbid dm akan lebih mudah mengalami kegagalan. puskesmas patrang memiliki kasus tb bta terkonfirmasi bakteriologis dan dm tertinggi di kabupaten jember selama tahun 2014 sampai 2016. penelitian ini bertujuan untuk mengetahui prevalensi dm pada pasien tb dan mengetahui hubungan faktor risiko dm pada pasien tb terhadap kejadian tb-dm di puskesmas patrang kabupaten jember tahun 2017. penelitian ini menggunakan metode penelitian analitik observasional dengan pendekatan cross sectional. teknik pengambilan sampel menggunakan metode simple random sampling dengan jumlah sampel sebanyak 47 orang. variabel bebas penelitian meliputi karakteristik responden (usia, jenis kelamin, jenis tb, kategori pengobatan, dan riwayat dm pada keluarga), obesitas sentral dan perilaku merokok. sedangkan varibel terikat penelitian yaitu status dm. hasil penelitian menunjukkan bahwa prevalensi dm pada pasien tb di puskesmas patrang kabupaten jember yaitu sebesar 23,4%. faktor yang berhubungan secara signifikan dengan kejadian tb-dm yaitu usia (p-value = 0,012), riwayat dm pada keluarga (p-value = 0,003), dan status merokok (p-value = 0,035). sedangkan faktor yang tidak berhubungan secara signifikan dengan kejadian tb-dm yaitu jenis kelamin (p-value = 0,731), jenis tb (p-value = 0,170), kategori pengobatan tb (p-value = 0,560), obesitas sentral (p-value = 0,435), jumlah rokok yang dihisap (p-value = 1,000) dan lama merokok (p-value = 80 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 79–85 1,000). faktor yang paling berhubungan terhadap kejadian tb-dm adalah riwayat dm pada keluarga dengan risiko sebesar 10.850 kali untuk menderita tb-dm dibandingkan pasien tanpa riwayat dm pada keluarga. kata kunci: tb, dm, faktor risiko dm introduction tuberculosis (tb) is infectious disease that caused by mycobacterium tuberculosis (m.tb) which can attack various organs espescially the lungs.1 tb is one of 10 causes of death in the world and the leading cause of death in the world in infectious disease domain.2 diabetes mellitus (dm) is a metabolic disorder disease characterized by elevated blood sugar levels due to decreased insulin secretion by pancreatic beta cells and or insulin function i.e. insulin resistance. diabetics are three times more likely to have tb than people without dm.3 tb patients with dm were at risk for the failure of sputum conversion after undergoing tb treatment, 1.69 times higher risk for treatment failure, 1.24 times higher for anti tuberculosis drug resistance, 3.89 times higher for relapse, and 4.95 times higher risk for death during treatment than those without dm.4 previous tb-dm studies have found that age, sex, pulmonary tb, and family history of dm are associated with tb-dm incidence.5 other studies were found that age, family history of dm, alcohol consumption, and central obesity were associated factors with tb-dm incidence.6,7 tb-dm is a problem in tb endemic countries with lower middle income because 70% of dm patients live in tb endemic countries and 80% of dm patients live in lower middle income countries.8,9 indonesia is in 4th highest tb cases below india, china and south africa with 48,000 cases.10 east java is in second place of highest bacteriologically confirmed bta tb cases after west java and is included in the top 10 of dm patients in indonesia.11,12 jember has the second largest number of bacteriologically confirmed bta tb cases after surabaya and has dm patients of 8619 people by 2012.13,14 puskesmas patrang has the highest cases of tb bta confirmed bacteriology and dm cases with 111 cases of tb bta confirmed bacteriology and 371 dm cases by 2016. studies on the effect of dm on tb epidemiology in 13 high tb burden countries were showed that 6 million tb cases and 1.1 million tb deaths can be avoided through dm control.8 previous studies have suggested that risk factors for dm incidence in tb patients include race, age, sex, type of tb, treatment category, family history of dm, central obesity, smoking, and alcohol. race is thought to be due to genetic factors that contribute to the incidence of insulin resistance. people who have black skin are more susceptible to dm than people with white skin. tb-dm patients are generally occur in old age especially after the age of 40 years. the aging process causes the ability of pancreatic beta cells in producing insulin reduced.15,16 tb-dm is more common in men than women as a result of risk factors such as smoking and alcohol consumption.17 based on type of tb, dm is more common in pulmonary tb patients than extrapulmonary tb.18 the prevalence of recurrent tb is higher in tb-dm patients than tb without dm. tb-dm patients are 3.9 times more likely to get tb relapse than tb without dm.19 dm tends to be inherited so the family members of patients with dm have the possibility of 2-6 times suffering dm compared with family members who do not suffer from dm.20 another factor is central obesity which can lead to insulin resistance due to excess fat that makes insulin work difficult so that blood sugar levels rise.15 based on the previous research, majority of tb-dm patients had ever use tobacco (78,3%). the effect of nicotine causes a decrease in insulin release due to catecholamine hormone activation, a negative effect on insulin action, impairment of pancreatic β cells and leads to insulin resistance.21 alcohol can affect the endocrine glands by releasing epinephrine that leads to transient hyperglicemia and hyperlipidemia so that alcohol consumption is contraindicated with diabetes.22 the problem of tb-dm in jember especially in puskesmas patrang tends to be in the top three highest cases of bacteriologically confirmed bta tb and dm during 2014-2016. a bacteriologically confirmed bta tb cases for three consecutive years i.e 94, 107 and 111 and dm cases for three consecutive years i.e 2049, 1011 and 371 cases. the declining of dm cases may be caused by undiagnosed patients. dm patients are usually asymptomatic but it is still a risk of tb-dm incidence. moreover, tb-dm problem in jember has not been studied more deeply, therefore we need to know the risk factors of dm incidence in tb patient at puskesmas patrang jember in 2017 for preventing and controlling dm incidence in tb patient considering its impact if tb and dm occur together. the purpose of this research is to know the risk factors of dm incidence in tb patients at puskesmas patrang jember in 2017. material and method this research uses observational analytic method with cross sectional approach. the research was conducted at puskesmas patrang jember in august 2017. the number of samples were 47 tb patients using simple random sampling technique. the variables of this research consist of independent and dependent variables. the independent variables include respondent characteristics (age, sex, type of tb, medication category, and family history of dm), central obesity and smoking behavior. while the dependent variable is the dm status. 81sasmita, et al.: prevalence and risk factors of diabetes mellitus table 1. prevalence of diabetes mellitus in tuberculosis p a t i e n t a t p u s k e s m a s p a t r a n g i n 2 0 1 7 dm status frequency (people) percentage (%) tb-dm 11 23.4 tb 36 76.6 sources of research data consist of primary data and secondary data. primary data was obtained from interview and measurement of abdominal circumference in tb patients. secondary data was obtained from the data of quarterly reports of new cases of tb bta confirmed bacteriologic finding in health office jember district during 2014-2016, dm report data at health office jember district during 2014-2016 and form tb 01 at puskesmas patrang jember district in 2017. data collection techniques are conducted through interview, measurement and observation. instrument of data collection was determined using questionnaire and meter measuring tool. data analysis techniques used univariate, bivariate analysis using chi-quare, and multivariate using logistic regression. result and discussion prevalence of diabetes mellitus in tuberculosis patient at puskesmas patrang jember in 2017 patients with tb as respondents in the study were 47 respondents. the respondents were divided into tb-dm patients and tb patients. tb-dm patients were tb patients who also were diagnosed by health professionals suffering from dm or taking dm drug while tb patients were those who only suffer tb. prevalence of dm in tb patient can be seen in table 1. in table 1, the prevalence of dm in tb patients is 23.4%. this figure exceeds the prevalence of dm only in the population in indonesia that is 3.2%. in addition, alisjahbana found that dm is more common in patients with tb than in control subjects. of 454 tb patients, 60 patients (13.2%) have dm rather than of 556 non tb patients, 18 patients (3.2%) have dm.23 guptan and shah (2000) were stated that impaired glucose tolerance (igt) in tb is much higher than overt dm. according to the national diabetes data group of nih, one to five per cent of patients with igt may progress to overt dm. in 1990, fong y et al was stated that glucose intolerance due to tb occurs because of increased cytokines il-1 and tnf alpha which can stimulate the anti insulin hormones. in addition, in 1993, sinurova et al was stated that cortisol which controls blood sugar levels also decrease. even, tb germs are able to release toxins through the bloodstream or tb germs directly attack the pancreas that causes the production of amyloid (abnormal protein) in the pancreas which further inflict dm.24,25 the mechanism is the reason why dm is more prevalent in people with tb than in general population. the exceeds of the dm prevalence in table 2. distribution of tuberculosis patient characteristics by age, gender, type of tb, treatment category, and family history of dm in puskesmas patrang jember in 2017 characteristics frequency (people) percentage (%) age ≥ 45 years old < 45 years old 18 29 38,3 61,7 sex male female 26 21 55,3 44,7 type of tb pulmonary extrapulmonary 39 8 83,0 17,0 treatment category category 2 category 1 3 44 6,4 93,6 family history of dm yes no 12 35 25,5 74,5 tb patient rather than in general population also occurs in countries where tb and dm cases are most prevalent.8 another research which conducted by raghuraman et al in india also found higher prevalence of dm in tb patients that is 29% than the prevalence of dm in the population. the population is 7% because india is included in high load tb cases that have a growing number of dm cases.6 relationship between respondent characteristics with tb-dm incidence in tuberculosis patients at puskesmas patrang jember in 2017. characteristics are the attributes or things possessed by an element. characteristics of respondents in this study include age, sex, type of tb, treatment category, and family history of dm. the distribution of tb patient characteristics can be seen in table 2. based on table 2, out of 47 patients were enrolled for this research, most tb patients are < 45 years old (61,7%). indonesia’s health profile (2017) was also stated that the highest proportion of tb cases are found in the < 45 years old. during 2012-2016, the proportion is 61.48%, 60.96%, 60.26%, 60.31%, and 59.91%.26 tb can attack anyone, especially the productive age (15-50 years old). people with productive age are easy to interact with others, high mobility makes it possible to transmit bacteria to others. infant or younger than 4 years old also have greater risk of experiencing the progression of tb infection because the t-cell mediated immune response of young children is not fully developed (immature) so it works inefficiently to antigen challenge. 27-29 this research was also found that most tb patients are males (55,3%). males are at higher risk of developing tb than females because they have smoking habit that makes lung function impaired, damages the macrophages as cells that can eat bacteria, making it easier to get tb.28,30 based on national health indicator survey (sirkesnas) in 2016, the prevalence of smoking in male is 59% and female is 1.6%.26 82 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 79–85 table 2 also was showed that most of respondents had pulmonary tuberculosis (83%). according to the definition, tb is a contagious infectious disease caused by mycobacterium tuberculosis that can attack various organs especially the lungs. tb transmission occurs through sputum sparks containing tb bacteria that are transmitted when people with tb cough or sneeze, so that pulmonary tb is the most common clinical manifestation compared to other organs.1,31 most tb patients in this research are in undergoing category 1 treatment (93,6%) especially tb bta confirmed bacteriologically. the number of tb bta confirmed bacteriology cases can be used as an indicator of tb patient discovery to achieve national indicators. national indicators are indicator of tb programs that can measure the success of a program.32 it means more getting tb bta confirmed bacteriologically patients among suspected sputum examination, getting treated faster. the result was showed that the majority of tb patients have no family history of dm. in this study, family history of dm was not associated with the incidence of tb. patient who has family history of dm is 2-6 times higher to get dm. dm is characterized by hyperglycaemia due to insulin secretion defect, insulin work or both. hyperglycaemia is capable of causing immune disorders i.e decreased gamma interferon function (ifn γ) which serves to stimulate macrophages to kill m.tuberculosis through the mechanism of endocytosis into the macrophages. this indicates that in this study there are other risk factors that cause tb such as table 3. bivariate analysis of the relation between respondent characteristics and tb-dm incidence in tuberculosis patients at puskesmas patrang jember in 2017 characteristics status dm p-value or (95% ci)tb-dm tb n n age ≥ 45 < 45 8 3 10 26 0,012* 6,933 (1,525-31,515) 1 sex male female 7 4 19 17 0,731 1,566 (0,389-6,298) 1 type of tb pulmonary ep 11 0 28 8 0,170 treatment category category 2 category 1 1 10 2 34 0,560 1,700 (0,139-20,749) 1 family history yes no 7 4 5 31 0,003* 10,850 (2,304-51,101) 1 ep = extrapulmonary *significant ≤ α (0,05) close contact with untreated tb patients, lack of nutritional status, low immunity, and smoking habits.28,33 the relation between the characteristics of respondents with tb-dm incidence could be identified through bivariate analysis. the related factors of tb-dm incidence were age and family history of dm. the unrelated factors of tb-dm incidence were sex, type of tb, and treatment category. table 3 gives the bivariate analysis of the relation between respondent characteristics and tb-dm incidence at puskesmas patrang jember in 2017. table 3 was showed that tb-dm was associated with older age. this result is in line with research by raghuraman et al (2014) that found the median age of tb-dm patients that was 49.5 ± 11.8.6 while mansuri et al (2015) found the average age of tb-dm patients is 47 ± 16.7 tb-dm tends to be experienced by people with old age as a result of dm cases often found in old age.8,34 the risk of developing dm usually appears after 45 years old. at that age there is a physiological decline in humans that affects the reduced ability of pancreatic beta cells in the production of insulin.15,16 family history of dm was also significantly related to tb-dm incidence. most patients with family history of dm tend to suffer from tb-dm (14.9%) than suffering tb (10.6%). these results are in line with other research that the family history of dm is associated with tb-dm incidence.6 dm tends to be inherited rather than transmitted. family members of patients with diabetes (diabetics) are 2 to 6 times more likely to suffer dm compared to family members who do not have dm.20 this research has not found any significant association between sex and tb-dm. similar results were reported by researches by raghuraman et al and mansuri et al.6,7 but the other study by suwanpimolkul et al was stated that men was associated with tb-dm. men are 1.66 times more likely to have tb-dm than women.35 in this research, most of the men (40.4%) and women (36.2%) only had tb. type of tb was found to be statistically insignificant. previous research also was found that the type of tb was not significantly related to tb-dm incidence.7 different results were found by workneh et al (2016) that pulmonary tb was significantly associated with tb-dm incidence. the prevalence of pulmonary tb among tb-dm patient was higher (64.2%) than tb patient (58.1%).5 while in this research, patients with pulmonary tb tend to suffer from tb (59.6%) than tb-dm (23.4%). it is also known that all extrapulmonary tb patients suffer from tb with percentage of 17%. treatment category was also found to be statistically insignificant. there are two treatment category i.e category 1 that consists of tb bta confirmed bacteriology, pulmonary tb patients diagnosed clinically, and extrapulmonary tb and category 2 that consists of relapse, drop out, and fail treatment.32 this result is in line with other researchers that also found that treatment category 2 was not significantly associated with tb-dm.6,7 but, research which was conducted by mi et al (2013) found that category 2 83sasmita, et al.: prevalence and risk factors of diabetes mellitus while the number of cigarettes (p-value = 1,000) and duration of smoking (p-value = 1,000) are not significantly related to tb-dm incidence. research by viswanathan et al was stated that smoking was risk factor for tb-dm. people that smoking is at risk 1.92 times higher than that of non-smokers.17 smoking causes inflammation and oxidative stress in the cells of the body thus increasing the risk of developing dm. nicotine content in cigarettes can lead to decreased insulin secretion, impaired function in pancreatic beta cells, increased beta cell apoptosis and risk of insulin resistance by binding and activating nicotinic acetylcholine receptors (nachrs) which are part of the transmembrane group of protein chanel ions in the central nervous system and the peripheral nervous system. activation of nachrs results in the increase and release of catecholamines in the bloodstream which can result in increased blood pressure, free fatty acid, and blood sugar mobilization/in contrast to the effects of insulin. exposure to nicotine in a short time (more than 1 μmol/l) and within 48 hours can inhibit the release of insulin. the risk of people who smoke is 30-40% higher for dm than nonsmokers.21,38. most associated factor with tb-dm in tuberculosis patients at puskesmas patrang jember in 2017 the most associated factor with tb-dm in tb patients in patrang health center in jember regency in 2017 could be known through multivariate analysis. based on bivariate analysis, variables that could be analyzed using multivariate analysis were age, family history of dm, and smoking status. the result of multivariate analysis aims to get the most dominant independent variable related to the dependent variable by looking at the biggest or value. the analysis can be seen in table 6. based on table 6, it can be seen that the history of dm in the family was the most associated factor with tb-dm incidence. tb patients who had a family history of dm is at risk 18.250 times higher for getting tb-dm rather than tb patients with no family history of dm. table 4. bivariate analysis of the relation central obesity and tb-dm incidence in tuberculosis patients at puskesmas patrang jember in 2017 central obesity dm status p-value or (95% ci)tb-dm tb n n yes no 4 7 8 28 0,435 2,000 (0,465-8,597) 1 *significant ≤ α (0,05) significantly associated with tb-dm. they found a higher prevalence of treatment category 2 in patients with tb-dm (13.8%) than tb patients (7.9%). they also were stated that the risk of patient with category 2 treatment was 1.7 times more likely suffering tb-dm than patient with category 1 treatment.19 in this research, the prevalence of patient with treatment category 2 among tb group (4.3%) is higher than tb-dm group (2.1%). furthermore, the prevalence of patient with treatment category 1 among tb group (72.3%) is higher than tb-dm group (21.3%). relationship between central obesity and tb-dm incidence in tuberculosis patient at puskesmas patrang jember in 2017 cental obesity is fat accumulation in the abdominal area that exceeds normal value. respondents were classified as central obesity if the abdominal circumference is ≥90 cm for men and ≥80 cm for women. the relation between central obesity and tb-dm incidence is shown in table 4. in table 4, central obesity is not significantly related to tb-dm incidence (p-value = 0.435). this result is in line with a study by raghuraman et al (2014) who found central obesity was not statistically related to tbdm incidence.6 in central obesity occurs fat accumulation in the body exceeds the normal value in the abdominal area.36 futhermore, fat in the stomach causes fat cells to secrete proinflammatory chemicals that make the body less insensitive to insulin by disrupting the function of insulin responsive cells and the ability of cells to respond insulin. the more fat deposits in the stomach, the more difficult the insulin works so that blood sugar rises.15,37 but, in this research, majority of respondent or 74.5% are not categorized to central obesity that respectively is 59,6% for tb patients and 14,9% for tb-dm patients. research by raghuraman et al was also found that majority of respondent were not categorized to central obesity (90,3%).6 relationship between smoking behaviour with tb-dm incidence in tuberculosis patients at puskesmas patrang jember in 2017. tb-dm risk factors also were influenced by smoking behaviour variables. smoking behavior variables in this study include smoking status, number of cigarettes, and smoking duration. the results of the bivariate analysis between smoking behaviour and tb-dm incidence are listed in table 5. in table 5, only smoking status was associated with the incidence of tb-dm (p-value = 0.035). table 5. bivariate analysis of smoking behaviour with tb-dm incidence in tuberculosis patients at puskesmas patrang jember in 2017 smoking behaviour dm status p-value or (95% ci)tbdm tb n n smoking status yes no 3 8 1 35 0,035* 13,125 (1,203-143,233) 1 the number of cigarettes > 10 ≤ 10 1 2 1 0 1,000 smoking duration > 20 years ≤ 20 years 1 2 1 0 1,000 *significant ≤ α (0,05) 84 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 79–85 table 6. analysis of most associated factor with tb-dm incidence in tuberculosis patient at puskesmas patrang jember in 2017 variable coefficient p-value or confidence interval 95% age ≥ 45 < 45 2,875 0,016* 17,717 1 1,710-183,506 family history yes no 2,904 0,014* 18,250 1 1,813-183,708 smoking status yes no 1,841 0,174 6,303 1 0,444-89,439 *significant ≤ α (0,05) this result is similar with research by sirait et al that the risk of dm was 2.4 times greater in those whose parents had a history of dm compared with those whose parents had no history of dm.39 the magnitude of family history is possible because of a genetic defect of pancreatic β cells and is possible due to genetic abnormalities in the action of insulin in the presence of abnormalities in insulin receptors.40 conclusion the conclusion of this research is the prevalence of dm in tb patients in puskesmas patrang higher than prevalence of dm in general population in indonesia. in indonesia, east java is in the second place of highest tb cases under west java. futher the second city of highest tb cases in east java is found in jember especially in puskesmas patrang which not only tend to be in the top three highest cases of tb but also tend to be in the top three highest cases of dm. factors associated with tb-dm incidence are age, family history of dm and smoking status. factors unassociated with tb-dm incidence were gender, type of tb, treatment category, central obesity, number of smoked cigarettes and duration of smoking. factors most associated with tb-dm incidence is family history of dm. acknowledgement i would like to thank to the national unity and politics board of jember district, health office jember district, patrang health center of jember district and tb patients as my respondents who have helped my research process. references 1. kementerian kesehatan republik indonesia. infodatin tuberkulosis. jakarta: pusat data dan informasi kementerian kesehatan republik indonesia; 2016. 2. who. global tuberculosis report. geneva: who; 2016. 3. kementerian kesehatan republik indonesia. petunjuk teknis penemuan pasien tb-dm di fasilitas kesehatan rujukan tingkat lanjut. jakarta: kemenkes ri dirjen pengendalian penyakit dan penyehatan lingkungan; 2015. 4. baker ma, harries ad, jeon cy, hart je, kapur a, lӧnnroth k, et al. the impact of diabetes on tuberculosis treatment outcomes: a systematic review. bmc medicine. 2011 jul 1;9(81):1-15. 5. workneh mh, bjune ga, yimer sa. prevalence and associated factors of diabetes mellitus among tuberculosis patients in southeastern amhara region, ethiopia: a cross sectional study. pai m, editor. plos one. 2016 jan 25;11(1): e0147621. 6. raghuraman s, vasudevan kp, govindarajan s, chinnakali p, panigrahi kc. prevalence of diabetes melitus among tuberculosis patients in urban puducherry. journal of medical sciences. 2014 jan 27;6(1): 30-34. 7. mansuri s, chaudhari a, singh a, malek r, viradiya, r. prevalence of diabetes among tuberculosis patients at urban health centre, ahmedabad. international journal of scientific study. 2015 jul;3(4):115-118. 8. restrepo bi. diabetes and tuberculosis. microbiology spectrum. 2016 dec;4(6):1-19. 9. arliny y. tuberkulosis dan diabetes mellitus implikasi klinis dua epidemik. jurnal kedokteran syiah kuala. 2015 apr 1;15(1):3643. 10. lӧnnroth k, roglic g, harries ad. improving tuberculosis prevention and care through addressing the global diabetes epidemic: from evidence to policy and practice. lancet diabetes endocrinal. 2014 sep; 2(9):730-739. 11. kementerian kesehatan republik indonesia. profil kesehatan indonesia tahun 2016. jakarta: pusat data dan informasi kementerian kesehatan republik indonesia; 2017. 12. dinas komunikasi dan informatika jawa timur. masih tinggi, prevalensi diabetes di jatim [online]. surabaya: dinas komunikasi dan informatika pemerintah provinsi jawa timur; 2015 [cited 2017 jun 29]. available from:http://kominfo.jatimprov.go.id 13. dinas kesehatan provinsi jawa timur. profil kesehatan provinsi jawa timur tahun 2013. surabaya: dinas kesehatan provinsi jawa timur; 2014. 14. azila aa. gambaran kualitas hidup pasien diabetes mellitus tipe 2 di poli interna rsd dr.soebandi jember. skripsi. jember: program studi ilmu keperawatan universitas jember; 2016. 15. tandra, h. strategi mengalahkan komplikasi diabetes. jakarta: pt gramedia; 2014. 16. singal g, katuuk me, bataha yb. hubungan pengetahuan tentang terapi insulin dengan inisiasi insulin pada pasien diabetes melitus tipe 2 di rumah sakit pancaran kasih gmim manado. e-journal keperawatan (e-kp). 2017 mei; 5(1):1-7. 17. viswanathan v, kumpatla s, aravindalochanan v, rajan r, chinnasamy c, srinivasan r, et al. prevalence of diabetes and prediabetes and associated risk factors among tuberculosis patients in india. pai m, editor. plos one. 2012 jul 26; 7(7): e41367. 18. noureen. diabetes mellitus and associated risk factors among tuberculosis patients in rawalpindi, pakistan. biochem int j. 2017 mar 8; 1(4):1-6. 19. mi f, tan s, liang l, harries ad, hinderaker sg, lin y, et al. diabetes mellitus and tuberculosis: patern of tuberculosis, twomonth smear conversion and treatment outcomes in guangzhou, china. tropical medicine and international health. 2013 nov; 18(11): 1379-1385. 20. shabella r. terapi herbal buah sayuran untuk diabetes. klaten: cable book; 2013. 21. thapa b, paudel r, thapa p, poudyal ak. prevalence of diabetes among tuberculosis patients and associated risk factors in kathmandu valley. saarc journal of tuberculosis, lung diseases & hiv/aids. 2015 feb; 12(2):20-27. 22. irawan d. prevalensi dan faktor risiko kejadian diabetes melitus tipe 2 di daerah urban indonesia (analisa data sekunder 2007). tesis. depok: fakultas kesehatan masyarakat universitas indonesia; 2010. 23. alisjahbana b, van crevel r, sahiratmadja e, den heijer m, maya a, istriana e, et al. diabetes mellitus is strongly associated with tuberculosis in indonesia. int j tuberc lung dis. 2006 jun; 10(6):696-700. 85sasmita, et al.: prevalence and risk factors of diabetes mellitus 24. guptan a & shah a. tuberculosis and diabetes: an appraisal. indian journal of tuberculosis. 2000; 47(1):3-8. 25. broxmeyer, l. diabetes mellitus, tuberculosis, and the science of denial [online]. us: us library of congress; 2011 [cited 2017 jul 18]. available from: https://lawrencebroxmeyer.wordpress. com/2011/01/26/diabetes-mellitus-tuberculosis-and-the-science-ofdenial-by-dr-lawrence-broxmeyer/. 26. kementerian kesehatan republik indonesia. profil kesehatan indonesia tahun 2016. jakarta: kementerian kesehatan ri; 2017. 27. wahyu rt. hubungan kondisi fisik rumah dan praktik kesehatan dengan kejadian tb paru di puskesmas masopati kabupaten magetan [tidak diterbitkan]. 2008. 28. departemen kesehatan republik indonesia. buku saku kader program penanggulangan tuberkulosis. jakarta: direktorat jenderal pengendalian penyakit dan penyehatan lingkungan departemen kesehatan republik indonesia; 2009. 29. kartasasmita cb. epidemiologi tuberkulosis. sari pediatri. 2009 agt 2; 11(2):124-129. 30. ruswanto b. analisis spasial sebaran kasus tuberkulosis paru ditinjau dari faktor lingkungan dalam dan luar rumah di kabupaten pekalongan. tesis. semarang: magister kesehatan lingkungan universitas diponegoro; 2010. 31. mayasari r. kejadian tuberkulosis paru pada pekerja di laboratorium mikrobiologi rumah sakit umum pusat persahabatan jakarta. naskah publikasi. 2016 mar 14. 32. kementerian kesehatan republik indonesia. pedoman nasional pengendalian tuberkulosis. jakarta: dirjen pengendalian penyakit dan penyehatan lingkungan kementerian kesehatan ri; 2014. 33. alatas a. prevalensi tuberkulosis paru dengan bta positif pada penderita diabetes melitus tipe 2 di rumah sakit umum kota tangerang selatan tahun 2013. skripsi. jakarta: fakultas kedokteran dan ilmu kesehatan uin syarif hidayatullah; 2013. 34. mihardja l, lolong db, ghani l. prevalensi diabetes melitus pada tuberkulosis dan masalah terapi. jurnal ekologi kesehatan. 2015 des 9; 14(04):350-358. 35. suwanpimolkul g, grinsdale ja, jarlsberg lg, higashi j, osmond dh, hopewell pc, et al. association between diabetes mellitus and tuberculosis in united states-born and foreign-born populations in san francisco.caylà ja, editor. plos one. 2014 dec 5; 09(12): e114442. 36. djausal an. effect of central obesity as risk factor of metabolicsyndrome. medical journal of lampung university. 2015 jan; 4(3):19-22. 37. the global diabetes community. diabetes and obesity; 2017 [cited 2017 may 22]. available from: diabetes.co.uk. 38. ario md. effect of nicotine in cigarette for type 2 diabetes mellitus. j majority. 2014 des; 3(7):75-80. 39. sirait am, sulistiowati e, sihombingm, kusuma a, idayani s. insiden dan faktor risiko diabetes melitus pada orang dewasa di kota bogor studi kohor prospektif faktor risiko penyakit tidak menular. buletin penelitian sistem kesehatan. 2015 apr 8; 18(2):151160. 40. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care. 2010 jan; 33(1):562-569. 115 vol. 7 no. 5 may-august 2019 detection of tumor necrosis factor- (tnf-) gene promoters polymorphism among liver cirrhosis patients with chronic hepatitis b virus (hbv) infection in surabaya, indonesia citrawati dyah kencono wungu1,α, mochamad amin2, s. eriaty n. ruslan2, priyo budi purwono3, ulfa kholili4, poernomo boedi setiawan4, maria inge lusida2,3, soetjipto1,2, retno handajani1,2 1 department of medical biochemistry, medical faculty of universitas airlangga, surabaya 2 institute of tropical disease, universitas airlangga, surabaya 3 department of medical microbiology, medical faculty of universitas airlangga, surabaya 4 department of internal medicine, medical faculty of universitas airlangga dr. soetomo general hospital, surabaya α corresponding author: cicit.biokimia@gmail.com abstract polymorphisms in tnf-α gene promoter region are known of its role in the production of tnf-α which may influences the pathogenesis of liver disease. snps in positions 238 and 308 of tnf-α gene promoters may affect the production of these cytokines. this study was aimed to detect single nucleotide polymorphism (snp) on -238 and -308 positions in the tnf-α gene promoter among liver cirrhosis patients with hbv infection in surabaya, indonesia. this was descriptive exploratory research with cross sectional study design using serum liver cirrhosis patients with hbv infection in endoscopy outpatient clinic dr. soetomo general hospital, surabaya from april-may 2017. snps at -238 and -308 on tnf-α gene promoter (rs361525 and rs1800629 respectively) were detected using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) with primers specific for the tnf-α promoter region and restriction enzymes ncoi and mspi. the genotypes of tnf-α gene promoter were assessed according to the length of the fragments produced in rflp. serum tnf-α levels was measured by commercial elisa. in this study, as much as 149 positive hbsag patients was found in endoscopy outpatient clinic, dr. soetomo general hospital, surabaya. from those amount, as much as 30 liver cirrhosis patients with positive hbsag were obtained. from 2/30 (6.7%) patients showed the ga heterozygote snp either position -238 or -308. no patient had the aa genotype. median blood tnf-α level in women (38 ng / l) was higher than in men (33 ng / l). tnf-α levels in patients with ga heterozygote genotype at -238 and -308 in this research was not different than wild-type (gg genotype). among patients with liver cirrhosis due to chronic hbv infection in surabaya, indonesia, surabaya, we found ga polymorphisms the tnf-α promoter gene at positions -238 and -308 in 6.7% patients, and did not find homozygous aa polymorphisms. further studies including larger numbers of patients from various ethnic backgrounds in indonesia are needed to provide robust data on tnf-α gene promoter polymorphisms and their role in the pathogenesis of liver cirrhosis with hbv infection in this country. keywords: liver cirrhosis, hepatitis b virus, snp, tnf-α, pcr-rflp abstrak polimorfisme pada promotor gen tnf-α diketahui berperan pada produksi tnf-α yang selanjutnya berperan dalam patogenesis penyakit hepar, penyakit infeksi, serta inflamasi. snp gen tnf, terutama pada posisi 238 dan 308 dari promotor gen tnf-α telah diidentifikasi dapat memengaruhi produksi sitokin tersebut. penelitian ini merupakan penelitian pendahuluan yang dilakukan untuk mendeteksi single nucleotide polymorphism (snp) promotor gen tnf-α posisi -238 dan -308 dari sampel penderita sirosis hati dengan infeksi vhb di poli endoskopi rsud dr. soetomo, surabaya. jenis penelitian ini adalah penelitian deskriptif eksploratif laboratorik dengan rancangan penelitian cross sectional study yang mengambil sampel pasien penderita sirosis hati dengan infeksi vhb di poli endoskopi rsud dr. soetomo surabaya dari bulan april-mei 2017. snp posisi -238 dan -308 (rs361525 dan rs1800629) promotor gen tnf-α dideteksi menggunakan teknik polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) research report 116 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 115–121 dengan primer yang spesifik untuk daerah promotor gen tnf-α dan enzim restriksi endonuklease ncoi dan mspi. selanjutnya dilakukan penentuan genotipe promotor gen tnf-α sesuai dengan panjang fragmen yang dihasilkan pada rflp. kadar tnf-α serum diukur dengan menggunakan metode elisa. dalam penelitian ini, didapatkan sebanyak 149 penderita dengan hbsag positif di poli endoskopi rsud dr. soetomo surabaya. dari jumlah tersebut, didapatkan sebanyak 30 penderita sirosis hati dengan hbsag positif. dari 30 serum sampel penelitian ini, didapatkan sebanyak 6,67% menunjukkan genotipe snp heterozigot ga untuk promotor gen tnf-α baik posisi -238 maupun -308. tidak ditemukan genotipe aa pada penelitian ini. median kadar tnf-α pada pasien wanita (38 ng/l) lebih tinggi dibandingkan dengan laki-laki (33 ng/l). kadar tnf-α pada pasien dengan genotipe snp heterozigot ga pada posisi -238 dan -308 tidak berbeda secara signifikan dibandingkan dengan wild-type (genotipe gg). pada penderita sirosis hati dengan infeksi vhb di poli endoskopi rsud dr. soetomo, surabaya ditemukan snp heterozigot ga (6,67%) dan tidak ditemukan snp homozigot aa promotor gen tnf-α (-238 dan -308). studi lebih lanjut pada berbagai ras diperlukan untuk memberikan data yang jelas mengenai snp promotor gen tnf-α pada sirosis hati dengan infeksi vhb. kata kunci: sirosis hati, virus hepatitis b, snp, tnf-α, pcr-rflp introduction about 240 million people of the world are infected with chronic hbv and about 600,000 people die each year from diseases related to hbv infection and hepatocellular carcinoma (hcc)(1). the 5-year cumulative incidence of cirrhosis ranges from 8–20% in untreated chronic hbv patients and, among those with cirrhosis, the 5-year cumulative risk of hepatic decompensation is 20%2. although it is not fully understood, there are several factors suspected to affect the progression of hbv infection, including viral factors, environmental factors, and host genetic factors3. from these various factors, research on host factors has begun to be widely developed to understand the difference in progression and outcome of hbv infection in each patient4. under conditions of chronic hbv infection, th1 cytokines primarily played by tnf-α play a dominant role, especially in the immune clearance, inactive carrier, and reactivation phases5. chronic inflammation and hepatic infiltration of leukocytes increase the production of cytokines including tnf-α that trigger cell death thus increasing hepatic damage. high production of tnf-α can cause liver fibrosis through upregulation of timp-1 and prevent apoptosis of hepatocytes6. polymorphisms in tnf-α promoter genes are known to play a role in the pathogenesis of liver disease, infectious diseases, and inflammation3. several previous studies have identified the presence of multiple single nucleotide polymorphisms (snps) in the tnf gene group. tnf-α promoter polymorphisms can affect the transcriptional rate and, consequently, tnf-α cytokine production. g nucleotide transition to a in the promoter position -238 and -308 is known to affect the production of tnf-α7,8. data on factors related to the incidence of liver cirrhosis in patients with chronic hbv infection in indonesia are limited, eventhough this data is needed to understand the pathophysiology of liver cirrhosis development, so this study was conducted to detect the tnf-α gene promoter snp in liver cirrhosis patients due to chronic hbv infection in indonesia. materials and methods sampling. this research was a descriptive cross-sectional study and the samples were from liver cirrhosis patients with positive hbsag who visited the endoscopy outpatient clinic dr. soetomo general hospital, surabaya in aprilmay 2017. the inclusion criterias of this study were: chronic hepatitis b patients with a history of positive hbsag ≥ 6 months, with ultrasound diagnosed results from an internist who showed cirrhosis of the liver, the willingness to participate in all study subjects and must sign informed consent, adult patients (≥16 years), in conscious condition, and not in an emergency condition. exclusion criterias in this study were: patients coinfected with hcv or hiv, and received immunosuppressant therapy. the study was conducted after obtaining approval from the research ethics committee of dr. soetomo general hospital, surabaya. blood collection was taken from cubital vein with 4 ml of blood put on a venoject tube with edta and 3 ml of blood put on a venoject tube without edta. blood samples were then taken to the laboratory of hepatitis in institute of tropical disease (itd) of universitas airlangga for laboratory examination. blood samples in the venoject tube with edta, plasma separation, pbmc isolation, and host genome extraction for pcr-rflp examination were performed, while blood samples in the venoject tube with edta, serum separation was performed for the tnf-α elisa examination. pbmc isolation. after plasma separation, the remaining part of blood was performed pbmc isolation using phosphate buffer saline (pbs) and ficoll-histopaque10779. the obtained pbmc was then transferred into a 1.5 ml eppendorf tube and stored in the -30°c at freezer. examination of serum tnf-α levels. serum tnf-α was examined using diagnostic kit: human tnf-α elisa kit with cat. no. e0082hu (bioassay technology laboratory, china). elisa was performed according to the procedures listed in the kit. optical density was measured using microplate reader: imark (biorad) s / n 117wungu, et al.: detection of tumor necrosis factor- (tnf-) gene promoters polymorphism 12908. tnf-α serum level was calculated by using online software: elisaanalysis.com. dna host extraction. the dna host was extracted using the qiaamp dna extraction kit (qiagen, inc., hilden, germany) with cat.no.51104 using procedures in accordance to the kit. controls were treated as the same as the sample. pcr tnf-α gene promoters. a total of 5 μl dna was used for amplification by pcr-rflp technique, using a pcr 2x pcr master mix solution (intron®) kit with ref no.25027. the pcr-rflp process for tnf-α gene promoter was carried out using: forward: 5’aggcaataggttttgagggccat -3 ‘and reverse primers: 5’-tcctccctgctccgattccg-3’ to identify -238 snp as well as the forward: 5’agaagacccccctcggaacc-3 ‘and reverse primers: 5’atctggaggaagcggtagtg -3’ to identify -308 snp. reaction mixture was made in 0.2 ml eppendorf tube with total volume of 50 μl for 1 sample. pcr was performed using the dna thermal cycler: applied biosystem veriti 96 well. for -238 snp, in the initial stage an initial denaturation was performed with 94°c for 5 min, followed by 40 pcr cycles in accordance to jamil et al with modifications: denaturation at 94°c for 30 s, annealing at 60°c for 30 seconds and elongation at 72°c for 40 seconds. at the end of the process, the final extension was done at 72°c for 7 minutes. for -308 snp, at the initial stage an initial denaturation was performed at 94°c for 5 minutes, was followed by 40 pcr cycles with the following details: denaturation at 94°c for 30 seconds, annealing at 58.5°c for 30 seconds and elongation at 72°c for 40 seconds. at the end of the process, the final extension was performed at 72°c for 7 minutes9. detection of pcr products with electrophoresis. pcr product was examined by electrophoresis using 2% agarose gel which indicated the expected band, ie 107 bp for -308 snp and 152 bp for -238 snp. 100bp ladder marker, the negative control, and the samples were put into agarose gel. electrophoresis results were visualized in the uv light and documented. incubation with restriction endonucleases. the pcr products of tnf-α gene promoter -238 was incubated with mspi restriction enzyme, while tnf-α gene promoter -308 was digested with ncoi restriction enzyme. incubation with restriction enzyme used protocols from manufacturers (new england biolabs) with a total reaction volume of 50 μl. incubation was performed at 37°c overnight. d e t e c t i o n o f p c r r f l p p r o d u c t s w i t h electrophoresis. pcr-rflp products were examined using 3% agarose gel. 20bp and 100bp ladder markers, pcrrflp products from samples, as well as negative control were incorporated into agarose gel wells. electrophoresis gel apparatus was run on 100 volts for approximately 25 minutes, then viewed under uv light and documented with doc printgraph gel ae-6933fxcf. snp analysis of tnf-α gene promotor. pcr-rflp product of tnf-α gene at -238 region showed a fragment of 152 base pair (bp) if there is snp (a allele) and fragment 132 and 20 bp if it is normal allele (g). if a band of 152 bp was found, the sample had homozygous aa allele. but, if the samples showed 152, 132, and 20 bp bands, the sample has ga heterozygous alleles. when two bands 132 and 20 bp were found, the sample has gg homozygous allele. pcr-rflp product of tnf-α gene at -308 region showed a fragment of 107 base pair (bp) if there is snp (a allele), and fragment 87 and 20 bp if it has normal allele (g). when there was a 107 bp band in the sample, the sample has homozygous aa allele. while the samples were showed 107, 87, and 20 bp, the samples has ga heterozygous alleles. if the sample showed two bands 87 and 20 bp, the sample has gg homozygous allele. results in this study, as many as 149 positive hbsag patients were cared for in the endoscopy outpatient clinic, dr. soetomo general hospital, surabaya. they were further screened to meet the inclusion and exclusion criteria resulting in blood samples of 30 liver cirrhosis patients with positive hbsag for more than 6 months table 1. sex and age characteristics of the patients sex number of patients median age age distribution < 40 41–50 51-60 >60 male 24 (80%) 49 6 7 6 5 female 6 (20%) 54 1 2 1 2 total 30 (100%) 7 9 7 7 among the patients with cirrhosis of the liver with hbv infection in the endoscopy outpatient clinic, dr. soetomo general hospital, surabaya in this study the youngest patient was 30 years old and the oldest patient was 71 years old. as shown at table 1, male patients were dominating (80%), especially in the 41–50 years age range. female patients in this study had a higher median with age than male, yet the number of female patients in this study was lower than male. table 2. ethnicity of the patients and time diagnosed with cirrhosis sex ethnicity time diagnosed (years) javanese other < 1 1–3 >3 male 23 1 12 9 3 female 6 0 4 2 0 total 29 1 16 11 3 as shown at table 2, samples in this study were predominantly obtained from patients of javanese ethnicity (97%). only 1 male patient was non-javanese (batak). most 118 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 115–121 patients, 16/29(53%), had also just recently been diagnosed with liver cirrhosis (<1 year). after separation of pbmc and hbv dna isolation, pcr was performed to amplify the tnf-α gene promoter of the -238 and -308 positions. all samples were showed positive pcr results. the result of pcr on tnf-α -238 region gene promoter gave positive result of 152 bp band (figure 1), while the result of pcr on tnf-α -308 region gene promoter gave positive result of 107 bp band (figure 2). figure 1. example of electrophoresis product of pcr promoter gene tnf-α position -238 description: lane 1: negative control; lane 2-8: samples with positive results; lane 9: marker figure 2. example of electrophoresis product of pcr promoter gene tnf-α position -308 description: lane 1: marker; lane 2-16: samples with positive results; lane 17: negative control furthermore, all samples were incubated with endonuclease restriction enzyme using mspi for -238 snp, and ncoi for -308 snp. incubation was performed overnight at 37°c, then the rflp product was visualized in 3% agarose gel. the determination of tnf-α gene promoter genotypes both in the -238 and -308 positions was based on the dna fragment formed. in the pcr-rflp position -238, the perfect cutting of mspi result that yielded the 132 and 20 bp fragments was showed the wild-type gg homozygous alleles, mspi partial cutting yielded three fragments 152, 132, and 20 bp were indicated ga heterozygotes, while intact 152 bp dna fragments which was not digested with mspi indicated homozygous aa. as shown at figure 3, in this study, we found samples with gg genotype (132 and 20 bp fragments) and ga genotype (152, 132, and 20 bp fragments), while no aa genotype was found. at the -308 pcr-rflp position, the perfect ncoi cutting yield of 87 and 20 bp fragments showed a wildtype gg homozygous allele, ncoi partial cutting yielded of three fragments 107, 87 and 20 bp were indicating ga heterozygotes, while intact 107 bp dna fragments undigested with ncoi showing homozygous aa. as shown at figure 4, in this study, we found samples with gg genotype (87 and 20 bp fragments) and ga genotype (107, 87, and 20 bp fragments), while no aa genotype was found. figure 3. pcr-rflp products of tnf-α promoter on -238 position description: lane 2-5, 7, 8, 11 and 12: gg genotype with 2 fragments (132 bp and 20 bp); lane 6 and 10: ga genotype with 3 fragments (152 bp, 132 bp, and 20 bp); lane 1: dna marker 100 bp; lane 9: dna marker 20 bp figure 4. pcr-rflp products of tnf-α promoter on -308 position description: lane 1-5, 7, 8, and 11: gg genotype with 2 fragments (87 bp and 20 bp); lane 9 and 10: ga genotype with 3 fragments (107 bp, 87 bp, and 20 bp); lane 6: dna marker 100 bp; lane 12: dna marker 20 bp from 30 patients in this study, 2 (6.7%) patients were showed ga heterozygote genotype on -238 tnf-α promoter snp. no sample was found with aa homozygous snp. the remaining 28 samples were showed genotype of wild-type gg. for -308 positions, 2 of 30 samples (6.7%) showed ga heterozygote snp genotype. these were not the same patients that had ga at position -238. no sample was found with aa homozygous snp genotype. the remaining 28 samples were showed a genotype of wild-type gg alleles. ga heterozygote samples for the -238 and -308 snp were sampled with different numbers. no sample was found with two snp-228 and -308 simultaneously in this study. 119wungu, et al.: detection of tumor necrosis factor- (tnf-) gene promoters polymorphism table 3. tnf-α levels by sex in liver cirrhosis patients with hbv infection sex number of patients median (ng/l) iqr male 24 (80%) 33 31,85 female 6 (20%) 38 627,525 table 4. tnf-α levels based on snp genotype of tnf-α gene promoter in liver cirrhosis patients with hbv infection genotype median (ng/l) iqr -238 gg 35 30,51 ga 41 -308 gg 35 28,36 ga 30 in this study, higher levels of tnf-α were obtained in women than in men. in 1 sample, tnf-α level even reached 1118.78 ng/l (table 3). patients with liver cirrhosis with gg genotype for both -238 and -308 positions in this study had no different levels of tnf-α than ga genotypes (table 4). discussion we were able to detect the presence of tnf-α promoter snp -238 and -308 in patients with liver cirrhosis due to chronic hbv infection using pcr-rflp. tumor necrosis factor-α is a major cytokine in the inflammatory response to infection. tnf-α normally functions to activate cellular immunity and to provide protection against microbes, but excessive levels of this cytokine will result in severe tissue damage, septic shock and even death10. schwabe11 suggests that in liver, tnf-α can induce cell death as well as hepatocyte proliferation. in hbv infection, tnf-α levels tend to increase and are associated with inflammation, fibrosis, and hepatic damage. it is also said that tnf-α can be used as a predictor of liver inflammation12. it was found that most liver cirrhosis patients with hbv infection in this study was male (80%). this is consistent with the demographic data in previous studies suggesting that patients with cirrhosis of the liver due to chronic hbv infection are more frequent men than women13–15. this may due to a protective role of estrogen that is prevents damage to the liver because estrogen inhibits the proliferation of hepatic stellate cells and fibrogenesis that play an important role in the course of cirrhosis. in animal models with liver cirrhosis, estradiol administration leads to decreases in type i and iii collagen and stellate cell proliferation16. polymorphisms at tnf-α promoters -238 and -308 have been associated with various diseases associated with severe inflammation, infection, and malignancy. researches on snp of tnf-α gene promoters in patients with hbv infection reported conflicting results. a study in china showed a low a allele frequency, which was 4.6% for position -238 and 7.4% for position -30817. another study in china also showed that no aa genotype found18. this distribution is different from that observed in tunisia, where the ga and aa genotype frequencies in liver cirrhosis with hbv infection are quite high, ie 38.8% g/a and 44.5 a/a for -308 snp, and 44.5% g/a and 33.3% a/a for -238 snp19. a study in turkey showed that aa genotype was found in healthy control individuals (6.7%), but none in hepatitis patients (0%).20 tnf promoter polymorphism was known to be ethnic-specific, so each region might has unique distribution of tnf-α snps21. in a recent study in brazil, among patients with hepatitis c (hcv) infection with mild fibrosis, a -308 a/a snp was found in 1.9% of the cases, and a g/a snp in 26.1%. in patients with severe fibrosis, -308 a/a snp in 0.8% while g/a snp was present 21% patients22. genotype aa is a rare genotype compared to genotype gg and ga23. also in this study the aa genotype was not found. it is said that the frequency of a alleles is much lower in asia than in other regions of the world.24 for indonesia itself, this is the first time that tnf-α promoter genes polymorphism was studied in hepatitis patients. however, there have been several studies on tnf-α promoter gene snp (-238, -308) in patients with other diseases such as acne vulgaris25, chronic obstructive pulmonary disease (copd) 26, and down syndrome27. in these studies, the frequency of aa genotype was also very low, even undetectable in in certain cases. also, the ga genotype was found in frequently. however, in all those studies, the level of tnf-α was not measured. the low frequency of snp in these researches could be due to the influence of indonesian race and ethnicity27. several previous studies on tnf-α snp in cirrhosis gave controversial results. some studies were showed that the g-308 allele is associated with low levels of tnf-α both in vitro 28 and in vivo29 however, some other studies did not support this phenomena30–32. studies of snp at position -238 have likewise produced conflicting results with regards to tnf-α levels. some studies were showed that having an a allele at this position is associated with elevated levels of tnf-α33, but other studies were showed that there is no relationship between the two34. in addition, studies have shown that a allele is associated with lower tnf-α levels17,35. the expression of tnf-α, just like other cytokines is tightly regulated both at the transcriptional and post-transcriptional level. the polymorphisms located within the regulatory regions of tnf-α have been reported to influence the expression and secretion of this cytokine35. in tnf-α -238 and -308 snp, the presence of the a-allele increases the binding of transcription factor to the promoter region of tnf-α, thereby altering its expression36. nonetheless, several studies in various infectious diseases have shown the importance of tnf g>a in disease susceptibility, albeit with currently unknown molecular mechanism37. in this study, patients with liver cirrhosis with the gg genotype both for the -238 and -308 positions actually had 120 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 115–121 levels of tnf-α comparable to those with the ga genotype. despite the associations between tnf promoter snps and disease, a direct impact of tnf promoter polymorphisms upon tnf transcription has not been conclusively demonstrated.35 it was also said that tnf-α expression may not be directly related to tnf-α promotor gene polymorphism, but requires further variation in adjacent genes. this is due to the location of the tnf-α gene close to the hla allele. as a result, pathological conditions might be caused by the variation in a linked gene that regulates the expression of this cytokine rather than due to polymorphism within the tnf-α gene itself.24 the presence of other polymorphisms in the tnf-α promoter region of the gene may also contribute to tnf-α expression34. finally, contradictory results may also be due to ethnic differences.17,28,34 given that in indonesia harbours various ethnic groups, there is a possibility of variation based on ethnic groups studied in indonesia, so further research is needed on the subject. conclusion in this pilot study, 2/30 of liver cirrhosis patients with chronic hbv infection in dr. soetomo general hospital, surabaya were ga heterozygote for the tnf-α promoters at positions -238 and -308. 2 patients with snp at position -238 and 2 patients with snp at position -308 were different samples. no genotype aa was found. tnf-α level in this study was found higher in women than men. tnf-α level in patients with ga heterozygote genotype at -238 and -308 tnf-α gene promoters in this study was not different than wild-type (gg). different results might also be due to racial or ethnic differences in each study. further studies including larger numbers of patients from various ethnicities are needed to provide solid evidence on the prevalence tnf-α promoters polymorphisms in liver cirrhosis patients with hbv infection in indonesia, and their relationship with the expression of tnf-α. conflict of interest the authors state that there is no conflict of interest in this study. acknowledgements we acknowledge universitas airlangga for research funder through medical faculty of universitas airlangga. the authors wish to thank each and every patients with liver cirrhosis who have contributed to give blood in this study. we also thank the staffs and medical providers at internal medicine department in dr. soetomo general hospital, surabaya. thank you also for the technicians and staffs in institute of tropical disease (itd) universitas airlangga which has helped the course of research, and various parties involved in this research that we can not mention one by one. references 1. yano y, utsumi t, lusida mi, hayashi y. hepatitis b virus infection in indonesia. world j gastroenterol. 2015;21(38):10714–20. 2. easl. easl 2017 clinical practice guidelines on the management of hepatitis b virus infection. j hepatol. 2017;67(2):370–98. 3. mathew s, abdel-hafiz h, raza a, fatima k, qadri i. host nucleotide polymorphism in hepatitis b virus associated hepatocellular carcinoma. world j hepatol. 2016;8(10):485–98. 4. zeng z. human genes involved in hepatitis b virus infection. world j gastroenterol. 2014;20(24):7696–706. 5. balmasova ip, yushchuk nd, mynbaev oa, alla nr, malova es, shi z. immunopathogenesis of chronic hepatitis b. world j gastroenterol. 2014;20(39):14156–71. 6. seki e, schwabe rf. hepatic inflammation and fibrosis: functional links and key pathways. hepatology. 2015;61(3):1066–79. 7. farid s, rashid l, swelam s. tumour necrosis factor-alpha gene expression in chronic hepatitis c virus infection . egypt j hosp med. 2013;51(april):395–404. 8. li x, liang c, parkman v, lv z. the association between tnfa 238a/g and 308a/g polymorphisms and juvenile idiopathic arthritis. medicine (baltimore). 2018;97(43):1–11. 9. jamil k, jayaraman a, ahmad j, joshi s, yerra sk. tnf-alpha -308g/a and -238g/a polymorphisms and its protein network associated with type 2 diabetes mellitus. saudi j biol sci [internet]. 2016;1–9. available from: http://dx.doi.org/10.1016/j. sjbs.2016.05.012 10. abbas ak, lichtman ah, pillai s, baker dl, baker a. cellular and molecular immunology. 8th ed. philadelphia: elsevier saunders; 2015. 11. schwabe rf. mechanisms of liver injury. i. tnf-alpha-induced liver injury: role of ikk, jnk, and ros pathways. ajp gastrointest liver physiol [internet]. 2006;290(4):g583–9. available from: http:// ajpgi.physiology.org/cgi/doi/10.1152/ajpgi.00422.2005 12. mourtzikou a, alepaki m, stamouli m, pouliakis a, skliris a, karakitsos p. evaluation of serum levels of il-6, tnf-alpha, il10, il-2 and il-4 in patients with chronic hepatitis. inmunología. 2014;33(2):41–50. 13. bobek v, kolostova k, pinterova d, kacprzak g, adamiak j, kolodziej j, et al. a clinically relevant, syngeneic model of spontaneous, highly metastatic b16 mouse melanoma. anticancer res. 2010;30(12):4799–804. 14. lee ss, byoun y-s, jeong s-h, kim ym, gil h, min b-y, et al. type and cause of liver disease in korea: single-center experience, 2005-2010. clin mol hepatol. 2012;18:309. 15. başyigit s, asiltürk z, sapmaz f, kefeli a, yeniova aö, uzman m, et al. hepatitis b virus is still the most common etiologic factor of liver cirrhosis: results from a single center in turkey. dicle med j / dicle tip derg [internet]. 2015;42(4):416–21. available from: http:// dergipark.gov.tr/doi/10.5798/diclemedj.0921.2015.04.0601 16. brady cw. liver disease in menopause. world j gastroenterol. 2015;21(25):7613–20. 17. wang b, wang j, zheng y, zhou s, zheng j, wang f, et al. a study of tnf-alpha-238 and -308 polymorphisms with different outcomes of persistent hepatitis b virus infection in china. pathology [internet]. 2010;42(7):674–80. available from: http://www.ncbi.nlm.nih.gov/ pubmed/21080879 18. xie hy, wang wl, yao my, yu sf, feng xn, jin j. polymorphisms in cytokine genes and their association with acute rejection and recurrence of hepatitis b in chinese liver transplant recipients. arch med res. 2008;39:420–8. 19. sghaier i, zidi s, mouelhi l, dabbech r, ghazouani e, brochot e, et al. the relationship between tnf alpha gene polymorphisms (-238/-308), tnf rii vntr (p75) and outcomes of hepatitis b virus 121wungu, et al.: detection of tumor necrosis factor- (tnf-) gene promoters polymorphism infection in tunisian population. gene [internet]. 2015;568(2):140–5. available from: http://dx.doi.org/10.1016/j.gene.2015.05.029 20. basturk b, karasu z, kılıc m, ulukaya s. association of tnfa à 308 polymorphism with the outcome of hepatitis b virus infection in turkey. infect genet evol. 2008;8:20–5. 21. luo m, yang y, luo d, liu l, zhang y, yang j, et al. tumor necrosis factor-alpha promoter polymorphism 308 g / a is not significantly associated with esophageal cancer risk: a literature search. oncotarget. 2016;7(48). 22. aroucha dc, carmo rf, silva lrv, lima re, mendonca furtado t, arnez le, et al. tnf-a and il-10 polymorphisms increase the risk to hepatocellular carcinoma in hcv infected individuals. j med virol. 2016;88(9):1587–95. 23. xia q, zhou l, liu d, chen z, chen f. relationship between tnfalpha gene promoter polymorphisms and outcomes of hepatitis b virus infections: a meta-analysis. plos one. 2011;6(5):1–8. 24. banday mz, balkhi hm, hamid z, sameer as, chowdri na, haq e. tumor necrosis factor-alpha (tnf-alpha)-308g/a promoter polymorphism in colorectal cancer in ethnic kashmiri population a case control study in a detailed perspective. meta gene [internet]. 2016;9:128–36. available from: http://dx.doi.org/10.1016/j. mgene.2016.06.001 25. mathar nrs. hubungan polimorfisme gen tumor necrosis factor alpha (tnf-αlpha) dengan akne vulgaris ringan di makassar [internet]. 2011 [cited 2017 nov 7]. available from: http://med.unhas. ac.id/ikkk/?p=273 26. tarigan ap, syafiuddin t, yunus f, suradi. association of tumor necrosis factor alpha and lymphotoxin alpha gene polymorphisms with the presence of chronic obstructive pulmonary disease. acta medica indones indones j intern med [internet]. 2015;47(4):283–90. available from: http://ajrccm.atsjournals.org/content/163/2/420. short 27. nurhajjah s, ratnaningrum sd, mundhofir fep, faradz sm. polimorfisme gen tnf-α -308 g>a pada penderita sindrom down. mka. 2014;37(1):44–9. 28. zhuang l, ma w, cai d, zhong h, sun q. associations between tumor necrosis factor-α polymorphisms and risk of psoriasis: a meta-analysis. plos one. 2013;8(12):1–7. 29. baghel k, rn s, a c, j a, hr k. tnf-alpha, il-6 and il-8 cytokines in relation to tnf-alpha-308 g/a polymorphism in postoperative sepsis [internet]. vol. 15, surgical infections. 2014. p. s11--s12. available from: http://ovidsp.ovid.com/ovidweb.cgi?t=js%7b&% 7dpage=reference%7b&%7dd=emed12%7b&%7dnews=n% 7b&%7dan=71437581 30. marotte h, arnaud b, diasparra j, zrioual s, miossec p. association between the level of circulating bioactive tumor necrosis factor α and the tumor necrosis factor α gene polymorphism at −308 in patients with rheumatoid arthritis treated with a tumor necrosis factor α inhibitor. arthritis rheum [internet]. 2008;58(5):1258–63. available from: http://doi.wiley.com/10.1002/art.23430 31. o’dwyer mj, mankan ak, ryan aw, lawless mw, stordeur p, kelleher d, et al. characterization of tumour necrosis factor-alpha genetic variants and mrna expression in patients with severe sepsis. int j immunogenet. 2008;35(4–5):279–85. 32. vikram nk, bhatt sp, bhushan b, luthra k, misra a, poddar pk, et al. associations of -308g/a polymorphism of tumor necrosis factor (tnf)-α gene and serum tnf-α levels with measures of obesity, intraabdominal and subcutaneous abdominal fat, subclinical inflammation and insulin resistance in asian indians in north india. dis markers. 2011;31(1):39–46. 33. scardapane a, breda l, lucantoni m, chiarelli f. tnf-α polymorphisms in juvenile idiopathic arthritis : which potential clinical implications? int j rheumatol. 2012;2012:1–16. 34. vázquez-huerta di, alvarez-rodríguez ba, topete-reyes jf, muñoz-valle jf, parra-michel r, fuentes-ramírez f, et al. tumor necrosis factor alpha -238 g/a and -308 g/a polymorphisms and soluble tnf-α levels in chronic kidney disease: correlation with clinical variables. int j clin exp med. 2014;7(8):2111–9. 35. tahan rr el, ghoneim am, mashad n el. tnf-α gene polymorphisms and expression. springerplus. 2016;5:1508–14. 36. arbab m, tahir s, niazi mk, ishaq m, hussain a, siddique m, et al. tnfa genetic predisposition and higher expression of inflammatory pathway components in keratoconus. inflamm pathw keratoconus onset. 2017;58(9):3481–7. 37. putri s, rasmiyyah s, amalia e, razari i, wicaksono bd. relationship between tnf-238g > a polymorphism and predisposition to pulmonary tuberculosis infection in the indonesian population ( a pilot study ). j kedokt yars. 2015;23(1):1–11. 57 vol. 7 no. 4 january-april 2019 research report prevalence of trichomoniasis in asymptomatic pregnant women population in bandung, west java, indonesia pati aji achdiat1, reiva farah dwiyana1, vina feriza1a, rasmia rowawi1, rm rendy ae1, oki suwarsa1, hendra gunawan1 1 dermatology and venereology department, medicine faculty, universitas padjadjaran a corresponding author: dr.vinaferiza@gmail.com abstract about 81% of pregnant women with trichomoniasis are asymptomatic, while trichomoniasis in pregnant women can increase the risk of complications, include premature rupture of membranes, preterm birth, and babies with low birth weight. trichomoniasis can also increase the risk of other sexually transmitted infections (stis) and human immunodeficiency virus (hiv) transmission. trichomoniasis case in pregnant women could be influenced by demographic characteristics,, the sexual behavior, and also the diagnostic method used. until now, there is no data about prevalence of trichomoniasis in pregnant women in indonesia. the aim of this research was to determine the prevalence of trichomoniasis in pregnant women in bandung, west java, indonesia. a descriptive cross-sectional study was performed in december 2016 until january 2017. the study participants were 50 pregnant women who visit antenatal care to obstetric and gynecology clinic of ’rumah sakit khusus ibu dan anak kota bandung’, and meet the inclusion and exclusion criteria, through consecutive sampling. the study participants had a history taking, venereological examination, and trichomonas rapid test from vaginal swabs. trichomoniasis in this study was diagnosed based on trichomonas rapid test, a test that uses color immunochromatographic, capillary flow, dipstick technology, and has high sensitivity and specificity in diagnosing trichomoniasis. almost all participants in this study were low risk pregnant women to have sti based on demographic characteristics and sexual behaviour. the positive trichomonas rapid test result was found from one of 50 study participants. in conclusion, prevalence of trichomoniasis in pregnant women in bandung was 2%. trichomoniasis case in low-risk pregnant women population is still found. keywords: pregnant women, trichomoniasis, trichomoniasis prevalence, trichomonas rapid test, sexually transmitted infection abstrak sekitar 81% ibu hamil dengan trikomoniasis tidak memberikan gejala, sedangkan kejadian trikomoniasis pada ibu hamil dapat meningkatkan risiko timbulnya berbagai komplikasi antara lain ketuban pecah dini, persalinan prematur, dan bayi dengan berat badan lahir rendah. trikomoniasis juga dapat meningkatkan risiko terkena infeksi menular seksual (ims) lain dan transmisi (human immunodeficiency virus) hiv. kejadian trikomoniasis pada ibu hamil dapat dipengaruhi oleh karakteristik demografi, perilaku seksual, dan metode diagnostik yang digunakan. sampai saat ini belum terdapat data mengenai prevalensi trikomoniasis pada ibu hamil di indonesia. tujuan penelitian ini dilakukan ialah untuk mengetahui prevalensi trikomoniasis pada ibu hamil di bandung, jawa barat, indonesia. penelitian deskriptif dengan desain potong lintang telah dilakukan pada bulan desember 2016 hingga januari 2017. peserta penelitian adalah 50 ibu hamil yang melakukan kunjungan kontrol kehamilan ke poliklinik kebidanan dan kandungan rumah sakit khusus ibu dan anak kota bandung, memenuhi kriteria inklusi dan eksklusi, berdasarkan urutan kedatangan. pada peserta penelitian dilakukan anamnesis, pemeriksaan venereologis, dan tes cepat trichomonas dari apus vagina diagnosis trikomoniasis pada penelitian ini ditegakkan menggunakan tes cepat trichomonas, yaitu suatu tes yang menggunakan teknologi ’dipstick’ berbasis imunokromatografi warna, serta memiliki sensitivitas dan spesifitas yang tinggi untuk mendiagnosis trikomoniasis. hampir seluruh peserta penelitian merupakan ibu hamil yang berisiko rendah terkena infeksi menular seksual berdasarkan karakteristik demografi 58 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january-april 2019: 57–62 dan perilaku seksual. hasil tes cepat trichomonas positif didapatkan pada satu dari 50 peserta penelitian. kesimpulan dari penelitian ini, prevalensi trikomoniasis pada populasi ibu hamil di bandung sebesar 2%. masih ditemukan kasus trikomoniasis pada populasi berisiko rendah. kata kunci: ibu hamil, trikomoniasis, prevalensi trikomoniasis, test cepat trichomonas, infeksi menular seksual introduction trichomoniasis is sexually transmitted infection (sti) caused by a parasite, trichomonas vaginalis (tv).1,2 based on the world health organization (who) meta analysis study in 2012, it is known that trichomoniasis is the world’s most common non-viral sti and one of four curable sti.3 in addition, the center for disease control and prevention (cdc) established trichomoniasis as one of the five neglected parasitic infections that became the priority of public health programs.4 in women, the disease causes inflammation primarily in the vagina,1 causing clinical abnormalities of dense, foul-smelling, yellow vaginal discharge, which may be accompanied by abdominal pain and dysuria.2,5,6 based on a study which conducted in 2013 at antenatal care (anc) clinics in iran, it is known that only 19% of pregnant women with trichomoniasis are symptomatic.7 in sustainable development goals (sdgs) established by the who in 2015, maternal health as well as the prevention of human immunodeficiency virus (hiv) infection/ acquired immunodeficiency syndrome (aids) are the main targets.8 if not adequately treated, trichomoniasis in pregnant women could cause complications in pregnancy, such as premature rupture of membranes (premature delivery), preterm labor, and low birth weight babies (lbw).1,9,10 trichomoniasis may also increase the risk of other stis and hiv transmission.4,6,7,11-13 based on prevalence studies which are conducted in 2005 from commercial sex workers population in ten cities/districts of indonesia, namely medan, tanjung pinang, palembang, west jakarta, semarang, banyuwangi, surabaya, bitung, jayapura, and bandung, the prevalence of trichomoniasis were between 3-33%, with the prevalence of commercial sex workers in bandung was 18%.9 there has been no report of trichomoniasis prevalence in pregnant women in indonesia. clinical diagnosis of trichomoniasis in women is difficult because of variation in signs and symptoms and the similarity to other stis, it really requires laboratory testing. laboratory tests used to diagnose trichomoniasis are microscopic examination of wet preparation, culture, rapid tests, and nucleic acid amplification tests (taan).1,7,10 trichomonas rapid test is a point-of-care examination with an immunochromatography-based detection system using monoclonal-specific antibodies to detect tv antigen. trichomonas rapid test results can be found within 10 to 30 minutes.10 in a previous study in women with trichomoniasis, the sensitivity of the examination based on trichomonas rapid test is 83%, culture 90%, and microscopic examination 56%. based on a study which is conducted by campbell, it is concluded that the trichomonas rapid test has good specificity and requires fewer human resources, that is why it is recommended for screening of low prevalence patient population.14 osom® trichomonas rapid test has been recognized by the united state (us) food and drug administration (fda) since 2004.10 this study was therefore conducted to determine the prevalence of trichomoniasis in pregnant women in bandung, west java, indonesia. material and method study methods the study was carried out in rumah sakit khusus ibu dan anak (rskia) bandung, west java, indonesia, which is the main maternity hospital in bandung. this study was a descriptive study using cross-sectional design conducted from december 29, 2016 to january 7, 2017. study participants the study participants were pregnant women who visited for anc in rskia bandung regardless of the age of pregnancy, and willing to follow the study after being given an explanation by signing the informed consent form. the selection of the study participants was done by consecutive sampling until the samples were met. based on the sample size formula, the study needs minimum 29 participants. pregnant women who has been used vaginal cleansers (vaginal douche) in the last three days, and who received metronidazole therapy in the last two weeks were excluded from the study. study procedure the study participants who had previously been examined based on rskia anc procedure, then performed: 1. history and replenishment of medical records of the study. 2. physical examination, venereological examination, and sampling of vaginal swab for trichomonas rapid tests. o s o m ® t r i c h o m o n a s r a p i d t e s t u s e s c o l o r immunochromatographic, dipstick technology. if tv is present in the sample, it will form a complex with the primary anti-trichomonas antibody coated on the nitrocellulose membrane. the positive result is indicated by the visible blue line along with the red control line (figure 1). 59achdiat, et al.: prevalence of trichomoniasis in asymptomatic pregnant women figure 1. trichomonas rapid test dipstick: the positive and negative result result and discussion participants of the study consisted of 50 pregnant women. trichomonas rapid test results based on demographic data and sexual behavior characteristics of study participants are shown in table 1 and 2. study participant with a positive test result of trichomonas positive was 24 years old, junior high school, unemployed (housewife), family earning above regional minimum wage (rmw) of bandung, and in first trimester of pregnancy. based on the venereological examination, the study participant had witish yellow and thick vaginal discharge. the youngest participant was 18 years old and the oldest was 43 years old. almost all participants were married (98%), most were senior high school graduated (42%), unemployed (76%), had family income less than bandung rmw (40%), and were in third trimester of pregnancy (66%). table 1. trichomonas rapid test results based on demographic data of study participants variable total n=50 trichomonas rapid test (+) (-) total n=1 % total n=49 % age (year old) >16-26 >26-45 20 30 1 0 100 0 19 30 38,78 61,22 education elementary school graduated junior high school graduated senior high school graduated university graduated 9 13 21 7 0 1 0 0 0 100 0 0 9 12 21 7 18,37 24,49 42,86 14,29 occupation unemployee employee civil servant commercial sex worker 38 10 1 1 1 0 0 0 100 0 0 0 37 10 1 1 75,51 20,41 2,04 2,04 income < rp 2.600.000 rp 2.600.000 – 4.500.000 ≥ rp 4.500.000 20 18 12 0 0 1 0 0 100 20 18 11 40,82 36,73 22,45 recent gestational age first trimester second trimester third trimester 3 14 33 1 0 0 100 0 0 2 14 33 4,08 28,57 67,35 table 2. trichomonas rapid test results based on sexual behaviour of study participants variable total n=50 trichomonas rapid test (+) (-) total n=1 % total n=49 % coitarche < 20 year-old ≥20 year-old 18 32 1 0 100 0 17 32 34,69 65,31 sexual partner 1 partner >1 partners 43 7 0 1 0 100 43 7 87,76 14,29 sexual partner stable partner husband boyfriend 49 1 1 0 100 0 48 1 97,96 2,04 not stable partner yes no 1 49 0 0 0 0 1 49 2,04 100 condom used no 45 1 100 44 89,80 yes: routine not routine 0 5 0 0 0 0 0 5 0 10,20 sexual orientation heterosexual bisexual lesbian 50 0 0 1 0 0 100 0 0 49 0 0 100 0 0 narcotic, smoking, & alcohol use no alcohol smoking 45 3 5 0 1 1 0 100 100 45 2 4 91,83 4,08 8,16 most (64%) of the study participants had coitarche at ≥20 years old. coitarche at 15 years old was the earliest and at 30 years old was the latest. there were seven (14%) participants who had more than one sex partners in life, six of them were known to marry twice, and only one participant (who is a commercial sex worker) had multiple sexual partners. the positive result of the trichomonas rapid test examination of this study was obtained in one (2%) of 50 study participants. this result is similar in studies which conducted by olowe et al.5 in 2012 on 100 pregnant women who obtain anc at the university of ladoke akintola university in nigeria, which trichomoniasis prevalence was 2%. in that study, the diagnosis of trichomoniasis was obtained by microscopic examination of wet preparations.5 the prevalence of trichomoniasis in pregnant women from various countries shows varying prevalence rates. the lowest prevalence has been reported for pregnant women was in south korea in 2013, which the prevalence of trichomoniasis (based on microscopic examination of wet preparations) was 0.6%,15 and the highest prevalence reported in the population of pregnant women in zambia, which the prevalence of trichomoniasis (based on polymerase chain reaction/pcr) was 32.2%.16 the results of trichomoniasis prevalence in pregnant women in other countries are varied, ranging from 3% in south korea in 2013 (pcr),15 3.3% in iran in 2010 (microscopic and culture),17 7.7% in brazil in 2009 (taan),18 8% in india in 2014 (microscopic 60 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january-april 2019: 57–62 wet preparation),19 9.9% in african republic in 1990 (culture),20 10.3% in nigeria in 2013 (microscopic wet preparation),21 to 41.4% in south africa in 1990 (taan).20 the prevalence of trichomoniasis in pregnant women is influenced primarily by demographic characteristics22 and the participants’ sexual behavior.23 in addition, the diagnostic method used may also affect the prevalence of trichomoniasis.13 some of the demographic factors significantly associated with trichomoniasis occurrence in pregnant women include active sexual age,22,24 low levels of education,22 as well as occupation as a prostitute.16 high rates of trichomoniasis in the active sexual population are associated with higher sexual activity, lack awareness of stis, as well as changes in vaginal microbiota (especially during menstrual periods).25 in a study of pregnant women in papua new guinea, it was found that pregnant women aged 24 years or older had twice the risk of having trichomoniasis compared to older adults.26 in this study, only 40% of participants were in active sexual age population. this fact illustrated that the study population of this study was not a high risk population. nonetheless, study participant with a positive test result of trichomonas was a 24 years old woman. this fact showed that the result of the study was in accordance with the characteristics of pregnant women at risk of trichomoniasis. low levels of education are associated with a high incidence of trichomoniasis. this is due to a relation between low levels of education with unsafe sexual behavior and the number of multiple sexual partners.26 based on study by allsworth et al., romoren et al., and miranda et al., pregnant women with education less than senior high school,27 junior high school,22 or only for eight years,18 were at higher risk of tv infection.18,22,27 in this study, more than 50% of participants had education higher than senior high school. this fact illustrated that the study population was not a high risk population based on education level. study participant with a positive trichomonas rapid test result in this study was known to have junior high school education, which was consistent with the characteristics of pregnant women at risk of trichomoniasi. based on the results of several studies, it is known that in unemployed pregnant women were at risk of tv infection. housewives belong to low-risk groups contracting stis. however, transmission can be obtained by sexual partners who act as bridging populations because they are associated with commercial sexual worker/prostitute (core population).29 based on studies conducted by madhivan et al.23 in 2006 in south india, it was found that as many as 74% of women with trichomoniasis were housewives. job that is considered to be at high risk for stis was commercial sex worker which is associated with unsafe sexual behavior.29 in a study performed by crucitti et al.16 in zambia, it was found that commercial sex workers were more likely to be infected with tv.16 about 76% of this study participants were housewives. participant with a positive test result trichomonas positive was a housewife. there was one participant who had a job as a prostitute but no trichomoniasis was found. further study is needed for trichomoniasis in relation with housewife work. trichomonas vaginalis infection can be a marker of high-risk sexual behavior.30 early coitarche may increase the risk of greater cumulative sexual exposure, thus increasing the risk of becoming infected with tv.23 in a study by madhivanan et al. 23 in 2006 in south india, twothirds of the women who had positive trichomoniasis had coitarche when less than 19 years old. participants in this study were (64%) coitarche at age ≥19 years. this fact illustrated that the study population was largely not a risk population based on coitarche. study participant with positive trichomonas rapid test result had coitarche in aged 18 years. this finding was suitable with the characteristic risk of trichomoniasis. based on the study of allsworth et al,27 it was concluded that women who had 3-5 sexual partners throughout their lives had a risk of trichomoniasis nearly nine times greater. rogers et al.12 showed the results of his study in baltimore in 2009 that women with trichomoniasis who had two or more previous sexual partners had a trichomoniasis risk almost three times higher than those with one sexual partner. most of the study participants (86%) have only one sexual partner. it also explains the low prevalence of trichomoniasis in this study. study participant with a positive trichomonas rapid test result in this study had a total sexual partner number of more than one person (three persons), which was fit with the characteristics of pregnant women at risk of trichomoniasis, more sexual partner–more risk of trichomoniasis. based on study by miranda et al.,18 it is known that pregnant women who have persistent sexual partners, are more at risk of being infected with tv. in this study, most of the study participants (98%) had one stable sexual partner (husband), including participant with a positive trichomonas positive test result. the relationship between trichomoniasis and the characteristics of sexual partners needs to be further investigated. based on univariate analysis by ambrozio et al.31 in 2016 from 19 municipalities in southern brazil, it was found that the absence of condoms during intercourse increased the risk of being infected with tv. in a study by swartzendruber et al.32 of african-american women with trichomoniasis in atlanta, it was found that there was no correlation between condom use and tv-infected risk. most (90%) study participants never used condoms during intercourse. participant with a positive test result of trichomonas rapid test, was known to never use condom. further study is needed on the use of condoms and the risk of trichomoniasis. it was found that pregnant women who used narcotics were nearly eight times more likely to be infected with tv than those who did not.18 based on studies of african61achdiat, et al.: prevalence of trichomoniasis in asymptomatic pregnant women american women, it was found that smoking and alcohol consumption was associated with an increased incidence of trichomoniasis. smoking is thought to affect the condition of the vagina to become susceptible to infection,16 whereas alcohol consumption may increase the risk of sti infection because the effects after taking it can increase sexual desire.30 most (90%) of the study participants had no history of smoking, alcohol consumption, or other drugs. this explains the participants in this study were at low risk of trichomoniasis. in this study, participant with a positive trichomonas rapid test was known to have a history of smoking and alcohol consumption. this finding was in line with the findings of the study described before. as known before, trichomoniasis prevalence in pregnant women is influenced by demographic characteristics,22 the participants’ sexual behavior,23 and the diagnostic method used.13 in this study, almost all participants were low risk pregnant women to have trichomoniasis based on demographic characteristics and sexual behaviour. however, the study participant with a positive test result of trichomonas rapid test was a woman with trichomoniasis risks: in active sexual age (24 years old), had low level of education (junior high school graduated), had early coitarche (at 18 years old), and had multiple sexual partners throughout her life (three partners). the diagnostic method that used (trichomonas rapid test) in this study was performed according to recommended procedures. this revealed that the method used in this study was probably not a factor that affects the low value of trichomoniasis prevalence. conflict of interest we have no conflict of interest to declare. acknowledgement this research is received funding from the faculty grant. conclusion trichomoniasis case in low-risk population is still found. considering trichomoniasis complications in pregnant women (premature delivery, preterm labor, and lbw) and other stis and hiv transmission risk after trichomoniasis infection, screening and treatment of trichomoniasis are necessarily included in anc program. reference 1. hobbs mm, sena ac, swygard h, schwebke jr. trichomonas vaginalis and trichomoniasis. dalam: holmes kk, sparling pf, stamm we, piot p, wasserheit jn, corey l, penyunting. sexually transmitted disease. edisi ke-4. new york: mcgraw-hill; 2008. h. 771-93. 2. kissinger p. trichomonas vaginalis: a review of epidemiologic, clinical and treatment issues. bmc infect dis. 2015;15:1-8. 3. newman l, rowley j, hoorn sv, wijesooriya ns, unemo m, dkk. global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting. plos one. 2015;10:10143304. 4. secor we, meites e, starr mc, workowski ka. neglected parasitic infections in the united states: trichomoniasis. am j trop med hyg. 2014: 800-4. 5. olowe oa, makanjuola ob, olowe r, adekanle da. prevalence of vulvovaginal candidiasis, trichomoniasis and bacterial vaginosis among pregnant women receiving antenatal care in southwestern nigeria. euro j micro immuno. 2014;4:193-7. 6. donbraye e, donbraye-emmanuel oob, okonko io, okedeji io, alli ja, dkk. detection and prevalence of trichomonas vaginalis among pregnant women in ibadan, southwestern nigeria. world appl sci j. 2010;1512-7. 7. manshoori a, mirzaei s, valadkhani z, arababadi mk, rezaeian m, zainodini n, dkk. a diagnostic and symptomatological study on trichomoniasis in symptomatic pregnant women in rafsanjan, south central iran in 2012-13. iran j parasitol. 2015;10;490-7. 8. united nations, department of economic and social affair. sustainable development goals. [diunduh 9 september 2016]. tersedia dari: http://www.un.org/sustainabledevelopment/ sustainable-development-goals/ 9. gerakan nasional penanggulangan hiv/aids. prevalensii infeksi saluran reproduksi pada wanita penjaja seks di medan, tanjung pinang, palembang, jakarta barat, bandung, semarang, banyuwangi, surabaya, bitung, jayapura, indonesia, 2005. [diunduh 31 maret 2016]. tersedia dalam: http://aids-ina.org/files/ publikasi/rti10kota2005.pdf. 10. hobbs mm, sena ac. modern diagnosis of trichomonas vaginalis infection. sex transm infect. 2013;89:434-8. 11. avidime s, sulayman hu, adesiyun ag. prevalence of trichomonas vaginalis and hiv co-infection among asymptomatic pregnant women in zaria, northern nigeria. j health res rev. 2014;1: 4953. 12. rogers sm, turner cf, hobbs m, miller wc, tan s, dkk. epidemiology of undiagnosed trichomoniasis in a probability sample of urban young adults. plos one. 2014;9:1-10. 13. saleh am, abdalla hs, satti ab, babiker sm, gasim gi, dkk. diagnosis of trichomonous vaginalis by microscopy, latex agglutination, diamond’s media, and pcr in symptomatic women, khartoum, sudan. diagnostic pathology. 2014;9:49. 14. campbell l, woods v, lloyd t, elsayed s, church dl. evaluation of the osom trichomonas vaginalis vaginitis in specimens from women with a low prevalence of infection. j clin microbiol. 2008;46:3467-9. 15. goo y, shin w, yang h, joo s, song s, ryu j, et al. prevalence of trichomonas vaginalis in women visiting 2 obstetrics and gynecology clinics in daegu, south korea. korean j parasitol. 2016;1:75-80. 16. crucitti t, jespers v, mulenga c, khondowe s, vandepitte j, et a. trichomonas vaginalis is highly prevalent in adolescent girls, pregnant women, and commercial sex workers in ndola, zambia. sex transm dis. 2010;37: 223-7. 17. nourian a, shabani n, fazaeli a, mousavinasab sn. prevalence of trichomonas vaginalis in pregnant women in zanjan, northwest of iran. jundishapur j microbiol. 2013;6:e7258. 18. miranda ae, pinto vm, gaydos ca. trichomonas vaginalis infection among young pregnant women in brazil. braz j infect dis. 2014;18:669-71. 19. deivam s, rajalakshmi r, priyadharshini s, seethalaksmi rs, balasubramanian n, brinda t, dkk. prevalence of trichomonas vaginalis infection among patients that presented to rural tertiary care hospital in tiruchirapalli, india in 2011 and 2013. int j pharm res health sci. 2013;2:255-60. 20. world health organization (who). global prevalence and incidence of selected curable sexually transmitted infections: overviews and estimates. geneva, switzerland, who. 2001. 62 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january-april 2019: 57–62 21. etuketu im, mogaji ho, alabi om, adeniran aa, oluwole as, ekpo um. prevalence and risk factors of trichomonas vaginalis infection among pregnant women receiving antenatal care in abeokuta, nigeria. j infect dis. 2015;9:51-5. 22. romoren m, velauthapillai m, rahman m, sundby j, klouman e, dkk. trichomoniasis and bacterial vaginosis in pregnancy: inadequately managed with the syndromic approach. bulletin of who. 2007. 85:297-305. 23. madhivanan p, bartman mt, pastuti l, krupp k, arun a, dkk. prevalence of trichomonas vaginalis infection among young reproductive age women in india: implications for treatment and prevention. sex health. 2009;6:339-44. 24. kementrian kesehatan, direktorat jendral pengendalian penyakit dan penyehatan lingkungan. pedoman nasional penanganan infeksi menular seksual. jakarta: kementrian kesehatan. 2011. 25. ambrozio cl, nagel as, jeske s, bragang gcm, borsuk s, villela mm. trichomonas vaginalis prevalence and risk factors for women in southern brazil. rev inst med trop sao paulo. 2016;58:61. 26. badman sg, vallely lm, toliman p, kariwiga g, lote b, pomat w, dkk. a novel point-of-care testing for sexually transmitted infections among pregnant women in high-burden settings: results of a feasibility study in papua new gunea. bmc infect dis. 2016;16:1-6. 27. allsworth je, ratner ja, peipert jf. trichomoniasis and other sexually transmitted infections: results from the 2001-2004 national health and nutrition examintaion surveys. j sex transm dis. 2009;36:738-44. 28. marconi c, duarte ts, da silva mg, marcolino ld, polettini j, goncalves ap, dkk. trichomonas vaginalis and chlamydia trachomatis prevalence, incidence and associated factors in pregrant adolescents from belem city, in the brazilian amazon. open journal of obstetrics and gynecology. 2015;5:677-87. 29. centers for disease control and prevention. sexually transmitted diseases treatment guidelines. morbidity and mortality weekly report (mmwr). department of health and human services; 2010. 30. verscheijden mma, woestenberg pj, van bethem bhb. sexually transmitted infections among female sex workers tested at sti clinics in netherlands, 2006-2013. emerg themes epidemiol. 2015;12:142. 31. ambrozio cl, nagel as, jeske s, bragan gcm, borsuk s, et a. trichomonas vaginalis prevalence and risk factors for women in southern brazil. rev inst med trop sao paulo. 2016;58:61. 32. swartzendruber a, sales jm, brown jl, diclemente rj, rose es. correlates of incident of trichomonas vaginalis infections among african american female adolescents. sex transm dis. 2014;41:240-5. ijtid vol 6 no 4 jan-maret 2017.indd 88 vol. 6. no. 4 january–april 2017 research report the antibacterial effect of roselle (hibiscus sabdariffa) extract against staphylococcus epidermidis in vitro terrence timothy evan lusida1a, bambang hermanto2, sudarno3 1 faculty of medicine, universitas airlangga, surabaya 2 department of pharmacology, faculty of medicine, universitas airlangga, surabaya 3 department of biochemistry, faculty of medicine, universitas airlangga, surabaya a corresponding author: terencelusida@gmail.com abstract infection of staphylococcus epidermidis is still a common problem in many hospitals. factor determining biofilm formation makes it harder for atibiotics to cure the infection. roselle (hibiscus sabdariffa), a well known traditional medicine plant, is a potential candidate as a drug againts infectious disease. the purpose of this research is to investigate the antibacterial effect of ethanol extract from roselle (hibiscus sabdariffa) calyx againts the growth of staphylococcus epidermidis. assessment for antibacterial effect is performed using broth diffusion method. the extract is made by maceration of the calyx of roselle in 96% ethanol. extracts with concentration of 125, 62.5, 31.25, 15.63, 7.81, 3.90, 1.95, 0.97, 0.48, 0.24 mg/ml are added into separated mueller-hinton broths (mhb), which have already been inoculated by staphylococcus epidermidis. as for bacterial growth control, we used mhb with bacterial inoculation, while sterility control we used mixture of extract and mhb. then from each broth, the solutions are added into separated nutrition agar plates. replications are done three times. clarity and bacterial growth are observed after 24 hours of incubation. however, clarity cannot be observed in 36 broth, but bacterial growth is observed on the plate for concentration 0.97, 0.48, and 0.24 mg/ml. therefore minimum inhibitory concentration (mic) cannot be determined because the extract’s color interfere the observation. while minimum bactericidal concentration (mbc), the last concentration before the concentration where the bacteria are still viable, is 1.95 mg/ml. based on the result of the research, the roselle calyx ethanol extract (hibiscus sabdariffa) through dilution method with a concentration of 1.95 mg / ml can kill staphylococcus epidermidis and in order to find mic in collored and turbid solution (before being incubated in incubator), we can consider using agar dilution methode or microdilution methode. keywords: hibiscus sabdariffa, antibacterial, staphylococcus epidermidis, biofilm, flavonoids abstrak infeksi staphylococcus epidermidis masih merupakan masalah umum yang ditemukan di banyak rumah sakit. kemampuan bakteri untuk membuat biofilm mempersulit atibiotik untuk menyembuhkan infeksi. rosella (hibiscus sabdariffa), tanaman obat tradisional yang umum beredar di masyarakat, adalah bahan yang berpotensial untuk dikembangkan menjadi obat untuk mengatasi infeksi. tujuan dari penelitian ini adalah untuk mengetahui efek antibakteri dari ekstrak etanol kelopak rosella (hibiscus sabdariffa) terhadap pertumbuhan staphylococcus epidermidis. penelitian ini merupakan penelitian eksperimental laboratorik dengan metode dilusi. ekstrak rosella dibuat dengan cara maserasi dari tampuk rosella dengan menggunakan etanol 96%. kemudian dilakukan pengenceran sebanyak 10 kali didalam 10 tabung. konsentrasi yang didapatkan adalah 125, 62.5, 31.25, 15.63, 7.81, 3.90, 1.95, 0.97, 0.48, 0.24 mg/ml. masing-masing tabung sudah berisi bakteri. sebagai kontrol tumbuh bakteri digunakan campuran bakteri dengan mueller-hinton broth dan kontrol sterilitas menggunakan cairan ekstrak dengan mueller-hinton broth. kemudian dari ke-12 tabung, dilakukan streaking pada media nutrient agar plate untuk melihat pertumbuhan dari bakteri. replikasi percobaan dilakukan sebanyak 3 kali hasil percobaan diamati setelah inkubasi selama 24 jam. hasil penelitian yang didapat, dari 36 tabung tidak dapat diamati kejernihan dari tabung. hal ini disebabkan warna dari ekstrak mengganggu dari kejernihan pengamatan sehingga tidak dapat ditentukan nilai dari konsentrasi hambat minimal (khm). kemudian dari nutrient agar plate, didapatkan pertumbuhan bakteri pada 89lusida, et al.,: the antibacterial effect of roselle with the increase in staphylococcus resistance and roselle medical potential, we need further research regarding the antibacterial effect of this plant. in this study, researcher aims to investigate the antibacterial effect of roselle extract on the growth of staphylococcus epidermidis in vitro. material and method plant material extraction the flowers of hibiscus sabdariffa were purchased from upt materia medica in batu small town, east java. the plant materials were taxonomically identified by a botanist at the same location. calyces of the plant were separated and ground to a fine powder. about 250 g dried powder was taken and soaked with 96% ethanol. the wet powder is put in a jar and as much as 1500 ml 96% ethanol was poured into the jar. the jar was then closed tightly for 24 hours and placed on the shaker with 50 rpm. after that the suspension was filtered and was placed into erlenmeyer. the sediment from the filtration was re-maceration with the same technique (1500 ml 96% ethanol). the ethanolic extract was evaporated with rotary evaporator for 2 hours and water bath for 1 hour. the final result from the extraction was 77 ml extract with concentration 250 g/ml. antibacterial assay prior to the experiment, a colony of s. epidermidis was subcultured in mueller hinton agar (mha) and incubated for 24 hours at 37 ºc. the bacteria were then adjusted by adding normal saline to be equivalent to 0.5 mcfarland standard which comprised 1.0 x 108. susceptibility testing procedure the experiment was repeated 3x. the extract was dispensed in 1 ml volume in each sterile tube with decreasing concentration starting from 125 g/ml. each tube was then inoculated by 1 ml volume of diluted s. epidermidis. the growth control tube consists of 1 ml inoculum and extract free medium while the sterility control contains 1 ml extract and 1 ml medium. all tubes were incubated at 37 ºc and mics were read after 24 hours of incubation. introduction bacterium is the microorganism that is most frequently found in human body. this microorganism often causes infection and medical problems. one of them is staphylococcus epidermidis. normally, this bacterium is present in healthy people and doesn’t cause infectious disease, however in certain condition like immunodeficiency syndrome, it can cause infectious disases.1 because of this condition, staphylococcus epidermidis is a common cause of nosocomial infections. as many as 40-90% of nosocomial infections associated with hospital tools are caused by this bacterium.2 this increases the patient’s health expenditure and duratiuon of staying in hospital. in america, 50-70% of the 16,000 cases of bacteremia by catheter infection in icu are caused by s. epidermidis with an additional cost of 37.000-39.000 us$ for each person.3 besides the high incidence rate, many strains of staphylococcus have antibiotics resistance like methicillin and vancomycin. this resistance is also associated with the bacteria’s ability to make biofilms.4 biofilm is an ability possessed by a certain kind of bacteria to bind and create a complex structure which is formed by its colonization. it has the ability to allow bacteria to develop resistance to host immune responce and antibiotics.5 therefore, the discovery of treatments to infectious diseases, in particular caused by s. epidermidis is very important. in addition to drug development, the use of medical plants as natural antimicrobial agents is gaining popularity. roselle (hibiscus sabdariffa) is a tropical and sub-tropical plant with a potential candidate in herbal medicine. it is commonly used to make form drink and pickle and it is used in folk medicine infor treatment of hypertention, liver disease and fever.6 several studies have been conducted on rosella and have shown various benefits for medical purpose. a research was conducted by majorie et al. bioactive substances in hibiscus like alkaloids, flavonoids, phenolics and biterpenoids ) may have antibacterial effect agains esherichia coli.7 anthocyanin and protocatechuic acid compounds also isolated from dried flower of hibiscus sabdariffa demonstrate protective effects against oxidative agents.6 moreover, a research also conducted found that the protocatecuic acid also inhibited the growth of methicillinresistant s. aureus, klebsiella pneumonia, pseudomonas aeruginosa, and acinetobacter baumanniliu, tsao, yin).8 konsentrasi 0.97, 0.48, dan 0.24 mg/ml. hal ini berarti bahwa konsentrasi bunuh minimal (kbm) dari ekstral rosella adalah 1.95 mg/ml. berdasarkan hasil penelitian yang dilakukan, ekstrak tampuk rosella dengan menggunakan metode dilusi dapat membunuh bakteri staphylococcus epidermidis pada konsentrasi1.95 mg / ml dan untuk memperoleh hasil khm pada larutan yang berwarna dan keruh (sebelum diinkubasi dalam inkubator). dapat dipertimbangkan untuk menggunakan metode dilusi agar atau mikrodilusi. sehingga rosella memiliki efek antibakterial dan memiliki potensi yang besar sebagai bahan antimikroba. kata kunci: hibiscus sabdariffa, antibacterial, staphylococcus epidermidis, biofilm, flavonoids 90 indonesian journal of tropical and infectious disease, vol. 6. no. 4 january–april 2017: 88–91 result a total of 30 tubes were tested for mic. the result of the mic is shown in table 1. overall, by using macrodilution methode the mic couldn’t be determined. this result happens because the extract’s red color and turbidity interfere with the observation and the assessment. the same conditon could also be seen in sterility conttrol tube because of that it couldn’t be used as a comparation to determine sterility. so in order to determine the extract’s efficacy, each of the 30 tubes was streaked into nutrient agar plate to determind mbc value which was shown in table 2. from the experiment, it can be determind that the mbc of roselle extract against s. epidermidis is 1.95 mg/ml (1.56%). discussion roselle is a great plant to be used for medical purpose. first, it is easily grown in tropical country like in indonesia and has many properties. the time required to grow is around 4 to 8 months with the lowest temperature 20°c, 13 hours lighting, and 130 to 250 mm of rainfall for each month.9 with these condition, people can easily get the plant and cultivate it. second, it is known to have many good effects. roselle has antimicrobial, anti-parasite, anticancer, anti-pyretic, anti-inflamation, anti-oxidant, nephroprotective, hepato-protective, diuretic, anti-cholesterol, antidiabetic, and antihypertensive.6, 10-11 from the result, the mic in the experiment couldn’t be indentified because of the extract’s red colour and turbidity. to find out the result of the mic, extract roselle can be tested with another dilution methode. in nigeria, research was conducted by mary12. she did mic testing by agar dilution methode. she examined the antimicrobial effect of roselle against staphylococcus aureus, bacillus stearothermophilus, micrococcus luteus, serratia mascences, clostridium sporogenes, escherichia coli, klebsiella pneumoniae, bacillus cereus, and pseudomonas fluorescence with mic 0.30 ± 0.2 1.30 ± 0.2 mg/ml. a similar research was conducted by sulistyani13 and her research group with microdilution methode. they tested antimicrobial activity against mouth pathological bacteria that could make biofilm. these bacteria were streptococcus mutans, streptococcus sanguinis, lactobacillus casei, actinomyces naeslundii, aggregatibacter actinomycetemcomitans, fusobacterium nucleatum, porphyromonas gingivalis and prevotella intermedia with mic and mbc 7.2 mg/ml to 28.8 mg/ ml and 14.4 to >57.6 mg/ml. interestingly, roselle extract also has the ability to inhibit biofilm formation on the concentration of the mic.13 the formation of biofilm is also found in s. epidermidis.4 the overal mechanism how roselle extract has antibacterial effects is still not completely comprehanded. in usa, marjorie, janak, jacqueline, shurrita, and leonardo were conducted a research about the antimicrobial activity of hibiscus sabdariffa against e. coli. by using disk diffusion method, they concludedgain conclusion that all concentration (10%, 5%, and 2.5%) of h. sabdariffa could table 1. roselle extract’s mic extract concentration 100% (1) 50% (2) 25% (3) 12.5% (4) 6.25% (5) 3.12% (6) 1.56% (7) 0.78% (8) 0.39% (9) 0.19% (10) g+ s1 x x x x x x x x x x x x 2 x x x x x x x x x x x x 3 x x x x x x x x x x x x g+: growth control s-: sterility control x: can't be assessed table 2. roselle extract’s mbc extract concentration 100% (1) 50% (2) 25% (3) 12.5% (4) 6.25% (5) 3.12% (6) 1.56% (7) 0.78% (8) 0.39% (9) 0.19% (10) g+ s1 + + + + 2 + + + + 3 + + + + g+: growth control s-: sterility control +: viable bacteria -: no viable bacteria 91lusida, et al.,: the antibacterial effect of roselle references 1. madigan mt, martinko jm, bender ks, buckley dh, stahl da. brock biology of microorganisms. 14th ed. boston: benjamin cummings; 2014. 2. du x, zhu y, song y, li t, luo t, sun g, et al. molecular analysis of staphylococcus epidermidis strains isolated from community and hospital environments in china. yam w-c, editor. plos one. 2013 may 13;8(5):e62742. 3. lyte m, freestone ppe, neal cp, olson ba, haigh rd, bayston r, et al. stimulation of staphylococcus epidermidis growth and biofilm formation by catecholamine inotropes. lancet (london, england). 2003 jan 11;361(9352):130–5. 4. vuong c, otto m. staphylococcus epidermidis infections. microbes infect. 2002 apr;4(4):481–9. 5. vu b, chen m, crawford rj, ivanova ep. bacterial extracellular polysaccharides involved in biofilm formation. molecules. 2009 jul 13;14(7):2535–54. 6. da-costa-rocha i, bonnlaender b, sievers h, pischel i, heinrich m. hibiscus sabdariffa l. a phytochemical and pharmacological review. food chem. 2014 dec 15;165:424–43. 7. fullerton m, khatiwada j, johnson ju, davis s, williams ll. determination of antimicrobial activity of sorrel (hibiscus sabdariffa) on escherichia coli o157:h7 isolated from food, veterinary, and clinical samples. j med food. 2011 sep;14(9):950–6. 8. liu k, tsao s, yin m. in vitro antibacterial activity of roselle calyx and protocatechuic acid. phytother res. 2005 nov;19(11):942–5. 9. sallam mn, plotto a. post-harvest operations fao. hibiscus postproduction manag improv mark access. 2005;2–20. 10. w, itharat a. antipyretic activity of the extracts of hibiscus sabdariffa l. calyces in experimental animals. songklanakarin j sci technol. 2007;29(suppl. 1):29–38. 11. hh, aty oaa-e, morgan en, s.youssaf sm, mackawy amh. biochemical and ultra structure studies of the antioxidant effect of aqueous extract of hibiscus sabdariffa on the nephrotoxicity induced by organophosphorous pesticide (malathion) on the adult albino rats. j am sci. 2011;7(12):407–21. 12. tolulope m. cytotoxicity and antibacterial activity of methanolic extract of hibiscus sabdariffa. j med plants res. 2007;1(1):9–13. 13. sulistyani h, fujita m, miyakawa h, nakazawa f. effect of roselle calyx extract on in?vitro viability and biofilm formation ability of oral pathogenic bacteria. asian pac j trop med. 2016 feb;9(2):119–24. 14. alshami i, alharbi ae. antimicrobial activity of hibiscus sabdariffa extract against uropathogenic strains isolated from recurrent urinary tract infections. asian pacific j trop dis. 2014 aug;4(4):317–22. inhibit e. coli activity in from food, veterinary, and clinical samples and showed that the most effective concentration was at 10%, whereas the least effective concentration was at 2.5%. they were stated that the antimicrobial effect of h. sabdariffa might come from its flavonoids chemical. the structure of flavonoids have the ability to form combined complex with bacterial walls. besides that, the number of hydroxyl groups present on the phenolic ring helps the antimicrobial activity of the extract. due to the increase of hydroxyl group, the hydroxylation would accelerate and cause the increase of antimicrobial activity.7 issam and ahmed alsowere stated a similar discussion that phenolic compounds including flavonoids and cyaniding contribute to antimicrobial activity. they added that flavonoid with its phenolic chain could decreases iron level and increases hydrogen level, which deactivates bacterial enzymes.14 moreover sulistyani et al. reported that flavonoids are also thought to have the ability to inhibit the formation of bacterial biofilms. this capability is possible because the phenolic group in the extract capable to bind strongly to proteins and enzymes from the bacteria. this makes the bacteria unable to produce biofilms.13 this effect is important considering s. epidermidis and some grampositive and gram-negative bacteria capable of producing biofilms. based on the research that has been conducted, roselle calyx extract can be used as a alternative treatment for infections caused by staphylococcus epidermidis. conclusion the roselle calyx ethanol extract (hibiscus sabdariffa) through dilution method with a concentration of 1.95 mg / ml can kill staphylococcus epidermidis and in order to find mic in collored and turbid solution (before being incubated in incubator), we can consider using agar dilution methode or microdilution methode. indonesian journal of tropical and infectious disease this journal is a peer-reviewed journal established to promote the recognition of emerging and reemerging diseases spesifically in indonesia, south east asia, other tropical countries and around the world, and to improve the understanding of factors involved in disease emergence, prevention, and elimination. the journal is intended for scientists, clinicians and professionals in infectious diseases and related sciences. we welcome contributions from infectious disease specialists 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ijtid@itd.unair.ac.id 73 vol. 7 no. 3 september–december 2018 the risk factors for drug induced hepatitis in pulmonary tuberculosis patients in dr. soetomo hospital soedarsono1a, sari mandayani1, kinasih prayuni2, rika yuliwulandari2 1 department of pulmonology and respiratory medicine, faculty of medicine, universitas airlangga, dr. soetomo general hospital, surabaya, indonesia 2 biomolecular laboratory, yarsi university, jakarta, indonesia a corresponding author: ssoedarsono@gmail.com abstract tuberculosis (tb) is still a major public health problem in indonesia. anti-tuberculosis drug-induced hepatotoxicity (dih) is common side effect leading to changes in treatment regimens, and the less effective second-line treatments. several risk factors such as age, sex, body mass index (bmi) and acetylization status for hepatotoxicity were suggested in previous studies but in the fact, those are often not related to dih incidence after receiving standard tb treatment regimen. the aim of this study was to asses the role of risk factors in the dih incidence in pulmonary tb patients receiving standard tb treatment regimen in dr. soetomo hospital, surabaya. study design was analytic observational with case control. the subjects were 30 tb dih patients and 31 tb non-dih patients receiving standard national tb program therapy. dih severity was divided based on international dih expert working group. demographic data and bmi status were taken from medical records. the age classification are ≥35 years old and <35 years old as one of the risk factors studied. dna sequencing was used to assess single-nucleotide polymorphisms in nat2 coding region to evaluate acetylator status from blood samples. the risk factors were evaluated using chi-square test and mantel-haenszel test. significant association between low bmi and dih in general was identified (or=3.017; 95% ci=1.029-8.845) and more significant association between low bmi and moderate dih (or=15.833; 95% ci=1.792-139.922). age, sex, and acetylization status has no significant correlation with dih incidence in general. significant association between slow acetylator phenotype and incidence of moderate dih was identified (or=7.125; 95% ci= 1.309-38.711). in conclusion, some risk factors were correlated to dih incidence in pulmonary tb patients receiving standart tb treatment regimen. keywords: tuberculosis, drug-induced hepatitis, anti-tb drugs abstrak tuberkulosis (tb) masih merupakan masalah besar di indonesia. hepatitis imbas obat (hio) tb merupakan efek samping yang banyak terjadi dan dapat menyebabkan perubahan regimen pengobatan, dan penggunaan obat tb lini kedua yang kurang efektif. beberapa faktor risiko timbulnya hepatotoksitas seperti umur, jenis kelamin, indeks massa tubuh (imt) dan status asetilator telah diteliti pada penelitian-penelitian sebelumnya, namun pada kenyataannya, kadang faktor risiko tersebut tidak terbukti berhubungan dengan kejadian hio setelah mendapat pengobatan dengan regimen standar tb. studi ini dilakukan untuk menilai peranan faktor risiko tersebut terhadap insidensi hio pada pasien tb paru yang mendapat pengobatan regimen standar obat tb di rs dr. soetomo, surabaya. desain studi adalah observasi analitik dengan case control. subjek terdiri dari 30 pasien tb hio dan 31 pasien tb tanpa hio yang mendapatkan terapi standar obat anti tuberkulosis (oat) sesuai program nasional tb. derajat keparahan hio dibuat berdasarkan international dih expert working group. data demografi dan imt dikumpulkan dari rekam medik. kami membagi usia menjadi ≥35 tahun dan <35 tahun sebagai salah satu faktor risiko yang diamati. sekuensing dna single-nucleotide polymorphism regio nat2 dilakukan untuk menentukan status asetilator dari sampel darah. evaluasi faktor risiko dilakukan dengan menggunakan uji chi-square dan uji mantel-haenszel. korelasi signifikan terjadi antara imt rendah dengan timbulnya hio secara umum (or=3.017; 95% ci=1.029-8.845) dan antara imt rendah dengan timbulnya hio derajat sedang (or=15.833; 95% ci=1.792-139.922). usia, research report 74 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 73–79 jenis kelamin dan status asetilator tidak berkorelasi signifikan terhadap timbulnya hio secara umum. korelasi signifikan terjadi antara asetilator lambat dengan kejadian hio derajat sedang (or=7.125; 95% ci=1.309-38.711). kesimpulan pada penelitian ini adalah beberapa faktor risiko berhubungan dengan terjadinya hio pada pasien tb paru yang mendapatkan terapi regimen standar. kata kunci: tuberkulosis, hepatitis imbas obat, obat anti tb introduction drug induced hepatitis (dih) is a severe side effect of oral anti tuberculosis. anti tuberculosis drugs are metabolized mainly by the liver, and therefore, are potentially hepatotoxic. in dih, there is an elevated liver enzyme, such as aspartate amino transaminase (ast), alanin transaminase serum (alt), and total bilirubin increase as much as 3 times from their normal ranges.1 dih due to anti tuberculosis drugs, not only causing morbidity and mortality, but also changes the regimen and the less effective second-line treatment which eventually causing drugs resistance. tb dih is generally unpredictable and happens to a small number of patients, even after the patients received the standard dose. there are several factors which play roles in the susceptibility of a patient to develop dih such as age, sex, body mass index (bmi), and genetic like acetylator status of nat2 phenotype. the prevalence of dih is much higher in developing countries owing to several factors such as acute or chronic liver disease, alcoholism, malnutrition, indiscriminate drug use, advanced tb, and other co-existing chronic illness. anti tuberculosis drugs may cause hepatotoxicity ranging from a transient asymptomatic rise in liver enzymes to acute liver failure. the reported mortality from dih after the development of jaundice varies from 4% to 12%. it is noted that the frequency of dih in different countries varies widely from 2% to 39%.2 advanced age has been noticed to be associated with increased risk of dih after receiving anti tb drugs.3 a case control study is showed increased incidence of dih in the age group of 35-65 as opposed to the younger population.4 naqvi, et al5 has reported that age >35 years is one of important risk factors for tb dih. some studies have implicated female sex to be at increased risk for tb dih which were also shown from data analysis of various international registries.4,6 a few studies showed female patients were significantly higher than male patients.7,8 malnutrition contributes to increased incidence of dih after receiving anti tb drugs. malnutrition which measured in terms of hypoalbuminemia (serum albumin levels <3.5 g/dl) is predicted two-fold higher incidence of dih.4 makhlouf9 also reported that lower body mass index (bmi) of 20 kg/m2 or less were independent predictors of tb dih. isoniazid (inh) is one of hepatotoxic drugs in the standard tb treatment regimen. one of the gene which is important in the metabolism of isoniazid is nat2 (n-acetyltransferase 2) gene. nat2 codes the acetyltransferase enzyme which has part in the process of isoniazid acetylization by the hepatic enzyme. polymorphism in the different metabolism locus could cause a different pharmacologic response towards every individuals. pharmacogenetics and n-acetyltransferase are historically related, and in vivo variation in nat activity is one of the pharmacokinetics patterns, which is first recognized. the first action of nat2 enzyme was identified as the advanced step, generally controlled for isoniazid metabolism. the slow decay of acetylhidrazin, a toxic substance, from patients who are affected is known as the slow acetylator.10 the study based on genotyping of nat2 showed that slow acetylators had increased the risk of hepatotoxicity than rapid acetylators. furthermore, slow acetylators had more severe hepatotoxicity in comparison with rapid acetylators. this basis can be explained by the fact that slow acetylators also convert the toxic intermediate monoacetyl hydrazine to diacetyl hydrazine slowly which increases the risk of hepatotoxicity.11 a reviewed article written by saha et al12 was stated that isoniazid (inh), pyrazinamide (pza), and rifampicin (rif) used in tb dots (directly observed treatment short-course) as the main drugs are potentially hepatotoxic and may lead to dih. inh mechanism has been known in the incidence of dih through acetylator status, while the toxicity mechanism of rif and pza are still unknown. an article reviewed by tostmann et al2 stated that the enzymes involved pza-toxicity is not exactly known. a study reported that the risk of hepatotoxicity increases during the addition pza to inh and rif.13 in this perspective, we designed this study to know the possible risk factors such as age, sex, bmi, and acetylator status for the development of dih in patients receiving antitubercular treatment as per national tb control programme (ntp) strategy in dr. soetomo general hospital, one of the top referral in east indonesia. material and method the study was an analytical observational, using the case control design. samples were collected from all of pulmonary tb patients who suffered from dih, as well as those who did not have dih which had been treated with full term of standard regimen in dr. soetomo general hospital surabaya. all of pulmonary tb patients were tested for blood plasma, liver and kidney function as a 75soedarsono, et al.: the risk factors for drug induced hepatitis baseline test to make sure that there was no liver function impairment in all patients before receiving standard tb treatment regimen. according to the following biochemical criteria, hepatitis was defined as: 1) elevation of serum alanine transaminase (alt) and/or aspartate transaminase (ast) 3 times the upper limit of normal (uln), or 2) elevation of serum bilirubin uln and alt and/ or ast 2 uln (the uln was defined as 41 u/l for ast, 50 u/l for alt in men and 35 u/l in women, and 1.0 mg/dl for bilirubin in our laboratory) with hepatitis signs and symptoms. patients were not categorized as dih if during the whole tb treatment, there was no hepatitis occurred. pulmonary tb patients which have hepatitis a, b, and c, liver cirrhosis, hepatoma, cholelithiasis, and also hiv were not included in this study. dih pulmonary tb patients were divided into mild dih and moderate dih, based on the severity. the severity of dih was graded in accordance with international dih expert working group,1 where severity of dih was graded as followed: mild (elevated alanine amino transferase or alkaline phosphatase concentration reaching criteria for dih but bilirubin concentration <2 upper limit of normal (uln) and moderate (elevated alanine amino transferase or alkaline phosphatase concentration reaching criteria for dih and bilirubin concentration ≥2 uln, or symptomatic hepatitis). demographic data such as age, sex, weight and height on the initial treatment were collected from the medical records. liver function laboratory tests were done when the symptoms occurred such as nausea, and or followed by jaundice and an impaired liver function according to the dih criteria. blood serum from pulmonary tb patients who suffered from dih was sent to the biomolecular laboratory in yarsi research center jakarta to examine the nat2 genotype sequencing. this examination aim is to evaluate single-nucleotide polymorphism. non dih pulmonary tb patients were collected from the pulmonary tb patients who had been on full term of standard regimen and did not have dih. in the end of tb treatment, the blood serum was taken to examine the nat2 genotype sequencing. statistic test used in this research was chi-square test to analyze the significance of variables on dih occurrence. result and discussion the number of sample collected was 30 pulmonary tb patients with dih and 31 patients without dih. based on the sex, number dih and non dih pulmonary tb patients were found a bit higher in men than women, that were 17 (56.7%) and 19 (61.3%) for men, respectively. the average age and weight in pulmonary tb patients with dih were slightly higher compared than non dih pulmonary tb patients. the pulmonary tb patients with dih were averagely 45.10±14.31 years old, while non dih pulmonary tb patients were averagely 49.84±12.85 years old. the height of the pulmonary tb patients with dih was averagely 1.59±0.08 m and the non dih pulmonary tb patient’s height were 1.57±0.08 m in average. the average weight of dih pulmonary tb patients (43.83±6.65 kg) was lower than the patients without dih (47.83±8.77 kg). from the body mass index (bmi), the dih pulmonary tb patients were 17.24±2.73 kg/cm2, including 23.3% patients with low bmi, 43.3% with severely low bmi, and 33.3%with normal bmi. while, non dih pulmonary tb patients were 19.24±2.83 kg/cm2 in average, including 12.9% of the patients with low bmi, 25.8% with severely low bmi, and 61.3% with normal bmi (table 1). based on the nat2 haplotype reconstruction, 9 nat2 haplotype could be identified in which each of them consisted of 6 snp. each were suitable with several studies, table 1. demography profile and bmi status of dih and non dih tb patients character dih (n=30) non dih (n=31) sex (men/women) 17/13 19/12 age (year) 45.10 ± 14.31 (17-66)* 49.84 ± 12.85 (26-68)* height (meters) 1.59 ± 0.08 (1.45-1.9)* 1.57 ± 0.08 (1.45-1.75)* weight (kg) 43.83 ± 6.65 (30-58)* 47.83 ± 8.77 (33-65)* bmi (kg/m2) 17.24 ± 2.73 (10.53-22.66)* 19.24 ± 2.83 (13.28-25.11)* * mean ± sd (range) table 2. slow and rapid allele frequency no haplotype nomenclature code cases control case frequency control frequency p value allele prediction 1 ttcaag nat2*6a *6a 16 11 0.27 0.18 0.2816 slow allele 2 ttcagg nat2*6c *6c 12 10 0.20 0.16 0.6441 slow allele 3 ttcgaa nat2*7b *7b 10 10 0.17 0.16 1 slow allele 4 ttcgag nat2*13 *13 0 1 0.00 0.02 1 rapid allele 5 ttcgga nat2*7c *7c 0 1 0.00 0.02 1 slow allele 6 ctcagg nat2*6f *6f 1 1 0.02 0.02 1 slow allele 7 ctcgag nat2*4 *4 5 5 0.08 0.08 1 rapid allele 8 ctcggg nat2*12a *12a 9 19 0.15 0.31 0.0514 rapid allele 9 cctggg nat2*5b *5b 7 4 0.12 0.06 0.3625 slow allele total 60 62 1 1 76 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 73–79 were enough to predict the nat2 phenotype based on the database14 among all patients. the determination of slow and rapid acetylator was based on several references, it is known that nat2* 6a, nat2*6b, nat2*6c, nat2*6j, nat2*7b, nat2*5b, nat2*5c, nat2*5d, nat2*5j were slow acetylators, while nat2*4a, nat2*12a, nat2*12b, nat2*12c, nat2*13b, nat2*11a and nat2*13a were rapid acetylator.14,15 the frequent each haplotype was shown in table 2. the nat2*6a frequency was higher but not significant (p 0.2816). as well as nat2* 12a which was higher in non dih pulmonary tb patients, compared to the dih pulmonary tb patients (p 0.0514) as shown at table 2. in distribution bimodal model, the frequency of nat2 phenotype in dih pulmonary tb patients was 63.3% slow acetylator and 36.7% rapid acetylator. the nat2 frequency in non dih pulmonary tb patients was 38.7% slow acetylator and 61.3% rapid acetylator. ast profile in dih pulmonary tb patients were averagely 141.69±96.023 u/l, while the ones without dih were 33.67±12.95 u/l in average, in which statistically showed a significant difference with p=0.001. alt profile in dih pulmonary tb patients were averagely 101.02±129.329, while the ones without dih were averagely 28.8±18.68 with p=0.004. total bilirubin in dih pulmonary tb patients were 5.17±18.97 in average, while the non dih were 0.37±0.15 in average with p=0.005. ast, alt, and total bilirubin value were the criteria to define hepatitis. from the age and sex, statistically, there was no significance between dih and non dih pulmonary tb patients. but from ast, alt, and total bilirubin, there were significance with each p value as many as 0.001, 0.004, and 0.005. while from the acetylator status, there was also no significance between dih and non dih pulmonary tb patients. the data analysis above is revealed in table 3. a few studies showed the same result, such as where men were higher than women in getting dih, although it was not significant.5,16 several different studies reported that female sex were observed to be independent risk factors for the development of dih after receiving anti tb drugs.3,7,8 one study is showed that patient’s sex was not significantly associated with anti-tb-dih.17 review article table 3. statistic analysis of demography, bmi, laboratory liver function and acetylator status profile between dih and non dih pulmonary tb patients variables dih (n=30) non dih (n=31) p value sex (women/men) 13/17 12/19 0.714 age (≥ 35 y.o/<35 y.o) 22/8 27/4 0.176 bmi (low/normal) 20/10 12/19 0.029 ast* 141.69 ± 96.023* 33.67 ± 12.95* 0.001 alt* 101.02 ± 129.329* 28.8 ± 18.68* 0.004 direct bilirubin* 2.04 ± 5.68* 0.62 ± 0.25* 0.079 total bilirubin* 5.17 ± 18.97* 0.37 ± 0.15* 0.005 acetylator status (sa/ra) 19/11 12/19 0.054 * mean ± sd ; sa = slow acetylator; ra = rapid acetylator ; p value based on chi-square test written by devarbhavi18 stated that women had a generally higher risk for dih. studies from japan and sweden found women contributed to the number of dih cases as much as 58% and 56% respectively and may indicate demographic peculiarity of those countries. this was not corroborated by studies which are conducted from spain (49%), usa (48%) and india (42%).18 however, women have a higher risk for dih in most studies. the study which conducted by kumar et al19 stated that there is an unclear reason for female preponderance, but another epidemiological study reported that compared with age-matched men, reproductive-aged women had a faster progression of tuberculosis, from infection into active disease. other than that, females’ activity of cyp3a was higher compared with males, which could explain why females were more susceptible to anti tuberculosis drug induced hepatitis (atdh).2 in indonesia, tb is significantly more common among men than among women.20 therefore the susceptibility of tb dih in men was higher than woman in this study, although it was statistically not significant, suggesting that different sex was not correlated with the incidence of dih in this study. reviewed article written by ramappa and aithal21 showed that age has been associated with an increased risk of dih. age over 60 years was associated with a 3.5 fold risk of dih due to the anti tb drugs. in a case control study, patients who developed dih on anti-tb drugs were older (39 years) compared to those who did not (32 years). the incidence of hepatotoxicity was 17% in patients below 35 years of age and 33% in age above 35 years; in a multivariate analysis, age >35 years was the only independent variable for predicting anti-tb dih.21 elderly is associated with decreased liver blood flow, drug distribution and metabolism by cyp450 enzymes changes, resulting in a potentially lower effective clearance of the drugs.2 however, the susceptibility of elderly to adverse events and drug-induced liver disease is highly variable and could be different from the younger people.22 the difference between patients who were above and below 35 years old was not significant in the incidence of dih in general as well as in groups in mild or moderate dih. we suggest that the incidence of dih in this study may be correlated with other factors than age. 77soedarsono, et al.: the risk factors for drug induced hepatitis based on bmi status as stated in table 3, incidence of dih was associated with low bmi with p-value 0.029. other studies also collected the same data, where the low bmi subjects5 and those who had malnutrition2,8 were on the higher risk of getting dih after receiving standard tb treatment regimen. decreased xenobiotic clearance and higher plasma levels are the results of malnutrition or lower bmi results.2 besides, this might be due to glutathione stores depletion, which causes patients to be more vulnerable to oxidative injuries.16 inh has been known as one of the anti tb drugs which can cause dih through its acetylization metabolism. the study which was conducted by sistanizad et al11 showed that there was a different response in several individuals to the inh acetylization process, dih incidence was more frequent in patients who has slow acetylator than rapid acetylator. individuals who were categorized as slow acetylators in fact had a very slow n-acetyltransferase enzyme activity, caused by the genetic variation from the gene coding the expression of n-acetyltransferase enzyme. for individuals who had the abnormality caused by autosomal recessive allele, manifested as the polymorphic variation, the nat enzyme activity became very slow. thus, the ability for inh to be excreted in the form of inactive acetyl-inh was slow, making the inh had a long term of work, with the risk of an impaired function.4 previous reported studies showed that nat2 slow acetylator phenotypes are associated with disease risks and drug toxicity.23 the nat2 slow acetylator phenotypes have been investigated to have association with isoniazid-induced hepatotoxicity in tuberculosis treatment.24,25 in this study, the most nat2 of dih pulmonary tb patients had was nat2*6a, which was the slow acetylators haplotype, but statistically, there was no significant difference compared to the rapid acetylators with the incidence of dih. there were 11 of dih pulmonary tb patients who have rapid asetylator, assumed that other tb drug like pirazinamide (pza), rifampicin (rif) may played role in the incidence of dih. according to the previous research, rif plasma levels were higher in cases with dih than in controls and independently predicted subsequent develop ment of dih compared with inh and pza.26 in this study, there was no measurement of the rif concentration in dih patient’s blood plasma after receiving the standard tb regimen. review article written by tostmann et al2 stated that the mechanism of rifampicin-induced hepatotoxicity is unknown and unpredictable. there is also no evidence for the toxic metabolite presence. rifampicin is a dominant inducer of the hepatic cyp450 system in the liver and intestine, by that, it increases metabolism of many other compounds. the usage of rifampicin and isoniazid combination has been correlated with a higher risk of hepatotoxicity. rifampicin induces isoniazid hydrolase, causing an increased hydrazine production when rifampicin is combined with isoniazid (especially in slow acetylators), which may explain the higher toxicity of the combination. p z a h a s b e e n s h o w n t o i n c r e a s e t h e r i s k o f hepatotoxicity, adding pza to inh and rif increased the risk of hepatotoxicity appreciably.13 a study reported that among the 17 patients with hepatotoxicity, 12 patients are showed anti-tb dih. ten patients showed pzarelated hepatotoxicity and 2 showed inhor rif-related hepatotoxicity.27 although, review article written by tostmann et al2 stated that pza is converted to pyrazinoic acid and further oxidized to 5hydroxypyrazinoic acid by xanthine oxidase. the serum half-life of pyrazinamide is not related to the length of treatment, indicating that pyrazinamide does not induce the enzymes responsible for its metabolism. the mechanism of pyrazinamide-induced toxicity is unknown; it is unknown whether enzymes are involved in pyrazinamide-toxicity and whether toxicity is caused by pyrazinamide or its metabolites. in a rat study, pyrazinamide inhibited the activity of several cyp450 isoenzymes (2b, 2c, 2e1, 3a), but a study in human liver microsomes showed that pyrazinamide has no inhibitory effect on the cyp450 isoenzymes. dih as the side effect of pza and rif cannot be avoided, but it was difficult to decide which drug causing the incidence of dih because all tb patients who were included in this study were taking a combination of four anti-tb drugs: inh, rif, pza, and emb. therefore, we exclude the role of rif and pza, and focused on gene nat2 which is important in the metabolism of inh. the role of bmi, sex and ages which from several studies are known as the factors causing dih in patients receiving standard tb treatment regimen, even though it is still controversial and not one of them mentioned the specific correlation with one of the tb drugs. based on the severity, pulmonary tb patients with mild dih is higher than moderate dih, that are 19 patients (63.3%) and 11 patients (36.7%), respectively, shown in table 4. table 4. characteristic of and non dih and mild/moderate dih pulmonary tb patients based on demography, bmi and acetylator status profiles variables non dih (n=31) mild dih (n=19) moderate dih (n=11) p value p value sex (women/men) 12/19 10/9 0.336 3/8 0.496 age (≥ 35 years old/< 35 years old) 27/4 14/5 0.231 8/3 0.272 bmi (low/normal) 12/19 10/9 0.336 10/1 0.003 acetylators (slow/rapid) 12/19 10/9 0.336 9/2 0.014 78 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 73–79 based on the dih severity, there was still no significant difference between age and sex in both mild and moderate. but acetylator status showed a significance in moderate dih, while low bmi showed a more significance compared to dih in general with p 0.029 (table 3 and 5). according to mantel-haenszel test low bmi has a significant correlation in the incidence of moderate dih with p 0.003<0.05 (or=15.833; 95% ci: 1.792-139.922). likewise with the acetylator status and the incidence of moderate dih with p 0.014<0.05 (or=7.125; 95% ci: 1.309-38.711) as shown in table 5. the different study results with the previous study24 which confirms the significance of the association between slow-acetylator nat2 variants and susceptibility to atdh generally in an indonesian population may be caused by a greater number of samples used in previous study. while the study by lv et al28 did not find significant association between nat2 genotype and atdh in community-based chinese population, and based on sistanizad et al11 study also showed that slow acetylators are prone to develop more severe hepatotoxicity than rapid acetylators. this study has limited information data. there were no other risk factors which could affect the incidence of dih after standard tb treatment regimen, such as smoker, alcoholic, and the albumin level in each individual. also, as mentioned above, there was no measurement of rif concentration in patient’s blood plasma, as well as the pza metabolism measurement which was suspected as toxic to the liver. conclusion from this study, it was concluded that the low bmi rate is the factor which affects the most in the incidence of dih after receiving standard tb regimen. acetylization status in inh metabolism pathway, as in inh, which is one of the standard tb regimen in this study does not affect the dih in general, but still plays role in the incidence of moderate dih. further studies with a higher number of hepatotoxicity cases and simultaneous analysis of more risk factors factors such as smoking, alcohol consumption, and albumin levels, rif concentration measurement and pza metabolites in patient’s blood plasma should be carried out in different ethnic populations and in different regions of indonesia. from this study, we suggest that at least the practical aspect of tb treatment are used with regular monitoring of liver function on tb patients receiving standard tb regimen especially in patients with low bmi. references 1. aithal gp, watkins pb, andrade rj, larrey d, molokhia m, takikawa h, et al. case definition and phenotype standardization in drug-induced liver injury. clin pharmacol ther. 2011 jun; 89(6):806–15. 2. tostmann a, boeree mj, aarnoutse re, de lange wcm, van der ven ajam, dekhuijzen r. antituberculosis drug-induced hepatotoxicity: concise up-to-date review. j gastroenterol hepatol. 2008 feb; 23(2):192–202. 3. gaude gs, chaudhury a, hattiholi j. drug-induced hepatitis and the risk factors for liver injury in pulmonary tuberculosis patients. j fam med prim care. 4(2):238–43. 4. singla r, sharma sk, mohan a, makharia g, sreenivas v, jha b, et al. evaluation of risk factors for antituberculosis treatment induced hepatotoxicity. indian j med res. 2010 jul;132:81–6. 5. naqvi ih, mahmood k, talib a, mahmood a. antituberculosis drug-induced liver injury: an ignored fact, assessment of frequency, patterns, severity and risk factors. open j gastroenterol. 2015;5(12):173–84. 6. chalasani n, fontana rj, bonkovsky hl, watkins pb, davern t, serrano j, et al. causes, clinical features, and outcomes from a prospective study of drug-induced liver injury in the united states. gastroenterology. 2008 dec;135(6):1924–34, 1934-4. 7. an h, wu x, wang z, xu j, zheng s, wang k. the clinical characteristics of anti-tuberculosis drug induced liver injury in 2457 hospitalized patients with tuberculosis in china. african j pharm pharmacol. 2013;7(13):710–4. 8. lucena mi, andrade rj, kaplowitz n, garcía-cortes m, fernández mc, romero-gomez m, et al. phenotypic characterization of idiosyncratic drug-induced liver injury: the influence of age and sex. hepatology. 2009 jun;49(6):2001–9. 9. makhlouf ha, helmy a, fawzy e, el-attar m, rashed hag. a prospective study of antituberculous drug-induced hepatotoxicity in an area endemic for liver diseases. hepatol int. 2008 sep; 2(3):353–60. 10. mohan n, kumar j, chakrawarty a, ranjan p. comprehensive review of anti-tubercular treatment induced liver injury. int j basic clin pharmacol. 2015;397–403. 11. sistanizad m, azizi e, khalili h, hajiabdolbaghi m, gholami k, mahjub r. antituberculosis drug-induced hepatotoxicity in iraniantuberculosis patients: role of isoniazid metabolic polymorphism. iran j pharm res ijpr. 2011;10(3):633–9. 12. saha a, shanthi f x m, winston a b, das s, kumar a, michael js, et al. prevalence of hepatotoxicity from antituberculosis therapy: a five-year experience from south india. j prim care community health. 2016;7(3):171–4. 13. chang kc, leung cc, yew ww, lau ty, tam cm. hepatotoxicity of pyrazinamide: cohort and case-control analyses. am j respir crit care med. 2008 jun 15;177(12):1391–6. table 5. analysis of risk factors for dih p value (dih vs non dih) or 95% ci p value (moderate dih vs non dih) or 95% ci low bmi 0.029 3.017 1.029-8.845 0.003 15.833 1.792-139.922 slow acetylators 0.054 2.587 1.884-7.568 0.014 7.125 1.309-38.771 * odd ratio based on chi-square test with cochran’s and mantel-haenszel statistics 79soedarsono, et al.: the risk factors for drug induced hepatitis 14. nats: human na. greece: department of molecular biology and genetics democritus university of thrace. database of arylamine n-acetyltransferases (nats): human nat2 alleles [online].; 2013. 15. khan n, pande v, das a. nat2 sequence polymorphisms and acetylation profiles in indians. pharmacogenomics. 2013;14(3):289– 303. 16. devarbhavi h, dierkhising r, kremers wk, sandeep ms, karanth d, adarsh ck. single-center experience with drug-induced liver injury from india: causes, outcome, prognosis, and predictors of mortality. am j gastroenterol. 2010 nov;105(11):2396–404. 17. wondwossen abera, waqtola cheneke, gemeda abebe. incidence of antituberculosis-drug-induced hepatotoxicity and associated risk factors among tuberculosis patients in dawro zone, south ethiopia: a cohort study. int j mycobacteriology. 2016 mar;5(1):14–20. 18. devarbhavi h. an update on drug-induced liver injury. j clin exp hepatol. 2012 sep;2(3):247–59. 19. kumar r, shalimar, bhatia v, khanal s, sreenivas v, gupta sd, et al. antituberculosis therapy-induced acute liver failure: magnitude, profile, prognosis, and predictors of outcome. hepatology. 2010 may;51(5):1665–74. 20. kementrian kesehatan republik indonesia. the joint external tb monitoring mission (jemm tb). 2010;71–5. 21. ramappa v, aithal gp. hepatotoxicity related to anti-tuberculosis drugs: mechanisms and management. j clin exp hepatol. 2013 mar;3(1):37–49. 22. mitchell sj, hilmer sn. drug-induced liver injury in older adults. ther adv drug saf. 2010 dec;1(2):65–77. 23. thorn cf, aklillu e, mcdonagh em, klein te, altman rb. pharmgkb summary: caffeine pathway. pharmacogenet genomics. 2012 may;22(5):389–95. 24. yuliwulandari r, susilowati rw, wicaksono bd, viyati k, prayuni k, razari i, et al. nat2 variants are associated with drug-induced liver injury caused by anti-tuberculosis drugs in indonesian patients with tuberculosis. j hum genet. 2016 jun;61(6):533–7. 25. teixeira rldf, morato rg, cabello ph, muniz lmk, moreira a da sr, kritski al, et al. genetic polymorphisms of nat2, cyp2e1 and gst enzymes and the occurrence of antituberculosis druginduced hepatitis in brazilian tb patients. mem inst oswaldo cruz. 2011;106(6):716–24. 26. satyaraddi a, velpandian t, sharma sk, vishnubhatla s, sharma a, sirohiwal a, et al. correlation of plasma anti-tuberculosis drug levels with subsequent development of hepatotoxicity. int j tuberc lung dis. 2014 feb;18(2):188–95, i–iii. 27. jeong i, park j-s, cho y-j, yoon h il, song j, lee c-t, et al. druginduced hepatotoxicity of anti-tuberculosis drugs and their serum levels. j korean med sci. 2015 feb;30(2):167–72. 28. lv x, tang s, xia y, zhang y, wu s, yang z, et al. nat2 genetic polymorphisms and anti-tuberculosis drug-induced hepatotoxicity in chinese community population. ann hepatol. 11(5):700–7. 145 vol. 6 no. 6 september–december 2017 research report l o w c d 4 l y m p h o c y t e c o u n t r e l a t e d r i s k t o pneumocystis jiroveci pneumonia in hiv/aids patients from bronchoalveolar lavage specimens using real time pcr detection alicia m. widya1a, ni made mertaniasih2, arthur pohan kawilarang2, isnin anang marhana3 1 clinical microbiology study program, faculty of medicine universitas airlangga surabaya 2 department of medical microbiology, faculty of medicine universitas airlangga, dr. soetomo general hospital surabaya 3 department of pulmonology & respiratory medicine, faculty of medicine universitas airlangga, dr. soetomo general hospital surabaya a corresponding author: alicia.widya@yahoo.com abstract hiv and opportunistic infections remain a big problem especially in developing country. pneumocystis jiroveci pneumonia is a prevalent infection in hiv infected patient with high mortality rate. diagnosis of pneumocystis jiroveci pneumonia is mainly based on clinical evidence. microbiological diagnosis is quite challenging since this microorganism cannot be cultured and is mainly based on microscopic examination. microscopic examination with special staining is still a gold standard diagnosis for p. jiroveci infection. the objectives of this study was to describe cd4 lymphocyte profile and establish microbiological diagnosis with recent molecular method in pjp suspected hiv positive patients. fiberoptic bronchoscopy of hiv infected patients with lower respiratory tract infection in dr. soetomo general hospital surabaya were performed to collect bronchoalveolar lavage specimens from december 2016 to april 2017 for identification of pneumocystis jiroveci using real time pcr assay. positive samples were then evaluated for microscopic examination with gommori methenamine silver staining for comparison. patient’s cd4 lymphocyte count were gathered prior of admission. cd4 lymphocyte count from this study were very low with 61% of the patients were below 50 cells/ µl. there were five of total thirteen patients (38,5%) with positive real time pcr assay (msg gene) and one patient was also positive with gms staining showing characteristic cysts shape with dark centered area of p. jiroveci. patient with positive microscopic examination showed no history of prophylactic therapy. low cd4 lymphocyte count remains a strong risk factor of p. jiroveci pneumonia in hiv/aids patients. real time pcr assay shows high value in detection of p. jiroveci regarding patient’s prophylactic status. keywords: hiv/aids, pneumocystis jiroveci, pneumonia, low cd4 count, dr. soetomo hospital surabaya abstrak hiv dan infeksi oportunistik masih merupakan masalah yang banyak ditemui terutama pada negara berkembang. pneumocystis jiroveci pneumonia merupakan infeksi yang banyak ditemui pada pasien hiv dan memiliki angka kematian yang tinggi. diagnosis pneumocystis jiroveci pneumonia terutama berdasarkan gejala klinis. diagnosis mikrobiologi merupakan suatu tantangan tersendiri karena mikroorganisme ini tidak dapat dikultur dan diagnosis utamanya bergantung dari pemeriksaan mikroskopis. pemeriksaan baku emas untuk diagnosis infeksi p. jiroveci adalah dengan pemeriksaan mikroskopis dengan pewarnaan khusus. tujuan dari studi ini adalah untuk menggambarkan profil jumlah hitung limfosit cd4 pada pasien hiv dan menegakkan diagnosis mikrobiologis pjp dengan metode molekular terbaru pada pasien yang dicurigai infeksi pjp. pemeriksaan bronkoskopi fiber optik dilakukan pada pasien hiv dengan infeksi saluran nafas bawah di rsud dr. soetomo untuk mengumpulkan spesimen broncoalveolar lavage, spesimen klinis dikumpulkan dalam rentang waktu desember 2016 hingga april 2017 untuk dilakukan identifikasi p. jiroveci dengan pemeriksaan real time pcr. sampel dengan hasil positif kemudian dilakukan pemeriksaan mikroskopis dengan pewarnaan gommori methenamine silver sebagai perbandingan. pemeriksaan hitung jumlah limfosit cd4 dilakukan pada awal pasien masuk rumah sakit. hitung jumlah yang didapatkan pada studi ini didapatkan hasil sangat rendah, yaitu 61% didapatkan hitung jumlah limfosit cd4 di bawah 50 sel / µl. lima 146 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 145–149 introduction human immunodeficiency virus (hiv) is a virus that attack immune cells and weaken the immune system. cummulative data reported from indonesia ministry of health showed that there were 191,073 persons with hiv infection and 77,940 persons with aids.1 by 2014, there were estimated 37,600 persons with new hiv infection in usa. opportunistic infection rarely occur at early stage of hiv infection, the use of antiretroviral therapy can reduce the viral load in the patient and maintain immune system.2 in indonesia, antiretroviral therapy coverage is quite low (11.67%) compared with high income countries where the coverage is expected to be more than 45%.3 several studies shows that at least 33.3% hiv patients with antiretroviral therapy will experience at least one time opportunistic infection during the study period and the general prevalence of opportunistic infection in hiv persons is 42.8% including recurrent infection.2,4 pneumonia is one of the most prevalent opportunistic infection in hiv patients, it covers around 22.1% of the total opportunistic infection.4 the frequency of opportunistic infection may vary on each country because differences in genetic factor, environmental factor, and the people social background such as discrimination and stigmata which remain a potential difficulty in diagnosis and treating infection.5 pneumocystic jiroveci is an opportunistic pathogen that often occur in immunocompromised persons with high mortality rate and strongly related with hiv/aids condition.6 the chance of patient with hiv/aids will experience pneumocystis jiroveci pneumonia (pjp) was 75% in their course of the disease.7 multicenter study in korea showed that prevalence of pjp in hiv/aids patient were 11.1%. pjp is the third most prevalent opportunistic infection in hiv patient after candida infection and tuberculosis infection.8 chemoprophylaxis in pjp suspected hiv/aids patients is directed to prevent pjp infection but even with routine prophylaxis, the death of pjp related in hiv/aids patient is around 12% to 33% depending on resources and facility of the hospital where the patient admitted.9 material and method clinical specimens f i b e r o p t i c b r o n c h o s c o p y i n o r d e r t o c o l l e c t bronchoalveolar lavage is an invasive and expensive procedure especially for patient with respiratory problem. they were performed by pulmonologist and to minimize the risk factor for the development of adverse effects, the patients should have had minimal prerequisite lung function status, arterial blood gas recent data, platelet count and prothrombin time. a total of 13 bronchoalveolar lavage specimens from hiv/aids patients with pneumonia were collected in 5 month period from december 2016 until april 2017. the patient’s blood was taken and sent to the clinical pathology laboratory for cd4 lymphocyte count using flowcytometry method. this research has been approved by the hospital ethic committee no. 401/panke. kke/vi/ 2017. specimens processing bal specimens were centrifuged at 3000g for 15 minutes. supernatant were removed. after the removal of supernatant, 1 ml sedimentation were resuspended with mixed pipetting, aliquote of the sediment were smear on an object glass for gms staining and the rest of the sediment were transferred in a microcentrifuge tube and kept in -80°c freezer until dna extraction were performed. gms staining the bal smears were fixed in alcohol 95% overnight and then ready to be stained according to the staining procedure. real time pcr assay before the pcr assay, dna extraction were done with qiaamp dna mini kit (qiagen) according to the manufacturer’s procedure. the real time pcr assay were performed by roche molecular system using roche light cycler 2.0. the primers used were specific for major surface glycoprotein (msg) gene. the sequence of the primers were as follows: forward primer 5’caaaaataacaytsacatcaacragg-3’, reverse dari total 13 pasien didapatkan hasil positif pemeriksaan real time pcr (gen msg) dan salah seorang diantaranya didapatkan hasil positif dari pemeriksaan mikroskopis yaitu ditemukannya bentukan karakteristik kista dengan area kehitaman pada bagian tengah dengan pewarnaan gms. pasien dengan hasil positif pemeriksaan mikroskopis tidak memiliki riwayat terapi profilaksis sebelumnya. hitung jumlah limfosit cd4 merupakan faktor risiko yang kuat terhadap terjadinya p. jiroveci pneumonia pada pasien hiv/aids. pemeriksaan real time pcr menunjukkan nilai yang tinggi dalam mendeteksi p. jiroveci tanpa memandang status profilaksis pasien. kata kunci: hiv/aids, pneumocystis jiroveci, pneumonia, hitung jumlah limfosit cd4 rendah, rsud dr soetomo surabaya 147widya, et al.: low cd4 lymphocyte count related risk to pneumocystis jiroveci primer 5’-aaatcatgaacgaaataaccattgc–3’, and probe 5’– tgcaaaccaaccagtgcacgacagg – 3’. master mix was prepared, aliquote of the master mix was pipette 15 µl and added by 5 µl extracted sample. the reaction was consist of one cycle of denaturation in 95°c for 10 minutes, continue with 45 cycles of annealing at 95°c for 10 seconds and extention at 58°c for 1 minutes. all reactions were run simultaneously with positive and negative controls. prevention of contamination prevention of contamination including the use of aerosol barrier pipette tips, the use of separate areas of the laboratory for master mix preparation and specimens dna extraction. result and discussion of the 13 specimens tested, 61% of the blood specimens showed very low cd4 lymphocyte count below 50 cells/ µl (figure 1). the mean cd4 lymphocyte value was 82,69 cells / µl (table 1). this result is similar to a multicenter study in korea which stated that 65% patient with pjp showed very low count below 50 cells / µl.8 low cd4 lymphocyte count is a risk factor for pjp infection, other risk factor of pjp are p. jiroveci past infection, oral candidiasis, recurrent bacterial pneumonia, loss of body weight, high hiv viral load10 and genotipic relationship with mannose-binding lectin.11 real time pcr assay were performed on all bal specimens and 5 (38.5%) were positives (table 2). one specimen was positive with pcr assay and microscopy examination with gms staining (figure 2). bal is the best specimen for detection of p. jiroveci cyst because lavage in each lung segment can overcome more than 1 million alveoli, it is estimated that up to 3% of the lung tissue can be sampled.12 real time pcr assay has sensitivity up to 96% in detecting 70 cases of pjp with negative microscopy examination and 94% in detecting 71 cases of pjp with positive microscopy examination, this assay rarely resulting false positive.13 positive results must be interpreted carefully since this assay has high sensitivity value, a positive result without evident clinical symptoms and negative microscopy examination might be colonization or asymptomatic carrier of this microorganism.11 negative pcr assay can be concluded true negative.14 pjp-hiv/ aids patients with negative microscopy examination often are colonized with p. jiroveci.15 this might be true in this research since among the positives pcr result, there is only 1 positive microscopy examination. real time pcr is a semi quantitative method. the result of this assay can be concluded negative when there are no dna detected and no increase of cycle threshold (ct) value, on the other hand positive when the targeted dna is detected and there is an increase of cycle threshold value below 45 cycles. the less cycle the increasing of ct indicated the more targeted dna load in the sample.16 cycle threshold value positively correlated with the microorganism density in the sample.17 a cut off ct value of 32 cycle can be applied in differentiating colonization to p. jiroveci infection with sensitivity 72% and specificity 75%.17 table 2. real time pcr assay of bal specimens from hiv/ aids patients with pneumonia in dr. soetomo hospital, period december 2016 – april 2017 real time pcr pjp n % positive 5 38.5 negative 8 61.5 total 13 100 table 3. association of cd4 lymphocyte count with real time pcr p. jiroveci from hiv/aids patients with pneumonia in dr. soetomo hospital, period december 2016 – april 2017 real time pcr p. jiroveci total positive negative cd4 < 200 5 0 5 cd4 > 200 0 8 8 total 5 8 13 cd4 lymphocyte count and p. jiroveci as an agent of pneumonia show significant relationship with p value 0.002. opportunistic infection with low cd4 count below 200 cells / µl was dominated with p. jiroveci infection compare to other causes such as cryptococcus and toxoplasmosis.18,19 it is clinically evident that cd4 lymphocyte count can be use table 1. characteristic of cd4 lymphocyte count in hiv/aids patients with pneumonia in dr. soetomo hospital surabaya from december 2016 to april 2017 n mean median cd4 lymphocyte count 13 82.69 cells/ µl 15 cells/µl figure 1. distribution of cd4 lymphocyte count in hiv/aids patients with pneumonia from december 2016 to april 2017 real time pcr assay were performed on all bal specimens and 5 (38,5%) were positives (table 2). one specimen was positive with pcr assay and microscopy examination with gms staining (figure 2). bal is the best specimen for detection of p. jiroveci cyst because lavage in each lung segment can overcome more than 1 million alveoli, it is estimated that up to 3% of the lung tissue can be sampled.12 real time pcr assay has sensitivity up to 96% in detecting 70 cases of pjp with negative microscopy examination and 94% in detecting 71 cases of pjp with positive microscopy examination, this assay rarely resulting false positive.13 positive results must be interpreted carefully since this assay has high sensitivity value, a positive result without evident clinical symptoms and negative microscopy examination might be colonization or asymptomatic carrier of this microorganism.11 negative pcr assay can be concluded true negative.14 pjphiv/aids patients with negative microscopy examination often are colonized with p. jiroveci.15 this might be true in this research since among the positives pcr result, there is only 1 positive microscopy examination. real time pcr is a semi quantitative method. the result of this assay can be concluded negative when there are no dna detected and no increase of cycle threshold (ct) value, on the other hand positive when the targeted dna is detected and there is an increase of cycle threshold value below 45 cycles. the less cycle the increasing of ct indicated the more targeted dna load in the 61% 1, 8% 3, 23% 1, 8% cd4 lymphocyte count in hiv/aids patients with pneumonia < 50 sel/µl 50 100 sel/µl 100 200 sel/µl > 200 sel/µl figure 1. distribution of cd4 lymphocyte count in hiv/aids patients with pneumonia from december 2016 to april 2017 148 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 145–149 as a biomarker for immunodeficient condition and related to opportunistic infection.19 yanagisawa and nojima also stated that pjp prevalency in hiv/aids patient is 50% higher in cd4 lymphocyte count below 200 cells/ µl.11 trimethoprim-sulfamethoxazole is one of few prophylactic therapy used for pjp, prophylactic is usually started when hiv/aids patient with low cd4 lymphocyte count is admitted in the hospital, especially when this patient come with clinical symptom and radiologic supporting pneumonia. of all 13 patients, more than 50% already administered trimethoprim-sulfamethoxazole for prophylactic therapy, the fiber optic bronchoscopy procedure was performed after more than 2 days of prophylactic therapy. among the 7 patients with therapy, positive pcr result was found in 4 patients (table 4). one positive pcr result was found in non prophylactic patient. figure 2. gms staining of bal smear from positive real time pcr assay showing characteristic cyst shape with dark centered area the diagnosis of pjp relies on microscopy detection of characteristic shape of p. jiroveci with special staining such as gms, giemsa or immunofluorescence, this microscopy examination is difficult and quite challenging especially when the fungal load is low, false negative result is often detected.20 microscopy examination with gms staining has the same sensitivity on bal or induction sputa, table 4. real time pcr positivity value against prophylactic therapy in hiv/aids patients with pneumonia in dr. soetomo hospital surabaya n nilai rt pcr positif nilai rt pcr negatif terapi cotrimoxazole profilaksis 4 3 / 75% 1 / 25% terapi cotrimoxazole definitif 3 1 / 33.3% 2 / 66.7% tidak mendapatkan terapi 6 1 / 16.7% 4/ 83.3% this specimens are specimen of choice in establishing microbiological diagnosis of pjp.21,22 p. jiroveci can colonize patient with high risk condition such as chronic obstructive pulmonary disease, it is important to differentiate p. jiroveci as a infective agent or colonizer.23 trimethoprimsulfamethoxazole therapy might interfere with microscopy examination result because non viable microorganism might be not detected since the characteristic cyst shape is destroyed during therapy. the p. jiroveci dna can be detected even after prophylactic therapy but the microscopy examination can be difficult to achieve.16 conclusion the majority cd4 lymphocyte count in hiv/aids patient in dr. soetomo hospital is below 200 cells/ µl. lower cd4 lymphocyte count is a strong risk factor of p. jiroveci pneumonia in hiv/aids patients. real time pcr p. jiroveci is a valuable diagnostic method with 57% positivity detection for p. jiroveci on patients receiving prophylactic and definitive therapy. acknowledgement we are grateful to anis karuniawati, dr., sp. mk(k) and clinical microbiology laboratory universitas indonesia jakarta for the support to finish this research. we declare no conflict of interest. references 1. infodatin. situasi penyakit hiv/aids di indonesia. kementerian kesehatan ri. jakarta selatan; 2016. 2. moges na. prevalence of opportunistic infections and associated factors among hiv positive patients taking anti-retroviral therapy in debremarkos referral hospital, northwest ethiopia. j aids clin res. 2014;5(5). 3. gbd 2015 hiv collaborators. estimates of global, regional, and national incidence, prevalence, and mortality of hiv, 1980-2015: the global burden of disease study 2015. lancet hiv. 2016 aug;3(8):e361–87. 4. bhaumik p, debnath k, sinha b. spectrum of opportunistic infections among hiv / aids patients of tripura. journal, indian acad clin med. 2013;14:218–21. 5. gobel fa. stigma dan diskriminasi terhadap odha, tugas dan tanggungjawab siapa? [internet]. 2014. available from: http://www. kebijakanaidsindonesia.net/id/artikel/artikel-kontribusi/1005-stigmadan-diskriminasi-terhadap-odha-tugas-dan-tanggungjawab-siapa 6. mussini c, manzardo c, johnson m, monforte a d’arminio, uberti-foppa c, antinori a, et al. patients presenting with aids in the haart era: a collaborative cohort analysis. aids. 2008 nov 30;22(18):2461–9. 7. phair j, muñoz a, detels r, kaslow r, rinaldo c, saah a. the risk of pneumocystis carinii pneumonia among men infected with human immunodeficiency virus type 1. multicenter aids cohort study group. n engl j med. 1990 jan 18;322(3):161–5. 8. kim yj, woo jh, kim mj, park dw, song j-y, kim sw, et al. opportunistic diseases among hiv-infected patients: a multicenternationwide korean hiv/aids cohort study, 2006 to 2013. korean j intern med. 2016 sep;31(5):953–60. 149widya, et al.: low cd4 lymphocyte count related risk to pneumocystis jiroveci 9. bennett je, dolin r, blaser mj. mandell, douglas, and bennett’s principles and practice of infectious diseases. 8th ed. philadelphia: elsevier saunders; 2015. 10. morris a, lundgren jd, masur h, walzer pd, hanson dl, frederick t, et al. current epidemiology of pneumocystis pneumonia. emerg infect dis. 2004 oct;10(10):1713–20. 11. k y, y n. hiv and pneumocystis pneumonia (pcp): an upto date. j infect dis ther. 2015;3(6). 12. meyer kc. the role of bronchoalveolar lavage in interstitial lung disease. clin chest med. 2004 dec;25(4):637–49. 13. alvarez-martínez mj, miró jm, valls me, moreno a, rivas p v, solé m, et al. sensitivity and specificity of nested and real-time pcr for the detection of pneumocystis jiroveci in clinical specimens. diagn microbiol infect dis. 2006 oct;56(2):153–60. 14. fillaux j, malvy s, alvarez m, fabre r, cassaing s, marchou b, et al. accuracy of a routine real-time pcr assay for the diagnosis of pneumocystis jirovecii pneumonia. j microbiol methods. 2008 oct;75(2):258–61. 15. huang l, crothers k, morris a, groner g, fox m, turner jr, et al. pneumocystis colonization in hiv-infected patients. j eukaryot microbiol. 2003;50 suppl:616–7. 16. unnewehr m, friederichs h, bartsch p, schaaf b. high diagnostic value of a new real-time pneumocystis pcr from bronchoalveolar lavage in a real-life clinical setting. respiration. 2016;92(3):144–9. 17. fauchier t, hasseine l, gari-toussaint m, casanova v, marty pm, pomares c. detection of pneumocystis jirovecii by quantitative pcr to differentiate colonization and pneumonia in immunocompromised hiv-positive and hiv-negative patients. j clin microbiol. 2016;54(6):1487–95. 18. masur h, ognibene fp, yarchoan r, shelhamer jh, baird bf, travis w, et al. cd4 counts as predictors of opportunistic pneumonias in human immunodeficiency virus (hiv) infection. ann intern med. 1989 aug 1;111(3):223–31. 19. crowe sm, carlin jb, stewart ki, lucas cr, hoy jf. predictive value of cd4 lymphocyte numbers for the development of opportunistic infections and malignancies in hiv-infected persons. j acquir immune defic syndr. 1991;4(8):770–6. 20. robert-gangneux f, belaz s, revest m, tattevin p, jouneau s, decaux o, et al. diagnosis of pneumocystis jirovecii pneumonia in immunocompromised patients by real-time pcr: a 4-year prospective study. j clin microbiol. 2014 sep;52(9):3370–6. 21. caliendo am, hewitt pl, allega jm, keen a, ruoff kl, ferraro mj. performance of a pcr assay for detection of pneumocystis carinii from respiratory specimens. j clin microbiol. 1998;36(4):979–82. 22. pinlaor s, mootsikapun p, pinlaor p, phunmanee a, pipitgool v, sithithaworn p, et al. pcr diagnosis of pneumocystis carinii on sputum and bronchoalveolar lavage samples in immuno-compromised patients. parasitol res. 2004 oct;94(3):213–8. 23. maskell na, waine dj, lindley a, pepperell jct, wakefield ae, miller rf, et al. asymptomatic carriage of pneumocystis jiroveci in subjects undergoing bronchoscopy: a prospective study. thorax. 2003 jul;58(7):594–7. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 � vol. 1. no. 1 january–april 2010 research report medical audit of the management of patients with sepsis in the intermediate care unit of department internal medicine school of medicine airlangga university/dr. soetomo hospital usman hadi and erwin astha triyono department of internal medicine airlangga university/dr.soetomo hospital abstract sepsis and septic shock is one of the highest causes of death in patients treated in hospitals. research objectives: to evaluate the quality of the management of sepsis patients in intermediate care unit, department of internal medicine dr. soetomo hospital surabaya, indonesia. this study was a retrospective study to re-evaluate the patient medical record. the number of patients treated: 275 patients, sepsis patients: 80 patients, the number of patients who entered the study 50 patients, 30 (60%) female and 20 (40%) men, most age groups aged 60 -70 years (32%), mean 54 median 56. diagnostic accuracy according to the criteria of sepsis 45 (90%) patients, compliance with taking blood culture 2 (4%) patients, appropriate antibiotic selection 49 (98%) patients, the number of patients who should not be given antibiotics (4 patients), but given the antibiotic is 3 (75%) patients, the number of patients who should be given antibiotics (46 patients) but were not given antibiotics 1 (2%) patient. the reason of patients discharged: 27 had died (54%), 13 cured (26%), not yet recovered 10 (20%). the reason of patients discharged forcibly: to feel recovered 2 (20%), the condition gets worse 3 (30%), no cost 2 (20%), not clear 3 (30%). patients with sepsis remain a big problem, and it still needs to improve the management of sepsis on existing guidelines. key words: sepsis, guideline, medical audit introduction sepsis and septic shock is one of the highest causes of death in patients treated in hospitals. severe sepsis mortality ranges between 28–50%. in 1991, american college of chest physicians/society of critical care medicine made a term and definition to describe a more precise sepsis is systemic inflammations response syndrome (sirs), sepsis, severe sepsis and septic shock. sirs criteria consisted of: 1). hyperthermia or hypothermia (> 38° c or < 36° c); 2). tachycardia > 90/min; 3). tachypnea > 20/min; and 4). leucocytosis or leucopenia (> 12,000 cells/cmm or < 4000 cells/cmm) or the existence of immature neutrophil > 10%. and this definition is used as criteria for sepsis consensus conference on sepsis and organ failure.1 the term sepsis is now recognized as a systemic response to an infection of the body. new approach in handling patients with sepsis may be a more appropriate management of host factors, environmental factors and factors of microorganisms. handling problems should be a priority of microbes, particularly those involving resistance to antimicrobial agents, in addition to handling problems that patients who enter hospital with a fever which is also still a problem and needs special attention.6 various efforts have been made to suppress the mortality of sepsis patients, including by publishing a manual handling of patients admitted with fever in 2003. the purpose of this study was to evaluate the quality of the management of sepsis patients in the intermediate care unit, department of internal medicine dr. soetomo, hospital, surabaya. methods design research this study is a retrospective study to re-evaluate the management of patients with sepsis in the intermediate care unit, department of internal medicine, by looking back the medical record of patients with diagnosis sepsis, after the patient are discharged from the hospital. � indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 1-4 materials research patients in the study were patients admitted with diagnosis in sepsis, the time period january 2007 s/d april 2007. data collection patient characteristics (gender, age), demographic data, working diagnoses, signs and symptoms, laboratory results (hemoglobin, white blood cell count, platelet count, urine sediment, leukocyte examination and parasites, and radiology tests serological tests). the use of antibiotics recorded by reviewing medical records after patients were discharged from hospital information about the use of antibiotics and clinical symptoms and signs of each case were collected from medical records. the quality of antibiotic use was assessed by agreement to flow-chart view of fever management guideline. table 1. patient’s age grouppatient’s age group patient’s age group n (%) 10–20 1 (2) 21–30 2 (4) 31–40 5 (10) 41–50 12 (24) 51–60 11 (22) 61–70 16 (32) 71–80 2 (4) 81–90 1 (2) total 50 (100) measurement results a) adherence to the guidelines; b) percentage of patients who receives antibiotic therapy in accordance with the guidelines; c) flow-chart management of fever patients on the first day they were treated in the intermediate care unit were evaluated to identify patients who need or who do not need antibiotics 5 (picture 1); d) the percentage of antibiotic selection was in accordance with the clinical diagnosis2; e) the percentage of patients who take blood cultures; f) mortality; g) the percentage of patients who died during sepsis treated in hospital. results number of patients treated in the intermediate care unit in the study period: 275 patients. the number of sepsis patients: 80 patients. number of patients included in the study 50 patients, 30 (60%) female and 20 (40%) men. most age groups 60–70 years of age (32%), mean 54 median 56 (table 1). primary disease in people with sepsis: diabetes mellitus 27 (54%); 7 chronic kidney disease (14%) chronic liver disease 11 (22%) source of infection: urinary tract infection (uti) 10 (20%); cholesistitis 2 (4%); peritonitis (sbp) 6 (12%); not clear 32 (64%). severe complications found: gastrointestinal bleeding 5 (10%); 4 pulmonary edema (8%); 18 septic shock (36%); multiple organ dysfunction syndrome 6 (12%). sirs criteria: patients who did not have sirs criteria 1 (2%), 1 criteria 4 (8%) patients, 2 criteria 12 (24%) patients, 3 criteria 27 (54%) patients and 4 criteria 6 (12%) patients condition of the patients when discharged from the hospital: 27 died (54%) patients, 13 cured (26%), not yet recovered (forcibly discharged) 10 (20%) patients. the reason forcibly discharged: to feel recovered 2 (20%) patients, the condition gets worse patient 3 (30%) patients, there is no cost 2 (20%) patients, the reason is not clear 3 (30%) patients. antibiotics used: ceftazidime 18 (36%), ceftriaxone 19 (38%), cefotaxime 7 (14%), ciprofloxacin 4 (8%), moxifloxacine 1 (2%) patients, did not receive antibiotics 2 (4%) microbiology test results: p aeruginosa 1 patient (25%), acinetobacter sp. 1 (25%), staphylococcus sp. 1 (25%), klebsiela spp. 1 (25%) patient. suitability of patient handling guidelines fever/sepsis: the accuracy of diagnosis: 1 according to the criteria of sepsis patients 45 (90%) patients; 2. compliance with taking blood samples for microbiological examination 2 (4%) patients; 3. the percentage of appropriate antibiotic selection sepsis guidelines/clinical diagnosis: 49 (98%) patients; 4. number of patients who should not be given antibiotics (4 patients), but were given antibiotics is 3 (75%) patients; 5. number of patients who should be given antibiotics (46 patients) but were not given antibiotics 1 (2%) patients. discussion intermediate care unit is a ward that serves to handle patients who require strict monitoring, which have facilities in between intensive care unit and general ward. during the study period was sepsis patients treated in this room reach 80 (29%) of the total 275 patients. this indicates that infectious diseases especially those that have led into the condition of sepsis is still a cause for concern regarding both diagnostic and maintenance procedural problems. of the 50 patients included sepsis in this study sample was obtained groups of women suffering more from the group of male patients. while most age groups are dominated by people aged 60–70 years (32%). this can be explained due to age factors that contribute greatly to the patient’s immune system. related data that underlie diseases such sepsis patients who had diabetes mellitus was found that the highest (27 patients or 54%) followed by chronic kidney disease (7 patients or 14%) and chronic liver disease (11 patients or 22%). this indicates that metabolic diseases are chronic sufferers which affect the immune system so that the group of patients with the disease susceptible to the condition of sepsis. data on the complications found in sepsis patients include gastrointestinal bleeding (5 patients or 10%), pulmonary �hadi and triyono: medical audit of the management of patients with sepsis figure 1. protocol management new patient with sepsis or fever fever routine laboratory examinations: haemoglobin, leucocytes count platelet count, urine sediment, stool microscopy. blood culture sirs1 criteria ? yes no source of infection identified ? source of infection identified ? yes (sepsis) not clear (suspected sepsis or sepsis due to non infection) yes (infection without sirs) not clear (suspected infection without sirs or other infection without sirs) treat according to identified infection: tonsillitis pneumonia uti2 (urosepsis) typhoid fever amoebic abscess cholecystitis diarrhea skin infection meningitis bone infection pelvic/genital infection empirical antibiotic therapy except for clinical dhf3, morbilli, and varicella (no antibiotics, observation every day). after 72 hours blood culture (-), and fever still (+): 1. no infection ? 2. repeat routine lab. 3. stop antibiotics. 4. search for other causes (sle 4, carcinoma ?) 5. other examination: radiology, serology, etc. treat according to identified infection tonsillitis pneumonia uti2 (urosepsis) typhoid fever amoebic abscess cholecystitis diarrhea skin infection meningitis bone infection pelvic/genital infection no antibiotic therapy antipyretic therapy after 72 hours blood culture (-), and fever still (+): 1. no infection ? 2. repeat routine lab. 3. search for other causes (sle, carcinoma ?) 4. other examination: radiology, serology etc. 1 sirs: systemic inflamatory response syndrome 2 uti: urinary tract infection 3 dhf: dengue haemorrhagic fever 4 sle: systemic lupus erythematosus oedema (4 patients or 8%), septic shock (18 patients or 36%), mods (6 patients or 12%). this shows that it is not easy to solve problems of sepsis and its complications posed. it takes a good understanding of the diagnostic aspect, the optimal procedure, teamwork both internally and externally as well as financially sometimes beyond the reach of patients. and the things that we conclude on patient conditions associated with discharged time where people who died 27 (54%), recovered 13 (26%) and have not recovered (forcibly returned) 10 (20%). the reason home forcibly felt quite varied as cured, the condition gets worse, and there is no cost and no obvious reason. completeness monitoring of the results from physical examination and laboratory criteria for diagnosis of sepsis supporting were good enough 98–100% (table 2), this shows that sepsis patients actually � indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 1-4 m o n i t o r e d a n d c a r r i e d o u t i n t e n s i v e t r e a t m e n t . research sample of 50 patients 45 were filled more than or equal to 2 criteria of sirs and only 5 that only meet less than 1 or sirs criteria. the results of this study indicate that there are 5 patients (10%) the criteria for diagnosis of sepsis was not in accordance with the guidelines, it can happen because the clinicians used to make the diagnosis of sepsis if the patient met the condition to appear severe, although judging from the criteria for sepsis is not enough met, in this case it may take special examinations such as procalcitonin as a diagnostic criteria sepsis.3 important problems encountered in this study is the low adherence of clinicians to perform microbiological tests before giving antibiotik,4 where many factors that affect it, such as the lack good preparation for facilities or personnel in making preparations for the examination, in addition to low awareness about the importance of examination microbiology in patients with sepsis.4 in the guidelines for the use of antibiotics in sepsis patients is ceftriaxon, compliance ceftriaxon use as empirical therapy is quite good (98%) it can happen because of the availability of sufficient ceftriaxon easy and relatively affordable price, the problem is whether this drug is useful or not, where the treatment here is not guided by the results of microbiological tests and the pattern sensitivity of germs. if you see the number of treated patients mortality is high (> 50%) then the problem of sensitivity of germs to antibiotics used to be an important consideration. the accuracy of antibiotic treatment indications in patients who should receive antibiotics were good enough, those 46 patients had indications the use of antibiotics, but only 1 patient who was not given antibiotics. conversely there are patients 3 patients given antibiotics from 4 patients who should not have any indication of the use of antibiotics. this shows the weakness is still the possibility of health workers to the understanding of the existing guidelines so that the supervision, evaluation and monitoring is expected to continue to be done on an ongoing basis. conclusion patients with infectious diseases who became sepsis remain a significant problem in dr.soetomo hospital, where the mortality rate is still high. still need to improve compliance with existing guidelines of management patients admitted with sepsis or fever, as well as enhanced ability to support microbiology laboratory in supporting the treatment of sepsis patients. references 1. bone rc, sibbald wj, sprung cl. the accp-sccm consensus conference on sepsis and organ failure. chest 1992; 101(6): 1481– 1483. 2. dellinger rp, carlet jm, masur h, et al. surviving sepsis campaignsurviving sepsis campaign guidelines for management of severe sepsis and septic shock. crit care med 2004; 32(3): 858–73. 3. giamarellos-bourboulis e.j., p g. should procalcitonin be introduced in the diagnostic criteria for the systemic inflammatory response syndrome and sepsis? j of crit care 2004; 19: 152–157. 4. cohen j, brun-buison c, tores a, j j. diagnosis of infection in sepsis: an evidence-based review. crit care med 2004; 32: s466–s490. 5. hadi u, keuter m, van asten h, et al. optimizing antibiotic usageoptimizing antibiotic usage in adult admitted with fever by a multifaceted intervention in an indonesian governmental hospital. trop. med and int. health 2008; 13: 888–99. 6. hadi u, duerink d, lestari es, audit of antibiotic prescribing in two governmental teaching hospitals in indonesia. clin microbiol and infect 2008; 14: 698–707. table 2. percentage of clinical and laboratory examination of sepsis patients. examination n (%) examination n (%) temperature 50 (100) blood pressure 50 (100) heart rate 50 (100) respiratory rate 50 (100) haemoglobin 49 (98) leukosit 49 (98) trombocyte 49 (98) blood sugar 48 (96) blood urea nitrogen 48 (96) electrolite serum 36 (72) blood gas analysis 31 (62) chest x ray (1 x) 40 (80) chest x ray (2x) 13 (26) chest x ray (3x) 3 (6) usg abdomen (1x) 25 (50) usg (2x) 5 (10) sgot/sgpt 48 (96) albumin 43 (86) globulin 28 (56) bilirubin 30 (60) ijtid vol 1 no 1 jan-apr 2010.3.pdf ijtid vol 1 no 1 jan-apr 2010.4.pdf ijtid vol 1 no 1 jan-apr 2010.5.pdf ijtid vol 1 no 1 jan-apr 2010.6.pdf �� vol. 2. no. 1 january–march 2011 association between atypical depolarization in celldyn ��00 and the presence of plasmodium spp in blood in dr. soetomo hospital surabaya jusak nugraha1,2, esti rohani1,2 1 department of clinical pathology, faculty of medicine, airlangga university 2 dr. soetomo hospital surabaya, indonesia abstract background: malaria is a parasitic disease worldwide with a high morbidity and mortality. a rapid and accurate method is needed to detect the presence of malaria parasites in blood. a flagging system atypical depolarization (atypdep) in cbc results from cell-dyn 3200 has been related with malaria infection. materials and methods: an observational cross sectional approach with 48 samples obtained from inpatients of the dr.soetomo hospital, surabaya. samples were screened by cell-dyn 3200 analyzer for atypdep flagging in cbc. positive samples were later confirmed by microscope to detect malaria parasites. results: from 48 samples with atypdep flagging, 7 samples were malaria positive on peripheral blood smear (13.1%). most frequent atypdep flagging was seen in malignancy (18.7%), and approximately 54.6% of the samples were not accompanied by fever symptoms. lekositosis and anemia each were found in 20 samples (41.6%) and thrombocytopenia in 33.3%. conclusion: the presence of atypdep flagging in cell-dyn 3200 does not necessarily indicate the existence of malaria or it could be said that atypdep flagging is not always associated with presence of malaria infection. the usage of an atypdep flagging in non-endemic areas such as surabaya is just an alert sign to evaluate malaria infection rather than a screening method to detect malaria. key words: malaria, atypical depolarization, hematology analyzer introduction until now, malaria remains the most important parasitic disease worldwide and causes health problems especially for those living in endemic areas. early diagnosis relies crucially on clinical suspicion. a clinician suspecting the disease has to request explicitly malaria examination by blood smears. lack of clinical suspicion is a wellknown factor for a missed diagnosis, which contributes substantially to patient morbidity and mortality in this disease.1 of the 300 – 500 million cases of malaria infection which are estimated to occur annually, approximately 2–3 million of these are fatal.2 the high frequency of severe clinical complications and mortality in endemic regions is exacerbated by delayed or inefficient treatment, limited access to clinical and laboratory services and the increasing influence of drug resistance.3,4 the female anopheline mosquito transmits malaria parasites, and after infecting a new host, the parasites are carried in the blood to the liver where they undergo a hepatic stage of multiplication. after a period of 9 to 16 days, the parasites return to the bloodstream and infect red cells. the typical spiking fever of malaria occurs when the red cells rupture and release free parasites.3 patients with symptoms of fever and malaise in nonendemic areas will usually consult a clinician. however, in many countries malaria ranks as a relatively infrequent cause of pyrexia and thus may not be considered as part of the differential diagnosis. this is especially true if a complete clinical/travel history is not obtained. in such situations, clinicians may only initially request general screening tests such as a full blood count (fbc).3 while a diagnosis of malaria can be established by microscopic examination of thin and thick blood film4, although the investigation does not necessarily indicate the existence of parasites. microscopic investigation of stained thick and thin blood smears has been the reference standard for malaria detection and species identification for decades. recently, a number of alternative diagnostic approaches have evolved, �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 15-19 including detection of plasmodium species dna stained with acridine orange in a quantitative buffy coat analysis, pcr methods, and assays based on detection of circulating plasmodium species specific antigens. recent studies using automated hematology analyzers have demonstrated unexpected abnormalities in differential white blood cell plots and reticulocyte histograms from patients with malaria.6 in normal blood samples, the only depolarizing wbc events are eosinophils. with cell-dyn multiangle polarized scatter separation (mapss) analysis, normal eosinophils viewed in the polarized-90° versus depolarized-90° (neu eos) plot form a distinct cloud of events that are color coded green. the depolarization of these cells is due to a component of eosinophil granules (figure 1).3 figure 1. granularity (90° depolarization axis) versus lobularity (90° polarization axis) plot showing typical cell-dyn 3200 eosinophil depolarization pattern. normal eosinophils are located within the area demarcated by the yellow oval line, and the atypical depolarization region indicated by the red broken line does not normally contain any events.3 during the intraerythrocytic stage, a malaria parasite digests and breaks down hemoglobin to its constituent parts heme and globin. the globin is used as a protein source by the parasite and the heme is converted by an enzyme (heme polymerase) to hemozoin or malaria pigment. the parasite initiates this process because heme is toxic to the parasite whereas hemozoin is not. in contrast to nondepolarizing heme, hemozoin has a distinctive ability to depolarize light. in the malaria parasite cell cycle, the malaria-infected red cells rupture at the schizont stage and the parasites are released together with hemozoin aggregates into the plasma. by an as-yet-unknown mechanism, circulating phagocytic wbcs (monocytes and neutrophils) then ingest the liberated free hemozoin. consequently, normally nondepolarizing monocytes and neutrophils will depolarize light when they contain aggregates of hemozoin. this will cause appearance of abnormal dots on neu-eosin scatter plot.3 research on detection of hemozoin by hematology analyzer has been done1,2,3,4,5,6,7. it was reported that the presence of one or more atypical depolarizing events can be attributed to malaria. discovery of the abnormal depolarization pattern in patients with unknown fever should be considered to possibility of malaria infection, so microscopic examination by stained thick and thin blood smears as a confirmation needs to be done. a study in portugal by hanscheid et al5 reported that diagnosing of malaria by detection of hemozoin using hematology analyzer obtain 95% sensitivity and a 88% specificity. while a south african study found a 72% sensitivity and 96% specificity. in the dr.soetomo hospital, surabaya, atypical depolarizing events are often found in complete blood count results. surabaya is not a malaria endemic area, but dr. soetomo hospital is a referral hospital for the eastern indonesian region, so that the patients are estimated to come from various regions. most patients were examined with a diagnosis of other diseases, without any suspicion of malaria infection. based on the fact, the researchers wanted to know whether the presence of atypical depolarization was actually due to malaria infection. if this was true, is it possible that existence of atypical depolarization (atypdep) could be used a screening marker for malaria in non-endemic areas such as surabaya? is there any association between the presences of atypdep flagging with the plasmodium in the blood in non-endemic areas such as surabaya? materials and methods this research was done in the laboratory of the department of clinical pathology, dr.soetomo hospital surabaya during february to may 2010. samples were obtained by selecting cbc results of inpatients in dr. soetomo hospital. samples of venous blood with edta anticoagulant were examined for cbc with a cell-dyn 3200 hematology analyzer. cbc results showing atypdep flagging were included in this study. these samples were examined by thin blood smear examination with giemsa staining to find and determine the types of parasites. samples were considered positive when parasites were found in thin blood smears. the numbers of parasites were counted per 1000 erythrocytes, and samples were considered negative if in the 50 fields of emersion parasites were not found. examination was conducted by 2 persons, a laboratory technician and a medical doctor. this study design was a descriptive observational study through crosssectional approach, data and results were presented in the form of tables and figures. results during the period of the study 48 samples were obtained that fullfield the criteria (males 64.5%, females 35.5%) ��nugraha and rohani: association between atypical depolarization and the precence of plasmodium spp with a variety of diagnosis. most of the atypdep flagging was found in adult patients (60.4%) and also in 5 samples of neonates. lekositosis and anemia were each found in 20 samples (41.6%), while thrombocytopenia was found in 16 samples (33.3%). sample characteristics can be seen in table 1. of the 48 samples collected, only 7 samples were malaria positive with a thin smear examination, or about 13.1% only. of the positive samples, almost all of them showed fever and a history of malaria endemic areas. of malaria positive samples there were 5 samples showing anemia and thrombocytopenia (71.4%). of all samples collected, atypdep appears most in the group of malignancy or tumor disease as much as 9 people, or 18.7%. (table 2) there are some patterns of atypical depolarizing events, some of which can be seen in figure 2a,2b,2c. parasites were found among malaria positive patients in various phases (trophozoit, schizont, gametocytes). table 1. samples characteristics parameter number persentage (%) sex male female 31 17 64.5 35.5 age < 1 yr 1–< 18 yr 18–60 yr > 60 yr 5 7 29 7 10.4 14.5 60.4 14.5 hb level < 6 g/dl 6–8 g/dl > 8–11 g/dl > 11–18 g/dl > 18 g/dl 4 15 28 1 8.3 31.2 58.3 2.1 temperature < 38°c >= 38°c 31 17 64.5 35.5 platelet level < 150,000 150,000–450,000 > 450,000 16 24 8 33.3 50 16.6 wbc < 4,000 4,000–11,000 > 11,000 4 24 20 8.3 50 41.6 persentage of eosinophil <=7% > 7% 43 5 89.5 10.5 table 2. clinical diagnosis of the positive atypdep patients diagnosis no of sample persentage (%) trauma (traffic accident) urinary bladder diverticle and chronic colitis nephrotic syndrome post partum dhf malignancy and/tumor down syndrome ckd hydrocephalus sepsis bph kidney stone suspected malaria 5 1 1 1 6 9 2 2 1 3 1 2 4 10.5 2 2 2 12.5 18.7 4.1 4.1 2 6.2 2 4.1 8.3 combustio febris dm decubitus ulcer pancytopenia hirschprung’s disease uti hemolitic anemia 3 2 1 1 1 1 1 1 6.2 4.1 2 2 2 2 2 2 figure 2a. samples showing occasional atypical depolarizing purple events (within yellow broken boundaries). figure 2b. samples showing many atypical depolarizing purple events. �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 15-19 figure. 2c. malaria samples with mixture of abnormal depolarizing purple and green events that are not in the position normally associated with typical eosinophils figure 3. trophozoit phase of plasmodium falcifarum (banana form) discussion in this study, out of 48 samples with atypep flagging, only 7 samples were malaria positive (13.1%). these results are not in accordance with various previous studies that have been done in several countries reporting that the sensitivity and specificity of atypical depolarization in detecting malaria is very high. this could be due to differences in sampling population in previous studies, samples taken from patients with clinical symptoms of malaria, and also done mostly in endemic malaria areas. while in this study, samples were taken at random, just based on the presence of atypdep flagging on cbc results regardless of clinical symptoms and diagnosis. after confirmation by thin smear examination, only 7 samples were positive for malaria. this shows that for non-endemic areas such as surabaya (low prevalence), the appearance of atypdep is not yet certain in malaria infection .therefore, it is important especially for patients with fever whose cbc results show atypdep flagging to confirm this by thick or thin smears in order to prove the existence of malaria infection. in this study, percentage of atypdep flagging that appeared in diseases without fever and other symptoms of malaria was nearly 65% and many were shown in malignancies this proves that the presence of atypdep is not only caused by the presence of hemozoin or malaria pigment in monocytes or neutrophils, but there may be other causes such as small cell fractions that capable to depolarizing light in addition to eosinophils. changes of the parameters such as wbc, red blood cells and platelets in malaria patients are generally not specific. some studies reported that the occurrence of thrombocytopenia in patients with clinical symptoms of malaria is an important indicator of malaria. although the frequency of occurrence of thrombocytopenia reported was about 80%,3,9 but these results varied in different studies that have been conducted. in this study, thrombocytopenia was found in 5 out of samples from the 7 malaria positive samples or approximately 71.4%. similarly, anemia was found in 5 samples, while the number of wbc showed no characteristic changes . of the malaria positive samples, all were imported malaria. all positive samples did not come from endemic areas, however, there was a history of traveling to endemic areas. in this study, one sample showed a negative thin smear with a history of malaria therapy 1 week before. this is consistent with the theory that in patients who are in recovery where parasites can no longer be found in the blood, atypdep flagging can still occur because of atypical depolarizing clearance of malaria pigment is slow. in some individuals, this malaria pigment can remain in circulation until 3 weeks after recovery3. there is a reference reporting that pseudoeosinophilia is associated with the emergence of atypical depolarizing.8 however, in this study, eosinophilia was found just in 5 samples or 10.4%. also lekositosis as much as 20 samples (41.6%) raises a question, whether lekositosis may be related with the emergence of atypdep ? the emergence of atypdep in neonates as much as 5 samples also need to be considered, whether neonatal blood could influence the occurrence of atypdep flagging. a further study is needed to determine the factors that lead to the emergence of atypdep flagging, so that atypdep flagging is not merely focused on the presence of malaria, but other possible causes as well. however, when atypdep flagging is found, it is important to confirm this by blood smear examination for malaria. further studies are needed to determine the factors causing the emergence of atypdep flagging because in this study there are several limitations, among others: • detection of plasmodium is influenced by the quality of the staining and the skills and expertise of examiners • positive results are influenced by the prevalence • more samples are needed. conclusion and recommendation it was found that the rise of atypdep flagging does not always indicate a malaria infection or it could be said that atypdep flagging is not always associated with the occurrence ��nugraha and rohani: association between atypical depolarization and the precence of plasmodium spp of malaria infection because from the 48 samples only 13.1% positive on blood smear. so the emergence of atypdep flagging on cell-dyn 3200 instrument can not be used as a screening of malaria in non-endemic areas such as surabaya. additional criteria for non-endemic areas such as surabaya are needed, when the existence of this atypdep flagging suspicious of malaria infection, for example: 1. frequency of atypdep appearance in the same patient 2. existence of thrombocytopenia 3. presence of clinical symptoms (fever, chills, etc.). this needs to be done by scoring and with a larger number of samples. moreover, further studies should be conducted to identify other factors leading to the emergence of atypdep flagging. acknowledgments the authors thank dr. yolanda probohoesodo, sp.pk(k) on the advice and assistance in preparing the paper into english. references 1. pasagna jf, nissapatorn v, 2005, malaria: the value of the automated depolarization analysis, southeast asian j trop med public health, vol 36 (suppl 4); 68–72. 2. mendelow bv, lyons c, nhlangothi p, tana m, munster m, et al, 1999, automated malaria detection by depolarization of laser light, british journal of haematology, 104: 499–503. 3. scott cs, zyl dv, ho e, meyersfeld d, ruivo l, et al., 2002, automated detection of wbc intracellular malaria-associated pigment (hemozoin) with abbott cell-dyn 3200 and cell-dyn 3700 analyzer: overview and results from the south african institute for medical research (saimr) ii evaluation, laboratory hematology, carden jennings publishing co., ltd, 8: 91–101. 4. dromigny ja, jambou r, scott cs, perrier-gross-claude jd, 2005, performance evaluation of automated depolarization anaysis for detecting clinically unsuspected malaria in endemic countries, transactions of the royal society of tropical medicine and hygiene, 99: 430–439. 5. hanscheid t, melo-cristino j, pinto bg, 2001, automated detection of malaria pigment in white blood cells for the diagnosis of malaria in portugal, am. j. trop. med. hyg., 64(5,6): 290–292. 6. wever pc, henskens ymc, kager pa, dankert j, gool t, 2002, detection of imported malaria with the cell-dyn 4000 hematology analyzer, journal of clinical microbiology, dec vol. 40, no.12: 4729–4731. 7. grobusch mp, hanscheid t, kramer b, neukammer j, may j, et al, 2003, sensitivity of hemozoin detection by automated flow cytometry in nonand semi-immune malaria patients, cytometry part b (clinical cytometry) 55b: 46–51. 8. huh j, jung j, yoon h, chung w, 2005, pseudoeosinophilia associated with malaria infection determined in the sysmex xe2100 hematology analyzer, ann hematol, 84: 400–402. 9. rathod da, patel v, kaur aa, patel vd, patel dd, 2009, diagnosis of acute malaria by laser based cell counter with comparison of conventional and recent techniques in indian scenario, indian journal of pathology and microbiology, 52(2): 185–188. 131 vol. 6 no. 6 september–december 2017 literature review a review on the chemistry and pharmacology of rennellia elliptica korth che puteh osman1,2a, nor hadiani ismail1,2 1 faculty of applied sciences, universiti teknologi mara, shah alam, selangor, malaysia 2 atta-ur rahman institute of natural product discovery, universiti teknologi mara, cawangan selangor, bandar puncak alam, selangor, malaysia. a corresponding author: cheputeh@salam.uitm.edu.my abstract rennellia elliptica, popularly dubbed as malaysian ginseng, is widely used in traditional medicine among the local jakun community in endau-rompin state park, pahang, malaysia. the decoction of the roots is traditionally taken for treatment of body aches, as postpartum tonic, as aphrodisiac and for the treatment of jaundice. in the effort of searching new botanical drugs and drug candidates from tropical rainforest, the team from this laboratory had conducted a sizeable phytochemical and biological screening program of tropical plant at endau rompin state park, pahang with the help from the indigenous people. r. elliptica showed strong antiplasmodial activity in vitro with the ic50 value of 4.04µg/ml. the comprehensive study on the root extract of r. elliptica in this laboratory yielded seventeen compounds from four different classes, including 2 new pyranoanthraquinones, one new anthraquinone, eleven known anthraquinones, one lactone triterpenoid, one coumarin and one phenolic acid. the chemical profile of the root extract was established using hplc and the selected marker compounds were used as external standards and quantified using standard calibration curve. nordamnacanthal 5, damnacanthal 7, 2-formyl-3-hydroxy-9,10-anthraquinone 6, 2-methyl-3-hydroxy-9,10-anthraquinone 11 and 1,2-dimethoxy-6-methyl-9,10-anthraquinone 3 were determined at 3.57, 10.32, 4.47, 12.18 and 4.09 µg/g, respectively. owing to the toxicity of dichloromethane, the extraction of the desired marker compounds was attempted using accelerated solvent extraction and soxhlet extraction using ethanol and water at different compositions. r. elliptica root extract and the isolated anthraquinones showed potential antiplasmodial activity, and the active compounds were probed for their mode of action. in addition, the dichloromethane root extract of r. elliptica and the selected anthraquinones were screened for anticancer, antioxidant, and a-glucosidase inhibitory activities as well as toxicity study in vitro. the review summarizes the findings on rennellia elliptica which includes phytochemistry, toxicity and its biological activities. the chemotaxonomic significance of rennellia elliptica is also discussed. keywords: rennellia elliptica, anthraquinone, rubiaceae, malaria, antiplasmodial introduction malaysia is the 12th most biodiversed nation in the world1 and is mainly covered by tropical rainforests. it was reported in 1953, there are about 550 genera of tropical plants containing over 1300 species possessing medicinal values in peninsular malaysia alone.2 tropical rainforests are rich source of flora and fauna, though they only cover 12% of earth’s land area, tropical rainforests are the home of 50–90% of world species. at least 25% of all modern drugs were discovered from rainforests even though less than 1% of tropical rainforests in the world are investigated for pharmacologically active metabolites.3 the great biodiversity of malaysian flora provides an immense source of chemically diverse bioactive metabolites for new lead candidates. malaysia is blessed with plethora of tropical plant species as well as indigenous knowledge on the traditional uses of medicinal plants. the uses of exotic tropical plants in traditional medicine are mostly confined within the local communities especially in the remote areas and are lost when the elders pass on. the destruction of tropical rainforests threatens the survival of tropical plants and without proper documentation and study, the knowledge of the traditional uses of these plants will be lost forever. thus, extensive phytochemical and biological assessments of our plant 132 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 131–140 species are of utmost importance to preserve the knowledge of our natural heritage for the next generation. in the effort for searching new botanical herbs and new drug candidates, large random plant collection program have been initiated by the malaysian government through various forms of funding at local research institutions and universities. various biodiversity centers have been established such as sarawak biodiversity centre and pahang biodiversity institute to conserve the flora and fauna as well as to promote research on the biological, pharmaceutical, medicinal and other applications of tropical rainforests. in conjunction with the national effort, the team from this laboratory had conducted a sizeable phytochemical and biological screening program of tropical plant at endau rompin state park, pahang with the help from the indigenous people in the search for new potential medicinal plants. rennellia elliptica was one of the most promising plants, and phytochemical and biological studies were carried out to assess its potential as new botanical herbs. rennellia elliptica korth. is a tropical shrub of about 1-2 m tall and can be found in lowland to hill forest to c. 500m above sea level. r. elliptica is locally known as ‘mengkudu rimba’ or ‘segemuk’ and popularly dubbed as malaysian ginseng probably due to the appearance of its yellow roots. among various malaysian ethnics, this plant is also known as ‘kayu penawar apow’ (dusun), ‘mengkudu hutan’ (iban), ‘akar bumi’, ‘urap gondor’ (sakai), ‘mengkudu gajah’, ‘lempedu tanah’ and ‘sekemang’ (jah hut, semelai). r. elliptica is native to south east asia and widely distributed in peninsular malaysia, southern thailand, borneo and indonesia.4 the decoction of rennellia elliptica is traditionally taken for the treatment of jaundice5 and body aches, as postpartum tonic and as aphrodisiac.6 during the random screening of selected malaysian tropical plants for antiplasmodial activity, r. elliptica showed promising activity (4.04 µg/ml) which warranted further investigation. following the screening program, extensive phytochemical study was carried out on the root extract yielding 17 compounds from four different classes in which four of them were found to possess strong antiplasmodial activity with ic50 values of less than 1 µm. 7 in order to establish the use of r. elliptica root extract of as potential herbal drug for the treatment of malaria, optimization of extraction methods, qualitative and quantitative hplc analyses of the extract as well as the investigation of the extract toxicity and possible mechanism of actions were warranted. the chemotherapeutic targets selected were inhibition against β-hematin formation via lipids and hrp2 catalyses. the anticancer, antioxidant, and antidiabetic activities of the crude extract and selected chemical compounds were also discussed. taxonomy of rennellia elliptica r.elliptica korth. was also previously known as r. elongata (king & gamble) ridl. recent phylogeny study revealed that r. elliptica and r. elongata are different species and are not synonym.8 this plant is a shrub of about 1-2 m tall. the leaf is somewhat narrow and rounded apex with tapering ends. it has leathery surface and slightly waxy with greyish-blue bloom below. the veins are purplish in colour when fresh. the inflorescences are terminal, consisting of head of flowers arranged along a rachis. the flower is violet in colour while the fruit is subglobose and unstalked.4,9 this shrub can be found in lowland to hill forest to c. 500m above sea level. r. elliptica is widely distributed from southern myanmar to west malaysia. the pictures of several parts of r. elliptica are shown in figure 1. phytochemistry of rennellia elliptica korth chemical constituents isolated from the root extract of r. elliptica the air-dried powdered roots of r. elliptica were extracted successively with n-hexane, dichloromethane and methanol and dried giving brown coloured crude extract (27g). the dichloromethane root extract was fractionated using column chromatography (60 cm x 5 cm) eluted with figure 1. rennellia elliptica korth 133osman and ismail: a review on the chemistry and pharmacology various compositions of solvents of increasing polarity (n-hexane-dichloromethane, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 100 ch2cl2 v/v; dichloromethane-methanol, 99:1, 95:5, 9:1 v/v) to give six fractions. purification using repeated column chromatography and preparative column chromatography yielded compounds 1-14, and 18. 7,10 subjecting the dichloromethane root extract (3g) to second fractionation using mplc packed with lichroprep (rp-18, 40-63µm) eluted with stepwise gradient (water: aceonitrile, 100% water, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 100% acetonitrile) using about 100 ml solvent for each solvent system to give nine fractions. fraction b (2-3) was further fractionated using radial chromatography (2 mm, f254, 40-60 mesh) eluted isocratically using dichloromethane to give 31 fractions. fractions b27-11 was purified using semi-preparative hplc [sunfire, c-18 column (250 mm x 5µm x 10 mm i.d.); water: acetonitrile (4:6→100% acetonitrile); flow rate 4.73 ml/min in 60 minutes; formic acid (0.01%) was added to mobile phase] to give compound 14 (1.5 mg), 16 and 17. the purification of the dichloromethane root extract yielded two new pyranoanthraquinones, one new anthraquinone, and eleven known anthraquinones along with a coumarin, a phenolic compound and a lactone triterpenoid from r. elliptica.7,10 their structures were elucidated as rennellianone a 1 and rennellianone b 2, 1,2-dimethoxy6-methyl-9,10-anthraquinone 3, nordamnacanthal 4, 2-formyl-3-hydroxy-9,10-anthraquinone 5, damnacanthal 6, 1-hydroxy-2-methoxy-6-methyl-9,10-anthraquinone 7, lucidin-ω-methyl ether 8, 3-hydroxy-2-methoxy-6methyl-9,10-anthraquinone 9, rubiadin 10, 3-hydroxy-2methyl-9,10-anthraquinone 11, rubiadin-1-methyl ether 12, 3-hydroxy-2-hydroxymethyl-9,10-anthraquinone 13, alizarin-1-methyl ether 14, scopoletin 15, 4-hydroxy-3,5dimethoxybenzaldehyde 16 and 3b-acetateoleanan-13b, 28-lactone 17.11-14 the triterpenoid lactone is reported for the first time from the family rubiaceae. figure 2 illustrates the chemical structure of compounds isolated from the root of r. elliptica. chemical profiling of r. elliptica root extract using high performance liquid chromatography preparation of standardized extract is an authentication of herbal preparation as means of controlling the quality of plant material used for product manufacturing. the standardized extract should have an acceptable content of bioactive metabolites and safe from toxic impurities.15 the dichloromethane root extract showed promising antiplasmodial and antioxidant activities. thus, the dichloromethane extract was profiled over waters 600 hplc on the sunfire column (c-18, 250 mm 5µm x 4.6 mm i.d.) to establish the chemical profile of the root extract. several combinations of mobile phases were attempted in order to obtain good chromatographic profile. the best minutes; formic acid (0.01%) was added to mobile phase] to give compound 14 (1.5 mg), 16 and 17. the purification of the dichloromethane root extract yielded two new pyranoanthraquinones, one new anthraquinone, and eleven known anthraquinones along with a coumarin, a phenolic compound and a lactone triterpenoid from r. elliptica.7,10 their structures were elucidated as rennellianone a 1 and rennellianone b 2, 1,2-dimethoxy-6-methyl-9,10-anthraquinone 3, nordamnacanthal 4, 2-formyl-3-hydroxy-9,10-anthraquinone 5, damnacanthal 6, 1-hydroxy-2methoxy-6-methyl-9,10-anthraquinone 7, lucidin-ω-methyl ether 8, 3-hydroxy-2-methoxy-6methyl-9,10-anthraquinone 9, rubiadin 10, 3-hydroxy-2-methyl-9,10-anthraquinone 11, rubiadin-1methyl ether 12, 3-hydroxy-2-hydroxymethyl-9,10-anthraquinone 13, alizarin-1-methyl ether 14, scopoletin 15, 4-hydroxy-3,5-dimethoxybenzaldehyde 16 and 3b-acetateoleanan-13b, 28-lactone 17.11-14 the triterpenoid lactone is reported for the first time from the family rubiaceae. figure 2 illustrates the chemical structure of compounds isolated from the root of r. elliptica. r1 r2 r3 r4 3 och3 och3 h ch3 4 oh och3 h ch3 5 oh cho oh h 6 h cho oh h 7 och3 cho oh h 8 oh ch2och3 oh h 9 h och3 oh ch3 10 oh ch3 oh h 11 h ch3 oh h 12 och3 ch3 oh h 13 h ch2oh oh h 14 och3 oh h h figure 2. chemical contituents isolated from the roots of rennellia elliptica korth. 134 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 131–140 chromatogram was achieved with combinations of water (solvent a) and acetonitrile (solvent b) buffered with 0.1 % formic acid (fa). the mobile phase was programmed consecutively in a linear gradient as follows: 0-20 min, 6035 % a; 21-40 min, 35-5 % a; 41-45 min, 5–0 % a; 46-60 min, 0 % a at a flow rate of 1.0 ml/min. the chromatogram of dichloromethane extract is given in figure 2. ten chemical constituents purified using conventional chromatographic techniques were used as external standard for qualitative peaks identification in the chromatogram. the known constituents were used as external standards to qualitatively distinguish the known constituents from unknown constituents. four compounds, 3-hydroxy-2hydroxymethyl-9,10-anthraquinone 13, rubiadin-1-methyl ether 12, rennellianone a 1 and rennellianone b 2 were not analyzed as standards due to limited compound availability. thus the unidentified peaks may belong to these metabolites. the study also included the establishment of the plant metabolites chemical profile of root extract using hplc analyses. nordamnacanthal 5, damnacanthal 7, 2-formyl3-hydroxy-9,10-anthraquinone 6, 2-methyl-3-hydroxy9,10-anthraquinone 11 and 1,2-dimethoxy-6-methyl-9,10anthraquinone 3 were selected as marker compounds due to their potent antiplasmodial activity 7. in order to determine the composition of each biomarker in the root extract, external calibration curves were constructed using five point concentrations. the concentration of compounds 5, 7, 6, 11, and 3 were determined at 3.57, 10.32, 4.47, 12.18 and 4.09 µg/g, respectively, with acceptable standard deviation (sd < 0.2) and coefficient of variance (cv<5%). it was evident from the chromatogram (figure 3), the marker anthraquinones present as major compounds in the root extract, thus it is submitted that the antiplasmodial action of the root extract is potentially due to the action of these metabolites. the potential antiplasmodial agents, 2-formyl3-hydroxy-9,10-anthraquinone 6, nordamanacanthal 5, damnacanthal 7, and 2-methyl-3-hydroxy-9,10anthraquinone 11 were the major constituents in the dichloromethane root extract. thus, it is postulated that the antiplasmodial action of the root extract is potentially due to the action of these metabolites. these metabolites can be used as biomarkers for standardization of the root extract as antiplasmodial agent. optimization of extraction d i c h l o r o m e t h a n e r o o t e x t r a c t o f r . e l l i p t i c a showed promising antiplasmodial activity, however, dichloromethane is not a suitable extraction solvent for herbal preparation owing to the toxic properties of the solvent. thus, the extraction of the dried root powder was attempted using ethanol and water in cold extraction, soxhlet and accelerated solvent extraction (ase). dichloromethane root extract was also prepared as a control to compare the presence of selected marker compounds. the extracts obtained from soxhlet and ase extractions were then analyzed for the presence of selected biomarkers using waters hplc system. the accelerated solvent extraction (20: 80, water: ethanol; 100°c) gave the comparable amount and quality of marker anthraquinones in the root extract as compared to dichloromethane root extract (table 1). the use of ethanol in cold and soxhlet extraction did not successfully extract the desired biomarkers compounds. the *the chromatogram was extracted at 276 nm. note: rubiadin (4.848), alizarin-1-methyl ether (7.815), 2-hydroxy-3-methoxy-6-methyl-9,10anthraquinone (22.477), 1-hydroxy2-methoxy-6-methyl-9,10-anthraquinone (23.279), 3-hydroxy-2-methyl-9,10-anthraquinone (24.443), 2-formyl-3-hydroxy-9,10anthraquinone (28.804), damnacanthal (28.435), lucidin-ω-methyl ether (34.406), 1,2-dimethoxy-6-methyl-9,10-anthraquinone (36.066), nordamna-canthal (40.278). the unknown peaks at 10.104, 14.094, 16.547, 19.994 and 30.676 could be due to as rennellianone a and rennellianone b, scopoletin, 4-hydroxy-3,5-dimethoxybenzaldehyde and 3b-acetateoleanan-13b, 28-lactone 10. source: osman et al. (2017) figure 3. hplc chromatogram of dichloromethane extract of r. elliptica korth. 135osman and ismail: a review on the chemistry and pharmacology ase can reduce the polarity of water and ethanol because high pressure and temperature will reduce the dielectric constant of water, which lowers its polarity and assists the extraction of more non-polar compounds.16, 17 biological activity of rennellia elliptica korth antiplasmodial activity the methanol and dichloromethane root extracts were screened against chloroquine sensitive p. falciparum (3d7). the methanol root extract displayed a stronger antiplasmodial activity (ic50=0.73 µg/ml) compared to the dichloromethane root extract (ic50 = 4.04 µg/ml). crude extracts with ic50 values of more than 50 µg/ml are considered effective as antiplasmodial agents.18 the percent inhibitions and ic50 values of root extracts of r. elliptica against p. falciparum are tabulated in table 2. the screening of dichloromethane and methanol root extracts for antiplasmodial activity in vitro showed promising activity. thus, the antiplasmodial activity of the extracts was further evaluated using rodent malaria, p. berghei (anka strain) in animal model. the dichloromethane root extract displayed stronger activity than the methanol root extract with an ed50 value of 1.23 µg/ml. methanol root extract also showed strong antiplasmodial activity with ed50 value of 27.57 µg/ml bw. the weaker activity of the methanol root extract was most potentially due to degradation of principle bioactive metabolites in the digestive tract. anthraquinones isolated from the root of r. elliptica were screened for antiplasmodial activity based on the promising screening results of the dichloromethane root extract (ic50 = 4.04 µg/ml). the in vitro antiplasmodial activity of anthraquinones isolated from r. elliptica against chloroquine sensitive strain of p. falciparum (3d7) is shown in table 3. compound 11 displayed the strongest inhibition activity, with an ic50 value of 0.34 µm, followed by compound 6 with an ic50 value of 0.63 µm. sittie et al. (1999) established that an aldehyde group at c-2 and a phenolic hydroxy group at c-3 on the anthraquinone skeleton enhance the activity of anthraquinones against the growth of p. falciparum. these results showed that a methyl group at c-2 together with a phenolic hydroxy group at c-3 as in compound 11 also gave significant activity. it should also be noted that both compounds 6 and 11 do not possess hydroxyl substituents at the peri positions. the new anthraquinone 3 also exhibited strong inhibition, with an ic50 value of 1.1 µm. interestingly, anthraquinone 4, which structurally differs only at c-1 (hydroxyl substituent instead of methoxyl substituent) did not show any significant activity. the position of substituents on the anthraquinone skeleton clearly influences the antiplasmodial activity, which warrants further investigation. one of the principle metabolites, compound 6 was also reported from the root extract of morinda lucida benth., an african medicinal plant widely used to treat malaria. many chemical constituents present in r. elliptica were also reported in morinda lucida. there is undocumented claim that r. elliptica is also taken from indigenous people to treat fever. thus, the data might support the traditional application of this plant. one of the important chemotheraupeutic targets in combating malaria infection is its food vacuole. the malaria parasite digests erythrocytes and releases heme19 along with oxygen.20 free heme is toxic owing to its detergentlike properties that destabilizes and lyses membranes.21,22 as well as inhibits the activity of several enzymes such as cysteine proteases22 and consequently leads to the death of table 1. optimization of extraction of root extract of rennellia elliptica type of extraction solvent/condition %yield cold extraction (10g) dichloromethane (3 days) 0.97 ethanol (3 days) 2.07 soxhlet extraction (10 g) dichloromethane (2 hours) 0.58 ethanol (2 hours) 2.28 accelerated solvent extraction (1g) 100: 0 (h2o:etoh), 60oc, 10 min 0.55 50: 50 (h2o:etoh), 60oc, 10 min 2.43 20: 80 (h2o:etoh), 60oc, 10 min 3.03 20: 80 (h2o:etoh), 80oc, 10 min 0.52 20: 80 (h2o:etoh), 100oc, 10 min 0.3 20: 80 (h2o:etoh), 140oc, 10 min 0.5 source: osman et al. (2017) table 2. inhibition against plasmodium faciparum (3d7) growth in vitro sample % inhibition at different dosage (µg/ml) ic50 (µg/ml) 100 10 1 0.1 0.01 meoh extract 100.00 59.18 54.66 23.84 21.78 0.73 dcm extract 92.02 62.32 20.87 9.10 5.60 4.04 136 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 131–140 the parasite. the mechanism of heme detoxification can be broadly classified into two types; primarily via dimerization into hemozoin and secondarily via degradation of heme by gluthatione and hydrogen peroxide.23 histidine-rich protein ii (hrp2)20,22 and lipids23 are proposed to catalyze the reaction but there are other evidences that the hemozoin formation may be spontaneous24 and autocatalytic.25 drugs such as quinine and chloroquine which targeted the prevention of β-hematin formation have a longer lifespan of effective use against malarial parasite. the parasite seems to have difficulties in finding alternative processes for haemoglobin utilization and heme detoxification as compared to other chemotherapeutic targets.26 in this study, the biomarkers were probed for their possible mode of action against β-hematin formation. nordamnacanthal 5 and damnacanthal 7 showed significant inhibition against hemozoin formation via hrp2 and lipids catalyses (table 4). it is interesting to note that the nordamnacanthal 5 and damnacanthal 7 showed weaker activity when tested against plasmodium falciparum (3d7 strain) in vitro as compared to 2-formyl-3-hydroxy9,10-anthraquinone 6 and 2-methyl-3-hydroxy-9,10anthraquinone 11.7 2-formyl-3-hydroxy-9,10-anthraquinone 6 and 2-methyl-3-hydroxy-9,10-anthraquinone 11 showed the strongest antiplasmodial activity in vitro and their mode of action are yet to be discovered. toxicity study the toxicity study was carried out to determine the selectivity of the dichloromethane root extract and marker compounds against the hepatocyte cell. the dichloromethane root extract showed mild toxicity with cc50 value of 318.0 µg/ml (table 4). for both in vitro and in vivo studies, the selectivity indexes were determined at 78.7 and 258.3, respectively. the selected biomarkers showed no toxicity except 2-formyl-3-hydroxy-9,10-anthraquinone, nordamnacanthal and damnacanthal which showed moderate toxicity with cc50 values of 181.34, 908.96 and 338.65 µm, respectively, with moderate selectivity index (table 4). anticancer activity anthraquinones are known chromophore for anticancer. they act mainly via dna intercalation27 and induce lipid peroxidation via free radical chain reaction and consequently induce oxidative stress on cancerous cells.28 oxidative stress can cause permanent modification of genetic material29 which represents the first step involved in mutagenesis, carcinogenesis and various disorders such as alzheimer, hungtinton’s disease, diabetes, and parkinson.30,31 in the previous report, nordamnacanthal was found to enhance cytotoxic effect of tamoxifen in treating human table 3. antimalarial activity of anthraquinones from r. elliptica korth. compounds r1 r2 r3 r4 ic50 (µm) 3 och3 och3 h ch3 1.10 4 oh och3 h ch3 na † 5 oh cho oh h 72.46 6 h cho oh h 0.63 7 och3 cho oh h 51.28 8 oh ch2och3 oh h 2.10 9 h oh och3 ch3 nt ‡ 10 oh ch3 oh h na † 11 h ch3 oh h 0.34 12 och3 ch3 oh h na † 13 h ch2oh oh h nt ‡ chloroquine diphosphate 6.30a each sample was tested in duplicate; the ic50 values were obtained from average values of percent inhibition within a series of concentration; notes: na† –no activity; nt‡ – not tested; a unit in nm. table 4. the toxicity, β-hematin and hrp2 assays of the root extract and selected compounds. aq toxicity cc50 µm antiplasmodial in vitro ic50 µm selectivity index β-hematin ic50 µm hrp2 ic50 µm 3 >3546.1 1.1 3225 na na 5 908.96 72.46 12.5 67.16 ± 0.2 4.37 6 181.34 0.63 285.6 158.73 ± 0.2 nt 7 338.65 51.28 6.6 5.32 ± 0.2 11.77 11 >3968.3 0.34 12,500 138.65+-0.1 nt root 318.0† 4.04† 78.7 nt nt ntnot tested; nano activity † unit µg/ml 137osman and ismail: a review on the chemistry and pharmacology breast cancer mcf7.32 damnacanthal 6 enhanced the expression of p21 and caspase-7 subsequently increased apoptosis in human breast cancer mcf7 cell.33 in the present study, only three other major compounds screened for cytotoxic activities using mcf7 and 4t1 cell lines (table 5). the dichloromethane root extract did not show cytotoxic effect against the mcf7 and 4t1 cancer cell lines. the presence of known anticancer against mcf7 and 4t1 cell lines, nordamnacnathal 4 and damnacanthal 6 as major compounds in the root extracts of r. elliptica does not contribute its cytotoxicity. 2-formyl-3-hydroxy9,10 anthraquinones 5 and 2-methyl-3-hydroxy-9,10anthraquinones 11 showed moderate cytotoxic activity against human breast cancer mcf7 cell lines. when tested against 4t1 cancer cell, only 2-formyl-3-hydroxy-9,10anthraquinones 5 showed moderate activities. dichloromethane root extract did not show cytotoxicity against 3t3 cell lines when screened at 30 µg/ml. the major compounds from r. elliptica were also were also screened for cytotoxic activity against 3t3 cell lines at 30 µg/ml. 2-formyl-3-hydroxy-9,10anthraquinones 5 and damnacanthal 6 showed moderate activity with 74.15 % and 67.34%, respectively. other compounds showed weak cytotoxicity. the cytotoxic activity of the selected anthraquinones was tabulated in table 5. damnacanthal and nordamnacanthal are widely reported as anticancer agents and antioxidants, however their abundance presence in the root extract of r. elliptica do not contribute to the activity of the extract. the activity of the extract could be a result of synergism between matrices of other components and not on the major components alone.34 antioxidant activity the roots extracts were tested for lipid peroxidation inhibition activity using ftc and tba methods. ftc measures the primary product of lipid peroxidation while tba method quatifies malondialdehyde (mda), the secondary product of lipid peroxidation that is commonly found as marker in oxidative stress related diseases.35 the dichloromethane root extract of r. elliptica showed stronger antioxidant activity than quercetin in both ftc and tba assays with 93.4 % and 90.6 %, respectively.36 the percent table 5. cytotoxicity of r. elliptica using 3t3, 4t1 and mcf7 cell lines compounds 3t3 % inhibition at 30 µg/ml 4t1 ic50 (µg/ml) mcf ic50 (µg/ml) 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs 1 74.15 3.87 31.21 42.13 33.01 26.31 26.31 2 28.18 nt nt 3 67.34 nt nt 8 50.40 46.41 45.89 46.29 50.13 na 10 42.48 50.34 na 44.44 38.70 na dichloromethane extract nt na na each sample was tested in triplicate; na – no activity, nt – not tested inhibitions of lipid peroxidation were calculated based on the final day of ftc assay when the absorbance of the control drops. the daily absorbance of ftc experiment is plotted in figure 1. the major anthraquinones from r. elliptica such as nordamnacanthal 5, damanacanthal 7, rubiadin 10, rubiadin-1-methyl ether 12 and lucidin-ωmethyl ether 8 were not tested because their antioxidant activities were widely reported.37,38 the radical scavenging assay was performed using dpph radicals. the method was modified from reported literature38,39 the absorbance values of samples were compared to quercetin as positive standard. the ic50 values of quercetin (~ 10-20 µm) were comparable with those reported in literature at same dpph final concentration of 300 µm.40,41 dpph radical was purple in colour and upon reduction via hydrogen acceptance; the purple colour is bleached to yellow and pale yellow.42 however, dpph assay is often affected by colour of sample solution which lead to underestimation of actual radical scavenging activity.43 when tested for the radical scavenging activity against dpph radicals, the methanol root extract of r. elliptica showed stronger activity than dichloromethane extract with ic50 values of 39.0 µg/ml and 250 µg/ml, respectively. based on these observations, the dichloromethane extract showed antioxidative role by inhibiting lipid peroxidation and has potential as a preventive antioxidant. the lipid peroxidation inhibition could be due to the presence of nordamanacanthal37 and damnacanthal38 as major compounds in the root extract. on the other hand, the methanol extract may play antioxidative role by competitive figure 4. daily uv absorbance of r. ellptica extracts in ftc assay. 138 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 131–140 reaction in which antioxidant and substrate compete for radicals in biological system. several anthraquinones were screened for dpph radical scavenging activity. the compounds were screened at the concentration of 100 µg/ml (table 6). all anthraquinones isolated from the root extract were generally weak radical scavengers. these observations were consistent with the dpph radical scavenging data reported by several authors37,38 anthraquinones showed weak radical scavenging activity probably due to the stability of anthraquinone radicals which could not form uncharged ions with other radicals.44,45 α-glucosidase activity when screened for α-glucosidase inhibitory activity at 10 µg/ml, the dichloromethane root extract did not show any activity. the anthraquinones showed weak activity and the moderate activity was shown by 1,2-dimethoxy6-methyl-9,10-anthraquinone 10 and damnacanthal 3 with 21.3 % and 19.9 %, respectively (table 6). there is no correlation observed between antioxidant and antidiabetic activities of anthraquinones from r. elliptica. even though the extract is a good antioxidant, the result does not echo in antidiabetic assay. chemotaxonomic significance the family rubiaceae was divided into three subfamilies, 43 tribes and 637 genera with over 13000 plant species. at least 70 genera and 555 species of rubiaceous plants were reported in malaysia.4 the genus rennellia belongs to family rubiaceae and subfamily rubiodeae. rennellia comprises of eight species which are native to south east asia.4,46 five species of rennellia are endemic to peninsular malaysia.4 the genus rennellia is characterized by cup-like stipule tube without ridges around the leaf stalk bases and the prominent secondary veins that loop at the leaf margin.4 the inflorescence is often rather large, but always at the ends of the stems. the calyx tubes are joined together as in morinda, but the head of flower, with only a few in each, set along a spike at the ends of the shoots.47 rennellia was initially placed in the tribe morindeae on the basis of morphology4 and phylogeny,48 however recent molecular study8,49 and wood anatomy study49 support the new placement of rennellia and prismatomeris in the tribe prismatomerideae. both genera are distinguished from the tribe morindeae by the occurrence of solitary vessels and the absence of axial parenchyma bands49 which is exclusive to the tribe prismatomerideae. close investigation of the tribe morindeae suggested close alliance between four genera, morinda, prismatomeris, rennellia and motleya despite the disputes over their tribal classification in the subfamily rubiodeae. to date, no phytochemical report on other species of the genus rennellia has been recorded. this review highlights the chemotaxonomic significance between the genera prismatomeris, morinda and rennellia. most of the anthraquinones (5, 6, 7, 6, 8, 10, 12, 14) identified from r. elliptica are common rubia type anthraquinones that can be found in the genera morinda50-53 and prismatomeris.54-57 rubia anthraquinones are characterized by substitution patterns on ring c only and substitutions on ring a are introduced at a later stage of biosynthetic pathways.58 this finding affirms the close alliance between morinda, prismatomeris and rennellia and support the placement of prismatomeris and rennellia in the tribe prismatomerideae. anthraquinones from the genera prismatomeris and morinda are typically substituted at c-1, c-2, c-3 and c-1 and c-2. compound 5, 7, 10, and 12 which substituted at c-1, c-2 and c-3 are reported in almost all species from morinda and prismatomeris. these anthraquinones present as major constituents especially plants from the genus table 6. radical scavenging activity of anthraquinones from r. elliptica at 100 µg/ml concentration compound dpph percent inhibition (%) at 100 µg/ml α-glucosidase activity percent inhibition (%) at 10 µg/ml 5 2.87 9.4 6 3.26 6.6 11 3.99 6.8 7 9.18 19.9 3 1.30 21.3 10 4.87 3.6 12 nt 6.4 8 3.91 na 13 nt na 4 nt na 9 nt na quercetin 15a each sample was tested in triplicate; the data was recorded as average percent inhibition at 100 µg/ml and 10 µg/ml. nt not tested, na = no activity. aunit in µm. source: osman et al. (2017) 139osman and ismail: a review on the chemistry and pharmacology morinda. the anthraquinones are also present abundantly in r. elliptica, however the presence of 2,3-disubstituted anthraquinones are more prevalent in this plant. 3-hydroxy-2-methoxy-6-methyl-9,10-anthraquinone 9 was only reported from hedyotis diffusa (huang et al., 2008). in addition, the new anthraquinone, 1,2dimethoxy-6-methyl-9,10-anthraquinone 3 and 1-hydroxy2-methoxy-6-methyl-9,10-anthraquinone 4 were also isolated and characterized from the root extract of r. elliptica. anthraquinones 3, 4 and 9 have a rare methyl substitution at c-67 differing from the anthraquinones of prismatomeris and morinda which are typically hydroxyl or methoxy substituted at c-658 and often followed by similar substitution at c-5. the presence of these anthraquinones could serve as taxonomic markers for r. elliptica. summary phytochemical study on the root extract of rennellia elliptica yielded 17 compounds from four different classes of natural products. the dichloromethane root extract showed strong antiplasmodial and antioxidant activities. the activities could be contributed by the presence of major compounds in the dichloromethane root extract. however, the dichloromethane root extract did not show significant anticancer activities against 4t1 and mcf breast cancer cell lines despite the major presence of nordamnacantahal and damnacanthal, the potent anticancer agents. the dichloromethane root extract showed mild toxicity will moderate selectivity against hepatocyte cell. the presence of rubia type anthraquinones in r. elliptica affirms the close alliance between morinda, prismatomeris and rennellia and support the placement of prismatomeris and rennellia in the tribe prismatomerideae. references 1. abd aziz r. siri syarahan perdana professor. skudai: universiti teknologi malaysia; 2003. 27 p. 2. burkill ih. a dictionary of the economics of the malay peninsular. kuala lumpur: ministry of agriculture; 1935. 3. kong j-m, goh n-k, chia t-f. recent advances in traditional plant drugs and orchids. acta pharmacologica sinica. 2003; 24(1): 17-21. 4. wong km. rubiaceae (from the genus rubia). in: ng fsp, editor. tree flora of malaya; a manual for foresters. 4: longman malaysia; 1989. p. 324-37, 404-5. 5. ismail i, linatoc ac, mohamed m, tokiman l. documentation of medicinal plants traditionally used by the jakun people of endaurompin (peta) for treatments of malaria-like symptoms. jurnal teknologi. 2015;77(31):63-9. 6. yusoff ni, latip j, liew hl, latiff a. kajian fitokimia awal tumbuhan taman negeri endau rompin, pahang: antrakuinon daripada akar rennellia elliptica korth. (rubiaceae). in: mohamad ismail s, mat isa m, w. ahmad wy, ramli mr, latiff a, editors. taman endau rompin: pengurusan persekitaran fizikal dan biologi. siri kepelbagaian biologi hutan: jabatan perhutanan semenanjung malaysia; 2004. 7. osman cp, ismail nh, ahmad r, ahmat n, awang k, jaafar fm. anthraquinones with antiplasmodial activity from the roots of rennellia elliptica korth. (rubiaceae). molecules. 2010; 15(10): 7218-26. 8. jalaluddin s, bruhl jj. testng species limits in rennellia (prismatomerideae, rubiodeae, rubiaceae). taxon. 2008;57(1): 43-52. 9. suratman. the indonesian species of rennellia korth. (rubiaceae). biodiversitas. 2008;9(4):259-63. 10. osman cp, ismail nh, wibowo a, ahmad r. two new pyranoanthraquinones from the root of rennellia elliptica korth. (rubiaceae). phytochemistry letters. 2016;16:225-9. 11. pereda-miranda r, gascón-figueroa m. chemistry of hyptis mutabilis: new pentacyclic triterpenoids. journal of natural products. 1988;51(5):996-8. 12. katai m, terai t, meguri h. triterpenoids of the bark of pieris japonica d. don (japanese name: asebi). ii. 13c nuclear magnetic resonance of the.gamma.-lactones of ursaneand oleanane-type triterpenes. pharmaceutical bulletin. 1983;31(5):1567-71. 13. imai f, itoh k, kishibuchi n, kinoshita t, sankawa u. constituents of the root bark of murraya paniculata collected in indonesia. chem pharm bull. 1989;37(1):119-23. 14. m.g.al-hazimi h, miana ga, deep msh. terpenoids from salvia lanigera. phytochemistry. 1987;26(4):1091-3. 15. jamal ja. malay traditional medicine; an overview of scientific and technological progress. tech monitor. 2006:37-49. 16. rodríguez-meizoso i, marin fr, herrero m, señorans fj, reglero g, cifuentes a, et al. subcritical water extraction of nutraceuticals with antioxidant activity from oregano. chemical and functional characterization. journal of pharmaceutical and biomedical analysis. 2006;41(5):1560-5. 17. ju zy, howard lr. effects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin. journal of agricultural and food chemistry. 2003;51(18):5207-13. 18. kohler i, jenett-siems k, siems k, hernandez ma, ibarra ra, berendsohn wg, et al. in vitro antiplasmodial investigation of medicinal plants from el savador. z naturforsch. 2002;57c:277-8. 19. egan tj. haemozoin (malaria pigment): a unique crystalline drug target. targets. 2003;2(3):115-24. 20. sullivan dj. theories on malarial pigment formation and quinoline action. international journal for parasitology. 2002;32(13): 1645-53. 21. cowman af, foote sj. chemotherapy and drug resistance in malaria. international journal for parasitology. 1990;20(4):503-13. 22. kumar s, guha m, choubey v, maity p, bandyopadhyay u. antimalarial drugs inhibiting hemozoin (β-hematin) formation: a mechanistic update. life sciences. 2007;80(9):813-28. 23. rathore d, jani d, nagarkatti r, kumar s. heme detoxification and antimalarial drugs known mechanisms and future prospects. drug discovery today: therapeutic strategies. 2006;3(2):153-8. 24. egan tj, chen jy-j, de villiers ka, mabotha te, naidoo kj, ncokazi kk, et al. haemozoin (beta-haematin) biomineralization occurs by self-assembly near the lipid/water interface. febs letters. 2006;580:5105-10. 25. kumar s, bandyopadhyay u. free heme toxicity and its detoxification systems in human. toxicology letters. 2005;157(3):175-88. 26. olliaro pl, yuthavong y. an overview of chemotherapeutic targets for antimalarial drug discovery. pharmacology & therapeutics. 1999;81(2):91-110. 27. beretta gl, zunino f. molecular mechanisms of anthracycline activity. in: krohn k, editor. anthracycline chemistry and biology ii. 283. heildelberg: springer; 2007. p. 1-20. 28. lai j-m, chang jt, wen c-l, hsu s-l. emodin induces a reactive oxygen species-dependent and atm-p53-bax mediated cytotoxicity in lung cancer cells. european journal of pharmacology. 2009;623(13):1-9. 29. valko m, rhodes cj, moncol j, izakovic m, mazur m. free radicals, metals and antioxidants in oxidative stress-induced cancer. chemicobiological interactions. 2006;160(1):1-40. 140 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 131–140 30. niki e, yoshida y, saito y, noguchi n. lipid peroxidation: mechanisms, inhibition, and biological effects. biochemical and biophysical research communications. 2005;338(1):668-76. 31. berg d. redox imbalace: in the triad genetic disturbances and mitochondrial dysfunction in parkinson's diease. in: qureshi a, parvez sh, editors. oxidative stress and neurodegenerative disorders. amsterdam: elsevier; 2007. p. 183-200. 32. subramani t, yeap sk, ho wy, ho cl, osman cp, ismail nh, et al. nordamnacanthal potentiates the cytotoxic effects of tamoxifen in human breast cancer cells. oncology letters. 2015;9(1):335-40. 33. aziz mya, omar ar, subramani t, yeap sk, ho wy, ismail nh, et al. damnacanthal is a potent inducer of apoptosis with anticancer activity by stimulating p53 and p21 genes in mcf-7 breast cancer cells. oncology letters. 2014;7(5):1479-84. 34. wagner h. synergy research: approaching a new generation of phytopharmaceuticals. fitoterapia. 2011;82(1):34-7. 35. antolovich m, prenzler pd, patsalides e, mcdonald s, robards k. methods for testing antioxidant activity. analyst. 2001;127: 183-98. 36. osman cp, ismail nh, ahmad r, widyawaruyanti a, tumewu l, choo cy, et al. evaluation of rennellia elliptica as potential antiplasmodial herbal remedy. jurnal teknologi. 2017;79(6):37-43. 37. jasril, lajis nh, lim ym, abdullah ma, sukari ma, ali am. antitumor promoting and antioxidant activities of anthraquinones isolated from the cell suspension culture of morinda elliptica. asia pacific journal of molecular biology and biotechnology. 2003;11(1):3-7. 38. ismail nh, mohamad h, mohidin a, lajis nh. antioxidant activity of anthraquinones from morinda elliptica. natural product science. 2002;8(2):48-51. 39. habsah m, amran m, mackeen mm, lajis nh, kikuzaki h, nakatani n, et al. screening of zingiberaceae extracts for antimicrobial and antioxidant activities. journal of ethnopharmacology. 2000;72(3): 403-10. 40. pourmorad f, hosseini sj, shahabimajd n. antioxidant activity, phenol and flavonoid contents of some selected iranian medicinal plants. african journal of biotechnology. 2006;5(11). 41. mohamad h, abas f, permana d, lajis nh, ali am, sukari ma, et al. dpph free radical scavenger components from the fruits of alpinia rafflesiana wall. ex. bak. (zingiberaceae). z naturforsch. 2004;59c:811-5. 42. molyneux p. the use of the stable free radical diphenylpicrylhydrazyl (dpph) for estimating antioxidant activity. songklanakarin journal science technology. 2003;26(2):211-9. 43. sharma op, bhat tk. dpph antioxidant assay revisited. food chemistry. 2009;113(4):1202-5. 44. ordoudi sa, tsimidou mz, vafiadis ap, bakalbassis eg. structure dpph scavenging activity relationships:parallel study of catechol and guaiacol acid derivatives. journal of agricultural and food chemistry. 2006;54(16):5763-8. 45. demirezer lö, kuruüzüm-uz a, bergere i, schiewe hj, zeeck a. the structures of antioxidant and cytotoxic agents from natural source: anthraquinones and tannins from roots of rumex patientia. phytochemistry. 2001;58(8):1213-7. 46. bisby f, roskov y, orrell t, nicolson d, paglinawan l, bailly n, et al. species 2000 & itis catalogue of life: 2008 annual checklist reading,uk2008 [cited 2000. available from: www.catalogueoflife. org/annual-checklist/2008/. 47. handerson mr. malayan wild flowers dicotyledons: the malayan nature society 1974. 48. bremer b, manen j-f. phylogeny and classification of the subfamily rubioideae (rubiaceae). plant systematics and evolution. 2000;225:43-72. 49. jansen s, lens f, ntore s, piesschaert f, robbrecht e, smets e. contributions to the wood anatomy of the rubioideae (rubiaceae). journal of plant research. 2001;114(3):269-89. 50. do qv, pharm gd, mai nt, phan tpp, nguyen hn, yea yy, et al. cytoxicity of some anthraquinones from the stem of morinda citrifolia growing in vietnam. tap chi hoa hoc. 1999;37:94-7. 51. ismail nh, ali am, aimi n, kitajima m, takayama h, lajis nh. anthraquinones from morinda elliptica. phytochemistry. 1997;45(8):1723-5. 52. rath g, ndonzao m, hostettmann k. antifungal anthraquinones from morinda lucida. international journal of pharmacognosy. 1995;33:107-14. 53. xiang w, song q-s, zhang h-j, guo s-p. antimicrobial anthraquinones from morinda angustifolia. fitoterapia. 2008;79(78):501-4. 54. feng zm, jiang js, wang yh, zhang pc. anthraquinones from the roots of primatomeris tetranda. chemical & pharmaceutical bulletin. 2005;53(10):1330-2. 55. hao j, feng s-x, qiu s-x, chen t. anthraquinone glycosides from the roots of prismatomeris connata. chinese journal of natural medicines. 2011;9(1):42-5. 56. kanokmedhakul k, kanokmedhakul s, phatchana r. biological activity of anthraquinones and triterpenoids from prismatomeris fragrans. journal of ethnopharmacology. 2005;100:284-8. 57. krohn k, gehle d, dey sk, nahar n, mosihuzzaman m, sultana n, et al. prismatomerin, a new iridoid from prismatomeris tetrandra. structure elucidation, determination of absolute configuration, and cytotoxicity. journal of natural products. 2007;70:1339-43. 58. han y-s, der heiden rv, verpoorte r. biosynthesis of anthraquinones in cell cultures of the rubiaceae. plant cell, tissue and organ culture. 2001;67:201-20. 99 vol. 7 no. 5 may-august 2019 research report acid-fast bacilli conversion of beijing and nonbeijing strain of pulmonary tuberculosis in south sulawesi syahridha1, ni made mertaniasih2, sustini florentina3, soedarsono4 1magister of tropical medicine, faculty of medicine, universitas airlangga 2department of microbiology, faculty of medicine, universitas airlangga 3departemen of publich health science and prevent medicine, faculty of medicine, universitas airlangga 4departement of pulmonology and respiratory, universitas airlangga-dr, soetomo general hospital 5doctor of soetomo general hospital surabaya a corresponding author: email: nmademertaniasih@gmail.com, ni-made-m@fk.unair.ac.id abstract beijing strains are a major part of the mycobacterium tuberculosis asian phylogenetic lineage. beijing strains represent about 50% of all tb strains in east asia and at least 13% of strains worldwide. beijing strain of mycobacterium tuberculosis is presumed as the factor of the increase in bacteria virulence and drug resistance, and the contributor in treatment failure. the aim of this study was to analyze the association between acid-fast bacilli conversion with strain genotipe beijing and non-beijing of pulmonary tuberculosis in south sulawesi. the design of research was observational analytic with prospective approach. the sampling technique used consecutive sampling. data were taken from active pulmonary tuberculosis patients’ medical record in balai besar kesehatan paru masyarakat makassar (pulmonary health center of makassar) and community health center in gowa regency, south sulawesi from march to june 2018. collected sputum samples were screened for afb and identified as beijing strain and non beijing strains using multiplex pcr in tropical disease institute of universitas airlangga. the results is showed that the characteristics of the respondents consisted of 12 respondents (33.3%) aged 56-65 years, 25 respondents (69.4%) men and 28 respondents (77.8%) had low category gradation of afb smear. univariate analysis showed 6 respondents (16.7%) with beijing strains, 30 respondents (83.3%) with nonbeijing strains, 32 respondents (88.9%) conversion sputum afb and 4 respondents (11.1%) non conversion sputum afb. bivariate analysis with chi-square statistical test shows that p value 0.022 < 0,05, that means there was association of beijing strains with bta conversion. microscopic examination of bta can be used to monitor and evaluate the treatment of new pulmonary tb patients undergoing treatment and the beijing mycobacterium tuberculosis strain has a significant correlation with the treatment failure of anti-tuberculosis drugs in south sulawesi. keywords: afb conversion, beijing strain, mycobacterium tuberculosis, pulmonary tuberculosis, treatment failure abstrak strain beijing merupakan bagian utama dari garis keturunan filogenetik mycobacterium tuberculosis asia. strain beijing mewakili sekitar 50% dari seluruh strain tb di asia timur dan setidaknya 13% dari strain seluruh dunia. strain beijing dari mycobacterium tuberculosis diprediksi penyebab peningkatan virulensi bakteri, resistensi terhadap pengobatan, dan berkonstribusi dalam kegagalan pengobatan. penelitian ini bertujuan menganalisis asosiasi konversi bta mikroskopis dengan strain beijing dan non beijing pada pasien tuberculosis baru di sulawesi selatan. jenis penelitian adalah analitik observasional dengan rancangan penelitian prospektif. teknik pengambilan sampel menggunakan konsekutif sampling. data diperoleh dari rekam medis pasien dengan tb paru aktif yang dirawat di balai besar kesehatan paru masyarakat makassar dan puskesmas di kabupaten gowa, sulawesi selatan pada maret sampai dengan juli 2018. sampel sputum yang dikoleksi dilakukan pemeriksaan bta kemudian diidentifikasi strain beijing dan non beijing dengan metode multiplex pcr di institute penyakit tropis universitas airlangga. hasil penelitian menunjukkan karakteristik responden terdiri dari 12 responden (33,3%) berusia 56-65 tahun, 25 responden (69,4%) laki-laki dan 28 responden (77,8%) memiliki 100 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 99–104 gradasi bta kategori low. analisis univariat menunjukkan 6 responden (16,7%) dengan strain beijing, 30 responden (83,3%) dengan strain non-beijing, 32 responden (88,9%) mengalami konversi sputum bta dan 4 responden (11,1 % tidak mengalami konversi sputum bta). analisis bivariat dengan uji statistik chi-square menunjukkan p value 0,022, terdapat assosiasi strain beijing dengan konversi bta. pemeriksaan bta dengan mikroskopis dapat digunakan untu monitoring dan evaluasi pengobatan pasien tb paru yang baru mejalani pengobatan serta strain beijing mycobacterium tuberculosis memiliki korelasi yang signifikan dengan kegagalan pengobatan obat anti tuberculosis di sulawesi selatan. kata kunci: konversi bta, strain beijing, mycobacterium tuberculosis, tbc paru, kegagalan pengobatan introduction pulmonary tuberculosis (tb) is both an acute and chronic disease which caused by mycobacterium tuberculosis that attacks the lungs. the disease spreads from one individual to another through the respiratory system. in 2015, it was estimated that there were 10.4 million new cases of tb worldwide; of which 5.9 million are found in adult male, 3.5 million in adult female, and 1 million in children. some countries in south and east asia such as the democratic republic of korea, indonesia, myanmar, bangladesh, india, and thailand comprise 46.5% of the total global number. india and indonesia alone make up one-third of the worldwide tb number, each having 23% and 10% respectively1. global efforts on tb control were required integrated and coordinated responses to monitor and assess global outbreak of tb, drug resistance surveillance, and high-risk population. an integrated approach is a must, combining networks of laboratories, tb control programs, and application of state-of-the-art molecular techniques to identify and report tuberculosis bacilli clones occurring around the world2. tuberculosis occurrence in indonesia is estimated at 1,020,000 cases, with only 330,729 of those are reported. of all the cases in indonesia, 93% are pulmonary tb with 64% of those are diagnosed through microscopic evaluation of acid-fast bacilli smear. the large number of untreated tb will become the source of further infections, creating urgency in having continuous preventive and curative measures. the success of a tb countermeasure program is linked to the success in dots treatment. such treatment is deemed to be a success if it is evidenced by sputum afb conversion at the final phase of the intensive treatment (2 months) reaching at least 80% and recovery rate at the end of treatment reaching more than 85%3. acid fact bacilli conversion is a strong predictor and the initial success of therapy in pulmonary tb4. sputum conversion in pulmonary tb was determined based on the absence of acid fact bacilli (afb) in sputum taken at the end of the 2nd month of treatment. sputum conversion in pulmonary tb cases occurred at the end of the first month (60-80%) and at the end of the second month (95%)4. afb conversion is one of the methods used in indonesia which have high accuracy, fast and economical costs. another method was chest x ray but the results are not specific to diagnosis tuberculosis5. in addition there are culture methods that have a higher sensitivity but the cost not effective and needed long time for know the result of diagnosis. the research of wang x, et al was showed that by using direct smear method ziehl nielsen staining obtained a sensitivity of 40%, then in the same sample culture method was carried out so the sensitivity value would increase by 65% 6. sputum afb conversion can be affected by previous medical history. mycobacterium tuberculosis exposed with antibiotics might undergone genetic mutation as a result of its biological trait to defend itself. mutation might occur to a specific gene targeted by the medication, creating risk of drug resistance, which in turn resulting in sputum afb conversion failure7. another factor correlating with sputum afb conversion is the mycobacterium tuberculosis’ genotype. the genotype of mycobacterium tuberculosis that attack tb patients in indonesia are different from one province to another as a result of the geographical differences of each province8. different strain has different characteristics; one of which is the tendency to develop antibiotics resistance. mycobacterium tuberculosis complex (mtbc) strains which are known to be easier to treat are mycobacterium africanum, mycobacterium bovis, mycobacterium microti, mycobacterium canneti and m. tuberculosis9. molecular epidemiology studies have pointed out that mycobacterium tuberculosis’ genotype is varied based on geographical distribution10. some pathogen hosts have undergone adjustments while specific genotype has evolved to increase its pathogenicity and virulence. in some east asian countries such as china and hong kong, 86% of the occurring genotype is beijing strain11. in contrast, indonesia has more varied distributions. south sulawesi, for instance, is still dominated by beijing strain. beijing strain genotypes have specific characteristic such as contribute to the spread of drug-resistant tb and clinically related to treatment failure12. thai studies is showed infection with beijing genotype is a significant risk factor for bacterial persistence in treatment that results in treatment failure or relapse inside 2 years13. lisdawati’s research on beijing strain distribution in indonesia is showed that in palembang and lampung, beijing strain occurrence number is 31.48%; serang, jakarta, bandung, and surabaya have 28.83%; banjarmasin and pontianak have 16.18%; and makassar has 25.93%. beijing strain distribution in male is 24.2%, while in female is 15.3%. specifically in makassar, other occurring 101syahridha, et al.: acid-fast bacilli conversion of beijing and non-beijing strain strains are sub-type eail (1 pattern), h1 (1 pattern), lam (2 patterns), ea12 (2 patterns), u (3 patterns), ulikes (1 pattern), ea15 (1 pattern), ulikely lam (1 pattern), manu2 (1 pattern), and h3 (2 patterns). the sample also holds 1 isolate which was showed pattern of m. bovis14. identifying beijing genotype can be done through various methods, such as spoligotyping, is6110 rflp, pcr, and sequencing15. these methods focus on various types of protein in specific target genes. nikajima’s research points out that mycobacterium tuberculosis genome h37rv is able to identify beijing strain, specifically the rv0679c protein with the size of 163bp15. data from south sulawesi provincial health office in the year 2016 shows that there were 13,659 tb cases with 12,965 cases of tb (7,180), 97 cases of mdr tb, and 597 cases of tb in children. in the cases of afb positive tb, treatment success rate was 86%, less than the targeted 90%. beside that, success rate just 82,5% (target more > 90%)16. method this is an analytical observational study with crosssectional analysis design. the research was done in the city of makassar and gowa regency from march to june 2018. the samples used were 36 new patients diagnosed with pulmonary tb, with an age of 15 to 65 years. ethical clearance was issued by ethical committee of universitas airlangga numbered 82/ec/kepk/ fkua/2018. tuberculosis patients were diagnosed using zielh neelsen afb smear method. conversion occurred if there was a change in the result of afb smear: from positive to negative during the 2-month treatment. detection of beijing strain was done by decontamination using petroff alkali method and extraction of dna using qiagen kits. amplification of beijing strain was done with multiplex pcr. the pcr mix had a total volume of 20 μl which were comprised of 10 μl of pcr buffer, 1 μl of on-1002 (fw), on-1258 (r1)α, and on-1127 (r2) primers, 6 μl of dw, and 2 μl of template dna. the next step was a 25-cycle of pre-denaturation at 95°c for 1 minute, denaturation at 95°c for 10 seconds, annealing at 66°c for 10 seconds, and extension at 72°c for 15 seconds. upon finishing, a final extension was done at 72°c for 3 minutes and an elongation at 15°c for 5 minutes16. data analysis was performed using spss 17. correlation between sputum afb conversion with beijing strain was assessed using bivariate analysis by chi-square test. result a preliminary survey was performed to collect data of respondents’ characteristics such as age, sex, and afb gradation. the results are presented in the table 1: mycobacterium tuberculosis was isolated from 50 patients are afb positive, but only 36 fit the criteria. beijing strain is identified through multiplex pcr with the result showing 6 samples (16.7%) are beijing strain and 30 samples (83.3%) are non-beijing. treatment success rate is identified by microscopic examination after 2 months of treatment. as much as 32 patients (88.9%) are found to have undergone conversion, while the other 4 (11.1%) are not. the results are presented in the table 2. chi-square test yields p-value of 0.010 < 0.05, meaning that there is a significant correlation between beijing strain genotype with sputum afb conversion. the results are presented in the table 3. table 1. respondent distribution based on age, sex, and afb gradation (n=36) no characteristic frequency (n) percentage (%) 1 age (years old) 16-25 26-35 36-45 46-55 56-65 6 5 5 8 12 16.7 13.9 13.9 22.2 33.3 2 sex male female 25 11 69.4 30.6 3 afb gradation low medium high 28 3 5 77.8 8.3 13.9 table 2. frequency distribution of genotype strain and afb smear conversion no research variables frequency (n) percentage (%) 1 genotype beijing strain non-beijing strain 6 30 16.7 83.3 2 afb conversion conversion non-conversion 32 4 88.9 11.1 table 3. correlation between genotype and sputum afb conversion mtb genotype afb conversion p-value ornon-conversion conversion total n % n % n % beijing 3 75.0 3 9.4 6 16.7 0.010 15.0 (95%, ci 1.8-120.8) non-beijing 1 25.0 29 90.6 30 83.3 total (n) 4 11.1 32 89.9 36 100 102 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 99–104 discussion this research was showed that afb conversion rate of new patients with afb positive is 89.9%. according to who, the conversion rate must be more than 80%17. diagnosis and conversion are performed with afb method using microscopes. examination on two sputum specimens is sufficient to identify the majority (95-98%) of afb-positive tb patients. the policy of who recommend to using two spesimen sputum on case finding using microscopy to get setting with appropriate external quality assessment and high-quality documentation. the microscopy of both specimens must be screened. in settings with appropriate quality assurance procedure, a case is defined as a person with one positive smear—meaning at least 1 afb in 100 microscopic fields18. in 2010, who confirmed the accuracy of microscopy of two consecutive sputum specimens on the same day for tb diagnosis, allowing treatment to take place since the first visit to a health center facility17. microscopy benefits in terms of simplicity and cost efficiency, allowing immediate detection of the most infectious pulmonary tb. sputum specimen from patients with pulmonary tb, especially those having cavity diseases, often have enough bacteria to be detected using microscopes. microscopy is also suitable for peripherylevel laboratories, or higher. it can be performed safely even in laboratories with low alert value, reducing the risk of tb infection in laboratories. it is a simple, speedy, and inexpensive test needed as a follow up measure for patients with high risk of tb. despite the benefits, microscopy also has limitation for afb. it is unable to distinguish mycobacterium tuberculosis complex from non-tuberculous mycobacteria. it cannot tell the difference between organisms that should be exterminated from those that actually pose no threat. it also cannot differentiate drug-sensitive strains from the resistant ones18. sputum afb conversion in pulmonary tb patients takes place after intensive phase of the treatment. afb conversion is the change in sputum smear from afb positive to afb negative after undergoing intensive treatment19. based on table 2, it can be seen that afb conversion occurs in 32 samples (88.9%) and does not occur in 4 samples (11.1%). this research was showed non conversion rate 11,1 %, based on intervew with health worker in the public health centre, the pasient who non conversion after 2 month treatment have comorbid diseaeses such as diabetes melitus. garrido research was showed that there was relationship between diabetes melitus with treatment failure20. this research was showed a conversion rate of 88.9%, fitting the indicator from the ministry of health of >80%. conversion acts as an indicator of treatment success. success of a treatment is also influenced by age, sex, medical history, presence of antibiotic resistance and bilateral radiological lession21, 22. smear acid bacilli, retreatment, comorbid diseases, and education23. beijing genotype strains have specific properties such as the effects of the bcg vaccine, increased virulence, and risk of treatment failure. clinically, there were no differences in signs and symptoms of the beijing and non-beijing genotype strains 24, but parwati research is showed that the proportion of patients in the beijing strain who had fever symptoms was higher than the strain non beijing25. at the molecular level, beijing strains have specific properties in terms of protein and lipid structure and their interaction with the human defense system. finally, the beijing genotype has polymorphisms in immune genes, showed the co-evolutionary of human-mycobacteria. the emergence of the beijing genotype family may represent the response of m tuberculosis to vaccination or antibiotic treatment, with a negative reaction to tuberculosis control26. the findings are showed that the result of sputum multiplex pcr of the respondents mostly (30 samples, or 83.3%) are non-beijing strain and only a few (6 samples, or 16.7%) are beijing strain. research done by octavian using spoligotyping shows that there are 8% beijing strain in papua19. research by lisdawati points out a proportion of beijing strain in makassar to be as much as 25.93%13. according to researcher’s assumption , the percentage of beijing in makassar is quite high compared to other regions in the eastern region of indonesia, makassar society has a higher level of mobilization and geographically located in central indonesia making it easier to move to other area. data analysis using chi-square test yields p-value of 0.022 < 0.05, meaning that there is a significant correlation between beijing strain genotype and sputum afb conversion. this finding is also advocated by parwati, claiming that there is a correlation between beijing strain and treatment failure26. this research is different from hang’s which focuses on beijing strain’s sublineage of modern, ancient, and non-beijing. hang’s research also shows that there is no correlation between the strain of mycobacterium tuberculosis and treatment failure27.a study examining clinical pathways and treatment outcomes depends on the genotype of the mycobacterium (mtb) strain among various age groups of tb patients. this study was conducted on 6 strains including beijing, lam, haarlem, ural / uganda i, s, africanum and showed that beijing was included in the group with a low treatment success rate28. mourik’s research is showed that there is still a presence of active bacteria in the first two months of treatment on test animals infected with mycobacterium tuberculosis beijing strain29. beijing strains was also associated with the incidence of mdr and recurrence of tb patients. vyazovaya’s research was showed mutations in rpob531 were most commonly found among beijing isolates (60 of 72 isolates with rpob mutations), five isolates (four of which were beijing) were showed multiple or triple mutations in rpob. inh resistance mutations, the katg315 mutation is also the most common in beijing isolates30. studies from estonia, china, korea and taiwan were showed a significantly higher mdr frequency among beijing descendants31. a study conducted in sweden which identified 13% of beijing strains for 15 years. the proportion of strains with mdr was significantly higher among beijing strains than in non-beijing strains31. 103syahridha, et al.: acid-fast bacilli conversion of beijing and non-beijing strain different with rutaihwa’s research, the existence of beijing strains in africa is not related to anti-tuberculosis drug resistance 32. this study was supported soolingen’s study of the relationship between genotype and resistance between unclear treatment failure33. conclusion although it is true that afb microscopy is vital in monitoring and evaluating one-line anti-tuberculosis treatment in pulmonary tb patients in tb endemic areas, early care management for pulmonary tb patients also needs to consider the lineage of the mycobacterium tuberculosis strain, e.g. beijing strain. at the molecular level, beijing strains have specific properties in terms of protein and lipid structure and their interaction with the human immune system. this becomes imperative given the fact that the strain of mycobacterium tuberculosis is a determinant of treatment success of the patients and examine the implications for future strategies. acknowledgement the authors would like to show their gratitude to the respondents of the research and the staff and officials of public health services in the city of makassar and gowa regency, south sulawesi for their contribution in data collection. this research was funded by lembaga pengelola dana pendidikan (education funds management board). references 1. world health organization (who), bending the curve ending tb: annual report 2017, (world health organization, regional office for south-east asia, 2017). 2. viedma dg, mokrousov, rastogi n. innovations in the molecular, epidemiology of tuberculosis. enfermedades infecciosas microbiologia clinica 2010; 29:8-13. 3. kementerian kesehatan ri. pedoman nasional penanggulangan tuberkulosis, (kementerian kesehatan ri 2010, jakarta: 20-21) 4. aliyah nur, emmy h, basti. gambaran konversi sputum bakteri tahan asam (bta) dan vitamin d pada penderita tuberculosis paru kasus baru. j chest crit and emerg 2016; 3: 1-6. 5. ryu yon ju, ‘diagnosis of pulmonary tuberculosis: recent advances and diagnostic algorithms’, (2015) 78 tuberculosis respiratory diseases. [64-71]. 6. wang x, zhao l, yu x, li y, ma y, dong l, huang h. bead capture increases the sensitivity of sputum microscopy for the diagnosis of tuberculosis in beijing, china. transactions of the royal society of tropical medicine and hygiene 2013:1-3. 7. andrade soraya, gales ana cristina, sader helio silva, antimicrobial resistance in gram-negative bacteria from developing countries, (spinger 2010)[249-266] 8. parwati i, reinout, mirawati sudiro, bachti alisjahbana. mycobacterium tuberculosis population structures differ significantly on two indonesian island. journal of clinical microbiology 2008; 46:3639– 3645. 9. supply philip, michael marceau, sophie mangenot. genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of mycobacterium tuberculosis. nature genetics 2012; 45:172-180. 10. borgdorff, soolingen van. the re-emergence of tuberculosis: what have we learnt from molecular epidemiology?. clinical microbiology and infection 2013; 19: 889–901. 11. couvin david, nalin rastogi. tuberculosis-a global emergency: tools and methods to monitor, understand, and control the epidemic with specific example of the beijing lineage. elseiver tuberculosis 2015; 95:177-189. 12. merker m, blin c, mona s, duforet-frebourg n, lecher s, willery e, blum mg,rusch-gerdes s, mokrousov i, aleksic e, allix-beguec c, antierens a, augustynowicz-kopec e, ballif m, barletta f, beck hp, barry ce, 3rd, bonnet m, borroni e, camposherrero i, cirillo d, cox h, crowe s, crudu v, diel r, drobniewski f, fauvilledufaux m, gagneux s, ghebremichael s, hanekom m, hoffner s, jiao ww, kalon s, kohl ta, kontsevaya i, lillebaek t, maeda s, nikolayevskyy v, rasmussen m, rastogi n, samper s, sanchezpadilla e, savic b, shamputa ic, shen a, sng lh, stakenas p, toit k, varaine f, vukovic d. evolutionary history and global spread of the mycobacterium tuberculosis beijing lineage. nat genet 2015; 47: 242-249. 13. thai phan v k, ha dang t m, hanh t nguyen, day jeremy, dunstan sarah, quynh nguyen thi, kiet vo sy, lan nguyen huu, huy dung nguyen, thi ngoc lan nguyen. bacterial risk factors for treatment failure and relapse among patients with isoniazid resistant tuberculosis. bmc infectious diseases 2018; 112:1-9. 14. lisdawati vivi, puspandari nelly, parwati ida. studi pemetaan awal dna mycobacterium tuberculosis complex secara spoligotyping pada hasil isolasi dahak pasien tuberkulosis paru dari 10 ibu kota propinsi. buletin penelititian kesehatan 2010; 38:208 – 224. 15. nakajima chie, tamaru aki,rahim zeaur, poudel ajay,maharjan bhagwan, saw aye khin, ling hong, hattori toshio, iwamoto tomotada, fukushima yukari, haruka suzuki, yasuhiko suzuki, takashi matsuba. simple multiplex pcr assay for identification of beijing family mycobacterium tuberculosis isolates with a lineagespecific mutation in rv0679c. journal of clinical microbiology 2013; 5:2025–2032. 16. dinas kesehatan provinsi sulawesi selatan, profil kesehatan sulawesi selatan tahun 2017 (dinas kesehatan sulawesi selatan 2017). 17. world health organization, treatmen of tuberculosis guidelines fourth edition (world health organization 2010). 18. global laboratory initiative, laboratory diagnosis of tuberculosis by sputum microscopy (sa phatology, 2013) 19. oktavian antonius, chaidir lidya, krismawati hana, suebu melda, karapa oktofianus. distribusi genotipe mycobacterium tuberculosis dan transmisi tb di kota jayapura provinsi papua. institute of research and development for biomedicine papua 2013: 70-85. 20. garrido ms, penna ml, perez-porcuna tm, souza ab, marreiro ls, albuquerque bc, martínez fe,. factors associated with tuberculosis treatment default in an endemic area of the brazilian amazon: a case control-study. plos one 2012; 7:1-7. 21. babalik a, kilicaslan z, caner ss, gungor g, ortakoylu mg, gencer s, mccurdy sa. a registry-based cohort study of pulmonary tuberculosis treatment outcomes in istanbul, turkey. japan journal infectiuos diseases 2013; 66: 115-120. 22. babalik a, kilicaslan z, kiziltas s, gencer s, ongen g. a retrospective case-control study, factors affecting treatment outcomes for pulmonary tuberculosis in istanbul, turkey. balkan medicine journal 2013; 30: 204-210. 23. sengul aysun, akturk ulku, aydemir yusuf, kaya nurullah, durmus nagihan, tasolar fatma. factors affecting successful treatment outcomes in pulmonary tuberculosis: a single-center experience in turkey, 2005–2011. the journal of infection in developing countries 2015; 8: 821-828. 24. feng jy, su wj, tsai cc, chang sc. clinical impact of mycobacterium tuberculosis w-beijing genotype strain infection on aged patients in taiwan. j clin microbiol 2008; 46: 3127–3129. 25. parwati i, alisjahbana b, apriani l, rista d, ottenhoff t, zanden a, meer j. mycobacterium tuberculosis beijing genotype is an independent risk factor for tuberculosis treatment. the lancet infectious diseases 2010; 10: 103-111. 104 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 99–104 26. parwati ida, reinout, dick soolingen. possible underlying mechanisms for successful emergence of the mycobacterium tuberculosis beijing genotype strains. lancet infection diseases 2010; 10:103–111. 27. hang nguyen t.l, maeda shinji, keicho naoto, pham h. thuong, hiroyoshi endo. sublineages of mycobacterium tuberculosis beijing genotype strains and unfavorable outcomes of anti-tuberculosis treatment. tuberculosis 2015; 95: 336-342. 28. konstantynovska olha, poteiko petro, synenko tetiana, kuznietsova iryna, rohozhyn anton, solodiankin oleksii, liashenko oleksandr. pulmonary tuberculosis among elderly and senile-aged patients depending on mycobacterium strains. european respiratory journal 2017; 50:2724. 29. mourik bas c, knug j de gerjo, verbon annelies, mouton johan, bax kannelore, steenwinkel jurriaaan. assessment of bactericidal drug activity and treatment outcome in a mouse tuberculosis model using a clinical beijing strain. american society for microbiology 2017; 61:1-9. 30. anna vyazovaya, igor mokrousov, natalia solovieva, alexander mushkin, olga manicheva, boris vishnevsky, viacheslav zhuravlev, olga narvskaya. tuberculous spondylitis in rhussia and prominent role of multidrug-resistant clone mycobacterium tuberculosis beijing b0/w148. american society for microbiology 2015: 1-14 31. hu y, ma x, graviss ea, wang w, jiang w, xu b. a major subgroup of beijing family mycobacterium tuberculosis is associated with multidrug resistance and increased transmissibility. epidemiol infect 2011; 139: 130-138. 32. rutaihwa liliana k, menardo fabrizio, stucki david, sebastian m. gygli, serey d ley, malla bijaya, feldmann julia, borrell farnov sonia, beisel christian, middelkoop keren. multiple introductions of the mycobacterium tuberculosis lineage 2 beijing into africa over centuries. cold spring harbor laboratory 2018; 1-21 33. buu tn, huyen mn, van soolingen d, lan nt, quy ht, tiemersma ew, et al. the mycobacterium tuberculosis beijing genotype does not affect tuberculosis treatment failure in vietnam. clin infect dis 2010; 51: 879-886. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 75 vol. 7 no. 4 january-april 2019 expression of four cytokine/chemokine genes in peripheral blood mononuclear cells infected with dengue virus sri masyeni1a, usman hadi2, kuntaman2, benediktus yohan3, nur ita margyaningsih3, r. tedjo sasmono3 1 faculty of medicine and health science, warmadewa university, denpasar-bali, indonesia, 2 faculty of medicine, universitas airlangga, surabaya, indonesia 3 eijkman institute for molecular biology, jakarta, indonesia a corresponding author: masyeniputu@yahoo.com abstract overproduction of numerous pro-inflammatory cytokines, during dengue virus (denv) infection, has been related to plasma leakage in the vascular endothelium and studied elsewhere with conflicting results. the current study objective is to evaluate the expression of four cytokine/chemokine genes following denv-2 infection within peripheral blood mononuclear cells (pbmc) isolated from a healthy donor. venous blood was drawn, and pbmcs were isolated using ficoll density gradient centrifugation. cells were maintained in culture medium and infected with indonesian isolate of denv-2. cells were harvested and followed by total rna extraction and reverse-transcription into cdna using oligo d(t) primers and reverse transcriptase enzyme system. the sybr greenbased quantitative qrt-pcr was used to calculate the relative expression of il-6, il-8, ip-10 and mip-1ßencoding genes during infection time points, compared to uninfected cell controls. the observation of the cytokine was on the 6 and 18 hours post-infection. the different expression profiles of cytokines/chemokines were observed. the up-regulation of gene expression was observed for il-8 and ip-10. in contrast, the down-regulatory of il-6 and mip-1ß genes expression was documented during the infection period. the cytokine il-8 and ip-10 are potent chemoattractants in the recruitment of neutrophil, basophil, and lymphocytes in response to an infection. the highlight of this study is on the up-regulation of il-8 and ip-10 genes expression which may confirm the roles of these chemokines in the pathogenesis of dengue infection. keywords: dengue, gene expression, cytokine, chemokine, pbmc abstrak produksi sitokin pro-inflamasi berlebihan pada infeksi virus dengue yang dihubungkan dengan terjadinya kebocoran plasma pada endotel vaskular telah diteliti dengan hasil yang bervariasi. penelitian ini bertujuan untuk mendeteksi ekspresi empat gen pengkode sitokin atau kemokin pada sel mononuklear darah tepi yang di-infeksi virus dengue serotipe-2. sel mononuklear darah tepi (pbmc) diisolasi dari donor sehat dengan menggunakan metode sentrifugasi gradient ficoll. ekstraksi rna dilakukan terhadap sel mononuklear darah tepi, kemudian sintesis cdna dilakukan dengan menggunakan primer oligo d(t) dan sistem enzim reverse transcriptase. dengan menggunakan quantitative real-time polymerase chain reaction (qrt-pcr) berbasis sybr-green, ekpresi gen penyandi il-6, il-8, il-10 dan mip-1ß dibandingkan antara sel yang terinfeksi dan tidak terinfeksi virus dengue serotipe-2. observasi dilakukan pada waktu pengamatan jam ke-6 dan waktu pengamatan ke-18 dari infeksi. ekspresi gen penyandi il-8 dan ip-10 ditemukan lebih tinggi, sebaliknya ekspresi gen penyandi sitokin il-6 dan mip-1ß lebih rendah pada sel mononuklear darah tepi yang diinfeksi virus dengue serotipe-2 dibandingkan dengan kontrol interleukin-8 dan ip-10 adalah sitokin yang bersifat sebagai kemotaktik untuk memicu kemotaksis dari sel netrofil, basophil dan limfosit sebagai respon dari suatu inflamasi. hasil penelitian ini menunjukkan bahwa ekspresi kedua gen penyandi kemokin yang meningkat setelah infeksi virus dengue serotipe-2 mungkin berperan pada patogenesis terjadinya kebocoran plasma pada infeksi virus dengue. kata kunci: dengue, ekspresi gen, sitokin, kemokin, pbmc research report 76 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 75–78 introduction infection caused by dengue virus is still the major cause of acute febrile illness in the world, particularly in the tropical and subtropical area, including southeast asian countries.1 the widely-spread dengue virus (denv) and the aedes mosquitoes vector have now become a major problem, and more than 125 countries are known to be dengue-endemic regions, including indonesia.2 the high denv expansion is related to climate change, globalization effect, traveling communities, socioeconomics, settlement and viral evolution.2 dengue is a complex disease, caused by an rna virus, entailing of four antigenically similar but with immunologically distinct serotypes (denv-1 to denv-4).3,4 a serotype-specific denv infection confers life-long immunity with only partial protective immunity for the other serotypes hence people in endemic countries can be infected up to four times by different denvs.5 the clinical manifestation varies from asymptomatic to the severe, life-threatening manifestation.6 the majority of dengue clinical manifestation is mild, asymptomatic dengue or mild fever. in the lesser incidence, the more severe form of dengue is dengue hemorrhagic fever (dhf) with various degrees and dengue shock syndrome (dss), in which the fatality rate may exceed more than 5% in special populations.4,5 expanded dengue syndrome or unusual dengue syndrome with a high mortality rate can be appeared without any sign of plasma leakage, the hallmark of severe dengue.7 the detailed pathogenesis of dengue is not yet entirely understood. the existence of cytokine storm-induced endothelial dysfunction in denv infection has been published over the past decades.8 the excessive release of various cytokines recognized as cytokine storm has been regarded as the underlying mechanism of plasma leakage in denv infection.9 the role of diverse cytokines and chemokines were observed during the more severe manifestation of dengue might be considered to be correlated with the infecting denv serotype.10 at the different phase of illness, cytokines/chemokines profiles were found to be increased in patients at the febrile phase.11 several studies were also reported the increased expression of cytokines/ chemokines within in vitro in human monocytes12 or epithelial13 cells or in vivo in dengue patients’ serum.14 the dual role of both innate and inflammatory pathways were activated during dengue disease and revealed the involvement of immune mediators.14 utilizing dengue human cell line infection model, the up-regulated cytokines/ chemokines gene expression profiles during denv serotypes infection have been described and among them were il-6, il-8, and ip-10.13 other report is highlights the induction of mip-1β by dengue virus.15 in this study, we are reported the expression profiles of four genes encoding cytokines and chemokines in the peripheral blood mononuclear cells (pbmcs), infected with indonesian isolate of denv-2. material and method ethical considerations the ethical considerations of this study have been reviewed and approved by the institutional review board of udayana university, bali, indonesia (document no. 2072/un.14.2/kep/2017). blood collection and peripheral blood mononuclear cells (pbmcs) isolation thirty ml of venous blood was drawn from a healthy donor and subjected to pbmc isolation using ficoll histopaque-1077 (sigma-aldrich, st. louis, mo) density gradient centrifugation. isolated pbmcs were maintained in 1 rpmi medium supplemented with 10% of fetal bovine serum (fbs), 1% of antibiotic/antimycotic, and 2 mm of l-glutamine (all from gibco-thermo fisher scientific, carlsbad, ca). cells were seeded at 1 106 cells per well of 24-well plates (corning, ny). the seeded cells were allowed to rest during overnight incubation at 37°c incubator with 5% co2 supplementation. denv infection the denv-2 virus strain smg-se001 was isolated from a severe dengue patient from semarang in 201216 (eijkman’s collection). cells were infected with denv-2 using the multiplicity of infection (moi) of 1 (theoretical calculation of one virus pfu per cell), prepared in 1 rpmi medium-2% fbs, performed in duplicate. to achieve the moi of 1 (theoretically one virus particle infecting one cell), a number of 106 plaque forming unit (pfu) of denv-2 was inoculated into 106 pbmc cells. the denv pfu titre was measured using a standard plaque assay method, as described elsewhere, 16 meanwhile uninfected cells controls were inoculated using the addition of medium only. plates were then incubated for 1 hour at 37°c, 5% co2 to facilitate virus infection. the incubation period was continued and cells were harvested at 0, 6, and 18 hours post-infection to represent the early phase of dengue illness. total rna extraction and qrt-pcr infected cells were harvested at the designated time points and subjected to the total rna extraction using mircury rna isolation kit – cell and plant (exiqon, vedbaek, denmark), as described in the manufacturer’s instructions. total rna quantity was measured using qubit 3.0 fluorometer and qubit rna br assay kit (life technologies-thermo fisher scientific, eugene, or). an amount of 100 ng of total rna was used in cdna synthesis performed using oligo d(t) primer and goscript reverse transcription system (promega, madison, wi). the resulting cdna was then amplified by quantitative realtime pcr using gotaq qpcr master mix (promega) and primers as listed in table 1. the relative gene expression analysis was performed using the equation of 2-∆∆ct of normalized ct value to human β-actin. 77masyeni, et al.: expression of four cytokine/chemokine genes result and discussion the increased expression of il-8 and ip-10 was observed during infection of denv-2, with the highest level seen at 18 hours post-infection (figure 1). by contrast, the relative expression of il-6 and mip-1β genes was relatively decreased along the infection time. the upregulation of il-8 and ip-10 was reaching more than twofold at 18 hours post-infection, relative to the uninfected by macrophages and other cell types, such as endothelial cells, fi broblasts, and synovial cells.17 it has been reported that increased levels of il-8 cytokine in the sera of dengue patients were correlated with the severe form of dengue.18 the level of il-8 was signifi cantly higher in samples from severe dengue cases and lower in cases of dengue without warning signs than in healthy controls. samples that were positive for anti-dv igg antibody had higher levels of il-8.18 the ip-10 is a member of cc-motif chemokine in response to interferon-γ in infectious diseases.18 the previous study result on the induced expression of ip-10 chemokine has been well-documented in dendritic cells and other primary cell lineages in response to in vitro dengue infection19 as well as in pbmcs of dengue patients.20 the level of ip-10 has been found to be increased in serum of dengue-infected patients in studies in venezuela19 and singapore.21 moreover, the increased level of il-8 and ip10 cytokines/chemokine was also observed in a549 human lung epithelial cells infected with denv.13 we observed the reduction trend in both il-6 and mip1β gene expressions during denv-2 infection of pbmcs. the increased level of il-6 has been reported in dengue patients.22 in addition, the increased level of il-6 has been correlated to disease severity with higher level observed in the more severe form of dengue manifestation, through the up-regulation of inflammatory responses in macrophages and induction of b cell maturation.23 dengue ns1 protein was found to significantly increase the production of il-6 thoroughly activation of tlr-2 and tlr-6 in pbmc infected with denv.24 the higher expression of mip-1β was reported in pbmc from denv-infected patients.11 however, in this study we did not found the elevated level of gene expressions for il-6 and mip-1β within 18 hours of denv infection. a systematic review on markers of dengue disease severity revealed that increased level of il-6 and mip-1β was observed in samples taken more than figure 1. the relative cytokine/chemokine genes expression of denv-2 infected-pmbcs compared to uninfected controls during three infection time points. control. the reduction of il-6 and mip-1β genes expression to the level of nearly half-fold was apparent at the same time points. we observed the up-regulation of il-8 and ip-10 in pbmcs infected with denv-2, relative to the uninfected controls. the il-8 is the pro-infl ammatory cytokines, a member of cxcl chemokine family and the factor of a neutrophil chemotactic factor, which have been widely investigated and found to increase at the protein level.17 the cytokine il-8 is a neutrophil chemoattractant produced table 1. primers used in cytokine/chemokine relative gene-expression analysis using qrt-pcr. target gene direction sequence (5’-3’) il-6 sense gaggataccactcccaacagacc antisense aagtgcatcatcgttgttcataca il-8 sense tgccaaggagtgctaaag antisense ctccacaaccctctgcac ip-10 sense ttcaaggagtacctctctctag antisense ctggattcagacatctcttctc mip-1β sense ctgtgctgatcccagtgaatc antisense tcagttcagttccaggtcataca β-actin sense catctcttgctcgaagtcca antisense atcatgtttgagaccttcaaca 78 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 75–78 48 hours after onset of fever.25 the results from this study may be related to the difference in the experimental setup of genes expressions analysis and the designated sample collection period to represent the early phase of dengue disease development during 18 hours of virus infection. however, the decreasing levels of il-6 and mip-1β have been observed in dengue with warning signs during disease progression from febrile to convalescence phase.11 the current study result concordant to another study result where increased serum levels of il-6 and il-8 were detected in patients with dengue haemorrhagic fever but not dengue fever.26 more in-depth research is needed to study the kinetics of these cytokines during dengue infection. conclusion it has been reported here the dynamic of four cytokines related to an early phase of denv infection. the upregulation of il-8 and ip-10, chemokines with profiles that highly correlated with dengue than other febrile diseases and have the potential to be used as a biomarker of dengue, since high expression of these cytokines at the earlier phase of observation in this study. acknowledgement this work was supported by the faculty of medicine and health science, university of warmadewa. we would like to thank prof. aryati, prof. yoes prijatna dachlan, dr. sunaryo, dr. h. muchlis a. udji, and dr. gondo mastutik, for their critical inputs and suggestions to the study. conflict of interest the authors declare that they have no conflict of interest. references ooi e-e, gubler dj. dengue in southeast asia: epidemiological 1. characteristics and strategic challenges in disease prevention. cad saude publica. 2009;25:s115–24. murray nea, quam mb, wilder-smith a. epidemiology of dengue: 2. past, present and future prospects. clin epidemiol. 2013;5:299– 309. guzman mg, halstead sb, artsob h, buchy p, farrar j, gubler 3. dj, et al. dengue: a continuing global threat. nat rev microbiol. 2010;8(12):s7–16. guzman mg, harris e. dengue. lancet. 2015;385(9966):453–65. 4. katzelnick lc, harris e, baric r, coller b-a, coloma j, crowe jr 5. je, et al. immune correlates of protection for dengue: state of the art and research agenda. vaccine. 2017;35(36):4659–69. martina bee, koraka p, osterhaus adme. dengue virus 6. pathogenesis: an integrated view. vol. 22, clinical microbiology reviews. 2009. p. 564–81. who. comprehensive guidelines for prevention and control of 7. dengue and dengue haemorrhagic fever [internet]. who regional publication searo. 2011. 159-168 p. available from: http://scholar. google.com/scholar?hl=en&btng=search&q=intitle:comprehensiv e+guidelines+for+prevention+and+control+of+dengue+and+den gue+haemorrhagic+fever#1 srikiatkhachorn a, mathew a, rothman al. immune-mediated 8. cytokine storm and its role in severe dengue. semin immunopathol. 2017 jul;39(5):563–74. basu a, chaturvedi uc. vascular endothelium: the battlefield 9. of dengue viruses. vol. 53, fems immunology and medical microbiology. 2008. p. 287–99. mangione jna, huy nt, lan ntp, mbanefo ec, ha ttn, bao lq, 10. et al. the association of cytokines with severe dengue in children. trop med health. 2014 dec;42(4):137–44. rathakrishnan a, wang sm, hu y, khan am, ponnampalavanar 11. s, lum lcs, et al. cytokine expression profile of dengue patients at different phases of illness. plos one. 2012;7(12). puerta-guardo h, sandino ar, gonzález-mariscal l, rosales vh, 12. ayala-dávila j, chávez-mungía b, et al. the cytokine response of u937-derived macrophages infected through antibody dependent enhancent of dengue virus disrupts cell apical junctional complexes and increase vascular permeability. j virol. 2013;jvi-00085. yohan b, kendarsari ri, mutia k, bowolaksono a, harahap ar, 13. sasmono rt. growth characteristics and cytokine/chemokine induction profiles of dengue viruses in various cell lines. acta virol. 2014;58(1):20–7. costa vv, fagundes ct, souza dg, teixeira mm. inflammatory 14. and innate immune responses in dengue infection: protection versus disease induction. am j pathol. 2013 jun;182(6):1950–61. bozza fa, cruz og, zagne smo, azeredo el, nogueira rmr, 15. assis ef, et al. multiplex cytokine profile from dengue patients: mip-1beta and ifn-gamma as predictive factors for severity. bmc infect dis. 2008;8:1–11. fahri s, yohan b, trimarsanto h, sayono s, hadisaputro s, 16. dharmana e, et al. molecular surveillance of dengue in semarang, indonesia revealed the circulation of an old genotype of dengue virus serotype-1. plos negl trop dis. 2013 aug;7(8):e2354. griffith jw, sokol cl, luster ad. chemokines and chemokine 17. receptors: positioning cells for host defense and immunity. annu rev immunol. 2014;32:659–702. tsai yt, chang sy, lee cn, kao cl. human tlr3 recognizes 18. dengue virus and modulates viral replication in vitro. cell microbiol. 2009;11(4):604–15. becerra a, warke k, xhaja k, de bosch n, rothman al, bosch 19. i. gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo. j med virol. 2009 aug;81(8):1403–11. ubol s, masrinoul p, chaijaruwanich j, kalayanarooj s, 20. charoensirisuthikul t, kasisith j. differences in global gene expression in peripheral blood mononuclear cells indicate a significant role of the innate responses in progression of dengue fever but not dengue hemorrhagic fever. j infect dis. 2008 may;197(10):1459–67. fink j, gu f, ling l, tolfvenstam t, olfat f, chin kc, et al. host 21. gene expression profiling of dengue virus infection in cell lines and patients. plos negl trop dis. 2007;1(2):e86. kanwal f, lu c, qadri i, sohail m, zia-ur-rahman f. interleukin-6; 22. a promising disease severity index for dengue virus infection. asian pacific j trop dis. 2017;7(5):266–9. scheller j, chalaris a, schmidt-arras d, rose-john s. the pro-and 23. anti-inflammatory properties of the cytokine interleukin-6. biochim biophys acta (bba)-molecular cell res. 2011;1813(5):878–88. chen j, ng mml, chu jjh. activation of tlr2 and tlr6 by 24. dengue ns1 protein and its implications in the immunopathogenesis of dengue virus infection. plos pathog. 2015;11(7). lee yh, leong w-y, wilder-smith a. markers of dengue severity: 25. a systematic review of cytokines and chemokines. j gen virol. 2016;97(12):3103–19. huang yh, lei hy, liu hs, lin ys, liu cc, yeh tm. dengue 26. virus infects human endothelial cells and induces il-6 and il-8 production. am j trop med hyg. 2000;63:71–5. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 11 no. 1 january–april 2023 original article electronic nose (e-nose) for quality detection of tuna (thunnus thynnus) contaminated bacteria suryani dyah astuti1* , alfian baggraf muhamad1, akif rahmatillah1, ahmad khalil yaqubi2 , yunus susilo3 , angger krisna aji4 1department of physics, faculty of science and technology, universitas airlangga, surabaya, indonesia 2doctorate program, faculty of science and technology, universitas airlangga, surabaya, indonesia 3faculty of engineering, universitas dr soetomo, surabaya, indonesia 4magister of biomedical engineering, department of physics, faculty of science and technology, universitas airlangga, surabaya, indonesia received: september 22nd, 2022; revised: february 16th, 2023; accepted: march 30th, 2023 abstract tuna (thunnus thynnus) is a food that is often consumed raw to support raw food diet activities, so it has the potential to be contaminated with salmonella typhi bacteria. fish can be contaminated by bacteria due to their high water and protein content. indonesia is the world's main tuna producer. salmonella typhi detection in fresh tuna in indonesia must be negative for salmonella microbial contamination in order to meet food safety requirements. microbial testing has drawbacks, such as long delays. an electronic nose was used to detect salmonella typhi bacteria in tuna fish. the sample used consisted of 3 kinds of samples: salmonella typhi bacteria, tuna, and tuna with salmonella typhi contamination. the research was conducted with a shelf life of 48 hours and a sensing period every 6 hours with a sensor array of 8 sensors. the sensor output data is processed using the pca (principal component analysis) method. through the pca method, each variation of bacterial treatment can be classified. the result of the cumulative percentage variance of the two main components (pc) in the classification test between salmonella typhi, tuna, and tuna with salmonella typhi bacteria contamination was 90.5%. the most influential sensors in this study are tgs 825 for pc1 with a loading value of 0.625 and tgs 826 for pc2 with a loading value of -0.753. therefore, it can be concluded that an electronic nose can classify between pure tuna and tuna contaminated with salmonella typhi bacteria. keywords: array gas sensor; electronic nose; principal component analysis; salmonella typhi; tuna (thunnus thynnus) highlights: the most influential sensors in this study are tgs 825 for pc1 with a loading value of 0.625 and tgs 826 for pc2 with a loading value of -0.753. therefore, it can be concluded that an electronic nose can classify between pure tuna and tuna contaminated with salmonella typhi bacteria. how to cite: astuti, s. d., muhamad, a. b., rahmatillah, a., yaqubi, a. k., susilo, y., aji, a. k. electronic nose (e-nose) for quality detection of tuna (thunnus thynnus) contaminated bacteria. indonesian journal of tropical and infectious disease. 11(1). 52–65. apr. 2023. doi: 10.20473/ijtid.v11i1.39206 * corresponding author: suryani-d-a@fst.unair.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0003-3000-0792 https://orcid.org/0000-0002-7189-8538 https://orcid.org/0000-0001-8774-6822 53 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 52–65 introduction people's habits and behavioral patterns have changed as a result of the times. knowledge of something, including information about food, enables a person to alter his behavior. humans require food to survive. food is therefore crucial for humans. the raw food diet is one change in lifestyle that can be influenced by food knowledge. a raw food diet is a way of eating that involves only consuming unprocessed, uncooked, or unheated food. due to the growing propensity of people to eat healthy foods and create a society that values health, this trend has spread more widely in recent years. this is in line with research on the perceptions of the surabaya population towards organic food.1 by using a quantitative exploratory research method and a multidimensional scaling technique, it was discovered that respondents' perceptions of the quality and safety of their food were, on average, 3.26, with the highest values occurring between the intervals of 2.6 and 3.4. this suggests that the surabaya population, or respondent, has perceptions of food quality that are very favorable. despite having a higher nutritional value than processed food, raw food has the potential to be contaminated with pathogenic bacteria, according to microbiological hazard identification research by the foodborne illness investigation (fii). the cleanliness and absence of pathogenic microorganisms that could potentially cause disease are indicators of good food quality. this illness is referred to as a foodborne illness. the majority of the bacteria identified as histamine producers are gram negative (87% of isolates), and the majority of these isolates (80%) are members of the enterobacteriaceae family. morganella morganii was the organism most frequently and actively producing histamine in canned tuna fish. along with several strains of enterobacter cloacae and enterobacter aerogenes, klebsiella oxytoca, klebsiella pneumoniae, and other potent histamineproducing bacteria were also discovered during the canning process. some workers have previously experienced similar outcomes. 73% of the former histamineproducing strains that were isolated and identified were from morganella and enterobacter spp. salmonella typhi bacteria is one of the microorganisms that frequently contaminate raw food. the maximum contamination limit for salmonella sp is negative per 25 grams of food. salmonella typhi is a gram-negative bacterium that causes typhoid fever. the disease may occur anywhere in the world, but is most prevalent in developing countries, including in indonesia. the incidence of typhoid fever in indonesia is thought to be between 300 and 810 cases per 100,000 people per year, with a range of 600,000 to 1,500,000 cases per year. effective prevention measures are required because this disease has a 1-5% patient mortality rate.3 salmonella typhi bacteria may be present in tuna (thunnus thynnus), which is one of the foods frequently consumed in a raw state to support raw food diet activities. additionally, due to the high water and protein content of fish meat, bacteria can easily contaminate fish.4 indonesia is the primary producer of tuna in the world, according to the food agriculture organization (fao). salmonella sp must test negative in every gram of fresh tuna for the specific type of salmonella microbial contamination test in order to meet quality and food safety requirements in indonesia. an organization called bkipm (fish quarantine agency, quality control and safety of fishery products) is in charge of vetting the safety and quality requirements for fishery products.5 microbiological tests in accordance with indonesian national standard (ins) are used as a detection, isolation, and confirmation mechanism for salmonella bacteria in tuna. however, due to the lengthy turnaround time (1-3 days) required for test results and the high absorption capacity of both the domestic and international tuna markets, there are a number 54 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license suryani dyah astuti, et al. electronic nose (e-nose) of weaknesses with the microbiological method of detection.6 the united states was indonesia's primary export market for fisheries products. the quantity of tuna products exported by indonesia to the united states from 8,504 tons in 2015 to 10,788 tons in 2016, the number of states increased. as of may 2017, indonesian tuna exports were registered at 65,875 metric tons, valued at usd 226 million. it was anticipated that the volume of fishery goods exported would grow, possibly leading to more products being returned. the primary cause of the rejection of shrimp commodities was microbial contamination. the most frequently reported microorganisms that caused the illnesses were salmonella, e. coli, and vibrio cholerae. the challenge faced by exporters from indonesia, the occurrence of variations in sample collection techniques and testing microorganisms between indonesian laboratories and destination nations. testing technology and laboratory infrastructure (methods and tools) change in order to provide results with varying degrees of precision.7 as anticipated, precooking tuna fish significantly reduced the numbers of all bacterial groups under investigation. however, the bacterial burden in tuna fish continued to rise following the precooking stage. for precooking tuna fish, high heat (100–105 °c for 110 min) is typically employed. the time-temperature link is sufficiently strong to significantly lower the bacterial load. after precooking, tuna was allegedly left at room temperature, allowing damaged germs to quickly recover, multiply, or get decontaminated by the environment. mesophilic and psychrotrophic bacteria counts rose concurrently during canning, but with a modest advantage for the latter. the temperature of the water where the tuna fish was caught (between 8 and 15 °c) and the length of time it was kept frozen are likely to be to blame for the larger number of psychrotrophic organisms. in frozen tuna fish, enterobacteriaceae and coliform counts have always been low and only made up 0.34 percent of the overall bacterial burden. but as the tuna was handled during the canning process, the number of enterobacteriaceae grew until it made up 2.18 percent of the bacterial load. an instrument called the electronic nose (e-nose) mimics how the sense of smell functions. as an alternative to olfactory receptors, which are responsible for detecting smells or scents, the e-nose is made up of a variety of gas sensors. the aroma picked up by numerous gas sensors will then take the form of a specific pattern.8 e-nose has applications in the areas of microbiological detection and food safety.9,10 e-nose has the benefits of being non-destructive, real-time, quick, and inexpensive. according to the research's conclusions, e-nose could differentiate between samples of beef, pig, or a combination of the two based on the fragrance pattern each sample created. e-nose has been extensively used in a variety of fields and industries, including those related to food, drink, chemicals, defense, health, etc.11 research on early detection and classification of pathogenic fungi that attack strawberry farming is one application of e-nose in the food industry for monitoring production processes.12 principal component analysis (pca) is one technique for analyzing the data produced by electronic nose. by using the pca method, it is possible to replace some of the original, correlated variables with a new, smaller set of uncorrelated variables. in order to make it simpler to interpret the data, the main goal of this method is to reduce the dimensions of the interconnected and numerous variables. the authors intend to conduct research on the pattern of data generated by the e-nose gas sensor array in an effort to detect the content of salmonella typhi in tuna (thunnus thynnus) using pca method because the national standard for microbial testing used to detect the content of salmonella typhi in tuna (thunnus thynnus) has a drawback, namely it takes a long time. 55 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 52–65 by using a gas sensor that can react to specific scents, the e-nose device imitates how a mammal's nose detects smells.13 as an alternative to olfactory receptors, which are responsible for detecting smells or scents, the e-nose is made up of a variety of gas sensors. the aroma picked up by various gas sensors will then take on a particular pattern. in order to analyze and identify the signal response produced by the e-nose to a specific scent, pattern recognition software will be used. enose has a wide range of uses, such as assessing food quality, tracking air pollution, and identifying various gases and toxins.14 e-nose uses the biological nose's operating principle to characterize various gas mixtures. the human smell system is divided into three layers, namely.15 1. a layer of approximately one billion olfactory cells 2. olfactory vesicles have three main functions: to control, regulate, and amplify messages from olfactory cells. 3. the brain's olfactory center, which defines signals and organizes the different types of smells that can be detected. the e-nose has three main components, sample handling, detection systems, and data processing systems.16 the e-nose operates on a similar principle to the human nose, which contains a variety of receptors for identifying scents. the sensors on the e-nose serve as a replacement for these receptors, and each remaining receptor reacts differently to the same aroma vapor. the stages in the e-nose system are signal pre-processing, signal processing, and pattern recognition system processing. the sensor array is initially exposed to the scent that needs to be detected. these sensors perform nearly as well as olfactory cells in humans. an analogue to digital converter (adc) will convert the analogue data from the sensor into digital data, which can then be saved to a computer and used for further analysis. preprocessing will be done on the adc data first. processing is used to get the signal ready so that a pattern recognition machine can process it quickly. similar to the vesicle layer in the human sense of smell, this stage performs almost the same functions. the pattern recognition system processes the data in the final step. this section seeks to categorize and forecast unidentified samples. this component's function is comparable to that of the brain's olfactory center.17 the following list of necessary components is provided by gardner and bartlett as a definition of an electronic nose device's fundamental requirements: 1. a sensor array system with an aroma delivery system that transfers volatile aromatic molecules from the source material.18 2. the environment in which the sensor is located: normally, the temperature and humidity are fixed, as this would prevent the aroma molecules from being absorbed otherwise. 3. electrical signals are transformed into chemical signals by electronic transistors. 4. a digital converter that transforms electrical (analogue) signals into digital. 5. a computer microprocessor that reads the digital signal and outputs it after statistical analysis is carried out to classify or identify a sample. each of the gas sensors in the e-nose will react to changes in smell or aroma. each gas sensor will respond to aroma or odor by changing its resistance.19each gas sensor's resistance will fluctuate, changing the voltage as a result. this voltage changes yielded data in the form of digital computer data. from this point forwards, a data processing device will be used to process the data. figure 1 displays the block diagram for the e-nose. figure 1. electronic nose block diagram 56 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license suryani dyah astuti, et al. electronic nose (e-nose) a sensor is a piece of technology used to identify symptoms or signals brought on by changes in energy, including electrical, mechanical, chemical, biological, and other types of energy. a transducer is a device that, when powered by an energy in a transmission system, transmits the energy to the following transmission system in the same form or in a different form. this energy transmission may be thermal, optical (radiation), mechanical, chemical, or electrical (heat). in other words, the sensor is a part that can be used to transform a certain quantity into an analogue unit so that an electronic circuit can read it. the sensor is the main part of a transducer, and the transducer is a supporting system that enables the sensor to have the desired output and to be directly readable at the output. principle component analysis (pca) is a mathematical technique that transforms a set of potential correlated variable observations into a set of principal components, which are linearly uncorrelated variable values.10 data from electronic nose output is processed using pca, which can classify data based on the type and concentration of bacteria. in order to obtain a smaller amount of data with the greatest data variation in the new coordinate system, pca reduces variable data by transforming it linearly. figure 2 displays thepca transformation's outcomes. figure 2. results of pca transformation.20 the pca method aims to reduce the dimensions of the observed variables, thus simplifying them. this is accomplished by changing the original independent variable into a new variable that is completely uncorrelated, thereby removing the correlation between the independent variables. these components become new independent variables once several components of the pca results that are independent of multicollinearity are obtained. one benefit of the pca method is that correlations can be effectively eliminated without reducing the number of initial variables. direct observation of the e-nose sensor output makes it challenging to distinguish between different samples. since the gas sensors used by the e-nose are non-selective and cross-sensitive, multivariable pattern recognition techniques like pca are required to represent the data for simple analysis. materials and methods materials the materials used by the bacteria in this study were salmonella typhi isolates, tuna fish (thunnus thynnus), cotton, plastic wrap, physiological graphic water, tsa, tsb, 70% alcohol, tissue, distilled water, aluminum foil. methods the first sample, specifically salmonella typhi bacteria, will be cultured and incubated for 48 hours until it forms a biofilm; once a biofilm has formed, the bacterial culture will release a more overpowering odor. both the second and final samples of tuna contain salmonella typhi bacteria. the electronic nose functions as a "sensing system" made up of three components: a sampling system, a chemical gas sensor array that produces a range of signals when exposed to gases, vapors, or scents, and a system for classifying the resulting pattern. the sensor in the enose will generate a voltage that varies depending on the sample time and the sensor's sensitivity in order to detect odors from the sample. at each data retrieval, a voltage will be measured and sent to a computer for analysis. the process for using the gas sensor array system and its basic operation is as follows: after turning on the power source, the tool 57 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 52–65 warms up the sensor for a minute before the sensor can be used to detect reactants. eight sensors are used, and each one will send a response voltage that is converted to digital form. there are 8 tgs sensors in this sensor array. a 10 ml beaker was used to hold the sample. eight gas sensors will then enter and pick up the aroma from the sample. excel will be used to plot the output voltage that is produced. in figure 3, systematic data collection is displayed. figure 3. systematics of data collection the target clean air will be inhaled by hose 3 during the preheating process as a control, and it will flow through the inlet hose into the chamber with the valve shutting off hoses 2 and 1 to prevent the clean air from mixing with the smell of the sample. because all sensors are in a steady state during that time, the preheating process takes 60 seconds. the valve closes hose 1 and opens hoses 2 and 3 during the sensing process to allow the target odor to enter the chamber. as the smell of the sample gradually fills the chamber, the sensor responds by outputting a specific voltage. the sensing process takes 300 seconds to complete. the valve closes hoses 1 and 2 and opens hose 3, draining the desired clean air into the chamber where it will be expelled through the outlet hose during the purging process. the gas inside the chamber is supposed to be cleaned with fresh air during the purging procedure. 120 seconds pass during this process. alternately flowing the target gas into the chamber through a number of processes. the sensing mechanism by the gas sensors kicks in when the target gas is in the chamber, allowing each gas sensor to generate an output in the form of a voltage. sensor response test tuna (thunnus thynnus), salmonella typhi biofilm, and a combination of the two were tested for sensor response with each sample being given a time variation of 0, 6, 12, 18, 24, 30, 36, 42, and 48 hour. sensor validity test the output data from the sensor is then tested to prove the validity of the data. the data validity test includes sensor precision tests and sensor accuracy tests. accuracy is the degree to which the results of a measurement closely resemble the actual value of the quantity being measured. in order to assess the correctness of the findings from the analytical tests that have been conducted, it is required to evaluate the percentage recovery (% recovery). at 10% recovery tolerance, or between 90% and 110%, accuracy is regarded as being good. data analysis the results of the sample test using the array of sensors are then processed using a personal computer, and the data is stored as a spreadsheet table in the form of a voltage value obtained from the output of the sensor series. the following are the steps in data processing: 1. feature extraction is the process of obtaining the most pertinent and instructive values that can represent the general characteristics of the sensor response. 2. data representation using radar graphs can show differences in the shape of the web between one and the other and serves to display data from 8 sensors in the gas sensor row. this type of radar chart displays a graph with the appearance of a spider's web. in comparison to other samples. the average value of the feature extraction results is used to create the radar graph. 3. using the principal component analysis (pca) technique, data on variations in the aroma of tuna (thunnus thynnus) were categorized. by reducing the number of variables, the pca method is used to 58 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license suryani dyah astuti, et al. electronic nose (e-nose) reduce the dimensions of the data. next, the variance value of each principal component (pc) is obtained. the initial data set used to create pca is the value obtained from feature extraction. orange data mining and minitab were the two pieces of software used in this study's pca analysis. pc data, eigenvectors, eigenvalues, and cumulative proportion of pca data are obtained using minitab software. the score plot graph is used as the final classification outcome and is used to represent the data using the principal component graph of the first and second principal components' values. results and discussion gas sensor response test results the goal of the sensor response test is to ascertain the e-nose sensor's response value when testing samples. in comparison to samples or compounds with weak aromas or low concentrations of gaseous compounds, e-nose sensors respond more strongly (signal amplitude) to samples with stronger aromas and higher concentrations of gaseous compounds. preheating gas sensor array before the sensor is used to detect and respond to gases that alter the output's resistance value, it is heated. a stable output during data collection is achieved by optimizing the preheating time of each sensor. to get the sensor ready for a steady state condition, preheating treatment is applied. preheating is done in a clean environment with a room temperature. figure 4 depicts the preheating process graph. figure 4. graph of preheating sensor time sensing is performed after 60 seconds because the sensor is ready to use at that point, as shown in figure 4 where all sensors produce a stable voltage output at 50– 60 seconds. sensing the gas sensor array to the sample the e-nose device's sample sensing treatment was performed based on the shelf life, which was as follows: 0 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, and 48 hours. two replications of each sensing data collection procedure were run on each sample for a total of 5 minutes. the information derived from the e-nose output includes stress on the smell of tuna (thunnus thynnus) with varying shelf life, stress on the smell of salmonella typhi bacteria with varying shelf life, and stress on the smell of tuna (thunnus thynnus) contaminated with salmonella bacteria. variations in the shelf life of typhi.21 the figure depicts the sensor array's response to varying time variations during the preheating, sensing, and purging processes for each type of sample. 59 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 52–65 following observation, it was determined that the sensor output response is stable between 100 and 200 seconds, so the output data between 100 and 200 seconds was used to analyze using pca after first being visualized as a line plot graph. the line plot graph is useful for displaying the range of data from each sensor. line plots with time variations are created in this study so that the range of data can be seen during each sensing period. each sample generates a unique voltage output, which results in a unique graphic pattern. radar graphs were used to visualize the data based on the sample type and shelf life. the radar graph interprets the sensor array's response for each sensing period. it is evident that the e-nose generates various sensor outputs for various sample types, resulting in various radar graphic patterns. the radar chart pattern of the three samples, however, is not noticeably different at 0 hour. the radar graph in figure 5 interprets the sensor array response for each type of sample. (a) (b) (c) figure 5. radar sensor graph for each sample type (a) salmonella typhi bacteria; (b) tuna (thunnus thynnus), and (c) tuna (thunnus thynnus) contaminated with salmonella typhi bacteria each sensing period saw an increase from the tgs 826 sensor in the tuna sample (thunnus thynnus). in the meantime, each sensing period saw an increase in the salmonella typhi bacteria sensor tgs 825 sample. it is clear from the radar graph that the sensor output response to the sample yields various values. each sample has a unique set of odor characteristics, which accounts for the variation in the radar chart pattern. because the sensor will produce a higher voltage when reacting to the target gas, which has a higher gas concentration as well, an increase in output voltage is obtained for the same sample with variations in shelf life at each shelf-life period. figure 6 displays the output value for each sensor in the sample with varying shelf lives. 60 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license suryani dyah astuti, et al. electronic nose (e-nose) figure 6. graph of voltage against time of each sensor the presence of protein damage in the sample is indicated by the production of ammonia. according to figure 6, tuna (thunnus thynnus) samples had higher ammonia production at peak times compared to samples of salmonella typhi and tuna (thunnus thynnus) with salmonella typhi contamination. ammonia production peaked for the tgs 826 sensor at 48 hours of storage. 61 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 52–65 when using the tgs 825 sensor to detect h2s, it was discovered that all samples produced the most h2s after 48 hours of storage, with salmonella typhi bacteria producing the most h2s overall. because ammonia and h2s are the main gases produced by samples of tuna and salmonella typhi bacteria, tgs 825 and tgs 826 also have sensor production peaks. sensor validation results accuracy is the closeness of conformity between the results of a measurement and the true value of the quantity measured. it is necessary to test the percentage recovery to measure the accuracy of the results from the analysis tests that have been carried out. accuracy is considered good at 10% recovery tolerance, or within the range of 90%–110%. the results of testing the accuracy of the h2s gas detected by the tgs 2602 and tgs 825 sensors are shown in table 1. table 1. sensor accuracy test results sensor recovery (%) 1 ppm 2 ppm 3 ppm 4 ppm 5 ppm tgs 2602 95.89 99.29 102.59 101.2 98.534 tgs 825 100.00 100.00 99.27 98.984 100.756 from table 1 it is known that the tgs 2602 and tgs 825 sensors, which function to detect h2s gas, meet the validation parameters, which are categorized as good because the percentage of h2s gas recovery ranges from 90% to 110%. the percent recovery value farthest from 100% is produced by the tgs 2602 sensor when it detects standard h2s gas with a concentration of 1 ppm and a recovery value of 95.899%, and the value closest to the standard concentration is produced by the tgs 2602 sensor when it detects h2s gas with a concentration of 2 ppm and a recovery value of 99.297 percent. principal component analysis (pca) results to find the correlation between each variable, the pca method searches for a covariance matrix. the eigenvalue of each variable is then determined using the covariance matrix. the data information formed at the new coordinates (principal component) is described by its eigenvalue. figure 7 depicts the connection between eigenvalues and principal components. figure 7. graph of eigenvalue relationship to principal component score plot a graph that displays where data clusters are located is the pca score plot graph. the similarity of grouped data can be displayed on the score plot graph. two or more data distributions are present when data are grouped together to form a cluster. the two variables, principal components 1 and 2, which are not correlated, are substituted for the eight sensor variables that are correlated with one another to create the score plot. 62 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license suryani dyah astuti, et al. electronic nose (e-nose) the graph shows the score plot of a sample of contaminated tuna (thunnus thynnus), salmonella typhi bacteria, and tuna fish. figure 8 illustrates a time variation of 0-48 hours with salmonella typhi bacteria. figure 8. pca score plot graph according to the type of sample, figure 8 depicts clusters forming among the samples. plotting differs for each type of sample with time variation. however, overlap was seen in samples of tuna and tuna that had salmonella typhi contamination at the 0th hour of shelflife variation. this is so because the sample's distinctive odor hasn't yet developed into a distinct or different characteristic. the organoleptic test classified the samples of tuna and tuna contaminated with salmonella typhi bacteria as fresh. the organoleptic values for pure tuna and tuna fish with salmonella typhi contamination were 8.4 and 8.3, respectively, making it impossible to tell the two samples apart based on their odor characteristics. the percentage of variance criterion is used to determine the maximum number of components that can be formed. the principal component is a linear combination of variables and a type of variable transformation.13 the number of main components that have a cumulative percentage of variance of at least 80% will be used in the cluster analysis. sensor output data can be classified using pca analysis according to sample type and sample time variation, with a total cumulative variance value of 90.5% and specifics for pc 1 and pc 2 variants of 73.9% and 13.6%, respectively. because each sample has different sensor output characteristics, each sensor's significance for the newly formed variable varies, resulting in the cluster on the score plot (principal component). the most significant variables are interpreted on the loading plot graph based on the relationship between their principal components. salmonella typhi must test negative in every gram of fresh tuna for the specific type of salmonella microbial contamination test in order to meet quality and food safety requirements in indonesia. microbiological testing for detection has a number of drawbacks, including a lengthy turnaround time (more than 10 days) for test results.14 utilizing an e-nose with a gas sensor can solve the issue of lengthy test times. each sensing period saw an increase from the tgs 826 sensor in the tuna sample (thunnus thynnus). given that the tgs 826 sensor measures ammonia content and that ammonia is one of the odors produced by bacteria that cause rot, it is obvious that the longer tuna fish are stored, the rottener tuna fish there will be. because it contains a lot of free amino acids, which are necessary for microorganism metabolism, ammonia production, biogenic amines, organic acids, ketones, and sulphur components, fish is known as a food that is both high in nutritional value and perishable.24 increasing storage time can accelerate bacterial growth. the rate of autolysis and the expansion of spoilage bacteria both decrease with increased handling speed. an instrument called the electronic nose (e-nose) mimics how the sense of smell functions. as an alternative to olfactory receptors, which are responsible for detecting smells or scents, the e-nose is made up of a variety of gas sensors. the aroma picked up by various gas sensors will then take on a particular pattern. eight semiconductor sensors from the tgs2620, tgs2611, tgs822, tgs832, tgs2602, tgs2600, tgs826 and tgs825 family of e-nose devices were used in this study. 63 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 52–65 when sno2 (tin dioxide) metal oxide crystals are heated to a high temperature in air, oxygen will be adsorbate on the crystal surface with a negative charge due to the presence of electron donors on the crystal surface. this negative charge is transferred to the adsorbate oxygen, creating a positively charged space layer. as a result, a surface potential will be created that has the ability to prevent the flow of electrons, which causes electrical resistance. the density of negatively charged oxygen adsorbed on the semiconductor surface of the sensor decreases in the presence of a reducing gas, which lowers the barrier height at the grain boundary. the resistance of the grain sensor in the gas environment decreases as the barrier height is reduced. the resistance value decreases as the gas concentration in free air increases. additionally, if a lower gas concentration value is detected in free air, a higher resistance value will be detected.22 principal component analysis (pca) is one technique for analyzing the data produced by e-nose. by using the pca method, it is possible to replace some of the original, correlated variables with a new, smaller set of uncorrelated variables. this method's primary goal is to reduce the dimensions of the variables that are connected and have a sufficient number of variables so that the data will be easier to interpret.23 fish gills and stomachs are where spoilage bacteria are most commonly found to accumulate. because so many organs in a fish's body degrade quickly to rot when it dies, the stomach and gills of fish are parts of the body that are very susceptible to microbial growth25. the largest source of microbes in the body is the stomach. the muscles, gills, and guts of fish are likely a source of bacteria because they naturally contain bacteria. with longer storage, there are more bacteria present. an ideal environment for bacterial growth that encourages optimum bacterial growth. strength and limitation the strength of this study was that the direct observation of the e-nose sensor output makes it challenging to distinguish between different samples the limitation of this study was since the gas sensors used by the e-nose are non-selective and crosssensitive, multivariable pattern recognition techniques like pca are required to represent the data for simple analysis conclusions the classification test between tuna fish, (thunnus thynnus), and tuna fish contaminated with salmonella typhi bacteria yielded results showing a cumulative variance of the two main components (pc) of 90.5%. tgs 825 for pc1 and tgs 826 for pc2 had loading values of 0.625 and -0.753, respectively, making them the most significant sensors in this study. thus, e-nose can tell the difference between tuna that is pure and tuna that has been tainted with salmonella typhi bacteria. acknowledgement this work was supported by airlangga university [grant numbers 1023/un3/2022]. funding this research was supported by grants from the institution of research and community service of widya mandala catholic university, surabaya, indonesia (assignment letter number 745/wm01.5/n/2022). conflict of interest the authors confirm that they have no conflict of interest. 64 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license suryani dyah astuti, et al. electronic nose (e-nose) author contribution conceptualization, methodology, writing review, validation, editing, funding acquisition, and supervision: sda. conceptualization, methodology, validation, original draft preparation: abm. writing review and editing, conceptualization, methodology, validation: ar. writing review and editing, conceptualization, validation: aky. conceptualization, methodology, validation original draft preparation: ys. conceptualization, methodology, validation: aka. references 1. richardo d, winarto o, japarianto e. analysis of surabaya society's perception of organic food. journal of hospitality and service management. 2015; 3(2): 260-273. 2. pan l, zhangw, zhu n, mao s, tu k.early detection and classification of pathogenic fungal disease in post-harvest strawberry fruit by electronic nose and gas chromatography–mass spectrometry. food research international. 2014; 62: 162-168. 3. lestari dw, prijo ta, astuti sd. optimation of 48 khz ultrasonic wave dose for the inactivation of salmonella typhi. indonesian journal of tropical disease. 2015; 5(4): 90-95. 4. tkaczewska j. peptides and protein hydrolysates as food preservatives and bioactive components of edible films and coatings-a review. trends in food science & technology. 2020; 106: 298-311. 5. wijaya dr, sarno r, zulaika e, sabila, si. development of mobile electronic nose for beef quality monitoring. procedia computer science. 2017; 124: 728-735. 6. chen a. the impact of sps measures on agricultural exports from developing countries: a case study of indonesian fishery industry (doctoral dissertation, thesis, world trade institute, swiss). 2014. 7. rahayu wp, prasetyawati c, arizona, y, adhi w. economic losses estimation due to rejection of indonesian exported food. journal of transportation & logistics management. 2020; 7(01): 13-24. 8. astuti sd, tamimi mh, pradhana aa, alamsyah ka, purnobasuki h, khasanah m, syahrom a. gas sensor array to classify the chicken meat with e. coli contaminant by using random forest and support vector machine. biosensors and bioelectronics. 2021; x, 9: 100083. 9. liu sf, moh lc, swager tm. single-walled carbon nanotube–metalloporphyrin chemiresistive gas sensor arrays for volatile organic compounds. chemistry of materials 2015; 27(10): 3560-3563. 10. astuti, sd, mukhammad y, duli, saj, putra ap, setiawatie em, triyana k. gas sensor array system properties for detecting bacterial biofilms. journal of medical signals and sensors. 2019; 9(3):158-164. 11. rosyad f, lenono, d. classification of beef purity based on electronic nose with principal component analysis method. ijeis (indonesian j. electron. instrum. syst, 2016; 6(1): 47. 12. papadopoulou os, panagou ez, mohareb fr, nychasgje. sensory and microbiological quality assessment of beef fillets using a portable electronic nose in tandem with support vector machine analysis. food research international. 2013; 50(1): 241-249. 13. hidayat sn, triyana k. optimized backpropagation combined with radial basic neural network for improving performance of the electronic nose: case study on the fermentation process of tempeh. in aip conference proceedings 2016, july; (vol. 1755, no. 1: p. 020001. aip publishing llc. 14. pradhana aas, astuti sd, khasanam, ardianti rkd. detection of gas concentrations based on age on staphylococcus aureus biofilms with gas array sensors. in aip conference proceedings. 2020, december; (vol. 2314, no. 1, p. 030012). aip publishing llc. 15. triyana k, taukhid subekti m, aji p, nur hidayat s, rohman a. development of electronic nose with low-cost dynamic headspace for classifying vegetable oils and animal fats. in applied mechanics and materials 2015; vol. 771: pp. 50-54. 16. peris m, escuder-gilabert, l. electronic noses and tongues to assess food authenticity and adulteration. trends in food science & technology. 2016; 58: 40-54. 17. fonollosa j, sheik s, huerta r, marco s. reservoir computing compensates slow response of chemosensor arrays exposed to fast varying gas concentrations in continuous monitoring. sensors and actuators b: chemical. 2015; 215: 618-629. 18. ansaloni l, rennemo r, knuutila hk, deng l. development of membrane contactors using volatile amine-based absorbents for co2 capture: amine permeation through the membrane. journal of membrane science. 2017; 537: 272282. 19. pepi m, leonzio c, focardi s, renzi m. production of methyl mercury by sulphatereducing bacteria in sediments from the orbetello 65 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 52–65 lagoon in presence of high macroalgal loads. ecological questions. 2020; 31(4): 21-40. 20. tharwat a. principal component analysis. an overview. pattern recognit. 2016; 3(3): 197-240. 21. mirzaee-ghaleh e, taheri-garavand a, ayari f, lozano j. identification of fresh-chilled and frozen-thawed chicken meat and estimation of their shelf life using an e-nose machine coupled fuzzy knn. food analytical methods. 2020; 13(3): 678-689. 22. triyana k, taukhid subekti m, aji p, nur hidayat s, rohman a. development of electronic nose with low-cost dynamic headspace for classifying vegetable oils and animal fats. in applied mechanics and materials 2015; vol. 771: pp. 50-54. trans tech publications ltd. 23. bolt lm, schreier al. student research collaboration as conservation education: a case study from the primate field school at maderas rainforest conservancy. american journal of primatology. 2022; 23414. 24. adom d. inclusion of local people and their cultural practices in biodiversity conservation: lessons from successful nations. american journal of environmental protection. 2016; 4(3): 67-78. 25. jamali sn, assadpour e, feng j, jafari sm. natural antimicrobial-loaded nanoemulsions for the control of food spoilage/pathogenic microorganisms. advances in colloid and interface science. 2021; 295: 102504. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 11 no. 1 january–april 2023 original article moringa oleifera leaf ethanol extract inhibits toxoplasma gondii tachyzoites replication laura wihanto1* , gladdy lysias waworuntu1, cecilia putri tedyanto1 , heni puspitasari2 1department of microbiology and parasitology, faculty of medicine, universitas katolik widya mandala surabaya, surabaya, indonesia 2toxoplasma study group, institute of tropical disease, universitas airlangga, surabaya, indonesia received: january 18th, 2023; revised: march 17th, 2023; accepted: march 17th, 2023 abstract the various infection routes of toxoplasma gondii that are close to daily life strongly support the incidence of toxoplasmosis. the emergence of drug-resistant toxoplasma gondii strains raises future concerns. moringa leaf ethanol extract has been shown to have several anti-pathogen activities, which could have an anti-toxoplasma effect. this research was conducted to analyze the anti-toxoplasma effect of moringa leaf ethanol extract against tachyzoites replication in toxoplasma gondii and the correlation between extract doses with the number of tachyzoites. mice were divided into five groups. the negative control group (group i) received cmc-na solution. the positive control group (group ii) received spiramycin 100 mg/kg bw. the treatment groups received moringa leaf ethanol extract 250 mg/kg bw (group iii), 500 mg/kg bw (group iv), and 1000 mg/kg bw (group v), respectively. mice were injected with 1 x 105 tachyzoites/0.1 ml/mice intraperitoneally on the first day. moringa leaf ethanol extract and spiramycin were given orally once daily for three days. the number of tachyzoites in the intraperitoneal fluid was calculated on the fifth day. the results have shown that there were significantly lower differences (p < 0.05) in group iv (p = 0.021) and group v (p = 0.022) compared to group i. there was also a significant negative correlation between the extract doses and the number of tachyzoites (p = 0.000; r = -0.781). moringa oleifera leaf ethanol extract has an anti-toxoplasma effect by inhibiting the tachyzoite replication at 500 mg/kg bw and 1000 mg/kg bw. keywords: moringa oleifera; tachyzoites; toxoplasma gondii highlights: this research provides the first study that proved the effectiveness of moringa oleifera leaf ethanol extract in inhibiting toxoplasma gondii tachyzoites replication. how to cite: wihanto, l., waworuntu, g. l., tedyanto, c. p., puspitasari, h. moringa oleifera leaf ethanol extract inhibits toxoplasma gondii tachyzoites replication. indonesian journal of tropical and infectious disease. 11(1). 35–43. apr. 2023. doi: 10.20473/ijtid.v11i1.42672 * corresponding author: laura@ukwms.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0001-7746-7678 https://orcid.org/0000-0003-3440-9886 https://orcid.org/0000-0002-0060-8820 36 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license laura wihanto, et al. moringa oleifera leaf ethanol extract introduction toxoplasma gondii is an obligate apicomplexan intracellular protozoan that causes toxoplasmosis in warm-blooded animals, including humans. it has been reported that approximately 30-50% of the world’s human population is infected by this parasite.1 the prevalence of toxoplasmosis seropositivity in humans has also reached 32,6% among 9 of 47 primary health centers in makassar, indonesia.2 the high incidence of toxoplasmosis makes this disease a global health problem that needs attention because it can cause severe clinical manifestations in immunocompromised patients and permanent fetal disability.3 the various infection routes of t. gondii that are close to daily life could strongly support the incidence of toxoplasmosis. this zoonotic infection can occur in several ways: accidentally ingesting cat faeces that contain oocysts; eating undercooked meat that contains tissue cysts; transplacental transmission from an infected mother to a fetus; and other possibilities, such as receiving a blood transfusion or an organ transplant from an infected donor.4 recent studies in clinical cases of toxoplasmosis have shown that drug resistance in t. gondii is ongoing. the emergence of drug-resistant t. gondii strains raises future concerns, not only in terms of treatment failure but also of increasing clinical severity in immunocompromised patients.5 using natural ingredients such as plants or fruits as herbal medicine can be an alternative. this alternative is also considered less toxic than synthetic drugs and is better in terms of economy, practicality, and accessibility. research on natural resources in indonesia should always be carried out due to the vast and abundant biodiversity in indonesia, which has excellent potential to bring benefits to the health sector. moringa (indonesian: kelor) or moringa oleifera is a plant often found in indonesia. parts of the plants that can be utilized are roots, stems, fruits, flowers, seeds, and leaves. in vitro study of m. oleifera seeds has been shown to inhibit the replication of tachyzoites.6 the leaves are part of the plant that is often consumed by indonesian people and have been shown to have antiinflammatory, antifungal, and antibacterial effects.7-9 it also acts as a larvicidal.10 a phytochemical analysis of m. oleifera leaf ethanol extract revealed alkaloids, phenolics, flavonoids, tannins, saponins, and terpenes as their bioactive compounds.8,11-13 it has rutin as its major flavonoid, gallic acid as its major phenolic acid, and lutein as its major carotenoid. several alkaloid compounds were also detected, such as pyrazoline alkaloids, piperidine alkaloids, and quinoline alkaloids.14,15 quinoline alkaloids are one of the typical deoxyribonucleic acid (dna) intercalating alkaloids that have cytotoxic and antiparasitic effects from their intercalating actions between the nucleotide pairs of the parasite.16 m. oleifera leaf ethanol extract could have an anti-toxoplasma effect through its dnadamaging compounds. therefore, this research was conducted to prove the potential of m. oleifera leaf ethanol extract as the new anti-toxoplasma drug against tachyzoites replication in t. gondii. materials and methods experimental materials and tools m. oleifera leaves were purchased from and identified by the technical implementation unit of the herbal laboratory, materia medica batu, east java, indonesia (reference number 074/656/102.20-a/2022). the rh strains of t. gondii tachyzoites were obtained from the institute of tropical disease, airlangga university, surabaya, indonesia. deutschland-denken-yoken (ddy) mice (male, 20-30 grams, 2-3 months old) were purchased from a certified local experimental animal breeder. other materials used in this research were spiramycin (500 mg, 37 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 35–43 spirasin®, sanbe, bandung, indonesia) and sodium chloride 0.9% (otsu®, pt otsuka indonesia, east java, indonesia). tools used in this research were neubauer counting chamber (0.1 mm depth, assistent®, germany), cover glass (22 x 22 mm, onelab®, indonesia), light microscope (nikon®, nikon corporation, japan), disposable syringe 3 cc (terumo®, terumo company, tokyo, japan), sterilized falcon tubes (nest®, nest biotech, china), micropipette (dlab®, dlab scientific co., ltd., beijing, china), and hand tally counter (maras®, togoshi seiki, taiwan). plant extraction m. oleifera leaves were washed, dried at 40oc temperature using an oven, and ground into powder. the powder was macerated with 96% ethanol for 24 hours while being stirred occasionally. the first maceration results were filtered, and the residue was remacerated with the same stage until the second maceration results were obtained. both maceration results were mixed and evaporated using a rotary evaporator at 40oc until they became a dense mass.17 phytochemical analysis table 1. chemical reaction tests for some bioactive compounds from m. oleifera leaf ethanol extract7,17 constituent method alkaloids mayer test flavonoids ammonium test phenolics ferric chloride test steroids lieberman-burchad test saponins froth test tannins ferric chloride test the phytochemical analysis was performed to determine secondary metabolites (alkaloids, flavonoids, phenols, steroids, saponins, and tannins) present in the m. oleifera leaf ethanol extract using the color and precipitate reaction methods (table 1). thin-layer chromatography analysis figure 1. retention factor (rf) values calculation formula in thin-layer chromatography analysis. the thin-layer chromatography (tlc) analysis was performed to isolate the specific compounds present in the m. oleifera leaf ethanol extract. the extract was applied on silica gel 60 f254 plates as the stationary phase. the mobile phases were (chloroform: methanol: water) (50:65:10) for alkaloids and steroids, (n-butanol: acetic acid: water) (4:1:5) for flavonoids, phenols, and tannins. after leaving the developed plates to dry, they were observed under ultra-violet (uv) light at both 254 nm and 366 nm, then sprayed with iodine reagents to detect the bands.18 the movement of the separated compounds was expressed by retention factor (rf) values, which were calculated by the formula (figure 1). animals all the mice have been declared healthy by the veterinarian. the mice were acclimated for one week under laboratory conditions in wire-covered cages with paddy husk as bedding at a temperature of 24±4oc, relative humidity of 44-56%, and 12 hours of light and dark cycle. four mice per cage were given free access to distilled water and standard mouse food. 38 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license laura wihanto, et al. moringa oleifera leaf ethanol extract parasite culture the tachyzoite culture was performed in vivo on male ddy mice. they were maintained by routine intraperitoneal passage every 72 hours. the number of tachyzoites was determined by counting them in a counting chamber, then diluted to sodium chloride 0.9% solution before being inoculated into the experimental mice.19 the toxoplasmosis induction used in this research was 1 x 105 tachyzoites/0.1 ml/mice. toxoplasmosis drug reference this research used spiramycin as the toxoplasmosis drug reference.20 it was administered orally at a dose of 100 mg/kg bw. the tablets were crushed into powder and dissolved with sodium carboxymethyl cellulose (cmc-na) solution until they became a homogenous suspension. experimental design and protocol mice were divided into five t. gondiiinfected groups (n = four mice for each group). the infected group were divided as follows: negative control group (group i) received cmc-na solution 0.5 ml/mice orally as a placebo, positive control group (group ii) received spiramycin 100 mg/kg w, group iii received m. oleifera leaf ethanol extract 250 mg/kg bw, group iv received m. oleifera leaf ethanol extract 500 mg/kg bw, and group v received m. oleifera leaf ethanol extract 1000 mg/kg bw. mice in the infected group were injected with 1 x 105 tachyzoites/0.1 ml/mice intraperitoneally on the first day. m. oleifera leaf ethanol extract and spiramycin were diluted into 0.5 ml of cmc-na solution and given orally once daily for three days from the second to the fourth day. on the fifth day, all mice were sacrificed with cervical dislocation, and the intraperitoneal fluid was collected to count the tachyzoites. intraperitoneal fluid collection the outer skin of the peritoneum was cut using scissors and tissue forceps, then gently pulled back to expose the inner skin lining the peritoneal cavity. the peritoneal cavity was washed with 3 ml of normal saline. the abdomen was shaken slowly to dislodge the tachyzoites into the saline solution. aspiration of the intraperitoneal fluid was carried out using a syringe.21 count of parasites the number of parasites was carried out by blind-direct examination using a counting chamber at 400x magnification of a light microscope.19 blind-direct examination means the counter does not know from which group the sample was taken. the mean of tachyzoites was expressed in a multiplication factor of 104. statistical analysis the research results were analyzed using statistical product and service solutions software (ibm corp., armonk, ny) version 25. the significant differences were statistically determined using the kruskalwallis test, followed by the mann-whitney test. values at p < 0.05 are considered significant. the pearson correlation coefficient (r) was used to determine the correlation between extract doses and the number of tachyzoites. 39 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 35–43 results and discussion phytochemical analysis table 2. phytochemical analysis results of m. oleifera leaf ethanol extract constituent result interpretation alkaloids development of cream-yellow precipitate positive flavonoids development of red or pink color positive phenols development of dark green color positive steroids development of blue color positive tannins development of brownish-green color positive saponins no formation of stable foam negative the phytochemical analysis of m. oleifera leaf ethanol extract revealed the absence of saponins and the presence of alkaloids, flavonoids, phenols, steroids, and tannins (table 2). these results were not aligned with the previous research, which revealed that the m. oleifera leaf ethanol extract contained saponins as its secondary metabolite compound.7,10,12 the absence of saponins in this research could have occurred due to several factors that affected the extraction process. factors influencing the maceration process results are temperature, solvent types and concentration, duration, and other factors.22 the extraction of m. oleifera leaves using methanol as a solvent with 72 hours of maceration showed positive saponin results on the phytochemical screening.7 positive saponin results were also obtained in the extraction using ethanol as the solvent with a maceration time of 72 hours.12 another study using ethanol as a solvent with 48 hours of maceration time showed negative screening results for saponins, which were the same as the results of the phytochemical analysis in this research using the same type of solvent but with 24 hours of maceration time.11 figure 2. tlc results of alkaloids under uv light (a) 254 nm (b) 366 nm figure 3. tlc results of flavonoids under uv light (a) 254 nm and (b) 366 nm figure 4. tlc results of phenols and tannins under uv light (a) 254 nm and (b) 366 nm figure 5. tlc results of steroids under uv light (a) 254 nm and (b) 366 nm (a) (b) (a) (b ) (a) (b) (a) (b) 40 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license laura wihanto, et al. moringa oleifera leaf ethanol extract table 3. rf values and colors of peaks of each compound of m. oleifera leaf ethanol extract constituent rf values colors of peaks alkaloids 0.63 red 0.73 blue 0.81 blue flavonoids 0.35 yellow 0.43 yellow 0.48 yellow 0.80 yellow 0.88 yellow phenols 0.78 blackish green steroids 0.75 yellowish blue 0.88 yellowish blue 0.93 yellowish blue tannins 0.78 blackish green the positive bioactive compound results were also confirmed with tlc analysis as shown in figure 2–5. alkaloids were detected with rf values of 0.63, 0.73 and 0.81. flavonoids were detected with rf values of 0.35, 0.43, 0.48, 0.80, and 0.88. phenols were detected with rf values of 0.78. steroids were detected with rf values of 0.75, 0.88, and 0.93. tannins were detected with rf values of 0.78 (table 3). number of tachyzoites figure 6. t. gondii-infected group. tachyzoites (black arrows) on intraperitoneal fluid from the infected group under a light microscope with 400x magnification. identification of tachyzoites was carried out based on its distinctive morphology, which is a crescent-like shape, sharp at the anterior and blunt at the posterior, with a length of about 4-8 µm and a width of about 2-3 µm. the color of tachyzoites in the intraperitoneal fluid preparation was clear and transparent, accompanied by movement, indicating that the protozoa are still alive and have motility (figure 6). table 4. tachyzoites count results in the intraperitoneal fluid of the t. gondii-infected group (n = four mice per group) group infection mean±sd (x 104) i + 8.91±3.45 ii + 2.88±2.14* iii + 6.41±1.25 iv + 3.03±1.08* v + 2.28±0.12* group i: negative control group; group ii: positive control group; group iii: treatment group 1; group iv: treatment group 2; group v: treatment group 3; +: infected; sd: standard deviation; *: p < 0.05 compared to group i figure 7. simple scatter plot of tachyzoites count results (y-axis) by the m. oleifera leaf ethanol extract doses (x-axis). the number of tachyzoites tends to increase as the extract doses decrease, with p = 0.000 and r = -0.781 the highest tachyzoites were in group i with a total of 8.91±3.45 x 104. group ii was significantly lower than group i, with a total of 2.88±2.14 x 104. there were two extract treatment groups with a significantly lower number of tachyzoites compared to group i: group iv with a total of 3.03±1.08 x 104 and group v with a total of 2.28±0.12 x 104. the number of tachyzoites in group iii, with a total of 6.41±1.25 x 104, was not significantly different compared to group i as shown in table 4. the toxoplasmosis intraperitoneally induction used in this research was 1 x 105 tachyzoites/0.1 ml/mice. tachyzoites can invade almost all the host nucleated cells 41 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 35–43 and replicate rapidly.3 the cell will eventually suffer damage and rupture due to this state, and the released tachyzoites will continue to look for other cells, perpetuating the cycle. the severity of the clinical symptoms depends on the degree of tachyzoite replication, which means that an individual's immune system is crucial in defining the clinical manifestations.23 administration of spiramycin at 100 mg/kg bw orally for three days effectively inhibited tachyzoite replication, as proved by the significantly lower difference in the mean number of tachyzoites present in the peritoneal fluid between group ii and group i (table 4). spiramycin is an antibiotic and an antiparasitic macrolide agent that is considered the drug of choice against t. gondii in pregnancy. the mechanism of action of the drug was to inhibit the synthesis of proteins and the growth of protozoan cells.24 its effectiveness as a toxoplasmosis drug has also been proven through previous research, which could reduce the number of t. gondii cysts in brain tissues.20 the doses of m. oleifera leaf ethanol extract used in this research were 250 mg/kg bw, 500 mg/kg bw, and 1000 mg/kg bw. the tachyzoite count differences were significantly lower in groups iv and v compared to group i. there was no significant difference in the mean number of tachyzoites between group iii and group i (table 4). the results revealed that m. oleifera leaf ethanol extract at doses of 500 mg/kg bw and 1000 mg/kg bw could inhibit tachyzoite replication, but the dose of 250 mg/kg bw could not. these results occurred because the concentration of chemical properties in m. oleifera leaf ethanol extract increased with increasing doses (figure 7). our results are also align with other research on plasmodium yoelii, in which the higher the dose of m. oleifera leaf ethanol extract, the greater the inhibitory activity against the parasites.25 quinoline alkaloids (3-methylquinoline) are typical dna intercalating compounds found in the m. oleifera leaf ethanol extract.15 their antiparasitic effect occurred through their hydrophobic, aromatic, and planar properties, which allow them to intercalate between the nucleotide pairs of the parasite. these cause mutations, such as deletions or frame-shift mutations, which will disrupt the replication of the parasite.16 if the mutation occurs in an essential protein-coding gene, it causes the death of the parasite.13 this theory also aligns with previous research, which proved that the moringa seeds extract promotes apoptosis-like death in t. gondii tachyzoites in vitro.6 a simple scatter plot showed a negative correlation between the extract doses and the number of tachyzoites (figure 7). the decrease in tachyzoite count results, along with increasing doses of m. oleifera leaf ethanol extract, occurred because the concentration of alkaloids in the extract is directly proportional to the dose. the higher concentration of alkaloids causes more intercalated dna in the parasites, resulting in more disruption in the replication of the tachyzoites. this research proved the effectiveness of m. oleifera leaf ethanol extract in inhibiting tachyzoites replication. future research needs to conduct more specific studies on the effect of the extract as an anti-toxoplasma, whether isolating the specific antiparasitic bioactive compound and examining the histopathological variables on t. gondii target organs or other variables of its pathway of antioxidant properties. strength and limitation the blind-direct examination method in this research provided minimal occurrence of bias due to the subjective perspective of the researcher. this research also verified and explained the antiparasitic mechanism of m. oleifera leaf ethanol extract through the combination of phytochemical screening, tlc analysis, and the count of parasites. although we adopt the blind-direct examination for the count of parasites method, it does not provide full objective 42 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license laura wihanto, et al. moringa oleifera leaf ethanol extract results in this research. more objective parameters, such as histopathological or hematological variables, in order to examine the impact of the t. gondii tachyzoites replication, should be carried out in future study to support the results of this research. conclusions the m. oleifera leaf ethanol extract has an anti-toxoplasma effect by inhibiting the tachyzoite replication at 500 mg/kg bw and 1000 mg/kg bw. acknowledgement the authors thank the institution of research and community service of widya mandala catholic university, surabaya, indonesia, for providing grants and the medical faculty of widya mandala catholic university, surabaya, indonesia, for the valuable support throughout this research. ethical clearance the research protocol was approved by the health research ethics committee (hrec) of the medical faculty of widya mandala catholic university, surabaya, indonesia (reference number 0326/wm12/kepk/dsn/t/2022). this research was carried out following the ethical principles outlined in the council for international organizations of medical sciences (cioms) and world health organization (who) international ethical guidelines for health-related research involving humans. funding this research was supported by grants from the institution of research and community service of widya mandala catholic university, surabaya, indonesia (assignment letter number 745/wm01.5/n/2022). conflict of interest the authors confirm that they have no conflict of interest. author contribution experimental design: lw, glw, cpt. extract preparation: glw, cpt. materials preparation: lw, cpt. research implementation: lw, cpt, hp. research supervision: lw, hp. data analysis: lw, glw. manuscript writing: lw, cpt. manuscript editing: lw, glw, cpt, hp. references 1. flegr j, prandota j, sovičková m, israili zh. toxoplasmosis a global threat. correlation of latent toxoplasmosis with specific disease burden in a set of 88 countries. plos one. 2014;9(3):e90203. 2. polanunu nfa, wahyuni s, & hamid f. seroprevalence and associated risk factors of toxoplasma gondii infection among pregnant mother in makassar, indonesia. plos one. 2021;16(6):e0245572. 3. wang d, liu h, ma x, ma y, li y, yang b, et al. toxoplasma gondii infection in immunocompromised patients: a systematic review and meta-analysis. front microbiol. 2017;8:389. 4. centers for disease control and prevention (cdc). epidemiology & risk factors of parasites toxoplasmosis. global health, division of parasitic diseases and malaria. 2018. 5. montazeri m, mehrzadi s, sharif m, sarvi s, tanzifi a, aghayan sa, et al. drug resistance in toxoplasma gondii. front microbiol. 2018;9(2857):1-12. 6. nishi l, sanfelice rads, da silva bortoleti bt, tomiotto-pellissier f, silva tf, evangelista ff, et al. moringa oleifera extract promotes apoptosis-like death in toxoplasma gondii tachyzoites in vitro. parasitol. 2021;148(12):1447-1457. 7. mittal a, sharma m, david a, vishwakarma p, saini m, goel m, et al. an experimental study 43 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 35–43 to evaluate the anti-inflammatory effect of moringa oleifera leaves in animal models. int j of basic clin pharmacol. 2017;6(2):452-457. 8. ahmadu t, ahmad k, ismail si, rashed o, asib n, omar d. antifungal efficacy of moringa oleifera leaf and seed extracts against botrytis cinerea causing gray mold disease of tomato (solanum lycopersicum l.). braz j biol. 2021;81(4):1007-1022. 9. fouad ea, abu elnaga asm, kandil mm. antibacterial efficacy of moringa oleifera leaf extract against pyogenic bacteria isolated from a dromedary camel (camelus dromedarius) abscess. vet world. 2019;12(6):802-808. 10. yasi r, restiani sh. the larvicidal activity of moringa oleifera extract leaf to the larva’s aedes aegypti mortality. j agromedicine med sci. 2018;4(3):159-164. 11. kashyap p, kumar s, riar cs, jindal n, baniwal p, guiné rpf, et al. recent advances in drumstick (moringa oleifera) leaves bioactive compounds: composition, health benefits, bioaccessibility, and dietary applications. antioxidants. 2022;11(2):402. 12. putra iwdp, dharmayudha aago, sudimartini lm. identification of chemical compounds ethanol extract leaf moringa (moringa oleifera l.) in bali. indonesia medicus veterinus. 2016;5(5):464-473. 13. syahputra ra, sutiana a, silitonga pm, rani z, kudadiri a. extraction and phytochemical screening of ethanol extract and simplicia of moringa leaf (moringa oleifera lam.) from sidikalang, north sumatera. int j sci technol manag. 2021;2(6):2072-2076. 14. rani nza, husain k, kumolosasi e. moringa genus: a review of phytochemistry and pharmacology. front pharmacol. 2018;9:108. 15. kadhim ej, al-shammaa da. phytochemical characterization using gc-ms analysis of methanolic extract of moringa oleifera (family moringaceae) plant cultivated in iraq. j chem mater res. 2014;6(5) 16. wink m. medicinal plants: a source of antiparasitic secondary metabolites. molecules. 2012;17(11):12771-12791. 17. tedyanto cp, wihanto l, hendrata ap. hepatoprotective effect of dried red jujube fruit extract against acetaminophen-induced acute hepatotoxicity. cureus. 2023;15(1):e33272. 18. nishu, jee c. preliminary phytochemical screening and thin layer chromatography of selected extract of moringa oleifera leaf. int j chem stud. 2020; 8(5):2407-2409. 19. nguyen tt, kamyingkird k, phimpraphai w, inpankaew t. viability of toxoplasma gondii tachyzoites in different conditions for parasite transportation. vet world. 2022;15(1):198-204. 20. omar m, abaza be, mousa e, ibrahim sm, rashed he, farag ti. effect of spiramycin versus aminoguanidine and their combined use in experimental toxoplasmosis. j parasit dis. 2021;45: 1014-1025. 21. jesus ad, pusec cm, nguyen t, keyhani-nejad f, gao p, weinberg se, et al. optimized protocol to isolate primary mouse peritoneal macrophage metabolites. star protoc. 2022;3(4):101668. 22. mehganathan p, rosli na. a review on extraction of bioactive compounds from moringa oleifera leaves: their principle, advantages, and disadvantages. med aromat plants. 2022;11(1):430. 23. naumov av, wang c, chaput d, ting l, alvarez ca, keller t, et al. restriction checkpoint controls bradyzoite development in toxoplasma gondii. asm. 2022;10(3):e0070222. 24. mccharthy js, wortmann gw, kirchhoff lv. drugs for protozoal infections other than malaria. mandell, douglas, and bennett's principles and practice of infectious diseases, 8th ed. elsevier. 2014;41:510-518. 25. shrivastava m, prasad a, kumar d. evaluation of anti-malarial effect moringa oleifera (lam) in plasmodium yoelii infected mice. indian j pharm sci. 2021;83(6):1221-1228. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 vol. 9 no. 2 may–august 2021 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 research article antibacterial activity of ethanol extract of kemuning (murraya paniculata) against klebsiella pneumoniae esbl by in vitro test illona okvita wiyogo1, pepy dwi endraswari2, yuani setiawati3 1faculty of medicine, universitas airlangga, surabaya indonesia 2departement of microbiology, faculty of medicine, universitas airlangga, surabaya indonesia 3departement of pharmacology, faculty of medicine, universitas airlangga, surabaya indonesia received: 07th august 2018; revised: 08th th march 2021 abstract klebsiella pneumoniae extended-spectrum β-lactamase (esbl) was one of the microorganism that cause nosocomial infection which resistant to beta-lactams antibiotics. orange jessamine (murraya paniculata) was traditional medicine which believed has antibacterial components, such as: fl avonoids, alkaloids, essential oils, coumarins, terpenoids, tannins, and saponins. in the previous studies, there was antibacterial activity in ethanolic extract of murraya paniculata againsts e.coli, k.pneumoniae, s.typhi, e.faecalis, p.aeruginosa, s.fl exneri, s.aureus, and s.sonneii with concentration 200 mg/ ml. there has not experiment about ethanolic extract of murraya paniculata against klebsiella pneumoniae esbl yet. the aim of this study was to fi nd out the in vitro antibacterial activity of ethanol extracts of murraya paniculata against klebsiella pneumoniae esbl broth dilution method with concentration 200 mg/ml, 100 mg/ml, 50 mg/ml, 25 mg/ml, 12,5 mg/ml, 6,25 mg/ml, and 3,125 mg/ml were used for the determination of the minimal inhibitory concentration (mic). while the minimal bacterial concentration (mbc) was assessed using streaking method in nutrient agar plate. the highest concentration in this study was obtained from 100 g of murraya paniculata leaves dissolved in 500 ml of 40% ethanol. the study was carried out 4 times replication. at the time of the sterility test extract, germ growth appeared on nutrient agar plate media, so the extract was fi ltered before being used for research. after incubation at 37 °c for 24 hours, growth of bacterial colonies on all agar plates was observed. the concentration of the ethanol extract of murraya paniculata (200 mg/ml) did not inhibit the growth of klebsiella pneumoniae esbl. the ethanol extracts of murraya paniculata in concentration 200 mg/ml had no antibacterial activity against klebsiella pneumoniae esbl. keywords: antibacterial, ethanol extracts, klebsiella pneumoniae, esbl, murraya paniculata leaves abstrak klebsiella pneumoniae extended-spectrum β-lactamase (esbl) merupakan salah satu mikroorganisme yang menyebabkan infeksi nosokomial yang resisten terhadap antibiotik beta-laktam. oranye jessamine (murraya paniculata)adalah obat tradisional yang diyakini memiliki komponen antibakteri, seperti: fl avonoid, alkaloid, minyak esensial, kumarin, terpenoid, tanin, dan saponin. dalam studi sebelumnya, ada antibakteri aktivitas ekstrak etanol murraya paniculata melawan e.coli, k.pneumoniae, s.typhi, e.faecalis, p.aeruginosa, s.fl exneri, s.aureus, dan s.sonneii dengan konsentrasi 200 mg/ ml. belum ada eksperimen tentang ekstrak etanol murraya paniculata terhadap klebsiella pneumoniae esbl. tujuan dari penelitian ini adalah untuk mengetahui aktivitas antibakteri in vitro ekstrak etanol murraya paniculata terhadap klebsiella pneumoniae esbl. metode pengenceran kaldu dengan konsentrasi 200 mg/ml, 100 mg/ml, 50 mg/ml, 25 mg/ ml, 12,5 mg/ml, 6,25 mg/ml, dan 3,125 mg/ml digunakan untuk penentuan minimum inhibition concentration (mic), sedangkan minimal bacterial concentration (mbc) dinilai menggunakan metode goresan pada pelat agar nutrien. konsentrasi tertinggi dalam penelitian ini diperoleh dari 100 g daun murraya paniculata yang dilarutkan dalam 500 ml etanol 40%. penelitian dilakukan 4 kali replikasi, dimana pada saat ekstrak uji sterilitas pertumbuhan kuman muncul pada media agar nutrien agar, sehingga ekstrak disaring sebelum digunakan untuk penelitian. setelah inkubasi pada 37 ° c selama 24 jam, pertumbuhan koloni bakteri pada semua * corresponding author: illonawi@gmail.com july 2020; accepted: 19 103illona okvita wiyogo, et al.: antibacterial activity of ethanol extract of kemuning ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 sehingga piring diamati. konsentrasi ekstrak etanol murraya paniculata (200 mg/ml) tidak menghambat pertumbuhan klebsiella pneumoniae esbl. ekstrak etanol murraya paniculata dalam konsentrasi 200 mg/ml tidak memiliki aktivitas antibakteri terhadap klebsiella pneumoniae esbl. kata kunci: anti bakteri, ekstrak etanol, klebsiella pneumoniae, esbl, daun murraya paniculata how to cite: wiyogo, i., endraswari, p., & setiawati, y. (2021). antibacterial activity of ethanol extract of kemuning (murraya paniculata) against klebsiella pneumoniae esbl by in vitro test. indonesian journal of tropical and infectious disease, 9(2) 102-107 introduction the percentage of nosocomial infection in haji adam malik hospital, medan in 2010 is 6-16% and the mean is 9.8%.1 the most frequently detected infection is nosocomial pneumonia (both ventilator and non-ventilator associated), and followed by urinary tract infection and central venous catheter associated bloodstream infections respectively.2 ventilator-associated pneumonia (vap) is the most common nosocomial infection among critical patients. nosocomial infection is handled diff erently from the non-nosocomial one since nosocomial infection is generally due to multidrug-resistant bacteria. in developing countries, antibiotics are often used in irrational dose, hence the increased prevalence of antibiotic-resistant bacteria in hospitals.3 the prevalence of imipenem-resistant acinetobacter, imipenem-resistant p.aeruginosa, and oxacillin-resistent s.aureus are 67.3%, 27.2% and 82.1% respectively. several bacteria are categorized as multidrug-resistant. the bacteria’s high level of resistance might limit therapy options.4 bacteria producing extended-spectrum β-lactamase (esbl) cannot be overcome with penicillin, cephalosporin, and monobactam aztreonam, such as several k. pneumoniae strains.5 the infection case of k. pneumoniae esbl in the group of nosocomial infection is 11 times higher than those of the communityacquired infection group.6 indonesia is a tropical country with abundant medicinal plants, one of which is orange jessamine (murraya paniculata). medicinal plants generally contain phenolic compounds, i.e. phenolic acids, fl avonoids, tannins, stilbenes, curcuminoids, coumarins, lignans, quinones, etc.7 the leaves of orange jessamine (murraya paniculata) contain bioactive compounds which are secondary metabolites, such as alkaloids, fl avonoids, saponins, terpenoids, and tannins.8 since 1970, fl avonoids and coumarins have been isolated from murraya paniculata.9 evaluations on the synthesis of nitro coumarins with or without the substitution of methyl or methoxy group has shown antibacterial activity against staphylococcus aureus, escherichia coli and candida albicans strains.10 other studies report that murraya paniculata has many benefits including anti-platelet aggregation, antiamoebic, anti-giardial, insecticide, pain relief, antidiabetic, antioxidant, antifungal, lipoxygenase and respiratory burst inhibitor.11 this study aims to test antibacterial activity of orange jessamine extract against multidrug-resistance (mdr) bacteria, klebsiella pneumoiae producing esbl. methods this is a quasi-experimental study with convenience post test controlled design, aimed to fi nd out the antibacterial activity of the leaves of orange jessamine against klebsiella pneumoniae esbl with dilution method. the orange jessamine (murraya paniculata) leaf extract were obtained from materia medica laboratory, batu, while antibacterial activity was tested in microbiology laboratory of faculty of medicine universitas airlangga. the independent variable is ethanol extract of orange jessamine (murraya paniculata) leaf extract concentration, while the dependent 104 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 102–107 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 variable is inhibition effect of klebsiella pneumoniae esbl bacteria growth in each tube containing concentration of ethanol extract orange jessamine leaf the control variable are temperature and incubation time of sensitivity test with dilution method. the suspension treatment groups of klebsiella pneumoniae esbl was exposed to ethanol extract orange jessamine leaf. the groups were t1 (100%), t2 (50%), t3 (25%), t4 (12.5%), t5 (6.25%), t6 (3.125%) and t7 (1.5625%). the control groups consisted of k1 (liquid medium and bacteria) and k2 (liquid medium and ethanol extract orange jessamine leaf). minimum inhibitory concentration (mic) is the lowest concentration that is still able to inhibit bacterial growth, whereas, minimum bactericidal concentration (mbc) is the lowest concentration that is able to kill bacteria. a further observation to determine mbc can be conducted upon the obtaining of growth inhibitory eff ect. the test for antibacterial activity in ethanol extract orange jessamine leaf against klebsiella pneumoniae esbl is conducted with dilution method to determine mic and mbc, which are analyzed descriptively and statistically using analysis of variance (anova). result this study used orange jessamine leaf extract obtained through maceration method. the highest concentration in this study was obtained from 100 g of murraya paniculata dissolved in 500 ml of 40% ethanol. the compared extract concentrations were 200 mg/ml, 100 mg/ml, 50 mg/ml, 100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml and 3.125 mg/ml. in this study, replications were carried out four times. during extract sterility test, there was bacterial growth in the medium nutrient agar plates. thus, the extract was fi ltered prior to the use in this study (table 1). the use of dilution method was aimed to assess the mic. there were seven tubes with different concentration of extract ethanol of murraya paniculata leaves and two tubes as the positive and negative control. the positive control tube contained bacteria and liquid mueller hinton broth (mhb), while the negative one contained extract and liquid mhb. from all the conducted replications, all tubes were unable to identify as the extract color tended to appear dark (figure 1). thus, streaking was carried out on nutrient agar plates to directly observe if there was any inhibition in the growth of klebsiella pneumoniae esbl bacteria by extract ethanol of murraya paniculata leaves. after incubated in the temperature of 37°c for 24 hours, bacterial growth appeared in al nutrient agar plates (figure 2). the adding of extract up to the highest concentration showed that there was still bacterial growth in nutrient agar plates table 1. data on minimum inhibitory concentration showing no antibacterial eff ect on the extract concentration of 3.125-200 mg/ml. replication extract concentration (mg/ml ) 200mg/ml 100 mg/ml 50 mg/ml 25 mg/ml 12.5 mg/ml 6.25 mg/ml 3.125 mg/ml 1 2 3 4 note: (-) bacterial growth appeared figure 1. results of dilution test of orange jessamine leaf extract using ethanol as solvent 105illona okvita wiyogo, et al.: antibacterial activity of ethanol extract of kemuning ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 figure 2. results of streaking on nutrient agar plates, showing bacterial growth in all extract concentration of murraya paniculata leaf extract. analysis of the results this study on antibacterial activity used extract ethanol of murraya paniculata leaves dissolved in 40% ethanol. this study was conducted by comparing the antibacterial activity of extract ethanol of murraya paniculata leaves with ethanol as the solvent in several concentrations, ranging from 200-3.125 mg/ml. mhb media were put in all tubes (except for the one with 200 mg/ml concentration) to reduce the extract concentration to half of the initial concentration. after all tubes were fi lled with the required amount of concentration, the bacterial suspensions measured using mcfarland’s 0.5 standard were added to each tube. furthermore, the tubes were incubated in the temperature of 37°c for 24 hours. the results can be seen in tube 1 to 7 (the tubes appeared turbid, resembling the positive control tube). similar thing occurred to the other tubes. due to the turbidity, observation could not be carried out for mic. therefore, streaking was conducted on nutrient agar plates to confi rm the obtained results. after incubated in 37°c temperature for 24 hours, there were bacterial colony growths in all agar plates. it was concluded that adding extract ethanol of murraya paniculata leaves extract up to the concentration of 200 mg/ml is unable to inhibit the klebsiella pneumoniae esbl bacterial growth. discussion dilution was carried out by preparing seven tubes containing mhb which then added with extract in certain concentrations and bacterial suspension. after incubated in the temperature of 37°c for 24 hours, the tubes were observed. bacterial growth still occurred if the solution appeared more turbid than in the negative control. the extract was made from 100 grams of murraya paniculata and 500 ml of 40% ethanol. this study was carried out four times according to federer’s formula measurement. there were four mechanisms of bacterial resistance against β-lactam enzyme: β-lactam inactivation by β-lactamase enzyme; penicillin binding protein (pbp) production with lower affi nity against antibiotics; changes in porin channel leading to decreased permeability against antibiotics; and effl ux pump that encourages antibiotics to escape the cells.12 klebsiella pneumoniae esbl is a bacterium that is able to product β-lactamase enzyme, the enzyme that resist to all penicillin and cephalosporins, including the sulbactam and clavulanic acid combinations and monobactams such as aztreonam.13 the expression of β-lactamase enzyme induced by muropeptides, which is a product from cell wall metabolism of gramnegative bacteria.14 according to the phytochemical assay, ethanol extract contains more secondary metabolite compounds than water extract does. secondary metabolite compounds comprise alkaloids, fl avonoids, saponins, triterpenoids, steroids, and tannins.15 active ingredients of extract ethanol of murraya paniculata leaves and are fathomed to have antibacterial effect are volatile oil, flavonoids, alkaloids, coumarins, terpenoids, saponins, and tannins. volatile oil contains a compound acting as antibacterial by interrupting the forming of membranes or cell walls.16 flavonoids, which is derived from phenol, show antibacterial activity since its penetration into cells causes protein precipitation, protein denaturation, protein coagulation, structure damage, and membrane lysis.17 alkaloids interrupt peptidoglycan components in bacterial cells, causing cell walls not to form well and the cell itself to die.18 coumarins show antibacterial activity due to its lipophilic structures and planar molecules that contribute to the penetration to cell 106 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 102–107 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 membrane or wall. adding methyl or o-methyl group in the c6 or c7 position into coumarin aromatic core maintains the antibacterial activity in gram-negative bacteria.25 terpenoids as a antimicrobial compounds whose mechanism of action is membrane disruption, could be futuristic biocide properties. it can be used in conjunction with other products such as antibiotics at sub-eff ective concentrations therefore it can confer bacterial resistance to antibiotics.19 saponins extract of the a. articulate have antimicrobial activity on ranges of gramnegative antibiotic-resistant isolates.20 saponin compound in acacia arabica extract has antimicrobial activity against diarrheagenic e.coli.21 saponin-rich extracts from guar meal and quillaja exhibited antibacterial activity against s.aureus.22 tannins has phenolic group which can be as antimicrobial and formulation based on tannin-rich plants have been used as diarrhea treatment.23 the previous study proved antibacterial eff ect of murraya paniculata extract. ethanol extract in murraya paniculata inhibits the growths of e.coli, k.pneumoniae, s.typhi, e.faecalis, p.aeruginosa, s.fl exneri, s.aureus, and s.sonneii in 200 mg/ml concentration. (24) meanwhile, 200 mg/ml concentration of ethanol extract in murraya paniculata nonsignifi cantly inhibits e.coli, p.mirabilis, s.typhi and e.aerogenes.25 the results show that extract ethanol of murraya paniculata leaves fail to inhibit and terminate klebsiella pneumonia esbl bacterial growth. this might be due to several matters, including: inhibition and termination of klebsiella pneumonia esbl require concentration of >200 mg/ml; combination with other antibiotics is required for optimum inhibition of klebsiella pneumonia esbl bacterial growth; further extraction is required until pure compound is obtained, enabling adjustment to optimum solvent. in a study using murraya paniculata ethanol extract with 300 mg/ml concentration, the growth of e.coli, p.mirabilis, s.typhi, dan e.aerogenes were inhibited signifi cantly.25 other study reported that total alkaloids extracted from sophorea alpecuroides l. combined with cefotaxime or ceftazidime against e. coli esbl has mics of 12.5 mg/ml.26 total alkaloids increase bacterial susceptibility to cefotaxime and ceftazidime by 8-16 times. natural flavonoid combined separately with amoxicillin. clavulanic acid, ampicillin/sulbactam and cefoxitin synergically inhibit the activities of klebsiella pneumoniae esbl that is still susceptible to imipenem and cefmetazole.26 t h e t h r e e m o s t s t u d i e d c o u m a r i n s i n c l u d e a u r a p t e n e , u m b e l l i p r e n i n a n d 7-isopentenyloxycoumarin.27 auraptene inhibits bacterial activity producing β-lactamase class a.28 in conclusion, the six flavonoids: 5,7dimethoxyfl avanone-4′-o-β-d-glucopyranoside; 5,7-dimethoxyflavanone-4′-o-[2″-o-(5‴-otrans-cinnamoyl)-β-d-apiofuranosyl]-β-dglucopyranoside; 5,7,3′-trihydroxy-fl avanone4′-o-β-d-glucopyranoside; naringenin 7-o-β-dglucopyranoside; rutin; and nicotifl orin, inhibit the of klebsiella pneumoniae esbl growth.29 conclusion extract ethanol of murraya paniculata leaves with concentration 200 mg/ml shows no antibacterial eff ects against the growth of klebsiella pneumoniae esbl. the mic of orange jessamine leaf extract is indeterminable. acknowledgement we thank you to department of microbiology faculty of medicine universitas airlangga. references 1. jeyamohan d. angka prevalensi infeksi nosokomial pada pasien luka operasi pasca bedah di bagian bedah di rumah sakit umum pusat haji adam malik, medan dari bulan april sampai september 2010. microbiology and management of hospital infection 2011. 2. dasgupta, sugata et al. nosocomial infections in the intensive care unit: incidence, risk factors, outcome and associated pathogens in a public tertiary teaching hospital of eastern india. indian journal critical care medicine. 2015;19(1):14-20. 107illona okvita wiyogo, et al.: antibacterial activity of ethanol extract of kemuning ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 3. kuntaman. analysis of microbiology results for managing hospital acquired infection eff ectively surabaya: universitas airlangga; 2011 [cited 2014 27th may]. available from: http://kuntaman-fk.web.unair. ac.id/artikel_detail-35548-umum-microbiology%20 a n d % 2 0 m a n a g e m e n t % 2 0 o f % 2 0 h o s p i t a l % 2 0 infection.html. 4. chung dr, song jh, kim sh, thamlikitkul v, huang sg, wang h, et al. high prevalence of multidrugresistant nonfermenters in hospital-acquired pneumonia in asia. american journal of respiratory and critical care medicine. 2011;184(12):1409-17. 5. munoz-price ls, jacoby ga. extended-spectrum betalactamases 2014 [cited 2014 9th august ]. available from: http://www.uptodate.com/contents/ extended-spectrum-beta-lactamases. 6. tsai ss, huang jc, chen st, sun jh, wang cc, lin sf, et al. characteristics of klebsiella pneumoniae bacteremia in community-acquired and nosocomial infections in diabetic patients. chang gung medical journal. 2010;33(5):532-9. 7. huang wy, cai yz, zhang y. natural phenolic compounds from medicinal herbs and dietary plants: potential use for cancer prevention. nutrition and cancer. 2010;62(1):1-20. 8. syahadat rm, aziz sa. pengaruh komposisi media dan fertigasi pupuk organik terhadap kandungan bioaktif daun tanaman kemuning (murraya paniculata (l.) jack) di pembibitan. 2012. 9. ng mk, al e. bioactivity studies and chemical constituents of murraya paniculata (linn) jack. international food research journal. 2012;19(4):1307-12. 10. matos mj, vazquez-rodriguez s, santana l, uriarte e, fuentes-edfuf c, santos y, et al. looking for new targets: simple coumarins as antibacterial agents. medicinal chemistry. 2012;8(6):1140-5. 11. xiang jl. kamuning 2013 [cited 2014 1st jun ]. available from: http://stuartxchange.com/kamuning. html. 12. rao s. extendedspectrum beta-lactamases2015. 13. parven, rm et al. extended-spectrum beta-lactamase producing klebsiella pneumoniae from blood cultures in puducherry, india. indian journal of medical research. 2011;134(3)392-395paterson dl, bonomo ra. extended-spectrum beta-lactamases: a clinical update. clinical microbiology reviews. 2005;18(4):657-86. 14. zeng, ximin & lin, jun. beta-lactamase induction and cell wall metabolism in gram-negative bacteria. frontiers in microbiology. 2013;4:128 15. iswantini d, al e. zingiber cassumunar, guazuma ulmifolia, and murraya paniculata extracts as antiobesity: in vitro inhibitory eff ect on pancreatic lipase activity. hayati journal of biosciences. 2011;18(1):6-10. 16. parwata im, oka a, dewi pfs. isolasi dan uji aktivitas antibakteri minyak atsiri dari rimpang lengkuas (alpinia galanga l.). jurnal kimia. 2008;2(2):100-4. 17. rahayu md. uji efektivitas antibakteri ekstrak lengkuas merah (alpinia purpurata k. schum) terhadap bakteri escherichia coli secara in vitro2012. 18. juliantina fr, citra da, nirwani b, nyrmasitoh t, bowo et. manfaat sirih merah (piper crocatum) sebagai agent anti bakterial terhadap bakteri gram positif dan bakteri gram negatif. jurnal kedokteran dan kesehatan indonesia. 2009:10-9. 19. jasmine r, selvakumar bn, daisy p. investigating the mechanism of action of terpenoids and the eff ect of interfering substances on an indian medicinal plant extracr demonstrating antibacterial activity. international journal of pharmaceutical studies and research. 2011;2:19-24 20. maatalah bm et al. antimicrobial activity of the alkaloids and saponin extracts of anabasis articulata. journal of biotechnology and pharmacheutical reserarch. 2012;3(3):54-57. 21. biswas d, roymon mg. validation of antibacterial activity of saponin against diarrheagenic e.coli isolated from leaves and bark of acacia arabica. journal of phytochemistry.2012;4:21-23 22. hasan sm, byrd ja, cartwright al, bailey ca. hemolytic and antimicrobial activites diff er among saponin-rich extract from guar, quillaja, yucca, and soybean. applied biochemistry and biotechnology. 2010;162:1008-1017. 23. omojate godstime c, enwa felix o, jewo augustina o, eze christopher o. mechanisms of antimicrobial actions of phytochemicals against enteris pathogens-a review. journal of pharmaceutical, chemical and biological sciences. 2014;2(2):77-85 24. gautam mk, al e. in-vitro antibacterial activity on human pathogens and total phenolic, fl avonoid contents of murraya paniculata linn. leaves. asian pacifi c journal of tropical biomedicine. 2012:1660-3. 25. sundaram m, al e. studies on in vitro antibacterial, antifungal property and antioxidant potency of murraya paniculata. pakistan journal of nutrition. 2011;10(10):925-9. 26. zhou xz, jia f, liu xm, yang c, zhao l, wang yj. total alkaloids from sophora alopecuroides l. increase susceptibility of extended-spectrum beta-lactamases producing escherichia coli isolates to cefotaxime and ceftazidime. chinese journal of integrative medicine. 2013;19(12):945-52. 27. mahdi askari, amirhossein sahebkarm mehrdad i r a n s h a h i . s y n t h e s i s a n d p u r i f i c a w t i o n o f 7-prenyloxycoumarins and herniarin as bioactive natural coumarins. iranian journal of basic medical sciences. 2009;12(2):63-69 28. safdari h, neshani a, sadeghian a, ebrahimi m, iranshahi m, sadeghian h. potent and selective inhibitors of class a beta-lactamase: 7-prenyloxy coumarins. the journal of antibiotics. 2014;67(5):373-7. 29. orhan dd, ozcelik b, ozgen s, ergun f. antibacterial, antifungal, and antiviral activities of some fl avonoids. microbiological research. 2010;165(6):496-504. 86 vol. 7 no. 4 january-april 2019 research report prevalence of soil transmitted helminthiasis among elementary children in sorong district, west papua natalia yuwono1a, soraya salle pasulu2, dominicus husada3, sukmawati basuki4 1master program of tropical medicine, faculty of medicine, universitas airlangga 2pediatric program, faculty of medicine, universitas airlangga/pediatric department, dr. soetomo hospital 3pediatric department, dr. soetomo hospital/faculty of medicine, universitas airlangga 4parasitology department, faculty of medicine, universitas airlangga acorresponding author: sukmawati basuki (sukmab@fk.unair.ac.id), dominicus husada (dominicushusada@yahoo.com) abstract soil transmitted helminthiasis are common in the world and cause illness, especially in developing countries. it can cause infection in humans by contact with parasitic eggs or larvae that live in moist and warm soil. soil-transmitted helminthiasis is often caused by ascaris lumbricoides, trichuris trichiura, ancylostoma duodenale, and necator americanus. in indonesia, soil transmitted helminthiasis prevalence is still high in some places. the tropical climate and high humidity support for the development of worms like in sorong district, but there was no data. the purpose of this study is to identify the presece of soil transmitted helminthiasis in primary school children in sorong district. a cross-sectional study was conducted in two elementary schools located in sorong district, west papua, indonesia. the two elementary schools are sdn 22 in klain village and sd inpres 24 in sub-district mayamuk. once collected, the pot that has contained stool is given formalin 10%. stool examinattion using direct smear method to determine the presence of soil transmitted helminthiasis. researchers get the subject as many as 147 children. the proportion of elementary school children by sex consists of 72 boys (49%) and 75 girls (51%). the prevalence of soil transmitted helminthiasis as a whole was 30.6% (45/147) with 40.1% (18/45) single infections and 59.9% (27/45) mixed infections. the single infection that most frequent is trichuris trichiura, then followed by ascaris lumbricoides. soil-transmitted helminthiasis mostly found in girl than boy and mostly found in 6-9 years age group. the worm species that infect elementary school children in the district is ascaris lumbricoides, trichuris trichiura, hookworm, and strongyloides stercoralis. this is probably related with the climate and low sanitation level. to eliminate soil transmitted helminthiasis among elementary school children, in addition to routine treatment also needs intensive counseling about the importance of maintaining personal hygiene and the environment. keywords: prevalence, soil transmitted helminthiasis, direct smear method, elementary school, sorong district abstrak soil-transmitted helminthiasis sering terjadi di dunia dan penyebab kesakitan khususnya di negara berkembang. hal ini dapat menyebabkan infeksi pada manusia melalui kontak dengan telur parasit atau larva yang tinggal di tanah lembab dan hangat. soiltransmitted helminthiasis sering disebabkan oleh ascaris lumbricoides, trichuris trichiura, ancylostoma duodenale dan necator americanus. di indonesia, prevalensi soil transmitted helminthiasis masih tinggi di beberapa tempat. iklim tropis dan kelembaban yang tinggi mendukung untuk perkembangan cacing seperti di kabupaten sorong, tetapi belum ada data. tujuan dari studi ini adalah untuk mengidentifikasi keberadaan soil-transmitted helminthiasis pada anak sekolah dasar di kabupaten sorong. studi cross-sectional dilakukan di dua sekolah dasar yang terletak di kabupaten sorong, papua barat, indonesia. dua sekolah dasar tersebut adalah sdn 22 berada di desa klain dan dan sd inpress 24 berada di sub-distrik mayamuk. setelah terkumpul, pot yang berisi tinja kemudian diberi formalin 10%. pemeriksaan feses menggunakan metode pemeriksaan langsung direct smear untuk mengetahui adanya soil transmitted helminthiasis peneliti mendapatkan subjek penelitian sebanyak 147 anak. proporsi anak-anak sekolah dasar berdasarkan jenis kelamin terdiri dari 72 laki-laki (49%) dan 75 perempuan (51%). prevalensi soil transmitted helminthiasis secara keseluruhan adalah 30,6% 87yuwono, et al.: prevalence of soil transmitted (45/147) dengan infeksi tunggal 40,1% (18/45) dan 59,9% (27/45) infeksi campuran. infeksi tunggal yang sering ditemukan adalah trichuris trichiura diikuti dengan ascaris lumbricoides. soil-transmitted helminthiasis sebagian besar ditemukan pada perempuan daripada laki-laki dan lebih banyak ditemukan pada kelompok usia 6-9 tahun. spesies cacing yang menginfeksi anak sekolah dasar di kabupaten tersebut adalah ascaris lumbricoides, trichuris trichiura, cacing tambang dan strongyloides stercoralis. hal ini mungkin terkait dengan iklim dan tingkat sanitasi yang rendah. untuk mencegah kejadian soil transmitted helminthiasis, selain perawatan rutin juga perlu dilakukan konseling intensif tentang pentingnya menjaga kebersihan diri dan lingkungan. kata kunci: prevalensi, soil-transmitted helminthiasis, pemeriksaan tinja mikroskopis cara langsung, kabupaten sorong introduction the worms parasite have infected humans in the world more than 1 billion. who estimates that more than 1.5 billion people worldwide or 24% of the world’s population are infected with worms and widely distributed in tropical and sub-tropical areas, mostly in sub-saharan africa, the americas, china and east asia. more than 270 million preschool children and over 600 million school-aged children live in endemic areas and require preventive therapy and intervention1. in 2011, indonesian health ministers said about 195 million indonesians live in worms endemic areas, including 13 million pre-school children and 37 million school-aged children2. soil transmitted helminthiasis is often caused by ascaris lumbricoides, trichuris trichiura, ancylostoma duodenale and necator americanus(1). soil transmitted helminthiasis is transmitted through eggs collected with the patient’s stool. female worms live in the human intestine and produce thousands of eggs daily. in areas with low sanitation levels, the eggs will contaminate the soil. things that may cause ascaris and trichuris eggs to be swallowed human beings include eating egg contaminated vegetables, while the vegetables are not cooked and washed properly, eggs contaminate drinking water and eggs are swallowed by children who play the soil and do not wash their hands thoroughly. hookworm and strongyloides stercoralis hatch in the soil, releasing filariform larvae that can infect humans through penetration of the larvae on human skin. penetration usually subdistrict mayamuk sorong district sd inpress 24 sdn 22 figure 1. map of sub-district mayamuk and klain village in sorong district, showing the location of sdn 22 and sd inpres 24. occurs through the skin of the foot that is not covered by footwear(3). worms produce eggs or larvae in very large quantities and have high reproductive capacity, which can lead to high incidence of infection in humans when the condition of the host is conducive to infections such as in the marginal areas in tropical countries(4). material and method subject of research the research is conducted by cross sectional study on two elementary schools in sorong district, west papua, indonesia. the two elementary schools are sdn 22 in klain village, and sd inpres 24 in sub-district mayamuk (see figure 1). sorong district lies in the coordinates of 000 33 ‘42’ ‘010 35’ 29 ‘’ south latitude and 1300 40 ‘49’ ‘1320 13’ 48 ‘’ east longitude with an area of 12,159.42 km2, which consists of land area of width 11,644.77 km2 and the sea area of 514.65 km2. sorong district consists of 19 sub-districts with 18 urban villages and 135 villages. one of the sub-districts is mayamuk. the research conducted in sub-district mayamuk. large sample calculations using lemeshow formula with unknown population. from the calculation, the sample size is at least 96, but to anticipate the error result, the researcher adds 10% of the minimum sample size. 88 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 86–91 data collection samples are collected on august 2017. students are given an explanation of how to collect and store stool samples. once collected, the pot that has contained stool is given formalin 10%. stool samples were taken by researchers to be examined at the department of parasitology faculty of medicine, universitas airlangga, surabaya. stool examination using direct smear/wet mounting method to determine the presence of soil transmitted helminthiasis. pots containing stools, stirred using sticks to be homogeneous, then stool taken and mixed on a object glass that has been given a drop of lugol 1% and then leveled. the rest of the food and crude fiber are removed with a stick and then closed with a cover glass and check under microscope systematically. the preparations are examined under a microscope with magnification 10x then 40x. data analysis results of direct-smear data then analyzed descriptively to determine the prevalence of infection and the type of worm infection. result and discussion researchers get the subject of research as many as 147 children. the distribution of male and female students in both primary schools is almost the same, with the total of male students being 72 students while the total female students are 75 students. by age group, the highest group was age 6–7 years (44.9%) while the lowest was 11-12 years old (2.7%) (see table 1). the results of microscopic examination showed 45 (30.6%) students from 147 students infected, with soil table 1. demographic characteristics of research subjects at sdn 22 and sd inpres 24 in sorong district. sdn 22 (n=55) n (%) sd inpres 24 (n=92) n (%) n=147 n (%) age (years) 6-7* 8-9* 10-11* 11-12 23 (41,8) 22 (40) 8 (14,5) 2 (3,7) 43 (46,7) 43 (46,7) 4 (4,4) 2 (2,2) 66 (44,9) 65 (44,2) 12 (8,2) 4 (2,7) grade level i ii iii iv v 16 (29,1) 13 (23,6) 6 (10,9) 14 (25,5) 6 (10,9) 40 (43,5) 23 (25) 27 (29,3) 2 (2,2) 0 56 (38,1) 36 (24,5) 33 (22,4) 16 (10,9) 6 (4,1) sex boy girl 29 (40,3) 26 (34,7) 43 (59,7) 49 (65,3) 72 (49) 75 (51) * some subjects are unknown of date of birth so the age of the subject is classified according to grade level. table 2. characteristic of soil transmitted helminthiasis cases by sex in children of sdn 22 and sd inpres 24 in sorong district. sdn 22 (n=24) n(%) sd inpres 24 (n=21) n (%) n (45) n (%) sex boy girl 13 (59,1) 11 (47,8) 9 (40,9) 12 (52,2) 22 (48,9) 23 (51,1) transmitted helminthiasis. prevalence in girls is higher than boys (see table 2). the highest prevalence is in level grade 1 (see figure 2). mixed soil transmitted helminthiasis was more common than single infection with 62.2% (28/45) prevalence compared to 37.8% (17/45). the single infection that most frequent is trichuris trichiura infection, followed by ascaris lumbricoides. the most common mixed infection are ascaris lumbricoides and trichuris trichiura also ascaris lumbricoides, trichuris trichiura and hookworm. in one microscopic stool examination sample found many mature ascaris lumbricoides eggs and ascaris 0 2 4 6 8 10 12 grade level 1 grade level 2 grade level 3 grade level 4 grade level 5 sdn 22 sd inpress 24 figure 2. soil-transmitted helminthiasis based on grade level. figure 3. ascaris lumbricodes larvae goes out the egg (arrow), 40 x obj 89yuwono, et al.: prevalence of soil transmitted lumbricoides larvae that came out of the egg (see figure 3). in this sample also found trichuris trichiura eggs containing embryos or in an infective stage (see figure 4). in this study hookworm eggs mostly found in stadium that containing 2-8 cell embryos, but there are also an advanced stage that containing larvae (see figure 5). strongyloides stercoralis infection is not found in a single infection but there was in mixed infection (see figure 6). students with mixed infection of strongiloides figure 4. trichuris trichiura egg, 40x obj a b figure 5. (a) hookworm egg. (b) an advance stage of hookworm egg, 40 x obj. figure 6. strongyloides stercoralis rhabditiform larvae, 40 x obj. table 3. results of stool examination of children of sdn 22 and sd inpres 24 in sorong district. sth infection sdn 22 (n=55) n (%) sd inpres 24 (n=92) n (%) n = 147 n (%) single infection ascaris lumbricoides (al) trichuris trichiura (tt) hookworm (hw) strongyloides stercoralis (ss) 0 6 (10,9) 4 (7,3) 0 5 (5,4) 2 (2,2) 0 0 5 (3,4) 8 (5,4) 4 (2,7) 0 mixed infection al – tt al – hw al – tt – hw al – tt – ss al – tt – hw – ss tt – hw tt – hw – ss 1 (1,8) 1 (1,8) 3 (5,5) 1 (1,8) 1 (1,8) 5 (9,1) 2 (3,6) 6 (6,5) 0 4 (4,3) 0 0 2 (2,2) 2 (2,2) 7 (4,8) 1 (0,7) 7 (4,8) 1 (0,7) 1 (0,7) 7 (4,8) 4 (2,7) not infected 31 (56,4) 71 (77,2) 102 (69,3) stercoralis were 6 students, 4 of whom were from sdn 22. the description of stool examination result of the sample was explained on table 3. from table 2 it can be concluded that the prevalence of soil transmitted helminthiasis is more common in girls than in boys. this may be due to female hygiene in this area is less good compared with male. bestari(5) in 2015 doing research in surakarta city also found the same results. most of the infected subjects were 6-9 years old. the prevalence of high soil transmitted helminthiasis at 6-10 years of age can be attributed to habit factors of play. generally children at that age play more outside home and contact with the ground which is a medium of worm transmissions. transmission can occur among school students through holding hands while playing with students who often play outside the house and contact with the ground(6). the types of worms found in stool examinations vary, from ascaris lumbricoides, trichuris trichiura, hookworm to strongyloides stercoralis. several surveys in indonesia show that often high prevalence of ascaris is accompanied by a high prevalence of trichuris as well(6,7). in this study, it was found similar but the prevalence of trichuris trichiura was higher than ascaris lumbricoides. sorong district areas include tropical areas that have a hot and humid climate(8). this becomes one of the risk factors because trichuris trichiura spread mainly in hot and humid areas(9,10). tropical climate with high air humidity as well as fertile soil are the optimal environtment for worm life. these two types of worms often lead to mixed infection because their habitats and life cycles equally require soil media(9,11). the number of found mixed infection indicates the level of 90 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 86–91 hygiene and sanitation in the children’s environment is very bad(6). the main factors that cause the occurence of soil transmitted helminthiasis is a behaviour factor that reflects low personal hygiene such as not washing hands with soap before eating and after defecation, cleanliness of the nails that are not maintained, eating foods which cleanliness is questioned, fingernail biting, not wearing footwear during outdoor activities, defecate in the open area12–14. the spread of soil transmitted helminthiasis is strongly influenced by the occurence of faeces contamination on the soil and water, so the defecation habits will be very decisive. in this study, hookworm infection is also commonly found. generally, the prevalence of hookworm is more common in adults. hookworm infections often occur in areas where human faeces are used as fertilizer or where defecation onto soil happens15. this may explained higher prevalence is found in plantation areas as well as in mining16,17. most of the sorong district community has major jobs in agriculture, plantation and forestry18. there was possibility of an infected adult defecating outside (near bush, in a garden, or field) then a mature hookworm egg and hatch, releasing larvae (immature worms)15. the larvae became mature into a form that can penetrate childern’s skin. many elementary school children do not wear footwear. this can increase the risk of getting infected with hookworm and strongyloides stercoralis19–21. strongyloides stercoralis infection was found in 6 students with mixed infection. this is probably related with the climate22,23. sorong’s climate is humid, the himidity ranges between 81-87%8. this tropical climate and high humidity support the development of strongyloides stercoralis. knowing the prevalence of worms can be useful for worm management strategies and can be used for basic data on allergic correlation research with worm infection. the prevalence of allergies is increasing dramatically in the world, both in developing and developed countries, especially in lowand middle-income countries. it is estimated that 30-40% of the world’s population is exposed to one or more allergic conditions. this increase is especially true in children. there is much debate about the interaction between helminths and allergic disease. some epidemiologic studies suggest that helminth infections induce or increase the severity of atopic diseases. other studies report children with soil transmitted helminthiasis have lower prevalence and milder atopic symptoms. although there have been many recent studies, the relationships between allergic and helminth infections remains controversial. the “hygiene hypothesis” is a very popular concept among scientists. the so-called “hygiene hypothesis” which posits that allergic phenomena arise from the sanitized living conditions of the developed world has been one of the major theories to account for this remarkable difference in prevalence of allergy. multiple mechanisms may account for the hygiene hypothesis, but there is considerable evidence to suggest that helminth infection plays a central role24. to eliminate soil-transmitted helminthiasis among elementary school children, in addition to routine treatment or deworming through mass drug administration also needs intensive counseling about the importance of maintaining personal hygiene and the environment. interventions water, sanitation and hygiene (wash) and health education could sustain the benefits of antihelmintic therapy25,26. they play an extremely important role in breaking the cylce of transmission and preventing infection. there was a study in sri lanka showed that even after 10 years of antihelmintic therapy, prevalence can be restored after discontinuation of preventive deworming, if the initial force of transmission is strong and other long-term control measures are not concurrently implemented25. conclusion prevalence of soil-transmitted helminthiasis among elementary school children in sorong district is 30,6% (45/147) with 40.1% (18/45) single infections and 59.9% (27/45) mixed infections. the single infection that most frequent is trichuris trichiura, then followed by ascaris lumbricoides. the worm species that infect elementary school children in the sorong district is ascaris lumbricoides, trichuris trichiura, hookworm and strongyloides stercoralis. soil-transmitted helminthiasis mostly found in girl than boy and mostly found in 6-9 years age group. acknowledgement the work describe in this publication is supported by universitas airlangga and the financial is supported by universitas ciputra. references 1. who. soil-transmitted helminths infections. world health organization. 2017. 2. tan m, kusriastuti r, savioli l, hotez pj. indonesia: an emerging market economy beset by neglected tropical diseases (ntds). plos negl trop dis. 2014 feb;8(2):e2449. 3. soedarto. buku ajar parasitologi kedokteran. jakarta: sagung seto; 2011. 4. maguire jh. intestinal nematodes (roundworms). in: bennet je, dolin r, blaser mj, editors. mandell, douglas, and bennett’s principles and practice of infectious disease volume 1. eight edit. philadelphia: elsevier ltd; 2015. p. 1–4908. 5. bestari rs, supargiyono, sumarni, suyoko. derajad eosinofilia pada penderita infeksi soil-transmitted helminth (sth). biomedika. 2015;7(2):27–34. 6. hairani b, waris l, juhairiyah. prevalensi soil transmitted helminth (sth) pada anak sekolah dasar di kecamatan malinau kota kabupaten malinau provinsi kalimantan timur. epidemiol zoonosis j. 2014;5(1):43–8. 7. sasongko a, irawan hsjy, tatang rs, subahar r, purnomo, margono ss. intestinal parasitic infections in primary school children in pulau panggang and pulau pramuka, kepulauan seribu. makara j heal res. 2002;6(1):8–11. 8. bps kota sorong. rata-rata kelembaban udara di kota sorong, 2000-2015. sorong: badan pusat statistik kota sorong; 2017. 91yuwono, et al.: prevalence of soil transmitted 9. ideham b, pusarawati s. helmintologi kedokteran. surabaya: universitas airlangga press; 2007. 10. areekul p, putaporntip c, pattanawong u, sitthicharoenchai p, jongwutiwes s. trichuris vulpis and t. trichiura infections among schoolchildren of a rural community in northwestern thailand: the possible role of dogs in disease transmission. asian biomed. 2010;4(1):49–60. 11. meliyanie g, andiarsa d. the differences of atopic status between child with and without helminths infection of student at kampung baru elementary school, kusan hilir subdistrict, tanah bumbu regency, kalimantan selatan. epidemiol zoonosis j. 2014;5(2):81–6. 12. chadijah s, sumolang ppf, verdiana nn. hubungan pengetahuan, perilaku, dan sanitasi lingkungan dengan angka kecacingan pada anak sekolah dasar di kota palu. media litbang kesehat. 2014;24(1):50–6. 13. jodjana e, majawati es. gambaran infeksi cacing trichuris trichiura pada anak di sdn 01 pg jakarta barat. j kedokt meditek. 2017;23(61):32–40. 14. sofiana l, sumarni s, ipa m. fingernail biting increase the risk of soil transmitted helminth (sth) infection in elementary school children. heal sci j indones. 2011;2(2):81–6. 15. cdc. parasites hookworm. central for disease control and prevention. 2013. 16. rusmartini t. parasitologi kedokteran ditinjau dari organ tubuh yang diserang. jakarta: egc; 2009. 17. walana w, aidoo enk, tay sck. prevalence of hookworm infection : a retrospective study in kumasi. asian pasific j trop biomed. 2014;4(suppl 1):158–61. 18. pemkab sorong. portal resmi pemerintah kota sorong. 2016. 19. amoah as, hamid f, smits hh, yazdanbakhsh m. environmental risk factor for allergy: helminth infection. in: akdis ca, agache i, editors. global atlas of allergy. switzerland: european academy of allergy and clinical immunology; 2014. p. 138–40. 20. maguire jh. introduction to helminth infections. in: bennet je, dolin r, blaser mj, editors. mandell, douglas, and bennett’s principles and practice of infectious disease volume 1. eight edit. philadelphia: elsevier saunders; 2015. p. 3196–8. 21. sandy s, sumarni s, soeyoko. analisis model faktor risiko yang mempengaruhi infeksi kecacingan yang ditularkan melalui tanah pada siswa sekolah dasar di distrik arso kabupaten keerom, papua. media litbang kesehat. 2015;25(1):1–14. 22. widyaningsih i. strongyloides. j ilm kedokt wijaya kususma. 2017;30(september):94–101. 23. suparli t, margono ss, abidin san. nematoda usus. in: susanto i, ismid is, sjarifuddin pk, sungkar s, editors. buku ajar parasitologi kedokteran. keempat. jakarta: badan penerbit fkui; 2015. p. 6–25. 24. stein m, greenberg z, boaz m, handzel zt, meshesha mk, bentwich z. the role of helminth infection and environment in the development of allergy : a prospective study of newly-arrived ethiopian immigrants in israel. plos negl trop dis. 2016;1–14. 25. gunawardena k, kumarendran b, ebenezer r, sanjeewa m, pathmeswaran a, silva n de. soil-transmitted helminth infections among plantation sector schoolchildren in sri lanka : prevalence after ten years of preventive chemotherapy. 2011;5(9). 26. speich b, croll d, f??rst t, utzinger j, keiser j. effect of sanitation and water treatment on intestinal protozoa infection: a systematic review and meta-analysis. lancet infect dis. 2015;87–100. 40 vol. 7 no. 2 may–august 2018 factor related to anti-tuberculosis drug resistency on pulmonary tuberculosis patients in labuang baji hospital makassar sapriadi1a, syahridha2 1 department of nursing, stik famika makassar, south sulawesi 2 department of tropical medicine, faculty of medicine, universitas airlangga a corresponding author: email: immjepot@yahoo.com abstract tuberculosis become crucial diseases in the world. the disease can spread rapidly since it spreads through the air. the transmission through coughing and sneezing from droplet tb patients. containing mycobacterium tuberculosis can be exposed to anyone who is around the patient. the aim of this research is to analyze the factors that affect the anti-tuberculosis drug resistance in patients with pulmonary tuberculosis in labuang baji hospital makassar. the research design is cross sectional approach. this study is conducted in june–july 2017 with a total sample of 60 respondents. based on the data analysis, it is found that there were 34 respondents (56.7%) from 15–34 years old group and 26 respondents (43.3%) from 35–54 years old group. there were 35 female respondents (58.3%) and 25 male respondent(41.7%), is respondents with positive vct test result and 45 respondensts (74.0%) with negatif vct test, 54 respondents (90,0%) who have treatment history previously, 45 respondents (78.3%) have history with tb contact, 45 respondents (75.6%) who had good knowledge. 51 respondents (95%) with anti tuberculosis drug resistance. the result of statistical test using chi-square test is showed that there was a significant correlation between treatment history and anti tuberculosis drug resistance with p = 0.024 < α = 0.05. but, there was no relationship between sex and knowledge with anti-tuberculosis drug resistance with p value 0.56 and 0.566 (p value > α = 0.05). it is recommended to health worker to give health education for patient and their family, so they can do their treatment until the patients are cured. it can prevent the patient to be anti tuberculosis drug resistance patient. keywords: tuberculosis, anti-tuberculosis drug resistance, treatment history, sex, knowledge abstrak tuberkulosis adalah salah satu penyakit menular yang membahayakan kesehatan. penyakit ini dapat menyebar dengan cepat karena menyebar melalui udara yang terjadi saat penderita tuberkulosis batuk dan bersin sehingga droplet yang mengandung mycobacterium tuberculosis dapat terpapar pada siapapun yang berada di sekitar penderita. tujuan umum dari penelitian ini adalah untuk menganalisis faktor-faktor yang memengaruhi resistensi obat anti tuberkulosis pada pasien tuberkulosis paru di rumah sakit labuang baji makassar. desain penelitian dengan pendekatan cross sectional. penelitian ini dilakukan pada bulan juni–juli 2017. dengan total sampel sebanyak 60 responden berdasarkan kriteria inklusi dan eksklusi. berdasarkan analisis data, ditemukan bahwa kelompok usia 15–34 tahun sebanyak 34 responden (56,7%) dan 35–54 tahun sebanyak 26 responden (43,3%), jenis kelamin sebanyak 35 responden (58,3%) perempuan dan 25 responden (41,7%) laki-laki, hasil tes positif vct sebanyak 15 responden (25,0%) dan 45 responden (74,0%) dengan uji vct negatif, responden yang memiliki riwayat pengobatan sebelumnya ada 54 responden (90,0%), responden yang memiliki riwayat kontak dengan tb sebanyak 45 responden (78,3%), yang memiliki pengetahuan sebanyak 45 responden (75,6%) responden dengan resistansi obat anti tuberkulosis sebanyak 57 responden (95%). hasil uji statistik dengan uji chi-square menunjukkan ada hubungan yang signifikan antara riwayat pengobatan dengan resistensi obat anti tuberkulosis yang memiliki nilai p = 0,024 < α = 0,05. namun, tidak ada hubungan antara jenis kelamin dan pengetahuan dengan resistensi obat anti tuberkulosis, nilai p 0,56 dan 0,566 (p value > α = 0,05). dianjurkan kepada petugas kesehatan untuk memberikan pendidikan kesehatan bagi pasien dan keluarga mereka, sehingga mereka dapat menjalani pengobatan hingga dinyatakan sembuh. hal tersebut dapat mencegah pasien menderita resistansi obat anti tuberkulosis. kata kunci: tuberkulosis, resistensi obat anti tuberkulosis, riwayat pengobatan, jenis kelamin, pengetahuan research report 41sapriadi and syahridha: factor related to anti-tuberculosis drug resistency introduction tuberculosis become crucial diseases in the world. this disease can be transmitted quickly because of its spread through the air. this deployment occurs when the tb patients are coughed and sneezed so that the droplets containing mycobacterium tuberculosis can be exposed to anyone who is around the patient. in 1993 the world health organization (who) is declared tb as very important and serious public health problem. who (1993) is stated that the disease causes pulmonary tuberculosis is a global emergency because the disease could not well-controlled yet in most countries in the world. this is due to the many patients who are not successfully treated, and it will cause death due to by infectious diseases.1 every year there are 9 million new cases and death cases nearly 2 million people. in 2015, indonesia has 2nd rank after india with tb incident reaching 330,910 cases. the incidence of tb continues to increase, the government is implemented the dots strategy (directly observed treatment short-course) optimally to combat tb. dots is a medical supervision directly in a short period which is expected to perform early diagnosis and appropriately to unexpected2 tb through microscopic examination. this strategy has proven to be the most cost effective1. this strategy focuses on finding and healing tb patients by breaking the tb chain of transmission as well as to reduce the incidence of tb in the community. implementation of dots strategy is proved able to decrease tb cases in indonesia. but other problem arised is related to tb in indonesia today, which is the increasing cases of mdr-tb (multi drug resistant). mdr-tb is a resistant form of tb two most potent anti tuberculosis drugs, namely rifampicin and isoniazid.1 data who annual report 2017 has put indonesia in 9th rank of the 27 countries with the highest burden of mdr-tb patients in the world with an estimated 32000 cases. estimated mdr-tb cases number of among notified pulmonary cases are 10000 cases. there are 2,8% of tb cases with mdr-tb among new cases. in 2013, there are 809 notified case in indonesia and 51% have success treatment.3 based on medical record from labuang baji hospital makassar, there were 162 cases of mdr-tb in south sulawesi from makassar and other districts in south sulawesi.4 most cases of mdr-tb is a chronic cases caused by the failure of the first categories of treatment. the results of the 2010 study from tehran, iran, are stated that there were 78 patients with mdr-tb, 46.8% of afghan patients and 2.1% in bangladesh. the patient's status is immigrant or refugee. that means if the resident patient becomes a risk factor for mdr-tb incidence. this suggests that there is a possibility that tb patients who become immigrants and they have drop out of treatment can be mdr-tb case. the case of mdr tb in hiv-infected tb patients is twice than non-hiv-infected tb patients. with the increasing cases of hiv infection, it is no doubt that it will stimulan the higher occurrence of mdr tb in indonesia.5 unfavorable treatment of tb health services in china in 2012 is also a risk factor for mdr-tb. this includes the availability of tb drugs in the health care center and treatment monitoring center of patients 95% did not meet the standards, including the availability of medicines in health care center so that the patients possible dropped out from the treatment.6 according to dedi research (2010), there were 23.8% of patients who receive late tb treatment although having good communication, information and education about tb.7 the correct information will be accept from healthcare worker through health education to increase knowledge of the patients. knowledge of the patients can support their self to do the treatment. factors associated with loss of follow up during treatment for multi drug resistant tb (mdr-tb) are tb knowledge of the patient, health care worker, and vomiting as adverse drug reaction.8 however, different research by mekonnen (2015), is stated that there was no significant relationship between sex, age, educational status, history of smoking, bcg vaccination, with mdr-tb status among pulmonary tb cases, but there is a significant correlation between history of previous treatment with mdr-tb status.9 other reported associated factors include occupation, education status, rural residence,10,11 older age, male sex,10,12 underlying health conditions such as hiv infection and diabetes mellitus.13 labuang baji hospital is the referral center for mdrtb examination in south sulawesi, while the number of mdr-tb patients reaches 162 cases, the mdr-tb case continues to increase.4 as an mdr-tb referral center, patients from various regions came to confirm the type of tb suffered. based on this background, the authors are interested in conducting research on factors related to resistance of anti tuberculosis drugs in patients with pulmonary tuberculosis at labuang baji makassar hospital. method this study is use descriptive analytic research design with cross sectional study approach to determine the factors of antituberculosis drug resistance from tb patients in labuang baji hospital. the population is tb patients at labuang baji hospital since 2016 until july 2017. the sample size in this research is determined by using categorical analytic sample based on research result about the influence of drug adherence in tuberculosis patients with success therapy in great comunity health center for lung in surakarta, patients who adhered to treatment had 76% efficacy therapy while non-adherent only 4%. large samples are required with type i error of 5%, type ii error of 10% using two-way hypothesis. thus, the sample size of each group is 30, so the sample required in this study is 60 people. 42 indonesian journal of tropical and infectious disease, vol. 7 no. 2 may–august 2018: 40–44 the sample in this research is pulmonary tuberculosis patients from labuang baji hospital. the sample is selected through simple random sampling technique. the study sample is tb patient who have positif examination of bta, and are stated mdr through genxpert examination seen on the patient's medical record. patients given informed consent to be respondents and structured interviews are conducted based on questionnaires. measurements of knowledge according to patient perception are done through instrument made by the researcher using guttman scale. true statement given score 1 and wrong statement given score 0. good knowledge is performed if score more than mean (average value). in the history of treatment variables are conducted interviews directly to respondents to see the history of previous treatment including category i or category ii. variable history of tb contact is conducted through interviews with respondents to find out that the people closest to tb patients maybe from the family, neighbors or colleagues and friends of respondents. while the hiv and anti tuberculosis resistant are known from the respondent's medical records provided by health worker. result and discussion univariate analysis univariate analysis in this study will describe the frequency distribution of the variables in this research. the frequency distribution of age, sex, hiv infection, treatment history and contact with tb patients, the officer's behavior and resistance frequency distribution anti tuberculosis drug are the variables in the research. bivariate analysis bivariate analysis is performed by using chi-square test to see the relationship of gender, history of treatment and knowledge with resistance of anti tuberculosis drug in pulmonary tuberculosis patient at labuang baji makassar hospital. the relationship between sex and anti-tuberculosis drug resistance in tb patients in rsud labuang baji from the research result it can be seen that female respondents with positive anti tuberculosis drug resistance were 34 respondents (56.7%), while men with positive anti tuberculosis drug resistance were 23 respondents (38.3%). the respondents with negative anti tuberculosis drug resistance in women were 1 respondent (1.7%) and 2 respondents (3.3%) in men. the studies in georgia which reveal that female were at higher risk of mdr-tb compared with male. the reason is postulated by the study, such as the role of women as care givers which may have predisposed them to developing mdr-tb as they have longer contact at home with sick mdr-tb patients than men. especially when the mdr-tb treatment was not widely available in the country.14 based on chi-square statistic test with fisher's exact test derived value p = 0,565 > (α = 0,05). thus it can be concluded that there is no significant relationship between gender with anti tuberculosis drug resistance from tuberculosis patient in labuang baji hospital. this result supported by omar research who stated that there is no significant relationship between mdr-tb incidence and gender.15 other reported that there is associated sex and cases of resistent tb.16 in the current global drug resistance survey report, the association between sex and mdr-tb was not clearly demonstrated. some countries in the former soviet union reported a higher prevalence among males compared to females. this was thought to be related to alcohol dependency and imprisonment status where more men than women are involved.17 the relationship between treatment history and antituberculosis drug resistance in tb patients in rsud labuang baji from the results in table 2 it can be seen that respondents with positive anti tuberculosis drug resistance and never had a history of treatment as much as 53 respondents (88.3%). they do not have history of treatment as much as 4 respondents (6.7%), as for respondents with negative anti tuberculosis drug resistance and 1 treatment (1.7%) and 2 respondents (3.3%) did not have a history of treatment. based on the results of the statistical test chi-square with fisher's exact test is showed that there were significant association between history of treatment with resistance of anti tuberculosis drug in tb patient in labuang baji hospital (pvalue = 0.024 < α = 0.05). others reported associated that treatment history of tuberculosis is strong risk factor for bacterial resistence to anti tuberculosis drugs.18,19 table 1. univariate variable frequency distribution in rsud. labuang baji makassar year 2017 variables frequency (n) percentage (%) age 15–34 years old 35–54 years old 34 26 56.7 43.3 sex male female 25 35 41.7 58.3 hiv infection negative positive 45 15 74.0 26.0 treatment history yes no. 54 6 90.0 20.0 contacts tb yes no. 47 13 78.3 21.7 knowledge poor good 15 45 75.0 25.0 anti-tuberculosis resistent negative positive 3 57 5.1 95.0 43sapriadi and syahridha: factor related to anti-tuberculosis drug resistency the patients who have previous of treatment history had 9,49 times to be mdr-tb than without previous of treatment history.20 it is well understood that bacterial factor play great role in the spread of mdr-tb. with the population mtb, spontaneous mutation in genes responsible for drug resistance for all first line and some second line drugs, thus scenarious are highly pronounced by misuse of drugs result in rapid selection of drug resistant mutants.21 the relationship between knowledge and anti-tuberculosis drug resistance in tb patients in rsud labuang baji based on table 2, we can explain if respondents with positive oat resistance have good knowledge are 42 respondents (70,0%), while having knowledge less are 15 respondent (25,0%). the respondents with negative oat resistance with good knowledge are 3 respondents (5.0%). based on chi-square statistic test with fisher's exact test, we get value p = 0,566> (α = 0,05). thus it can be concluded that there is no significant relationship between knowledge with anti-tuberculosis drug resistance in tb patients in labuang baji hospital makassar. according to the researchers' assumptions, some respondents who experience positive oat resistance but lack knowledge, this is caused of a better understanding of treatment and mr-tb is a close relative. this research is different with marahatta's research which states that there is a relationship between knowledge with mdr-tb.22 a lack of knowledge about the treatment in progress have an impact on disease transmission and treatment outcomes. health education must be done to prepare the patient about knowledge of tb, disease-causing agent, transmission, and treatment that will be undertaken. conclusion there is correlation between treatment history and anti tuberculosis drug resistance in tb patient at rsud labuang baji makassar. recommendation to nursing profession in order continuously improve knowledge, attitude and skill in assisting patient on tb treatment and motivate patient to adherance treatment are needed. references 1. the ministry of health. guidance a national countermeasures tuberculosis. indonesia: ministry of health of the republic of indonesia; 2014. p. 1–6. 2. ministry of health. profile health of indonesia 2015. indonesia: ministry health 2016; 2016. p. 160–7. 3. tb e, region whosa. bending bending the curve the curve ending tb ending tb in the who south-east asia region bending the curve -ending tb in the who south-east asia region. 4. rsud labuang baji makassar. medical record rsud labuang baji makassar. 2017. 5. weird pb. risk factor associated with multidrug-resistant. tanaffos. 2015;17–21. 6. zhao p, li xj, zhang sf, wang xs, liu cy. social behaviour risk factors for drug resistant tuberculosis in mainland china: a metaanalysis. j int med res. 2012;40(2):436–45. 7. nofizar d, nawas a, burhan e. ’risk factors identification tubercolosis multidrug resistance (mdr-tb). megazine of medicine indonesia. 2010;12. 8. tupasi te, garfin amcg, kurbatova e v, mangan jm, orillaza-chi r, naval lc, et al. factors associated with loss to follow-up during treatment for multidrug-resistant tuberculosis, the philippines, 2012-2014. emerg infect dis. 2016 mar;22(3):491–502. 9. mekonnen f, tessema b, moges f, gelaw a, eshetie s, kumera g. multidrug resistant tuberculosis: prevalence and risk factors in districts of metema and west armachiho, northwest ethiopia. bmc infect dis. 2015 dec 26;15(1):461. 10. dobler cc, korver s, batbayar o, nyamdulam b, oyuntsetseg s, tsolmon b, et al. multidrug-resistant tuberculosis in patients for whom first-line treatment failed, mongolia, 2010-2011. emerg infect dis. 2015 aug;21(8):1451–4. 11. mulisa g, workneh t, hordofa n, suaudi m, abebe g, jarso g. multidrug-resistant mycobacterium tuberculosis and associated risk factors in oromia region of ethiopia. int j infect dis. 2015 oct;39:57–61. 12. m a, salih, a m, merza. risk factors for multi-drug resistant tuberculosis: a review. duhok med j. 2010;4:1–7. 13. marahatta sb, kaewkungwal j, ramasoota p, singhasivanon p. risk factors of multidrug resistant tuberculosis in central nepal: a pilot study. kathmandu univ med j. 2012 jun 5;8(4). 14. lomtadze n, aspindzelashvili r, janjgava m, mirtskhulava v, wright a, blumberg hm, et al. prevalence and risk factors for multidrugtable 2. relationship of category gender, treatment history and knowledge in anti tuberculosis drugs resistance on tb patients in hospitals labuang baji tahun 2017 variables anti tuberculosis drug resistance pnegative positive amount n % n % total % gender man women 2 1 2.7 1.4 23 34 38.4 57.5 25 35 41.7 58.3 p = 0,56 treatment history yes no 1 2 1.7 3.3 53 4 88.3 6.7 54 6 90.0 10.0 p = 0.024 knowledge good less 3 0 5.0 0 42 15 70.0 25.0 45 20 75.0 25.0 p = 0.566 44 indonesian journal of tropical and infectious disease, vol. 7 no. 2 may–august 2018: 40–44 resistant tuberculosis in the republic of georgia: a population-based study. int j tuberc lung dis. 2009;13(1):68–73. 15. elmi os, hasan h, abdullah s, mat jeab mz, bin alwi z, naing nn. multidrug-resistant tuberculosis and risk factors associated with its development: a retrospective study. j infect dev ctries. 2015 oct 29;9(10):1076. 16. fregona g, cosme lb, moreira cmm, bussular jl, dettoni v do v, dalcolmo mp, et al. risk factors associated with multidrug-resistant tuberculosis in espírito santo, brazil. rev saude publica. 2017;51. 17. daniel o, osman e. prevalence and risk factors associated with drug resistant tb in south west, nigeria. asian pac j trop med. 2011 feb;4(2):148–51. 18. espinal ma, laserson k, camacho m, fusheng z, kim sj, tlali e, et al. determinants of drug-resistant tuberculosis: analysis of 11 countries. int j tuberc lung dis. 2001;5(10):887–93. 19. faustini a. risk factors for multidrug resistant tuberculosis in europe: a systematic review. thorax. 2006 feb 1;61(2):158–63. 20. günther g, van leth f, alexandru s, altet n, avsar k, bang d, et al. multidrug-resistant tuberculosis in europe, 2010-2011. emerg infect dis. 2015 mar;21(3):409–16. 21. andrade ss, sader hs. antimicrobial resistance in developing countries. springer. 2010;249. 22. sb m. risk factors of multidrug resistant tuberculosis in central nepal. kathmandu universi ty med j. 2010;32:392–7. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 150 vol. 7 no. 6 september-december 2019 detection of helicobacter pylori infection in chronic gastritis biopsy specimen using warthin-starry and modified giemsa stain in dr soetomo hospital surabaya willy sandhika 1a 1 department of pathology, faculty of medicine universitas airlangga/ dr. soetomo general hospital surabaya, indonesia a corresponding author: willysand@fk.unair.ac.id abstract helicobacter pylori is a bacteria that commonly cause chronic gastritis. identification of its infection is essential for eradication treatment. detection of h.pylori bacteria in gastric biopsy specimen by histology method is a diagnostic tool that widely accepted because it is superior to serology examination. although the bacteria can be seen in routinely hematoxylin-eosin staining, modifiedgiemsa and whartin-starry stain was commonly used to identify the bacteria more clearly. whartin-starry stain gives more contrast to the bacteria but modified-giemsa stain is preferable at many centres because it is a cheaper and simple method. this study aim is to explore the differences of two stain method to identify h.pylori. paraffin blocks from gastric biopsy patients with chronic gastritis in the year 2017 were retrieved from anatomic pathology laboratory dr.soetomo hospital surabaya. thirty paraffin blocks were taken randomly and were made into microscopic slides for staining with warthin-starry and modified-giemsa stain concomitantly. specimen with whartin-starry stain found 19 out of 30 were positive for h.pylori while in modified-giemsa stain only 16 out of 30 specimen were positive for h.pylori. detection of h.pylori warthin-starry stain give more chance to obtain positive result because it use silver technique that coat the bacteria making it is more clearly visible in microscopic examination. keywords: h.pylori detection, h.pylori infection, warthin-starry, modified giemsa, chronic gastritis. abstrak helicobacter pylori adalah bakteri penyebab gastritis khronis yang umum dijumpai. identifikasi infeksi h.pylori diperlukan untuk acuan terapi eradikasi. deteksi h.pylori pada spesimen biopsi gaster dengan metode histopatologi merupakan teknik diagnostik yang diterima secara umum mengingat teknik ini lebih akurat dibandingkan dengan pemeriksaan serologi. walaupun bakteri h.pylori dapat terlihat pada pengecatan rutin hematoxylin-eosin, akan tetapi umumnya digunakan pengecatan tambahan modified-giemsa atau whartin-starry untuk melihat bakteri dengan jelas. pengecatan modified-giemsa lebih disukai di banyak sentra laboratorium oleh karena lebih murah dan lebih mudah dikerjakan akan tetapi pengecatan whartin-starry dapat melihat bakteri lebih jelas dengan kontras yang lebih baik. penelitian ini bertujuan untuk membandingkan adanya perbedaan hasil identifikasi h.pylori pada kedua jenis pengecatan tersebut. blok parafin biopsi lambung diambil dari pasien dengan gastritis kronis yang diperiksa di laboratorium patologi anatomik rumah sakit dr.soetomo surabaya pada tahun 2017. tiga puluh blok parafin diambil secara acak untuk dibuat slide mikroskopis dan dilakukan 2 jenis pewarnaan yakni warthin-starry dan modified-giemsa. pada pengecatan whartin-starry bakteri h.pylori terdeteksi pada 19 dari 30 spesimen sedangkan pada pengecatan modified-giemsa hanya 16 dari 30 spesimen yang menunjukkan adanya h.pylori. deteksi infeksi h,pylori dengan pengecatan whartin-starry memberikan hasil positif yang lebih banyak dibandingkan dengan pengecatan modified-giemsa. pereaksi perak pada reagen whartin-starry dapat membuat bakteri menjadi terlihat lebih jelas pada pengamatan mikroskopis. kata kunci: deteksi h.pylori, infeski h.pylori, pewarnaan warthin-starry, pewarnaan modified giemsa, gastritis kronis. research report 151sandhika.: detection of helicobacter pylori infection introduction h.pylori is a bacteria that closely related with chronic gastritis and dyspepsia. chronic gastritis case and dyspepsia are commonly found in daily practice. beside causing chronic gastritis, h.pylori infection plays some important role in the presence of gastric malignancy either in the form of gastric carcinoma or gastric lymphoma.1,2 according to the american college of gastroenterologists guidelines on management of h.pylori infection3, the diagnostic method h.pylori infection detection comprises of urea breath test and gastric endoscopic biopsy. serology method should be avoided. nevertheless if the serologic test gave positive results, it should be confirmed with a test that identify an active infection such as the urea breath test or stool antigen test. urea breath test, stool antigen test, histology examination with special staining for h.pylori organisms, and bacteria culture are considered to be the gold standard tests for diagnosis of h.pylori infection.4,5 culture is not routinely used for initial diagnosis of h.pylori infection but is required for antibiotic susceptibility testing if physicians suspect antibiotic resistance in patients who have previously failed therapy.5 for stool antigen test, mayo medical laboratories utilizes the pocone infrared spectrophotometer. performance characteristics for this instrument have not been established for persons under age 3. for patients 3 to 17 years, age, weight and height must be included in test request for appropriate result interpretation.6 the gold standard diagnostic test for h.pylori infection is to find the bacteria through direct smear or h.pylori culture.4 cross reaction with other antigens often encountered in serology test with posibly to obtain false positive result. several staining methods of biopsy specimen are proposed to detect h.pylori infection, usually start with routinely stain hematoxylin-eosin to more specific stain using monoclonal antibody for immunohistochemistry testing.7 whartin-starry stain is used mainly to detect spirochetes bacteria with silver impregnation methods. whartin starry has some advantage compare to other methods because it gives more contrast color (bacteria stained black in yellow background).8 this method can improve sensitivity of its detection. on the other hand, giemsa stain is a common method for examining blood smear. in tissue, giemsa stain can detect microorganism that appears dark blue in pinkpale blue background. a modification of giemsa stain was proposed to reduced the staining time and it was known as modified-giemsa technique.9 modified-giemsa stain is a non-silver stain that commonly use to detect h pylori infection in many health centre including dr.soetomo general hospital. the staining methods are quite simple and the reagent is easy to obtain and cheap. sometimes it fails to detect microorganism due to lack of contrast in dirty background. it must be used by experienced experts. there are still a debate which stain should be used to detect h. pylory as a routine procedure.10 this study aim is to compare the result of these two staining methods for detection of h.pylori infection in gastric biopsy specimens and to find out if there is difference result of h.pylori identification in gastric biopsy tissue using whartin–starry stain compared to modified-giemsa stain. material and method thirty paraffin blocks from patients who diagnosed clinically as chronic gastritis were retrieved from anatomic pathology laboratory archives at dr soetomo hospital surabaya in the year 2017. two microscopic slides were made from each paraffin blocks by slicing the tissue 6 μm thick and placed on object glass. the first slide was stained with modified-giemsa technique according to bancroft9 which have done routinely in the laboratory while the other slides stained with warthin-starry for spirochetes (bio-optica cat no.04-040903). in modified-giemsa stain, the h.pylori bacteria were identified as a reddish purple microorganism in the background of other cells that was stained blue and pale-blue. warthin-starry stain give more contrast colour. the bacteria were stained black while the background cells are stained yellow. the two microscopic slides were assessed by one pathologist and h.pylori infection scoring was made according to updated sidney classification system.11 h.pylori infection in gastric tissue biopsy were score as follows: score +1 when there were sparse bacteria found in specimen (mild density of bacteria), score +2 when there were some bacteria (moderate density) and score +3 when there were many bacteria in tissue (marked bacteria were found).12 this study has been approved by health-research ethical commission in dr. soetomo general hospital. result and discussion on examination of 30 gastric biopsy specimens with warthin-starry stain, 19 tissue biopsies were found positive for h.pylori bacteria while specimens with modifiedgiemsa stain only 16 biopsies tissue were found positive for h.pylori (table 1). table 1. comparison of h.pylori bacteria examination with warthin-starry and modified-giemsa stain warthin-starry with positive bacteria warthin-starry with negative bacteria modified-giemsa with positive bacteria 16 0 modified-giemsa with negative bacteria 3 11 152 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 150–154 detection of h.pylori in gastric biopsy specimen has been accepted worldwide to diagnose h.pylori infection. culture method is gold standard for infection detection but h.pylori culture did not routinely used due to its complexity and need a longer period to obtain the result.4 until this date, majority of microbiologic laboratory in the world are not equipped to perform h.pylori culture. instead of culture, pcr technique for detection of h.pylori may be considered as a gold standard, but pcr technique also has a limitation due to genetic sharing of other microbes which can gives false positive result and the presence of pcr inhibitors can give false negative result in specimen with low bacterial count.13 histological examination from gastric biopsy specimen has been a method of choice for identifies h.pylori infection. there are several staining methods that can be used to identify the presence of the bacteria which can be classified into two groups: silver-based stain and common histochemistry stain. silver-based stain such as whartin-starry stain has an advantage over other stain since it can detect h.pylori bacteria even if its morphology has been altered by proton pump inhibitor and antibiotic administration, which can alter the bacteria morphology to cocoid and short bacillary forms. proton pump inhibitor administration can cause h.pylori migrate into deeper portion of oxyntic glands, making its detection is imposible without a silver-based or immunohistochemical stain.14 modified-giemsa stain is a histochemistry stain that has been performed in lots of laboratory because it is cheap and simple method. the lack of contrast is a disadvantage of this technique. silver-based stain such as h.pylori silver stain and whartin-starry stain give more clearly visible bacteria. so it can be detected easily on histological examination. the disadvantage of this technique it is more expensive than modified-giemsa stain and it takes longer period to do the staining protocol.15 immunohistochemistry with specific antibody has been proposed to be used as a gold standard to detec h.pylori infection.8 but according to the study of patel, et al. there is no difference result for h.pylori detection by immunohistochemistry technique compared with modifiedgiemsa stain. the limitation of immunohistochemistry technique is more expensive than any other stain including whartin-starry and it took longer period than any other stain. it also needs a control specimen to be done with every slide making it is more complicated.13 table 1 is showed that there were 3 cases of h.pylori infection detected by warthin-starry stain which did not detected by modified giemsa stain. the discordance occurs due to lack of contrast between the color of micro-organism and background color. using whartin-starry stain one can easily direct to get the presence of the bacteria because it provides black color of bacteria in yellow background. furthermore, the silver reagent in whartin-starry stain gives a good result for detecting h.pylori because the organism are coated with the silver stain and therefore look larger, making their identification easier.15 assessment of h.pylori infection in gastric biopsy specimen has performed according to updated sidney system scoring. the method is to detect h.pylori bacteria throughout entirely biopsy specimen and making the score by counting the number of bacteria in one high power field which give most number of bacteria.11 the result of h.pylori scoring according to updated sidney system was presented in figure 1. figure 1. result of h.pylori bacteria examination with warthinstarry dan modified giemsa stain. score 0 = no bacteria detected, score +1 = only sparse bacteria detected, score +2 = moderate bacteria count detected, score +3 = many bacteria detected. figure 1 is showed that warthin-starry stain gives more results of identified bacteria compared to modified-giemsa stain. three cases show h.pylori score 0 on modified giemsa staining (no bacteria detected) while these cases show sparse h.pylori bacteria (score +1) on warthin-starry staining. there were also 1 case which gives h.pylori score +1 with modified giemsa staining while it gives score +2 with warthin-starry staining. h.pylori infection can be easily detected with whartinstarry stain compare to that of modified giemsa stain either in low density or high density bacteria as shown at figure 2 and figure 3. figures 2 and figure 3 are showed various h.pylori score in gastric biopsy specimen. this study was showed that there were some cases when the presence of h.pylori infection is either undetectable or detectable in smaller amounts in modified-giemsa stain compare to whartinstarry stain. in warthin-starry stain the spirochete bacteria wall were react with silver nitrate impregnation so that the bacteria will appear black within a yellow background. according to glickman, h.pylori bacteria can not be detected if it present in a small number.16 the gold standard diagnosis of h.pylori infection in gastritis is made by finding h.pylori bacteria in gastric tissue. gastric tissue specimens are generally obtained by endoscopic biopsy techniques. this technique is a minimally invasive but it can see directly at the morphology of the gastric tissue followed by tissue endoscopic biopsy for pathology examination.17 the presence of h.pylori bacteria in gastric biopsy can be detected by culture, pcr technique and h.pylori 153sandhika.: detection of helicobacter pylori infection figure 2. gastric biopsy specimen with high density of h.pylori bacteria (+3). specimen stianed with whartin-starry (left) and modified giemsa (right). thebacteria were found on gastric mucous layer ( ) (400 x magnification). figure 3. gastric biopsy specimen with low density of h.pylori bacteria (+1). specimen stianed with whartin-starry (left) and modified giemsa (right). thebacteria were found on gastric mucous layer ( ) (400 x magnification). visualization techniques with histochemical stain to indentify directly h.pylori by light microscope.7 the histochemical technique of gastric biopsies has an advantage over culture methods because it can directly see h.pylori in a relatively simple and faster way. therefore, culture methods are not commonly used for h.pylori detection. histochemical staining techniques are also easier to perform and have less cost than pcr molecular techniques. the histochemical technique has become the standard diagnostic of h.pylori infection in many health centers.4 routine hematoxyllin-eosin staining has been used in h. pylory examination but in several cases the bacteria can not be visualized without special stain.18 histochemical special stain for h.pylori detection include gimenez, toluidine blue, romanowski, genta and giemsa stain which can be used for visualization of with various modifications (modified-giemsa) for a more rapid workup time.4 silver-based stain such as warthin-starry stain has advantage compare to common histochemistry stain since it uses silver impregnation technique to give black color to h.pylori bacteria. in warthin-starry, h.pylori bacteria will appear dark brown, making them easier to see and can increase the sensitivity of h.pylori detection in gastric biopsy tissue.14 this study was performed on gastric endoscopic biopsy specimens in patients with chronic gastritis. in this study, 19 samples (63.33%) were detected as h.pylori positive on warthin-starry stain from 30 biopsy specimens of chronic gastritis patients. adlekha, et al reported h.pylori infection in 329 of 530 (62%) chronic gastritis patients in kerala india.19 the positivity of h.pylori from india is similar to this study. adlekha took a gastric biopsy specimen from patients with dyspepsia complaints as many as 530 patients in 2010 2012. on microscopic slides biopsy material performed hematoxylin-eosin and modified-giemsa staining. h.pylori examination was performed by a pathology specialist and the results were 154 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 150–154 presented in grading (mild, moderate and severe) infections in accordance with the updated sydney grading system.11 immunohistochemistry technique using monoclonal antibody anti-h.pylori can gives more specific result. however, this technique rarely be done in many pathology laboratory due to higher cost of antibody-based detection.20 according to rodger haggit recommendation: immunohistochemistry stain should not be use as a routine procedure. it was only performed in gastric biopsy specimen with chronic inflammation with negaive finding on h.pylori.21 detection of h.pylori bacteria by special stain has been served as a routinely h.pylori detection in tissue specimen.13 the results of this research showed difference of h.pylori detection due to increased sensitivity detection by warthin-starry stain compared to modified-giemsa stain. warthin-starry was found more specimens with positive result than modified-giemsa stain in 3 out of 30 cases. the presence of h.pylori that is undetected by the observer is largely due to lack of color contrast in modified-giemsa stain. bacteria are missed from observation in h.pylori infection with mild intensity due to the unclear color. in infections with moderate to severe intensity, this result found 100% concordance result of h.pylori stained with modified-giemsa compared to warthin-starry. conclusion a study has been conducted to find the difference of helicobacter pylori detection in gastric biopsy tissue with warthin-starry and modified giemsa staining. detection of h.pylori warthin-starry stain give more possibility to obtain positive result because it use silver technique that coat the bacteria making it is more clearly visible in microscopic examination. acknowledgement this research was funded by 2017 dr soetomo hospital research grant. the author thanks gilda hartecia for her help in writing assistance of proposal manuscript. conflict of interest. there is no conflict of interest for this research. the manuscript has not been published previously. references 1. watari j, chen n, amenta ps, fukui h, oshima t, tomita t, et al. helicobacter pylori associated chronic gastritis, clinical syndromes, precancerous lesions, and pathogenesis of gastric cancer development. world j gastroenterol. 2014; 20(18): 5461–5473. 2. barbosa aja. detection of h.pylori in endoscopic gastric biopsies: a routine research that goes far beyond the laboratory limits. j brasileiro de patologia e medicina lab. 2015;51(2):70-71. 3. chey wd, leontiadis gi, howden cw, moss sf. acg clinical guideline:treatment of helicobacter pylori infection. am j gastroenterol 2017 feb; 112: 212–238. 4. talebi bezmin abadi a. diagnosis of helicobacter pylori using invasive and noninvasive approaches. j pathog. 2018 may 22; 2018:9064952. doi: 10.1155/2018/9064952.. 5. garza-gonzález e, perez-perez gi, maldonado-garza hj, bosquespadilla, fj. a review of helicobacter pylori diagnosis, treatment, and methods to detect eradication. world j gastroenterol. 2014 feb; 20(6): 1438–1449. 6. shimomaya t. stool antigen tests for the management of helicobacter pylori infection. world j gastroenterol. 2013 dec; 19(45): 81888191 7. ramis ib, de moraes ep, fernandes ms, mendoza-sassi r, rodrigues o, juliano crv, et al. evaluation of diagnostic methods for the detection of helicobacter pylori in gastric biopsy specimens of dyspeptic patients. braz j microbiology. 2012; 43(3):903–908. 8. lee jy and kim n. diagnosis of helicobacter pylori by invasive test: histology. ann transl med. 2015; 3(1):10-17. 9. morris gb, ridgway ej, suvarna sk. traditional stains and modern techniques for demonstrating microorganisms in histology. in: suvarna sk, layton c, bancroft jd, editors. bancroft’s theory and practice of histological techniques 8th ed. philadelphia: churchill livingstone/elsevier; 2013, p.263. 10. smith sb, snow an, perry rl, qasem sa. helicobacter pylori: to stain or not to stain? am j clin pathol. 2012; 137:733-738. 11. sipponen p, price ab. the sydney system for classification of gastritis 20 years ago. j gastroenterol hepatol. 2011 jan;26 suppl 1:31-4. 12. rugge m, pennelli g, pilozzi e, fassan m, ingravallo g, russo vm, di mario f. gastritis: the histology report. dig liver dis. 2011 mar;43 suppl 4:s373-84. 13. patel sk, pratap cb, jain ak, gulati ak, nath, g. diagnosis of helicobacter pylori: what should be the gold standard? world j gastroenterol. 2014 sep; 20(36): 12847–12859. 14. genta rm and lash rh. helicobacter pylori-negative gastritis: seek, yet ye shall not always find. am j surg pathol. 2010; 34(8):e25e34. 15. farouk wi, hassan nh, ismail tr, daud is, mohammed f. warthinstarry staining for the detection of helicobacter pylori in gastric biopsies. malays j med sci. 2018;25(4):92–99. 16. glickman jn, noffsinger a, nevin dt, ray m, lash rh, genta rm. helicobacter infections with rare bacteria or minimal gastritis: expecting the unexpected. dig liver dis, 2015; 47(7):549-55. 17. wang yk, kuo fc, liu cj, wu mc, shih hy, wang ssw, et al. diagnosis of helicobacter pylori infection: current options and developments. world j gastroenterol. 2015 oct; 21(40): 11221– 11235. 18. versalovic j. helicobacter pylori: pathology and diagnostic strategies. am j clin pathol. 2003 mar;119(3):403-12. 19. adlekha s. chadha t, krishnan p, sumangala b. prevalence of helicobacter pylori infection amongst patients undergoing upper gastrointestinal endoscopy in a medical college hospital in kerala, india. ann med heatlh sci res. 2013 oct-des; 3(4): 559-563. 20. wang xi, zhang s, abreo f, thomas j. the role of routine immunohistochemistry for helicobacter pylori in gastric biopsy. ann diagn pathol. 2010 aug;14(4):256-9. 21. batts kp, ketover s, kakar s, krasinskas am, mitchell ka, wilcox r, et al. appropriate use of special stains for identifying helicobacter pylori: recommendations from the rodger c. haggitt gastrointestinal pathology society. am j surg pathol. 2013 nov;37(11):e12-22. 109 vol. 7 no. 5 may-august 2019 overview of nuclear factor-kb (nf-kb) and nonstructural protein 1 (ns1) in patients with dengue fever in premier hospital, surabaya ni nyoman budiutari1a, yoes prijatna dachlan2, dan jusak nugraha3 1department of immunology postgraduate school, universitas airlangga, surabaya, east java, indonesia 2departement of parasitology, faculty of medicine, universitas airlangga surabaya, east java, indonesia 3departement of patology clinic, faculty of medicine, dr. soetomo hospital, surabaya, east java, indonesia acorresponding author: budiutari2212@gmail.com abstract dengue fever (df) is an acute viral fever caused by rna virus that is transmitted by aedes aegypti and aedes albopictus mosquitoes. df is also called viral arthropod-borne disease and is accompanied by headaches, joint and muscle pain. the main target of dengue infection is macrophages or monocytes and dendritic cells (dc). infected dc is caused the viral replication and the endocytosis into endosomal, easier, thus inducing the activation of nf-ĸb transcription factor to produce proinflammatory cytokines such as tumor necrosis factor-α (tnf-α), interleukin-1 (il-1), il-6, il-12 and chemokine. nf-kb is one of the transcription factors involved in the regulation of the expression of various cytokines, chemokines and anti/pro-apoptotic proteins during infection and act as indicator of disease severity. infected dc cells are secreted ns1 protein which is the co-factor needed for viral replication and can be detected in the first eight days. the level will be higher in the initial phase of fever. the purpose of this study was to analyze the description of nf-kb and ns1 levels in the serum of patients with dengue fever through observational analytic studies through a cross-sectional approach. this study was done on 40 patients with dengue fever and 10 healthies people as negative controls. ns1 was analyzed in serum of panbio rapid test and nf-kb level were measured by sandwich elisa. the results are showed positive and negative ns1 results in dengue fever patients. the average nf-kb serum level in dengue fever patients was found to be higher than the control. nf-ĸb level in negative ns1 was higher than the ns1 positive group. it is showed that ns1 is detected both in the acute phase. the detection of nf-ĸb is showed the involvement of transcription factors in the development of dengue virus infection and has a protective role for host cells. keywords: dengue fever, nuclear factor-ĸb, ns1 protein, viral infection, tropical disease. abstrak demam dengue (dd) merupakan penyakit demam virus akut yang disebabkan oleh virus rna, ditularkan oleh nyamuk aedes aegypti dan aedes albopictu. dd disebut juga sebagai penyakit arthropod-borne viral dan disertai sakit kepala, nyeri otot dan sendi. target utama infeksi dengue adalah makrofag/monosit dan sel dendritik (dc). dc yang terinfeksi akan mempermudah replikasi virus dan endositosis ke dalam endosomal, dimana hal tersebut dapat menginduksi aktivasi faktor transkripsi nf-ĸb untuk menghasilkan sitokin proinflamasi seperti tumor necrosis factor-α (tnf-α), interleukin-1 (il-1), il-6, il-12 dan kemokin. nf-kb merupakan salah satu faktor transkripsi yang terlibat dalam regulasi ekspresi berbagai sitokin, kemokin, protein anti/pro-apoptosis selama infeksi dan menjadi tanda tingkat keparahan penyakit. sel dc yang terinfeksi akan mengsekresikan protein ns1 yang merupakan co-factor yang diperlukan untuk replikasi virus. ns1 terdeteksi baik dalam delapan hari pertama serta kadarnya akan lebih tinggi pada fase awal demam. tujuan penelitian ini adalah menganalisis gambaran kadar nf-kb dan ns1 pada serum pasien demam dengue melalui studi observasional analitik melalui pendekatan cross sectional. pada penelitian ini menggunakan 40 sampel penderita demam dengue dan 10 sampel orang sehat sebagai control negatif. ns1 dianalisa pada serum menggunakan panbio rapid test dan kadar nf-kb menggunakan sandwich elisa. hasil menunjukkan pada pasien demam dengue ditemukan hasil ns1 positif maupun negatif. rerata kadar nf-kb serum pada pasien demam dengue ditemukan lebih tinggi daripada kontrol dan kadar nf-ĸb pada ns1 negatif lebih tinggi dibandingkan research article 110 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 109–114 kelompok ns1 positif. hal ini menunjukkan bahwa ns1 terdeteksi baik pada fase akut dan terdeteksinya nf-ĸb menunjukkan adanya keterlibatan faktor transkripsi pada perkembangan infeksi virus dengue dan mempunyai peran proteksi untuk sel host. kata kunci: demam dengue, nuclear factorĸb, protein ns1, infeksi virus, penyakit tropis. introduction dengue is an endemic disease caused by rna viruses which transmitted by aedes aegypti and aedes albopictus mosquitoes.1 this disease is endemic throughout tropical and subtropical regions which are affected by rainfall, temperature and unplanned urbanization.2 world health organization (who) is recorded from 1968 to 2009 indonesia as the country with the highest dhf cases in southeast asia. in indonesia, it was first discovered in the city of surabaya in 1968 with 58 people infected and 24 people dead. in 2015 there were 126,675 sufferers in 34 provinces in indonesia.3 in surabaya in 2015 there was an increase in the number of cases by 46%. dengue fever (dd) is an endemic disease which is also called viral arthropod-borne disease. the first infection to the host’s body raises various pathological levels, ranging from asymptomatic mild symptoms (dengue fever), dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss).4 dengue is classified as a pediatric disease in southeast asia, but at this time dengue patients are recorded not only in children but also in adults.5 dengue virus (denv) is a genus of flavivirus in the family flaviviridae which has four different antigenic serotypes namely denv-1, denv-2, denv-3 and denv4.6 the denv genome consists of positive single-stranded rna with a length of about 11 kb which encodes ten proteins, ie no protein structural capsules (c), envelope (e), pre-membrane (prem) and seven non-structural proteins (ns1, ns2a, ns3, ns4, ns4a, ns4b, ns5).2 non-structural 1 (ns1) protein is an important glycoprotein and a co-factor needed for viral replication, although its role in replication is largely unknown. ns1 is detected during the acute phase and will slowly decrease to an undetectable level on days 5-6. the presence of this protein is associated with the severity of the disease and the progression towards dhf.6 the main target in dengue infection is towards macrophages/monocytes and dendritic cells.7 dendritic cells were infected with dengue virus will facilitate viral replication and endocytosis to endosomal, which can induce activation of the nf-ĸb transcription factor.8 nf-ĸb is a transcription factor that is involved in the regulation of gene expression to encode cytokines, chemokines, proproteins and antiapoptosis. activation of nf-ĸb during viral infection is explained as a host protective response to pathogens. nf-ĸb is a weapon used by hosts to control viruses, but viruses can use them differently to block apoptosis and increase viral replication. activation of the nf-ĸb pathway produces various cytokines such as tumor necrosis factor-a (tnf-α), interleukin-1 (il-1), il-6, il-12. nf-ĸb is one of the most widely exploited pathways for gene regulation by denv, but how the role of the nf-ĸb pathway in dengue pathogenesis is not completely clear.9 this study is based on the picture of nf-ĸb levels and ns1 protein in serum of dengue fever patients and controls, where an increase in nf-ĸb levels is indicated as a sign that a more severe prognosis is dengue shock syndrome (dss). material and method study population this study was done in the institute of tropical diseases, universitas airlangga on august 2018. the study was included 40 patients with dengue fever and 10 healthy people as control who were selected from surabaya premier hospital. patients who were selected were in accordance with the inclusion criteria in patients with clinical symptoms such as that had onzet fever day 1–4 and without age limitation. patients who had only clinical symptoms of dengue, but dengue infection had not found in diagnosis laboratory were excluded because of the difficulty of evaluating the extent of the disease. the control group was included 10 apparently healthy subject with comparable age characteristics, no positive history of dengue infection disease, no fever for 1 month prior to the study. the clinical disease severity was classified according to the 2011 world health organization (who) dengue diagnostic criteria.10 patients with sharp temperature and is frequently associated with a flushed face and headache, the body temperature is between 39-40°c and lasting 5-7 days in the majority of cases and the other common symptoms including anorexia were classified as df. patients with increase of hematocrit greater than 20% compare with the baseline hematocrit or clinical/ultrasound scan evidence of plasma leakage were classified as having dhf. shock was defined as having cold clammy skin, along with a narrowing of pulse pressure of 20 mmhg. according to the who 2011 disease classification, 40 patients were classified as df. ethics statement ethics approval was obtained by ethical review committee at faculty of dental medicine, universitas airlangga. all patients were recruited following informed written consent. blood samples samples were collected into tubes. serum from patients was obtained after centrifugation for 10 minutes at 10,000 111budiutari, et al.: overview of nuclear factor-kb (nf-kb) and non-structural protein 1 (ns1) rpm. then the samples were stored and frozen at -80°c until used. laboratory diagnosis dengue serum ns1 were detected using panbio rapid test (immunochromatography) from panbio dengue early rapid test with positive and negative qualitative results. nf-ĸb levels were measured using the human nf-ĸb p65 sandwich-elisa kit from elabscience followed procedures in the protocol. this is a commercial enzymelinked immunosorbent assay for detecting nf-ĸb level against dengue virus in human serum or plasma.11,12 statistical analysis in this study, were we analyzed the levels of nf-ĸb and ns1 in patients with dengue fever are determined clinically. we are use the mann-whitney test to see the difference between ns1 positive and ns1 negative in patients with dengue fever. statistic package for social sciences (spss) was used for data entry, processing and statistical analysis at the end of the study. p-values less than 0.05 were considered significant. result and discussion study population a total of 40 patients suffering from dengue fever and fulfilling the inclusion criteria were enrolled in the study. serum ns1 examination results from 40 patients found 30 patients with positive ns1 and 10 ns1 patients negative. as a control, 10 healthy people without dengue infection with various ages. table 1. age and gender distribution of the participants (n=50) control (n=10)/% ns1 positive (n= 30)/% ns1 negative (n=10)/% total /% age (mean±sd) 4.66±1.722 age group 0-5 0/ 0 1/ 3.3 0/ 0 1/ 2.0 6-11 0/ 0 2/ 6.7 1/ 10 3/ 6.0 12-16 1/ 10 5/ 16.7 0/ 0 6/ 12.0 17-25 8/80 8/ 26.7 3/ 30 19/ 38.0 26-35 1/ 10 5/ 16.7 1/ 10 7/ 14.0 36-45 0/ 0 4/ 13.3 2/ 20 6/ 12.0 46-55 0/ 0 2/ 6.7 2/ 20 4/ 8.0 56-65 0/ 0 2/ 6.7 1/ 10 3/ 6.0 >65 0/ 0 1/ 3.3 0/ 0 1/ 2.0 total 10/100 30/ 100 10/ 100 50/ 100 gender male 3/30 13/14.3 6/60 22/44 female 7/70 17/56.7 4/40 28/56 total 10/100 30/100 10/100 50/100 table 1 gives the age and gender distribution of the participants. the age grouping used was based on the ministry of health (2009) (mean 4.66 and sd = 1.722). majority of the dengue fever were between the age group of 17 to 25 years (38.0%), the lowest were toddlers (0-4 years) and elderly (> 65 years old), namely 3.3%. there were 28 (56.0%) males and 22 (44.0%) females with various age, the females was found more than males. ns1 serum ns1 was examined in the serum of dengue-infected patients collected from days 1-4 of the onset of fever and also in healthy controls. the method used for ns1 examination uses the panbio rapid test of the 40 serum samples of dengue fever patients, 30 ns1 samples were positive and 10 ns1 samples were negative. in 10 controls ns1 was negative. examination the levels of nf-ĸb nf-ĸb levels were determined in serum samples in dengue fever patients collected from fever patients day 1 to 4 onsets of fever. the level of nf-ĸb in serum from negative control is very low (table 2). nf-ĸb levels in serum from dengue fever patients were found to be high (>10 ng/ml); higher than the nf-ĸb level of control in the negative control. however, the average emission in the group of patients with negative ns1 (mean=13.165) was found to be higher compared to patients with ns1 positive (mean=10.013), but there were no significant differences in nf-ĸb levels from 40 patients (figure 1). in the negative control sample, there was low nf-ĸb level with mean value of 1.646. nfĸb levels tend to increase linearly in patients of all clinical values when the disease develops, but there is no significant difference between cases of dengue fever with ns1 positive and ns1 negative (p=0.187) (ρ>0.05). table 2. nf-ĸb in dengue fever patient control (n=10)/% ns1 positive (n= 30)/% ns1 negative (n=10)/% total/% nf-ĸb (mean ± sd) 1.646 ± 1.294 10.013 ± 4.808 13.165 ± 5.805 9.002±5.919 0,1–5,0 ng/ml 10/100 2/6.7 0/0 12/24 5,1–10 ng/ml 0/0 14/46.7 3/30 17/34 >10 ng/ml 0/0 14/46.7 7/70 21/42 total 10/100 30/100 10/100 50/100 the average emission value of nf-ĸb levels in positive ns1 was found to be higher than in the negative ns1 group. the highest nf-ĸb levels were found to reach 25000 ng/ ml. in the box plot the control group is at the minimum value of the dengue fever group. 112 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 109–114 discussion our results were showed that in patients with dengue fever did not always show positive results on ns1 examination in serum, this was clear in this study of 40 samples of dengue fever patients found 30 patients with ns1 positive results and 10 patients with ns1 negative. the results in nf-ĸb showed that the mean levels in serum of dengue fever patients were higher compared to controls. homogeneity of the sample in this study was carried out by limiting the duration of fever patients with days 1-4 fever. this restriction is based on a theory which states that in dengue fever patients will experience a fever phase for 2-7 days and on days 1-4 is the first fever phase where fever will appear high enough to 40°c and is the time well in the diagnosis of dengue fever where the antigen from the dengue virus can be detected at a higher level in the acute phase or in the first eight days.13,14 dengue diagnosis is difficult to enforce at the beginning of the disease because the signs and symptoms are not specific so it is often difficult to distinguish from influenza virus infection, measles and typhoid fever. viremia or the dengue virus in the bloodstream will last for 1 week. ns1 examination is an examination that detects the body parts of the virus and does not wait for the body’s response to infection, so the best time to do the best examination is day 0 to day 4 and can be detected before the decline in platelets.13 in our study there were several patients with negative ns1 results when examined using a panbio rapid test. in, patients with dengue fever 1-4 days with ns1 positive results show that there is a dengue infection in the body. conversely, patients dengue fever with ns1 negative do not close the possibility of dengue infection but ns1 is detected at low levels causing false negative and further examination is needed. to detect the viral protein is needed sufficient levels of the amount of virus circulating, while in the initial phase there are not enough viruses, but if it takes the sample after the appearance of antibodies the level of dengue virus will also decrease.15 dengue virus (denv) has 4 serotypes denv-1, denv-2, denv-3, and denv-4. this can cause the emergence of negative results on the examination of ns1 in patients with dengue fever is suspected to be associated with the dengue virus serotype that infects. it recommends that ns1 examination must be repeated on the seventh day later to provide more valid results.16 ns1 is associated with the duration of the disease and is less sensitive to illness that lasts over 3 days or the level detected will decrease. they associate ns1 with the severity of the disease and the risk of progression leading to dengue hemorrhagic fever (dhf).6 ns1 is a glycoprotein secreted by cells infected with the dengue virus, mainly in the serum supernatant of infected patients, but not in the virus.17 ns1 is found in primary and secondary infections and can be detected in the first eight days of fever and levels will be high at the start of fever and decrease before defervescence and become negative in some cases. in tambunan et al. they found that ns1 antigen sensitivity was higher on the third day of fever.18 variable antigen levels were showed that ns1 secretion is not persistent (stable). the results of research by blacksell and dussart stated that ns1 which binds to soluble endothelium (soluble) and collected in the hepatocyte causes circulating ns1 secretion of blood to become slow.19,20 this can affect the level of ns1 detected in the blood.14 ns1 is found in high concentrations in patients with severe degrees.17 as a reaction from the host to a viral infection that occurs in the body is the activation of one of the transcription factors namely nf-ĸb which will secrete various cytokines and chemokine which play a role in infection. the activation of nf-ĸb is triggered by the genome and protein of the virus which induces the nf-ĸb signaling pathway recognized by the host prr through tlr3, tlr7, tlr8 which then activates myd88 and irak-4 which subsequently leads to irak-1 bonding with traf6 and lead to phosphorylation figure 1. (a) nf-ĸb levels in serum from patients with dengue fever (n = 30) were determined clinically and in control (n = 10). (b) the mean value of nf-ĸb levels in patients with dengue fever ns1 is positive and ns1 is negative 113budiutari, et al.: overview of nuclear factor-kb (nf-kb) and non-structural protein 1 (ns1) and degradation of ikb and cause translocation of nf-ĸb to the nucleus.21 activation of nf-ĸb will cause the secretion of proinflammatory cytokines such as il-1, il-6, tnf-α. under normal conditions without infection, nf-ĸb is bound to the iĸb in various cells and is inactive.22 in the event of nf-ĸb stimulation will be activated and detached from iĸb. because of protein exposure and viral genome nf-ĸb activation can also trigger extrinsic apoptotic pathways directly or with their products and cause endothelial cell death which results in bleeding. this is consistent with the results of lin et al. an experimental study using mice and they found dengue virus protease induce cell apoptosis through its interaction with iĸbα, iĸbβ and cause the development of bleeding.23 dengue viral protease was found to cleave iĸbα and iĸbβ, activating ikk and triggering activation of nf-ĸb which causes caspase-mediated endothelial cell apoptosis. activation of nf-ĸb usually leads to the production of antiapoptotic proteins and protected cells from apoptosis. in dengue infection, nf-ĸb has played a pro-apoptotic role. the possibility of nf-ĸb-mediated endothelial apoptosis is triggered by dengue virus proteases through increased recruitment of macrophages and through increased sensitivity of endothelial cells to tnf by expressing tnf receptors.23 however, the underlying mechanism activation of nf-ĸb caused by infection with the dengue virus or viral protein remains were not defined yet. in yi-lin cheng et al. states that there is an increase in nf-ĸb activation found in hepatomas induced by dengue virus. research using raw264.7 cells is showed that dengue virus protein is needed for nf-ĸb activation found in endothelial cells and cells undergo apoptosis within 48 hours after infection. in addition, they were found two pathways that could increase nf-ĸb activation in dengue infection through excessive tnf-α production and through host prr, tlr3.24 based on the results of data analysis were showed that the levels of nf-ĸb in dengue fever patients were higher than those of healthy people. this shows that patients with dengue fever experience more nf-ĸb activation than healthy people. this occurs because of the large presence of dengue viruses that can activate nf-ĸb which plays a role in innate defenses which then secrete various proinflammatory cytokines and chemokine that function to fight dengue virus and lead to activation of the humoral immune response. in addition, it was found that patients with negative ns1 had higher nf-kb levels than positive ns1, but did not have a significant difference. in patients who showed negative ns1 results, it did not rule out the possibility that dengue infection was occurring in the body, but the number of dengue viruses and ns1 levels in the blood was low, so that there was no detection and further examination was needed. this is not uncommon in some case and the virus can directly exploit the immune system by activating nf-ĸb and replicating. in this study, 13 cases of patients who died because of dengue infection, it was showed that neither dengue ns1 or dengue ns1 antibodies were detected in the endothelium, questioning the role of ns1 antibodies in the pathogenesis of acute dengue.25 adikari et al. showed that acute ns1 infection was found to correlate with serum il-10 levels and that ns1 contributed to the pathogenesis of dengue infection by inducing the production of immunosuppressive cytokines from primary monocytes.26 based on this, that high amounts of ns1 can induce the production of anti-inflammatory cytokines and vice versa, proinflammatory cytokines which result in an increase in the number of dengue virus replication. but how the nf-ĸb pathway in the pathogenesis of dengue is still unclear. conclusion in conclusion, this study was analyzed the picture of serum nf-ĸb and ns1 in dengue patients where there was an increase in nf-ĸb activation and this is known from the high levels of nf-ĸb detected in serum which is thought to occur due to host cell apoptosis. and ns1 as a marker of early diagnosis of dengue infection that has been widely used was showed good results in the initial infection. but how the path of activation and involvement of nf-ĸb is still need further research. acknowledgement author would like to thank gina khairinisa for pooled dengue serum; prof. dr. jusak nugraha for providing clinical data of the patients at surabaya premier hospital; iswahyudi adam for primers and help in performing elisa examination; prof. dr. yoes prijatna dachlan for his comments throughout the research project. references 1. who. dengue and severe dengue. from world health organization. 2017. available from: http://www.who.int/mediacentre/factsheets/ fs117/en/. 2. dow b. mosquito wars: an overview of dengue fever virus. modern biol and chemic threats. wordpress. 2017. philadelphia, pa. 3. kementerian kesehatan ri. hari demam berdarah dengue: situasi dbd. infodatin pusat data dan informasi kementerian kesehatan ri. 2016. available from: http://www.depkes.go.id/resources/download/ pusdatin/infodatin/infodatin-demam-berdarah.pdf 4. tan ty, chu jjh. dengue virus-infected human monocytes trigger late activation of caspase-1, which mediates pro-inflammatory il-1β secretion and pyroptosis. j gen virol. 2013;94(part10):2215-20. 5. aamir m, masood g, aamir v, rasheed a, et al. gender difference in patients with dengue fever admitted in a teaching hospital lahore. pjmhs. 2014:08. 6. alcala ac, medina f, gonzales-robles a, salazar-villatoro l, fragoso-roriano rj, vásquez c, et al. the dengue virus nonstructural protein 1 (ns1) is secreted efficiently from infected mosquito cells. virology. 2016:488:278-87. 7. sun p, kochel tj. the battle between infection and host immune responses of dengue virus and its implication in dengue disease pathogenesis. sci world j. 2013. 114 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 109–114 8. zhao jun, minassian a, shanping he, et al. recent advances on viral manipulation of nf-ĸb signalling pathway. curr opin virol. 2015:15:103-111. 9. silva bm, sousa lp, gomes-ruis ac, leite g, et al. the dengue virus nonstructural protein 1 (ns1) increase nf-kb transcriptional activity in hepg2 cells. arch virol .2011:156:1275-79. 10. world health organization. comprehensive guidelines for prevention and control of dengue fever and dengue haemorrhagic fever. who. 2011:196 (cited 15 september 2018). available from, http://www. searo.who.int/entity/vector_borne_tropical_diseases/documents/ searotps60/en/. 11. soegijanto s. demam berdarah dengue. surabaya. airlangga university press; 2012:2. 12. muller da, alexandra ci, young pr. clinical and laboratory diagnosis of dengue virus infection. the infec diseases j. 2017:215:s89-95. 13. kalayanarooj s. clinical manifestations and management of dengue/ dhf/dss. trop med health. 2011:39:83-87. 14. aryati a, trimarsanto h, yohan b, wardhani p, fahri s, sasmono rt. performance of commercial dengue ns1 elisa and molecular analysis of ns1 gene of dengue viruses obtained during surveillance in indonesia. bmc infec diseases. 2013. 15. megariani, rinang m, et al. uji diagnostik pemeriksaan antigen nonstruktural 1 untuk deteksi dini infeksi virus dengue pada anak. padang: ilmu kesehatan anak universitas andalas. 2014:6. 16. aryati, rini p. sd dengue duo® (ns1, igg, igm) rapid test dalam menunjang diagnosis infeksi virus dengue. indonesian j clin patholog and med lab. 2010. 17. david gn. the relationship of interacting immunological component in dengue pathogenesis. virol j review. 2009. 18. tambunan ba, aryati, husada d. evaluation of plateliatm. dengue ns1 antigen assay for early diagnosis of acute dengue infection. presented at 17th european congress of clinical microbiology and infection diseases, icc, munich, germany. 2011. 19. blacksell sd, mammen mpjr, thongpaseuth s, gibbons rv, jarman rg, et al. (2008). evaluation of the panbio dengue virus non-structural 1 antigen detection and immunoglobulin m antibody enzyme-linked immunosorbent assays for the diagnosis of acute dengue infections in laos. virolog. p43-49. 20. dussart p, petit l, labeau b, bremand l, leduc a, et al. evaluation of two new commercial test for the diagnosis of acute dengue virus infection using ns1 antigen detection in human serum. plos negl trop dis. 2008. 21. murphy k, weaver c. janeway’s immunology 9th edition. new york and london. garland science. 2017. 22. dev a, iyer s, razani b, cheng g. nf-ĸb and innate immunity. curr top microbiol immunol. 2011. 23. lin jc, shih-ching l, cen wy, yen yt, lai cw, et al. dengue viral protease interaction with nf-kb inhibitor α/β result in endothelial cell apoptosis and hemorrhage development. j immunol. 2014. 24. cheng, yl, lin ys, chen cl, wan sw, ou yd, et al. dengue virus infection cause the activation of distinct nf-kb pathways for inducible nitric oxide synthase and tnf-α expression in raw264.7 cells. mediators inflamm.. 2015(part2015):274025. 25. aye ks, charngkaew k, win n, et al. pathologic highlights of dengue hemorrhagic fever in 13 autopsy cases from myanmar. hum pathol. 2014:45: 1221-33. 26. adikari tn, gomes l, wickramasinghe n, salimi m, et al. dengue ns1 antigen contributes to disease severity by inducing interleukin (il)-10 by monocytes. clin exp immunol. 2015:1:90-100. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 9 772085 110066 e issn 2356-0991 p issn 2085-1103 vol. 7 ● no. 2 may–august 2018 e issn 2356 0991 p issn 2085 1103 e-journal.unair.ac.id/index.php/ijtid indexed by: soil-transmitted helminth infection and eosinophil level among waste collectors in banda aceh effectiveness of meniran (phyllanthus niruri linn) as antibacterial for antibiotics resistance enterotoxigenic escherichia coli factor related to anti-tuberculosis drug resistency on pulmonary tuberculosis patients in labuang baji hospital makassar onychomycosis finger nail by cryptococcus laurentii, trychophyton spp the effectiveness of herbal mosquito coils “morizena” against aedes aegypti death e issn 2356 0991 p issn 2085 1103volume 7 number 2 may–auguts 2018 editorial team of indonesian journal of tropical and infectious disease editor in chief prihartini widiyanti, indonesia editorial board mark alan graber, united states kazufumi shimizu, japan masanori kameoka, japan hak hotta, japan fumihiko kawamoto, japan nasronudin nasronudin, indonesia maria inge lusida, indonesia puruhito puruhito, indonesia indropo agusni, indonesia retno handajani, indonesia kuntaman kuntaman, indonesia soegeng soegijanto, indonesia bambang prajogo, indonesia ni nyoman sri budayanti, indonesia achmad fuad hafid, indonesia tri wibawa, indonesia irwanto irwanto, indonesia marcellino rudyanto, indonesia yulis setiya dewi, indonesia laura navika yamani, indonesia secretariat zakaria pamoengkas firda fatma hamzah secretariat office publishing unit of indonesian journal of tropical and infectious disease, institute of tropical disease universitas airlangga kampus c, jalan mulyorejo surabaya 60115, jawa timur – indonesia. phone 62-31-5992445-46 faximile 62-31-5992445 e-mail: ijtid@itd.unair.ac.id homepage: e-journal.unair.ac.id/index.php/ijtid contents page printed by: universitas airlangga press. (rk 223/05.18/aup-77e). kampus c unair, mulyorejo surabaya 60115, indonesia. telp. (031) 5992246, 5992247, fax. (031) 5992248. e-mail: adm@aup.unair.ac.id 1. soil-transmitted helminth infection and eosinophil level among waste collectors in banda aceh teuku romi imansyah putra, ricke loesnihari, merina panggabean ...................................... 27–34 2. effectiveness of meniran (phyllanthus niruri linn) as antibacterial for antibiotics resistance enterotoxigenic escherichia coli sri hidanah, emy koestanti sabdoningrum, retno sri wahyuni, arini rahmi dewi, erma safitri ...................................................................................................................................... 35–39 3. factor related to anti-tuberculosis drug resistency on pulmonary tuberculosis patients in labuang baji hospital makassar sapriadi, syahridha ......................................................................................................................... 40–44 4. onychomycosis finger nail by cryptococcus laurentii, trychophyton spp dhelya widasmara, diane tantia sari .......................................................................................... 45–49 5. the effectiveness of herbal mosquito coils “morizena” against aedes aegypti death rina priastini susilowati, win darmanto, nanik siti aminah ................................................... 50–55 volume 7 number 2 may–august 2018 e issn 2356 0991 p issn 2085 1103 69 vol. 7 no. 4 january-april 2019 research report effect of african leaf (vernonia amygdalina) to il-6 and il-10 level on staphylococcus aureus infection lidwina tri kristanti setiawan1, jusak nugraha2,3a, pudji lestari4, restry sinansari5, lisa soegianto5, luh putu trys monika handayani5, stephanie beatrix5, wahyu dewi tamayanti5 1 master of immunology, postgraduate school, universitas airlangga 2 faculty of medicine, widya mandala catholic university 3 clinical pathology, faculty of medicine and institute of tropical disease, universitas airlangga 4 public health departement, faculty of medicine, universitas airlangga 5 faculty of pharmacy, widya mandala catholic university a corresponding author: jusak.nugraha@yahoo.com abstract currently, infectious disease is increase in world wide. the african leaf (vernonia amygdalina) – va is used to antimicrobial treatment. it may protect the host against microbial attack in several ways. this plant has attracted the interest of researchers in recent decades because of the constituents have important roles in modulating immune system in bacteria infection. the aim of study is to analyze the prophylactic activity of va’s ethanol extract in modulating the levels of il-6 and il-10 as well as the number of bacteria in male wistar rats that were (staphylococcus aureus) – sa – infected. there were as many as 30 rats were divided into 5 treatment groups: negative control (nc) was treated by cmc na 2% (w/v); positive control (pc) was treated by 9mg/200g body weight (bw) of cephadroxil; t1; t2; and t3 were respectively treated with ethanol extract of va of doses 20mg/200g bw; 40mg/200g bw and 80mg/200g bw. after the oral treatment was administered, all the rats were infected with 0.25ml (3x108cfu) sa via intra peritoneal route. their blood was drawn in order to identify the il-6 and il-10 levels by elisa. furthermore, their peritoneal fluid was also taken to count the number of survived bacteria by pour plate method. the results are showed median of il-6 and il-10 levels as well as bacterial number respectively in nc 370.530pg/ml; 67.044pg/ml; 7.4x103cfu/ml; in pc 234.556pg/ml; 42.839pg/ml; 6.8x103cfu/ml,; in t1 164.019pg/ml; 17.240pg/ml; 1.1x104cfu/ml; in t2 49.291pg/ml; 2.961 pg/ml; 6.3x103cfu/ml and in t3 43.342pg/ml; 13.235pg/ ml; 7.1x103cfu/ml. these results are implied that va’s ethanol extract is effective as a prophylactic agent to suppress the bacterial invasion at dose of 40mg/200g bw in wistar rat particularly shown by the decrease level of il-6 and the number of bacteria. keywords: vernonia amygdalina, il-6, il-10, bacterial number, staphylococcus aureus. abstrak saat ini, penyakit infeksi meningkat di dunia. daun afrika (vernonia amygdalina) – va digunakan sebagi terapi antimikroba. va dapat melindungi host terhadap serangan mikroba melalui berbagai macam cara. tanaman ini menarik perhatian para peneliti pada dekade terakhir karena va memiliki berbagai kandungan yang berperan penting dalam memodulasi sistem imun pada infeksi bakteri. pada penelitian ini, dilakukan analisa terhadap kemampuan profilaksis dari ekstrak etanol va dalam memodulasi kadar il-6 dan il-10 serta jumlah bakteri pada tikus wistar jantan yang terinfeksi (staphylococcus aureus) – sa. tikus wistar sebanyak 30 ekor dibagi ke dalam 5 kelompok uji yaitu kontrol negatif (nc) yang diterapi dengan cmc na 2% (b/v), kontrol positif (pc) yang diterapi sefadroksil 9mg/200g bb, kelompok t1, t2 dan t3 yang diterapi dengan ekstrak etanol va dengan dosis: masing-masing 20mg/200g bb; 40mg/200g bb; dan 80mg/200g bb, secara berurutan. setelah diberikan terapi, tikus diinfeksi dengan bakteri sa sebanyak 0,25ml (3x108cfu) secara intra peritoneal. darah tikus dikoleksi untuk dianalisa kadar il-6 dan il-10 menggunakan elisa dan dikoleksi pula cairan peritoneal untuk dihitung jumlah bakteri yang bertahan hidup menggunakan metode tuang. hasil median dari kadar il-6, il-10 dan jumlah bakteri secara berurutan adalah sebagai berikut: pada kontrol negatif (nc) 370,530pg/ml; 67,044pg/ml; 7,4x103cfu/ml; pada kontrol positif (pc) 234,556pg/ml; 42,839pg/ml; 6,8x103cfu/ml; pada t1 164,019pg/ml; 17,240pg/ml; 1,1x104cfu/ml; pada t2 49,291pg/ml; 2,961 pg/ml; 6,3x103cfu/ml; pada 43,342pg/ml; 13,235pg/ml; 7,1x103cfu/ml. hasil penelitian ini mengimplikasikan 70 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 69–74 bahwa ekstrak etanol va efektif sebagai senyawa profilaksis yang mencegah invasi bakteri, khususnya pada dosis 40mg/200g bb tikus karena pada dosis tersebut terjadi penurunan kadar il-6 dan jumlah bakteri. kata kunci: vernonia amygdalina, kadar il-6, kadar il-10, jumlah bakteri, staphylococcus aureus. introduction globally, there is an increase in infectious disease especially in bacteria infection. it was caused of host body defense mechanism can’t control the immune system.1 therefor, it is an evident from human history that we have to some new product to modulate immune system against bacteria infection. one of which is medicinal plants that it have been utilized as therapeutic agents in variety of disease including infection diseases. medicinal plants are through to be mediated through inhibition and modulating cell-signaling pathways in immune system.2 the immunomodulating characteristic of medicinal plants is safety, effectiveness, minor side effect and cultural acceptability.3 vernonia amygdalina (va) is one of medicinal plants4 and a member of the asteraceae family. this plant is a small tree in 2-5 m of size. vernonia amygdalina leaf is ellipse in form and about 6 mm of diameter. the green leaves are showing bitter taste and odor characteristics.5 this bitter taste serves as a protection of animals attack such as insects and microbes.6 bitter taste is due to the flavonoids and sesquiterpenes lactones contained in va.7 vernonia amygdalina contains many other constituents such as: tannins,8 saponins, alkaloids, terpenoids, stigmastanetype steroid glycosides, coumarin, phenolic acids, lignans, xanthones, anthraquinones and edotides.9 several constituents of va that have been reported to function as antimicrobial agent and modulate the immune system is known as luteolin5 and myricetin; andrographolide;9 and chlorogenic acid.10 previous studies were illustrated the antimicrobial effects of va in ethanol extract. this ethanol extract is more effective to exhibit antimicrobial effects than water extract.4,11-13 previous in vitro studies is proved that ethanol extracts of va showed high potency and effectivy against staphylococcus aureus (sa).11,13 staphylococcus aureus is a pathogenic bacterium14 that belong to gram-positive bacteria with coccal (round) form and also known as a facultative anaerobes. it has a complex cell wall consisting of murein, teichoic acids and surface proteins.15 in the human body, lipoteichoic acid (lta) on the surface of sa’s cell wall was known as pathogen associated molecular patterns (pamps) which generally be recognized by pattern recognition receptors (prrs) such as toll like receptors (tlrs)-1/2 or tlrs-2/6. the prrs is the property of the immune cells such as macrophages, dendritic cells, endothelial cells, mast cells, eosinophils and b cells.16,17 prr will exhibit a signal that may attract the nuclear factor κappa b (nfκb) transcription factor to enter the nucleus and synthesize pro-inflammatory cytokines such as interlukin (il)-1β, tumor necrosis factor (tnf)-α, il-6,18 il-12 and il-8 or cxcl8.19 il-6 is known as a pleitropic cytokines found in each organ system. it is synthesized by mononuclear phagocytes, dendritic cells, vascular endothelial cells, fibroblasts and other cells in response to pamps and il-1 and tnf stimulation.17 il-6 production immediately increases in the acute inflammatory condition that occurs due to infection, injury, trauma and other stress conditions. these cytokines retain extracellular and intracellular growth from sa but excessive production can lead to systemic inflammation with damaging effects rather than protection of the host.18 in order to balance the il-6 effects, another cytokine, il-10 is produced and is served as an anti-inflammatory agent. this cytokine is produced by immune cells including macrophages and active dendritic cells, t regulators (tregs), t helper (th)-1, and th2 cells. il-10 is also produced by several b lymphocytes which show immune suppression function, called regulatory b cells. il-10 is also an important cytokine that regulate the immune responses in infection diseases.20 in infection caused by sa, generally, immune cells are stimulated to promote pro-inflammatory cytokine (such as: il-6) dan anti-inflammatory cytokine, such as il-10.21 however, until recently, there is no study identify ethanol extract of va activity in modulating these cytokines in microbial infections. therefore, this study was conducted in vivo to study the va’s ethanol extract activities in immune system in wistar rats which infected by sa. further, this study is aimed to analyze prophylactic activity of va’s ethanol extract in modulating levels of il-6, il-10 as well as the decrease of the number of bacteria in male wistar rats that were sa-infected. material and method i. collection of plant materials fresh leaves of vernonia amygdalina were obtained from jember, east java, indonesia. the leaves were dried in an oven at 60°c and then blended into powder. 71setiawan, et al.: effect of african leaf (vernonia amygdalina) ii. preparation of ethanol extract vernonia amygdalina powder was macerated with 96% ethanol (1: 5) for 72 hours was followed by filtration with whatman no.1 filter paper and was evaporated in a rotary evaporator, the thick extract was yielded. the thick extract dissolved in cmc na 2%. iii. standardization and phytochemistry screening standardization of the extract organoleptic organoleptic examination includes examination of color, odor, and taste.22 total ash content thickened extracts were weighed 2 to 3 grams and were placed into the incandescented and were tared silicate crucible and were heated in furnace at 600oc for 3 hours. then, it was cooled and weighed. if charcoal stays, hot water was added, then was stirred and was filtered with ash-free filter paper. the filtering residue and filter paper were applied to the same crucible. the filtrate was added to the crucible, evaporated and incandescented until the weight was fixed, then weighed. total ash content was calculated against the weight of thick extract expressed in% b/b.23 acid insoluble ash content the ash was boiled with 25ml of diluted sulfuric acid for 5 minutes, was collected parts which were not soluble in acid, filtered through glassy crust or ash-free filter paper, was washed with hot water, incandescented until the weight remained, then was weighed. then the ash content which is insoluble in acid against thick extract was calculated in % b/b.23 loss on drying the dried-shrinkage method was determined as follows: carefully weigh 1 to 2 grams of substances which have previously been heated at 105oc for 30 minutes and have been tared. before weighing, the extract was flattened in a bottle and then put into a drying chamber and dried at 105oc to a fixed weight. let the bottle be closed and cooled in the desiccator to room temperature. then note the fixed weight obtained to calculate the percentage of drying loss.23 phytochemistry the phytochemical screening of dried simplicia and ethanol extract of va: alkaloids, flavonoids, saponins, tannins was analyzed using the standard methods as described by soetarno (2008);24 steroids and terpenoids, antraquinones by kristanti et al (2008);25 phenol and glycoside by harbourne (2008).26 iv. collection of staphylococcus aureus isolate of staphylococcus aureus atcc 25923 from department of microbiology, widya mandala catholic university. these bacteria were rejuvenated in nutrient broth medium and were incubated at 37°c for 24 hours. then the isolate was mixed in 0.9% nacl and standardized to mcfarland iv (1.2x109cfu/ml). v. determination of antimicrobial effect thirty male wistar rats were divided into 5 groups : negative control (nc): rats were treated cmc na 2% positive control (pc): rats were treated 9mg/200g bw of cefadroxyl antibiotics t1: rats were treated 20mg/200g bw of vernonia amygdalina ethanol extract t2: rats were treated 40mg/200g bw of vernonia amygdalina ethanol extract t3: rats were treated 80mg/200g bw of vernonia amygdalina ethanol extract the rat in both nc, t1, t2 and t3 groups were treated with 1 ml va ethanol extract orally 3 times a day. the next day, the pc group was administered 1ml of cefadroxyl orally. then all rats were injected with 0.25ml sa suspension in 0.9% nacl (3x108cfu) intraperitoneally. after 24 hours of bacteria injection, all rats were sacrificed and blood was withdrawn intracardially to measure il-6 and il-10 levels. the peritoneal fluid was collected to count the number of survived bacteria. vi. elisa elabscience rat il-6 elisa kit (catalog no. e-elr0015) and elabscience rat il-10 elisa kit (catalog no. e-el-r0016) were used to quantify the il-6 and il-10. vii. bacterial counting the fluid from peritoneal rat was identified by pour plate method. the 0.1ml peritoneal fluid was withdrawed and was placed in a tube containing 9.9ml of sterile aquadest (tube 1). then, 1ml from the tube 1 was taken and it into 9ml sterile aquadest (tube 2) and so on until tube 3 (twice replication was performed). after that, from each tube, 1ml was taken and placed in a petri dish, which subsequently was added 10ml of nutrients sterile agar (at 50oc) and was rotated in order to obtain to mixture of bacteria. all petri dish were incubated at 37oc for 24 hours. afterwards, the number of colonies was calculated.27 result and discussion the standardization results were showed that va’s ethanol extract (table 1) have a dark green color, charateristic odor and bitter taste. the total ash content shows high mineral content such as calcium, chlorine, chromium, copper, iron, potassium, magnesium, manganese, nickel, phosphorus, potassium, sodium in this plant28,29 while the acid insoluble ash content was showed the contamination of fine particles from sand and soil from the environment.30 furthermore, 88.36% of the compounds lost during the drying process.22 phytochemical screening was indicated that both simplicia and ethanol extract of va contained flavonoids, saponins, tannins, steroids, terpenoids, phenols and 72 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 69–74 glycosides, while alkaloids and anthraquinones was not detected (table 2). this might be due to geographical differences where the plants grow. in this study, it was observed the effectiveness of va in modulating immune system in wistar rat that was infected by sa. generally, in infectious condition induced by bacteria, the body generates a defence mechanism in reaction to the encountered microbes or their products. the defence is preceded by the presence of immune cells such as macrophages, dendritic cells, neutrophils, natural killer cells, and limfoid cells. they were resolved the microbes through two main actions: the first is recruiting phagocytes and other leukocytes to destroy the microbes (indicates inflammatory reaction) and secondly by limiting microbial replication or killing microbial-infected cells without inflammatory reaction.17 during inflammation process, immune cells recognized the molecular structure produced by sa through a binding between tlrs-1/2 or tlrs-2/6 with lipoteichoic acid (lta). this binding was activated the transcription factor, nfκb, to produce high amounts of il-6 levels.1618 this cytokine was needed when inflammation occurs to increase the formation of neutrophils in bone marrow and recruitment of neutrophils to the site of infection to replace the leukocyte cells that died during inflammation.17 however, high levels of il-6 also was stimulated a negative impact which is correlated with disease progression31 and is contributed in exacerbating inflammation so it triggered to autoimmune diseases.17 therefore, the effectivity of the va’s ethanol extract in reducing production of il-6 levels (figure 1) and may be potential to reduce inflammation. the va’s constituents that are playing role as the il-6 reducing agents are luteolin and myricetin, they are belongs to the flavonoid groups. luteolin inhibits nf-κb activation by blocking the degradation of iκbα and phosphorylation of p65.18 whereas, myricetin works by inhibiting the activation of p38 and extracellular signals from tlr2/6 and also blocking the degradation of iκb. myricetin which given as prophylactic agents can significantly reduce iκb degradation,32 furthermore, viljoen et al. (2016) were reported that myricetin also inhibits the activation of erk-1/2, akt and p38 induced by lta,33 thus these mechanisms were blocked the production of proinflammatory cytokines, il-6.32 the other constituents of va ethanol extract may exhibit molecular function in regards of il-6 reduction such as andrographolide that belongs to terpenoids groups. andrographolide also plays a role in decreasing il-6 by inhibiting nfκb activation, suppressing inos, and preventing oxygen radicals produced by neutrophils.34 furthermore, tannin, that is one of va’s constituents has ability to reduce intracellular kinase phosphorylation and inhibit nfκb at p65 and its catalytic activity, therefore those processes may decrease the il-6 levels.35 moreover, chlorogenic acid in va’s ethanol extract belongs to phenol group suppresses the expression of the nfκb signaling pathway inhibits the activation of this signaling pathway and reducing inflammatory cytokines production. a previous study was confirmed that taking chlorogenic acid can reduce levels of nfκb p50 and ikkα/β.36 on the other hands, in presence of microbial infections, immune cells were also produced anti-inflammatory cytokines such as il-10 to reduce inflammation.18 in table 1. standardization results of vernonia amygdalina determination ethanol extract organoleptics color: dark green odor: characteristic taste: bitter total ash content 16.18 ± 0.48% acid insoluble ash content 0.822 ± 0.18% loss on drying 88.36 ± 0.74% table 2. phytochemical screening of vernonia amygdalina determination simplicia ethanol extract alkaloids flavonoids + + saponin + + tannin + + steroid + + terpenoids + + antraquinone phenol + + glycoside + + description: (+) = identified and (-) = not identified groups nc pc t1 t2 t3 figure 1. graphic of il-6 levels distribution. nc: given cmc na 2%; pc: given 9mg/200g bw of cefadroxyl antibiotics; t1: given 0mg/200g bw of vernonia amygdalina ethanol extract; t2: given 40mg/200g bw of vernonia amygdalina ethanol extract; t3: given 80mg/200g bw of vernonia amygdalina ethanol extract. 73setiawan, et al.: effect of african leaf (vernonia amygdalina) contrast, this study was found that il-10 levels decreased after va’s ethanol extract administration (figure 2). this might be occured due to the myricetin and chlorogenic acid which were suppressed the expression of jak/stat signaling pathways34 and thus were inhibited the production of il-10 in wistar rats’s immune cells.17 cheng and iyer (2012) were reported that the majority of intracellular infections were better controlled or were cleaned quickly in a no il-10 state. decreasing il-10 signaling leads to increase host survival after infection and to increase adaptive immune response, including cd4+ t cells that produce interferon (ifn)-γ.37 similar to cheng and iyer’s report, riley et al. (2008) were observed that il-10 is an important regulator component in almost all infections.20 this statement is clarified through a research conducted by mcloughlin et al. (2017), that during systemic acute infection was induced by sa, il-10 was regulated local and systemic proinflammatory responses that prevented the host from immunopathology condition caused by bacteria spreading.38 in infections which were caused by sa, the decreased of il-10 levels can increase ifn-γ,39 il-17, il-22 and cxcl1. therefore it was stimulated the increasement of th1 cells as well as activated the phagocytes to clear the bacteria.38 in addition, the il-10 decreased levels may increase the expression of costimulators and major histocompatibility complex (mhc) ii molecules and il-12 production in macrophages and dendritic cells. il12 is the main cytokine that stimulate adaptive immune response, th1 cells, which will secrete ifn-γ. ifn-γ plays an important role in the reaction of innate and adaptive immune cells against intracellular microbes.17 therefore, il-10 deficiency in intracellular infections can reduce the nc pc t1 t2 t3 groups figure 2. graphic of il-10 levels distribution. nc: given cmc na 2%; pc: given 9mg/200g bw of cefadroxyl antibiotics; t1: given 0mg/200g bw of vernonia amygdalina ethanol extract; t2: given 40mg/200g bw of vernonia amygdalina ethanol extract; t3: given 80mg/200g bw of vernonia amygdalina ethanol extract. number of microbes by activating the adaptive immunity to kill bacteria.38 moreover, figure 3 shown that va’s ethanol extract also plays a role in reducing the number of bacteria. this extract occurred due to the content of andrographolide and luteolin. andrographolide had a bacteriostatic effect. andrographolide will weaken dna synthesis of sa so it will produced inhibition on biosynthesis pathway of intracellular dna in staphylococcus aureus.40 in addition, luteolin also had antibacterial effects by inhibiting the activity of staphylococcus aureus bacteria in dna topoisomerase i and ii which will result in a decrease in nucleic acid and protein synthesis.41 conlusion this study was indicated that the optimum dose of va’s ethanol extract in exhibiting il-6 and il-10 modulation in wistar rats is 40mg/200g bw. this dose can decreased il-6 levels and bacterial numbers which tent to will decrease the inflammation. it may imply the effectivity of this plant as a prophylactic agent to prevent sa infection. references nfambi j, bbosa gs, sembajwe lf, gakunga j and kasolo jn. 1. immunomodulatory activity of methanolic leaf extract of moringa oleifera in wistar albino rats. jbcpp. 2015;26(6):603–11. ramalingum n and fawzi m. the therapeutic potential of medicinal 2. foods. aps. 2014;2014:1–18. nc pc t1 t2 t3 groups figure 3. graphic of bacterial numbers distribution. nc: given cmc na 2%; pc: given 9mg/200g bw of cefadroxyl antibiotics; t1: given 0mg/200g bw of vernonia amygdalina ethanol extract; t2: given 40mg/200g bw of vernonia amygdalina ethanol extract; t3: given 80mg/200g bw of vernonia amygdalina ethanol extract. the maximum value in t1 group is 210000 and the maximum value in t4 group is 1300000 74 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 69–74 shrestha g, st clair ll and o’neil kl. the immunostimulating 3. role of lichen polysaccharides: a review. pr. 2015;29(3):317– 22. udochukwu u, omeje fi, uloma is and oseiwe fd. phytochemical 4. analysis of vernonia amygdalina and ocimum gratissimum extracts and their antibacterial activity on some drug resistant bacteria. ajrc. 2015;3(5):225–33. audu sa, taiwo ae, and ojuolape ar. a study review of 5. documented phytochemistry of vernonia amygdalina (family asteraceae) as the basis for pharmacologic activity of plant extract. jnsr. 2012;2(7):1–7. idowu ab and idowu oa. effect of food plants on the volume 6. of repellent secretion obtained in adult zonocerus variegates (orthoptera: pyrgomorphidae). rbt. 2001; 49(2): 679–84. nangendo g, stein a, gelens m, de gier a and albricht r. 7. quantifying differences in biodiversity between a tropical forest area and a grassland area subject to traditional buring. fem. 2002;164:109–20. ghamba pe, balla h, goje lj, halidu a and dauda md. in vitro 8. antimicrobial activities of vernonia amygdalina on selected clinical isolates. ijcmas. 2014;3(4):1103–13. egharevba c, osayemwenre e, imieje v, ahomafor j, akunyuli 9. c, udu-cosi aa, theophilus o, james o, ali i and falodun a. sigmificance of bitter leaf (vernonia amygdalina) in tropical disease and beyond: a review. mcce. 2014;3(120):1–10. johnson ce, lin lz, harnly jm, oladeinde fo, kinyua am, 10. michelin r and bronner y. identification of the phenolic components of vernonia amygdalina and russelia equisetidormisi. jnp. 2011;4:57–64. adetunji co, olaniyi oo and ogunkunle atj. bacterial activity of 11. crude extracts of vernonia amygdalina on clinical isolates. jma. 2013;5(6):60–64. anibijuwon ii, oladeko bo, adetitun do and kolawole om. 12. antimicrobial activities of vernonia amygdalina against oral microbes. gjp. 2012;6(3):178–85. oshim io, desmond co, nwobu rau, ezugwu um and urama 13. eu. kinetics of minimum inhibitory concentration, minimum bactericidal concentration and minimum fungicidal concentration of va (bitter leaf) on microorganisms isolated from wound infections. ijsr. 2016;5(1):8–14. peterson ml, anderson mj, lin yc, gillman an, parks pj and 14. schlievert pm. alpha-toxin promotes staphylococcus aureus mucosal biofilm formation. fcim. 2012;2(64):1–10. patel h, vaghasiya y, vyas brm and chanda s. antibiotic-resistant 15. staphylococcus aureus: a challenge to researchers and clinicians. bj. 2012;2(2):23–45. murphy k and weaver c. janeway’s immunobiology. 916. th edition. usa: garland science; 2017:2, 89, 95, 111, 346, 375, 377. abbas ak, litchman ah and pillai s. cellular and molecullar 17. immunology. 9th edition. philadelphia: elsevier; 2018: 1–7, 9–10, 53, 59–61, 82–84, 93, 228–229. guo my, guo yf, xu nn, sun w, zhao y and li cy. luteolin 18. reduces inflammation in staphylococcus aureus-induces mastitis by inhibitong nf-κb activation and mmps expression, oj, 2017;8(17):28481–93. roitt im, delves pj, martin sj and burton dr., roitt’s essential 19. immunology. 13th edition. uk: willey blackwell; 2017:20-22, 26, 30-31. riley em, couper kn and blount dg. il-10: the master regulator 20. of immunity to infection. ji. 2008;180:5771–7. yang m, wang j, chen c, ma y, he s and wang c. the expression 21. and significance of il-6 and il-10 in the process of clinical common bacteria bloodstream infection in the mouse models analyzed by the luminex® xmaptm system, flm1, 2017;55–8. depkes ri. parameter standar umum ekstrak tumbuhan obat. 22. jakarta: depkes ri; 2000:31. depkes ri. farmakope herbal indonesia edisi i. jakarta: depkes 23. ri; 2008:169, 171. soetarno s. persiapan ekstraksi bahan alam, prosiding temu ilmiah 24. nasional bidang farmasi v. bandung: itb; 2008:30. kristanti an, aminah ns, tanjung m dan kurniadi b. buku ajar 25. fitokimia. surabaya: universitas airlangga press; 2008:48–50. harbourne jb. phytochemical methods. 326. rd edition. london: chapman and hall; 2008: 60, 135–203. sgm. basic practical microbiology. uk: educational departement; 27. 2006:1–48. gbaruko bc and friday ou. bioaccumulation of heavy metals in 28. some fauna and flora. ijest. 2007;4:197–202. oboh g. nutritive value and haemolytic properties (in vitro) of 29. the leaves of vernonia amygdalina on human erythrocyte. nh. 2006;18:151–60. park s, kim d, kim b, yun e, kim j and chae y. statistical quality 30. control of total ash, acid-insoluble ash, loss on drying, and hazardous heavy metals contained in component medical herbs of “ssanghwatang”, a widely used oriental formula in korea. jnm. 2012;63(1):27–35. dembic z. the cytokines of the immune system. the role of 31. cytokines in disease related to immune response. usa: elsevier; 2015:172. venegas gg, luna oa, arroyo jav and bermúdez ch. 32. myricetin supresses lipoteichoic acid-induced interleukin-1β and cyclooxygenase-2 expression in human gingival fibroblast. mi. 2013;57(12):849–56. viljoen a, semwal dk, semwal rb, and combrinck s. myricetin: 33. a dietary molecule with diverse biological activities. n. 2016;8(90):1–31. levita j, nawawi a, mutalib a and ibrahim s. andrographolide: 34. a review of its anti-inflammatory activity via inhibition of nfkappab activation from computational chemistry aspects. ijp. 2010;6(5):569–76. clinton c. plan tannis a novel approach to the treatment of 35. ulcerative colitis. nmj. 2009;1(11). chai l, lou l, zhou j, liu y, wei y, zhao j, deng j, dong b, 36. zhu l, wu a and yang y. chlorogenic acid induces apoptosis to inhibit inflammatory proliferation of il-6-induced fibroblast-like synoviocytes through modulating the activation of jak/stat and nf-κb signaling pathways. etm. 2016;11:2054–60. cheng g and iyer ss. role of interleukin 10 transcriptional 37. regulation in inflammation and autoimmune disease. cri. 2012;32(1):23–63. mcloughlin rm, leech jm, lacey ka, mulcahy me and medina e. 38. il-10 plays opposing roles during staphylococcus aureus systemic and localized infections. ji. 2017;198:2352–65. montgomery cp, daniels m, zhao f, alergre m-l, chong as and 39. daum rs. protective immunity against recurrent staphylococcus aureus skin infection requires antibody and interleukin-17a. ii. 2014;82(5):2125–34. mukherjee sk, banarjee m, parai d and chattopadhyay s. 40. andrographolide: antibacterial activity against common bacteria of human health concern and possible mechanism of action. fm. 2017:1–8. wang q and xie m. antibacterial activity and mechanism of 41. luteolin on staphylococcus aureus. ams, 2010;50(9):1180–84. 404 not found not found the requested url was not found on this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ vol. 11 no. 1 january–april 2023 original article portable and battery-operated isothermal amplification device validation for onsite analysis of m. tuberculosis “dna hunter” hatice esra agel biomaterials, biomechanics, and bioelectronics center of excellence, tubitak mrc, gebze kocaeli, turkey received: november 11th, 2022; revised: february 22nd, 2023; accepted: march 10th, 2023 abstract point-of-care (poc) devices play an important role in the protection of public health by providing rapid diagnosis of infectious diseases, patient management, and effective treatment. fast, easy-to-interpret, environmentally resistant, and cost-effective poc tests that can be used practically in the field are gaining more and more importance every day. there is a need for portable devices that will enable rapid diagnosis kits to be used in the field for early diagnosis and treatment. the aim of this study is to evaluate the dna hunter device that was developed in terms of providing the required temperature for m. tuberculosis (mtb) diagnosis of the loopmediated isothermal amplification (lamp) assay and visually evaluating the analysis results. the device in this study; handheld (total weight 430 g, outer dimensions 70 x 175 x 80 mm), the average operating time can reach a maximum temperature of 110 degrees in 2 minutes with a fully charged battery, and the processing time is about 90 minutes without being connected to electricity. it can display the pre-evaluation result on the screen with the full digital color sensor. the device can be adjusted to the desired reaction temperature and time. it also has software where sample registration numbers can be entered. dna hunter can be used for all analyses performed by the lamp method and the results can be evaluated colorimetrically, thus it is well suited for poc testing. keywords: handheld device; loop-mediated isothermal amplification (lamp); m. tuberculosis (mtb); point of care (poc) highlights: a portable device has been developed that allows an important public health pathogen such as tuberculosis infection to be screened with the lamp method without the need for complex laboratory infrastructure. the most important aspect of this device is that it is small enough to fit in the palm and can work independently of electricity for a certain period of time. how to cite: agel, h. e. portable and battery-operated isothermal amplification device validation for onsite analysis of m. tuberculosis “dna hunter”. indonesian journal of tropical and infectious disease. 11(1). 1–11. apr. 2023. doi: 10.20473/ijtid.v11i1.40482 * corresponding author: esra.agel@tubitak.gov.tr https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-8434-3232 2 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license h. esra agel. portable and battery-operated isothermal amplification device validation introduction analyses that can be performed quickly outside of the laboratory are known as pointof-care (poc) tests.1 poc tests are required for disease screening in the diagnosis of infectious diseases, particularly in countries with limited laboratory facilities, a high disease burden, and a low income. the need for poc tests for the rapid screening of infectious diseases is rapidly expanding.2 the main advantages of poc tests are to reduce procedures, and costs associated with hospitalization and prevent the risk of hospital-acquired infections by determining the infection factor detected during hospitalization, rapid diagnosis in epidemics and pandemics, and the ability to work with fewer samples compared to traditional methods.3 poc tests, as well as antimicrobial use control, rapid treatment initiation, and outbreak monitoring and control, all contribute to the investigation of unknown pathogens.4 with a compound annual growth rate of 11.4%, the global poc market was valued at usd 29.5 billion in 2020 and is expected to reach usd 50.6 billion in 2025. the production of devices for poc analysis has economic and commercial importance, as shown by these statistics.5 tuberculosis is one of the oldest known diseases, is an infectious disease caused by the mycobacterium tuberculosis complex (m. tuberculosis, m. bovis, m. africanum, m. microti). this disease is characterized by the presence of granulomas in infected tissues involving the respiratory tract or other organs. although tuberculosis has fluctuated in its incidence over thousands of years of human history, it has remained a permanent threat to public health.6 tuberculosis disease treatment takes a long time, it can be transmitted from patients with positive sputum smears via respiration to healthy people and can cause mortality. the fight against tuberculosis requires a continuous and disciplined public health practice. because of droplet infection, each patient should be diagnosed early and treated effectively to protect public health.7,8 nucleic acid amplification methods are widely used to identify m. tuberculosis (mtb), which is difficult to see by microscopy and takes a long time to produce in culture.9,10 in parallel with the developments in molecular techniques such as polymerase chain reaction (pcr), real-time pcr, and transcriptionbased amplification (tma) have been developed for the diagnosis of tuberculosis.11;13 however, the most important disadvantage of molecular methods is that they mostly require a laboratory environment and cannot be applied as the poc tests.14;16 nucleic acid amplification tests (pcr, real-time pcr) can detect trace amounts of genetic material (dna or rna) of various pathogens in the early stage of the disease. however, the thermal cycling condition adds complexity to the way pcr devices operate.15,17 recently, various isothermal amplification methods have been developed, such as rolling circle amplification (rca), recombinase polymerase amplification (rpa), and loop-mediated isothermal amplification (lamp). among these methods, lamp is the most popular isothermal nucleic acid test for detecting viruses, bacteria, fungi, and parasites due to its low cost and operation at a single temperature.16;19 loop-mediated isothermal amplification (lamp) reaction is performed with four or six primer sets for dna/rna amplification.18 the most important advantage of the lamp method is; it provides the opportunity to reproduce target nucleic acid sequences under isothermal conditions (60-65°c) in a miniaturized environment with low energy consumption.20 proliferation; turbidity can be monitored with dyes that show the amount of fluorescence or free magnesium bound to nucleic acids. therefore, it does not require any expensive device, allowing the results to be evaluated with a simple optical system or with the naked eye. the lamp method gives more sensitive results than other amplification 3 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 1–11 methods because it provides sequence-specific visual detection of the 4/6 region on the target gene.21 however, lamp assay requires a heating block system in order to be operated as in other diagnostic techniques. therefore, new generation smart devices are needed to perform analysis in places without laboratory infrastructure. the portability of these devices, low cost, robustness, ease of use, less need for trained personnel, easy-tointerpret results, and ability to produce accurate and reliable results quickly are important.22 the aim of this study was to develop a hand-held portable device (dna hunter) for rapid and accurate diagnosis of mtb and enabling analysis without the expert in the field as point-ofcare testing. dna hunter (total weight 430 g, outer dimensions 70 x 175 x 80 mm) can be adjusted to the desired reaction temperature and time also has software where sample numbers can be entered. the device has 6 aluminum chambers and the average operating time can be realized around 90 minutes with a fully charged battery, without being connected to electricity. reservoir and the cover section have a heating function reaching a maximum temperature of 110°c in 2 minutes. the color change is measured and evaluated positively and negatively by a full digital color sensor and the result can be displayed and compared with the reference values on the screen. materials and methods materials the 93 sputum samples (68-culture positive, 25-culture negative) were used in this study, and the m. tuberculosis h37rv pasteur institute standard strain was provided by atatürk chest diseases hospital within the scope of the tubitak project (115r002). arb staining, löwensteinjensen culture (bd, new jersey, usa), and geneexpert (cepheid, california, usa) analyses were routinely performed by the institutional laboratory where sputum samples were obtained. the qiaamp dna mini kit was purchased from (qiagen, hilden, germany). lamp amplification reagents (warmstart colorimetric lamp 2x master mix) were purchased from new england biolabs (massachusetts, usa) and loopamp mtbc detection kit was purchased from eiken chemical co., ltd. (tokyo, japan). lamp primers used in this study were synthesized microsynth ag (balgach, switzerland) as hplc grade. methods lamp primers targeted specifically for the mtb is6110 gene (genbank accession number: x17348) were designed using the primerexplorer v5 program. the primers were optimized according to the protocol described in the previous study.23 the lamp primers consisted of two outer primers (f3 and b3) and two inner primers [fip (f1c + f2) and bip (b1c + b2)], and two loop primers [flp: (forward loop primer) and blp (backward loop primer)].21 for the preparation of samples, the standard strain of m. tuberculosis h37rv was used. h37rv pure culture produced in lowenstein jensen (lj) broth was homogenized with pbs (ph 6.8) in a sterile glass beaded tube, its density was adjusted according to mcfarland 1 and accepted as the main dilution. it was then diluted up to 101 in 10-fold serial dilutions starting with the main dilution. arb negative sputum samples were spiked with main stock and its serial dilution from 1/10 to 1/100000. a nonspiked sputum sample was used for negative control. dna of all sputum samples was extracted using qiaamp dna mini kit, according to the manufacturer’s instructions. the purity and quality of dna were controlled using the implen nanophotometer (implen gmbh, germany). then extracted dna was stored at –20°c until used. for the colorimetric assay, neb warmstart colorimetric lamp 2x master mix kit (table 1) was used and the process was performed according to the manufacturer’s instructions. 4 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license h. esra agel. portable and battery-operated isothermal amplification device validation table 1. colorimetric lamp reaction mixture content stock const. final const. 1x (µl) 2x master mix 2 µl 1 12,5 10x primer mix 10 µl 1 2,5 dna 40 ng/µl 1 ultrapure water 9 total 25 the limit of detection (lod) of the m. tuberculosis lamp test was determined as 102 cfu/ml in the previous study.23 in this study to test whether the dna hunter device performed the lamp reaction correctly, five different concentrations (101-105 cfu/ml) were studied above and below the lod limit. a bacteria-free sputum sample was used for negative control. the colorimetric lamp method works on the principle of changing the color of the ph indicator dye added to the reaction with the hydrogen ions formed during the reaction changing the ph of the environment.24 lamp results were evaluated based on color change, with yellow color indicating positive and pink color examined as negative in visual evaluation. however, the ph-sensitive dye in the colorimetric lamp reaction mix may not be seen as clear yellow or pink due to the amount of nucleic acid in the sample or some substances that come with the sample. the especially orange color formation can cause problems in evaluating the test. after lamp reaction orange color is formed, which is considered an intermediate result in some studies even though the reaction color change from pink to yellow was not clear. in order to interpret the unclear results, a color scale was created and shown in figure 1.25,26 figure 1. lamp reaction results; positive: yellow, negative: pink (--) strongly negative, (-) negative, (±) positive/ negative (unclear), (+) positive, (++) strongly positive.25,26 fabrication of dna hunter device the lamp method requires a constant temperature (approximately 65 degrees) during the analysis. in the first design of the device, peltier was used as the heater. however, the peltier tends to generate an excessive amount of heat, so instead of using the peltier as a heater, it was decided to use a resistor. the heater is designed to reach a maximum temperature of 110°c in 2 minutes at room temperature. the mechanical and electronic materials used were chosen to withstand temperatures of 150°c for a short period of time (maximum 2 minutes) and 120°c for an indefinite time. the temperature resistance between the top of the heater and the cover is very high. in order to keep the top cover temperature low a teflon plate with a thickness of 4 mm was used. it is one of the materials with a very low thermal conductivity coefficient (5.10-4 cal/c m. s. degree). 5 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 1–11 to prevent the heat from affecting the motherboard and graphics cards, the internal heat dissipation should be designed well. for this reason, the device’s internal structure was changed several times and the air outlet was adjusted with the appropriate fan placement. active cooling time was measured as 30 seconds while the fans were running, and the cooling time on its own (passive) was more than 10 minutes due to thermal insulation. it was observed that the warm-up cooling time was less than 2 minutes between 20 degrees and 60 degrees. dna hunter device was designed with 6 wells using solidworks® software (dss solidworks 2016) and shown in figure 2. figure 2. design of the device (a), off mode (b), and open mode (c), processor card of the device system (d), and the completed device (e) results and discussion battery and display features since dna hunter was designed to be handheld, it was equipped with a li-ion battery with a low power consumption feature. with a fully charged battery, the total power drawn was about 10 w on average, and the running time was about 90 minutes. every measuring cell was equipped with six highprecision full-color digital sensors. as a result of the system's evaluation, the color change was measured during the test and compared to the reference values that were displayed on the screen. the total weight of the unit is 430 g, and the exterior dimensions are 70 x 175 x 80 mm. the device does not require expertise to use; test protocols can be accessed by simply entering the transaction code, thanks to the 15 different protocol storage processes. it assures also three different temperature set values and warm-up times for each test process. the user is also provided with temporary or permanent correction options. test program features protocols can be quickly accessed by entering transaction codes, owing to the program's memory capacity for 15 different operations. the protocols determine three separate temperature set values and warm-up times for each test process (figure 3). figure 3. program features (a) and (b), process selection page (c), and adding new process (d) the user is given the option of temporary or permanent correction if appropriate. the results of the tests can be transferred to a microsd card and then to the host machine. in order to enter sample information on the device, a touch screen suitable for alphanumeric information entry and appropriate software was arranged. test recipes and procedures can be entered on the device with the help of the same touch screen. in order to record the date and time of the tests, a battery-protected real-time clock was used. 6 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license h. esra agel. portable and battery-operated isothermal amplification device validation monitoring temperature changes of dna hunter mtb-lamp assay requires a constant temperature which is at 65°c for 30 minutes (figure 4). to check the temperature stability of the device, every 6 wells were tested separately and also 10 repetitions were read for each well. temperature adjustment is provided in the device with an accuracy of ± 0.1°c between 50-100°c. figure 4. temperature curve of “dna hunter” device after 30 minutes, the device turned off the heating and indicated the end of the operation with an alarm sound before turning itself off at the end of the run. visualization of mtb the colorimetric lamp method works on the principle of changing the color of the ph indicator dye depending on the hydrogen ions formed during the reaction. in this study, a colorimetric lamp master mix kit was used according to the optimized protocol.23 lamp assay was performed by a thermal cycler (biorad, hamburg, germany) at the same time as carrying out the dna hunter device. lamp reaction operated under the same conditions (65°c for 30 minutes). as shown in figure 1 the color change from pink to yellow in the tubes was considered positive, and the absence of color change (pink) was considered negative at the end of the result.24 in our study, (orange), that is, unclear color formation, which creates problems in the evaluation of the results colorimetrically, was not observed. lod values on both devices were remarkably similar, which was 102 cfu/ml to monitor the performance stability of dna hunter 6 pcr wells in the device, were studied with samples contaminated with 5 different concentrations of bacteria, to evaluate whether there was a performance difference between the wells.23 for this study, 10 readings were repeated for each well. the performance measurement chart is shown in table 2. table 2. performance evaluation results of the device wells 1st well 2nd well 3rd well 4th well 5th well 6th well ★ ▼ ■ ● □ △ ▼ ■ ● □ △ ▼ ■ ● □ △ ▼ ■ ● □ △ ▼ ■ ● □ △ ▼ ■ ● □ △ 1 + + + + + + + + + + + + + + + + + + + + + 2 + + + + + + + + + + + + + + + + + + + + + 3 + + + + + + + + + + + + + + + + + + + + + 4 + + + + + + + + + + + + + + + + + + + 5 + + + + + + + + + + + + + + + + + + 6 + + + + + + + + + + + + + + + + + + 7 + + + + + + + + + + + + + + + + + + 8 + + + + + + + + + + + + + + + + + + + 9 + + + + + + + + + + + + + + + + + + 10 + + + + + + + + + + + + + + + + + + + ★ number of readings, ▼(-) strongly negative, ■(-) negative, ●(±) unclear, □ (+) positive, △(++) strongly positive 7 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 1–11 sample analysis arb staining, löwenstein-jensen culture, and genexpert analyses were performed by the institutional laboratory where sputum samples were obtained. these sputum samples were subjected to dna extraction using the qiaamp dna mini kit in our laboratory. all isolates were analyzed in parallel with the thermal cycler using the eiken loopamp kit and the dna hunter using the in-house lamp method.23 the results were demonstrated in table 3 and table 4. table 3. sputum sample results sample no *arb **lj culture gene expert eiken loopamp/ thermal cycler inhouse lamp/ dna hunter 1 ++ + + + + 2 + + + + + 3 ++ + + + + 4 5 ++ ++ + + + 6 + ++ + + + 7 ++ ++ + + + 8 ++ ++ + + + 9 10 11 ++ + + + 12 ++ ++ + 13 ++ + + 14 + ++ + + 15 ++ + + + + 16 ++ ++ + + + 17 + 18 ++ ++ + + + 19 ++ + 20 ++ + + + + 21 ++ + + + 22 + + + + 23 24 ++ + + + + 25 + + + + 26 + ++ + + + 27 + + + + 28 + + + 29 ++ + + + 30 ++ + + + 31 32 33 ++ + + + + 34 ++ + + + + 35 + 36 + + + + 37 ++ + + + + 38 ++ + + + 39 + + 40 + + + + 41 42 + + + + 43 + + + + + 44 + + + + + 45 ++ ++ + + + 46 ++ ++ + + + 47 48 49 50 + 51 ++ ++ + + + 52 + + + + + 53 ++ + + + + 54 ++ ++ + + + 55 56 + + + + + 57 + + + + + 58 + + + + 59 ++ ++ + + + 60 61 ++ ++ + + + 62 ++ + + + + 63 ++ ++ + + + 64 65 ++ ++ + + + 66 ++ + + + + 67 + + + 68 + + + + + 69 ++ ++ + + + 70 + + + + 71 + + + + 72 ++ ++ + + + 73 74 75 + + + + 76 ++ ++ + + + 77 + + + + + 78 ++ + + + + 79 + + + + 80 81 82 +++ + + + + 83 ++ ++ + + + 84 + ++ + + + 85 ++ + + + + 86 87 ++ ++ + + + 88 ++ + + + + 89 90 ++ + + + + 91 + 92 ++ ++ + + + 93 + + + + + (+) positive, (++) strongly positive, (-) negative *microscopy results; 100 microscope scanned area: no arb (-), 1-9 arb (+),10-99 arb (++).27 **culture media (lj) results; 50-100 cfu (1+), 100-200 cfu (2+).28 8 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license h. esra agel. portable and battery-operated isothermal amplification device validation table 4. analysis results of sputum samples in different techniques results arb staining lj culture gene expert eiken loopamp dna hunter positive 68 56 63 62 64 negative 25 25 23 19 22 false positive 2 6 2 false negative 12 5 6 5 total 93 93 93 93 93 according to the arb staining method of the sputum samples obtained, it was found 68/93 positive, and 25/93 negative. compared with the arb results; with the lj culture, 12 samples were obtained as false negatives. the geneexpert method detected 5 arb positive samples as negative. the false negative detection rate of the dna hunter device was the same as the gene expert method. finally, 6 false positives and 6 false negative samples were detected by the eiken loopamp lamp kit. the dna hunter device with the in-house lamp method was successful with only 2 false positives and 5 false negatives results. although various articles were published about the detection of pathogenic microorganisms by lamp assay, the use of the lamp method is not limited to pathogens, its application in the diagnosis of allergens, gmos, and cancer was reported. the importance of the lamp method increased especially during the covid19 pandemic period. the popularity of lamp depends on its ability to operate at a constant temperature. for this reason, simple heaters have been developed by researchers for usage in the field. papadakis et al. developed a realtime quantitative colorimetric lamp (qclamp) device. their device is 3dmanufactured and operates via an in-house developed smartphone application. the size and weight of this device are (11×10×10 cm;370 g.) the device employed a mini digital camera for monitoring in real-time the transition during colorimetric lamp amplification. the device’s clinical evaluation is demonstrated in cancer mutations-analysis and covid-19 testing.29 kaygusuz et al. also developed a device called diamond which is used for gmo detection. the device features are 108 g, 6 × 6 × 3 cm. the physical parts of the device were manufactured by using a 3d printer. in this device, peltier is used as a heating element, different from our study. the lamp reaction result was evaluated colorimetrically using hnb.30 liang et al. developed a handheld, automatic, and detection system-free thermal digital microfluidic (dmf) device for lamp assay. droplet manipulation and real-time temperature control systems were integrated into a handheld device31 called lampport that performed detection of trypanosoma brucei, a blood parasite (table 5). in addition to that hu et al., studied salmonella contamination on eggs with the lamp method by using genie ii (optigene, uk) instrument. this device is commercially available for lamp analysis and products can be visualized under uv light.32 table 5. comparison of isothermal amplification devices and lamp applications lamp applications devices portable monitoring of lamp results mtb in sputum dna hunter yes colorimetric cancer mutationsanalysis, covid-19 testing (papadakis et al., 2022) qclamp yes colorimetric gmo in soybean (kaygusuz et al.,2019) daimondna yes colorimetric trypanasoma brucei in blood (liang et.al.,2019) lampport yes uv light salmonella ser. enteritidis in egg products (hu et al., 2018) genie iii optigene yes uv light zika virus detection (song et al., 2016) disposible cassette yes colorimetric fecal bacteria analysis in water (lee et al.,2019) lamp pcr device yes colorimetric song et al. reported a simple, easy-to-use, lamp assay for the detection of the zika virus. the system has a disposable cassette that carries out all the unit operations from sample introduction to detection. the device reported in this study is different from the other devices shown in table 5, it can operate independently of electricity, as in our device.33 lee et al., studied lamp assay on a 9 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 1–11 portable device for the detection of indicator microorganisms in environmental water samples.34 in our device, the ability to work for 90 minutes independently without electricity is provided by a low-power consumption li-ion battery. this feature of our device provides important benefits besides being portable for analysis in the field. dna hunter was successfully used in our other study on the detection of streptococcus type a (gas).35 strength and limitation the strength of this work was supported as a research project and developed over a 48month period. there was no time or financial hardship. the lamp method used in device tests was completed as a project work package and the results were published in other articles. the weakness of the study is that the article writing phase is delayed and takes a long time after the project is completed. conclusions mtb lamp assay with the portable device; it can be used in natural disasters and war situations and/or in places with insufficient laboratory infrastructure. dna hunter can also be used as a screening test for those who stay in prisons, immigrants, refugees, asylum seekers, come from other countries with a high incidence of tuberculosis, and the homeless. mtb-lamp is a simple molecular assay that requires less than one hour to perform and can be analyzed by the naked eye. following a review of the latest research, who suggests that mtb-lamp can be used as a replacement for microscopy in the diagnosis of pulmonary mtb in adults with signs and symptoms of tuberculosis. acknowledgement i would like to thank ismail ceyhan for the sample supply, mikronet electric company for technical support, and hasan sagcan for formatting the manuscript. funding this research was supported by tubitak (the scientific and technological research council of turkey) under project 1003 115r002 entitled ‘‘development of integrated microfluidic chip based diagnostic kit for sensitive and rapid diagnosis of tuberculosis infection’’ conflict of interest the author declare that she has no conflict of interest. author contribution as a project coordinator and researcher, i carried out the relevant studies and completed the article writing. references 1. shrivastava s, trung tq, lee ne. recent progress, challenges, and prospects of fully integrated mobile and wearable point-of-care testing systems for self-testing. chem soc rev. 2020;49(6):1812–66. 2. chen h, liu k, li z, wang p. point of care testing for infectious diseases. clin chim acta. 2019;493:138–47. 3. kozel tr, burnham-marusich ar. point-of-care testing for infectious diseases: past, present, and future. j clin microbiol. 2017; 55(8):2313–20. 4. park s, zhang y, lin s, wang th, yang s. advances in microfluidic pcr for point-of-care infectious disease diagnostics. biotechnol adv. 2011; 29(6): 830-839. 10 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license h. esra agel. portable and battery-operated isothermal amplification device validation 5. markets and markets market research reports. 2021. [internet]. available from: https://www.researchandmarkets.com 6. suárez i, fünger sm, kröger s, rademacher j, fätkenheuer g, rybniker j. the diagnosis and treatment of tuberculosis. dtsch aerzteblatt int. 2019;116(43). 7. drain pk, bajema kl, dowdy d, dheda k, naidoo k, schumacher sg, et al. incipient and subclinical tuberculosis: a clinical review of early stages and progression of infection. clin microbiol rev. 2018;31(4):e00021-18. 8. palomino jc. nonconventional and new methods in the diagnosis of tuberculosis: feasibility and applicability in the field. eur respir j. 2005;26(2):339–50. 9. parsons lm, somoskövi á, gutierrez c, lee e, paramasivan cn, abimiku a, et al. laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities. clin microbiol rev. 2011;24(2):314–50. 10. nurwidya f, handayani d, burhan e, yunus f. molecular diagnosis of tuberculosis. chonnam med j. 2018; 54(1): 1–9. 11. dahiya b, prasad t, singh v, khan a, kamra e, mor p, et al. diagnosis of tuberculosis by nanoparticle-based immuno-pcr assay based on mycobacterial mpt64 and cfp-10 detection. nanomedicine. 2020;15(26):2609–24. 12. dorn-in s, gareis m, schwaiger k. differentiation of live and dead mycobacterium tuberculosis complex in meat samples using pma qpcr. food microbiol. 2019;84:103275. 13. ramezani r, moghadam mf, rasaee mj. development of sensitive and rapid rna transcription-based isothermal amplification method for detection of mycobacterium tuberculosis. avicenna j med biotechnol. 2019;11(2):169. 14. castan p, de pablo a, fern dez-romero n, rubio jm, cobb bd, mingorance j, et al. point-of-care system for detection of mycobacterium tuberculosis and rifampin resistance in sputum samples. j clin microbiol. 2014;52:502–507. 15. wei z, zhang x, wei c, yao l, li y, zhang x, et al. diagnostic accuracy of in-house real-time pcr assay for mycobacterium tuberculosis: a systematic review and meta-analysis. bmc infect dis. 2019;19(1):1–11. 16. zhang k, zhang h, cao h, jiang y, mao k, yang z. rolling circle amplification as an efficient analytical tool for rapid detection of contaminants in aqueous environments. biosensors. 2021;11(10):352. 17. de paz hd, brotons p, muñoz-almagro c. molecular isothermal techniques for combating infectious diseases: towards low-cost point-ofcare diagnostics. expert rev mol diagn. 2014;14(7):827-43. 18. lobato im, o’sullivan ck. recombinase polymerase amplification: basics, applications and recent advances. trac trends anal chem. 2018;98:19–35. 19. notomi t, mori y, tomita n, kanda h. loopmediated isothermal amplification (lamp): principle, features, and future prospects. j microbiol. 2015;53(1):1–5. 20. aglietti c, luchi n , pepori al, bartolini p, pecor if, raio a , et al. real-time loop-mediated isothermal amplifcation: an early-warning tool for quarantine plant pathogen detection. amb expr. 2019; 9:50. 21. de paz hd, brotons p, muñoz-almagro c. molecular isothermal techniques for combating infectious diseases: towards low-cost point-ofcare diagnostics. expert rev mol diagn. 2014;14(7):827-43. 22. quyen tl, van ngoc h, chidambara va, bang dd, anders wolff. loop –mediated isothermal amplification (lamp) based point-ofcare for rapid detection of pathogens recent development and future out look, annals of clinical and medical case reports. 2021;17(19). 23. agel e, sagcan h, ceyhan i, durmaz r. optimization of isothermal amplification method for mycobacterium tuberculosis detection and visualization method for fieldwork. turk j med sci. 2020;(50):1-7 24. tanner na, zhang y, evans jr tc. visual detection of isothermal nucleic acid amplification using ph-sensitive dyes. biotechniques. 2015;58(2):59–68. 25. babaa m m, bitewb m, fokamc j, leloe e a, ahidjoa a, asmamaw k. diagnostic performance of a colorimetric rt -lamp for the identification of sars-cov-2: a multicenter prospective clinical evaluation in sub-saharan africa. eclinicalmedicine. 2021;(40):101101. 26. thi vld, herbst k, boerner k, meurer m, kremer l, kirrmaier d. screening for sarscov-2 infections with colorimetric rt-lamp and lamp sequencing. medrxiv and biorxiv. 2020;(10):1101. 27. who, laboratory services in tuberculosis control, part ii: microscopy. who, geneva, 1998. (who/tb/98.258). 28. world health organization. services in tuberculosis control culture. part iii. geneva: who, 1998. 29. papadakis g, pantazis a k, fikas n, chatziioannidou s, tsiakalou v, michaelidou k, et.al. portable real time colorimetric lamp device for rapid quantitative detection of nucleic 11 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license indonesian journal of tropical and infectious disease, vol. 11 no. 1 january–april 2023: 1–11 acids in crude samples. nature.scientifc reports. 2022;(12):3775. 30. kaygusuz d, vural s, aytekin a o, lucasc s c, elitas m. daimondna: a portable, low-cost loop-mediated isothermal amplification platform for naked-eye detection of genetically modified organisms in resource-limited settings. biosensors and bioelectronics. 2019; (141):111409. 31. liang w, jie g, tianlan c, cheng d, haoran l, yan-zi w, et.al. lampport: a handheld digital microfluidic device for loop-mediated isothermal amplification (lamp). biomed microdevices. 2019;(21):9. 32. hu l, ma m l, zheng s, he x, hammack t s, brown e w, zhang g.development of a novel loop-mediated isothermal amplification (lamp) assay for the detection of salmonella ser. enteritidis from egg products. food control. 2018;(88):190-197. 33. song j, mauk m g, hackett b a, cherry s, bau h h, liu c. instrument-free point-of-care molecular detection of zika virus. anal. chem. 2016;(88):7289−7294. 34. lee s, ling v k s, medriano c a d, lee t, park s y, bae s. rapid and in-situ detection of fecal indicator bacteria in water using simple dna extraction and portable loop-mediated isothermal amplification (lamp) pcr methods. water research.2019;(160); 371-379. 35. toptan h, agel e, sagcan h, ertunç y m, elmas b, koroglu m, sengil a z, altindis m. rapid molecular diagnosis of group a streptococcus with a novel loop mediated isothermal amplification method. clin lab. 2022;(8)1:68. 36. world health organization. the use of loopmediated isothermal amplification (lamp) for the diagnosis of pulmonary tuberculosispolicy guidance. geneva: world health organization. 2016. isbn-13: 978-92-4-151118-6.