ijtid vol 8 no 2 may-agustus 2020_newfromsarah.indd vol. 8 no. 2 may–august 2020 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 original article first line anti-tuberculosis drug resistance pattern in multidrugresistant pulmonary tuberculosis patients correlate with acid-fast bacilli microscopy grading soedarsono1*, ni made mertaniasih2, titiek sulistyowati3 1department of pulmonology and respiratory medicine, faculty of medicine, universitas airlangga, surabaya, east java, indonesia 2department of clinical microbiology, faculty of medicine, universitas airlangga, surabaya, east java, indonesia 3surabaya health laboratory center, surabaya, east java, indonesia received: 10nd july 2019; revised: 29th october 2019; accepted: 19th december 2019 abstract multidrug-resistant tuberculosis (mdr-tb) is a global public health crisis. acid-fast bacilli (afb) gradation in sputum examination is an important component in pulmonary tuberculosis (ptb) diagnosis and treatment outcome monitoring. previously treated pulmonary tb patients with a higher afb smear gradation may have higher rates of acquired resistance. patients with a higher afb grade indicate a higher bacillary load and had higher rates of acquired resistance. this study aims to evaluate the correlation between afb gradation and fi rst-line anti-tb drug resistance patterns in mdr pulmonary tb patients. this was a retrospective study conducted from august 2009 to april 2018 in dr. soetomo hospital. sputum samples were taken from mdr ptb patients. sputum smear examination was done using ziehl–neelsen staining and gradation was measured according to iuatld criteria. samples with positive smear were evaluated for resistance patterns based on culture and resistance tests using the mgit 960 bactec system. there were 433 sputum samples with afb positive collected from mdr ptb patients. resistance to rhes was found in 22 (14%) afb +1, 19 (15%) afb +2, and 29 (20%) afb +3. resistance to rhs was found in 22 (14%) afb +1, 12 (9%) afb +2, and 13 (9%) afb +3. resistance to rhe was found in 39 (25%) afb +1, 38 (29%) afb +2, and 35 (24%) afb +3. resistance to rh was found in 74 (47%) afb +1, 61 (47%) afb +2, and 69 (47%) afb +3. statistic analysis by spearman test showed that there was no signifi cant correlation between afb gradation and fi rst-line anti-tb drug resistance patterns. acquired resistance to rhes can also found in lower bacillary load afb +1. keywords: mdr pulmonary tb, afb grading, fi rst line anti-tb drug resistance pattern abstrak tuberkulosis multidrug-resistant (tb-mdr) merupakan salah satu masalah kesehatan utama di dunia. pemeriksaan basil tahan asam (bta) pada sampel dahak merupakan komponen yang penting dalam diagnosis dan pemantauan hasil pengobatan pasien tb paru. pasien tb paru dengan jumlah bta yang lebih tinggi memiliki potensi tinggi terjadi resistensi obat. pasien dengan jumlah bta yang lebih tinggi menunjukkan jumlah basil yang lebih banyak dan memiliki potensi terjadi resistensi yang lebih tinggi. penelitian ini bertujuan untuk mengevaluasi hubungan antara gradasi bta dan pola resistensi obat anti-tb lini pertama pada pasien tb paru mdr. studi ini merupakan studi retrospektif yang dilakukan di rumah sakit dr. soetomo pada bulan agustus 2009 hingga bulan april 2018. sampel dahak diambil dari pasien tb paru mdr. pemeriksaan dahak dilakukan menggunakan pewarnaan ziehl-neelsen dan jumlah bta diukur sesuai dengan kriteria iuatld. sampel bta positif dilakukan evaluasi pola resistensi obat anti-tb lini pertama berdasarkan uji kultur dan resistensi dengan sistem bactec mgit 960. terdapat 433 sampel dahak dengan bta positif dari pasien tb paru mdr. resistensi terhadap rhes ditemukan pada 22 (14%) bta +1, 12 (9%) bta +2, dan 13 (9%) bta +3. resistensi terhadap rhe ditemukan pada 39 (25%) bta +1, 38 (29%) bta +2, dan 35 (24%) bta +3. resistensi terhadap rh ditemukan pada 74 (47%) bta +1, 61 (47%) bta +2, dan 69 (47%) bta +3. analisis statistic dengan uji spearman menunjukkan bahwa tidak terdapat * corresponding author: ssoedarsono@gmail.com 84 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: 83–89 hubungan yang signifi kan antara gradasi bta dan pola resistensi obat anti-tb lini pertama. pola resistensi rhes juga dapat ditemukan pada jumlah basil yang lebih rendah bta +1. kata kunci: tb paru mdr, gradasi bta, pola resistensi obat anti-tb lini pertama how to cite: soedarsono, mertaniasih nm, sulistyowati t. first line anti-tuberculosis drug resistance pattern in multidrug-resistant pulmonary tuberculosis patients correlate with acid-fast bacilli microscopy grading. indonesian journal of tropical and infectious disease, 8(2), 1–8. introduction drug-resistant tuberculosis (dr tb) continues to be a public health crisis. in 2017, around 558,000 people in the world developed rifampicin-resistant tb (rr-tb) and 82% had multidrug-resistant tb (mdr-tb).1 mdr-tb is defi ned as tb which caused by strain mycobacterium tuberculosis resistant at least to isoniazid (h) and rifampicin (r), two of the main fi rst-line anti-tb drugs.2 first-line anti-tb drugs consist of isoniazid (h), rifampicin (r), pyrazinamide (z), ethambutol (e), and streptomycin (s). globally, indonesia is the 7th rank in the estimated incidence of rrtb cases in 2017 is 23.000 people with mdr percentage among rr-tb cases was 91%.1 from all of tb cases, 2.4% of new tb cases and 13% of previously treated cases had mdr/ rr-tb. this means the miss management of tb cases is still dominant as the cause of dr tb. drug resistance occurs when drug-susceptible tb (ds tb) patients receive inadequately or interrupted therapy which leads to the selection of drug-resistant bacteria and ‘acquired’ drug resistance. infectious patients who are infected by resistant strain mycobacterium tuberculosis could spread through airborne droplets as transmitted drug resistance.3 a c i d f a s t b a c i l l i ( a f b ) m i c r o s c o p y examination is a common simple tool for the diagnosis and treatment outcome monitoring of pulmonary tb.4 patients with higher afb grade indicates higher bacillary load and increasing baseline drug resistance had higher rates of acquired resistance.5 the recent dogma stated that the level of resistance to inh and rif (required for mdr-tb) was caused by the individual mutation rates for inh and rif; that is, in the order of 10-6. for the evolution of mdr strains, a total population of at least 106 bacilli must be present in each infected person.6 the possibility that a single drug-resistant mutant may arise earlier after infection, and could replicate to a large enough population from which the possibility of a second drug-resistance mutation will not be too slow.7 the potential drug-resistant mutation is varied in each drug, ranging from around 1 in 108 bacilli for rifampicin, to about 1 in 106 bacilli for isoniazid, streptomycin, and ethambutol. besides, mycobacterium tuberculosis consists of various phylogenetic lineages,8 that could have some intrinsic drug resistance character in the bacilli population of the ptb patients. on the other hand, mdr-ptb cases with several an active disease process with afb bacilli production in sputum with many population characteristics of anti-tb resistance that related to multi factors. some clinicians assume that more amount of afb can cause acquired more drug resistance. this study aims to determine the drug resistance pattern of all positive smear in mdr ptb patients and evaluate its correlation with afb microscopy grading. materials and methods study defi nition patients were divided by a history of previous tb treatment according to who guideline9 1. new cases: who have never been treated for tb or have taken anti-tb drugs for less than 1 month. 2. previously treated patients have received 1 month or more of anti-tb drugs in the past. they are further classifi ed by the outcome of their most recent course of treatment: 85soedarsono, et al.: first line anti-tuberculosis drug resistance pattern copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 a. relapse patients have previously been treated for tb, were declared cured or treatment completed at the end of their most recent course of treatment, and are now diagnosed with a recurrent episode of tb (either a true relapse or a new episode of tb caused by re-infection). b. treatment after failure: patients are those who have previously been treated for tb and whose treatment failed at the end of their most recent course of treatment (who category i regimen or who category ii regimen). who category i regimen: 2 (hrze)/ 4(hr)3 or 4(hr) who category ii regimen: 2 (hrze)s/ (hrze)/ 5(hr) 3e3 or 5(hr)e c. treatment after loss to follow-up: patients have previously been treated for tb and were declared lost to follow-up at the end of their most recent course of treatment (these were previously known as a treatment after default patients). d. other previously treated patients are those who have previously been treated for tb but whose outcome after their most recent course of treatment is unknown or undocumented. study subjects and design this was a retrospective study. samples were collected from all mdr pulmonary tb (mdr ptb) patients who are treated from august 2009 to april 2018 in dr. soetomo hospital. the medical records of enrolled patients were reviewed to obtain their microbiological examinations. sputum samples were taken from new and previously treated mdr ptb patients. sputum smear examination was done using ziehl–neelsen staining. direct smears were made from each sputum sample and were stained with ziehl-neelsen (zn) stain according to the who recommendation. afbs identifi ed were graded according to the international union against tuberculosis and lung disease (iuatld) and the who smear grading scale. findings were scored as follows: 1–9 afb/100 fi elds (1+); 1–9 afb/10 fi elds (2+); and 1–9 afb/ fi eld (3+). each slide was examined by three independent readers to ascertain the presence of afb and grade positive smears. the slide readers were blinded on the clinical and laboratory diagnoses of the participants whose samples were studied. samples with positive smear were evaluated for resistance pattern based on culture method using mgit 960 bactec system for determinate the sensitivity to rifampicin (r), isoniazid (h), ethambutol (e), and streptomycin (s). examination of microscopic sputum smears, culture method for identification and drug sensitivity test were carried out at the surabaya health laboratory center which has been certifi ed by who. statistic analysis using the spearman test was used to analyze the signifi cance of afb grading and resistance pattern. results and discussion there were 433 mdr-tb patients with positive smear, 253 (58.4%) men and 180 (41.6%) women in mdr-tb clinic care of dr. soetomo hospital from august 2009 to april 2018. the number of mdr ptb patients were higher in men than women in this study with 253 (58.4%) and 180 (41.6%) women, respectively. another study also found that the mdr/rr tb strains were three times more common in men than women.10 being a man or woman can be a factor to develop drug resistance; however, the fi ndings vary on the subject. a global prevalence study did not fi nd sex to be a risk factor for mdr-tb.11 the average age of mdr ptb patients was 43.82 years old and most mdr tb patients were productive with age range 15-49 year-old with a count of 291 (67.2%). globally, there were cases in all countries and age groups but overall 90% were adults (aged ≥15 years).1 a study in switzerland reported that age <35 years old increased risk of resistance to fi rst-line drugs (or=1.5; 95% ci 1.0–2.3).12 based on tb treatment history, mdr ptb patients were divided into new cases and previously treated cases (relapse, return after 86 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: 83–89 default, failure of the who category i, failure of the who category ii, and other cases such as unstandardized treatment). most of mdr ptb patients were ones with previously treated with 426 (98%). relapse cases were dominant with 160 (36.9%), followed by failures of the who category i regimen with 110 (25.4%), and return after default with 91 (21%). this result was shown in table 1. there were 426 (98%) of mdr-tb patients were coming from patients with the previous history of tb treatment in this study. previously treated tb patients were a risk factor for mdrtb.13 previous anti-tb treatment was by far a solid predictor of drug resistance.14 previously treated tb patients had a higher chance as many as 8.1 times to develop an mdr-tb infection compared to newly diagnosed tb patients.15 in this study, relapse cases were the most common with 160 cases (36.9%), followed by failures of the who category i regimen with 110 cases (25.4%). relapse cases were dominant among patients with mdr-tb in this study. the previous study reported that most of drug-resistant tb were relapse cases with 123/290 patients (42.4%), followed by treatment failures with 123/290 (34.8%).16 the dominance of relapse cases among mdr-tb patients may caused by inadequate treatment and less compliance of patient during previous treatment resulted dormant mdr-tb. subsequently, the survival of dormant mdr-tb increased the risk of tb relapse.17 the dominance of relapse cases also happened because tb recurrence resulted from either relapse or reinfection was remained defi ned as relapse according to the who guideline. to defined relapse or reinfection cases, the examination of mycobacterium tuberculosis strain was needed to know whether it was relapse of an original infection or exogenous reinfection with a new mycobacterium tuberculosis strain. in the previous study, 51.4% of relapse happened in ≤2 years and 48.6% of relapse happened in >2 years, while 57.1% of reinfection happened in >2 years and 42.9% reinfection happened in ≤2 years.17 although new tb diagnosing technologies have been improved, the use of afb microscopy still the main of the diagnostic18 and patients with positive afb are often considered as mdr-tb due to greater afb leads the bacterial mutation. patients with higher bacterial load are more potential for drug-resistant mutations and have a greater risk of developing mdr-tb.19 initial afb sputum smear ≥3+ was correlated with acquired drug resistance.5 of the 433 sputum samples with afb positive collected from mdr ptb patients, resistance to rhes was 14% in afb +1, 15% in afb +2, and 20% in afb +3. resistance to rhs was 14% in afb +1, 9% in afb +2, and 9% in afb +3. resistance to rhe was 25% in afb +1, 29% in afb +2, and 24% in afb +3. resistance to rh was 47% in afb +1, 47% in afb +2, and 47% in afb +3. based on statistic analysis by spearman test, there was no signifi cant correlation between afb gradation and resistance pattern with p-value 0.786 as presented in table 2. the results in table 2 showed that resistance to more drugs was also happened by the lower afb grading (afb +1) and indicated that the grade of afb might not represented the number of mycobacterium tuberculosis. afb-positive smears may be because of the presence of table 1. history of tb treatment profi le of mdr tb patients in dr. soetomo hospital. variable r+h r+h+e r+h+s r+h+e+s total new cases 3 (43%) 3 (43%) 0 (0%) 1 (14%) 7 previously treated cases 201 (47%) 109 (26%) 47 (11%) 69 (16%) 426 • failure treatment with who category ii regimen 19 (34.5%) 16 (29%) 8 (14.5%) 12 (22%) 55 • failure treatment with who category i regimen 53 (48%) 29 (26%) 8 (7%) 20 (18%) 110 • relapse 84 (52.5%) 39 (24%) 17 (11%) 20 (12.5%) 160 • return after default 43 (47%) 22 (24%) 14 (15%) 12 (13%) 91 • other case 2 (20%) 3 (30%) 0 (0%) 5 (50%) 10 87soedarsono, et al.: first line anti-tuberculosis drug resistance pattern copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 nonviable mycobacterium tuberculosis bacilli or nontuberculous mycobacteria (ntm).20 our study found that the afb grading did not represent the resistance pattern of fi rst-line anti-tb drugs. afb +1, which was the lower bacillary load, also showed resistance to rhes. based on statistical analysis using the spearman test, afb grading was not correlated with the resistance pattern of mdr tb patients with p 0.786. this result showed that the bacillary load did not aff ect the resistance to some tb drugs. a diff erent result was shown by another study that reported higher smear grade (+2 and +3) has a higher rate of mdr-tb/ rif resistance with 76/256 (29.7%) compared with smear grades of +1, scanty positive and negative with 61/301 (20.3%) (p-value = 0.01).10 there was no reveal the correlation of the first-line anti-tb drug resistance pattern with afb grading in this study. resistance to more drugs (rhes) also found in patients with afb +1. analysis of correlation between afb grading and every treated group showed that there was not a signifi cant diff erence with a p-value of 0.895 as presented in table 3. the defi nition of each group has been described in the methodology. the results in table 3 showed that the afb grading was not aff ected by the history of tb treatment. actually, afb smear can be used to assess tb treatment outcome, but careful examination of microbiologic status, including culture and drug susceptibility testing were also needed to confirm the afb smear examination.4 greater afb grading is often considered associated with the incidence of drug resistance. a higher afb grading represented higher bacilli and it possible to acquired drug resistance. acquired resistance to rifampicin was estimated by mutation of 108 bacilli and acquired resistance to isoniazid, streptomycin, and ethambutol by mutation of 106 bacilli.21 this rate might also be aff ected by the drug concentration in the medium, the drug resistance profi le of the strain and its genetic background.22 drug resistance-associated genes were katg and inha in isoniazid, rpob in rifampicin, rpsl in streptomycin, and embb in ethambutol.23 previous studies reported that there were varies drug resistance patterns among sputumsmears positive; mdr-tb, non-mdr two drug resistance, and resistance to any one of the fi rst line of drugs (isoniazid, ethambutol, and rifampicin).24 table 2. analysis of correlation between afb grading and the fi rst line anti-tb drug resistance pattern. afb grading resistance pattern total p value r+h+e+s r+h+s r+h+e r+h +++ 29 (20%) 13 ( 9%) 35 (24%) 69 (47%) 146 ( 34%) 0.786 ++ 19 (15%) 12 ( 9%) 38 (29%) 61 (47%) 130 ( 30%) + 22 (14%) 22 (14%) 39 (25%) 74 (47%) 157 ( 36%) total 70 (16%) 47 (11%) 112 (26%) 204 (47%) 433 (100%) *p value based on spearman test. correlation coeffi cient (0.013). table 3. correlation between afb grading vs. every treated group. history of tb treatment afb p-value + ++ +++ new cases (n=7) 2 (28.5%) 2 (28.5%) 3 (43%) 0.895 failure treatment with who category ii regimen (n=55) 18 (32.7%) 18 (32.7%) 19 (34.6%) failure treatment with who category i regimen (n=110) 43 (39%) 31 (28%) 36 (33%) relapse (n=160) 53 (33%) 49 (31%) 58 (36%) return after default (n=91) 37 (40%) 27 (30%) 27 (30%) other case (n=10) 4 (40%) 3 (30%) 3 (30%) 88 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: 83–89 acquired resistance to more drugs may correlate with mycobacterium tuberculosis strain in mdr pulmonary tb patients. different strain of mycobacterium tuberculosis also represented diff erent frequencies of genes which played role in drug resistance. the prevalence of specifi c drug resistance-associated mutations also varies within the lineage, such as the frequencies of the rpob s531l and katg s315t mutations are greater in the modern (typical) beijing strains than in ancient (atypical) ones. there was a signifi cant variation in the mutation rates of strains, the study also showed that strains from lineage 2 of mycobacterium tuberculosis (includes beijing family of strains) acquire drug resistance in vitro rapidly than strains from lineage 4.22,25 conclusions there was no signifi cant correlation between the fi rst-line anti-tb resistance pattern of mdr ptb strain with afb microscopy grading. acquired resistance to rhes can also found in lower bacillary load afb +1. acknowledgement the authors would like to thank mrs. atika, m.sc., department of public healthpreventive medicine, faculty of medicine, universitas airlangga who helped us in data analysis. conflict of interest there is no confl ict of interest of this paper. references 1. world health organization. global tuberculosis report 2018.geneva: who; 2018. 2. world health 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mycobacterium tuberculosis mutation rate estimates from different lineages predict substantial diff erences in the emergence of drug resistant tuberculosis. nat genet. 2013; 45(7); 784–90. 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 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(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 ijtid vol 3 no 1 jan-maret 2012.indd 15 vol. 3. no. 1 january–march 2012 mycobacteria and other acid fast organisms associated with pulmonary disease in jos, nigeria pulmonary disease and acid fast organisms ani ae1, diarra b2, dahle ur3, lekuk c4, yetunde f4, somboro am2, anatole tounkara2, idoko j5 1 department of medical microbiology, university of jos, nigeria 2 hiv immunology and tb laboratory, serefo, univesity of bamako, mali 3 norwegian institute of public health, oslo 4 apin/pepfar laboratory, jos university teaching hospital, jos, nigeria 5 department of medicine, university of, jos, nigeria abstract objective: acid fast bacilli (afb) for sputum smear microscopy is the affordable method used for prompt diagnosis of tuberculosis in nigeria despite its lack of specificity and limited sensitivity. the study aims to identify mycobacterium tuberculosis and other acid fast organisms isolated from sputum of of hiv positive adult patients with pulmonary disease in jos, nigeria. methods: acid fast organisms isolated from 80 afb positive sputa of hiv positive adult patients suspected for tuberculosis in jos, nigeria were identified for members of m. tuberculosis complex (m tuberculosis, m bovis, m africanum, m canetti m. microti and m. caprae) by use of spoligootyping, multiplex gen probe, hain genotype assay and gene sequencing for spoligotype negative isolates. results: seven different spoligotypes of m. tuberculosis complex were identified from 70/80 (87.5%) total number of isolates. m. kansasii (1), m. dulvalii (1) nocardia species (1) and tsukamurella species (2) were detected from 5/10 spoligotype negative isolates. conclusion and recommendation: although m. tuberculosis is the dominant afb associated with chronic pulmonary disease in jos, nigeria, other clinically relevant mycobacteria were observed in the study. this suggests that other afb positive microorganisms associated with tuberculosis -like symptoms could be misdiagnosed and incorrectly treated as m. tuberculosis. it is therefore necessary for laboratories in tb high burden countries to step up diagnostic procedures beyond routine smear microscopy. key words: acid fast bacilli (afb) mycobacteria tuberculosis, other mycobacteria species introduction mycobacterium tuberculosis is a pathogenic species of the genus mycobacteriaceae and the agent of human classical tuberculosis. the less virulent non tuberculous mycobacteria (ntm) found in environments such as dust and running surface waters1–3 are morphologically indistinguishable from m. tuberculosis. although not transmissible from human to human, ntms cause opportunistic infection capable of multifocal organ involvement in humans, and more frequently chronic lung diseases.4-5,2 infection of the lungs may be similar to classical tuberculosis but more difficult to treat and if necessary, prolonged treatment periods may be required.6–7 hiv positive and severely immunocompromised persons are at high risk due to very low cd4 counts.8–10 the lack of sensitive identification methods in most clinical laboratories may predispose to misdiagnosis of ntm disease for tuberculosis especially in resource limited settings that rely only on afb smear microscopy for tb diagnosis. although ntms have been associated with primary disease in severe immunodeficiency conditions, it could also constitute a secondary infection in active tb or after tb therapy.11 it is therefore necessary to carryout comprehensive clinical and radiological investigations in infected persons, to understand the pathological role of ntm when isolated. establishment of referral centers including expert physicians in ntm treatment and management has been recommended. published studies on mycobacterium infections are scarce in nigeria in spite of high burden of hiv and tb and the prevalence of atypical mycobacteria associated research report 16 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 15−18 with pulmonary disease is not known. reports from other countries have demonstrated that atypical mycobacterial infections are associated with hiv positive persons, other immunocompromised patients and transplant receivers.12–13 conventional methods14-16 for identification of mycobacterium species are time consuming and often not specifically conclusive in species identification, while the newer biochemical (high performance liquid chromatography) and some of the highly specific molecular methods17-19 are not cost effective for use in routine clinical laboratories. spoligotyping,20 a simple pcr based method distinguishes members of m.tuberculosis complex in clinical specimens or culture. the procedure, though not cost effective for routine use, has been widely applied in molecular epidemiology and identification of m tuberculosis complex. we identified acid fast bacilli isolated from sputa in jos nigeria, where smear microscopy has been the most widely used laboratory method for tb diagnosis. the study examined 80 consecutive isolates from cases of pulmonary tuberculosis. materials and methods ethical consideration the study which was respectively approved by the ethical committee of the jos university teaching hospital and the plateau state hospital jos, nigeria, was descriptive of a bacterial collection and contained no material of human origin. personal data were removed from all bacterial cultures to protect the anonymity of the patients. ethical clearance was granted with no requirement for patient informed consent. eighty afb positive isolates from 94 afb positive sputa were identified by spoligotyping, genprobe, hain genotype and 16s ribosomal dna gene sequencing. the strains were isolated during january 2008 to december 2009 from 790 total number of hiv patients suspected for tuberculosis in jos, nigeria. sputum specimens were collected in 1ml solution of 1% cetyl pyridinum chloride (cpc) with 2% sodium chloride and processed for culture on lowenstein jensen (lj) medium.8 afb smear microscopy was used for preliminary identification of suspect isolates. afb positive cultures on lj slants were subcultured and preserved at -20° c and subsequently shipped to seefo nih tb/imunology laboratory mali for spoligotyping and multiplex geneprobe. spoligotyping was performed as described by kermerbeek et al.20 unidentified species were sent to the norwegian institute of public health oslo for sequencing. results seventy of the 80 (88%) total number of isolates were m. tuberculosis complex spoligotypes; latin america mediterranean family (lam) 75.6%, t (10%), haarlem (4.3%), m. africanum (2.9%) eai (5.7%), f (1.4%). only one (m. kansasii) of the 10 spoligotype negative isolates were identified by geneprobe, 4 others; m. duvalii (1), norcardia asteroids (1) and tsukamurella species (2) were detected by 16s rrna by gene sequencing while 5/10 isolates were lost to contamination. these results illustrate the importance of further investigation of afb cases to exclude other mycobacteria/ n o n m y c o b a c t e r i a l m i c r o o r g a n i s m s , e s p e c i a l l y in immunosuppressed patients suspected of having tuberculosis. table 1. genus actinomycetes isolated from sputa of pulmonary disease cases in jos, nigeria n = 80 no of isolates % m. tuberculosis 70 87.5 ntm 2 2.5 nocardia spp 1 1.2 tsukamurella spp 2 2.5 total 75* 93.7* *five isolates were lost to contamination table 2. spoligotypes of m tuberculosis complex isolated from jos, nigeria mtb family number % lam 10 47 67 lam 8 6 8.6 haarlem 3 4.3 eai 4 5.7 f 1 1.4 m 2 2.9 t 7 10 total 70 99.9 discussion the detection of 88.5% m tuberculosis complex by spoligotyping confirms that m. tuberculosis is the major cause of chronic pulmonary disease in jos nigeria and that the use of smear microscopy for prompt and presumptive diagnosis of m tuberculosis remains an effective and relevant tool especially in a resource limited setting lacking the more sensitive technological implements for more 17ani et al.: mycobacteria and other acid fast organisms associated accurate and rapid diagnosis. the findings in this study agrees with others in some countries where a declining incidences of tuberculosis have been reported following the practice of the directly observed treatment short course (dots).21-22 however, the emergence of drug resistance tb or the non eradication of acid fast bacilli after successful completion of therapy with first line anti tuberculosis drugs remains a concern. the prevalence of 10/80 (12%) afb positive and spoligotype negative isolates in this study calls to question the position of some of the cases that failed eradication with consistent acid fast positive smears after completion of treatment with first line anti tuberculosis drugs. the detection of m. kansasii (1), m. duvalii (1), nocardia spp (1) and tsukamurella spp (2) from the 5 available isolates may not be unrelated to such cases. the pathogenic relevance of the isolates could not be explained from the available data in this study even though all five isolates were from sputa of new cases which apparently qualified the patients for recruitment under the dots tb treatment program. m. kansasii could be clinically relevant as it has been known to cause tuberculosis -like pulmonary disease in humans.2,23-24 nocardi spp and tsukamurella spp have also been associated with pulmonary disease in humans.8,25-26 there are scare reports associating m. duvalli with human infection although it has been reported to have some antigenic relatedness with m. leprae27 and also was reported in hiv patient in india.28 all three genera (mycobacteria, nocardia, tsukamurella) belong to the same family actinomycetales with mycolic acid cell walls.29-30 further studies are intended to ascertain the followup treatment outcome of ntm isolates in cases treated with conventional anti tb regimen in jos nigeria. only 94 of 790 (12%) total number of patients suspected for tuberculosis had afb positive smear sputa. this is less than 25% estimated prevalence of tb in hiv positive cases in nigeria. it is possible that some of the patients were unable to expectorate detectable levels of bacilli in sputa due to hiv immunosuppression. hiv and tb endemic countries need to step up laboratory diagnostic facilities to include more sensitive detection methods such as the nucleic acid amplification test (naat) to enable effective detection and treatment of ntm as well as other non mycobacteria pulmonary diseases. this would prevent unnecessary rise in drug resistant mycobacteria species. the concept which suggests that non specific cross immunity develops due to latent tb against the atypical mycobacteria especially in m. tuberculosis endemic countries10 may not significantly apply in hiv/tb endemic communities like nigeria. the dominance of lam 10 family of m tuberculosis in this and a previous study31 needs to be investigated further to establish the transmission pattern of tuberculosis in jos. although lam is generally reported in other west african countries,32-34 the unique homogeneity of lam 10 seen in nigeria has not been reported elsewhere. we have previously suggested that the dominance of lam family in nigeria and west africa may be a result of the historic interactions between west africa and south america of which the nigerian sea coasts served as major export route.31 the limitations of the study included the inability to define the clinical relevance of other acid fast bacilli isolated. however, the results illustrate the importance of investigating for ntms and other non mycobacatrial afb in clinical specimens (sputa) especially in immunosuppressed patients. such organisms may colonize the airways and cause life threatening diseases. precise identification of some genera and species requires advanced methodologies which are not readily available in several high tb burden countries. acknowledgement part of this study was accomplished through fellowships granted by the west african health organization (waho) and the norwegian institute of public health (niph), oslo. waho and niph have no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. no additional external funding received for this study. we acknowledge the skilled technical staff of department of bacteriology and immunology, niph. conflict of interest: none author contributions aea and urd conceived and designed the study, cl and yf did the pre analytical processing of specimens and data arrangement, urd did the gene sequencing while aea, bd, urd, and sm performed the other assays and analyzed the data. aea, urd and ji wrote the report which was reviewed and approved by all authors. references 1. primm tp, christie a. lucero1, joseph o falkinham iii2. health impacts of environmental mycobacteria. clin microbiol rev 2004; 17: 98–106. 2. griffith de, aksamit t, brown-elliot ba, catanzaro a, daley c, gordin f, et al. an official ats/idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. am j respir crit care med 2007; 175: 367–416. 3. national jewish health (2009). lung line 1-800-222-lung. 4. o'brien rj, deiter lj, snider jr de. the epidemiology of non tuberculous mycobacterial disease in the united states. results of a national survey. am rev resp dis 1987; 135: 1007–14. 5. marras tk, chedore p, ying am, jamieson f (2007). isolation prevalence of non tuberculous mycobacteria in ontario 1997-2003. thorax 62: 661–6. 6. banks j, jenkins pa combined versus single antituberculosis drugs on the invitro sensitivity patterns of non tuberculous 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sola c, rastogi n, vincent v, gutierrez mcgenetic biodiversity of mycobacterium tuberculosis complex strains from patients with pulmonary tuberculosis in cameroon. j clin microbiol 2003 41: 2547–2553. 33. easterbrook pj, gibson a, murad s, lamprecht d, ives n, ferguson a, lowe o, mason p, ndudzo a, taziwa ahigh rates of clustering of strains causing tuberculosis in harare, zimbabwe: a molecular epidemiological study. j clin microbiol 2004, 42: 4536–4544. 34. eldholm v, matee m, mfinanga s, heun m, dahle ura. first insight into the genetic diversity of mycobacterium tuberculosis in dar es salaam, tanzania, assessed by spoligotyping. bmc microbiol 2006. 6: 76. 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. vol. 7 ● no. 5 may-august 2019 e issn 2356–0991 p issn 2085–1103 e-journal.unair.ac.id/index.php/ijtid indexed by: combined target site vgsc mutations play a primary role in pyrethroid resistant phenotypes of aedes aegypti as dengue vector from palu city, central sulawesi acid-fast bacilli conversion of beijing and non-beijing strain of pulmonary tuberculosis in south sulawesi anti-dengue type 2 virus activities of zinc (ii) complex compounds with 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen-4-one ligands in vero cells overview of nuclear factor-b (nf-b) and non-structural protein 1 (ns1) in patients with dengue fever in premier hospital, surabaya detection of tumor necrosis factor- (tnf-) gene promoters polymorphism among liver cirrhosis patients with chronic hepatitis b virus (hbv) infection in surabaya, indonesia microbial pattern and antibiotic susceptibility in pediatric intensive care unit dr. soetomo hospital, surabaya e issn 2356 0991 p issn 2085 1103volume 7 number 5 may–august 2019 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 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 196/04.19/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 5 may–august 2019 1. combined target site vgsc mutations play a primary role in pyrethroid resistant phenotypes of aedes aegypti as dengue vector from palu city, central sulawesi purwaningsih, sitti rahmah umniyati, budi mulyaningsih ....................................................... 93–98 2. acid-fast bacilli conversion of beijing and non-beijing strain of pulmonary tuberculosis in south sulawesi syahridha, ni made mertaniasih, sustini florentina, soedarsono ............................................. 99–104 3. anti-dengue type 2 virus activities of zinc (ii) complex compounds with 2-(2,4-dihydroxyphenyl)3,5,7-trihydroxycromen-4-one ligands in vero cells teguh hari sucipto, harsasi setyawati, siti churrotin, ilham harlan amarullah, sri sumarsih, puspa wardhani, aryati, soegeng soegijanto ..................................................... 105–108 4. overview of nuclear factor-b (nf-b) and non-structural protein 1 (ns1) in patients with dengue fever in premier hospital, surabaya ni nyoman budiutari, yoes prijatna dachlan, dan jusak nugraha .......................................... 109–114 5. 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 wungu, mochamad amin, s. eriaty n. ruslan, priyo budi purwono, ulfa kholili, poernomo boedi setiawan, maria inge lusida, soetjipto, retno handajani ...... 115–121 6. microbial pattern and antibiotic susceptibility in pediatric intensive care unit dr. soetomo hospital, surabaya i wayan putra, arina setyaningtyas, dwiyanti puspitasari, irwanto, agung dwi wahyu, ira dharmawati, abdul latief azis, kuntaman ........................................................................... 122–130 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. 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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. 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 article categories c-reactive protein and hepcidin in non-dialysis chronic kidney disease edward muliawan putera1, widodo1,2, nunuk mardiana1,2 1 nephrology division, internal medicine department, faculty of medicine, universitas airlangga, surabaya, indonesia; and 2 dialysis instalation, dr. soetomo general hospital, surabaya, indonesia; received: 22nd january 2019; revised: 22nd february 2019; accepted: 23rd february 2020 abstract complications such as anemia and its clinical consequences arise as chronic kidney diseases progress,. one renal anemia pathophysiology is a disruption of iron metabolism, regulated by the main iron exporter hormone, hepcidin. chronic kidney disease patients were constantly in an infl ammatory state, represented by an increased in c-reactive protein. this infl ammatory state would facilitate the liver to secrete hepcidin, which would subsequently follow a decrease of iron circulation, thus resulting in functional iron defi ciency. both acute phase reactants which used thoroughly as markers in tropical and infectious diseases, had their own roles in chronic kidney disease. the correlation of c-reactive protein and hepcidin in chronic kidney disease patients was still controversial. to analyse the relationship between c-reactive protein and hepcidin in non-dialysis chronic kidney disease patients. we conducted an observational cross-sectional study with 40 non-dialysis chronic kidney disease patients who met the inclusion and exclusion criteria. patients were enrolled with consecutive sampling and were examined for serum c-reactive protein and hepcidin levels.a total of forty subjects (67.5% male with mean age of 50.23 ± 1.04 years) were eligible for enrolment in this study. the most comorbid factor was hypertension (62.5%). the common stage for chronic kidney disease was stage 3 (40%). the mean hemoglobin value was 10.74 ± 0.36 g/dl, mean blood urea nitrogen was 39.98 ± 29.59 mg/dl, and serum creatinine of 4.12 ± 3.39 mg/dl. mean serum c-reactive protein levels were 3.52 ± 5.13 mg/l. mean hepcidin level were 94,03 ± 95,39 ng/ml. serum c-reactive protein levels correlated positively (r=0.487) and signifi cantly (p-value=0.001) with serum hepcidin value. c-reactive protein and hepcidin was signifi cantly correlated in non-dialysis chronic kidney disease patients. keywords: crp; hepcidin; ckd; non-dialysis; iron; liver abstrak progresivitas penyakit ginjal kronis akan membawa komplikasi anemia dengan berbagai konsekuensi klinis. salah satu patofi siologi anemia pada penyakit ginjal kronis dapat diakibatkan oleh gangguan metabolisme besi yang diatur oleh hormon eksporter utama besi yaitu hepsidin. pasien penyakit ginjal kronis berada dalam kondisi infl amasi, yang diwakili dengan peningkatan c-reactive protein. adanya infl amasi akan menyebabkan liver mensekresi hepsidin yang kemudian berdampak pada menurunnya kadar besi dalam sirkulasi yang dapat berdampak pada anemia defi siensi besi fungsional. kedua reaktan fase akut yang biasa digunakan dalam penyakit tropik dan infeksi, ternyata juga memiliki peran dalam penyakit ginjal kronis. hubungan antara c-reactive protein dengan hepsidin pada penderita penyakit ginjal kronis nondialisis masih menjadi kontroversi. menganalisis hubungan antara kadar c-reactive protein dengan hepsidin pada pasien penyakit ginjal kronis yang belum menjalani hemodialisis. studi ini adalah studi analisis observasional cross sectional, diikuti 40 pasien penyakit ginjal kronis yang belum menjalani dialisis yang sesuai dengan kriteria inklusi dan eksklusi. subjek penelitian di ambil secara konsekutif dan diperiksakan kadar c-reactive protein serum dan hepsidin serum. empat puluh subjek penelitian ini, terdiri dari 27 subjek laki-laki dan 13 subjek perempuan dengan rerata usia 50,23 tahun. penyakit komorbid terbanyak adalah hipertensi (62,5%). stadium terbanyak adalah stadium 3. rerata kadar hemoglobin pada penelitian ini sebesar 10,74 ± 0,36 g/dl, rerata blood urea nitrogen 39,98 ± 29,59 mg/dl, dan rerata serum kreatinin sebesar 4,12 ± 3,39 mg/dl. rerata kadar c-reactive protein serum sebesar 3,52 ± 5,13 mg/l. rerata kadar hepsidin serum sebesar 94,03 ± 95,39 ng/ml. pada * corresponding author: nunuk43mardiana@gmail.com 162 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: 161–167 penelitian ini diperoleh hubungan positif (r=0,487) yang signifi kan (p=0,001) antara c-reactive protein dan hepsidin. didapatkan hubungan positif yang signifi kan antara kadar c-reactive protein dan hepsidin pada pasien penyakit ginjal kronis non-dialisis. kata kunci: crp; hepsidin; pgk; non-dialisis; besi; liver how to cite: c-reactive protein and hepcidin in non-dialysis chronic kidney disease. putera, e. m., widodo, mardiana, n. indonesian journal of tropical and infectious disease, 8(3), 161–167. introduction hepcidin and c-reactive protein (crp) had their roles in infectious diseases for a period of time. hepcidin lowered mammal’s blood iron levels at the time the pathogen-infected the hosts. low blood iron level hindered pathogen’s growth so that infections might be stopped. c-reactive protein had its own roles in activating platelets, leukocytes, endothelial growth factors, complements and chemokines during infections to cease infection. however, the roles of these two markers in renal anemia in chronic kidney disease (ckd) have not been elucidated yet. c-reactive protein has been one of the sensitive inflammation markers which correlate with hepcidin in ckd. there have been substantial studies to backed up and come against it. chronic kidney disease, stated as a chronic state of lowgrade infl ammation, could initiate a chain of sequences that lead to secretion of crp and hepcidin. however, hepcidin was fi rst recognized by ganz, et al. as liver expressed antimicrobial peptide-1 (leap-1) secreted during infection or high-grade infl ammation, putting hepcidin into lower place in this chain of sequences than crp. c-reactive protein was proven to be inversely correlated with the estimated glomerular fi ltration rate (egfr) and stage in ckd. crp also correlated with other infl ammation markers such as interleukin-6 (il-6). interleukin-6 was directly correlated with the secretion of crp and hepcidin in the human liver. hepcidin is a major iron exporter hormone in mammals. it interacts with its receptor, ferroportin in gastrointestinal tracts and reticuloendothelial systems. degradation and internalizing process of ferroportin inhibits daily iron intake entering circulation from duodenum and traps intracellular storage iron. these processes create a hypoferremia state which results in functional iron defi ciency anemia. anemia brings clinical consequences such as a decrease of quality of life, deterioration of egfr, increased cardiovascular events, increased mortality rate, and even increased economical burden. high infl ammation state and other confounding factors (anemia, duration of dialysis) was seen in ckd patients on dialysis which lead to sample selection of non-dialysis patients. materials and methods s t u d y d e s i g n : t h i s w a s a n a n a l y t i c observational study with cross-sectional design in ckd patients in nephrology outpatient clinic at dr. soetomo general hospital, surabaya, indonesia. this research was ethically approved by health research ethics committee of dr. soetomo hospital. written informed consent was obtained from all subjects. chronic kidney disease, diagnosed using kdigo criteria, are abnormalities in kidney function or structure that have occurred for more than 3 months. the stage was determined based on the decrease in egfr with the chronic kidney disease epidemiology collaboration (ckd-epi) formula.1,2 inclusion criterias for the samples were non-dialysis stadium iii-v chronic kidney disease patients. patients with history of cancer, hepatitis b, hepatitis c, liver cirrhosis, diabetes mellitus, chronic inflammation (hiv-aids, obesity, rheumatic disease, geriatric patients), diagnosed with acute infection (urinary tract infection, respiratory infection, pneumonia, gastroenteritis), under oral and intravenous iron or erythropoietin 163edward muliawan putera, et al.: c-reactive protein and hepcidin in non-dialysis chronic kidney disease copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 stimulating agents (esa) therapy, hormonal therapy, history of blood transfusion, alcoholism, absolute iron defi ciency, gastrointestinal bleeding were excluded.1 data collection: consecutive sampling was done to complete an amount of 40 samples. direct interview was done by the author and blood samples were taken by professional healthcare and sent to dr. soetomo general hospital laboratory to be examined. infl ammations in this research were represented by serum crp. serum level of crp was measured using extended range c-reactive protein method with reagent siemens flex® reagent cartridge c-reactive protein extended range cat no. rcrp3749, an in vitro diagnostic test with a particle enhanced turbidimetric immunoassay (petia) technique, meant to quantitatively measure crp level in human serum. serum level of hepcidin was circulating level of hepcidin-25 in blood. serum hepcidin level was measured using the enzyme linked immunosorbent assay (elisa) method. serum were stored in a deep freezer at a temperature of -80°c until the hepcidin measurement was performed. the reagent used was drg hepcidin-25 (bioactive) elisa from cat no eia-5782, an enzyme immunoassay for in-vitro quantitative examination for hepcidin-25 peptide in serum and plasma.3,4 statistical analysis: all data was analysed by statistical package for the social sciences (spss ver 23). data was delivered in the form of analytic statistics. data analysis was provided in mean ± standard error of mean (se). correlation of serum hepcidin with ckd stage was calculated by pearson parametric test if it had normal distribution or spearman parametric test if the data distribution was not normal. it was said to be signifi cant if the p-value is <0.05. results and discussion patient characteristics a total of forty subjects (67.5% male with mean age of 50.23 ± 1.04 years) were eligible for enrollment in this study. this study was done in nephrology outpatient clinic, dr soetomo general hospital, surabaya, indonesia within the period of 1 june 2018 31 august 2018. the results of demographic and clinical characteristics of this study subjects were described in table 1 and table 2. twenty seven of 40 subjects were male (67.5%), the youngest is 27 years old and the oldest is 58 years old with mean age of 50.23 years old (se 1.04), mean of body mass index (bmi) was 22.54 (se 0.57). based on ckd stage, 16 patients (40%) of the total sample had stage 3 ckd. (table 1) table 1. demographic characteristics category result frequency sex male 27 (67.5%) female 13 (32.5%) age (years) mean ± se 50.23 ± 1.04 range (min max) 27–58 bmi (kg/m2) mean ± se 20.54 ± 0.58 range (min max) 14.57 – 24.38 ckd stage stage 3 16 (40%) stage 4 9 (22.5%) stage 5 15 (37.5%) table 2. clinical characteristics clinical data level frequency hemoglobin (g/dl) mean ± se 10.74 ± 0.36 range (min max) 7.30 – 15.70 bun (mg/dl) mean ± se 39.98 ± 4.68 range (min max) 11 – 125 creatinine serum (mg/dl) mean ± se 4.12 ± 0.54 range (min max) 1.22 – 16.53 comorbid disease hypertension (n) 25 (62.5%) urinary tract stone (n) 7 (17.5%) others 8 (20.0%) 164 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: 161–167 clinical characteristics mean hemoglobin level of 40 subjects was 10.74 g/dl with se of 0.36. mean of blood urea nitrogen (bun) levels was 39.98 mg/dl with se of 4.68. mean level of serum creatinine was 4.12 with se of 0.54. the most frequent comorbid factor was hypertension (62.5%) (table 2). distribution of crp levels by ckd stage results were mean crp 166 mg/l (range 0.10 9.40), 2.68 mg/l (range 0.10 8.90), 6.01 mg/l ( range of 0.30 21.10) in stage 3, 4, and 5 respectively. the overall mean of crp levels in this study was 3.52 mg/l with a range 0.10 21.10. (table 3) distribution of hepcidin value by ckd stage were mean hepcidin 27.24 ng/ml (range 0.12 70.14), 84.69 ng/ml (range 1.08 254.87), and 170.88 ng/ml (range 9.96 352.42) in stage 3, 4, and 5 respectively. hepcidin level overall mean was 94.03 ng/ml (range 0.12 352.42). (table 4) signifi cance and strength of crp and hepcidin value correlation in non-dialysis ckd patients. distribution of crp and hepcidin level data were used to analyze the correlation between crp and hepcidin in ckd patients. kolmogorovsmirnov normality test showed that the distribution of crp and hepcidin level data is abnormal (both p-value < 0.05). spearman correlation test was used to further analyze the correlation between crp and hepcidin levels. (table 5) analysis of crp and hepcidin value showed an association with a positive correlation coeffi cient of 0.487. the correlation of crp and hepcidin value in this study was signifi cant, indicated by the p-value = 0.001. the meaning of this positive correlation coeffi cient showed a unidirectional relation, if the crp level increase, the hepcidin level would be increased consequently. discussion c h r o n i c k i d n e y d i s e a s e s p r o g r e s s e d alongside complications such as anemia and its clinical consequences. one of the renal anemia pathophysiologies was disruption of iron metabolism, regulated by main iron exporter hormone, hepcidin. chronic kidney disease patients were constantly in an inflammatory state, represented by increased of crp. this infl ammatory state results in the liver secreting hepcidin, which subsequently followed a decrease in iron circulation, thus resulting in functional iron defi ciency. inclusion of stage 3 to 5 ckd patients was based on earlier studies that stated complications of ckd, particularly anemia, were more commonly seen in stage 3 to 5 ckd patients. non-dialysis ckd patients were selected to reduce confounding factor such as duration of dialysis in ckd patients.5,6 most of the study subjects were men with a percentage of 67.5%, similar to studies by toima, et al., mercadel, et al, elmenyawi, et al.5,6,7 higher male prevalence than female could be infl uenced by numerous factors like hypertension, hyperglycemia, lifestyle, kidney structure and hormonal diff erences.8 the mean age in this study was 50.23 ± 1.04 years old, similar to studies by mercadel, et al, table 3. crp level characteristics stage crp level (mg/l) p mean ± se median range (min-max) stage 3 1.66 ± 0.41 0.25 0.10 – 9.40 0.036 stage 4 2.68 ± 0.49 1.10 0.10 – 8.90 stage 5 6,01 ± 1.11 2.50 0.30 – 21.10 total 3.52 ± 0.81 1.10 0.10 – 21.10 table 4. hepcidin level characteristics stage hepcidin level (ng/ml) p mean ± se median range (min max) stage 3 27.24 ± 3.24 23.18 0.12 – 70.14 0.000 stage 4 84.69 ± 12.23 55.65 1.08 – 254.87 stage 5 170.88 ± 15.81 200.00 9.96 – 352.42 total 94.03 ± 15.08 53.98 0.12 – 352.42 table 5. result of spearman correlation test variable 1 variable 2 rs p crp hepcidin 0.487 0.001 165edward muliawan putera, et al.: c-reactive protein and hepcidin in non-dialysis chronic kidney disease copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 toima, et al., 2010, elmenyawi, et al.5,6,7 aging process infl uenced ckd progression and lesser function was expected from older nephrons.9 mean hemoglobin result in this study was 10.74 g/dl with se of 0.36. the results of this study were similar to other studies by toima, et al., peters, et al., and goyal, et al. 5,10,11 mean results of bun value were 39.98 mg/dl with se of 4.8. bun and creatinine serum levels found in this study were similar to study by toima, et al.5 the most frequent comorbid disease in this study was hypertension, at 62.5% of the total subjects. study by toima, et al., peters, et al., and goyal, et al., also mentioned hypertension as the most frequent comorbid disease found in ckd patient. 5,10,11 hypertension was the highest prevalent chronic disease in indonesia based on 2013 riskesdas study.12 hypertension risk factors were age, race, family history, obesity, high sodium intake, and smoking. 8 in this study, higher mean crp levels was seen in more advanced ckd stage. the mean total crp level in this study was 3.52 mg/l with a se of 0.81. this was similar to previous studies by toima et al. who found crp levels of 6.0 mg/l with a standard deviation of 0.9, elmenyawi et al. who found mean crp level was 4.28 mg/l with a standard deviation of 3.7, rasheed et al. who found crp mean levels were 7.59 mg/l in all ckd stages, and. 5,7,13 fluctuations in crp levels may also have been due to the highest staging diff erences in the population study. in a study by elmenyawi et al the most frequent ckd stage in the population was stage 3 with mean crp level at 3.52 and 4.28 mg/l.7 in the study of toima et al. and rasheed et al. the highest staging in the population was stage 5 and mean crp levels were, 6.0 and 7.59 mg/l.5,13 this study found that crp level increased along with a decrease in egfr, which were consistent with other studies. 14,16 total mean hepcidin found in this study was 94.03 ng/ml with se of 15.08. while toima, et al. found mean hepcidin level of 84 ng/ml with a standard deviation of 18.6, goyal, et al. and uehata, et al. found mean hepcidin levels of 65.0 ng/ml and 15.4 ng/ml respectively. 5, 11,17 analysis of crp and hepcidin levels in nondialysis ckd patients revealed a moderate to signifi cant relationship (correlation coeffi cient 0.487; p-value 0.001). this result indicated that an increase in crp levels would lead to a directly proportional increase of hepcidin value. the results of this study were in accordance with studies by toima et al., peters et al., and lee et al., who inferred a positive relationship between crp and hepcidin levels. 5,10,15 toima et al. organized a study in egypt regarding the importance of hepcidin role as a novel biomarker which refl ected iron status in ckd patients and its relationship with crp levels. thirty ckd patients and 10 healthy subjects, used as controls, were enrolled. the result showed a correlation in crp and hepcidin value with r of 0.68 (p = 0.001).5 patients who had iron or erythropoietin therapy for the previous 21 days were excluded. inclusion of diabetes mellitus patients might lead to a strong correlation found in this study. this study used the same method in crp and hepcidin level measurement as toima, et al.5 peters, et al. conducted an observational crosssectional study of factors aff ecting hepcidin in 83 non-dialysis ckd patients and 48 dialysis ckd patients in the netherlands. there was a weak positive relationship (r=0.21, p <0.001) between crp and hepcidin levels which was probably related to inclusion of patients who were under erythropoietin therapy. the method used to measure crp level was the same as in our study, but a diff erent method (light chromatography mass spectrometry / lc-ms) was used in measuring hepcidin level.10 lee, et al. in korea analysed whether hepcidin was a novel uremic toxin using multivariate analysis of various variables aff ecting hepcidin in 2090 non-dialysis ckd patients. they found a positive correlation between crp and hepcidin with r=0.23 (p <0.001). patients with intravenous iron, oral iron, and erythropoietin therapy were not excluded. these factors might have played a role as confounding factors to the weak correlation. this study used the same method in crp and hepcidin level measurement as lee, et al.15 the result of the uehata et al. study result was diff erent compared to this study. that study 166 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: 161–167 included 505 samples of non-dialysis ckd patients and found no association between crp and hepcidin levels (r = 0.03 and p = 0.4). patients with liver cirrhosis were not excluded, while liver cirrhosis can induce negative feedback on hepcidin and crp. the level of crp was measured using immunoagglutination detection method and hepcidin level was determined using lc-ms.17 another study by goyal, et al. in india analyzed the relation between crp and hepcidin levels in 100 non-dialysis ckd patients. they found similar results with this study, the correlation coeffi cient of 0.0001 and p = 0.896 between crp and hepcidin levels. patients having oral iron therapy were not excluded, while oral iron could induce positive feedback on hepcidin. c-reactive protein levels were measured using different eia kits which could influence the absence of association of crp and hepcidin.11 study by wagner, et al., which analysed predictive factors of mortality in patients with non-dialysis ckd patients, showed contradiction to this study by stating that crp level was not associated with hepcidin levels (correlation coeffi cient of 0.01 and p <0.001). their case control study stated that crp and hepcidin (measured hepcidin using ria method) were infl uenced by factors that change over time. the mean hemoglobin in their study was higher than this study (13.1 g/dl) while anemia could induce negative feedback on hepcidin.18 these factors might have played a role in the absence of a correlation. another study contrasting this study results was macdougall et al. in the netherlands who used a random sampling system and including patients with erythropoietin therapy which caused positive feedback on hepcidin. 19 there were diff erences in the results of this study compared to previous studies. diff erent methods in measuring crp and hepcidin levels could have contributed to this result. in earlier studies, the crp level was measured using the immunoturbidimetric assay, immunoagglutination, or eia method.5,10,15 in previous studies, hepcidin level was measured by elisa and lc-ms methods.20 studies conducted by mercadel, et al., macdougall, et al. used diff erent methods to determine hepcidin levels.6,21 hepcidin could be measured using ria, elisa, and mass spectrometry-based methods.22 measurement using ria detects hepcidin-25 greater than actual condition. measuring hepcidin-25 using elisa were accurate and cheap.3,23 mass spectrometrybased was indeed more accurate but not practical, requiring more instruments and too expensive. besides diff erences in measurement methods, there were also diff erences in this study subjects’ characteristics compared to previous studies.24 history of erythropoietin therapy, a history of blood transfusion, iron therapy, diabetes mellitus, and other factors infl uencing crp and hepcidin level were not excluded in previous studies, whereas it could have affected the results.6,11,14,25 this was a novel study of hepcidin and inflammation marker in ckd in surabaya, although we were aware that the small number of subjects might interfere with the study results. study with a larger, more homogenous sample, more markers of infl ammation and iron might be needed in the future. conclusion a signifi cant positive correlation with rs = 0.487, p = 0.001) was found between crp and hepcidin levels in non-dialysis ckd patients. if there was an increase in serum crp levels in non-dialysis ckd patients there was a tendency for an increase in serum hepcidin levels. conflict of interest there is no confl ict of interest of this paper. acknowledgement this project would have been impossible without the support of all the participants in this study, all nephrology division staff s and internal medicine department functional staff s of dr. soetomo general hospital surabaya. the authors would like to thank prodia laboratory surabaya 167edward muliawan putera, et al.: c-reactive protein and hepcidin in non-dialysis chronic kidney disease copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 for their cooperation in providing laboratory kit and medical analyst for this study, and indonesian australia language foundation for proofreading this project. references 1. kdigo, 2012. kidney disease: improving global outcomes (kdigo) clinical practice guidelines for anemia in chronic kidney disease. kidney international supplements, 2(4), pp. 279-335. 2. kdigo, 2013. kidney disease: improving global outcomes (kdigo) clinical practice guideline for the evaluation and management of chronic kidney disease. kidney international supplements, volume 3, pp. 1-150. 3. kemna, e., tjalsma, h., podust, v. & swinkels, d., 2007. mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications. clinical chemistry, 53(4), pp. 620-628. 4. swinkels, d., jorna, a. & raymakers, r., 2007. synopsis of the dutch multidisciplinary guideline for the diagnosis and treatment of hereditary haemochromatosis. the netherlands journal of medicine, 65(11), pp. 452-455. 5. toima, s., madkour, m., saleh, a. & hammam, o., 2010. hepcidin and iron status in chronic kidney disease. african journal of nephrology, volume 14, pp. 16-23. 6. mercadel, l., metzger, m., haymann, j. & thervet, e., 2014. the relation of hepcidin to iron disorders, infl ammation and hemoglobin in chronic kidney disease. plos one, 9(6), pp. 1-7. 7. elmenyawi, a., hassan, a. & sawar, s., 2017. relationship between hepcidin, ferritin and c-reactive protein in hemodialysis patients. the egyptian journal of hospital medicine , 69(2), pp. 1786-1793. 8. chang, p., chien, l. & chiou, h., 2016. risk factors of gender for renal progression in patients with early chronic kidney disease. medicine, 95(30), pp. 1-7. 9. wang, x., liu, s. & jin, y., 2014. correlation of serum high-sensitivity c-reactive protein and interleukin-6 in patients with acute coronary syndrome. genetics and molecular research, 13(2), pp. 4260-4266. 10. peters, h., laarakkers, c. & swinkels, d., 2010. serum hepcidin-25 levels in patients with chronic kidney disease are independent of glomerular fi ltration rate. nephrology dialysis transplantation, volume 25, pp. 848--853. 11. goyal, h., mohanty, s. & sharma, m., 2017. study of anemia in nondialysis dependent chronic kidney disease with special reference to serum hepcidin. indian journal of nephrology, 27(1), pp. 44-50. 12. trihono, 2013. riset kesehatan dasar, jakarta: badan penelitian dan pengembangan kesehatan. 13. rasheed, n., ali, s. & shami, a., 2013. serum hepcidin levels in anemia of chronic kidney diseases compared to iron defi ciency anemia and it's correlation with serum levels of hs –c reactive protein, interlukin-6 and ferritin. global journal of bio-science and technology, 2(1), pp. 43-50. 14. nand, n., aggarwal, h., yadav, r. & gupta, a., 2009. role of high-sensitivity c reactive protein as a marker of infl ammation in pre-dialysis patients of chronic renal failure. the journal, indian academy of clinical medicine, 10(1), pp. 18-22. 15. lee, s., kim, j. & sung, s., 2017. serum hepcidin may be a novel uremic toxin, which might be related to erythropoietin resistance. nature scientifi c reports, volume 7, pp. 1-7. 16. kumar, s., 2015. comparative study of hs-crp in chronic kidney disease. international organization of scientifi c research journal of pharmacy, 5(7), pp. 8-12. 17. uehata, t., tomosugi, n. & tsubakihara, y., 2012. serum hepcidin-25 levels and anemia in non-dialysis chronic kidney disease patients: a cross-sectional study. nephrology dialysis transplantation, volume 27, pp. 1076-1083. 18. wagner, m., ashby, d. & schramm, l., 2015. hepcidin-25 in diabetic chronic kidney disease is predictive for mortality and progression to end stage renal disease. plos one, 10(4), pp. 1-14. 19. putten, k., jie, k. & gaillard, c., 2010. hepcidin-25 is a marker of the response rather than resistance to exogenous erythropoietin in chronic kidney disease/ chronic heart failure patients. european journal of heart failure, volume 12, pp. 943-950. 20. babitt, j. & lin, h., 2010. molecular mechanisms of hepcidin regulation: implications for the anemia of ckd. american journal of kidney diseases, 55(4), pp.726-41. 21. macdougall, i., malyszko, j. & hider, r., 2010. current status of the measurement of blood hepcidin levels in chronic kidney disease. clinical journal of american society of nephrology, 5(9), pp. 1681-1689. 22. arezes, j. & nemeth, e., 2015. hepcidin and iron disorders: new biology and clinical approaches. int. jnl. lab. hem., 37(1), pp.92-98. 23. jairam, a. et al., 2010. iron status, infl ammation, and hepcidin in esrd patients: the confounding role of intravenous iron therapy. indian journal of nephrology, 20(3), pp.125-31. 24. kato, a., 2010. increased hepcidin-25 and erythropoietin responsiveness in patients with cardio–renal anemia syndrome. future cardiology, 6(6), pp.769-71. 25. miura, k. et al., 2008. hepatitis virus induced induced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity. hepatology, 48, pp.1420-29. 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. 2. no. 1 january–march 2011 biocompatibility of azitromicyn on connective tissue shafira kurnia s periodontal department faculty of medicines and health sciences airlangga university surabaya indonesia abstract background: periodontal disease is commonly caused by bacteria, especially actinomyces actinomycetemcomitans and porphyromonas gingivalis have an abilty enter epithelial cells objectives: to investigate systemic azithromycin as the antibiotic of choice for periodontal disease based on biocomptability test in connective tissue. material and methods: bhk 21 cell lines were exposed to 0.025%, 0.050%, 0.075%, and 0.1% azithromycin solution for seven times. samples were put in incubator for 24 hours. result: azitrromycin 0.050%-0.1% showed significant difference between life cells percentage and control, however, azithromycin 0.025% revealed insignificant difference with control. conclusion: 0.025% azithromycin was considered biocompatible with connective tissue and 0.050% was not. key words: azithromycin biocomptability, connective tissue, periodontal disease introduction until nowadays, infectious diseases still become very prominent diseases in many developing countries, including indonesia, and a lot of effort had been done to eliminate these problems. periodontitis is an infectious disease caused by bacterial accumulation on tooth surface that cause inflammation, bleeding on probing, pocket formation, periodontal attachment loss, tooth mobility, and tooth lost1. since it was known that most periodontal disease was caused by bacteria, the idea of antibiotics treatment was emerged, the periodontal pathogenic bacteria in the oral cavity will recolonize rapidly after scaling and root planning. the ability of actinobacillus actinomycetemcomitans to penetrate the soft tissue make it protected from scaling and root planing.1 this is the rationale for the need of antibiotics in the successful treatment of peridontal disease. some classes of antibiotics like penicillin, amoxicillin, erythromycin, azithromycin, tetracycline, metronidazole, and clyndamisin are widely used in dental treatment.2 azithromycin is the latest generation of macrolides, erythromycin-derived but slightly differs in chemical compounds.3 azithromycin is a broad spectrum antibiotics, works effectively to gram-positive aerobic, gramnegative aerobic, anaerobes obligates such as bacteroides fragilis, fusobacterium sp, and peptostreptococcus sp. azithromycin was stated to be effective againts actinobacillus actinomycetemcomitans and porphyromonas gingivalis.4 some researches supported this opinion by declaring that azithromycin was effective as adjuntive therapy to patients with advanced periodontitis and deep pocket.5 azithromycin was also effective for treating porphyromonas gingivalis-involved refractory periodontitis.1 systemically-administered azithromycin was shown to be 4–8 times more effective. its local usages are also expected to be more effective than erythromycin, and required a lower concentration than eritromisin.6 in order to minimize the side effects of systemic-administered azithromycin and to make it more economical and affordable, the authors consider to prepare azythromycin as a local preparations. however its biocompatibility test needs to be carried out to determine the optimum concentration of therapeutical doses which will not harm the gingival and the surroundings tissues. biocompatibility test can be performed in cell culture, tissue culture, or culture organ.7 connective tissue cells (fibroblasts) are one of cell types which is suitable for biocompatibility observation. ��kurnia: biocompatibility of azitromicyn on connective tissue connective tissue cells (fibroblasts), which is often used in cell culture techniques are the l-929 cells, from rat’s lung fibroblasts derivation, and bhk-21 cells, hamster’s kidney fibroblasts derivation.8 fibroblasts is the largest component of the pulp and periodontal ligament. in periodontal tissue, fibroblasts synthesize collagen and extracellular matrix that preserve the health of periodontal ligament.9 the question is whether the lower the concentration of azithromycin, the more biocompatible to the connective tissue cells? it is hypothetized that the l o w e r t h e c o n c e n t r a t i o n o f a z i t h r o m y c i n , t h e more biocompatible to the connective tissue cells. the purpose of this study is to determine the biocompatibility of azithromycin at various concentrations to connective tissue, as the basis to produce local azithromycin preparations research method preparation of azithromycin solution azithromycin powder was prepared to four different concentrations: 0.025%, 0.050%, 0.075%, and 0.1%. the powder is digitally weighed, and diluted in aqua bidestillata to reach certain concentration. these solutions were sterilized with ultra violet. preparation of cell culture the monolayer cell lines of bhk 21 with eagle’s mem medium was grown in culture bottles, then incubated with 37° for 2 × 24 hours. the goal was that cells can live and repopulate. after 2 × 24 hours, 21 bhk cells are harvested and in re-suspensed in eagles mem medium with approximate density 2 × 106 cells / ml. then it was divided in 35 petri dishes, for each concentration of azithromycin and control needed seven petri dishes. prepared azithromycin solution respectively 0.025%, 0.050%, 0.075%, and 0.1% was introduced to the petri dishes. for each concentration of azithromycin was 7 times replicated. a cell culture with eagles mem medium without azithromycin was used as a control. then the petri dishes was incubated for 24 hours. after 24 hours, eagle’s mem was discarded, then the petri dishes were washed with 20 ml pbs (phosphate buffer saline) twice to clean up the waste products of cell metabolism, only the cells would be left on the petri dishes. in order to observe and count the changes, the cells attached to the petri dishes ought to be removed with 0.1 ml trypsyn versene 0.25%. eagle’s mem medium were used again to obtain cell suspension. to count the cells, 0,1 ml were taken from each suspensions, and added with 0.9 ml added tryphan blue, then brough into the hemocytometer.7 the results were obtained by calculating the average number of living cells and dead cells of each box. the calculation were performed with the aid of a microscope with 100 times macnification. the living cells were brightly colored, while the dead cells will absorb the blue color. this calculation were performed for all concentration of azithromycin and control groups. the calculations of each concentration obtained were compared to control group, to determine the biocompatibility azithromycin at different concentrations results and data analysis the precentage of total cells in azithromycin administered with various concentrations after 24 hours can be seen in table 1 below (the results can be seen in appendix i) table 1. the average value and standard intersection number of living cells after treatment for 24 hours group n average (%) deviation control 7 100 0,00 0,025% 7 95,11 3,69 0,050% 7 94,76 3,25 0,075% 7 94,61 3,02 0,1% 7 86,23 12,18 the test results showed that after 24 hours the group with 0.025% azithromycin, had the highest percentage of living cells while the 0.1% group had the lowest. summary of test results can be seen in table 2 (see annex iii). table 2. summary of test results t-test kgroup kontrol 0,025% 0,050% 0,075% 0,1% kcontrol 0,025% 0,050% x 0,075% x 0,1% x description: “x” means no significant difference from table 2 it might be observed that treatment with azithromycin concentration of 0.050%–0.1% showed no significant difference in the percentage of living cells to control, whereas treatment with azithromycin 0.025% showed no significant difference against control. this means that azithromycin at concentrations of 0.025% was biocompatible to connective tissue cells, whereas azithromycin concentrations above 0.050% tend not to be biocompatible. �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 42-45 figure 1 image of bhk-21 cell lines when a head count by hemositometer description: a live cell = b = cell death discussion recently, research in antibiotics therapy has developed rapidly especially as an adjuvant in aggresive periodontal treatment. microbiology research data states that mechanical treatment (scaling and root planing) alone was not able to completely eliminate bacterial pathogens, such as actinobacillus actinomycetemcomitans from local and subgingiva, therefore antibiotic application was considered to be effective as an adjuvant.6 azithromycin is an effective antibiotic against gram-positive aerobic bacteria, gram negative, and strict anaerobes such as bacteroides fragilis, fusobacterium sp, peptostreptococcus sp. in addition, azithromycin also showed good activity against actinobacillus actinomycetemcomitans and porphyromonas gingivalis.4 its antimicrobial activity had been proven against oral infections such as periodontitis, periodontal abscess, acute infection of the oral cavity and others. azithromycin as adjuvant treatment, had shown to be beneficial in reducing deep pockets (> 6mm) in patients with severe periodontitis.5 azithromycin was used in this study, because its broad spectrum activity and effective for the periodontal disease, especially when resistance to tetracycline and erythromycin had been developed,1 while metronidazole would be effective when combined with the other antibiotics.3 however, the side effects of systemic adiministered azithromycin should not be nglected. in order to minimize the side effects and increase its effectivity, a new delivery route shall be developed. local administration of azythhromicin may be suitable for these purposes to support the periodontitis treatment. the local preparations shall not exceed the biocompatibility dose which had been proven not to harm the exposed tissues. for these reasons we do the biocompatibility test for azithromycin against connective tissue cells (fibroblasts). fibroblast cell was chosen as the object of exposure because it consists of 65% of gingival fibroblasts as connective tissue cells. in addition, the largest cellular component of the periodontal ligament is fibroblas.21 in this study using fibroblast cell cultures was prepared from baby hamster kidney (bhk-21) because according to ma’at (1999), the best culture material is derived from young tissue cells or embryonic fibroblasts and bhk-21 cells is able to grow and subcultured easily. in addition, bhk-21 fibroblast cells have been frequently used as materials in the dentistry for biocompatibility test. the biocompatibility of azithromycin at various concentrations on fibroblast cell cultures are presented in tables 1 and 2. it may be observed that azithromycin at concentrations more 0.050% were not biocompatible to the fibroblast cell culture. although at a concentration of 0.050% of the average percentage of living cells is still relatively high at 94.76 ± 3.25, but it was statistically different. in azithromicyn-tretaed groups with concentrations 0.025%, 0.050%, 0.075%, and 0.1% was the average number of living cells in a row is 95.1%, 94.76%, 94.61%, and 86.23%. this shows that the higher concentration of azithromycin, the more toxic to the cells. intensity of cell death depending on the levels of drugs that come into contact with cells, tissue or organ. cell death increased as a result of azithromycin at a high concentration can be caused by the nature and chemical structure of azithromycin that may interfere with the living cells15. azithromycin inhibits sythesis of bacterial protein at the ribosomal subunits 50 s16. increased doses of chemicals and drugs have altered some vital functions of cells, which manifests as changes in homeostatic mechanisms associated with protein synthesis and cause changes in membrane permeability.15 if the permeability change, it will cause an increase in intra-cell movement of to the extra cell, so that the it may lose metabolites necessary to preserve life. azithromycin dissolve in water which cause it to be more easily in penetrating the cell’s membrane, and may cause intra-cell disturbances and may cause cell death16. in this study, the dead cells absorb the blue color from blue tryphan because of the disruption in cells’ membrane permeability. azithromycin concentrations below 0.050% was shown to be biocompatible. azithromycin toxicity was increasing as the concentration increase. this means that the drug has the potential capability to cause tissue destruction. this is in accordance with the opinion stating that all substances may be considered toxic, depend on its dosages.20 conclusions and suggestions conclusion azithromycin was shown to be biocompatible to tissues at concentrations below 0.050%. suggestions more researches shall be done to demonstrate the effectivity of azithromycin in biocompatible concentration to inhibit bacteria that cause periodontal disease before preparing local azithromycin for clinical usages. ��kurnia: biocompatibility of azitromicyn on connective tissue references 1. newman mg, takei hh, carranza fa. clinical periodontology, 9th ed.wb saunders co. philadelphia. 2002. 67–69, 559-560, saunders co. philadelphia. 2002. 67–69, 559-560, 676–681. 2. winkelhoff aj & vandenbroucke cmje. principles of antimicrobial chemotheraphy in dental and orofacial infection. antibiotic and antimicrobial use in dental practice, 2nd ed. quintessence publishing co, inc. 2001. 3–11. 3. martindale. the complete drug reference, 32 nd ed. the pharmaceutical press. massachusetts .1999.155–156. 4. sanz m & herrera d. individual drugs. antibiotic and antimicrobial use in dental practice, 2nd ed. quintessence publishing co, inc. 2001. 33–51. 5. smith sr, foyle dm, daniels j, joyston bs, smlaes fc, sefton a, williams. a double-blind placebo-controlled trial of azithromycin as and adjunct to non-surgical treatment of periodontitis adults: clinical result. j clin periodontal. 2002. 29: 54–61.29: 54–61. 6. pajukanta r, asikainen s, saarela m, alaluusua s, & somer hj. in vitro activity of azithromycin compared with that of erythrommycinagainst actinobacillus actinomycetemcomitans. antimicrobial agents & chemotherapy.(1992). vol 36. no.6: 1241–1243. 7. soejono sk. laporan penelitian pengembangan teknik kultur jaringan yang berperan dalam sistem enzim dan faktor tropik. pau bioteknologi. ugm. yogyakarta. 1988. 8. freshney ri. culture of animal cells (a manual of basic technique), 2nd ed. alan r. liss inc. new york. 1987. 7–12. 9. grossman li, oliet s, & del rio ce. imun endodontik dalam praktek.rossman li, oliet s, & del rio ce. imun endodontik dalam praktek. alih bahasa abyono r. penyunting suryo s. edisi ke-11. egc jakarta. 1995. 47–48. 10. bird pr & forrester ft. basic laboratory techniques in cell culture. public health service centre of disease control. 1981. 33–36. 11. carranza fa & newman mg . clinical periodontology, 8th ed.wb saunders co. philadelphia.1996. 511–515. 12. informasi spesialite obat indonesia. edisi farmakoterapi. volvol xxxiii. ikatan sarjana farmasi indonesia.jakarta. 2000. 369. 13. levine jm & edgerton m.biocompatibility : its future in prosthodontics research. j prost dent. 1993. 69: 406–410.j prost dent. 1993. 69: 406–410. 14. ma’at s. kultur jaringan. program pasca sarjana universitas airlangga s-3. 1999. 26–34. 15. malizia t, tejada mr, gheraldi e, senesi s, gabriele m, giuca mr, blandizzi c, danesi r, compa m, tacca md. periodontal tissue disposition of azithromycin. j periodontal.1997. 68:1206–1209. 16. mombelli a& tonetti ms. topical antimicrobial agents: general principles and individual drugs. antibiotic and antimicrobial use in dental practice, 2nd ed.quintessence publishing co, inc. 2001. 53–57. 17. muller hp, holderrieth s, burkhardt u, & hoffler u. in vitro antimicrobial susceptibility of oral strain of actinobacillus actinomyceten\mcomitans to seven antibiotics. j clin periodontol. 2002. 29: 736–742. 18. pfizer labs. antibiotic efficacy ztthromax (azithromycin). pfizer inc, new york. 2004. 19. robbins sl & kumar vk. buku ajar patology 1, ahli bahasa staf pengajar laboratorium patology anatomi fk unair, egc. jakarta.laboratorium patology anatomi fk unair, egc. jakarta. 1995. 1–25. 20. steel rgd & torrie jh. prinsip dan prosedur statistik. suatu pendekatan biometrik. alih bahasa sumantri b. edisi ke-2. gramedia jakarta.1995.145. 21. timbrelll ja. principles of biochemical toxicology, 2nd ed. taylor and francis ltd, london, 1994. 216–227. 22. wilson tg and kornman ks.anatomy of the periodontium fundamentals of periodontics, 2nd ed. quintessence publishing co,inc. 2003. 32–33. 23. winkelhoff aj, pavivic mjamp, & graaf j. antibiotics in periodontal therapy, proceedings of the 1st ed. european workshop on periodontology. quintessence pub, co, ltd. 1993. 261–263. 105 vol. 7 no. 5 may-august 2019 anti-dengue type 2 virus activities of zinc (ii) complex c o m p o u n d s w i t h 2 ( 2 , 4 d i h y d r o x y p h e n y l ) 3 , 5 , 7 trihydroxycromen-4-one ligands in vero cells teguh hari sucipto1,α, harsasi setyawati2, siti churrotin1, ilham harlan amarullah1, sri sumarsih2, puspa wardhani1, aryati1, soegeng soegijanto1 1 dengue study group, institute of tropical disease, universitas airlangga, indonesia 2 department of chemistry, faculty of science and technology, universitas airlangga, indonesia α corresponding author: teguhharisucipto@staf.unair.ac.id abstract dengue virus (denv) is a disease that is transmitted through aedes aegypti and aedes albopictus mosquitoes, and is spread in tropical and sub-tropical regions. now, dengue or antiviral vaccines for humans do not yet exist, but there are great efforts to achieve this goal. complex compounds are reported to fungicidal, bactericidal and antiviral activity. antiviral activity against denv is an important alternative to the characterization and development of drugs candidate. the purpose of this study was to study zinc(ii) compounds with 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen-4-one ligand on denv-2 replication in vero cells. vero cell lines (african green monkey kidney) was used in this study, maintained and propagated in minimum essential eagle medium containing 10% fetal bovine serum at 37°c in 5% co2. the activity of dengue virus was carried out by enzyme-immunosorbent assay (elisa) method and celltiter96® non-radioactive proliferation. the value of activity inhibition (ic50) of complex compounds with variations of mol metal: ligand 1:2, 1:3, and 1:4 against dengue virus type 2 (denv2) was 2.44 μg/ml, 2.75 μg/ml, respectively and 2.00 μg/ml, also the toxicity value (cc50) of complex compounds with variation mol metal: ligand 1:4 for vero cells is 3.59 μg/ml. the results of this study were indicate that these properties have been shown to inhibit anti-dengue type 2 virus (denv-2), but are also toxic in vero cells. including previous study about complex compound interaction with dengue virus type 2 activity, zn(ii) more reactive compound then cu(ii), and co(ii). the comparison with cu(ii) complex compound, it has been revealed that co(ii) and zn(ii) is more toxic, was found to be nontoxic to human erythrocyte cells even at a concentration of 500 μg/ml. keywords: anti-denv2, complex compounds, zinc(ii), 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen-4-one abstrak virus dengue (denv) adalah penyakit yang ditularkan melalui nyamuk aedes aegypti dan aedes albopictus, serta didistribusikan di daerah tropis dan sub-tropis. kini, vaksin dengue atau antivirus untuk manusia belum disetujui secara klinis, meski telah ada upaya besar untuk mencapai tujuan ini. senyawa kompleks dilaporkan menunjukkan aktivitas fungisida, bakterisida, dan antivirus. aktivitas antivirus melawan denv merupakan alternatif penting untuk karakterisasi dan pengembangan obat-obatan. tujuan dalam penelitian ini adalah untuk investigasi aktivitas senyawa kompleks logam seng(ii) dengan ligan 2-(2,4-dihidroksifenil)-3,5,7-trihidroksikromen-4-on terhadap replikasi denv-2 pada sel vero. sel vero (african green monkey kidney) yang digunakan dalam penelitian ini, dipelihara dan diperbanyak dalam medium essential eagle yang mengandung 10% serum janin sapi pada 37°c dalam 5% co2. aktivitas senyawa kompleks virus dengue dilakukan dengan metode enzyme-immunosorbent assay (elisa) dan toksititas dengan metode celltiter96® non-radioactive proliferation. nilai penghambatan aktivitas (ic50) senyawa kompleks dengan perbandingan mol logam:ligan 1:2, 1:3, dan 1:4 terhadap virus dengue tipe 2 (denv2) masing-masing adalah 2.44 μg/ml, 2.75 μg/ml, dan 2.00 μg/ml, serta nilai toksisitas (cc50) senyawa kompleks dengan perbandingan mol logam:ligan 1:4 untuk sel vero adalah 3,59 μg/ml. hasil penelitian ini menunjukkan bahwa senyawa kompleks tersebut menunjukkan aktivitas penghambatan anti-dengue virus tipe 2 (denv-2), tetapi juga bersifat toksik pada sel vero. termasuk penelitian sebelumnya tentang interaksi senyawa kompleks dengan aktivitas virus dengue tipe 2, zn (ii) lebih research report 106 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 105–108 reaktif senyawa kemudian cu (ii), dan co (ii). perbandingan dengan kompleks cu (ii), telah diketahui bahwa co (ii) dan zn (ii) lebih toksik, ditemukan tidak beracun pada sel eritrosit manusia bahkan pada konsentrasi 500 μg / ml. kata kunci: anti-denv2, senyawa kompleks, seng(ii), 2-(2,4-dihidroksifenil)-3,5,7-trihidroksikromen-4-on introduction a major public health concern worldwide in recent years is a most prevalent mosquito-borne viral pathogen dengue virus (denv), was transmitted through aedes aegypti and aedes albopictus mosquitoes, and is spread in tropical and sub-tropical regions.1,2,3 presently around the world dengue is endemic in 112 countries.4 the incidence of denv has increased approximately 30-fold over the past 50 years.5 dengue virus, the causal agent of dengue, has been shown to induce apoptosis in vitro and in vivo.6,7 the mechanisms that trigger the apoptotic cellular responses, however, have not been thoroughly investigated.8 micronutrient homeostasis is a key factor in maintaining a healthy immune system. trace element zinc is a critical cofactor for many proteins involved in cellular processes like differentiation, proliferation and apoptosis that zinc is a nutritionally fundamental trace element and is second most abundant trace metal in the human body after iron.9 in previous study, zn2+ was suggested that denv-2 infection of vero cells and human breast adenocarcinoma cell line resulted in the induction of apoptosis.8,10 the complex compound from metal and organic compound reaction can be used an anti-denv-2, especially cu(ii) with 2,4,5-triphenylimidazole exhibited adsorption inhibitory activity at ic50 = 2.3 μg/ml. 11 a significant inhibitory activity to that of the complex co(ii) with 2-(2,4dihydroxyphenyl)-3,5,7-trihydroxycromen -4-one ligand was reported against the tested pathogenic denv-2 in vero cells 3.08 μg/ml.12 in the present study, the inhibitory activity of zinc(ii) with 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen-4one ligand against the replication of denv-2 in vero cells was investigated. material and methods chemicals and medium the chemical reagents used in this research were the zinc(ii)–2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen -4-one complex compound, dimethyl sulfoxide by merck 99.98%, germany, minimum essential eagle medium by sigma-aldrich (germany), dengue virus type 2 surabaya isolate (kt012513), vero cell by african green monkey kidney, celltiter96® non-radioactive proliferation reagent by promega (usa), and denv antibody (4g2) for enzyme-linked immunosorbent assay (elisa). vero cells preparation vero cell lines (african green monkey kidney) was used in this study, maintained and propagated in minimum essential eagle medium containing 10% fetal bovine serum. cultured vero cell lines were incubated at 37 °c, respectively in 5% co2. confluent monolayer of vero cells were detached with trypsin-edta and incubate cells at 37 °c for 5 minutes. add minimum essential eagle medium containing 10% fetal bovine serum, pipetting gently to break up any clumps of cells and counted using a hemocytometer. add cells in 96-well plate with 1 × 106 cells/10 ml and incubated in 37°c incubator with 5 co2. monitor cells daily or every other day, cells reach a >90 % confluent monolayer.13,14 anti-dengue type 2 virus assay confluent monolayers of vero cells were prepared on a 96-well plate (1 × 106 cells/10 ml), and the titer of denv-2 (2 × 104 ffu/well). the 50% inhibitory concentration (ic50) was calculated as follows: ic50 = (nc − ac) 100/ nc after incubating at 37°c for 2 days in 5% co2, where nc is the mean of the absorbance of negative controls and ac is the absorbance of the compound tested. the denv-2 inhibition of replication by compound was further investigated by using quantitative elisa at 415 nm.15 cytotoxicity assay the dye of celltiter96® non-radioactive proliferation reagent by promega is a modification of mtt assay method by mosmann. the assay is suitable to use in adherent and suspension cells. assay is very sensitive, it can detect 1,000 cells/well of a plate reader. vero cells (1×105 cells/ml) were seeded in 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 100 μl of cell proliferation reagent was added into each well, incubated for 4 hour at 37°c. the plate was read at 570 nm using elisa reader (imarktm microplate absorbance reader). result and discussion anti-dengue type 2 virus activity metal complex compounds are a promising class of drug leads, and the associated studies have attracted more attention. however, the systematic basic research of metal complex compounds is lagging behind, partly because very few efforts have been made to establish a set activity screening and subsequent evaluation system for the comprehensive investigations on the structure and activity relationship of metal complex compounds. the in vitro anti-dengue virus activity of the zinc(ii)–2(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen-4-one complex compound was tested against one type dengue virus (dengue virus type 2) by enzyme-linked immunosorbent 107sucipto, et al.: anti-dengue type 2 virus activities of zinc(ii) complex compounds assay (elisa).15 the susceptibility of the strains toward the present compounds was judged measuring the size of inhibition. zinc(ii)– 2-(2,4-dihydroxyphenyl)-3,5,7trihydroxycromen-4-one complex compound was further studied for their inhibitory effect on replication of the dengue virus type 2 in vero cells. the ic50 (inhibitory concentration 50) was determined from the dose response curve with variations of mol metal:ligand 1:2 (figure 1), 1:3 (figure 2), and 1:4 (figure 3). figure 1. inhibition curve of dengue virus type 2 at several concentrations of zn(ii) complex with variations of mol metal:ligand 1:2 figure 2. inhibition curve of dengue virus type 2 at several concentrations of zn(ii) complex with variations of mol metal:ligand 1:3 figure 3. inhibition curve of dengue virus type 2 at several concentrations of zn(ii) complex with variations of mol metal:ligand 1:4 the ic50 value with variations of mol metal:ligand 1:2, 1:3, and 1:4 against dengue virus type 2 was 2.44 μg/ml, 2.75 μg/ml, respectively and 2.00 μg/ml. the comparison of the complex compounds and the known anti-dengue virus type 2 activity showed that the 1:4 variation mol metal:ligand was more effective than 1:2 and 1:3. the bulky 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen -4-one ligand on binding to the metal cation reduces the polarity of the metal ion due to the ligand orbital overlap with the metal orbitals, resulting in a delocalization of positive charge.16 this increases the lipophilic character of the metal favors its permeation through the lipoid layer of the virus membranes.17 the effects of other compounds against cellular rna polymerases and formation of the complex with rna have reported suggesting that compound could also affect the similar replication enzymes.18 previous research was reported anti-dengue type 2 activity, especially cu(ii) with 2,4,5-triphenylimidazole exhibited adsorption inhibitory activity at ic50 = 2.3 μg/ml.11 a significant inhibitory activity to that of the complex co(ii) with 2-(2,4-dihydroxyphenyl)-3,5,7trihydroxycromen -4-one ligand was reported against the tested pathogenic dengue virus type 2 in vero cells 3.08 μg/ ml. the comparison of the other complex, zn(ii) complex compound more significant activity for inhibit dengue virus type 2 replication then cu(ii) complex, and co(ii) complex. as for the central ion (m2+), when chelated with ligand to form the complex, it has the following order in stability: zn2+ > cu2+ > co2+. besides that, cu(ii) free ligand more reactive to dengue virus type 2 up to 0.13 μg/ ml because cu2+ has stronger oxidative activity19 and react with cysteine residues on the surface of the protease.18 at molecular scale such complex compound interact directly with proteins and dna, leading to dysfunction and cleavage of the structure of macromolecular.20 cytotoxicity activity this compound was tested for cytotoxicity by modification of mtt assay method by mosmann assay on vero cell lines, zinc(ii)–2-(2,4-dihydroxyphenyl)3,5,7-trihydroxycromen-4-one complex compound showed cytotoxicity with cc50 at 3.59 μg/ml. the cc50 value was found to increase with an increasing concentration of the test compound, as shown in figure 4. figure 4. cytotoxicity of zn(ii) complex curve for vero cell lines at several concentrations 108 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 105–108 previous research was reported cytotoxicity other complex compounds to vero cell lines, cu(ii) with 2,4,5-triphenylimidazole at cc50 = 44.74 μg/ml11 and the complex co(ii) with 2-(2,4-dihydroxyphenyl)-3,5,7trihydroxycromen-4-one ligand at 3.36 μg/ml.12 the comparison with cu(ii) complex compound, it has been revealed that co(ii) and zn(ii) is more toxic, was found to be lower toxic to human mcf7 cell proliferation.21 conclusion metal complex compounds are a promising class of drug leads, and the associated studies have attracted more attention. including previous study about complex compound interaction with dengue virus type 2 activity, zn(ii) more reactive compound then cu(ii), and co(ii). acknowledgement this work was supported by the institute of tropical disease (itd) the center of excellence (coe) program by the ministry of research and technology (ristek) indonesia. references 1. carrington lb, simmons cp, 2014. human to mosquito transmission of 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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. 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 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. 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. 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. �� 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 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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. 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 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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 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. 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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, 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aureus. open j med microbiol. 2015; 5(june): 69–75. copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 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. 90 vol. 5. no. 4 january–april 2015 optimation of 48 khz ultrasonic wave dose for the inactivation of salmonella typhi dwi may lestari,1 tri anggono prijo,2 suryani dyah astuti2 1 bachelor of physics, faculty of science and technology, universitas airlangga, surabaya, indonesia 2 department of physics, faculty of science and technology, universitas airlangga, surabaya, indonesia abstract this study was aimed to determine the effect of ultrasonic dose exposure which could decrease the viability of salmonella typhi by using the variation of exposure time (15, 20, 25, and 30 minutes) and volume of bacterial suspension (2, 4, 6, and 8 ml) at constant power. the sample used was salmonella typhi. ultrasonic wave transmitter was a piezoelectric tweeter with 0,191 watts of power and 48 khz frequency generated by the signal generator. piezoelectric tweeter was a kind of transducer which converted electrical energy into ultrasonic energy. this research was an experimental laboratory with a completely randomized design. the decrease of bacterial percentage was calculated by using tpc (total plate count). data were analyzed by using one way anova. the results showed that the variation of exposure time and volume of bacterial suspension gave significant effect on the percentage of salmonella typhi kill. the most optimal of ultrasonic dose exposure to kill salmonella typhi was 281.87 j/ml with 100% bacterial kill. key words: ultrasonic dose exposure, ultrasonic wave, piezoelectric tweeter, salmonella typhi, total plate count abstrak penelitian ini bertujuan untuk menentukan efek dosis paparan ultrasonik yang dapat mengurangi viabilitas salmonella typhi dengan menggunakan variasi paparan waktu (15, 20, 25, and 30 menit) dan volume suspense bakteri (2, 4, 6, and 8 ml) pada kekuatan konstan. sampel yang digunakan ialah salmonella typhi. transmiter gelombang ultrasonik ialah tweeter piezoelectric dengan daya 0,191 watt dan frekuensi 48 khz yang dihasilkan oleh signal generator. tweeter piezoelectric ialah sejenis tranduser yang mengubah energi listrik menjadi energi ultrasonik. penelitian ini ialah percobaan laboratorium dengan desain random lengkap. pengurangan persentase bakteri dihitung dengan menggunakan teknik pengujian total bakteri. data dianalisis menggunakan anova satu arah. hasil menunjukkan bahwa variasi paparan waktu dan volume suspensi bakteri memberikan efek yang signifikan pada persentase salmonella typhi yang mati. dosis paparan ultrasonik untuk membunuh salmonella typhi yang optimal ialah 281.87 j/ml dengan 100% bakteri yang mati. kata kunci: dosis paparan ultrasonik, gelombang ultrasonik, tweeter piezoelectrik, salmonella typhi, pengujian total bakteri research report introduction food is an important requirement for organisms because food serves as a source of carbohydrates, proteins, fats, vitamins, minerals, and other essential substances needed by organisms for growing process, developing process, and repairing damaged cells. food and beverages consumed by humans must have good quality and free from pathogenic bacterial. pathogenic bacterial which often contaminate water, food, eggs and meat, fish and meat, milk and its processed products is salmonella typhi.1 salmonella typhi is very dangerous because it is pathogenic to humans and causes fever.2 most effort to obtain sterile food and beverage is using sterilization process. the method is used on sterilization process is heating. however, this method has the disadvantage because it reduces some nutrients contained 91lestari dm, et al.: optimation of 48 khz ultrasonic wave dose in the food during the sterilization process. besides heating, another sterilization method which is often used is ultraviolet radiation that can cause mutagenic damage to dna. ultraviolet radiation is very harmful for humans when exposed directly.3 therefore, other alternatives are needed in the sterilization process that is ultrasonic wave’s exposure. ultrasonic waves are very effective on materials sterilization process from bacterial,4,5,6,7,8,9 this method is very safe because it is free from chemical substances and selectively to reduce bacterial viability without giving bad effects to humans and environment. ultrasonic wave exposure on bacterial suspensions showed that exposure time is proportional to the decrease of the number of salmonella typhi colonies. the bacterial kill after ultrasonic wave’s exposure occurs due to cavitation effects. cavitation is the formation of bubble collapse which is a continuous stretch and eventually will be destroyed when it reaches the limit of its elasticity.9 ultrasonic wave’s exposure on bacterial with causes mechanical stress on the bacterial cell wall so the cell wall stretches beyond the limits of its elasticity. stretching of cell wall can lead to rupture of the cell wall, lyse, and ended in the death of the bacterial.7 cavitation occurs due to local pressure in the sound wave drops to a low enough pressure. it causes rupture of the cell as indicated by the following relationship: ρ is the acoustic pressure, p is the total pressure, and p0 is the average local pressure. p value is always positive in the gas medium so that the amplitude of the acoustic pressure must be less than atmospheric pressure. while the liquid has a specific volume and can withstand the negative pressure. when the pressure is very low, the liquid will break up and form small cavities such as the ball is called cavity.11 based on the characteristic of its formation, cavitation is distinguished on acoustic cavitation caused by ultrasonic wave and hydrodynamic cavitation caused by variations in fluid pressure.10 the mechanisms of cavitation started by the formation of bubbles which get pressure from the outside so that the bubbles are unstable and eventually rupture. cavitation causes free radicals because of molecular bonds damage. for example h2o molecule breaks into h–, oh–, and ho2 – and eventually form h2o2 which can damage the chemical structure of the bacterial cell wall so the cell wall is weak and broken and the liquid from the outside enter the cell and lyse is occurred resulting in death of the bacterial.8,11 besides cavitation, ultrasonic wave exposure can also increase temperature of the fluid due to acoustic energy imposed on a medium will be released back into heat. it causes temperature rising. experimental research sample the sample of salmonella typhi was obtained from institute of tropical desease, airlangga university, surabaya. the sample was grown in nutrient broth sterile medium for treatment and salmonella shigella agar for tpc. expossure equipment ultrasonic wave generator was a piezoelectric tweeter with 2 cm of diameter were fitted with 10 ohm resistor and generated by function generator (fg-350 iwatsu). voltage and frequency issued by function generator were detected by using an oscilloscope type protex 20 mhz (oscilloscope 6502a). scheme of the ultrasonic wave instrument is shown in figure 2. ρ = p – p0 (1) figure 1. the mechanism of cavitation.3 figure 2. scheme of ultrasonic wave instrument. 92 indonesian journal of tropical and infectious disease, vol. 5. no. 4 january–april 2015: 90–95 this research used square wave with 48 khz of frequency and ac current.4 the circuit of v2 in figure 1 is rc integrator circuit. rc integrator circuit is shown in figure 3 below: minutes, and 30 minutes12 with a fixed volume was 2 ml to determine the optimum time with 100% of bacterial kill7 at a fixed frequency that was 48 khz.4,7,13 each treatment was accompanied by the control group using 5 times replication. the percentage of bacterial kill was plotted in a graph to obtain the optimum time with 100% of bacterial kill by using linear regression equation (equation 3). where was the percentage of bacterial kill up to 100%, was the optimum time (minutes) required to kill the bacterial up to 100%. this optimum time used to do the second experiment to determine the optimum dose for the 100% of bacterial kill. the second experiments were performed using a variation of the volume; these were 2 ml, 4 ml, 6 ml, and 8 ml with a fixed exposure time. the bacterial growth salmonella typhi were cultured at luria bertani broth sterile (miller m1245-500 g). the bacterial cultures were incubated at 37°c of temperature for 18 hours. the dilution factor was qualify if the number of bacterial colonies that grew as much as 30–300 colonies, so this culture was incubated until od600nm = 0.7 and the value of dilution up to 10–6 dilution (30–300 colonies). bacterial dilution which was exposed by ultrasonic waves was cultured on sterile agar medium called salmonella shigella agar (oxoid cm 0099) and incubated at 37××°c of temperature for 18–24 hours. ultrasonic wave exposure on bacterial 2 ml of bacterial suspension with 10–6 bacterial concentration was poured into a glass with 3 cm diameter and 4 cm of height and exposed by ultrasonic waves with a variation of exposure time 15, 20, 25, 30 minutes. the height of that suspension was approximately 3 mm. exposure was done by dipping the piezoelectric tweeter into the bacterial suspension. the second experiments were performed using a variation of the volume, these were 2 ml, 4 ml, 6 ml, and 8 ml with a fixed exposure time which giving lethal dose 100% on bacterial. the bacterial in the treatment group and the control were grown on salmonella shigella agar medium. counting the number of bacterial colonies the bacterial colonies were counted by total plate count method using a quebec colony counter. the next was calculating the percentage of bacterial kill by using equation 4. (4) figure 3. rc integrator circuit.12 performance test which consist of calibration and measurement of fluid temperature rising were done before giving treatment on bacterial suspension. calibration was done to calculate the voltage. that voltage was used to determine the electrical power which was converted into ultrasonic power by the transducer. the power output was calculated by equation 2. (2) measurement of liquid temperature rising was performed to determine the effect of rising temperatures on the viability of salmonella typhi because the bacterial could be killed at certain temperature. this method was used to determine whether the bacterial actually killed caused by the mechanical vibrations of ultrasonic waves or due to the heating effect. if the number of live bacterial colonies derived from ultrasonic wave exposure is fewer than heating, it means that bacterial kill caused by mechanical vibrations of ultrasonic waves. the exposure of ultrasonic waves in liquids can increase the liquid temperature because acoustic energy imposed on a medium will be released back into heat, causing an increase in temperature of the fluid. besides heating, the exposure of ultrasonic waves in the sample can also cause mechanical vibration as the effects of cavitation. ultrasonic wave’s exposure on bacterial with causes mechanical stress on the bacterial cell wall so the cell wall stretches beyond the limits of its elasticity. stretching of cell wall can lead to rupture of the cell wall, lyse, and ended in the death of the bacterial.7 research methods this research conducted using completely randomized design. the first experiment was conducted using exposure time variation, these were 15 minutes, 20 minutes, 25 (3) statistical analysis this research data were analyzed by using spss (statistical package for social science) 20 that was one 93lestari dm, et al.: optimation of 48 khz ultrasonic wave dose way anova for determining the effect of each factor. multiple comparison post hoc was used to determine the factors that most influence the percentage of bacterial kill. results and discussion design and assembly tool the set up of experiment tool in this research is an integrator circuit which processes of charging and discharging capacitor were happened. the circuit has a time constant so that when the capacitor was not fully charged, the voltage vs has changed the sign to be negative. that leads to discharge the capacitor. the capacitor was charged by using negative charge up to -vp. before it was fully charged, the voltage vs changed the sign. the process occurred repeatedly and forms a triangular output signal.12 the results of electrical voltage measurements which were converted into an ultrasonic voltage by each of piezoelectric tweeter tabulated in a table (appendix 1). the next step was combining the entire of piezoelectric tweeters into one so that the distribution of the power supplied by each transducer was the same. the results tabulated in a table (appendix 2). based on the results of electrical voltage measurements which were converted into ultrasonic voltage by the entire of piezoelectric tweeters obtained an average power value in appendix 2 was 0.191 watt. measurement of liquid temperature rising these measurements were performed by means of ultrasonic wave’s exposure to the bacterial suspension and the rising of temperature occurred were measured. the ultrasonic exposure in 8 ml of bacterial suspension for 30 minutes increased temperature from 25°c to 27°c. results of liquid temperature rising was plotted in a graph (figure 4). suspension up to 27°c and obtained 0% of percentage killing of bacterial (didn’t cause killing effect on bacterial). based on these results, it was certain that the death of the bacterial was not due to the effect of rising temperature as a result of ultrasonic wave’s exposure but due to the cavitation effect caused by the ultrasonic waves. ultrasonic wave exposure on salmonella typhi the results of this study is the decrease of salmonella typhi colony due to exposure time variations of ultrasonic waves (15, 20, 25, and 30) min, volume variations (2, 4, 6, 8) ml, fixed frequency (48 khz), and fixed power (0.191 w). the results of the study are shown in figure 5 below. figure 4. graphic of rising temperature because of ultrasonic exposure. figure 5. graphic relationship of exposure time ultrasonic wave against percentage of salmonella typhi kill. equation 5 below is the linear regression equation obtained from figure 5: y = 3,4732 x – 28,282 (5) percentage of bacterial kill (y%) obtained for exposure time x minutes with a gradient of 3.4732 and a constant of 28.282. based on the linear regression equation, lethal dose 100% could be obtained by exposing for 36.94 minutes or 36 minutes 56 seconds. this time variation was the time that would be used in all subsequent experiments. the next experiment used variations of volume (2 ml, 4 ml, 6 ml, and 8 ml) with a fixed time (36 minutes 56 seconds). the results of the study are shown in figure 6. figure 6. graphic relationship between volume variations against percentage of salmonella typhi kills. bacterial exposure process using ultrasonic waves in that process has the potential to kill the bacterial salmonella typhi up to 55.9%. the next step was to warm the bacterial 94 indonesian journal of tropical and infectious disease, vol. 5. no. 4 january–april 2015: 90–95 the results were analyzed by using one way anova test to determine the effect of each factor. terms of anova test is interval and ratio scale data and normally distributed. normality test performed using the kolmogorov-smirnov 1 sample. the test is used to compare the distribution of the data of the study sample with a theoretical distribution. results of kolmogorov-smirnov test showed a significance value p = 0.809 for time variation and p = 1.14 for volume variations. the test results showed that the data were normally distributed as p > α (0.05). levene test results generated significant value for p = 0.101 and p = the time variation of 0.309 for variations in the volume so that it could be concluded that the variance of the data was homogeny, which means that the population had the same variance (uniform). summary of anova test in table 1 indicate that the time factor and the volume has a significance level of p = 0.000 is < 0.05, which means that the time factor and the volume effect on the decrease in the number of bacterial colonies. table 1. summary of one way anova test of ultrasonic exposure to percentage of salmonella typhi kill factor group n mortality percent (%) anova average sd significance summary time 15 mina 5 24 2.58135 0.000 there is a significant difference 20 minb 5 42 6.47927 25 minc 5 59 7.46598 30 mind 5 76 1.11437 volume 8 mla 5 46 0.61409 0.000 there is a significant difference 6 mlb 5 51 0.24234 4 mlc 5 70 0.83264 2 mld 5 82 0.76731 (6) explanations: d = dose (j/ml) e = energy (j) v = volume (ml) p = power (w) t = exposure time (s) based on salmonella typhi research data obtained the percentage of bacterial kill at various energy doses which are tabulated in table 2. dose of energy dose of energy is the energy of ultrasonic waves exposure which absorbed by the bacterial suspension. basically, the emitted energy is electrical energy which is converted into mechanical vibration by the transducer. but there is proportionality between the electrical energy emitted by the ultrasonic energy received by a medium with a constant of proportionality k. thus, ultrasonic energy received by the medium approaches the electrical energy emitted. mathematically, dose of energy is the result of power (p) times exposure time (t) divided by the volume of the bacterial suspension (v). the optimum dose exposure of ultrasonic wave on salmonella typhi inactivation obtained from equation 6 below. table 2. results of the percentage of salmonella typhi kills on the variation of ultrasonic wave exposure time and volume, frequency of 48 khz, and a power of 0.191 w time (min) volume (ml) energy (j) dose (j/ml) percentage of bacterial kill (%) 15,00 2 171,90 85,95 23,68 20,00 2 229,20 114,60 41,76 25,00 2 286,50 143,25 57,80 30,00 2 343,80 171,90 76,22 36,94 2 423,33 211,67 81,98 36,94 4 423,33 105,83 68,27 36,94 6 423,33 70,56 50,72 36,94 8 423,33 52,92 45,52 (6)d = –– = ––––v v e p ×××x t 95lestari dm, et al.: optimation of 48 khz ultrasonic wave dose figure 7. graphic of dose exposure at frequency 48 khz ultrasonic wave against the percentage of salmonella typhi kill at exposure time variations. the percentage of salmonella typhi kill at different doses ultrasonic waves with the volume variation plotted in linear regression to determine the dose with 100% bacterial kill. figure 8 gives the linear regression equation on equation 7 below. y = 0,2236 x + 36,974 (7) if y is the percentage of bacterial kill up to 100% and x is the desired dose, the dose can be used to kill bacterial up to 100% is 281.87 j/ml. conclusion the research results show that piezoelectric tweeter produces ultrasonic waves and the voltage generated by the signal generator. the optimum time exposure of ultrasonic waves which effectively decrease the viability of salmonella typhi up to 100% is 36.94 minutes. the optimum volume of bacterial suspension is 2 ml with 81.78% of bacterial kill. the optimum dose exposure of ultrasonic waves which effectively decrease the viability of the bacterial salmonella typhi is 281.87 j/ml with 100% of bacterial kill. the relationship between the dose of ultrasonic energy to the ultrasonic energy which produces mechanical vibration; ultrasonic power; and ultrasonic voltage are proportional. dose of ultrasonic energy is proportional to ultrasonic energy where ultrasonic energy is a product of ultrasonic power with long time exposure. ultrasonic power itself is the product of voltages generated by the transducer with long time exposure. references 1. supardi i dan sukamto. 1999. microbilogy in processing and food safety. mikrobiologi dalam pengolahan dan keamanan pangan. alumni. bandung. 2. mason cf., 1991. biology of freshwater pollution. second edition. john willey & sons, inc. new york. 3. endarko. 2013. design of decontamination water purification systems based on river bio sand filter and ultraviolet lamp. rancang bangun sistem penjernihan dan dekontaminasi air sungai berbasis biosand filter dan lampu ultraviolet. physics periodic journal. vol. 16 no. 3, juli 2013, page 75–84 issn: 1410-9662. physics department, fmipa, institut teknologi sepuluh november. surabaya. 4. arifin, syamsul, ni’matuzzahro, sugianto, r. apsari, suhariningsih. 2013. aquatic bacterial of pseudomonas aeruginosa growth model in tube ultrasonic. international journal of scientific & technology research volume 2, issue 8, august 2013, issn 2277-8616. 5. dehghani, mohammad hadi. 2005. effectiveness of ultrasound on the destruction of e. coli. american journal of environmental sciences 1 (3): 187–189, 2005. issn 1553-345x. department of environmental health engineering, school of public health center for environmental health research, tehran university of medical sciences, tehran, iran. 6. mahvi ah. 2009. application of ultrasonic technology for water and wastewater treatment. iranian j publ health, vol. 38, no. 2, 2009, pp. 1–17. school of public health and center for environmental research, tehran university of medical sciences, iran. 7. mansyur, mas. 2011. effect of ultrasonic wave dose exposure in the death of e. coli. dosis paparan gelombang ultrasonik terhadap kematian bakteri e. coli. the journal of lecturer in faculty of medicine, wijaya kusuma university surabaya. 8. mason tj, e. joyce, ss. phull, and jp. lorimer. 2003. the development and evaluation of ultrasound for the treatment of bacteriall suspensions. a study of frequency, power and sonication time on cultured bacillus species. ultrasonics sonochemistry 10 (2003) 315–318. sonochemistry centre, school of science and the environment, coventry university, coventry cv1 5fb, uk. 9. sayadi mh, mr. doosti, r. kargar. 2012. water treatment using ultrasonic assistance: a review. proceedings of the international academy of ecology and environmental sciences, 2012, 2(2): 96–110. environment and civil eng. dept., university of birjand, birjand, iran. 10. http://www.chm.bris.ac.uk/webprojects2004/eaimkhong/ultrasound. htm 11. ackerman, 1988. biophysics science. ilmu biofisika. translated by redjani and abdul basir. airlangga university press. surabaya. 12. sutrisno. 1986. electronics: elektronika: basic theory and its application 1st edition. teori dasar dan terapannya jilid 1. publisher itb. bandung. 13. josé, jackline freitas brilhante são and maria cristina dantas vanetti. 2011. effect of ultrasound and commercial sanitizers in removing natural contaminants and salmonella enterica typhimurium on cherry tomatoes. food control 24 (2012) 95e99. brazil. figure 8. graphic of dose exposure at frequency 48 khz ultrasonic wave against the percentage of salmonella typhi kill at suspension volume variations. the relationship between ultrasonic wave's doses exposures with the percentage of salmonella typhi kill clarified by figures 7 and 8 below. 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. 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 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. 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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 80 404 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 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. 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. 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(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/ �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 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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. 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. ijtid vol 8 no 2 may-agustus 2020_newfromsarah.indd vol. 8 no. 2 may–august 2020 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 original article relationship between level of serum adiponectin and frailty in elderly patients with chronic obstructive pulmonary disease erika marfi ani2a, jusri ichwani1, novira widajanti1, daniel maranatha3, muhammad amin2,3 1department of internal medicine, faculty of medicine, universitas airlangga, surabaya, east java, indonesia 2universitas airlangga hospital, surabaya, east java indonesia 3department of pulmonology and respiratory medicine, faculty of medicine, universitas airlangga, surabaya, east java, indonesia received: 7th november 2019; revised: 31st january 2020; accepted: 20th february 2020 abstract elderly are prone to the health eff ects of chronic obstructive pulmonary disease (copd). frailty is a geriatrics syndrome, adiponectin is an adipokine that regulates energy. adiponectin is aff ected by age. increased adiponectin can lead to muscle wasting which will further reduce body mass index (bmi), which indirectly increases the degree of frailty. the relationship between adiponectin with frailty degree in copd is still unknown. the aims of this study was to investigate the relationship between plasma adiponectin levels and frailty in copd elders. this was an observational analytic cross-sectional study. all anthropometric parameters, including weight, height, and bmi, were measured. adiponectin was measured by elisa methods obtained from venous blood samples. aged more than or equal to 60 years old, the patients underwent spirometry and the degree of frailty defi ned by the fried criteria. statistic analysis used rank spearman. thirty-eight male copd patients became the subject of the study. the average age was 70-74 years, with a total of 13 robust, 12 prefrails and 13 frail patients. level of adiponectin (mean and sd) in robust, prefrail, and frail were 6.84+ 2.66 , 6.58 + 4.27, and 11.62 + 4.90 respectively, p=0.015. further analysis showed that the level of adiponectin rose progressively with an increasing number of components of frailty. the degree of obstruction mostly with mild (42.1%), and no subjects with very severe. there was an increase in serum adiponectin levels in all subjects. in conclusion, the level of adiponectin serum correlates positively with the degree of frailty. keywords: adiponectin, copd, frailty abstrak lansia sangat rentan terhadap efek kesehatan yang merugikan dari penyakit paru obstruktif kronik (ppok). frailty adalah sindrom geriatrik yang penting, sedangkan adiponektin adalah adipokin yang mengatur homeostasis energi. adiponektin dipengaruhi oleh usia. peningkatan adiponektin dapat menyebabkan pengecilan otot yang selanjutnya akan mengurangi indeks massa tubuh (imt), yang secara tidak langsung meningkatkan derajat frailty. hubungan antara adiponektin dengan derajat frailty pada ppok usia lanjut masih belum diketahui. tujuan penelitian ini adalah untuk menentukan hubungan antara kadar adiponektin plasma dan frailty pada lansia dengan ppok. penelitian ini adalah penelitian cross-sectional analitik observasional. semua parameter antropometrik, termasuk berat badan, tinggi badan, dan imt, diukur. adiponektin diukur pada sampel darah vena dengan metode elisa. pasien yang berusia lebih dari atau sama dengan 60 tahun menjalani spirometri dan derajat frailty menurut kriteria fried. analisis statistik menggunakan rank spearman. tiga puluh delapan pasien ppok laki-laki menjadi subjek penelitian. usia rata-rata adalah 70-74 tahun, dengan total 13 pasien robust, 12 prefrail dan 13 frail. kadar adiponektin (rerata dan sd) pada kelompok robust, prefrail, dan frail masing-masing adalah 6,84 + 2,66, 6,58 + 4,27, dan 11,62+ 4,90, p=0,015. analisis lebih lanjut menunjukkan bahwa kadar adiponektin meningkat secara progresif seiring peningkatan jumlah komponen frailty. derajat obstruksi sebagian besar ringan (42,1%), dan tidak ada subjek dengan obstruksi berat. terdapat peningkatan * corresponding author: erika.marfi ani@fk.unair.ac.id 102 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: 101–107 introduction chronic obstructive pulmonary disease (copd) is a typical disease of aging with a prevalence of around 12% in the age group >64 years.1,2,3,4 in elderly who suff er from copd, the process of this disease can also increase the level of adiponectin through inspiratory muscle mechanism that is exercised continuously (chronic exercise), thereby increasing the ree (resting energy expenditure). as a result, an increase in fatty tissue activity will release adipokine and cause an increase in plasma adiponectin levels.5,6,7,8 increased severity and shortness of breath result in the inactivity of copd patients, which in turn results in loss of muscle strength, leading to mobility problems, which contribute to the high frequency of frailty in those patients.9,10,11 underweight patients have an increase in ree compared to overweight and normal-weight patients, which is associated with decreased serum and adipose tissue leptin. increased serum adiponectin also occurs, demonstrating the role of adipokines in cachexia-related energy imbalances in copd.12,13,14,15,16 this study was conducted to identify the relationship between serum adiponectin levels and the degree of frailty measured using the cardiovascular health study (chs) scoring system17, a scoring system that is most widely used and has the broadest validity to determine the degree of frailty in the population of copd elderly patients in surabaya. materials and methods this study was a cross-sectional analytic study to analyze diff erences in serum adiponectin levels between degrees of frailty in elderly kadar adiponektin serum pada semua subjek. sebagai simpulan, kadar serum adiponektin berkorelasi positif dengan derajat frailty. kata kunci: adiponectin, ppok, frailty how to cite: marfi ani, erika., ichwani,jusri., widajanti, novira., maranatha, daniel., amin, muhammad. relationship between level of serum adiponectin and frailty in elderly patients with chronic obstructive pulmonary disease. indonesian journal of tropical and infectious disease, 8(2), 1–8 copd patients. this study was conducted at the pulmonary and geriatric outpatient unit, dr. soetomo hospital, surabaya, indonesia. the study samples were subjects aged > 60 years at the outpatient unit, dr. soetomo hospital, surabaya, who fulfi lled the inclusion criteria, ie aged over or equal to 60 years old, a mini mental state examination (mmse) score of > 18, and was willing to follow the study by signing informed consent and information for consent. criteria for the exclusion of the subjects were in acute exacerbations, had a history of diabetes mellitus, had a malignancy or history of malignancy, and had a history of stroke with limited motor function. measurement of serum adiponectin adiponectin is a 30 kda glycoprotein that is secreted primarily by adipocytes and induces wide ranging paracrine and endocrine eff ects on metabolism and infl ammation. adiponectin circulates in the blood with a high concentration as total adiponectin18. adiponectin measurement in this study used a quantitative elisa method from venous blood samples in μg / ml units. blood samples were taken as much as 5 ml and put into vacuette z serum sep clot activator tubes and store inside the cooler box with a temperature of 2–4° c, to be processed and separated the serum part in less than 24 hours by centrifugation. the total adiponectin was measured using a commercial tool kit sekisui medical co., ltd. the normal value of adiponectin serum was a range between 2.54–6.06 μg / ml. type of data is a ratio data. samples were taken by consecutive sampling. a total of 38 samples were obtained19. all data were entered into the computer through the statistical program r version 3.1.2. data on general 103erika marfiani, et al.: relationship between level of serum adiponectin and frailty copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 characteristics of the samples according to age, sex, level of education, degree of copd, smoking history, and comorbid history were presented descriptively in tabular form. subjects’ specifi c characteristics data including body mass index, mmse score, handgrip strength, 15 feet walking test, and pase scores are presented in tables and graphs. types of data were ordinal (categorical) data for frailty degrees and ratio (numeric) data for serum adiponectin levels, so we used oneway anova test if the parametric statistical test requirements were met, or the kruskal-wallis test if the parametric statistical test requirements were not met. subanalysis was conducted to determine the relationship of serum adiponectin levels with fried’s fi ve frailty components. results and discussion general characteristics of the subjects the number of subjects in this study were 38 copd patients in the pulmonary and geriatric outpatient unit, dr. soetomo hospital, surabaya, table 1. general characteristics of the subjects characteristics total age. year (mean ± sd) (min-max) (70.26 ± 7.52) (60 84) education. n (%) no formal education elementary junior secondary senior seconday high education nutritional status (bmi) low (bmi<18.5) normal (bmi 18.5-25.0) high (bmi>25.0) smoking history yes no comorbidities hypertension heart disease renal disease liver disease degree of copd obstruction mild moderate severe very severe 3 ( 7.9%) 12 ( 31.6%) 7 ( 18.4%) 15 ( 39.5%) 1 ( 2.6%) 10 ( 26.3%) 21 ( 55.3%) 7 ( 18.4%) 38 (100%) 0 4 ( 10.5%) 1 ( 2.6%) 0 0 16 ( 42.1%) 14 ( 36.8%) 8 ( 21.1%) 0 table 2. particular characteristics of the subjects frailty components frequency percent fatigue (cesd) yes no 23 15 60.5 39.5 weight loss yes no 11 27 28.9 71.1 pase yes no 10 28 26.3 73.7 slowness (walking) yes no 11 27 28.9 71.1 muscular weakness (handgrip) yes no 0 38 0.0 100.0 indonesia, who had fulfi lled the inclusion and exclusion criteria. table 1 shows the general characteristics of the study subjects. most subjects were found in the 70-74 years age range. the mean age of the subjects in robust group was 69.69 ± 7.85 years, in prefrail group 70.50 ± 6.85 years, and in frailty group 70.85 ± 8.19 years. all of the subjects (100%) were male. the degree of copd obstruction used in this study was based on the 2014 gold criteria which divided into 4 groups, mild (gold 1), moderate (gold 2), severe (gold 3) and very severe (gold 4) obstruction20. we obtained mostly copd patients with mild obstruction degrees as many as 16 (42.1%) patients, and no subjects with copd had very severe obstruction degrees. increased serum adiponectin level was found in copd patients with severe obstruction. however, the comparative test did not show differences in adiponectin levels in various degrees of copd obstruction. table 3 shows that under frail conditions serum adiponectin levels increase. the comparative test showed diff erences in serum adiponectin levels between degrees of frailty with p=0.015 (p <0.05). further post-hoc analysis showed signifi cant diff erences in serum adiponectin levels between frail and prefrail patients, and between robust and frail patients. furthermore, analysis with spearman’s correlation between serum adiponectin levels and 104 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: 101–107 figure 1. relationship between the degree of copd obstruction and frailty frequency. figure 2. relationship between the degree of copd obstruction and adiponectin levels table 3. adiponectin levels at various frailty levels degree of frailty n adiponectin level p mean sd median minimum maximum robust 13 6.84 2.66 5.94a 3.68 11.59 0.015*prefrail 12 6.58 4.27 5.30a 2.70 18.31 frail 13 11.62 4.90 11.36b 2.97 17.56 frailty degrees showed spearman’s correlation coeffi cient rs=0.368 with p=0.023 (p <0.05), showing the relationship between serum adiponectin levels and frailty degrees. the analysis showed that the higher the degree of frailty, the higher the adiponectin level. 105erika marfiani, et al.: relationship between level of serum adiponectin and frailty copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 characteristics of the subjects this study was conducted to determine the relationship between serum adiponectin levels and degrees of frailty in copd patients from mild (gold 1) to very severe (gold 4) obstruction non-exacerbations with age limited to > 60 years. in this study, the mean age was 70.26 + 7.52 years with an age range between 60-84 years. according to fried frailty phenotype/chs system as many as 13 (34.2%) of the total 38 samples included in the robust group, 12 (31.6%) in the prefrail group, and 13 (34.2%) in the frail group. based on fried’s phenotype criteria and their various modified versions, the prevalence of frailty in adult populations aged 65 years or older in the united states ranges from 7% to 12% and increases according to the age group of 3.9% in 65 to 74 the age group, and increased to reach 25% in age group above 85 years.21 this is similar to the fi ndings in this study, that the robust group was found in the age range of 60-69 years while most of the frail group were over the age of 70 years. subjects in this study were all male, although the authors did not limit only one sex. in this study, 21 patients (55.3%) had normal bmi, 10 patients (26.3%) with low bmi, and 7 patients (18.4%) with high bmi. a study conducted by vestbo et al in 2008 also reported that 96.9% of the copd population had a normal or high bmi. we also fi nd similar fi ndings. a population-based epidemiological study conducted by de oca who examined bmi in copd patients conducted in 5 cities in latin america showed that most asian ethnicities had normal bmi, compared with less and more bmi. in this study the most comorbidity was hypertension, which was as much as 10.5%, followed by heart disease of 2.6%. other comorbidities such as diabetes mellitus were excluded in this study because diabetes mellitus can aff ect the results of adiponectin levels. in diabetes mellitus the level of adiponectin is low. in this study, various degrees of frailty were found in various degrees of copd, it was apparent that that prefrail and frail conditions were more common in copd subjects (table 2) determining the degree of frailty in elderly copd patients in this study, copd subjects were obtained with various degrees of frailty, both in copd with mild, moderate and severe obstruction. this shows that the higher the degree of obstruction, the higher the increase of prefrail and frail conditions. in a study conducted by lahouse in 2014 on the risk of frailty in elderly, as many as 28.8% of copd patients were found to be frail, 16.4% prefrail and 14.1% robust.22 this was diff erent from this study’s fi nding, where frail and robust had the same prevalence. this could be caused by age. the robust patients were mostly in the age range of 60 years while the frail ones were mostly in the age range of 70 years. in a study conducted by lahouse, the average age was 70 years. if the degree of obstruction was categorized based on gold classifi cation, out of 402 copd subjects, patients with mild obstruction were 200 subjects (49.8%), moderate obstruction 174 subjects (43.3%) and severe obstruction 28 subjects (7,0%). in this study, the prevalence of frailty was strongly related to the severity of copd, according to the degree of obstruction based on gold classifi cation. the higher the degree of copd obstruction, the frailer condition obtained, as compared to robust and prefrail conditions.22 measuring adiponectin level in elderly copd patients this study found elevated levels of adiponectin in copd patients, with a median of 7.55 μg/ ml in mild obstruction, 6.80 μg/ml in moderate obstruction, and 8.34 μg/ml in severe obstruction. the highest increase was found in copd with severe obstruction. chan, who examined serum levels of adiponectin in copd patients in 2010, found that copd subjects who smoked had signifi cantly higher levels of adiponectin, il-6 and crp than healthy smokers and nonsmokers. this study found that the higher the degree of copd, the higher the serum adiponectin level. serum adiponectin, il-6 and crp levels were negatively correlated with fev1 (% predicted) in copd patients and healthy smokers.23 similar 106 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: 101–107 to this study and chan’s study, tomoda et al. examined the levels of adiponectin in copd with low and normal body weight, also found increased levels of adiponectin in copd subjects.6 relationship between serum adiponectin levels and degree of frailty in elderly copd patients in this study, the median serum adiponectin levels in the robust, prefrail, and frailty groups were 5.9 μg/ml (3.68-11.59), 2.70 μg/ml (2.70-18,31), and 11.36 μg/ml (2.97-17.56) respectively. these results indicated that higher serum adiponectin levels are found at a higher degree of frailty. this study also found diff erences in adiponectin level between degrees of frailty and, in addition, also found a relationship between levels of adiponectin with degrees of frailty with spearman’s correlation coeffi cient of 0.368 and p=0.023 (p <0.05), showing a relationship between adiponectin levels and the degree of frailty. the analysis showed that the higher the level of adiponectin, the higher the degree of frailty. in a study conducted by tsai, who examined the relationship between adiponectin levels and frailty components, 168 subjects were found to be 65-90 years old, and 83 (49.4%) were male. serum adiponectin levels diff ered signifi cantly between the three subgroups (p=0.012). the results of the study showed that plasma adiponectin levels were positively related to an increase in frailty components in older men.10 in contrast to our study, the subjects in tsai’s study were elderly (>60 years), and tsai’s study as well as this study showed an increase in adiponectin levels. this indicates that in the elderly the adiponectin level is increasing. this study did not fi nd female respondents because female copd sufferers were rarely found. however, the data in this study, as those of tsai’s and huang’s fi ndings showed that sex was an important factor that could have aff ected not only blood adiponectin levels, but also the severity of frailty.24,25 conclusions serum adiponectin level in all subjects was found to increase with median in robust, prefrail, and frailty groups. the highest increase was found in severe degree copd. a weak positive relationship was found between adiponectin level and the degree of frailty. references 1. kirkwood tb, 2005. understanding the odd science of aging. cell; 120: 437–447. 2. halbert rj, natoli jl, gano a, badamgarav e, buist as & mannino dm, 2006. global burden of copd: systematic review and meta-analysis. european respiratory journal; 28: 523–532. 3. incalzi r, scarlata s , pennazza g, santonico m & pedon c, 2014. chronic obstructive pulmonary disease in the elderly. european journal of internal medicine; 25: 320–328. 4. kobayashi s, yanai m, hanagama m & yamanda s, 2014. the burden of chronic obstructive pulmonary disease in the elderly population. respiratory investigation; 52: 296–301. 5. fantuzzi g,2005. adipose tissue, adipokines, 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cohort study. thorax; 1–8 11. mittal n, raj r, ebtesam ataya islam ea & nugent k, 2015. the frequency of frailty in ambulatory patients with chronic lung diseases. journal of primary care & community health; 7(1): 10–15. 107erika marfiani, et al.: relationship between level of serum adiponectin and frailty copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 12. de oca mm, t'alamo c, perez-padilla r, b. jardim jr, muino a, lopez mv, valdivia g, pertuze j, moreno d, j. halbert r & b. menezes am, for the platino team, 2008. chronic obstruc tive pulmonary disease and body mass index in fi ve latin america cities: the platino study. respiratory medicine: 642–650. 13. brusik m, ukropec j, joppa p, ukropcova b, skyba p, balaz m, pobeha p, kurdiova t, klimes i, tkac i, gasperikova d & tkacova r, 2012. circulatory and adipose tissue leptin and adiponectin in relationship to resting energy expenditure in patients with chronic obstructive pulmonary disease. physiological research: 469–480. 14. breyer mk, rutten epa, locantore nw, watkins ml, miller be & wouters efm, 2012. dysregulated adipokine metabolism in chronic obstructive pulmonary disease. european journal clinical investigation; 42(9): 983–91. 15. mohamed na, fawzy ma, reda elgamry r , gad dm & ibraheem ha, 2013. role of adiponectin and other infl ammatory biomarkers in copd patients. egyptian journal of chest diseases and tuberculosis; 62: 45–50. 16. omar mm, isa ha, abdelsadek a & abd-elhamid ma, 2014. serum adiponectin level in obese and non-obese copd patients during acute exacerbation and stable conditions. egyptian journal of chest, diseases and tuberculosis; 63: 313–319. 17. rockwood k, song x, mcknight c, bergman h, hogan db & mc dowell i, 2005. a global clinical measure of fitness and frailty in elderly people. canadian medical association journal; 173: 489–495. 18. wang zv, scherer pe, 2016. adiponectin, the past two decades. j mol cell biol. apr; 8(2): 93–100. doi: 10.1093/jmcb/mjw011. epub 2016 mar 18. 19. global initiative for chronic obstructive lung disease (gold), 2014. chapter 2. diagnosis and assessment. in: global strategy for the diagnosis, management, and prevention of chronic obstructive ling disease updated 2014. 2014 global initiative for chronic obstructive lung disease inc. 20. fried lp, ferrucci l, darer j, williamson jd & anderson g, 2004. untangling the concepts of disability, frailty, and comorbidity: implications for improved targeting and care. the journals of gerontology. series a, biological sciences and medical sciences; 59a: 255–263. 21. lahousse l, maes b, ziere g, loth dw, verlinden vja, zillikens mc, uitterlinden ag, rivadeneira f, tiemeier h, franco oh, ikram ma, hofman a, brusselle gg, stricker bh, 2014. adverse outcomes of frailty in the elderly: the rotterdam study. european journal of epidemiology; 29(6): 419–427. 22. chan kh, yeung sc, yao tj, ip ms, cheung ah, chan-yeung ah, mak jc, and the copd study group of the hongkong thoracic society, 2010. elevated plasma adiponectin levels in patients with chronic obstructive pulmonary disease. international journal of tuberculosis and lung disease; 14: 1193–1200. 23. tsai js, wu ch, chen sc, huang kc, chen cy, chang ci, chuang lm & chen cy. 2013. plasma adiponectin levels correlate positively with an increasing number of components of frailty in male elders. plos one; 1–8. 24. huang c, niu k & momma h, 2014. inverse association between circulating adiponectin levels and skeletal muscle strength in japanese men and women. nutrition, metabolism & cardiovascular diseases; 24: 42–49. 25. huang c, momma h & niu k,2016. high serum adiponectin levels product incident falls among middleaged and older adults: a prospective cohort study. age and ageing; 45: 366–371. 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. 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 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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/ 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 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. 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 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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. 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ijtid@itd.unair.ac.id �� 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 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 original article prevalence of methicillin-resistant staphylococcus aureus (mrsa) carrier in hemodialysis patients at dr. soetomo academic general hospital eko oktiawan wicaksono1, artaria tjempakasari1*, widodo widodo1, kuntaman kuntaman2, usman hadi1 1.department of internal medicine, faculty of medicine universitas airlangga-dr. soetomo academic general hospital surabayaindonesia 2.department of clinical microbiology, faculty of medicine universitas airlangga-dr.soetomo academic general hospital surabaya-indonesia received: 8th april 2019; revised: 29th january 2020; accepted: 23rd april 2020 abstract chronic kidney disease (ckd) is now a global epidemic, and the prevalence is increasing worldwide. hemodialysis is one of the ways to treat by kidney function replacement. infection is the number two cause of death in patients with hemodialysis (hd). methicillin-resistant staphylococcus aureus (mrsa) is a common cause of bacteriemia in patients with dialysis. the epidemiological data of mrsa carriers in ckd in indonesia are still scarce. this study was to determine the prevalence of mrsa carriers in patients at the kidney and hypertension outpatient-clinic and hemodialysis installation at dr. soetomo academic general hospital, surabaya indonesia. the study design was descriptive-analytic with a crosssectional study design. sampling was collected consecutively. data on the general characteristics of the research subjects will be analyzed using a chi-squared test. there were 150 ckd stage fi ve patients included in this study, the number of patients has mrsa carrier were 6 (4%), among them, subjects underwent hd mrsa carrier were 2 subjects(2.7%), while for non-hd patients with mrsa were 4 subjects (5.3 %). there were no signifi cant diff erences in mrsa carriers between hd and non hd groups (p=0.404). comorbid factors that accompany mrsa carriers are diabetes mellitus, hypertension, kidney stones, gout, and systemic lupus erythematosus (sle). conclusions: this study found, there were no signifi cant diff erences in the incidence of mrsa carriers in stage fi ve ckd non hd or hd groups. mrsa colonization exists in stage fi ve ckd suff erers, so awareness of mrsa colonization. keywords: chronic kidney disease, hemodialysis, mrsa, diabetes mellitus, hypertension, indonesia. abstrak penyakit ginjal kronis (ckd) saat in menjadi epidemi global, dan prevalensi meningkat di seluruh dunia. hemodialisis adalah salah satu cara untuk terapi penggai ginjal. infeksi merupakan penyebab kematian nomor dua pada pasien dengan hemodialisis (hd). staphylococcus aureus yang resisten terhadap metisilin mrsa) adalah penyebab tersering bakteriemia pada pasien dengan dialisis. saat ini data epidemiologis pembawa mrsa pada penderita ckd di indonesia belum lengkap. penelitian ini untuk mengetahui prevalensi pembawa mrsa pada pasien-pasien di klinik rawat jalan ginjal dan hipertensi dan instalasi hemodialisis di rumah sakit umum dr. soetomo, surabaya indonesia. desain penelitian adalah deskriptif-analitik dengan desain penelitian cross-sectional. pengambilan sampel dikumpulkan secara berurutan. data karakteristik umum dari subjek penelitian akan dianalisis menggunakan uji chi-squared. terdapat 150 pasien ckd stadium lima yang masuk didalam penelitian ini, jumlah pasien yang menjadi pembawa mrsa ada 6 subjek (4%), di antara mereka, subjek yang menjalani hd sebagai pembawa mrsa ada 2 subjek (2,7%), sedangkan untuk pasien non-hd dengan pembawa mrsa ada 4 subyek (5,3%). tidak ada perbedaan yang signifi kan antara pembawa mrsa antara kelompok hd dan non hd (p = 0,404). faktor komorbid yang menyertai pembawa mrsa adalah diabetes mellitus, hipertensi, batu ginjal, asam urat, dan systemic lupus erythematosus (sle). penelitian ini mendapatkan, tidak ada perbedaan * corresponding author: okkiandra.29@gmail.com 190 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: 189–194 yang signifi kan pada kejadian pembawa mrsa pada stadium lima ckd non hd atau kelompok hd. kolonisasi mrsa ditemukan pada penderita ckd stadium lima, sehingga kesadaran pada kolonisasi mrsa. kata kunci: penyakit ginjal kronis, hemodialisis, mrsa, diabetes mellitus, hipertensi, indonesia. how to cite: prevalence of methicillin-resistant staphylococcus aureus (mrsa) carrier in hemodialysis patients at dr. soetomo academic general hospital. wicaksono, eo. tjempakasari, a. widodo, w. kuntaman, k. hadi, u. indonesian journal of tropical and infectious disease, 8(3), 189–194. introduction chronic kidney disease is a serious public health problem because there has been an increase in the number of patients, morbidity, and mortality.1 mrsa can be spreading from hospital to community, in addition to hospitalhospital transfers.2 in ckd patients with a history of hemodialysis with the use of vascular access, they should always be aware of the possibility of bacterial infections, it causes of death number 2 in patients with hemodialysis.3,4,5 vascular access is needed to obtain a large, enough blood fl ow. this access can be in the form of a fi stula (artery-vein), graft, or intravenous catheter, which functions to drain blood during hd. vascular access is one of the main risk factors for bacteremia (15.16%) and infections associated with frequent hospitalization and death (about 17.18%) in patients with hemodialysis.6 the percentage of fatalities in ckd stage 5 patients due to infection is quite large. patients who undergo hemodialysis are very susceptible to infection, especially methicillin-resistant staphylococcus aureus (mrsa). there was a meta-analysis study that estimates the prevalence of mrsa colonization in dialysis patients, the time and long-term risk of mrsa infection. from the data of 5596 dialysis patients, the prevalence of mrsa colonization was 6.2% (95% confi dence interval, 4.2% to 8.5%). prevalence increased over time but remained stable after 2000. over a long period (6-20 months), the likelihood of developing mrsa is around 19% in patients who have hemodialysis compared with patients with hemodialysis without mrsa colonization of about 2%.7 the infection will also worsen kidney function or will add a burden to the already poor kidney function, which will contribute to the increase in morbidity, mortality, and costs, therefore infection problems in stage fi ve ckd patients are critical.8 chronic kidney disease (ckd) is often a s s o c i a t e d w i t h s e v e r a l i m m u n o l o g i c a l abnormalities, both congenital immune system disorders, and adaptive immune systems and, therefore, can increase susceptibility to infection.9 infection enhanced with the use of a central catheter compared to av fistula. infections originating from the use of vascular access are often associated with microorganisms staphylococcus aureus and staphylococcus epidermidis.10 mrsa is a major nosocomial pathogen that aff ects inpatients. endemic mrsa strains originate from the hospital. most of the hd units have had patients with mrsa colonization of bacteria.5 bacteriemia or infection through the blood caused by s. aureus is a signifi cant cause of high morbidity and mortality.5,11 based on the above considerations, the researcher wants to examine the prevalence of mrsa carriers in ckd stage fi ve patients in the outpatient clinic and hemodialysis installation, as an essential data on the incidence of mrsa carriers. methods the study was a descriptive-analytic study with a cross-sectional design. the research subjects were obtained by consecutive sampling. the study was conducted in the outpatient clinic and hemodialysis installation of dr. soetomo general hospital, surabaya indonesia in july august 2018. there were two research groups, namely stage fi ve non-hd ckd and the stage fi ve ckd who had undergone hd treatment. 191eko oktiawan wicaksono, et al.: prevalence of methicillin-resistant staphylococcus aureus copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 the study sample was selected through the inclusion and exclusion criteria of the population of stage fi ve ckd patients in the hemodialysis unit and kidney and hypertension outpatient clinic. the requirements of participants: over 18 years old, willing to take part in this study, and signed informed consent. participants who were not included in the study, if one or more of the following criteria found: subjects with decreased consciousness or sepsis. specimens were collected by using sterile dry cotton swabs, and instructions were given on how to take swab samples from anterior nares and throat. one swab was used for both nostrils. all swabs were transported to the laboratory and directly inoculated into 5 ml of phenyl mannitol salt broth (difco), incubated overnight at 37oc and then subcultured onto mrsachromagar, and further identifi cation using vitex 2, as standard microbiological procedure in the microbiology laboratory at dr. soetomo general hospital.12,13,14 results there were 75 subjects in the hd group, and 75 subjects in the non hd group, prevalence mrsa carriers were found in 6/150(4%) of total samples. data on the general characteristics of research subjects in table 1. in the non hd group, there were 45 male subjects (60%), and 30 female subjects (40%). in the hd group, there were 36 male subjects (48%), and 39 female subjects (52%). in this study, the number of subjects with mrsa (+) carriers in patients who received hemodialysis was two (2.7%), and the number of mrsa carriers (+) in patients who have not undergone hemodialysis as many as four patients (5.3%). see table 2. carrier prevalence of mrsa with comorbid dm, there were 69 (46%) patients suff ering from dm, and 4 of them became mrsa carriers. in this study in the non hd group who suff ered ht as many as 65 (86.7%) with mrsa carrier incidence rates of 4 patients. whereas in the hd group, who suff ered ht as much as 64 (85.3%) with mrsa carrier incidence rate of 1 patient. in the non-hd group who suff ered kidney stones as many as 20 (26.7%), one patient with an mrsa carrier. in the hd group suff ering from kidney stones as many as 9 (12%), none of them with mrsa in this study in the non-hd group who suff ered from hepatitis b was 2 (2.7%). in the hd group subject suff ering from hepatitis b were 9 (12%), but from both groups, there were no carriers of mrsa in this study, the hepatitis c comorbid factor was not found, because the hd group with the hepatitis c comorbid factor was not willing to participate in this study. in this study, there were no mrsa carriers with comorbid factors in urinary tract infections in either group hd and non hd subjects. this study found only one patient with sle in the hd group, and also as a carrier of mrsa. table 1. general characteristics characteristics subject n (%) age (years) average ± sd 52.1±11.8 age range 19-78 co-morbid factors diabetes mellitus 69 (46%) hypertension 129 (86%) kidney stones 29 (19%) gout 30 (20%) hepatitis b 2(1.3%) sle 1(0,66%) cervical cancer 6(4%) hiv 1(0.66%) others (hepatitis c, ovarian cyst, uti) 0 (0%) types of vascular access av fi stula 74(49%) cvc 1(0.66%) hd frequency never been hd 75(50%) hd 1x / week 1(0,66%) hd 2x / week 74(49%) long hd <4 years 51(34%) 4-6 years 14(9.3%) > 6 years 10(6.660%) 192 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: 189–194 for other comorbid factors such as cervical cancer, ovarian cancer, ovarian cyst, and hiv, there were no mrsa carriers. the hd group that used double-lumen vascular access was one (1.33%) with an mrsa carrier occurrence rate of zero, while in the hd group who used av shunt vascular access as many as 73 (97.3%) with an mrsa carrier of two patients. the hd group who had done two times/week was 74 (97.3%) patients, with two mrsa carriers. there was no mrsa carriers found in hd group who had done one time/week. in the hd group with a length of time of hd less than 4 years as many as 51(68%) patients, with mrsa carriers of two (3.9%) patients, while the period of time undergoing hd 4-6 years was 14 (18.6%) patients, with mrsa carrier event of zero, while the length of time to experience hd more than 6 years was 10 (13.3%) patients, with mrsa carrier was zero. discussion patients undergoing dialysis are vulnerable to get mrsa infections, especially they have carries or colonization of mrsa in their nose or throat. this study found 150 patients, consist of 81 (54%) male and 69 women (46%), total mrsa carriers there were 6 (4%), in women as many as 3 (4.3%) patients, and 3 (3.7%) patients in male, where statistically was no signifi cant diff erence (p = 0.84). this result of mrsa carriers were lower than the result from a previous study in the same hospital in surgical and medical wards in the year 2016, there were 52 (8.1%) of 643 patients on admission were colonized with mrsa, this result was higher than our study, this is possible because most of the patients were referred from other hospitals.15 in this study, the prevalence of mrsa carriers who had not received hd treatment 4(5.3%) patients, compared to those who had received hd treatment 2(2.7%) patients, but the statistical diff erence was not signifi cant (p = 0.45). in the research of wang et al. found a little higher prevalence of mrsa carriers in patients with hd, the prevalence of s. aureus colonization in hemodialysis patients around 22.4% consisting of 16.5% mssa and 5.9% mrsa.16 this study had a mean age of 54 research subjects with the youngest age range of 19 years and the oldest 78 years in the non-hd group, with mrsa carrier incidence rates of four patients (50, 52, 61, and 76 years). as for the hd group, the average age of the study subjects was 49 years, with the youngest age range being 29 years and the oldest being 75 years with mrsa carrier incidence rates of two patients (ages 44 and 74 years). this research similar to the following study conducted by celik et al., 2011 which reported that patients with hemodialysis found higher mrsa carriers incidence rates in the age group between 55-64 years (30.55%) and mrsa carrier incidence rates that were the youngest with a range between 25-34 years.17 table 2. prevalence of mrsa carriers characteristics subject mrsa carriers (n) co-morbid factors diabetes mellitus 4(5.8%) hypertension 5(3.9%) kidney stones 1(3.4%) gout 2(6.67%) hepatitis b 0(0%) sle 1(100%) cervical cancer 0(0%) hiv 0(0%) others (hepatitis c, ovarian cyst, uti) 0 (0%) types of vascular access av fi stula 2(2.7%) cvc 0(0%) hd frequency never been hd 4(5.3%) hd 1x / week 0(0%) hd 2x / week 2(2.7%) long hd <4 years 2(3.9%) 4-6 years 0(%) > 6 years 0(%) 193eko oktiawan wicaksono, et al.: prevalence of methicillin-resistant staphylococcus aureus copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 non-hd group research subjects having comorbid diabetes mellitus as many as 45 people (60%) with an mrsa carrier occurrence rate of four patients. whereas for the hd group, who had dm comorbid factors as many as 24 patients (32%) with an mrsa carrier incidence rate of zero. other studies also reported similar data based on data from kang et al., 2012 found that out of a total of 296 research subjects. hd group with dm comorbid factors were 125 people.18 mrsa colonization was 11 patients (57.9%), while mrsa colonization was not found 114 (41.2%). research conducted by lai et al., 2011 found that of 306 research subjects with dm comorbid factors in hd patients with mrsa carriers as many as 11 people (37.93%), while subjects without mrsa carriers were 147 people (53.07%).19 another study yeoh et al., 2014, found that dm increases the high risk for mrsa colonization infection (odds ratio 4.2).20 research conducted by saxena et al., 2009 found that the prevalence of type 2 dm increases three times higher risk factors for mrsa nasal carriers compared to non-dms (72.4% vs. 24.6%) in patients with hemodialysis (rr. 2.97, p <0.0001). in nasal carriers, about 72.4% in dialysis patients with type 2 diabetes, 29% higher than in non-dm hd patients.21 this study found hypertension in the non-hd group was sixty-fi ve patients (86.7%) with an mrsa carrier occurrence rate of four patients. whereas for the hd group, who had hypertension comorbid factors as many as sixty-four patients (85.3%) with an mrsa carrier occurrence rate of one patients. a study conducted by kang et al., 2012, found that mrsa carriers in hd patients with hypertension as comorbid as many as 84.2%.18 this study found that hepatitis b comorbid factors in the non-hd group were two patients (2.7%) with an incidence of mrsa carrier of zero (0%). whereas for the hd group who had hepatitis b comorbid factors as many as nine patients (12%) with an mrsa carrier occurrence rate of zero (0%). this result concordance with research conducted by kang et al., 2012 found that of the subjects as many as 296 patients who had hepatitis b comorbid factors as many as 32 patients (10.8%) with mrsa carrier events as much as zero (0%).18 conclusion in this study, the prevalence of mrsa in subjects with stage fi ve ckd were 6/150 (4%) there were no significant differences in the incidence of mrsa carriers in stage fi ve ckd non hd or hd groups. this study shows that mrsa colonization exists in stage fi ve ckd suff erers who have or who have not received hd therapy. conflict of interest there is no confl ict of interest of this paper. acknowledgement thank for supporting of grant from dr. soetomo academic general hospital surabaya. references 1. stevens la, coresh j & selvin e, 2009. prevalence of chronic kidney disease in the united states. jama, 298, pp:2038-2047 2. huang yh, tseng sp, hu jm, tsai jc, hsueh pr, teng lj. 2007. clonal spread of sscmec type iv methicillin resistant staphylococcus aureus between community and hospital. clinmicrobiol infect; 13(7):717-24. 3. stone jcv, 1994. hemodialysis apparatus. in (jt daugirdas, ts ing, eds). handbook of dialysis, 2nd edition. london: little, brown and company, pp:3052 4. chen cc, chen cl, fang hc, lee pt, chang ty, chou kj, chung hm. 2005. the risk factors of staphylococcus aureus bacteremia related mortality among patients undergoing hemodialysis. acta nephrologica 18: 162-166. 5. levi j, morgan j & brown e, 2004 a practical guide to dialysis and how to manage end stage renal failure. oxford handbook 2nd ed 6. datta r, huang ss, 2008. risk of infection and death due to methicillin-resistant staphylococcus aureus in long-term carriers. clinical infection disease 47:17681 7. zacharioudakis im, zervou fn, ziakas pd, and mylonakis e. 2014. meta-analysis of methicillin194 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: 189–194 resistant staphylococcus aureus colonization and risk of infection in dialysis patients. j am soc nephrol. 25(9): 2131-2141. 8. agata emcd. 2018. addressing the problem of multidrug-resistant organisms in dialysisclin j am soc nephrol. 6; 13(4): 666–668. doi: 10.2215/ cjn.13781217 9. vanholder r, schepers e, meert n & lameire n, 2006. what is uremia? retention vs. oxidation blood purif. 2006;24, pp:33-38 10. dougirdas jt, blake pg & ing ts, 2015. infection. handbook of dialysis 5th ed. a lippincott william's & wilkins handbook. wolters kluwer pp:615-663 11. chatterjee a, rai s, guddattu v, mukhopadhyay c, and saravu k. 2018. is methicillin-resistant staphylococcus aureus infection associated with higher mortality and morbidity in hospitalized patients? a cohort study of 551 patients from south western india risk manag healthc policy. 11: 243–250.doi: 10.2147/rmhp. s176517 12. tiemersma ew, bronzwaer sl, lyytikäinen o. 2004. european antimicrobial resistance surveillance system participants. methicillin-resistant staphylococcus aureus in europe, 1999–2002. emerg infect dis2004; 10: 1627–1634. 13. nahimana i, francioli p, blanc ds. 2006. evaluation of three chromogenic media (mrsa-id, mrsa-select, and chromagar-mrsa) and orsab for surveillance cultures of methicillin-resistant staphylococcus aureus. clin microbiol infect.; 12(12):1168-74. 14. stoakes l, reyes r, daniel j, lennox g, john ma, lannigan r, hussain z. 2006. prospective comparison of a new chromogenic medium, mrsaselect, to chromagar mrsa and mannitol-salt medium supplemented with oxacillin or cefoxitin for detection of methicillin-resistant staphylococcus aureus.j clin microbiol 44(2):637-9. 15. kuntaman k., hadi u., setiawan f., koendori eb., rusli m., santosaningsih d & verbrugh ha. 2016. prevalence of methicillin-resistant staphylococcus aureus from nose and throat of patients on admission to medical wards of dr. soetomo hospital, surabaya. indonesia. southeast asian journal of tropical medicine and public health 2016, 47 (1), pp:66-70. 16. wang cy, wu vc, wang wj, lin yf, lin yh, chen ym, su ct, wang jy, wu kd & hsueh pr, 2012. risk factors for nasal carriage of methicillin-resistant staphylococcus aureus among patients with end-stage renal disease in taiwan. journal of the formosan medical association (2012) 111, 14-18 17. celik g, gulkan a, dikici n & gulcan e, 2011, prevalence of nasal staphylococcus aureus carriage in the patients undergoing hemodialysis and evaluation of risk factors and laboratory parameters. pp:494498 18. kang yc, tai wc, yu cc, kang yh, and huang yc. (2012). methicillin-resistant staphylococcus aureus nasal carriage among patients receiving hemodialysis in taiwan: prevalence rate, molecular characterization, and de-colonization. bmc infectious diseases 12:284. available in: http://www.biomedcentral.com/14712334/12/284. accessed 5th. dec. 2019 19. lai cf, liao ch, pai mf, chu fy, hsu sp, chen hy, yang jy, chiu yl, peng ys, chang sc, hung ky, tsai tj, wu kd. 2011. nasal carriage of methicillinresistant staphylococcus aureus is associated with higher all-cause mortality in hemodialysis patients. clin j am soc nephrol. 6(1):167-74 doi: 10.2215/ cjn.06270710 20. yeoh ly, and tan fl, willis gc & ooi st., 2014. methicillin-resistant staphylococcus aureus carriage in hospitalized chronic hemodialysis patients and its predisposing factors. hemodialysis international 2014, pp:142-147 21. saxena ak, panhotra br, venkateshappa ck, sundaram ds, naguib m, uzzaman, w & al mulhim k., 2002. the impact of nasal carriage of methicillinresistant staphylococcus aureus and methicillinsusceptible staphylococcus aureus (mrsa & mssa) on vascular access-related septicemia among patients with type-ii diabetes on dialysis. renal failure vol. 24, no 6, pp:763-777. 22. karanika s, zervou fn, zacharioudakis im, paudel s &mylonakis e., 2015. risk factors for methicillinresistant staphylococcus aureus colonization in dyalisis patients: a metaanalysis. journal of hospital infection xxx(2015) pp:1-7 23. p e r n e f r i , 2 0 0 3 . k o n s e n s u s d i a l i s i s . j a k a r t a : perhimpunan nefrologi indonesia 24. kotanko p, kuhlmann mk & levin nw, 2010. hemodialysis: principles and techniques in: j foege, rj johnson, j feehalli, eds. comprehensive clinical nephrology. 4th ed. st louis, missouri: elsevier; 2010. pp: 1053-9. 25. lerma ev, weir mr, 2017. immune dysfunction and low grade persistent infl amator practical guide to dialysis and how to manage end stage renal failure. oxford handbook 2nd ed. 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 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 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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 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, 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. 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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. 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. �� 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. 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 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. 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). 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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 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 ijtid vol 6 no 2 mei-agustus 2016_edit.indd 29 vol. 6. no. 2 mei–agustus 2016 research report production and characterization of immunoglobuline yolk as anti antigen membrane toxoplasma gondii yuliana praptiwi1, heni puspitasari2a, luciana t suwanti3, mufasirin 3 1 magister of veterinary, the faculty of veterinary medicine, universitas airlangga surabaya 2 toxoplasma study group, instutute of tropical disease, universitas airlangga surabaya 3 parasitology departement, the faculty of veterinary medicine, universitas airlangga surabaya a corresponding author: henipuspitasari486@gmail.com abstract toxoplasma gondii is an obligate parasite intracellular which can infection human and other mammalian. immunoglobulin y technology offers several advantages better than antibody production in mammals. this research is aimed to get immunoglobulin y from egg yolk, and to find the characterization of immunoglobuline y according to molecular weight by sds page and targeted protein with antibodies using western blot. this research divided from many step: culture tachyzoites of t. gondii fromintraperitoneal fluid, preparation of membrane antigen tachyzoite of t. gondii, then immunization laying hens with membrane antigen, extraction and purification immunoglobuline y from egg yolk and then protein analyzed by sds page and western blot. the result of this research showed that immunoglobulin y from egg yolk can produced antibody against protein membrane of t. gondiiandprofile protein immunoglobuline y according sds page has molecular weight 179,8 kda. immunoglobuline y was analyze by western blot can recognize antigen epitope of t.gondii on molecular weight 35,7kda and 78,8 kda. keywords: toxoplasma gondii, anti membrane t.gondii, immunoglobulin y anti membrane abstrak toxoplasma gondii merupakan parasit obligate intraselluler yang dapat menginfeksi manusia dan mamalia lain. pemanfaatan immunoglobulin y memberikan beberapa keuntungan dari pada antibodi yang diproduksi mamalia. tujuan dari penelitian ini adalah mendapatkan immunoglobulin y dari kuning telur dan menemukan karakterisasi immunoglobulin y berdasarkan berat molekul dengan sds page dan penargetan protein dengan antibody menggunakan westerblot. penelitian ini dibagi menjadi beberapa tahapan yaitu kultur takizoit toxoplasma gondii dari cairan intraperitoneal, preparasi antigen membran toxoplasma gondii, imunisasi ayam dengan protein membrane toxoplasma gondii, ekstraksi dan pemurnian kuning telur untuk mendapatkan ig y dan karakterisasi ig y dengan sds page serta penargetan protein westrn blott. hasil sds page ig y ditemukan pita protein 178 kda7038kda, kemudian analisa penargetan protein dengan westrn blottdapat mengenali antigen epitop toxoplasma gondii pada berat molekul 35,7kda dan 78,8kda. kata kunci: toxoplasma gondii, membran anti t. gondii, immunoglobulin y anti membran introduction toxoplasma gondii is an obligate intracellular parasite that can infect humans. definitive host of this parasite is the cat, while the intermediary hosts include mammals, birds and reptiles nation even fish. in the life cycle of this parasite can infect a host by 3 ways: through ingestion of tissue cysts by bradizoit, by ingestion of oocysts and congenital infection with tachizoit.1 cats infected with toxoplasma gondii in all excretions will spend millions of oocysts. when the oocyst is ingested by an intermediate host such as humans, cows, goats on the various tissues will be established intermediate host groups tropozoit actively dividing to form the rest of the stadium in the form of cysts 30 indonesian journal of tropical and infectious disease, vol. 6. no.2 mei–agustus 2016: 29−33 (bradizoit) on the network. at the intermediate host is not formed sexual stage but only just asexual stage. when cats eat mice containing cysts are formed in the sexual stage in the cat’s intestine.2 humans infected with the t. gondii occurs not only on those who keep cats or dogs but can also occur in other people who like to eat the food of undercooked meat containing tissue cysts, drinking fresh milk undercooked, water contaminated with raw vegetables and raw contaminated by disease-causing agents toxoplasmosis.2 incidence of toxoplasmosis has not significant changed in recent years, caution and attention to these diseases has increased dramatically. about 30-50% of the world population is estimated have been infected by toxoplasma gondii. according to chandra, gandhahusada research that conducted in 1995 showed that prevalence toxoplasmosis in humans ranges between 2–63%, 35–70% on cat, 75% in dogs, 11–61% in goats, 11–36% in pigs, and less than 10% on the cow.3 research results from fitria, showed that 46,66% pork intersection in rph surabaya positive toxoplasmosis.4 the negative impact on the human is very detrimental to the failure of pregnancy and abortion. in human and animal therapy for this disease is very expensive, the impact of livestock on the economic loss due to a decline in production. the administration of drugs such as pyrimethamine and a sulfonamide can kill tachizoit of stadium t. gondii, but these treatments are not effective on stage bradizoite. in addition, these drugs are toxic, so is not recommended for use in the long term. prevention by vaccination not fully provided protection. using antibodies for controlling is start assess, one of them is making antibodies from the egg yolk. antigens which used as the immune system host stimulation can come from different parts of the body t. gondii. one of them that can be used as an antigen is membrane of stadium tachizoit t. gondii. specific antigen in tachizoit surface are p30, sag-1, p22 (sag-2), p35, while the same protein membrane between tachizoit and bradizoite are p23 and p43 (sag-3).5 this membrane has immunogenic protein. some protein major of tachizoit such as p22, p23, p30 and p45 have molecular weight around 20–43 kda. membrane protein is able to provoke both cellular immune response (lymphocyte cell, nk cell) and humoral (immunoglobulin), so that many use as diagnostic kit and vaccine.6 in chronic toxoplasmosis cases, one of the immune system that had the play role is immunoglobulin g (igg). some efforts had done to multiply both polyclonal and monoclonal antibody using animal trial. serum from this animal should be taken to get its antibody and this animal should be sacrificed. it becomes consideration both animal welfare aspect and economics. the research of igy usage as passive immunization had been done by chalghoumi et al., on wilkie, by using salmonella enteridis that was changed into antigen and given to layer hens.7 the advantage of igy is not activated the complement, igy did not bind a and g protein, igy did not bind the mammals antibody, such as rheumatoid factor which is similar with auto-antibody that react with fc receptor in igg and hama (human anti-murine antibodies), igy did not bind fc receptor in the surface. the character of igy which is similar with igg in mammals, both from its structure and its function. material & method this research included in laboratory explorative research. research design was used descriptive analysis. the animals that were used for collecting igy were layer hens strain with 20 weeks of age, 5 hens were adapted during 2 weeks. toxoplasma gondii passage and cultivation in mus musculus strain balb/c with 3 months of age. toxoplasma gondii strain rh was used in this research, from department of parasitology, medical faculty, universitas gajah mada. tachizoit t. gondii cultivating and harvesting were done by infecting mice with t. gondii rh strain isolation by 103 for doses in 50 mice balb/c through intraperitoneally. protein membrane tachizoit of t. gondii was isolated by sonication and centrifugation. protein concentration was interpreted using spectrophotometry with 595 nm.6 in vitro cultivating and harvesting of tachizoit t. gondii were done by using infected mice with isolate t. gondii strain rh through 1 x 103 on 50 balb/c mice by intraperitoneal. isolation of protein membrane tachizoit t. gondii used sonication and centrifugation.6 protein concentration reader used spectrophotometry with 595 nm. protein was aliquot and saved on -20° c until it was used.6 protein membrane tachizoit of t. gondii was analyzed using sds page. the chicken that immunization using antigen from protein membrane tachizoit of t. gondii by 50 μg that was diluted on pbs and emulted using freund’s complete adjuvant through 1:1 of ratio until homogenous. emulsion injected through intra-muscular (on femur). immunization was repeated twice with 14-days interval. on repetition immunization, 50 μg of antigen was emulted using freund’s complete adjuvant then 14 days after second repetition. isolation of anti-toxoplasma antibody on yolk used combination of chloroform and ammonium sulfate precipitate method was the chosen method that produce antibody with high purity level.8 purification was done by using precipitation of ammonium sulfate 40% and using ratio between igy supernatant: ammonium sulfate 40% was 1:1. solution was precipitated one night on 4° c and was centrifuged with 10,000 rpm for an hour.9 precipitate was taken for re-suspension using pbs then it was purified and analyzed. immunoglobulin y precipitation was covered with specific plastic for sonication, then added 0.5m pbs and stirred using magnetic stirrer for 24 hours in 4° c. characterization was done by reacted antibody from purification with antigen membrane using elisa method and western bloth. igy antibody titter measurement using elisa. 31praptiwi, et al.: production and characterization of immunoglobuline yolk as anti antigen membrane immunoglibulin y protein analyzed using sds page. antigenic membrane protein of t. gondii was identified using western bloth method. igy antibody titter measurement using elisa. result and discussion the result of chicken immunization was read using optical density level on indirect elisa. igy measurement was done after the third immunization booster. elisa was done on two samples, blood serum and immunized yolk by membrane antigen. sample consist of yolk that taken before immunization and on the 7th, 14th and 28th day after immunization. the result of elisa on yolk between before and after immunization of t. gondii membrane antigen shows that od level increased and it significant difference between before and after immunization (p < 0.005). the 7th and 14th day after immunization, there were no differences. result of elisa od level can be seen on table 1. immunoglobulin y was gotten from egg yolk that was extracted with chloroform and precipitated with ammonium sulfate 40% then the protein was analyzed using sds page. marker used for sds page of immunoglobulin y could detect protein with molecular weight between 10 kda-260kda. protein molecular weight assessment had done using regression analysis between rf and bm log. on this research, protein band on marker had line equation y= -2.501x5 + 2.920x4 – 2.202x3 + 3.467x2 – 3.253x + 2.597. on the 2nd column, immunoglobulin without dilution, showed 6 protein bands with molecular weight 179.8kda, 130.4kda, 70.6kda, 59.1kda, 38.6kda and 25 kda. on the 3rd column, immunoglobulin y with dilution ration 1:5 showed 4 protein bands, 179.8 kda, 67.4 kda, 61.6 kda and 38.6 kda. on the 4th column, immunoglobulin y with dilution ration 1:10 showed 4 protein bands, 179.8 kda, 67.4 kda, 61.6 kda and 38.6 kda. column 5th, immunoglobulin y with dilution ration 1:20, showed protein bands with molecular weight were 179.8 kda dan 67.8 kda. concentration of immunoglobulin y protein from preparation was 0.16 μg/μl. the result of western blott, using antigen that were t. gondii, was reacted with polyclonal antibody from egg yolk which were ig y. then, it compared with antibody from rabbit that was already immunized using toxoplasma gondii proteins, igg. used marker on sds page could detect protein with 20 kda–120 kda of molecular weight. molecular weight assessment of immunoglobulin y had been done using regression analysis between rf and bm log. on this research, protein band on marker had equation as y = -3.103x4 + 5.506x3 – 2.515x2 – 0.936x + 2.246. the 1st column was antigen of t. gondii membrane which was reacted with rabbit ig g. this column showed protein band with molecular weight 35.7 kda. the 2nd and 3rd column were antigen of t. gondii membrane that was reacted with chicken igy and showed protein band with molecular weight 35.7 kda and 78.8 kda. p r e p a r a t i o n o f m e m b r a n e a n t i g e n p r o t e i n o f toxoplasma gondii on tachizoit stadium was continued with characterization using (sds page) method. this method was the common used of electrophoresis method. electrophoresis method was used for protein characterization based on molecular weight. the result of sds page analysis of t. gondii membrane protein showed 35.4 kda, 59.8 kda; 66.8 kda; 81.9kda; 86.8kda which were more than 118 kda (figure 2). based on chalghoumi, et al., (2009), 10-100 kda protein for vaccine were needed table 1. averages of igy od level on yolk that was immunized with t. gondii membrane antigen yolk od level average od level deviation before immunization 7th day after the 3rd immunization 14th day after the 3rd immunization 28th day after 3rd immunization 0.9856a 1.5332b 1.8873b 1.8303b 0.5424 0.2201 0.3788 0.3640 different superscript on the same column show significant difference (p < 0.005) figure 1. the result of immunoglobulin y protein preparation using sds page note: 1st marker 2nd immunoglobulin without dilution 3rd immunoglobulin with dilution ratio 1:5 4th immunoglobulin with dilution ratio 1:10 5th immunoglobulin with dilution ratio 1:20 on the 5th column, there is protein with 67.4kda and 179.8kda of weight 32 indonesian journal of tropical and infectious disease, vol. 6. no.2 mei–agustus 2016: 29−33 to provoke immune response. it fulfill requirement for immunization so hopefully could provoke immune response.7 from this result was in accordance with the suwanti’s research6 that major protein on membrane had molecular weight between 60 kda–200 kda. 66 kda–70 kda of protein also found in membrane and roptry t. gondii was proven by bonhomme et al. (1990) which was citated by suwanti.6 it was proven by protein characterization result of p22 recombinant from tachizoit membrane that showed protein band between 35 kda–40 kda.5 molecular weight of protein band above 118 kda could not be determined using regression equation between log of molecular weight and rf from marker, since those protein out of regession line. antibody was produced from induced egg yolk with immunization from membrane t. gondii protein. on this research, immunization was done via intra-muscular for three times using mix of complete freund adjuvant at the 1st immunization and incomplete freud adjuvant at the 2nd and 3rd immunization. the aim of cfa and ifa given was for induced the bigger immune response. it caused on cfa consist of protein from death micobactery or component from cell wall of bacteria which had ability to induce both cellular immune response and humoral response against injected protein antigen. therefore, cfa addition hopefully can form antibody against t. gondii antigen membrane. this research used indirect elisa model for detected ig y, thereby anti-igy conjugate was needed. the form of igy similar with igg was monomer, so the system of igy also had high affinity against antigen. fab (antibody fragment) on igy could recognize antigen epitope more than on igg. structure of heavy and light chain of igg and igy was relatively similar which was two heavy chains on igy had molecular weight 67–70 kda on each chain and two light chain with molecular weight 25 kda on each. the differences of igg and igy only on ch4 chain on fc.7,10 od level on elisa result both on yolk and on serum which was immunized with antigen of t. gondii membrane showed that there was significant different between before immunization and after the 3rd immunization. it showed that antigen of t. gondii membrane was immunogenic. based on abbas,10 forming of immunoglobulin antibody would increase on the 2nd antigen exposure with the same antigen type.11 b cell would produce immunoglobulin after 5 days after antigen exposure and immunoglobulin level would be kept on 23 days.7 if booster was done every 14 days, the increasing of antibody would occur after the 3rd immunization. on the screening of both yolk and serum on the 7th day and 14th day after the 3rd immunization did not show significantly difference. it indicated that between 7th day and 14th day after the 3rd immunization immunoglobulin y production was relatively constant on the chicken body. on the 28th day after immunization did not show significantly difference. antibody testing on chicken serum was done until the 14th day after the 3rd immunization. on this research, protein band with molecular weight 25 kda and 38.6 kda might be fragment from fab igy on light chain, while protein with molecular weight 59.1 kda, 61.6 kda, 67.4 kda and 70.6 kda were fragment from fab igy on heavy chain. this was in accordance with michael et al.,8 who said that two heavy chains on igy had molecular weight 67–70 kda on each and two light chain with molecular weight 25 kda on each.8 protein band which had molecular weight 130.4 kda, 179.8 kda was probably the fragment from complex bond of fc receptor igy (fcr-igy) and the whole molecule from igy. this was in accordance with he and bjorkman, which was said that fcr-igy complex and whole igy had molecular weight between 150 kda–180 kda.10 the result of western blot using antigen from t. gondii membrane which was reacted with polyclonal igy antibody showed protein band reaction with molecular weight 35.7 kda and 78.8 kda (figure 2). on 35.7 kda of western blot showed that igy antibody could recognize antigen apitop, that was shown by band reaction between protein from antigen which was 35.7 kda (sag 1) t. gondii with 38 kda of igy molecule (light chain fab igy). p30 t. gondii (sag1) was the major protein on rh strain and had molecular weight around 30–38 kda. surface antigen (sag) was protein which took role on attachments. sag was protein on the tachizoit surface that consist of glicosilphosphatidilinolsitol (gpi) and helpfully gave signal on attachment process between sag and ligan on the cell surface that would be infected.12 western blott result which had reacted with primary antibody of rabbit (igg) also showed protein band with molecular weight 82 kda. it proved that the recognizing epitope of t. gondii antigen against fab igg of figure 2. t h e r e s u l t o f t . g o n d i i m e m b r a n e p r o t e i n characterization using western blott. 1st column was t. gondii antigen which was reacted with rabbit igg antibody, 2nd and 3rd column were t. gondii antigen which were reacted with chicken igy antibody and the 4th column was marker. the red sign was protein with 78.8 kda of molecular weight, green sign was protein with 35.7 kda of molecular weight. 33praptiwi, et al.: production and characterization of immunoglobuline yolk as anti antigen membrane rabbit was occure. fab igg of mammalian on heavy chain had molecular weight 67-70 kda and light chain 25 kda.8 on the result of western blott using rabbit igg antibody showed 35.7 kda of molecular weight. it meant that fab igg could recognize protein of tachizoit membrane and proved there was similarity of fab structure from igg and igy. fragment from molecular antibody was antigen binding fragment and fc was crystalizable fragment (constant) as biology effector. on aves, igy fc receptor was known as fcry. in fact, aves fcry had similarity with fcrn igg on mammals, whereas fcrn also act as mhc1 which could bind with antigen peptide for t cell. the similarity of fcry and fcrn was could bind with immunoglobulin molecule on ph ≤ 6 and did not bind on ph ≥ 7. fcry which bind with the whole igy molecule had dimer structure with n terminal chain and cyctin receptor for binding peptide from antigen. fcry bonded on igy ch4 chain. the differences between fcry and fcrn were on recognizing ligand receptor of ch3-ch4 igy and ch2-ch3 igg whereas igy ligand had double ability than igg ligand. antigen epitope could be recognized by more igy molecule than mammalian immunoglobulin.10 conclusions to sum up briefly, the result from profil analysys of membrane protein of tachizoit t. gondii was protein with molecular weight 35.4 kda, 59.8 kda, 66 kda, 81 kda and 86 kda. immunoglobulin y from egg yolk could produce antibody anti protein of t. gondii membrane. based on western blot result, could be concluded that protection mechanism of immunoglobulin y was on fab which could recognize epitop of t. gondii antigen with molecular weight 35.7 kda and 78.8kda references 1. hanafiah, m., wisnu n, mufti k, dan fadrial k. 2009. produksi dan isolasi protein membran stadium bradizoit toxoplasma gondii: suatu usaha untuk mendapatkan material diagnostik dalam mendiagnosa toksoplasmosis. fakultas kedokteran hewan universitas syiah kuala. aceh. vol. 10 no. 3: 156–164. 2. hiswani. 2003. tesis: toxoplasmosis penyakit zoonosis yang perlu diwaspadai oleh ibu hamil. fakultas kesehatan masyarakat. universitas sumatera utara. chandra, g. 2001. toxoplasma gondii: aspek biologi, epidemiologi, diagnosis dan penatalaksanaannya. 3. chandra, g. 2001. toxoplasma gondii: aspek biologi, epidemiologi, diagnosis dan penatalaksanaannya. http://www.emedice.com. (juni 2011). 4. ardhiani, f. 2008. insidensi toxoplasmosis pada babi di rph pengirian surabaya dan rph gadang malang. fakultas kedokteran hewan universitas airlangga. surabaya. 5. arabpour, m., mojgan b. maryam n., seyyed h.a. 2011. african journal of biotechnology vol. 10(40): cloning and expression of toxoplasma gondii tachyzoite p22 protein. department of parasitology and mycology, school of medicine, shahid beheshti university of medical sciences, tehran, iran., pp. 7746–7750. 6. suwanti, l.t. 1996. identifikasi dan produksi antibodi monoklonal protein membran toxoplasma gondii stadium takizoit. tesis pascasarjana universitas gadjah mada. yogyakarta. 7. chalghoumi, r., b. yves, p. daniel and t. andre. 2009. hen egg yolk antibodies (igy), production and use for passive immunization against bacterial enteric infection in chicken. gembloux agriculture university. belgium. 295–308. 8. michael, a., s. meenatchisundaram, g. parameswari, t. subbraj, r. selvakumaran and s. ramalingam. 2010. chicken egg yolk antibodies (igy) as an alternative to mammalian antibodies. indian j. science technology. 3(4): 468–474. 9. ko k. and ahn d.u, 2007. preparation of immunoglobulin y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography. poultry science. 86: 400–407. 10. he, y and pamela j.b. 2011. strukture of fcry, an avian immunoglobuli receptor related to mammalian mannose receptor, and its compleks with igg. california institute of technology. usa. page: 12431–12436. 11. abbas, a.k., a.h. lichtman and j.s. pober. 2000. cellular and molecular immunology. w.b. saunders company, philadelphia. p p. 235–338. 12. carruthers, v.b. 2002. host cell invasion by the opportunistic pathogen toxoplasma gondii. acta trop. 81: 111–122. 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 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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. 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 a prototype n95 sterilizer: an alternative solution during personal protective equipment crisis muh. aprizal azhar1 , rosdiana natzir2 , rizalinda sjahril3,4 , elyas palantei5 , sudirman katu6 , najdah hidayah7 , muhammad nasrum massi3,4* 1master of biomedical science, graduate school universitas hasanuddin, makassar, indonesia 2department of biochemistry, faculty of medicine, universitas hasanuddin, makassar, indonesia 3department of microbiology, faculty of medicine, universitas hasanuddin, makassar, indonesia 4microbiology laboratory, hasanuddin university hospital, makassar, indonesia 5faculty of engineering, universitas hasanuddin, makassar, indonesia 6department of internal medicine, subdivision of tropical infectious diseases, faculty of medicine, universitas hasanuddin, makassar, indonesia 7postgraduate program, faculty of medicine, universitas hasanuddin, makassar, indonesia received: july 14th, 2022; revised: july 27th, 2022; accepted: september 12th, 2022 abstract the high demand for n95 masks, especially during the covid (coronavirus disease)-19 pandemic, has caused shortages worldwide. this study aimed to examine the sterilization ability of the portable sterilizer prototype for n95 masks and its effect on the filtration ability and changes in air resistance on the n95 mask in order to thrift personal protective equipment (ppe) use during a shortage. the sample used was an n95 mask type 1860. the mask was contaminated with 0.6-0.8 mfu staphylococcus aureus and escherichia coli. the sterilization methods used were ultraviolet germicidal irradiation (uvgi), heat at 75°c, and a combination of both from 1 to 120 minutes. next, the masks were cultured in a nutrient agar medium. for aerosol penetration and air resistance tests, masks were tested before and after the sterilization process, lasting from 5 to 60 minutes. this prototype sterilizer with heat effectively killed e. coli and s. aureus starting from 3 minutes. the filtration ability of the n95 mask was maintained at >95% even after the sterilization process with 75°c heat, uvc, or a combination of both for up to 60 minutes. there was also no significant difference in air resistance between new masks and masks that had been sterilized using a portable sterilizer. this prototype sterilizer with heat at 75°c can effectively sterilize against both gram-positive and negative bacteria in the n95 mask without reducing the aerosol filtration ability and changing the air resistance of the n95 mask. keywords: aerosol; filtration; n95; personal protective equipment; sterilization abstrak tingginya permintaan masker n95 terutama di masa pandemi covid (coronavirus disease)-19 menyebabkan kelangkaan masker di seluruh dunia. penelitian ini bertujuan untuk menguji kemampuan sterilisasi dari prototipe portable sterilizer masker n95 dan pengaruhnya terhadap kemampuan filtrasi dan perubahan hambatan udara pada masker n95 dalam rangka penghematan penggunaan alat pelindung diri (apd) pada saat terjadi kelangkaan. sampel yang digunakan adalah masker n95 tipe 1860. masker dikontaminasikan dengan 0,6-0,8 mfu (mcfarland unit) staphylococcus aureus dan escherichia coli. metode sterilisasi yang digunakan adalah ultraviolet germicidal irradiation (uvgi), panas pada suhu 75°c, dan kombinasi keduanya dalam durasi 1 hingga 120 menit. selanjutnya, masker dikultur dalam media nutrien agar. untuk uji penetrasi aerosol dan hambatan udara, masker akan diuji sebelum dan sesudah proses sterilisasi dengan durasi 5 hingga 60 menit. prototipe sterilizer dengan panas 75 oc ini efektif membunuh e. coli dan s. aureus mulai dari 3 menit waktu sterilisasi. kemampuan filtrasi * corresponding author: nasrumm@unhas.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-9682-8149 https://orcid.org/0000-0001-6444-7037 https://orcid.org/0000-0002-4678-9372 https://orcid.org/0000-0001-8876-4926 https://orcid.org/0000-0002-9788-3262 https://orcid.org/0000-0002-9811-2080 https://orcid.org/0000-0002-3347-6529 177 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license muh. aprizal azhar, et al. a prototype n95 sterilizer masker n95 tetap terjaga >95% meskipun telah melalui proses sterilisasi dengan panas 75°c, uvc, atau kombinasi keduanya hingga 60 menit. selain itu, tidak ada perbedaan yang signifikan dalam hambatan udara antara masker baru dan masker yang telah disterilkan menggunakan alat sterilisasi portabel. prototipe alat sterilisasi dengan panas pada suhu 75°c ini dapat secara efektif mensterilkan bakteri gram positif dan negatif pada masker n95 tanpa mengurangi kemampuan filtrasi aerosol dan mengubah hambatan udara masker n95. kata kunci: aerosol; alat pelindung diri; filtrasi; n95; sterilisasi how to cite: azhar, m. a., natzir, r., sjahril, r., palantei, e., katu, s., hidayah, n., massi, m. n. a prototype n95 sterilizer: an alternative solution during personal protective equipment crisis. indonesian journal of tropical and infectious disease. 10(3). 176–188. dec. 2022. introduction infectious diseases are one of the leading causes of death in the world. the world health organization (who) reports that lower respiratory tract infections are the fourth leading cause of death globally and the second most common cause of death in developing countries.1 the easy transmission of disease from animals to humans or fellow humans makes infectious diseases have a reasonably high incidence. one method of transmission that transmits very quickly is through aerosol or airborne. this transmission occurs when an infected person expels droplets or aerosols when talking, singing, coughing, or sneezing.2,3 one way to prevent this disease's transmission is using face masks. the use of face masks can reduce a person's chance of being infected by up to 90%.4 one type of face mask is recommended for health workers as personal protective equipment (ppe) on duty is the n95 mask. the high demand for n95 masks, especially during the covid-19 pandemic, has caused shortages worldwide.5 the center for disease control and prevention (cdc) publishes guidelines for reusing n95 masks during the ppe crisis. this reuse must pay attention to several things regarding n95 masks. some things that need to be considered in mask reuse are contamination and filtration performance. in addition, it is also necessary to pay attention to the mask damage and its fitting.6 many studies have been conducted on the sterilization methods of n95 masks for reuse. some methods studied were evaporation, dry heat, ultraviolet c (uvc), gamma radiation, hydrogen peroxide, boiling in water, and liquid disinfectants such as chlorine and alcohol.7–9 some of these methods damaged the mask, but uvc and dry heat were reported to maintain a safe mask's filtration performance.7 based on this problem, we designed a special portable sterilizer for n95 masks, which can eliminate pathogens but does not affect mask performance. this sterilizer is compact, easy to use, and can be used anywhere. materials and methods materials & tools the masks used were the n95 type 1860 3m masks. e. coli atcc 25922 and s. aureus atcc 6538 isolates were used to contaminate the masks. particle counter hti type ht9600, manometer ht-1890, and nebulizer omicron my-520a were used as aerosol generators. the particle counter could measure aerosol particles starting from 0.3 µm, 2.5 µm, to 10 µm. this device also had a maximum measurement capability of 107 piece/l particles and resolution up to 1 piece/l. methods prototype design of portable sterilizer this portable sterilizer was made using a 15 mm medium density fiberboard (mdf). dimensions were about 37 cm high, 21.8 cm wide, and 21 cm depth. inside the sterilization chamber, a wire mesh was placed as a base, and hangers were attached on the sides so that the masks could be hung vertically on the side 178 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: 176–188 walls so that all masks could get an even heat between one another. the prototype of the portable sterilizer is shown in figures 1a and 1b. figure 1. portable sterilizer (a) front view, (b) inside view of portable sterilizer prototype when operating this device used the heat method for sterilization resulting from converting electrical energy into heat. the heating element used was nichrome ni80 wire (80% nickel, 20% chromium). a 4w uvc lamp was also added to maximize the sterilization effectiveness. the power source of this tool used a 240 w power supply with a 12 v dc and 20 a current. this tool could produce 200 w of thermal energy to heat the sterilization chamber by radiation. a 9 cm fan would help circulate the hot air produced by the heating element, so the heat was more evenly distributed throughout the sterilizer chamber. the thermostat was used to control the heat. it was set on at 75°c and off at 75.5°c. a timer was used to adjust the duration of sterilization according to the treatment group. mask filtration efficiency tester design the mask filtration efficiency tester was designed as shown in figures 2a and 2b. this tool was tried to imitate the working principle of the standard tool for measuring mask filtration capability, tsi automated filter tester 8130a. this tool was made from polyvinyl chloride (pvc) tube with an inside diameter of about 6.35 cm (2.5 inches) and a length of about 65 cm. the aerosol produced by the nebulizer would be mixed with room air using a blower which would then be blown into the intake of the filtration tester tool. the nebulizer will automatically generate a variable-sized particle. these particles' sizes will be distinguished using particle counter and calculated in numbers. the vacuum fan would suck the air that had been mixed with the nacl aerosol. the mask would be placed in the middle of the tool to filter out aerosol particles that had been sucked. particle counters were placed in space before filtration occurred or in front of the mask (upstream) and space after the air was filtered or behind the mask (downstream). in addition, manometer sensors were also placed in the two chambers to measure the air pressure difference between chambers. 179 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license muh. aprizal azhar, et al. a prototype n95 sterilizer figure 2. the mask filtration efficiency tester (a) overall view, (b) the working principle of the mask filtration efficiency tester sterilization test the experiment was conducted in microbiology laboratory, hasanuddin university hospital, makassar, indonesia. the masks were artificially contaminated by the method used by ibáñez et al. 10 with modifications. airborne pathogens could not be used in this study due to limited laboratory biosafety availability. in this investigation, e. coli and s. aureus microorganisms were employed instead. the mask was cut into small pieces about 20 x 7.5 mm so that it would later fit into the microcentrifuge tube during the elution process. all mask samples were clamped using a wooden clamp, put in a clear plastic bag, and pre-sterilized at 90°c for 60 minutes to eliminate environmental contamination. the mask was removed from the plastic and contaminated with 100 µl of a 0.6-0.8 mfu (mcfarland unit) solution of s. aureus or e. coli. after that, the mask was put back into a plastic bag and the sterilization process was carried out using (1) uvc, (2) 75°c heat (temperature to inactivate s. aureus and e. coli),11,12 and (3) a combination of both in a duration of 1, 3, 5, 10, 30, 60, 90, and up to 120 minutes. for the control, we used an uncontaminated mask as a negative control and an unsterilized mask as a positive control. after the sterilization process was complete, the mask was drowned in 0.5 ml of saline solution in a microcentrifuge tube and vortexed to elute the bacteria contained in the mask. the saline solution was then dropped as much as 0.1 ml onto a nutrient agar medium and spread. the medium was incubated at 37°c for 24 hours. after 24 hours, bacterial growth would be observed. culture results showing more than 30 colonies were categorized as positive, while less than 30 were categorized as negative because they were too few to represent the sample. cultures that produced more than 300 bacterial colonies were considered too many to count (tmtc).13 180 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: 176–188 aerosol penetration and air resistant test in preparation, the stiff edges of the mask were cut to remove the rigid structure of the mask. this was intended to make it easier for the mask to be inserted into the filtration test device later. after that, the mask was tested for its filtration ability (f) by calculating the difference in the number of 0.3 µm sized particles between the upstream (us) and downstream (ds) spaces using the formula: 𝐹 = (𝑈𝑠 − 𝐷𝑠) 𝑈𝑠 𝑥100 air resistance was tested by calculating the difference in air pressure in the us and ds chambers. after obtaining the initial data, the mask was sterilized using uvc, 75°c heat, or a combination of both for 5, 10, 30, and 60 minutes, respectively. after the sterilization process, the mask was tested again for its sterilization ability and air resistance as in the previous method. the results were about 20 x 7.5 mm compared before and after the sterilization process. the air pressure difference data were entered into microsoft excel software and tested using a paired ttest to find their significance. this method was a modification of the method used by gobi et al.14 and vossen et al.15 to determine the mask's filtration efficiency and air permeability. briefly, the research flow is depicted in figure 3. figure 3. research flow results and discussion sterilization test most of the culture results made more than 300 colonies of bacteria and were considered too many to count (tmtc). more than 30 colonies were categorized as positive results, while less than 30 were categorized as negative because they were too few to represent the sample. some plates also showed the results of colonies stacking up on each other due to the uneven distribution of eluted solution during preparation. the results of mask culture after sterilization using the n95 prototype sterilizer are summarized in table i. using a portable sterilizer with the heat of 75°c gave negative culture results for both grampositive and gram-negative bacteria from 5 minutes to 60 minutes of sterilization duration, as shown in figure 4. 181 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license muh. aprizal azhar, et al. a prototype n95 sterilizer table 1. mask culture results after sterilization using the n95 prototype sterilizer sterilization method culture result e. coli (0.81 mfu) s. aureus (0.7 mfu) positive control positive positive negative control negative negative uvc 5 minutes positive positive uvc 10 minutes positive positive uvc 30 minutes positive positive uvc 60 minutes positive positive heat 75°c 5 minutes negative negative heat 75°c 10 minutes negative negative heat 75°c 30 minutes negative negative heat 75°c 60 minutes negative negative uvc + heat 75°c 5 minutes negative negative uvc + heat 75°c 10 minutes negative negative uvc + heat 75°c 30 minutes negative negative uvc + heat 75°c 60 minutes negative negative figure 4. mask culture results after sterilization using heat of 75°c we tried to reduce the duration of heat exposure to 1 and 3 minutes, respectively, to see the lower limit of this portable sterilizer's performance. at 1 minute, there was still colony growth, especially in e. coli culture, while in the 3 minutes group, the two groups of bacteria did not show any colony growth on the medium. in contrast, sterilization using uvc gave the opposite result. this portable sterilizer could not eradicate s. aureus and e. coli even with 60 minutes of sterilization. the documentation of the mask culture results after sterilization using the prototype n95 with the uvc method is shown in figure 5. 182 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: 176–188 figure 5. mask culture results after sterilization using uvc therefore, we added the duration of the mask's exposure to uvc rays to 90 to 120 minutes, but that still was not able to give negative culture from both bacteria, as shown in table 2. culture result documentation can be shown in supplementary figure 6. using a combination of heat at 75°c and uvc gave no different culture results than using heat alone. culture documentation is shown in figure 7. culture testing was done in a duplex to get more accurate results. there was no difference in culture results between the first and second experiments. table 2. mask culture results in shortened and extended sterilization durations 1only a colony was found sterilization method culture result e. coli (0.64 mfu) s. aureus (0.82 mfu) positive control positive positive negative control negative negative heat 75°c for 1 minute positive negative1 heat 75°c for 3 minutes negative negative uvc + heat 75°c 1 minute positive negative uvc + heat 75°c for 3 minutes negative negative uvc 90 minutes positive positive uvc 120 minutes positive positive 183 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license muh. aprizal azhar, et al. a prototype n95 sterilizer figure 6. mask culture results in shortened and extended sterilization durations figure 7. mask culture results after sterilization using both uvc and heat aerosol penetration and air resistance test the filtration ability of the n95 mask was maintained at >95% even though it had been through a sterilization process with 75°c heat, uvc, or a combination of both for up to 60 minutes figure 8. in terms of air resistance, there was also no significant difference (ρ=0.07–0.50) between new masks and masks that had been sterilized using a portable sterilizer as shown in supplementary figure 9. 184 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: 176–188 figure 8. comparison of n95 masks aerosol filtration efficiency before and after sterilization using the portable sterilizer figure 9. comparison of air resistance of n95 masks before and after sterilization using the portable sterilizer based on the sterilization ability test results, using a portable sterilizer with the heat method was more effective in eradicating s. aureus and e. coli. this method killed the bacteria on the mask pieces starting by heating for 3 minutes, while uvc still gave positive culture results even though the mask pieces had been exposed to uv light for 2 hours. this result shows that the use of heat in the portable sterilizer is more effective when compared to the use of uvc rays. the combination of heat and uvc also gave a 185 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license muh. aprizal azhar, et al. a prototype n95 sterilizer negative result on the culture results. thus there was no need to use both methods because it was just a waste of energy. one of the factors that increase heat sterilization capability is humidity. the use of moist heat is more effective than the use of dry heat.16 humidity in tropical countries like indonesia is relatively high. in this experiment, the humidity level in the room was around 55–60% rh. this high humidity environment increased the effectiveness of the heat sterilization capability of this portable sterilizer without the need for modification of the humidity level in the sterilization chamber. other studies have also shown that heat is more effective than treatment using uvc for the sterilization of n95 masks.17 bacteria's walls composed of protein have thermophobia characteristics. high temperatures will cause denaturation of these proteins and result in the death of these microorganisms.18 e. coli at 60°c will die within 2.9 minutes.19 meanwhile, s. aureus will die at a temperature of 60°c for about 4.8– 6.6 minutes.20 for comparison, heating containing sars cov-2 media at 65°c for 3 minutes is recommended to kill the covid19 virus.21 however, mycobacterium tuberculosis needs a higher temperature at 80°c to lose its viability.22 but, it should also be considered that too high temperatures can damage the structure of n95. the polypropylene layer on the n95 mask has a melting point of 130–171°c.23 if the temperature is too close, the structure will be damaged, impacting its filtration performance. heating at a temperature of 125°c can reduce the filtration ability of n95 up to 90%.7 we had tried to use an autoclave for the initial sterilization process to remove the environmental contamination of the mask sample before it was artificially contaminated. however, the mask sample showed a physical deformity like melting after being removed from the autoclave. although it has excellent germicide capabilities, using an autoclave was not recommended in sterilizing n95 masks.24 the filtration ability of n95 is obtained by utilizing a combination of polypropylene microfibre and electrostatic charges. the name n95 was given because this mask could filter at least 95% of solid and aerosol particles in laboratory trials. the letter n indicates that this mask cannot filter oil-based vapor.25 the n95 mask consists of several layers, one of which is a layer made of nonwoven polypropylene fiber with a diameter of 4.2 ± 3.9 µm, forming a layer with a thickness of 200-400 µm.26,27 the sterilization process must maintain the electrostatic charge of this membrane so that the mask filtration performance does not decrease. golovkine et al. showed a decrease of 3 log concentrations of sars cov-2 on the n95 surface after sterilization using uvc light at 1 mw/cm2 for 10 minutes.28 this result could be achieved because the uvc light source they used was very close and right on the mask's surface and back, leading to an adequate uvc exposure. in contrast to this portable sterilizer, the lack of effectiveness uvc is thought to be the result of the device configuration. the mask was placed in a hanging position on the side of the sterilization chamber, while the uv light source only came from above, resulting in uneven exposure to uvc rays and a blind spot in the sterilization process. in addition, this study did not measure the uvc radiation exposure dose, so that the uvc dose may be too low. however, exposure of the mask to uvc rays at an excessive dose can also reduce the filtering performance of the mask; thus, a precise dose is required.29 this sterilizer was designed because the heat source was located directly at the bottom of the sterilization chamber. if the mask was placed directly under the uvc source, in the bottom position, it was feared that the mask would melt because it was so close to the heating source. thus, the mask must be hung on the side. however, the advantages of this configuration make this tool capable of loading a total of four masks in one 186 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: 176–188 sterilization process, making it more efficient in operation. in terms of an aerosol penetration test, using a portable sterilizer did not reduce the mask's filtration performance below 95%, whether using uvc, heat, or a combination of both, even after being exposed for 1 hour. this study also showed no significant change in the air resistance of the n95 mask in all sterilization methods for 1 hour, so the user was still comfortable breathing when using a sterilized mask. another study also provided a similar result. xiang y et al. reported that exposure to dry heat to n95 at a temperature of 60°c and 70°c for 1 hour killed seven strains of bacteria and fungi, including e. coli and s. aureus, without reducing their filtration ability below 95%.30 even the use of heat up to 100°c for 5 minutes repeated 20 times did not affect the filtration ability of the n95 mask. 7 conclusions it can be concluded that this portable sterilizer was able to kill e. coli and s. aureus in the n95 mask using the 75 °c heat method for 3 minutes without negatively affecting the filtration performance and air resistance. although the effect was shown starting from 3 minutes, we recommend using this portable sterilizer with the heat method with a minimum duration of 5 minutes to compensate for the time that this tool takes to raise the temperature from room temperature (25 °c) to operational temperature (75°c). in addition, this mini sterilizer is only for emergencies, such as when there is a shortage of n95 masks. however, it is much safer to use a new mask than a mask that has undergone sterilization. the advantage of this tool is that it is smaller, compact, portable, and easy to use compared to the tools used in previous studies, which mainly used tools that were generally used on a commercial scale. the design itself still needs much improvement. form mask placement needs to redesign, so it is safe from heat sources. besides, the operating system needs to be changed from analog to digital so that the timer set becomes easier to set, and the exterior design must be updated to make it look contemporary. in addition, many more tests are needed regarding the effect of using a mini sterilizer on n95 masks, such as the impact of repeated use on masks, its effect on microscopic n95 fibers, the elasticity of mask strap rubber and fitment test, calculation of uvc doses, and the effect on various airborne pathogens such as m. tuberculosis and sars cov-2. acknowledgement the authors thank hasanuddin university hospital, which has provided laboratory facilities and the supply of bacteria strains. conflict of interest the authors declare that they have no conflict of interest. references 1. world health organization. the top 10 causes of death [internet]. 2020 [cited 2021 nov 29]. available from: https://www.who.int/newsroom/fact-sheets/detail/the-top-10 causes-ofdeath 2. dhand r, li j. coughs and sneezes: their role in transmission of respiratory viral infections, including sars-cov-2. am j respir crit care med. 2020;202(5):651–9. 3. coleman kk, tay djw, tan k sen, ong swx, koh mh, chin yq, et al. viral load of 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xiang y, song q, gu w. decontamination of surgical face masks and n95 respirators by dry heat pasteurization for one hour at 70°c. am j infect control. 2020;48(8):880–2. 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 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 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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 8:1-17 404 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, 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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. 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 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(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. �� vol. 2. no. 1 january–march 2011 r i s k f a c t o r o f b a c t e r e m i a i n c h i l d r e n w i t h pneumonia retno asih, zuhrotul aini, landia setiawati department of child health ,medical faculty airlangga university/dr. soetomo hospital surabaya abstract background: pneumonia is known as a frequent cause of morbidity and mortality among children in developing countries. in children,it caused predominantly by bacteria. bacteremia has been associated with severity and mortalitas of pneumonia. identify factors caused bacteremia important to prevent severity and mortalitas of pneumonia. objective: the objective of this study was to identify risk factors of bacteremia in children with pneumonia. methods: a retrospective study was conducted in children with pneumonia in dr. soetomo surabaya hospital from january 2007 to december 2008. blood cultures be performed on all of this patients. factors associated with bacteremia were identified following review of medical records include clinical features, laboratory , radiology and blood culture results. results: frequency of bacteremia was 8,2% (36 patients) of 438 children with pneumonia. interval from onset of symptoms to hospital admission more than 5 days (22.69 ci 95%), severe malnourished (or 9.05 ci 95%), anemia (or 2.44 ci 95%), leucocyt counts less than 5000/mm3 and more than 20.000/mm3 (or 2.41 ci 95%) and pao2 less than 80 mmhg (or 4.25 ci 95%) were at increased risk of bacteremia in children with pneumonia. conclusion: risk factors bacteremia in children with pneumonia included age under 1 year, symptoms more than 5 days, severe malnourished ,anemia, leucosyt counts less than 5000/mm3 and more than 20.000/mm3 and pao2 less than 80 mmhg. key words: risk factors, bacteremia, pneumonia introduction pneumonia is an acute inflammatory disease of the lung parenchyma involving the distal terminal bronchioles, respiratory bronchioles, alveoli ducts, alveoli sacs, and alveoli .. this disease is one of the most common infections in the pediatric age group. in developing countries, researchers estimate that more than 150 million new cases occur annually in children < 5 years. it is well known as a frequent cause of morbidity and a leading cause of mortality among children.1 one in five of childhood deaths in developing countries have been ascribed to acute respiratory tract infections (ari) and 90% of these deaths are due to pneumonia.2 a variety of microorganisms can cause pneumonia in children-bacteria, viruses or fungi. pneumonia in developing countries is caused predominantly by bacteria.1 the frequency of bacteremia in pneumonia patients varies from 4% to 18%.3 bacteremic pneumonia are potentially life-threating in children.4 predictor of bacteremia in pneumonia included recent antibiotic treatment, comorbidity disease,increase respiratory rate, temperature, pulse and laboratory abnormalities. 3 the objective of this study was to identify risk factors of bacteremia in children with pneumonia. a retrospective study was conducted in children with pneumonia in dr. soetomo surabaya hospital from january 2007 to december 2008. blood cultures be performed on all of this patients. factors associated with bacteremia were identified following review of medical records include clinical features, laboratory , radiology and blood culture results. identify factors caused bacteremia important to prevent severity and mortalitas of pneumonia subjects and methods a retrospective study was conducted in children age 1 months until 5 years with diagnosis community acquired ��asih, et al.,: risk factor of bacteremia in children pneumonia in dr. soetomo surabaya hospital from january 2007 to december 2008. this formed part of larger study investigating epidemiology study streptococcus pneumoniae at surabaya. the diagnosis of pneumonia in this study was made based on clinical symtomps presenting of lower respiratory tract infection. sex, age, clinical characteristics, laboratory and radiology findings were collected by medical records review. patients were defined as bacteremic if a blood culture drawn of presentation to the hospital before antibiotic treatment grew an organism, and not defined as a contaminant. from the time of admission, we noted duration of symptoms, clinical manifestation, nutritional state, laboratory and radiological findings. we classified temperature into 2 categories, less than 36,5o c, or more than 38,5o c and between 36.5o c and 38,5o c. nutritional state were classified into 3 categories, well nourished if ideal body weight more than 90%, moderate malnutrition if ideal body weight between 70%–90% and severe malnutrition if ideal body weight less than 70%. we collected laboratory finding includes hemoglobin level, leucocyte level, c reactive protein and blood gas analyse. anemia if hemoglobin level less than 10 gr/dl. leucocyt counts classified into less than 5000/cmm, between 5000/cmm and 20.000/cmm and more than 20.000/cmm.. x2 tests, fisher’s exact test and odds ratio and relative risks with 95% confidence intervals were used to determine whether an association was significant (p < 0.05). results we identified 440 patients with pneumonia who met the inclusion criteria for the study. blood culture was performed on 438 patients, bacteremia was detected in 36 patients (8.2%) and 73 patients (16.7%) had a contaminated bacteria (table 1) of the total cases, ratio male and women was 1.6:1. the mean age was 12.2 months with the majority at the age of 1–12 months as many as 293 (66.9%) children. the majority interval from initial symptoms until it is brought to the hospital was less than 3 days (51.6%) children. most nutritional status is well nourhised as much as 281 (64.2%) children. anemia was found in 251 (57.3%) children, levels of leukocytes less than 5000/mm3 or more than 20.000/mm3 many as 116 (26.5%) children, positive crp was found in 329 (75.1%) of children and acidosis obtained in 75 (17.1%) children. preview photos thoracic infiltrates was found in 354 (80.8%) children. there were no significant difference of based line characteristic children with bacteremia positive and bacteremia negative include sex ang age. (table 2 ) table 3 shows risk factors several variables on the occurrence of bacteremia in patients with pneumonia of children. from the table, the risk factors of pneumonia were the interval from onset of symptoms to hospital admission more than 5 days, severely malnutrition, anemia , leukocyte less than 5000/mm3 or more of 20.000/mm3 and po2 less than 80%. discussion the aetiology of pneumonia in developed countries is predominantly viral, associated with a low case fatality rate, whereas in developing countries bacteraemia is common and associated with a high case fatality rate. poor sanitation, table 1. characteristic of children with pneumonia at dr. soetomo hospital, january 2007–december 2008. age (months) mean ± sd (range) 1–12 month, n (%) 13–36 month > 36 month 12.2 ± 11.2 293 (66.9) 126 (28.8) 19 (4.3) sex ratio (male: female) 1.6: 1 interval from onset of symptoms to hospital admission , n (%) < 3 days ≥ 3–< 5 ≥ 5 226 (51.6) 199 (45.4) 13(3.0) nutritional state, n (%) well nourished moderate malnourished severely malnourished 281(64.2) 150 (34.2) 7 (1.6) temperature, n(%) < 36.5 c or > 38.5 c 36.5 c – 38.5 c 124 (28.3) 314 (71.7) blood culture, n (%) sterile bacteremia contaminant chest x-ray, n(%) normal lobar consolidation patchy infiltrate pleural effusion 329 (75.1) 36 (8.2) 73 (16.7) 60 (13.7) 21 (4.8) 354 (80.8) 3 (0.7) table 2. based line characteristic of children with pneumonia at dr. soetomo hospital january 2007–december 2008 variable bacteremia n (%) no bacteremia n (%) p value sex male female 25 ( 5.7) 11 (2.5) 248 (56.6) 154 (35.2) 0.169 age group (months) 1–12 month 13–36 month > 36 month 27 (6.2) 9 (2.1) 2 (0.5) 266 (60.7) 117 (26.7) 17 (38.8) 0.307 0.431 0.548 �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 34-37 overcrowding, inadequate nutrition, insufficient vaccination coverage, low levels of education, and accumulation of other diseases have been suggested as reasons for the differences in aetiology and mortality. 5 to reduce mortality from pneumonia in developing countries the problem has to be addressed from a number of aspects, social and environmental as well as medical.5 in this comparative study of bacteremic and non bacteremic patients,we found that bacteremia was detected in 8.2% children. the previous study reported frequency of bacteremia in patients pneumonia varies from as low as 4% to as high as 14 to 18%. 3. we found that bacteremic and non-bacteremic pneumonia patients not differed in baseline characteristics. the study by spooner et al from papua new guinea(png) identified firstborn children and female children to have increased bacteremia and mortality risk in children with pneumonia.5 meanwhile jover et al, in research on adult patients with pneumonia, bacteremia get no difference between men and women.6 we have identified six independent factors of bacteremia in children with pneumonia.. five of these significantly associated with bacteremia. include interval from onset of symptoms to hospital admission has significantly associated with bacteremia, the interval from onset of symptoms to hospital admission more than 5 days, severely malnutrition, anemia, leukocyte less than 5000/mm3 or more of 20.000/ mm3 and po2 less than 80%. spooner et al. identified that poor feeding, cyanosis, bronchial breathing, and a temperature > 38° c were all associated with bacteraemia.5 table 3. risk factors bacteremia of children with pneumonia at dr. soetomo hospital january 2007 – december 2008 variable bacteremia positive n (%) bcteremia negative n (%) or (95% ci) p value interval from onset of symptoms to hospital admission , n (%) < 3 days > 3–< 5 ≥ 5 36 (8.2) 31 (7.1) 8 (1.8) 199 (43.4) 168 (38.3) 5 ( 1.1) 1.35( 0.69-2.67) 0.97(0.48-1.97) 22.69(6.96-73.9) 0.386 0.933 0.000* nutritional state, n (%) well nourished moderate malnourished severely malnourished 26 (5.9) 8 (1.8) 3 (0.7) 255 (58.2) 142 (32.4) 4 (0.9) 1.52(0.71-3.23) 0.52(0.23-1.18) 9.05(1.94-42.13) 0.279 0.113 0.001* temperature < 36.5 c or > 38.5 c 8 (1.8) 116 (26.5) 0.70(0.31-1.59) 0.397 laboratory findings hb < 10 g/dl leucocyte < 5,000/cmm or > 20,000/cmm crp positive blood gas analysis ph < 7.35 po2 < 80 mmhg pco2 > 50 mmhg spo2 < 95% 21(4.8) 16(3.7) 25(5.7) 6(1.4) 3(0.7) 5(1.1) 7(1.6) 230(52.5) 100(22.8) 304(69.4) 69(15.8) 100(22.8) 27(6.2) 89(20.3) 2.44(1.04-5.71) 2.41(1.21-4.84) 0.67(0.26-1.72) 0.85(0.29-2.44) 4.25(1.18-15.4) 0.48(0.15-1.60) 1.19(0.27-5.31) 0.034* 0.011* 0.404 0.766 0.018* 0.236 0.858 chest x-ray lobar consolidation 7(1.6) 14(3.2) 1.19(0.27-5.310 0.833 interval from onset of symptoms to hospital admission more than 5 days has significantly associated with bacteremia. previous study reported that history of fever for more than 7 days significantly increased the chance of dying.5 the onset to hospital admission associated with prior use of antibiotic. metersky et al,reported that the risk of bacteremia could be predicted by assessing the prior use of antibiotics.3 malnourished children are particularly at risk as demonstrated in this study. a study in png reported that malnourished patients had a significantly higher risk of dying.5 in our study, bacteremic patients were more likely to be anemic than non-bacteremic patients and to have abnormal leucocyt counts and po2 less than 80%. brandeburg found that bacteremic patients were more likely to have anemia, lower albumin, and elevated blood urea and serum creatinine levels.7 while metersky reported that lood urea nitrogen more than 30 mg/dl (11 mmol/l) , sodium less than 130 mmol/l and wbc less than 5,000/mm3 or more than 20,000/mm3 were independent predictors of bacteremia in community-acquired patients with pneumonia.3 results of radiological findings in association with bacteraemia have been analysed. they showed that a peripheral homogeneous opacity was the best predictor of bacteraemia.5 jover reported that although not statistically significant, pleural effusion was more frequent in bacteremic patients.6 in this study,the most radiological finding was infiltrate. ��asih, et al.,: risk factor of bacteremia in children there are some limitations in this study. the main weakness of our study is its retrospective design. collection of some data was therefore incomplete. another limitation could be the lower number of non-bacteremic cases compared to bacteremic cases. conclusion risk factors bacteremia in children with pneumonia included age under 1 year,symptoms more than 5 days, severe malnourished ,anemia, leucosyt counts less than 5000/mm3 and more than 20.000/mm3 and po2 less than 80 mmhg. bacteremia has been associated with severity and mortalitas of pneumonia. identify factors caused bacteremia important to prevent severity and mortalitas of pneumonia. refferences 1. masria s. pattern of �acteria causing pneumonia inchildren and its sensitivity to some antibiotics. proc asea� congr trop med parasitol 2008; 3: 121–4. 2. �arayanan m, falade a�. clinical risk factors for death in children with pneumonia. international child health review collaboration. 13th october 200�. 3. metersky ml, ma a, �rratzler dw, houck pm. predicting �acteremia in patients with community-acquired pneumonia. am j respir crit care med 2004; 1�9: 342–7. 4. toikka p, virkki r, mertsola j, ashorn p, eskola j,ruuskanen o. �acteremic pneumococcal pneumonia in children. clin infect dis 1999; 29: 5�8–72. 5. spooner v, �arker j, tulloch s, lehmann d, marshall tf, kajoi m, alpers mp. clinical signs and risk factors associated with pneumonia in children admitted to �oroka hospital, papua �ew �uinea. j trop pediatr. 1989; 35: 295–300. �. jover f, cuadrado jm, andreu l, martinez s, canizares r, tabla vo et al. a comparative study of bacteremic and non-bacteremica comparative study of bacteremic and non-bacteremic pneumococcal pneumonia. eur j of intern med 2008; 19: 15–21. 7. �randenburg ja, marrie tj, coley cm, singer de, obrosky s, kapoor w�, et al. clinical presentation, processes and outcomes of care for patients with pneumococcal pneumonia. j �en intern med 2000;15: �38–47. ijtid vol 3 no 1 jan-maret 2012.indd 1 vol. 3. no. 1 january–march 2012 comparative study on the intensity of mycobacterium leprae exposure between household and nonhousehold contact of leprosy yuniarti arsyad1, friska jifanti1, muh. dali amiruddin1, anis irawan anwar1, dinar adriaty2, ratna wahyuni2, iswahyudi2, indropo agusni2, shinzo izumi2 1 department of dermatology, hasanudin medical faculty, makassar 2 institute of tropical disease, airlangga university, surabaya abstract leprosy stills a public health problem in west sulawesi which has a case detection rate (cdr) around 43.69/100.000 population. household contacts of leprosy are a high risk group to be infected, due to droplet infection mode of transmission of the disease. a nose swab examination and serological study was conducted to detect exposure of m. leprae of people who live in leprosy endemic area. detection of m. leprae in the nasal cavity will represent the exposure rate from outside and the measurement of specific antibody is represented the result of exposure to the immune system. two group of inhabitants (30 household contacts of leprosy and 30 nonhousehold contacts) were involved in the study. they live in banggae district, a leprosy endemic area of majene regency, west sulawesi. sixty nose swab samples and sixty capillary blood samples from the same invidividuals of the two groups were collected and sent to leprosy laboratory of the institute of tropical disease, airlangga university surabaya. a polymerase chain reaction (pcr) was performed to the nose swab samples for detection of m. leprae. the blood samples were examined serologically to measure the level of anti pgl-1 antibody. pcr examination of nose swab samples showed 1/30 positive result in the household contact group and also 1/30 positive result in non-household contact of leprosy (statistically no significant difference, p > 0.05). serological study showed higher sero-positive result in the household contact group (15/30 or 50%) compared to non-household contact (11/30 or 36%), but statistical calculation revealed no significant difference between the two groups (p > 0.05) on sero-positive results of leprosy. it is concluded that household and non-household contact in leprosy have the same risk to be affected by the disease. the term of household and non-household contact need to be redefined. the possible role of exposure from the environment was also discussed, especially from non-human resource of m. leprae. key words: leprosy – sero epidemiology – pgl-1 antibody introduction leprosy is a chronic infectious disease caused by m. leprae and primarily affect the peripheral nerves, secondary to skin and other organs. the complication of the disease can cause some disabilities and social problem in the community. close contact is one condition that increased the risk of transmission. from several contact surveys, it is reported that more leprosy patients found and live in the same house, indicates that household member of leprosy patient is a high risk group for affected the disease.1 droplet infection mode of transmission seems the main route of transmission.2 after the lepra bacilli enter the body, the immune response will be induced to eradicate the microorganism. specific antibody to m. leprae, the anti phenolic glycolipid-1 (pgl-1) antibody is also developed. the level of antibody is correlated with the antigen load of the bacilli, which means that level of antibody is represented the amount of m. leprae in the body.3 from this point of view, the intensity of m. leprae exposures to individual could be measured by examining the presence of m. leprae in the nasal cavity and study the level of specific antibody on the bacilli. research report 2 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 1−4 aim of study the aim of this study is to compare the intensity of m. leprae exposure between the healthy household contacts group and the non-household contacts of leprosy, by detection of m. leprae in the nasal cavity and measurement the specific antibody to m. leprae of the same individuals as an immunologic response to the infection. site of study figure 1. geographic area of the study material and method sixty adult healthy individuals from banggae subdistrict, majene, west sulawesi, (figure 1) consisted of 30 household contacts of leprosy patients (live in the same house with the leprosy patients) and 30 non-household contacts were involved in the study. nose swab specimen (for pcr study) capillary blood, dried in filter paper (for serological study) figure 2. collection of specimens 3akbar, dachlan: comparative study on the intensity of mycobacterium leprae from each patient a nose swab specimen was collected and 100 ul capillary blood was collected by finger tip punctured and dried in the filter paper (figure 2). these 60 pairs of specimens were brought to leprosy lab in the institute of tropical disease, airlangga university, surabaya. a polymerase chain reaction (pcr) test were performed to detect m. leprae in the nose swab specimens, while the dried capillary blood was examined serologically to measure the level of anti pgl-1 antibody using the elisa technique.4 the results will be analyzed to compare the positive pcr results of the nose swab specimens and also to compare the immunologic response to m. leprae between the two groups. the dried blood in filter paper are diluted in distilled water for two hours and shaked. this diluted blood was used as a specimens for elisa study to measure the level of igm anti pgl-1 antibody and using the conversion value, the results were converted to serum equivalence value.5 by biolise program in computer, the optical density (od) value was converted to unit.ml. using cut off value 605 u/ml, sero-positive result were established.6 results using the lp1-lp4 nested primer that amplify the rlep region of m. leprae dna (99 bp), the household contact group showed 1/30 positive pcr result, compared to 1/30 positive result in the non-household contact group. statistically there is no significant difference between the two goups in the positive pcr results (p > 0.05). in serological examination, after a conversion to achieve the serum equivalency and using the cut off 605 u/ml for igm anti pgl-1 (elisa), 15/30 samples from the household contact group showed sero-positive results, compared to 11/30 sero-positive in the non-household contact group. although the number of sero-positive is higher in household contact group, statistically no significant difference between the two groups in the sero-positive results. also when the two datas (pcr and serology of leprosy) are combined, still no significant difference between the two groups (p > 0.05). discussion the route of transmission in leprosy mainly by droplet infection, since multibacillary leprosy case will harbour many lepra bacilli in his nasal cavity.7 prolonged contact, intimate and continuously with leprosy patients are the condition for affected the disease.8 the existence of m. leprae in the nasal cavity could be either from outside, aspirated during respiration, or secretion from the nasal mucous as a secretion from leprosy lesion in the nasal cavity.9,10 household contacts of leprosy fulfill these criteria and become the high risk group. when the bacilli enter the body, the immune response will develop. although the anti pgl-1 antibody is not effective to eradicate the m. leprae infection, it is a useful parameter for monitoring the infection.11 the level of this specific antibody is correlated with the amount of m. leprae in the body.12 based on previous serological surveys in endemic and non-endemic areas, the cut off igm and igg anti pgl1 (elisa) can be calculated. the level 605 u/ml for igm anti pgl-1 and 615 for igg anti pgl-1 was used as the cut value. those who have the igm anti pgl-1 level >605 are considered as a sero-positive case. most of serological studies use serum samples, which originally from venous blood samples. the use of capillary blood which is dried on the filter paper has been introduced since 2007 and very useful for collecting blood samples from field that located long distance from the laboratory.13,14 using a conversion coefficient, the equivalence value of anti pgl-1 antibody in serum can be obtained.15 sub-clinical leprosy is a term for healthy individual who live in leprosy endemic area, with high level of igm anti pgl-1 in serological examination. these sub-clinical leprosy cases still show no sign of clinical leprosy, but they are potential to progress toward manifest leprosy.16 in this study the serological examination result showed around 43% of the inhabitants showed sero-positive, which means that they are already exposed to m. leprae and induced the humoral response. since the level of antibody correlates with the antigen load, once can assume the load of bacilli in the body is also more than normal people in other areas. although it is hypothesized that household contacts will get more m. leprae exposure than those non-household contact of leprosy, this study showed that by cross sectional study both groups of study only showed 1/30 pcr (+) for m. leprae in the nose swab cavity. this means that airborne infection of m. leprae in the household and non-household contacts is similar, or in other word the m. leprae infection source not only from leprosy patient in the house, but maybe from other patients or environment. from the serological study, the results showed the same phenomena, but the level of antibody in sero-positive cases showed a different pattern. household contacts with sero-positive anti pgl1 antibody showed a higher incidence and higher (figure 1). anti pgl-1 antibody level (u/ml) 0 1000 2000 3000 4000 5000 6000 0 5 10 15 house hold nonhouse hold xy (scatte r) 3 number of sero-positive cases figure 3. distribution of serological level of sero-positive cases among household and non-household contacts of leprosy 4 indonesian journal of tropical and infectious disease, vol. 3. no. 1 january–march 2012: 1−4 since the immune response need a certain duration before it is developed, once can assume that household contacts have more antigen load (m. leprae) in their body. prolonged contact with leprosy patient in the same house might cause the accumulation of antigen and induce high level of specific antibody production. those sero-positive contacts with high level of antibody (sub-clinical leprosy) need special attention to avoid progression towards manifest leprosy in the future.17 references 1. castro n, alzate j, hernandez r (2008). survey to identify m. leprae infected household contacts of patients from prevalent regions of leprosy in colombia. mem inst oswaldo cruz 103(4): 332–6. 2. bedi bms, narayanan e, sreevatsa et al. (1976). dispersal of m. leprae by leprosy patients while breathing. ann indian acad med sci 12: jan-march. 3. harboe m (1994). overview of host parasite relation.in hasting r (ed). leprosy. churchil livingstone. 4. gillis tp, and dl williams. 1991. polymerase chain reaction and leprosy. int. j. lepr. other mycobact. dis. 59: 311–316. 5. prakoeswa crs, adriaty d, wahyuni r et al. (2009). 6. frota c, freitas m, foss n (2010). seropositivity to anti pgl-1 in leprosy cases, contacts and no known contacts of leprosy inendemic and a non endemic area in northeast brazil. trans r soc trop med hyg 104: 490–95. 7. 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. 8. noorden sk (1994). the epidemiology of leprosy. (section 2) in hasting r and opromolla dva (eds) leprosy. edinburg, churchil livingstone. 9. castro n, alzate j, hernandez r (2008). survey to identify m. leprae infected household contacts of patients from prevalent regions of leprosy in colombia. mem inst oswaldo cruz 103(4): 332–6. 10. klatser 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. 11. beers s, izumi s,madjid b (1994).an epidemiological study of leprosy by serology and polymerase chain reaction. int j lepr 62(1): p 1–9. 12. kai m. (2011). serology of leprosy. (chapter 9) in in makino m, matsuoka m, goto m (eds) leprosy. science working towards dignity. tokai university press. 2011 13. prakoeswa crs, listiawan my, adriaty d. et al (2007). the use of finger tip capillary blood on filter paper to detect the level of igm and igg anti pgl-1 in leprosy patients. xi perdoski annual meeting, surabaya (abstract book). 14. agusni i, prakoeswa crs, listiawan my, et al. (2008). serological examination for leprosy from a drop of blood on the filter paper. 15. prakoeswa crs, listiawan my, adriaty d. et al (2007). correlation of igm anti pgl-1 antibody level between cubital vein blood and capillary blood vessel dried on filter paper. berkala ipkk 2007; 19(2): 115–119. 16. douglas j, cellona r, fajardo r et al.(2004). prospective study of serology conversion as a risk factor gor development of leprosy among household contacts. clin diag lab immunol 11: 897–900. 17. agusni, i (2011). clinical manifestation of leprosy. (chapter 11) in makino m, matsuoka m, goto m (eds) leprosy. science working towards dignity. tokai university press. 2011. 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 �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. 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 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 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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 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. 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 case report eff ect of zinc(ii)-2,4,5-triphenyl-1h-imidazole complex against replication denv-2 in vero cell aswandi wibrianto1, fatimah martak2, teguh hari sucipto3a, siti churrotin3, ilham harlan amarullah3, harsasi setyawati4, puspa wardhani3, aryati3, soegeng soegijanto3 1 undergraduate student, department of chemistry, faculty of science and technology, universitas airlangga, indonesia 2 department of chemistry, faculty of natural science, institut teknologi sepuluh nopember, indonesia 3 dengue study group, institute of tropical disease, universitas airlangga, indonesia 4 department of chemistry, faculty of science and technology, universitas airlangga, indonesia received: 8th april 2019; revised: 29th january 2020; accepted: 23rd april 2020 abstract dengue virus (denv) is a signifi cant pathogen emerging worldwide as a cause of infectious disease. denvs are transmitted to humans through female mosquitoes from aedes aegypti and aedes albopictus species. indonesia is one of the largest countries in the world in dengue endemic regions worldwide. dengue fever was occurred for the fi rst time as an outbreak in surabaya and jakarta in 1968. many eff orts have been made to prevent and treat denv infections, and clinical trials of a number of vaccines are currently underway. antiviral testing of denv is an important alternative for drug characterization and development. complex compounds are formed as a result of metal and organic complex reactions. complex compounds can be used as an anti-infl ammatory, antimicrobial antifungal, antibacterial, antivirus. the zn2+ ion can be used as an antiviral candidate. the purpose of this project was investigated zinc(ii)-2,4,5-triphenyl-1h-imidazole antiviral compound to be further tested for inhibitory eff ect on the replication of denv-2 in cell culture. denv replication was measured by antiviral activity assay and cytotoxicity assay. the inhibitory activity of zinc(ii)-2,4,5-triphenyl-1h-imidazole complex compound was determined by viral toxglotm assay. the cytotoxicity of zinc(ii)-2,4,5-triphenyl-1h-imidazole complex compound was determined by celltiter96® aquoeus assay. the inhibitory concentration (ic50) of zinc(ii)-2,4,5-triphenyl1h-imidazole against dengue virus type-2 was 34.42 μg/ml. the cytotoxic concentration (cc50) of compound against vero cell was <100 μg/ml. the results of this study demonstrate the antidengue serotype 2 inhibitory activity of investigated zinc(ii)-2,4,5-triphenyl-1h-imidazole complex and its high toxicity in vero cells. further studies are not required before investigated zinc(ii)-2,4,5-triphenylimidazole can be applied in the treatment of denv-2 infections. keywords: zinc (ii), complex compound, cytotoxicity, inhibitory activity, denv-2 abstrak virus dengue (denv) adalah patogen signifi kan yang muncul di seluruh dunia sebagai penyebab penyakit menular. denv ditransmisikan ke manusia melalui nyamuk betina dari spesies aedes aegypti dan aedes albopictus. indonesia adalah salah satu negara terbesar di dunia di daerah endemik demam berdarah di seluruh dunia. demam berdarah terjadi untuk pertama kalinya sebagai wabah di surabaya dan jakarta pada tahun 1968. banyak upaya telah dilakukan untuk mencegah dan mengobati infeksi denv, dan uji klinis sejumlah vaksin saat ini sedang berlangsung. pengujian antivirus denv adalah alternatif penting untuk karakterisasi dan pengembangan obat. senyawa kompleks terbentuk sebagai hasil dari reaksi kompleks logam dan organik. senyawa kompleks dapat digunakan sebagai anti-infl amasi, antimikroba antijamur, antibakteri, antivirus. ion zn2+ dapat digunakan sebagai kandidat antivirus. tujuan dalam proyek ini adalah menyelidiki senyawa antivirus zink(ii)-2,4,5-trifenil-1h-imidazol yang diuji lebih lanjut untuk efek penghambatan pada replikasi denv-2 dalam kultur sel. replikasi denv diukur dengan uji aktivitas antivirus dan uji sitotoksisitas. aktivitas penghambatan senyawa kompleks zinc(ii)-2,4,5-triphenyl1h-imidazol ditentukan dengan viral toxglotm assay. sitotoksisitas senyawa kompleks zinc(ii)-2,4,5-triphenyl* corresponding author: teguhharisucipto@staf.unair.ac.id 184 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: 183–188 1h-imidazol ditentukan dengan uji celltiter96® aquoeus. konsentrasi penghambatan (ic50) zinc(ii)-2,4,5-trifenil1h-imidazol terhadap virus dengue tipe-2 adalah 34,42 μg/ml. konsentrasi sitotoksik (cc50) senyawa terhadap sel vero adalah <100 μg/ml. hasil penelitian ini menunjukkan aktivitas penghambatan serotipe 2 antidengue dari zinc(ii)-2,4,5trifenil-1h-imidazol yang diteliti dan toksisitasnya yang tinggi dalam sel vero. studi lebih lanjut tidak diperlukan sebelum investigasi zinc(ii)-2,4,5-trifenil-1h-imidazol dapat diterapkan dalam pengobatan infeksi denv-2. kata kunci: seng (ii), senyawa kompleks, sitotoksisitas, aktivitas penghambatan, denv-2 how to cite: effect of zinc(ii)-2,4,5-triphenyl-1h-imidazole complex against replication denv-2 in vero cell. wibrianto, a. martak, f. setyawati, h. sucipto, th. churrotin, s. amarullah, ih. wardhani, p. aryati, a. soegijanto, s. indonesian journal of tropical and infectious disease, 8(3), 183–188. imidazole-1-β-ᴅ-ribofuranoside is examined for four diff erent types of viruses from the fl aviridae family in vitro, including hepatitis c virus (hcv), japanese viral encephalitis (jev), west nile virus (wnv), and dengue virus (denv) in vitro against ntpases/helicases. the compound showed activity highly active against wnv with ic50 was 23 μm.7 complex compounds are formed as a result of metal and organic compound reactions. complex compounds can be used as an anti-infl ammatory8, antimicrobial9, antifungal, antibacterial10, and antivirus11. based on previous research, copper(ii)-imidazole derivatives can be used as antidenv-2, can be used as low toxicity and potential as drug candidates. the compound exhibited adsorption inhibitory activity against denv-2 at ic50 = 2.3 μg/ml.12 the zn2+ ion can be used as an antiviral candidate.13 zn2+ ions can change the activity of various transcription factors and thus, patterns of cellular and viral gene expression.14 thus, the antiviral test of the compound zinc(ii)-2,4,5triphenyl-1h-imidazole was investigated. materials and methods chemicals and media chemical reagents used in this research were zinc(ii)-2,4,5-triphenyl-1h-imidazole complex compound, minimum essential eagle medium (sigma-aldrich, germany), dengue virus serotype 2 surabaya isolate (kt012509), vero cells (african green monkey kidney), viral toxglotm assay (promega, usa), celltiter96® introduction dengue virus (denv) is a virus carried by the fl avivirus genus of the family flaviviridae. dengue virus (denv) consists of four serotypes which is dengue virus type 1, dengue virus type 2, dengue virus type 3, and dengue virus type 4. dengue virus is transmitted to humans through female mosquitoes from aedes aegypti and aedes albopictus species. world health organization (who) reported 390 million dengue infections per year.1 indonesia is one of the largest countries in the world with dengue endemic areas. surabaya and jakarta were the cities where dengue disease was fi rst reported in indonesia in 1968.2 many studies have been conducted to overcome the threat of dengue virus infections, and clinical trials of a number of vaccines are currently on the way.3 antiviral testing on denv is a very important method in the development and characterization of drugs. supplementary to vaccines, inhibitors in each natural cycle of viral replication have the potential to cure dengue virus infection and indeed compounds such as rna replication inhibitors have been tested as such.4 however, there is no commercially available drug with antiviral activity for denv.5 l i g a n d 2 , 3 , 5 t r i p h e n y l 1 h i m i d a z o l e compound is a derivate of imidazole. imidazolecontaining drugs that have strong therapeutic properties have encouraged scientists to synthesize many novel chemotherapeutic agents consisting of these entities. n5-(4-fluorophenyl)-n4(2-(pyridin-4-yl)benzyl)-1h-imidazole-4,5dicarboxami-de, a derivate of imidazole, was reported anti-denv activity.6 4-carbamoyl-5(4,6-diamino-2,5-dihydro-1,3,5-triazin-2-yl) 185aswandi wibrianto, et al.: effect of zinc(ii)-2,4,5-triphenyl-1h-imidazole complex against replication copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 aquoeus one solution cell proliferation assay (promega, usa). 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 concentrations of zinc(ii)2,4,5-triphenyl-1h-imidazole were 50 μg/ml; 25 μg/ml; 12.5 μg/ml; 6.25 μg/ml; 3.13 μg/ ml; 1.57 μg/ml; 0.78 μg/ml; and 0.39 μg/ml with addition 100 μl viral toxglotm assay per well. the 50% inhibitory concentration (ic50) of denv-2 replication by each compound was further investigated by using glomax® discover system. cytotoxicity assay a cytotoxicity assay was performed using celltiter96® aq uoeus one solution cell proliferation reagent. the celltiter96® assay is a modifi cation of the mtt assay method portrayed by akter.15 the concentrations of zinc(ii)-2,4,5triphenyl-1h-imidazole were 100 μg/ml; 200 μg/ml; 400 μg/ml; 600 μg/ml; 800 μg/ml; and 1000 μg/ml. the medium was allowed to equilibrate for 1 hour; then 20μl/well of celltiter 96® aqueous one solution reagent was added. after 1 hour at 37°c in a humidifi ed, 5% co2 atmosphere, the absorbance at 490nm was recorded using glomax® discover system. viral detection by reverse transcriptasepolymerase chain reaction rna replication was estimated using the reverse transcriptase-polymerase chain reaction (rt-pcr). the purpose of this assay was to known rna replication after treatment. briefl y, denv-2 rna was extracted from the denv-2 infected cells and cell culture supernatant using rna extraction kit by qiagen, germany. the two-step kit (toyobo, japan) was used for cdna synthesis and polymerase chain reaction (pcr) following the manufacturer’s instructions. primer oligonucleotide sequences were as follows by bhatnagar et. al. 2012.16 amplifi cation condition was 54 °c for one minute (annealing temperature) and the amplifi ed product was the analyzed on 1.5% agarose gel. results and discussion the cytotoxicity of zinc(ii)-2,4,5-triphenyl1h-imidazole complex compound was determined by celltiter96® aquoeus assay and the recorded cc50 value is <100 μg/ml to vero cells. when compared with a previous study, copper(ii) was found to be nontoxic to human erythrocyte cells to concentrations of 500 μg/ml.17 cc50 is the cytotoxicity level of [cu(2,4,5-triphenyl-1himidazole)2]n (compound) to cause death to 50% of vero cells.12 the toxicity value of cobalt(ii) complex with 2,4,5-triphenyl-1h-imidazole ligand was 362.24 mg/l, which was not toxic.18 the toxicity value of 2-methyl-4,5-diphenyl1h-immidazole ligand compound was 192,3 μg/ ml.19 the toxicity of [mn(2-(4-chlorophenyl)4,5-diphenyl-1h-imidazole)2(h2o)2]·2h2o was >200 μg/ml which had less toxicity.20 zinc(ii)–2(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen4-one complex compound defi ned cytotoxicity with cc50 at 3.59 μg/ml.21 but, the metal-free imidazole more toxic for vero cells (cc50 = 5.03 μg/ml).22 activity against hiv-1 strain iiib and hiv-2 strain rod in mt-4 cells (cc50) by zinc(ii) complexes with hexyl-me2-cyclam (hmc; 3,14dimethyl-2,6,13,17-tetraazatricyclo(16.4.0.07,12)docosane) were >372 μm and >372 μm with selectivity index >35 and >3. activity against hiv-1 strain iiib and hiv-2 strain rod in mt-4 cells (cc50) by zn(ii)–hmc diacetate were 110.67 ± 12.67 μm and 110.67 ± 12.67 μm with selectivity index 32 and <1.23 the complex stability is highly dependent on both the metallic ion and the ligands. as for the central ion (m2+), zn(ii) more unstable than cu(ii), mn(ii), and co(ii). the zn(ii) complex has grater polarizability that that cu(ii), mn(ii), and co(ii) because it contains more d-electrons, and the zn(ii) complex produced more product ions soluble in water.24 this eff ect causes zn(ii) to be more toxic, because zn2+ in the medium are 186 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: 183–188 more numerous, so it damages the cell wall faster than complex compound that have high stability such as cu(ii), mn(ii), and co(ii). the percentage inhibition of the development of dengue virus type-2 by the test sample of zinc(ii)-2,4,5-triphenyl-1h-imidazole complex compound was shown on fi gure 1. the ic50 value was determined from the concentration–response curve (figure 1); the ic50 value was 34.42 μg/ml, r2 was 0.9196. based on the value of the ic50 zinc(ii)-2,4,5-triphenyl-1h-imidazole complex compound was a medium toxic compound. antiviral activity was also shown in figure 2, these fi ndings were corroborated by results obtained from rt-pcr which indicated signifi cant reduction in the amount of denv-2 genomic rna levels. the highest percentage of viral inhibition was observed after treating the infected cells with 50 μg/ml. based on the previous study, [cu(2,4,5triphenyl-1h-imidazole)2]n complex compound exhibited adsorption inhibitory activity against denv-2 at ic50 = 2.3 μg/ml. the inhibition at ic50 was not signifi cantly high (p<0.005) compared to that of the metal-free imidazole (ic50 = 0.13 μg/ ml).12 the maximal inhibitory concentration (ic50) of copper(ii)chloride dihydrate against denv-2 was 0.13 μg/ml.22 activity against hiv-1 strain iiib and hiv-2 strain rod in mt-4 cells (ic50) by zinc(ii) complexes with hexyl-me2-cyclam (hmc; 3,14dimethyl-2,6,13,17-tetraazatricyclo(16.4.0.07,12) docosane) were 10.51 ± 0.23 μm and 133.78 ± 14.10 μm. activity against hiv-1 strain iiib and hiv-2 strain rod in mt-4 cells (ic50) by zn(ii)– hmc diacetate were 3.50 ± 0.33 μm and >110.67 μm.23 anti-hiv-1 activity (ic50) in c8166/iiib, mt-4/gun1 and pbls/iiib were 8.0 μg/ml, 3.5 μg/ml, and 9.3 μg/ml, respectively. the ic50 value of the cobalt(ii)–morin complex for denv-2 was 3.08 μg/ml.25 mb21, a benzimidazole derivative, was found to be the most potential inhibitor of cloned proteases (ic50 = 5.95 μm).26 this study suggest that of zinc(ii)-2,4,5triphenyl-1h-imidazole complex compound can’t be an attractive antiviral option. it would be interesting to further investigate whether 2,4,5-triphenyl-1h-imidazole complex with other metal. the result of this study, zinc(ii)-2,4,5triphenyl-1h-imidazole complex compound more toxic than cu(ii)-2,4,5-triphenyl-1h-imidazole, this is caused by the zn (ii) complex being unstable compared to the cu (ii) complex. conclusion further studies are not required before zinc(ii)2,4,5-triphenyl-1h-imidazole can be applied in the medication of denv-2 infections. this study did not show the potential of the zinc(ii)-2,4,5triphenyl-1h-imidazole complex as a candidate for antiviral agents against denv-2 because it was shown to be toxic to vero cells. figure 1. inhibition of denv-2, at variation concentrations of zinc(ii)-2,4,5-triphenyl1h-imidazole complex compound 1000 bp 500 bp 100 bp (a) (b) (c) (d) (e) (f) (g) (h) figure 2. electrophoresis on 1.5% agarose of rtpcr after treatment, molecular weight marker (100 bp), (a) treatment with 50 μg/ml compound, (b) 25 μg/ml, (c) 12.5 μg/ml, (d) 6.25 μg/ml, (e) 3.13 μg/ml, (f) 1.57 μg/ml, (g) 0.78 μg/ml, and (h) 0.39 μg/ml 187aswandi wibrianto, et al.: effect of zinc(ii)-2,4,5-triphenyl-1h-imidazole complex against replication copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 conflict of interest there is no confl ict of interest of this paper. acknowledgement this research was supported by a research grant from mandat universitas airlangga (hrmua) 2019; 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;496(7446):504–7. 2. chi j, meng l, zhai x, guo y, pan s, zhou c, et al. tanshinon ii a attenuates 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virus type 2 protease inhibits the replication of all four dengue virus serotypes in cell culture. virol j. 2015;12(1):1–7. 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 80 404 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 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 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 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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. 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 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. 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 80 161 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. 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 �� vol. 1. no. 1 january–april 2010 research report risk factors of neonatal sepsis: a preliminary study in dr. soetomo hospital martono tri utomo department of child health medical school, airlangga university/dr. soetomo hospital abstract the risk factors of developing neonatal sepsis could be caused by maternal and neonatal risk factors. objective to determine the characteristics and risk factors for neonatal sepsis. study design was case control study. the data of neonates were taken from the medical record. neonates who were admitted in neonatal care unit of dr. soetomo hospital were included at january 2010 to february 2010, and divided into 2 groups, one group was sepsis cases and other group was non sepsis cases as a control. the risk factors that associated with sepsis were studied. chi square and logistic regression analysis were used to analyze the data. 97 patients were included and 31were sepsis cases and non sepsis case were 66. the risk factors that significantly cause sepsis are low birth weight (p=0.001 or 2.75, 95% ci 1.454–5.200) , prematurity (p=0.000, or 4.073, 95% ci 2.180–7.609), meconeal amniotic fluid (p=0.029, or 2.535, 95% ci 1.225–5.245) and c-section (p=0.032, or 1.895, 95% ci 1.087–3.303). the significant risk factors of the neonatal sepsis are low birth weight, prematurity, meconeal amniotic fluid, and caesarian section key words: risk factors, neonatal sepsis introduction newborn infection that is called neonatal sepsis can be very severe disease and lead to the high morbidity and mortality. neonatal sepsis is clinical syndrome of systemic illness accompanied by bacteremia occurring in the first month of life.1,2 the incidence of neonatal sepsis is approximately 7.1 to 38 cases per 1000 live births in asia, 3.5 to 8.9 per 1000 live birth in south america and the carribean.2,3 the incidence rate of neonatal infection in several referral hospitals in indonesia is approximately 8.76–30.29%, with the mortality rate is 11.56–49.9%.4 cipto mangunkusumo hospital reported at january-september 2005, the incidence of sepsis was 13,68% with the mortality rate 14,18%.5 dr soetomo hospital reported that 49 from 2416 patients showed bacterial positive blood culture (proven bacteremia sepsis).6 the clinical manifestation of neonatal sepsis can be varied from the subtle condition until very severe condition. the clinical manifestation of sepsis are hypoor hyperthermia, lethargy, irritability, or change in tone, mottling, pallor, petechiae, or jaundice, feeding intolerance, vomiting, diarrhea, or abdominal distention, tachypnea, respiratory distress, apnea, tachycardia, or hipotension, hypoor hyperglycemia, metabolic acidosis, focal infection.1,7 the latest criteria to diagnosis sepsis is if we found 1 of the fetal inflammatory response syndrome (firs), i.e. tachypnea, hypoor hyperthermia, crt > 3 seconds, wbc < 4.000 or > 34.000, crp > 10 mg/dl, il-6 or il-8 > 70pg/ml, positive 16srrna pcr, plus clinical variables.5,8 some conditions had been identified as the risk factors for developing neonatal sepsis. these conditions are:5 1. maternal risk factors are premature rupture of membranes (prom) especially more than 18 hours, infection and fever of the mother during labor, foul smell of amniotic fluid, turbidity and meconeal amniotic fluid, and multiple gestations. 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 frequent found in male than female, in black neonate, and in low social economy neonate. attack rate of neonatal sepsis increase significantly in low birth weight infants and the presence of maternal (obstetric) �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 23-26 risk factors especially with sign of chorioamnionitis such as prolonged rupture of membranes, maternal intrapartum fever (>37.5º c). the other potential factors are immunity risk factors that associate with male sex, congenital immune defect, congenital anomalies, omphalitis, and twinning; prematurity is a risk factor for both early onset and late onset sepsis.2,4,5,9,10 in the collaborative perinatal research study sponsored by the national institutes of health in boston city hospital at boston, low birth weight infants acquired sepsis three times more frequent than did term infant who weighed more than 2500 gram.2 the purpose of this study is to determine the risk factors for developing neonatal sepsis that associate with maternal or neonatal condition of the patients who were delivered or referred in the neonatal intensive care unit dr. soetomo hospital. method study design was case control study. the data were collected from the medical record of neonates who were admitted in neonatal care unit of dr. soetomo hospital between january 2010 to february 2010. technical sampling was purposive sampling. we reviewed data of all neonates who had been diagnosed as sepsis and collected the data of sample characteristic such as sex, gestational age, birth weight, mode of delivery and outcome. the risk factors that associate with sepsis such as sex, gestational age, birth weight, premature rupture of the membrane, turbid and meconeal amniotic fluid, asphyxia, and congenital anomaly were studied. the sample was grouped in the sepsis and non sepsis. we compare the risk factors between this two group. definitions prematurity are liveborn infants delivered before 37 weeks of pregnancy (based on the ballard score or from first day of the last menstrual period) low birth weight (lbw) neonate is neonate whose birth weight is less than 2,500 gram premature rupture of membrane (prom) is defined as the time from membrane rupture to onset of delivery was more than 18 hour. meconeal amniotic fluid was considered if the amniotic fluid was green in color or mixed with meconeal, or appears meconeal stained in the baby. congenital anomaly is defined as any abnormality of anatomy and morphology that found during physical examination. asphyxia is defined as apgar score less than 3 in the five minutes from delivery diagnosis of sepsis neonatorum based on clinical findings and supported by laboratory data (blood cell examination, value of c reactive protein and microbial blood culture). firs (fetal inflammatory response syndrome) defined as 1 of the tachypnea, hypoor hyperthermia, crt > 3 seconds, wbc < 4.000 or > 34.000, crp > 10 mg/dl, il-6 or il-8 > 70pg/ml, positive 16srrna pcr is found. statistical analysis data are presented in distribution tabulation and data analysis was performed with a computer assisted statistical package (spss ver. 12.0). chi square and logistic regression analysis were used to analyze the data. risk factors were calculated with odds ratio and 95% confidence interval, p values less than 0.05 was considered significant. results the collected data from 1 january 2010 until 28 february 2010 have been reviewed from all of medical record of the neonates that admitted in nicu ed dr soetomo hospital: 97 medical records sample of these neonates were studied, diagnosis sepsis were 31 patients, and no sepsis 66 patients. the distribution of characteristic samples between sepsis and non sepsis cases i.e., the referral cases is significantly found in sepsis group; the birth weight in sepsis cases were also significantly lower than non sepsis cases, but there were no difference in the mode of delivery between two groups. mortality rate in the sepsis cases were high (67%) (table 1). there were no significant difference among sepsis that associated with prom, turbid amniotic fluid, congenital malformation and asphyxia (p>0.05). potential risk factors of infection that significantly cause sepsis are low birth weight (lbw), prematurity, meconeal amniotic fluid, and caesarian section (p=0.001, p=0.000, p=0.029, and p=0.032 respectively). the risk factors which showed significant differences were analyzed by logistic regression analysis showed there were statistically significant association between the incidence of sepsis with prematurity and meconeal amniotic fluid (p=0.000; p=0.001 respectively). discussion neonatal sepsis is still the major problem in developing country that can cause the high morbidity and mortality.11 the diagnosis of sepsis neonatorum is still difficult, some effort has been developed by using the criteria of firs to diagnosis sepsis clinically.5 because of the morbidity and mortality of sepsis is high, and the difficult diagnosis, some effort to detect the risk factors for infection has been studied.11,12,13 ��utomo: risk factors of neonatal sepsis table 1. characteristics of neonated that admitted in dr soetomo hospital january–february 2010 characteristics of samples sepsis (+) n = 31 sepsis (-) n = 66 p value referral case 9 3 0.01* sex: male 21 39 0.413 female 10 27 birth weight (g) 2,091.9 2,720.4 0.00* gestasional age: premature 21 12 0.000* aterm 10 54 mode of delivery: spontaneous 14 14 0,18 breech delivery 3 3 manual aid 1 3 vaccum extraction 1 3 caesarian section 12 12 outcome: live 7 41 0.00* death 21 11 discharge on request 3 14 table 2. risk factors of infection in neonated that admitted in dr soetomo hospital january–february 2010 risk factors of infection p or 95% ci lbw 0.001* 2.75 1.454 – 5.200 prematurity 0.000* 4.073 2.180–7.609 prom > 18 hours 0.274 1.786 0.694–4.596 turbid amniotic fluid 0,805 0,854 0.237–3.077 meconeal amniotic fluid 0.029* 2.535 1.225–5.245 congenital malformation 0.983 0.981 0.169–5.707 caesarian section 0.032* 1.895 1.087–3.303 asphyxia 0.159 1.688 0.874–3.258 in this study, the determined diagnosis of sepsis based on the firs criteria that have been proposed by haque,8 beside the other examination such as bacterial blood culture and blood cell laboratory examination. the blood culture sometimes have the problem i.e the antibiotics already given before the blood culture was taken, the amount of blood that was taken, and desinfectan procedure before collecting the blood. this condition can influence the result of blood culture.14,15 risk factors that can lead to sepsis have been identified i.e.: maternal risk factors are premature rupture of membranes, infection and fever of the mother during labor, foul smell of amniotic fluid, turbidity and meconeal amniotic fluid, and multiple gestations; 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. other risk factors were more frequently found in male than female, in black neonate, and in low social economy neonate.5,11,13 the other study add the risk factors such as mode of delivery especially caesarian section.12 the risk factors that had been analyzed in this study were as follow: lbw, premature, prom > 18 hours, turbid amniotic fluid, meconeal amniotic fluid, congenital malformation, caesarian section and asphyxia. but the significant risk factors were lbw, premature delivery, meconeal amniotic fluid and caesarian section. in this study the lbw have the significant risk for the sepsis condition. the lbw babies have the risk to become sepsis 2.75 higher than non lbw babies. this result is similar to the other study by shah 200.11 preterm delivery is also contributed to sepsis. in this study the premature delivery had risk of 4 times higher than fullterm babies. the results this study was similar with the other study by shah and ladfors.11,12 the relatively immunodeficiency condition in the premature and lbw infant predisposed to the sepsis condition. these premature and lbw infants were also got some invasive procedure and monitoring leading to nosocomial infections.11 premature rupture of the membrane and prolonged leakage of the amniotic fluid can increase the risk of infection because of ascending bacterial from the urinary tract.9,11,12 in this study we didn’t find the correlation of premature rupture of the membrane and sepsis, some factors may contribute to this finding, such as history taking of the patient such as patient didn’t remember when the membrane had been ruptured. this condition is also indicated that post delivery antibiotic that was always given in the prom neonate as a standard procedure can decrease the risk of neonatal sepsis.1 �� indonesian journal of tropical and infectious disease, vol. 1. no. 1 january–april 2010: 23-26 the meconeal stained amniotic fluid can be caused by some infection in the uterine, prolonged fetal hypoxia in uterine and other stress condition of fetal in uterine. the choriamnionitis can produce the meconeal stained and foul smelling amniotic fluid because of inflammation reaction of the infection.1,9 in this study we found the significant risk factors of meconeal stained amniotic fluid to become sepsis with the risk of 2.5 times higher than non meconeal stained amniotic fluid. this finding is similar with the other study.11,12 the baby born from caesarian section have a risk 1,89 times higher than non caesarian section to become a sepsis. this finding is similar with the previous study12 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.16 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.17 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 understanding of the risk factors is important to determine the policy for the high risk babies with the risk factors to sepsis especially on the prevention. summary the risk factors of sepsis neonatorum in dr. soetomo hospital at january–february 2010 i.e. low birth weight, prematurity, meconeal amniotic fluid and caesarian section. references 1. gomella t, cunningham md, eyal fg. sepsis. in: gomella t, cunningham md, eyal fg., editor. neonatology management, procedures, on-call problems, diseases and drugs. 6th ed. new york: the mc graw-hill, co, inc; 2009. p. 665–72. 2. klein jo, 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. 3. vergnano s, sharland m, kazembe p, mwansambo c, heath pt. neonatal sepsis: an international perspective. arch dis child fetal neonatal ed. 2005;90:f220–4. 4. yu v, monintja h. infeksi sistemik pada neonatus. in: yu v, monintja h, editor. beberapa masalah perawatan intensif neonatus. 1st ed.beberapa masalah perawatan intensif neonatus. 1st ed. jakarta: balai penerbit fk ui; 1997. p. 217–30. 5. 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. 6. rahman t, utomo mt, etika r, indarso f, harianto a, damanik sm. sepsis neonatorum di rsu dr soetomo surabaya 2006. in: sadjimin t, juffrie m, julia m, wibowo t, editor. pit ika iii idai; 2007; yogyakarta: percetakan kita; 2007. p. 532. 7. puopolo k. bacterial and fungal infections. in: cloherty j, eichenwaldin: cloherty j, eichenwald ec, stark ar, editor. manual of neonatal care. 5th ed. philadelphia:manual of neonatal care. 5th ed. philadelphia: lippincott william & wilkins; 2008. p. 275–300. 8. haque kn. definition of blood stream infection in the newborn. pediatr crit care med. 2005;6:s45–9. 9. mccracken g, schleonka r, freij bj,. bacterial and viral infectionsbacterial and viral infections of the newborn. in: mcdonald m, mullen md, seshia mmk, editor. neonatology pathophysiology and management of the newborn. philadelphia: lippincott company; 2005. p. 1235–75. 10. stoll b. infections of the neonatal infant. in: kliegman r, behrman re, jenson hb, stanton bf, editor. nelson textbook of pediatrics. philadelphia: wb saunders co; 2007. p. 794–801. 11. shah g, budhathoki s, das bk, mandal rn. risk factors in early neonatal sepsis. kathmandu university medical journal. 2006; 4: 187–91. 12. ladfors l, tessin i, mattsson la, eriksson m, seeberg s, fall o. risk factors for neonatal sepsis in offspring of women with prelabor rupture of the membranes at 34–42 weeks. j of perinatal med 1998; 26: 94–101. 13. bhutta z, yusuf k. . neonatal sepsis in karachi: factors determining outcome and mortality j of tropical ped 1997; 43: 65–70. 14. weinstein m, towns ml, quartery sm. the clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology and outcome of bacteremia and fungemia in adults. clin infect dis 1997. 1997; 24: 584–602. 15. aminullah a. masalah terkini sepsis neonatorum. in: hegar b, trihono pp, ifran eb, editor. update in neonatal infections. jakarta:jakarta: departemen ilmu kes anak fkuirscm; 2005. p. 1–15. 16. bennel r, nord ce. development of the faecal anaerobic microfloradevelopment of the faecal anaerobic microflora after caesarean section and treatment with antibiotics in newborn infants. infection. 1987; 15: 332–6. 17. 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. ijtid vol 1 no 1 jan-apr 2010.25.pdf ijtid vol 1 no 1 jan-apr 2010.26.pdf ijtid vol 1 no 1 jan-apr 2010.27.pdf ijtid vol 1 no 1 jan-apr 2010.28.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 1 vol. 7 no. 1 january–april 2018 a study of correlation between agent, host, environment and vaccine factors with prevalence of rabies in indonesia 2015 tyas ika budi setyowati1, putri bungsu machmud2a 1 epidemiology department, public health faculty, universitas indonesia 2 epidemiology department, public health faculty, universitas indonesia a corresponding author: putri.bungsu10@ui.ac.id abstract a zoonotic disease has been global threat related to health and one of them is rabies. more than 150 countries around the world has infected by rabies disease problem and the case fatality rate (cfr) reaches 100%, which there are 55,000 people died every year because of rabies. in indonesia, there are 25 from 34 province, which has endemic of rabies disease. the purpose of this study is to know the correlation between the factors of the agent, host, and environment and also anti rabies vaccine with the prevalence of rabies in indonesia at 2015. the study used correlation design which using indonesian zoonotic reported data by ministry of health and also used other secondary data, which is provided by central bureau of statistic indonesia (bps). the provinces that included in this study are the endemic provinces associated with the rabies incident that reported to ministry of health and have the completeness of data in 2015. a total of 22 provinces was included in this study, which only use rabies cases from dog’s bite only. rabies that source from others animal’s bite could not included in this study because of data limitations. this study used simple linear of regression statistical tests through provincial as unit analysis. the result of this study showed that there were correlations between agent that have positive specimens (r=0.606, p value =0.0003), status of working participation rate (r=0.435, p value 0.004) and also coverage of rabies vaccine (r=-0.567, p value =0.041) with the prevalence of rabies disease. in summary, there are a positive correlation between positive specimen of agent and also status of working participant rate with the prevalence of rabies disease. however, rabies vaccine coverage has negative correlation. furthermore, there is no correlation between environment factors and prevalence of rabies disease in this study. it still need further research to be more research on a smaller level with variables that varied. keywords: rabies, agent, host, environment, arv abstrak penyakit zoonosis telah menjadi ancaman global dalam kesehatan, salah satunya adalah penyakit rabies. lebih dari 150 negara di dunia terjangkit rabies dan memiliki case fatality rate (cfr) sebesar 100% dimana sebanyak 55.000 orang meninggal setiap tahunnya akibat penyakit ini. di indonesia, terdapat 25 provinsi dari 34 provinsi endemis penyakit rabies. tujuan dari penelitian ini adalah mengetahui korelasi antara faktor agen, pejamu dan lingkungan serta vaksin anti rabies dengan prevalensi kejadian rabies di indonesia tahun 2015. desain studi yang digunakan adalah korelasi dengan menggunakan data yang bersumber dari laporan kementerian kesehatan bidang penyakit zoonotic serta data badan pusat statistik (bps) di indonesia. adapun provinsi yang diikutkan dalam studi ini adalah provinsi endemik terkait kejadian rabies yang mengirimkan laporan kepada kementrian kesehatan dan memiliki kelengkapan data yang dibutuhkan pada studi ini pada tahun 2015. sebanyak 22 provinsi yang diikutkan dalam studi ini sampai kepada tahap analisa data. pada studi ini hanya menganalisis kejadian rabies dari gigitan anjing saja sedangkan rabies yang berasal dari gigitan hewan lainnya tidak diikutsertakan oleh karena keterbatasan data. analisis yang dipergunakan yaitu uji statistik regresi linier sederhana dengan unit analisisnya adalah provinsi. hasil studi menggambarkan bahwa adanya korelasi antara antara agen dengan spesimen positif (r=0,606, pvalue=0,003) status kerja pada host (r=0,435, pvalue=0,004), dan cakupan vaksin anti rabies (r= -0,567, pvalue=0,041) dengan prevalensi rabies. dapat disimpulkan bahwa adanya korelasi positif antara spesimen positif dan status kerja research report 2 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 1–5 pada host. sedangkan cakupan vaksin anti rabies memiliki korelasi negative terhadap besaran prevalensi kejadian rabies. selain itu faktor lingkungan dinilai tidak memiliki korelasi pada studi ini. perlu penelitian lebih lanjut pada tingkat yang lebih kecil dengan variabel yang bervariasi. kata kunci: rabies, agen, pejamu, lingkungan, var introduction zoonotic disease has become a global threat because of its spread and allows the emergence of new infectious diseases or reappearance of old infectious diseases. in addition, zoonotic disease has a broad impact, not only on the health sector but also on the sectors of the economy, tourism and wildlife conservation. one of the zoonotic diseases that is a public health concern around the world is rabies.1 rabies is an acute infection of the central nervous system (brain), which is caused by lyssa-virus (rabies virus) and is transmitted to humans through the bites of rabid animals (dogs, apes, weasels, wild dogs, cats etc.). the rabies virus entered the body through a bite wound and persists for 2 weeks around the bite wound as well as replicates in the muscle tissue around the bite wound. the virus will travel to the central nervous system through peripheral nerves without clinical signs and symptoms. after reaching the brain, the virus replicates rapidly and spreads throughout the brain nerve cells/neurons, especially the cells of the limbic system, the hypothalamus and the brain stem. the virus runs toward the periphery through the efferent nerve fibers of both the voluntary and autonomic nervous systems after multiplying in the brain neurons. the virus attacks almost every organ and tissue in the body and will also multiply in tissues such as salivary glands, kidneys and others organ.2,3 about 150 countries in the world was infected with rabies and about 55,000 people died from rabies annually. the average number of human rabies cases per year in asian countries such as india 20,000 cases, china 2,500 cases, philippines 20,000 cases, vietnam 9,000 cases and indonesia 80,433 cases.4 in indonesia, there are 25 provinces out of 34 indonesian provinces was infected by rabies. number of animal bites related to rabies transmitters in indonesia increased by 86.3%, which was from 45,466 cases (2009) to 84,750 cases (2012) caused by rabies outbreaks in 2009-2012 in bali.4 as in other parts of the world, the incidence of human rabies in indonesia was influenced by age-related, sociodemographic and ecological factors. in addition, the risk of human rabies occurrence is directly proportional to the density of population.5,6 most of the human cases in indonesia caused by rabies was infected by dog bites (98%) and others by apes and cats.7 case fatality rate (cfr) of rabies was 100% and until now, there is no effective medicine to cure rabies. once clinical signs and symptoms occur, always ends as mortality. however, rabies can be prevented by early recognition of rabies-transmitted animal bites, washing the bites by soap/detergent under flowing water for 15 minutes, antiseptic around bite wounds and anti-rabies vaccine or anti-rabies serum.2,3 previous study showed that that in general 50% of human rabies occurs in children under 15 years of age and occurs mostly in men (sex ratio). men do more activities outside the room at night than women, which tends to be the reason of increased risk of rabies.5,8 the incidence of rabies is also influenced by employment (labor force participation rate). jobs make people stay outdoors more (especially in the night), so interaction with animals transmitting rabies will be higher than those who do not work.9 in addition, rabies is also associated with educational level (literacy rate). a high level of education and awareness is believed to reduce the incidence of rabies in humans.10 this study aim to determine the correlation of agent, host, environment and anti-rabies vaccine with the prevalence of rabies among humans in indonesia by the provincial level. the results of this study can be the basis for future analytic research at the individual level and can be used as a basis for the development of interventions for a larger research. material and method the study design was using descriptive correlation study. the study also included agent factor, host, environment, anti-rabies vaccine coverage as determine of rabies prevalence. the agent factor was portrayed by positive specimen variable. the host factor is depicted in the sex ratio variable and environmental factors were described in population density, labor force participation rates and literacy rates. the incidence of rabies is described in the prevalence of rabies. the study population was 25 provinces of rabies endemic area in indonesia 2015, which was set by the ministry of health, based on the provinces whose completed data. exclusion criteria were provinces whose have no complete data related to agent, host, environment and coverage of anti-rabies vaccine. based on the inclusion and exclusion criteria, there were 22 provinces of rabies endemic in indonesia. the rabies data of each province was obtained from the reported cases of animal bites and lyssa in the year of 2015 by ministry of health (zoonotic division), which provided the agent factor data (positive specimens) and the anti-rabies vaccine coverage. this study also explored 3setyowati and machmud: a study of correlation between agent, host, environment and vaccine the data related to host factors (sex ratio), environmental factors (population density, labor force participation rate and literacy rate) that obtained from provincial reports, figures were published by bps. data analysis was performed to obtain an overview of each variable (minimum, maximum, average, median, sd and 95% ci mean) and correlation picture (r, r2, line equation, p value and stock diagram) of each variable with rabies prevalence at provincial level. the correlation used is spearman correlation because the data is not normally distributed. this study already passed the ethical review issued by the public health faculty, university of indonesia with letter of reference number: 355 / un2.f10 / ppm.00.02 / 2017. result and discussion a total of 22 provinces (88%) out of 25 provinces in indonesia was examined in this study. the results of this study can only describe the condition of the province included in the investigation and cannot be generalized as conditions in the province outside the research area. tabel 1. distribution of rabies event, agent, host environment and anti rabies variabel mean sd 95% ci rabies prevalence 0,2 0,3 0,05 – 0,28 positive specimens 25,4 47,8 4,19 – 46,54 sex ratio 1,0 3,8 1,01 – 1,04 population density 181,2 276,1 58,82 – 303,63 labor force participation rate 66,3 3,9 64,50 – 68,01 literacy rate 98,6 1,3 98,06 – 99,21 anti-rabies vaccine coverage 0,8 0,2 0,75 – 0,88 table 1 showed the mean, median, sd and 95% ci mean values of all the variables studied. the highest prevalence of rabies in indonesia is 1.16 per 100,000 populations and there are some provinces were not sent the report related to rabies cases in 2015. the provinces with the highest rabies prevalence was north sulawesi, while provinces that reported zero rabies cases are lampung, banten, west sulawesi, south kalimantan and east kalimantan. based on table 1, it can be noticed that there are some difference condition among the provinces. based on correlation test results, there are several variables that showed significant correlation with rabies prevalence, i.e. positive specimen variable, labor force participation rate and anti-rabies vaccine coverage. the positive specimen variable has a strong relationship (r = 0.606) and is the positive pattern. this means that the higher the positive specimen, the higher the prevalence of rabies. figure 1. correlation of positive specimens on the agent on the prevalence of rabies in indonesia in the year 2015 the positive specimen data was used the number of rabies-transmitted animal specimens that were tested positive for rabies at the provincial level in indonesia. this study showed that rabies infected animals depicted with positive specimens had strong positive correlation (r = 0.606) and statistically there was a significant correlation (p-value=0.003) (figure 1). this figure shows that an increase in the number of positive specimens will be followed by an increased prevalence of rabies. this research is in line with research conducted by mau and desato in east nusa tenggara province (2011), which stated that rabies deaths cases in some (health facilities) puskesmas in ntt province are caused by high rabies positive dog population. the causative agent of rabies is the lyssa-virus that enters the body through an exposed skin. about 99% of deaths from rabies are caused by dog bites all over the world. some developing countries, dogs are the main reservoir for rabies, as well as in indonesia, which 80% source of rabies transmission of all rabies infected by dog bite.11 cases from other animals cases could not included in this cases, such as bat’s bite, rat’s bite, etc. positive animal specimens can be controlled by animals’ vaccinations that have the potential to transmit rabies. the failure to control rabies in developing countries was caused by the low of vaccination coverage.12 therefore, the cycle of rabies disease, especially in dogs cannot be broken yet. previous study in the bali province in 2012 revealed that the coverage of dog vaccination reached 91%, but it still cannot prevent the spread of rabies disease in this province because there are still many wild dogs that can spread rabies virus. 4 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 1–5 number of vaccination coverage should be increased to prevent the spread of rabies disease. vaccination programs should be conducted regularly so that immunity to rabies virus in proprietary dogs in endemic provinces can remain high.11 figure 2 describe about the coverage of anti-rabies vaccine has a strong relationship (r = -0.567) with negative pattern. this means that the higher of the coverage of antirabies vaccine, it will reduce the prevalence of rabies. figure 2. correlation of anti-rabies vaccine coverage against rabies prevalence in indonesia 2015 the anti rabies vaccine coverage is the amount of anti-rabies vaccine that given to people who have been potential transmitted by animal bites. this study showed that the anti-rabies vaccine described with anti-rabies vaccine coverage had strong negative correlation (r=0.567) and statistically there was a significant correlation (p-value=0,041) with rabies prevalence. this suggests that anti rabies vaccine coverage will be followed by a decrease in rabies prevalence. another research conducted in tanzania 2008 showed that 68% rabies-transmitted bite cases that did not receive anti rabies vaccine died of rabies. rabies infection cannot be treated, but the infection can be prevented by post-exposure prophylaxis using anti rabies vaccine. anti-rabies vaccine is almost 100% effective in preventing human deaths from exposure to rabies-transmitted animals.13 the labour force participation rate is the proportion of economically active worker in age population (the productive age group). this study revealed that labour force participation rate was moderately proportional to the correlation (r=0.44) and there is significant correlation (p-value=0,004) statistically. this suggests that an increase in labour force participation rate is followed by an increase in rabies prevalence. a previous study in new york and dallas in 1999 proved that someone who has a job with high mobility is more exposed to rabies. although everyone may be exposed to rabies, but in his research found that working as a courier is more risk to get rabies. courier work spends more time on the road on motorcycles or bicycles, thereby increasing the risk of being bitten by rabid animal. work as a courier is an outdoor job that is similar to the working conditions that is more often outdoors in indonesia, such as in the field of agriculture is more often outdoors. therefore, it is important to increase knowledge to outdoor workers about rabies in order to reduce the spread of rabies disease.14,15 other studies that conducted in bhutan in 2011 and iran in 2015 also showed that there was a significant relationship between work and rabies events (p-value <0.01). a worker will be more active outside, thus increasing the risk of being bitten by a rabies-transmitting animal.9,16 this study has some limitations especially in the related to correlation analysis, so there is a disruption in taking the causal conclusion of ecological bias due to the use of aggregate/group data as uni-analysis. therefore, this study can only be concluded in the population level (province), not in the individual level. conflict of interest there is no conflict of interest with relevant parties in this study. conclusion overall, in rabies endemic provinces in indonesia, there was a tendency for an increased number of rabies cases to be reported from rabies positive animal specimens and also an increase in the number of labour force (employment), therefore will increase the prevalence of rabies. in addition, there is also a tendency for a low anti-rabies vaccine coverage, which will tend to increase the prevalence of rabies. acknowledgement acknowledgments to prof. dr. nuning maria kiptiyah, mph, dr.ph, indonesian ministry of health, zoonosis division and bps indonesia as data provider of this study. figure 3. correlation of host work status based on labor force participation rate on rabies prevalence in indonesia 2015 5setyowati and machmud: a study of correlation between agent, host, environment and vaccine references 1. komisi nasional pengendalian zoonosis. laporan nasional komisi nasional pengendalian zoonosis. 2012. 2. who. who expert consultation on rabies. 2013. 3. kementerian kesehatan ri. laporan penelitian situasi dan analisis rabies. jakarta: pusat data dan informasi kementerian kesehatan ri; 2014. 4. kementerian kesehatan ri. buku profil kesehatan indonesia tahun. jakarta: kementerian kesehatan ri; 2015. 5. naipospos t. vaksin oral rabies. buletin veterinae, center for indonesian veterinary analytical studies. 2010; 6. mills j. ecologic studies of rodent reservoirs: their relevance for human health. emerg infect dis. 1998 dec;4(4):529–37. 7. kementrian kesehatan ri. buku saku petunjuk teknis penatalaksanaan kasus gigihtan hewan penular rabies di indonesia. jakarta : direktorat jenderal pencegahan dan pengendalian penyakit tular vektor dan zoonotik; 2016. 8. yibrah m, damtie d. incidence of human rabies exposure and associated factors at the gondar health center, ethiopia: a three-year retrospective study. infect dis poverty. 2015;4(1):3. 9. riabi hra. a three-year (2011–2013) surveillance on animal bites and victims vaccination in the south of khorasan-e-razavi province, iran. j clin diagnostic res. 2015; 10. yao h-w, yang y, liu k, li x-l, zuo s-q, sun r-x, et al. the spatiotemporal expansion of human rabies and its probable explanation in mainland china, 2004-2013. rupprecht ce, editor. plos negl trop dis. 2015 feb 18;9(2):e0003502. 11. istri t, cintya a, puja ik, kardena im. ekologi dan demografi anjing di kecamatan denpasar timur. 2012;1(2):160–72. 12. soeharsono. penyakit zoonotik pada anjing dan kucing. yogyakarta : kanisius; 2007. 13. hampson k, dobson a, kaare m, dushoff j, magoto m, sindoya e, et al. rabies exposures, post-exposure prophylaxis and deaths in a region of endemic canine rabies. kieny mp, editor. plos negl trop dis. 2008 nov 25;2(11):e339. 14. andresen m. an investigative study to develop an epidemiological description of reported dog bites that occurred in the five easternmost towns on long island, new york, during 1996 and 1997. 1999. 15. lambert tl. epidemiology of animal bites in the greater dallas/fort worth area, 1994-1998. 1999. 16. tenzin, dhand nk, gyeltshen t, firestone s, zangmo c, dema c, et al. dog bites in humans and estimating human rabies mortality in rabies endemic areas of bhutan. zinsstag j, editor. plos negl trop dis. 2011 nov 22;5(11):e1391. ijtid vol 6 no 2 mei-agustus 2016_edit.indd 49 vol. 6. no. 2 mei–agustus 2016 case report norwegian scabies in aids patient: a case report meita ardini pratamasari1, indropo agusni1a, cita rosita sigit prakoeswa1, linda astari1, willy sandhika2 1 department of dermatology & venereology 2 department of pathology anatomy faculty of medicine universitas airlangga/dr. soetomo general hospital a corresponding author: indropo49@gmail.com abstract scabies is a skin infection caused by sarcoptes scabiei var. hominis. this disease may present severe clinical manifestations in immune-compromised patient, well-known as norwegian scabies or crusted scabies.a 36-year old man with aids had chief complaint thick crust almost all over his body in this case. history of household member infected by scabies before was present. clinical findings show hyperpigmented macules unsharply marginated, covered with thick scales and accompanied by papules, fissures, and erotion. t cell cd4 level was 12 cell/μl. scraping examination showed scabies infection and so did the histopathology examination. this patient was treated by topical permethrin 5% combined with 2-4 ointment application in between permethrin usage. before topical scabicide was given, thick crust was previously treated by topical urea 10% and wet dressing by normal saline. on day 14 after the patient first came there was lesion improvement. key words: norwegian scabies, immunocompromised, aids, thick crusts abstrak skabies adalah suatu penyakit infeksi kulit yang disebabkan oleh tungau sarcoptes scabiei var. hominis. penyakit ini bisa bermanifestasi klinis yang hebat pada pasien dengan sistem imun yang rendah dan biasa disebut “norwegian scabies” atau skabies berkrusta. dilaporkan seorang laki-laki, usia 36 tahun, penderita aids, yang datang dengan keluhan keropeng yang tebal dan gatal pada sekujur badannya. beberapa anggota keluarga juga menderita gatal pada malam hari, namun tidak separah pasien. pemeriksaan klinis menunjukkan adanya bercak hiperpigmentasi yang menebal, disertai adanya erosi dan fisura pada beberapa tempat. pemeriksaan sel limfosit cd4 menunjukkan kadar yang rendah ( 12 sel/ul). pada pemeriksaan kerokan kulit ditemukan adanya infeksi scabies dan ditunjang oleh pemeriksaan histopatologi. pengobatan diawali dengan kompres nacl fisiologis dan salep urea 19%, selanjutnya diberikan salep permethrin 5% secara berkala, diselingi dengan kombinasi salep campuran asam salisilat dan sulfur (“ salep 2-4 “). setelah 14 hari diobati, lesi kulit berkurang dan menunjukkan banyak kemajuan. kata kunci: norwegian scabies, immunocompromised, aids, krusta tebal introduction acquired immunodeficiency syndrome (aids) is a group of clinical symptoms due to the decreasing of lymphocyte t-cd4+ cell count, caused by human immunodeficiency virus (hiv) infection. this virus belongs to genus lentivirus, family retroviridae or commonly known as the retroviral group. it destroys the lymphocyte t-cd4+ cells, causing the cell count to decrease below 200 cells/μl and the patients become prone to infection.1 one of infection that could affect hiv/aids patient is scabies. scabies is a disease caused by sarcoptes scabiei var. hominis parasite infestation, family sarcoptidae, class arachnida on the skin.2 this disease is one of major skin health problem in development countries associated with poverty, with estimated 300 million people worldwide are affected.3 the prevalence of scabies in indonesia varies from 2–65% and it relates with geographical, seasons and communities. high prevalence are reported in certain communities (pondok pesantren, 50 indonesian journal of tropical and infectious disease, vol. 6. no.2 mei–agustus 2016: 49−53 dormitory, jails).4 scabies infection is very contagious and could be the source of infection to the surrounding environment through direct skin or clothing contact.2,3 clinical symptoms such as itchy, especially at night time accompanied by papular skin eruptions. pathognomonic lesion in scabies infection is a burrow, which is a thin, thread-like, linear structure 1-10 mm in length. burrow is actually a tunnel caused by the movement of the mite in the stratum corneum, with predilection at interdigital webs, periumbilical, and genital areas.2,3 clinical findings of scabies infection in hiv patients is different with immunocompetent patient. lesions manifest as thick crusts so it’s called crusted scabies or commonly known as norwegian scabies.5 this type of infection has very enormous mite population so that it’s highly contagious even through casual contact. it also affects face, scalp, nail, with minimal pruritic symptom. this uncommon and hyperkeratotic type of scabies infection tends to affect immuno-compromised person due to lack of immune system ability to maintain the mite.6,7 case report a thirty-six year old male patient admitted to the dr. soetomo general hospital surabaya with chief complaint thick crusts almost all over his body since 1 week before. firstly it was some small papules over his thigh area, felt a bit itchy but no itchy sensation at nighttime. the papules then spread to all over his body, became thick crusts with some cracks in between and causing difficulty when moving. his wife and child ever had similar complaint 1 month before visitation, which were papules over their bodies accompanied with itchy sensation. they had been treated with permethrin cream and their lesions were getting better. meanwhile, the patient’s mother suffered from psoriasis but history of lesion on koebner area in this patient was denied. before his admission, he did the voluntary counseling and testing (vct) and hiv rapid test 2 months before and the result was positive, confirmed by three methods test hence he was diagnosed as aids. he had controlled routinely to the hiv outpatient clinic and consumed antiretroviral (arv) treatment for 1 month. at the outpatient clinic about 3 weeks before, he was diagnosed chronic dermatitis and got topical corticosteroid with emollient, and his complaint was getting better until the last complaint occurred 1 week before his admission. physical examination showed weak general condition was but good consciousness with normal vital sign. he had anemic conjunctiva and also slight enlargement of the liver. for the dermatological state, on auricular, axilla, colli, abdominal, inguinal, extremity (interdigitalis), and also gluteus regions there were large hyperpigmented macules, unsharply marginated and covered by thick crusts. there were also some fissures over the thick crusts, erosions, and we could see multiple papules over the eroded area over the scrotum (figure 1). the laboratory examination results were: white blood cells 3,600 cells/mm3, red blood cells 4.54 x 106 cells/ mm3, platelet count 128,000 cells/mm3, hemoglobin level 7.8 g/dl, sgot 126 u/l, sgpt 67 u/l, bun 7 mg/dl, creatinine serum 0.6 mg/dl, albumin level 2.1 g/dl, random blood glucose 86 mg/dl, natrium 130 mmol/l, kalium 3.8 mmol/l, chloride 100 mmol/l. the cd4 absolute count had been performed before with the result was only 12 cells/ μl.lesion scraping was done to find if there was any mite of sarcoptes scabiei, there were the adult mite with some eggs. histopathologic examination was done to exclude psoriasis. nevertheless, the diagnosis of scabies had been established by the scraping examination, so the therapy was soon started. first the wet dressing by normal saline solution was done to the thick crusts area in orderboth to decrease the crust andaddress the erosion, accompanied by urea 10% cream application. the thick crusts should figure 1. thick crusted lesion over abdomino-inguinal and interdigital region. 51pratamasari, et al., : norwegian scabies in aids patient: a case report be removed because it could inhibit topical antiscabies penetration, and the erosion should be treated soon because the applicationof topical antiscabies over the erosion could cause irritation. after 4 days of wet dressing treatment the thick crusts and the erosion were decreased, application of permethrin 5% cream once a week at night was started, with exception wet dressing and urea application were still done for area with thick crusts. besides dermatology therapy, the patient also continue the antiretroviral (arv) treatment, include terafovir, lamivudine and neviral; concomitant with supportive treatment. histopathology examination showed hyperkeratotic, parakeratosis, acanthosis and burrow in stratum corneum layer of epidermis. while in dermis there were capillary vessels with a little inflammatory cell, so the conclusion from the histopathology examination was scabies infection. after hospitalized for 1 week and there were progress of his lesion, this patient was discharged from hospital. before went home, he and his family were given education to repeat the use of permethrin 5% cream 1 week after the first use if there was any lesion persist either crusts or papules and to visit hospital afterwards, to wash all clothes, towels, and bedding by hot water. if there were any other household members that have the same complaint, they should be treated soon. discussion norwegian scabies or crusted scabies is a rare manifestation of scabies characterized by uncontrolled proliferation of mites in the skin. this disease was first described by boeck and danielssen among leprosy patients in norway in 1848.8,9 high risk groups for this infection such as they who are taking systemic glucocorticoid therapy or using potent topical glucocorticoid therapy, organ transplant recipient, having mental or physical disability, infected by hiv or human t-lymphotrophic virus-1, and also people with malignancy.10 this severe variant 2a 2b figure 2. sarcoptes scabiei mite from the lesion scraping: adult mite (2a), eggs (2b) figure 3. histopathology slides with 40x magnification 52 indonesian journal of tropical and infectious disease, vol. 6. no.2 mei–agustus 2016: 49−53 of scabies occurs as widespread hyperkeratotic crusted lesions, hence the name “crusted scabies” is preferred as the synonym of “norwegian scabies”.11 the causative agent, mite sarcoptes scabiei var. hominis, is an obligate parasite that lives in burrowed tunnels in the stratum corneum. in the skin, the mite survives on a diet of dissolved human tissue but does not feed on blood. it makes a sloping burrow, in the stratum corneum to the boundary of stratum granulosum every day. the mite lives in the burrow for a 30 day-period, consisting cycle as follows. the female mite lays 2 3 eggs daily and the eggs hatch in 10 days, then the young larva leaves the burrow to become mature adult mite in 14 – 17 days.2,3,9 in normal patient, it is estimated that only 10% of the eggs that develop into adults with total average mite is about 11. however, the number of the mites is very enormous in crusted scabies because of uncontrolled infection.8,9,10 recently there is increasing occurrence of this case due to various immunosuppressive agent and increasing case of hiv patient (figure 2). cutaneous manifestations of scabies are due to the burrowing of the female mite followed by humoral and delayed hypersensitivity of the host.2,3 the mite antigens that trigger the immune response are probably in the mite’s saliva. combined with scratching, the immune system in the healthy host will reduce the mite load but rarely eliminates the mite. the failure of the immune system to suppress the proliferation of the mite is considered to play role in crusted scabies development, though incidence of crusted scabies in australian aborigines with normal immunity has been reported.13 in this case, hiv stadium iv that the patient suffered from made the level of cd4+ t cells dropped until 12 cells/μl so that the patient was susceptible to infection. while less itchy sensation occurred as the result of inadequate immune system, a huge amount of mites makes this disease very contagious.7,8 definitive diagnosis of crusted scabies is equal with common scabies, which is the presence of mite, egg, eggshell or fecal material from skin lesion scraping, demonstrated by potassium hydroxide 10% solution under light microscopy examination. in this patient, we found the presence of the mite and egg so that antiscabies treatment could be initiated without waiting for the histopathology result. later, the histopathology examination revealed that there was burrow in the stratum corneum that is surrounded by inflammation cells, showed that cellular immunity plays role in this disease’s pathogenesis. burrow was pathognomonic sign that we can find in scabies infection (figure 3).2,3 scabies management involves the use of scabicide agent and mite control. antiscabies agent might be taken orally and topically as discussed above, meanwhile mite control needs education for the patient and his family. all family members that live together with the patient should be treated at the same time to prevent asymptomatic carrier’s reinfestation. if possible, during the time of application of the topical scabicide, all linens, bedding, and clothing in the house that has been used should be soaked with warm/hot water before washed, and then ironed with high temperature to eradicate mite.2,3 this patient was treated initially with wet dressing (2-3days), using normal saline combined with 10% urea cream to remove the thick crusts. then a 5% permethrin cream was applied intermittently combined with ointment contains 2% salicylic acid plus 4% sulfur (“2-4 ointment) daily in between the permethrin. after 14 days application of this topical medication there is mark improvements. theoretically oral ivermectine could be used since this drug acts on nerve synapses utilizing glutamate or γ-aminobutyric acid.14 but this oral medication could not penetrate into the thickness of the keratinous debris and this drug is not available in indonesia. conventional wet dressing method using nacl 0.9% solution was used, then followed by topical urea 10% use for softening the crusts. after the crusts already minimal and thinned, permethrine 5%, a topical antiscabies agent, was applied to this patient. this topical agent will be effective in such situation due to better absorption in the skin. scabies management involves the use of scabicide agent and mite control. antiscabies agent might be taken orally and topically as discussed above, meanwhile mite control needs education for the patient and his family. all family members that live together with the patient should be treated at the same time to prevent asymptomatic carrier’s reinfestation. if possible, during the application time of topical scabicide, all linens, bedding, and clothing in the house that has been used should be soaked with warm/hot water before washed, and then ironed with high temperature to eradicate mite.2,3 references 1. murtiastutik d. hiv & aids. in: barakbah j, lumintang h, martodihardjo s, editors. buku ajar infeksi menular seksual. surabaya: airlangga university press; 2008. p. 211–69. 2. burkhart cn and burkhart cg. scabies, other mites, and pediculosis. in: goldsmith la, katz si, gilchrest ba, paller as, leffell dj, wolff k, editors. fitzpatrick’s dermatology in general medicine. 8th ed, vol 2. new york: mcgraw-hill, 2012. p. 2569–77. 3. burns t, breathnach s, cox n, griffiths c, editors. diseases caused by arthropods and other noxious animal. in: burns t, breathnach s, cox n, griffiths c, editors. rook’s textbook of dermatology. 8th ed. west sussex: wiley-blackwell; 2010. 4. hilmy f. prevalensipenyakit scabies dan hubungannya dengan karakteristik santri pesantren x jakarta timur [skripsi]. fakultas ilmu kesehatan masyarakat, universitas indonesia; 2010. 5. chan cc, lin sj, chan yc, liao yh. infestation by norwegian scabies. cmaj. 2009, 181(5). 6. perna ag, bell k, rosen t. localised genital norwegian scabies in an aids patient. sex transm infect. 2004, 80: 72–3. 7. karthikeyan k. crusted scabies. indian j dermatol venereol leprol. 2009, 75: 340–7. 8. subramaniam g, kaliaperumal k, duraipandian j, rengasamy g. norwegian scabies in a malnourished young adult: a case report. j infect dev ctries. 2010, 4(5): 349–51. 9. fernandez-sanchez m, saeb-lima m, de la barrera ca, reyes-teran g. crusted scabies-associated immune reconstitution inflammatory syndrome. bmc infectious diseases. 2012, 12: 323. 53pratamasari, et al., : norwegian scabies in aids patient: a case report 10. binic i, jankovic a, jovanovic d, ljubenovic m. crusted (norwegian) scabies following systemic and topical corticosteroid therapy. j korean med sci. 2010, 25: 188–91. 11. davis js, mcgloughlin s, tong syc, walton sf, currie bj. a novel clinical grading scale to guide the management of crusted scabies. plos negl.trop. dis. 2013, 7(9). 12. workowski ka and berman s. sexually transmitted diseases treatment guidelines, 2010. in: centers for disease control and prevention [internet]. morbidity and mortality weekly report. [cited 2013 aug 20] available from: www.cdc.gov/mmwr 13. walton sf and currie bj. probles in diagnosing scabies, a global disease in human and animal populations. clin microbiol rev. 2007, 20(2): 268. 14. .ly f, caumes e, ndaw cat, ndiaye b, mahe a. ivermectin versus benzyl benzoate applied once or twice to treat human scabies in dakar, senegal: a randomized control trial. bull world health organ. 2009, 87: 424–30. 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 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 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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 4.0 404 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 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. 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 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 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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 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 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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. 62 vol. 7 no. 3 september–december 2018 rna isolation of dengue virus type 1 with different precipitation solvents: dimethyl sulfoxide, acetone, and ethanol 70% anisa maharani1a, teguh hari sucipto2, harsasi setyawati1, siti churrotin2, ilham harlan amarullah2, puspa wardhani2, aryati2, shuhai ueda3, soegeng soegijanto2 1 department of chemistry, faculty of science and technology, universitas airlangga 2 dengue study group of tropical disease, universitas airlangga 3 center for infectious disease, kobe university graduate school of medicine, japan a corresponding author: maharanisa77@yahoo.com abstract dengue hemorrhagic fever (dhf) is caused by dengue viruses that belong to flaviviridae. the disease is known to be caused by 4 types of dengue viruses, namely denv-1, denv-2, denv-3, and denv-4 associated with antigenic. dengue virus is a virus rna that causes illness with clinical manifestations of dengue fever, dengue hemorrhagic fever and dengue shock syndrome. the aim of research was to determine the effectiveness of dimethyl sulfoxide, acetone, and ethanol 70% as precipitation solvent in the process of rna isolation. the method used was reverse transcription polymerase chain reaction (rt-pcr) and polymerase chain reaction (pcr) with specific primers for dengue virus type 1 (denv-1). rna isolation can be done easily using an rna isolation kit. use of rna isolation kit results in a purer rna isolate from contaminants and from rna degradation. in generally the isolation is using cold ethanol / alcohol with concentration 90-95%. ethanol / alcohol does not dissolve rna and light density of alcohol lighter than water makes rna rise and hover on the surface. in rna isolation solvent precipitation that used are acetone, ethanol 70%, and dmso. in qualitative rna measurements using agarose gel electrophoresis and was examined under the uv light-illuminator and quantitative rna measurements using nanodrop spectrophotometry with absorbance ratio at 260/280 and 260/230 showed a good result indicated by the appearance of the band on electrophoresis results in pcr. while the measurement quantitatively is showed that there was still protein contamination but the results are quite good because it does not much different from the ratio set in the reference. acetone, ethanol 70%, and dmso can be used as a substitute of 96% ethanol in the process of rna isolation in denv-1 virus and can also be applied to other dengue virus because the structure of the 4th antigen serotype is very similar one with the other and no effect. key words: rna, precipitation solvents, dmso, acetone, ethanol abstrak demam berdarah dengue (dbd) disebabkan oleh virus dengue milik flaviviridae. penyakit ini diketahui disebabkan oleh 4 jenis virus dengue, yaitu denv-1, denv-2, denv-3, dan denv-4 yang terkait dengan antigenik. virus dengue adalah virus rna yang menyebabkan penyakit dengan manifestasi klinis demam berdarah dengue, demam berdarah dengue dan syok dengue shock. metode yang digunakan reverse transcription polymerase chain reaction (rt-pcr) dan polymerase chain reaction (pcr) dengan primer spesifik untuk virus dengue tipe 1 (denv-1). isolasi rna dapat dilakukan dengan mudah menggunakan rna isolation kit. penggunaan rna isolation kit menghasilkan isolat rna yang lebih murni dari kontaminan dan dari degradasi rna. pada umumnya isolasi menggunakan etanol/ alkohol dingin dengan konsentrasi 90-95%. etanol/alkohol tidak melarutkan rna dan kerapatan ringan alkohol lebih ringan daripada air membuat rna naik dan melayang di permukaan. pada pengendapan pelarut rna terisolasi yang digunakan adalah aseton, etanol 70%, dan dmso. pada pengukuran rna kualitatif menggunakan elektroforesis gel agarose dan diperiksa di bawah sinar-iluminator uv dan pengukuran rna kuantitatif menggunakan spektrofotometri nanodrop dengan rasio absorbansi pada 260/280 dan 260/230 menunjukkan hasil yang baik yang ditunjukkan oleh kemunculan band hasil elektroforesis pada pcr. sedangkan pengukuran secara kuantitatif menunjukkan bahwa masih ada sedikit kontaminasi protein namun hasil tersebut cukup research report 63maharani, et al.: rna isolation of dengue virus type 1 introduction dengue is appeared in two forms, classic dengue fever and severe form. in severe form, dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) can caused abdominal bleeding, hemorrhage and circulatory failure. if this severe form is not treated with prompt this will lead to the fatal cases.1 dengue hemorrhagic fever (dhf) caused by dengue viruses is still a major problem in tropical countries.2 dengue hemorrhagic fever (dhf) are the most rapidly spreading vector-borne diseases with approximately 50 million cases of infection worldwide.3 in the last decades, dengue has emerged as a prime health concern in tropical and subtropical expanse of the world resulting in increased mortality every year.4 indonesia is the one of the many tropical countries that are affected by the dengue virus (denv). the main clinical manifestations, namely dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss), are responsible for high morbidity and mortality rates every year. over 40% (2.5 billion) of the population in 100 tropical and subtropical countries continue to live under the threat of contracting dengue infection. it is estimated that 100 million cases of df, 500,000 cases of dhf, and 25,000 deaths are reported annually worldwide.5 increases in human population size, dengue vector-density and human mobility cause rapid spread of dengue virus in indonesia. dengue virus is transmitted through the bite of a female mosquito. transmission can occur when aedes female mosquitoes are sucking blood of people infected with dengue virus and the mosquito will soon bite others as well. the process of isolating rna was through several stages and in that stage, the role of solvent is very important that is to know the level of effectiveness of the solvent used and its impact on the results of the isolation. in a baik karena tidak berbeda jauh dari rasio yang ditetapkan dalam referensi. sehingga dapat disimpulkan hasil tersebut menunjukkan bahwa aseton, etanol 70%, dan dmso dapat digunakan sebagai pengganti etanol 96% dalam proses isolasi rna pada virus denv-1 serta dapat juga diaplikasikan pada virus dengue yang lain karena struktur antigen ke-4 serotipe ini sangat mirip satu dengan yang lain dan tidak berpengaruh. kata kunci: rna, presipitasi pelarut, dmso, aseton, etanol previous research, rna isolation protocol were based on cetyltrimethylammonium bromide (ctab).6,7,8,9 and two successive precipitations with 10 m lithium chloride (licl).10,11,12 the good parameters in ctab depend on several factors. first, the nacl concentration must be above 1.0 m to prevent the formation of ctab-rna complexes. then, extracts and ctab-containing cell solutions should be stored at room temperature since the ctab-dna complex is insoluble in temperatures below 15°c. and the last, the use of ctab with good purity will determine the purity of rna obtained and with very little polysaccharide contamination, with ctab method will also be obtained rna with a thin band located far below the dna band. the existence of the rna band depends on the extracted material.13 although licl is commonly used to precipitate rna, but this method has received little attention and not effective with low concentration. generally we are used ethanol or isopropanol in the precipitation stage. both of these compounds will precipitate rna in the aqueous phase so that the rna agglomerates to form a fiber structure and pellets are formed after centrifugation. in this research we used several kinds of solvents that can be an alternative to a solvent commonly used in rna isolation, the topic we are raising is rna isolation of dengue virus type 1 with different precipitation solvents: dimethyl sulfoxide (dmso), acetone, and ethanol 70%. dimethyl sulfoxide (dmso), acetone, and ethanol 70%which has lower constant dielectric compared to water increases the interaction of the salt and the coulomb force of attraction between the cation and the negatively charged nucleic acid backbone (that is, the resistance from the solvent’s electric field sufficiently diminishes to gain efficient interaction; the solvation shells surrounding the solute’s charges depletes).14 table 1. oligonucleotide primers used to amplify and type dengue viruses.15,16,17 primer sequence genome position size in bp, of amplified dna product (primers) d1 5’-tcaatatgctgaaacgcgcgagaaaccg-3’ 134-161 511 d2 5’-ttgcaccaacagtcaatgtcttcaggttc-3’ 616-644 511 ts1 5’-cgtctcagtgatccggggg-3’ 568-586 482 (d1 and ts1) ts2 5’-cgccacaagggccatgaacag-3’ 232-252 119 (d1 and ts2) ts3 5’-taacatcatcatgagacagagc-3’ 400-421 290 (d1 and ts3) ts4 5’-ctctgttgtcttaaacaagaga-3’ 506-527 392 (d1 and ts4) 64 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 62–66 material and method dengue virus type 1 (denv-1) was isolated from surabaya (genbank accession ab915377), acetone (merck, germany), dimethyl sulfoxide (dmso) (merck, germany), ethanol 96% (merck, germany), rna isolation kit (qiaamp mini kit) was supplied by qiagen from usa, master mix pcr was supplied by promega from usa, reagent for rt-pcr was supplied by invitrogen from germany, nuclease free water (nfw) was supplied qiagen from usa, ethidium bromide (merck, germany), tae buffer (promega, usa), agarose (promega, usa), and primer are demonstrated in the table 1. rna isolation for rna, isolation from vero infected cells has used concentration from the reagent. samples were taken from refrigerator and were melted. sample were mixed 2.24 ml avl and 22.4 µl ave, this composition was already included in the protocol of the kit that has been purchased. mixing this solution with vortex for 15 s. after centrifugation, the resulting rna pellet was washed with ethanol 560 µl. then, move 630 µl to mini column qiaamp. the mixture was centrifuged at 8,000 x g for 1 min, and the water was removed and combined with an equal volume of solvent to precipitate the rna. after that, add 500 µl buffer aw 1, centrifuged at 8,000 x g for 1 min. the next step is add 500 µl buffer aw 2, centrifuged at 14,000 x g for 3 min then remove the remaining water and move to the new tube and then centrifuged again at 14,000 x g for 3 min and after finishing put it down to the eppendorf tube. add with ave buffer 40 µl then centrifuged at 8,000 x g for 1 min. for the last, keep it in temperature -80°c. in this research we change precipitation solvents with dmso, acetone, and ethanol 70%. rna measurements were quantitatively performed using nanodrop spectrophotometry with an polysaccharides absorb most uv light at λ230 nm and protein at λ280 nm. reverse transcription-polymerase chain reaction (rt-pcr) rt-pcr process has used concentration from the reagent. mix dntp, primer, nfw, and rna then was centrifuged for 1 min. then, the mixture was put in thermocycle with temperature 65°c for 5 min. for the next step, master mix that consist of fs buffer, rnase out, dtt and superscript was made. the mixture was centrifuged at 8,000 x g for 1 min. the first master mix from thermocycle was mixed with the second master mix, then it was centrifuged for 1 min and was put in to thermocycle at 50°c for 60 min, and then the process was continued for temperature 85°c for 5 min. isolation of dna by pcr 12.5 µl master mix was mixed with 5.5 µl nuclease free water (nfw), 2 µl primer, and 5µl cdna from rt-pcr result. then, was put in to micro tube. the mixture was centrifuged at 8,000 x g for 1 min. after that the solution was inserted into the thermocycle. agarose gel electrophoresis some of the electrophoresis process steps mixing 1 µl marker, 6 µl tae buffer, and 3 µl dna. the gel was run for 30 min at 100 volt and was stained with ethidium bromide. the bands were visualized on an ultraviolet trans-illuminator. result and discussion in this report, we have described solvent that we have used for isolation of rna. in this research, we have been able to complete the rt-pcr assay, starting from rna isolation and completing with agarose gel analysis. in addition, dengue virus was consists of four serotypes (denv-1, denv-2, denv-3 and denv-4).18,19,20 in indonesia there were dominance serotype denv-2, followed denv-3 in 2003 to 2005.21 virus serotype can be demonstrated by molecular techniques such as pcr and rt-pcr. isolation of rna can be done through several stages as described in the research method. the result was obtained from the isolation process were used for the rtpcr process. figure 1 was showed a serum samples that were obtained from surabaya. the results were obtained throughout the sample is positive, there is no dengue virus dna bands appear, actually the bands were visible but not intensive. isolation of rna with pcr was done in a similar way to rt-pcr but with different temperature variations. rt-pcr was used temperature 45ºc whereas pcr used temperature 65ºc. the data which was obtained from isolation dna by pcr was showed the good results. when ethanol was removed and the pellet was drained in a tube, the remaining pellets in the tube were concentrated rna. the process of re-precipitation with ethanol before pellet was allowed to increase the degree of purity of isolated rna.22 after the isolation process, the obtained dna can be concentrated by precipitation. commonly was used ethanol or isopropanol in the precipitation stage. both of these compounds will precipitate rna in the aqueous phase so that the rna agglomerates to form a fiber structure and pellets were formed after centrifugation. at this precipitation stage, the precipitated rna will be separated from the remaining dna and protein residues. the residue also undergoes coagulation but there was no fiber structure and was in the form of granular precipitates.23 when solvents was removed and the pellet was dried in a tube, the remaining pellets in the tube were concentrated rna. the process of precipitation with solvents before the pellet was dried can increase the degree of purity of isolated rna. in the isolation process, the principle is to use organic solvents, carry out with a substance that is insoluble in water, dissolve in an organic solvent, mix with water, then add distilled water under certain conditions.24 ethanol was a versatile solvent, water soluble and other organic solvents. so, ethanol was the most important solvent option used in the rna isolation process. acetone, ethanol 70%,and dmso precipitation were efficient method for isolation 65maharani, et al.: rna isolation of dengue virus type 1 of rna. they are including in polar solvents and have almost the same solubility properties as ethanol. some important properties of solvents include ability to solubility, the velocity evaporates, boiling route, specific gravity, flashpoint. this equation was allowed them to be used as a replacement solvent when ethanol was absent. the appearance of the ribbon was clearly showed that acetone, ethanol 70%, and dmso can be used instead of ethanol. in addition, these solvents were easier to obtain than other organic solvents. the results were positive, they were showed dengue virus dna bands. positive control was used the dengue virus serotype denv-1 (482bp). the next step is to know rna measurements were quantitatively performed using nanodrop spectrophotometry with an absorbance ratio of 260/280 and 260/230. absorbance data was showed in table 2. the principle of nanodrop spectrophotometric work is pure rna which capable of absorbing ultraviolet light due to the presence of purine and pyrimidine bases.25 the presence of contaminants can also be known through spectrophotometer. polysaccharides were absorbed most uv light at λ230 nm and protein at λ280 nm. the level of purity of rna can be known by measuring the amount of sample absorbance at λ230 nm, λ260 nm, and λ280 nm, then measuring large comparison (ratio) a260 to a280 and ratio a260 against a230.26 pure rna isolate has an a260/a280 ratio of 2.0±0.1. a low a260/a280 ratio indicates protein contamination. from the test results can be known that at a260/280 nm much samples have a ratio of more than 2.0. it can be stated that the rna isolate is contaminated with proteins even it is not too much. in the above data can be seen nanodrop results for acetone on a260/280 nm lower than the other three solvents. in addition, if sorted from the yield of nanodrop, ethanol 70% has the highest value compared with the other three solvents so that the value of protein contamination can be said more than 96% ethanol, acetone, or dmso. the test results on a260/a230 was showed that no sample less than 1.5 which means the results of rna still contain other contaminants, pure rna isolate has an a260/a230 ratio of 2.0-2.4.27 so it can be concluded that 96% ethanol, acetone, and dmso can be used as precipitation solvents in rna isolation because the protein concentrations of nanodrop yields show good results. the qualitative results indicate that the three solvents can be used as precipitation of solvent in the process of rna isolation, which were indicated by the appearance of the band on the electrophoresis results of pcr. the results were showed that acetone, ethanol 70%, and dmso can be used as a substitute of 96% ethanol in the process of rna isolation in denv-1 virus and can also be applied to other dengue virus because the structure of the 4th antigen serotype is very similar one with the other and no effect. based on previous research on the epidemiology of dengue fever in indonesia, who south-east asia regional office (searo), indonesia did not report laboratory-confirmed data in period 2000-2013. also no active data were found for the study period, and very few dengue case were reported on the searo website, although dengue is considered to be endemic countries. another limitation is that we did not further elaborate on circulation of the predominant serotype, as only qualitative data were available from passive surveillance data.28 in surabaya-indonesia, 2012, denv-1 was reported dominant serotype with 90.7%, followed by denv-2 (9.3%), for denv-3 and denv-4 were not detected. in 2013, denv-1 was reported dominant serotype also with 66.7%, denv-2 (27.5%), denv-3 (5.8%), and denv-4 were not detected.18 in 2014, denv-2 was reported dominant serotype with 92.8%, denv-1 (7.2%), for denv-3 and denv-4 were not detected. the same time, madura island isolates was showed high nucleotide similarity to other indonesia isolates, indicating frequent virus circulation in indonesia.29 the results of the present study highlight the importance of help viral isolation in dengue endemic areas to obtain a cleare understanding of the dynamics of denv in indonesia. used ethanol or isopropanol in the precipitation stage. both of these compounds will precipitate rna in the aqueous phase so that the rna agglomerates to form a fiber structure and pellets were formed after centrifugation. at this precipitation stage, the precipitated rna will be separated from the remaining dna and protein residues. the residue also undergoes coagulation but there was no fiber structure and was in the form of granular precipitates.23 when solvents was removed and the pellet was dried in a tube, the remaining pellets in the tube were concentrated rna. the process of precipitation with solvents before the pellet was dried can increase the degree of purity of isolated rna. in the isolation process, the principle is to use organic solvents, carry out with a substance that is insoluble in water, dissolve in an organic solvent, mix with water, then add distilled water under certain conditions.24 ethanol was a versatile solvent, water soluble and other organic solvents. so, ethanol was the most important solvent option used in the rna isolation process. acetone, ethanol 70%,and dmso precipitation were efficient method for isolation of rna. they are including in polar solvents and have almost the same solubility properties as ethanol. some important properties of solvents include ability to solubility, the velocity evaporates, boiling route, specific gravity, flashpoint. this equation was allowed them to be used as a replacement solvent when ethanol was absent. the appearance of the ribbon was clearly showed that acetone,ethanol 70%, and dmso can be used instead of ethanol. in addition, these solvents were easier to obtain than other organic solvents. the results were positive, they were showed dengue virus dna bands. positive control was used the dengue virus serotype denv-1 (482bp). figure 1.results of electrophoresis of pcr with solvent (1) dmso, (2) ethanol 70%, (3) acetone, (4) ethanol 96%, and (m) marker. the next step is to know rna measurements were quantitatively performed using nanodrop spectrophotometry with an absorbance ratio of 260/280 and 260/230. absorbance data was showed in table 2. the principle of nanodrop spectrophotometric work is pure 3 2 2 4 4 1 m figure 1. results of electrophoresis of pcr with solvent (1) dmso, (2) ethanol 70%, (3) acetone, (4) ethanol 96%, and (m) marker. table 2. the data result of nanodrop spectrophotometry for rna samples sample a260/280 nm a260/a230 nm denv1 – ethanol 70% 3.28 0.38 denv1 – ethanol 96% 3.25 0.28 denv1 – acetone 3.16 0.39 denv1 – dmso 3.20 0.48 66 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 62–66 conclusion in this study, rna measurements were performed qualitatively using agarose gel electrophoresis and examined under a uv trans-illuminator. the quantitative rna measurement was used nanodrop spectrophotometry with absorbance ratio at 260/280 and 260/230. rna measurements were quantitatively performed using nanodrop spectrophotometry with an absorbance ratio of 260/280 and 260/230 was showed good results, ethanol 70% has the highest value compared to the other three solvents so that the value of protein contamination can be said more than 96% ethanol, acetone, or dmso.while the results on absorbance 260/230 was showed that no sample less than 1.5 which means the results of rna still contain other contaminants, pure rna isolate has an a260 / a230 ratio of 2.0-2.4. the qualitative measurements were showed that in pcr, the appearance of the ribbon was signified that acetone, ethanol 70%, and dmso can be used instead of ethanol, so if ethanol 96% is not available in the laboratory, we can be used dmso, acetone, and ethanol 70% as precipitation of solvent in the process of rna isolation in denv-1 virus and they can also be applied to other dengue virus because the structure of the 4th antigen serotype is very similar with the other and there was. acknowledgement thank you to institute of tropical disease universitas airlangga for research internship opportunity in the dengue laboratory, department of chemistry, faculty of science and technology universitas airlangga and for japan initiative for global research network on infectious disease (j-grid). references 1. gubler dj. dengue, urbanization and globalization: the unholy trinity of the 21(st) century. trop med health. 2011 dec;39(4 suppl):3–11. 2. saptawati l, febrinasari rp, yudhani rd, faza ag, ummiyati hs, sudiro tm, et al. in vitro study of eight indonesian plants extracts as anti dengue virus. heal sci j indones. 2017;8(1):12–8. 3. who wmo. atlas of health and climate change. health and environment series. 2012. 176 p. 4. sharma a, 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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. 21. aryati, soetjipto, hariadhi s, rantam fa ss. profile serotype virus dengue di indonesia tahun 2003–2005. maj ked trop ind. 2006;17(1):72–80. 22. bettelheim, f.a. & landesberg j.m. laboratory experiments for general, organic, and biochemistry. 2007. 23. irawan h. analisis dna. 2014. 24. anief m. farmasetika. gadjahmada university press; 2007. 110-111 p. 25. fatchiyah a, widyarti le rs. biologi molekular prinsip dasar analisis. malang: erlangga; 2011. 26. amanda ud. isolasi rna total dari mesokarp buah kelapa sawit (elaeis guineenssis jacq. var. tenera). 2015;1(april):171–6. 27. farrell re. rna methodologies: a laboratory guide for isolation and characterization. in: elsevier academic press. 3rd ed. burlington; 2005. 28. toan nt, rossi s, prisco g, nante n, viviani s. dengue epidemiology in selected endemic countries: factors influencing expansion factors as estimates of underreporting. trop med int health. 2015 jul;20(7):840–63. 29. sucipto th, kotaki t, mulyatno kc, churrotin s, labiqah a, soegijanto s, et al. phylogenetic analysis of dengue virus in bangkalan, madura island, east java province, indonesia. j trop med. 2018;2018:8127093. ijtid vol 6 no 1 jan-april 2016_revisi.indd 12 vol. 6. no. 1 january–april 2016 research report cellular immunity activation method by stimulating rd1 complex proteins as virulence marker on mycobacterium tuberculum to establish diagnosis on tuberculosis and latent tuberculosis infection rebekah j. setiabudi 1,a, ni made mertaniasih 2, didik handijatno 3, retno asih setyoningrum4 1,2 department of medical microbiology, faculty of medicine, universitas airlangga, surabaya, indonesia. 3 faculty of veterinary, universitas airlangga, surabaya, indonesia. 4 department of pediatrics, faculty of medicine, universitas airlangga, surabaya, indonesia. corresponding author : rebekahsetiabudi@gmail.com abstract this study was intended to invent a simpler and more affordable method to establish diagnosis on tuberculosis (tb) and latent tuberculosis infection (ltbi). similar to “quantiferon tb gold in tube” (qft-git) and t.spot.tb methods, the researchers also utilized “early secreted antigenic target 6kda” (esat-6) and “cultur filtrate protein 10kda” (cfp-10) proteins to be induced on the specimen. esat-6 and cfp-10 are commercial products used to induce interferon gamma (inf-γ) which were to be read using sophisticated and expensive equipment. this study was intended to conduct an analysis on effective cocktail protein modification, i.e. esat-6, cfp-10 and ag85a/b/c, with high validity to detect cellular immunity activity through in vitro examination on peripheral blood monocyte cells of tuberculosis-suspected patients or patients with latent tuberculosis infection. peripheral blood monocyte cells (pbmcs) activity on children tuberculosis patient or latent tuberculosis infection (ltbi), adult tuberculosis patient or ltbi, which induced by cocktail protein modification and not induced, were analyzed microscopically. the activity of pbmcs on children and adult tuberculosis patient or ltbi induced by rd1 secretory proteins: esat-6, cfp-10, ag85a/b/c was higher compared to pbmcs which had not been induced by the secretory proteins. cellular debris and monocyte cells with abnormal shapes were found on pbmcs which had been induced by rd1 secretory proteins at 8th day after culture. key words : cellular immunity activation, region of difference 1 (rd1) complex proteins stimulation, virulence markers, mycobacterium tuberculosis, diagnosis on latent tuberculosis infection (ltbi). abstrak penelitian ini bertujuan menemukan inovasi metode yang lebih sederhana dan terjangkau dalam hal biaya. hampir serupa dengan qft-git dan t-spot.tb assay, penelitian ini juga akan menggunakan protein “early secreted antigenic target 6kda” (esat-6) dan “cultur filtrate protein 10kda” (cfp-10) untuk induksi pada spesimen. kedua produk komersial tersebut, esat-6 dan cfp-10, telah digunakan secara komersial untuk menginduksi interferon gamma (inf-γ) yang kemudian akan dibaca dengan alat yang canggih dan berbiaya tinggi. studi ini bertujuan melakukan analisis modifikasi cocktail protein yang efektif, yaitu esat-6, cfp-10 and ag85a/b/c, dengan validitas tinggi untuk deteksi aktivitas imunitas seluler pada uji in vitro kultur sel monosit darah tepi dari pasien dengan suspek tuberkulosis atau infeksi tuberkulosis laten. aktivitas sel monosit peripheral blood monocyte cells (pbmcs) pada pasien tuberculosis aktif/tuberculosis laten pada anak/tuberculosis dewasa/ltbi yang diberi induksi dengan modifikasi cocktail protein dan yang tidak diberi induksi, dianalisis secara mikroskopis . aktivitas sel-sel monosit pbmcs dari pasien tuberculosis aktif/tuberculosis laten pada anak/tuberculosis dewasa/ltbi yang diberi induksi campuran protein sekretorik rd-1 : esat-6, cfp-10, ag85a/b/c lebih tinggi dibandingkan dengan sel-sel monosit pbmcs dari pasien tuberculosis aktif/tuberculosis laten pada anak/tuberculosis dewasa/ltbi yang tidak diberi induksi campuran protein sekretorik rd-1 : esat-6, cfp-10, ag85a/b/c. terdapat beberapa debris sel dan bentukan 13setiabudi, et al.: cellular immunity activation method by stimulating rd1 complex proteins abnormal dari sel monosit pada kultur hari kedelapan dari sel-sel monosit pbmc pasien tuberculosis aktif/tuberculosis laten pada anak/tuberculosis dewasa/ltbi yang diberi induksi campuran protein sekretorik rd-1 : esat-6, cfp-10, ag85a/b/c. kata kunci: aktivasi imunitas seluler, stimulasi protein kompleks rd-1, marka virulensi, mycobacterium tuberculosis, diagnosis infeksi tuberculosis laten. introduction tuberculosis (tb), an infectious contagious disease caused by mycobacterium tuberculosis still becomes one of global health issues especially in developing countries. world health organization estimated that almost a third of world population had ever infected by tb with 8.7 million new cases found and annual mortality rate as much as 1.4 million.1,2 according to indonesian ministry of health, indonesia positioned the fourth rank among countries with highest tb cases and pulmonal tb had infected many individuals at productive age and become the second highest cause of death in indonesia.3 the burden of tb in indonesia is increasing as the increase of new resistant tb cases found in indonesia. the new tb cases are resistant to standard medication regiment, such as multi drug resistant tb/mdr-tb, followed by emerging extended drug resistant tb/xdr-tb, and extreme extended drug resistant tb/xxdr-tb. the load of tb in indonesia becomes greater as the emerge of comorbid tb cases with human immunodeficiency virus (tb-hiv).1,3 immunocompromised condition caused by hiv might improve the risk of tb infection, reactivation of dormant mycobacterium tuberculosis on latent tuberculosis infection (ltbi) patients, and mortality rate caused by tb.1,4,5 who estimated about 10% of ltbi could develop to tb.2,6 therefore, tuberculosis was the main opportunistic infection in tb endemic areas, including indonesia.2,4 lower new tb cases found compared to expectancy rate of tb cases indicated that there were many tb cases which is happening on communities remained unidentified and has not been covered by tb governance.7 one of the causes of this problem was inaccurate and improper implementation of tb diagnosis establishment in indonesia. in order to establish diagnosis on tb, the patient should undergo laboratory examination in health care institutions. however, many people living in remote areas in indonesia found difficulties in accessing health facilities due to distance and length of time to be taken to reach health care centers. this problem became harder because of bad roads, limited means of transportation, limited electricity availability and coverage, limited health care facility, and low quality and quantity of human resources owned by health care facilities in indonesia.3 the first important step to be taken in conducting an effective and efficient tb prevention effort is improving case finding by applying proper method and establishing accurate diagnosis on tb. a quick and accurate tb diagnosis is the foundation in determining adequate medication.2 quick and accurate detection and identification on tb infection enables quick and adequate medication given to the patient in order to prevent pulmonal tissue damage and transmission of the disease. however, confirming diagnosis on tuberculosis was not easy, especially on primary tuberculosis cases, extra-pulmonal tuberculosis cases (i.e. pleural tuberculosis, cerebrospinal fluid (csf), pericardial tuberculosis, and ascetic tuberculosis), tb on children, and tb-hiv co-infection cases. confirming tb diagnosis becomes more difficult as the improvement of non-tuberculous mycobacteria (ntms) infection prevalence.8 common methods to diagnose tb such as through microscopic examinations on dyed acid fast bacilli (afb) and specimen culture require sophisticated health facility and skilled health facility operator. these methods also possess some disadvantages. microscopic examination on dyed afb has limited sensitivity and specificity while specimen culture takes too long to produce its results. the results of specimen culture can only be achieved 2-8 weeks later.8 tuberculin skin tests (tsts) have been used worldeide for more than a century as an aid in diagnosing both ltbi and active tuberculosis but a valid tsts requires proper administration by the mantoux method with intradermal injection of 0,1ml of tuberculin-purified protein derivative (ppd) into the volar surface of the forearm. in addition, patients must return to a health-care provider for test reading, and inaccuracies or bias can exist in reading the test.9 the most recent method, interferon gamma release assays (igra) is quite promising.7 this method offers accurate sensitivity and specificity in shorter time period.10,11 however, this method is still quite expensive and requiring sophisticated instruments making this method quite hard to be applied in health care centers in indonesia. a quick, accurate, and affordable method to diagnose active tuberculosis infection and latent tuberculosis infection (ltbi) becomes an urgent need.12 this study was conducted to invent a simpler and more affordable method in diagnosing tuberculosis infection and latent tuberculosis infection (ltbi). similar to qft-git and t-spot.tb assay, in this study, the researchers use esat-6 and cfp-10 proteins to be induced into patient peripheral blood monocyte cells (pbmcs) specimen.13,14 esat-6 and cfp-10 are commercial proteins which are used to induce interferon gamma (inf-γ) and the result will be examined using sophisticated and expensive equipment.15,16 different from previous studies, in this study the researchers do not only use esat-6 and cfp-10 proteins but also use 14 indonesian journal of tropical and infectious disease, vol. 6. no. 1 january–april 2016: 12−18 other proteins from region of difference 1 (rd1) protein family, such as ag85a/b/c to be induced into suspected tb patient and ltbi patient’s pbmcs suspension through in vitro manner.17,18 specific proteins from rd1 family can be found on all virulent strains and all clinical isolation of mycobacterium tuberculosis and mycobacterium bovis.19,20 rd1 protein family perform enzymatic function, namely to metabolize lipid on mycobacterium tuberculosis cell wall.21 these proteins were associated to virulence and immunogenicity of mycobacterium tuberculosis.22,23 after being induced, the monocyte cells undergo microscopic examination under light microscope to examine the activation of monocyte cells. material and method this study was categorized as laboratory experimental research. this study was conducted by comparing treatment groups consisting of peripheral blood monocyte cell (pbmcs) culture sample collected from tb-suspected patients and ltbi patients which had been induced by rd1 secretory protein compounds and control group consisting of monocyte cell which had not received treatment (i.e. rd1 secretory protein induction). pbmcs suspension samples were collected from children tb patients/ltbi,24,25,26 and adult tb patients/ltbi based on random consecutive method.27,28 the samples were collected by collecting 5-10 ml median cubital vein blood based on vena puncture method using syringe. the collected blood was put into flasks containing heparin anti-coagulant. the flasks were shaken slowly to mix the blood with the anti-coagulation agent and preventing the blood from coagulation. pbmcs preparation was conducted based on ficoll-histopaque 1077 technique. pbmcs culture was incubated for 7 to 10 days enabling the monocyte cells to differentiate into macrophage. this process produced 105 macrophages in each well. at the 4th day, treatment on pbmcs samples was conducted namely by inducing esat-6, cfp-10, ag85a/b/c protein compounds into several wells while the viability of pbmcs was observed using inverted microscope and giemsa colorization. rd1 secretory protein compounds (esat-6, cfp-10, and ag85a/b/c) were prepared by culturing mycobacterium tuberculosis h37rv on lowenstein jensen medium. the culture was incubated for 3-4 weeks enabling the culture to reach its logarithmic phase. 1 ose of mycobacterium colony was collected and put into 10 ml middle brook 7h9 medium which had been prepared before. the suspension was incubated in co2 incubator at 37 °c for 2-3 weeks with loosened cap. after 2-3 weeks incubation period, the flasks containing mycobacterium tuberculosis cultured in middle brook 7h9 medium were centrifuged for 30 seconds-1 minute until the mixture became homogenous. the mixture was rested for an hour until sedimentation formed. 200 μl supernatant was collected and used as “treatment” (tx) in this study. results and discussion this study was conducted from november 2014 to february 2015. the samples of this study consisted of peripheral blood monocyte cells (pbmcs) samples collected from healthy patients with negative tuberculin skin test (tst) result (k2), and pbmcs samples collected from child tuberculosis patients/ltbi, and adult tuberculosis patients/ltbi (k1). treatment (tx) was given to several k1 samples at the fifth day after incubation. the results were compared with k1 samples which had not received treatment. pbmc culture was cultivated daily by feeding (i.e. changing the medium daily). microscopic examination was conducted at the 8th day. table 1. results of peripheral blood monocyte cells activity examination result of examination k2 k1 (without tx) k1 (receiving tx) amount of monocyte/100lp 11.56 49.22 80.33 note: k1: pbmcs samples collected from children tuberculosis patients/ltbi, and adult tuberculosis patients/ltbi. k2: pbmcs samples collected from healthy individual with negative tuberculosis infection. tx: treatment (esat-6, cfp-10, ag85a/b/c secretory protein compounds induction). table 2. results of peripheral blood monocyte cells qualitative analysis microscopic examination k2 k1 (without tx) k1 (receiving tx) cellular debris, abnormality in monocyte cells morphological appearance negative negative positive note: k1: pbmcs samples collected from children tuberculosis patients/ltbi, and adult tuberculosis patients/ltbi. k2: pbmcs samples collected from healthy individual with negative tuberculosis infection. tx: treatment (esat-6, cfp-10, ag85a/b/c secretory protein compounds induction). 15setiabudi, et al.: cellular immunity activation method by stimulating rd1 complex proteins figure 1. result of microscopic analysis on mycobacterium tuberculosis h37rv suspension preparation cultured in middle-brook 7h9 medium (zn, 100x). acid fast bacilli are identified as red thin bacillus. mycobacterium tuberculosis h37rv suspension cultured in middle-brook 7h9 medium, was stained by ziehl nielsen staining for detection. it is seen that the morphology of mycobacterium tuberculosis is red thin rod/bacillus.29 figure 2. patient pbmcs culture preparation at the 5th day (before receiving treatment) (giemsa coloration, 10x): monocyte cell started growing (indicated by dark blue nucleus with round structure). pbmcs was cultured and incubated for 7 to 10 days enabling the monocyte cells to differentiate into macrophage. but we do the feeding of the pbmcs culture day by day to keep them alive. figure 3. patient pbmcs culture at the 5th day (giemsa coloration, 100x) before treatment: dark blue kidneyshaped monocyte nucleus started developing into macrophage. monocyte cell start to become a macrophage by developing the pseudopodia from its cellular membrane. figure 4. patient pbmcs culture at 8th day (after treatment) (giemsa, 100x): monocytes are interacting with macrophage. after treatment, there were a lot of active macrophages formed from monocytes. it is seen that the two macrophages is active and did the engulfment. 16 indonesian journal of tropical and infectious disease, vol. 6. no. 1 january–april 2016: 12−18 figure 5. patient pbmcs culture preparation at 8th day (after treatment) (giemsa, 100x): monocyte-lymphocyte cells are interacting with m. tuberculosis infected macrophage. after treatment, there were many monocyte and lymphocyte cells came to interact with macrophages which had been infected by mycobacterium tuberculosis. figure 6. p a t i e n t m a c r o p h a g e p r e p a r a t i o n a t 8 t h d a y (after treatment) (giemsa, 100x): macrophage nuclei undergoing pyknosis (probably caused by autophagy). after treatment, some of macrophages became pyknosis. pyknosis is the irreversible condensation of chromatin in the nucleus of a cell undergoing necrosis or apoptosis. interaction between macrophage and mycobacterium tuberculosis (i.e. the role of macrophage as response made by host) was very important in tuberculosis infection. complement receptors (cr1, cr2, cr3, and cr4), mannose receptors (mr), and other molecular receptors on the surface of the cells played significant role in binding the microorganism to the phagocytes. interaction between phagocyte molecular receptors and mycobacteria might be mediated by lipoarabinomannan (lam), glycoprotein found on the surface of mycobacteria. prostaglandin e2 (pge2) and interleukin 4 (il4), cytokines produced by t-helper 2 (th2) cells, expression regulation, the function of mr and cr receptors, and interferon γ (inf-γ) could reduce receptors expression resulting in reducing the ability of mycobacteria to attach to macrophage cells. surfactant proteins, cd14 receptors, and scavenger receptors also functioned in mediating mycobacteria attachment.30 microorganisms underwent phagocytosis were to be degraded through hydrolysis at acid condition after phagolysosome fusion. this process indicated a significant antimicrobial mechanism performed by phagocyte cells. meena ls and rajni (2010) proposed a hypothesis stating that phagolysosome fusion inhibition referred to a mechanism in which mycobacterium tuberculosis survived in macrophage cell.31 previous studies reported that mycobacterial sulphatides–a derivative of multiacylatedtrehalose 2-sulphatehad an ability to inhibit phagolysosome fusion.32,33 previous in vitro studies showed that mycobacterium tuberculosis produced a huge amount of ammonia which could be the factor affecting this inhibition process.34,35 there were several functions of macrophage antimicrobial effectors including improving reactive oxygen intermediates (roi), reactive nitrogen intermediates (rni), and other mechanisms mediated by cytokines.30 hydrogen peroxide (h2o2), one of roi produced by macrophage through oxidative reaction was identified as the first molecular effectors affecting mycobactericidal effect of mononuclear phagocyte cells. previous laboratory researches showed that mycobacterium tuberculosis infection might induce accumulation of macrophage on pulmonal tissue and h2o2 production. however, h2o2 production improvement by alveolar macrophage cells was not specific on tb infection. moreover, alveolar macrophage cells produced less h2o2 compared to blood monocyte cells.4 through interferon gamma (inf-γ) and tumor necrosis factor alpha (tnf-α), phagocyte cells produced nitric oxide (no) and other rni through inducible nitric oxide synthase (inos2) using l-arginine as substrate. the significance of these toxic nitric substances as host immune response against mycobacterium tuberculosis had been proven in in vitro examination, especially by using murine.30 another mechanism as the result of interaction between macrophage cells and mycobacterium tuberculosis was antimicrobial effect mediation by ifn-γ and tnf-α. previous reports indicated human ifn-γ macrophage effect on mycobacterium tuberculosis replication was varied from inhibition to enhancement. 1,25-(oh)2d3 itself (or combined with ifn-γ and tnf-α) might activate macrophage to inhibit and kill mycobacterium tuberculosis inside human body.36 other potential mechanism associated with macrophage defense response to mycobacterium tuberculosis was 17setiabudi, et al.: cellular immunity activation method by stimulating rd1 complex proteins apoptosis or “programmed cell death. lee et al (2009) showed that apoptosis by macrophage might reduce the viability of mycobacterium tuberculosis.37 until recently, biomolecular processes taking place in macrophage after mycobacterium tuberculosis infecting the host andhow the bacteria survive on these processes are still being studied.38 this study is attempted to compare the monocyte cells of healthy individuals and tb patient monocyte cells and tb patient monocyte cells which have been induced by esat-6, cfp-10, and ag85a/b/c secretory protein compounds through microscopic examination. the results indicated improvement on monocyte cell activity of tb patient peripheral blood sample after secretory protein induction compared to the activity of tb patients which had not received secretory protein compounds. the result also showed that monocyte cell activity of tb patient was higher than healthy individual (control group). the result also indicated abnormal monocyte cells morphological appearance of tb patient monocyte cells after receiving esat-6, cfp-10, and ag85a/b/c induction. the abnormal appearance was probably caused by macrophage/monocyte cells undergoing autophagy. conclusion monocyte cells activity of child tuberculosis patient, children with ltbi, and adult tuberculosis patient was higher than healthy individual monocyte cells activity. monocyte cells activity of child tuberculosis patient, children with ltbi, and adult tuberculosis patient receiving treatment (i.e. rd1 secretory proteins induction) was higher compared to monocyte cells which did not receive treatment. cellular debris and abnormal monocyte cell appearance were found at 8th day examination on child tuberculosis patient, children with ltbi, and adult tuberculosis patient monocyte cells sample after receiving rd1 secretory proteins (esat-6, cfp-10, and ag85a/b/c) induction. acknowledgement the writers would thanks to lppm unair, lpt unair, and indonesian ministry of research, technology, and higher education (kemenristek dikti ri) for support and assistance during the preparation of this study. references 1. corbett, e.l., watt, c.j., et al., 2003. the growing burden of tuberculosis:global trends and interactions with the hiv epidemic. arch. intern. med. 163 (9), 1009–1021. 2. world health organization. global tuberculosis report. available at, http://www.who.int/tb/publications/global_report/gtbr12_ executivesummary.pdf;2012 [accessed on 30.04.13]. 3. departemen kesehatan ri. riset kesehatan dasar 2007. jakarta: 2008 4. raja a. 2004. immunology of 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2005;174:3570-9 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. 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 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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. 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, 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. vol. 10 no. 1 january–april 2022 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ original article characteristics of chronic sinusitis based on non-contrast ct scan at the ent-head and neck surgery polyclinic of regional general hospital dr. zainoel abidin banda aceh teuku husni t.r1, teuku romi imansyah putra2*, hesti anandini sariningrum3, dhiatama endalif3 1faculty of medicine, syiah kuala university/ent-head and neck surgery dept-faculty of medicine syiah kuala university/rsud dr. zainoel abidin, banda aceh, indonesia 2department of parasitology faculty of medicine, syiah kuala university, banda aceh, indonesia 3faculty of medicine, syiah kuala university, banda aceh, indonesia received: 10th february 2022; revised: 8th march 2022; accepted: 20th march 2022 abstract chronic sinusitis is a long-term infl ammation that occurs in the nasal and paranasal mucosa for 12 weeks. non-contrast ct scan is gold standard in diagnosing chronic sinusitis. this study aims to determine the characteristics of chronic sinusitis based on non-contrast ct scan at the ent-head and neck surgery polyclinic of rsudza banda aceh in 2019. this research was a descriptive study with retrospective data, medical record. the sample of this study was taken by consecutive sampling method in october 2020 and obtained 111 samples. the results showed that most patients with chronic sinusitis were 30-39 years), as many as 42 people (37.8%). most of the sexes suff ering from chronic sinusitis were women, as many as 59 people (53.2%). based on the non-contrast ct scan, the location of the sinuses most aff ected was the maxillary sinuses, as many as 110 people (99.1%). the number of sinuses that were most aff ected was single sinusitis, which was 58 people (52.3%). most patients with chronic sinusitis without polyps were found, as many as 89 people (80.2%). the most common anatomical variation found was septal deviation as many as 25 people (22.5%). the conclusions in this study indicate that women, late adulthood, maxillary sinus, single sinusitis, chronic sinusitis without nasal polyps, and septal deviation are characteristics of chronic sinusitis patients based on non-contrast ct scan. keywords: chronic sinusitis, non-contrast ct scan, rsudza, aceh abstrak sinusitis kronis merupakan infl amasi jangka panjang yang terjadi pada mukosa nasal dan paranasal selama 12 minggu. pemeriksaan penunjang gold standard dalam menegakkan diagnosis sinusitis kronis adalah ct scan tanpa kontras. penelitian ini bertujuan untuk mengetahui karakteristik penderita sinusitis kronis berdasarkan gambaran ct scan tanpa kontras di poliklinik tht-kl rsudza banda aceh pada tahun 2019. jenis penelitian ini adalah penelitian deskriptif dengan rekam medis. sampel diambil dengan teknik consecutive sampling dan didapatkan 111 sampel. hasil penelitian ini mendapatkan bahwa penderita sinusitis kronis paling banyak dialami pada umur 30-39 tahun yaitu sebanyak 42 orang (37.8%). jenis kelamin yang paling banyak menderita sinusitis kronis yaitu perempuan sebanyak 59 orang (53.2%). berdasarkan gambaran ct scan tanpa kontras, letak sinus yang paling banyak terkena yaitu sinus maksilaris sebanyak 110 orang (99.1%). jumlah sinus yang paling banyak terkena yaitu single sinusitis sebanyak 58 orang (52.3%). penderita sinusitis kronis tanpa polip nasi paling banyak ditemukan yaitu sebanyak 89 orang (80.2%). variasi anatomi yang paling banyak ditemukan adalah deviasi septum nasi yaitu sebanyak 25 orang (22.5%). kesimpulan pada penelitian ini menunjukkan bahwa perempuan, usia dewasa akhir, sinus maksilaris, single sinusitis, sinusitis kronis tanpa polip nasi, dan deviasi septum merupakan karakteristik dari penderita sinusitis kronis berdasarkan gambaran ct scan tanpa kontras. kata kunci: sinusitis kronis, ct scan tanpa kontras, rsudza, aceh* corresponding author: teukuromiimansyahputra@unsyiah.ac.id ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 56 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 55–61 how to cite: husni, t., r., t., putra, t. r. i., sariningrum, h. a., endalif, d. characteristics of chronic sinusitis based on non-contrast ct scan at the ent-head and neck surgery polyclinic of regional general hospital dr. zainoel abidin banda aceh. indonesian journal of tropical and infectious disease, 10(1), p. 55–61, apr. 2022. introduction sinusitis, better known as rhinosinusitis, is an infl ammation that occurs in the paranasal sinuses.1 the cause can be due to infection, allergies, or autoimmune problems. in some case studies, viral infection was the most common cause and resolved within 10 days.2 sinusitis was classifi ed by duration as acute if less than four weeks and chronic if more than 12 weeks with or without acute exacerbations.3 chronic sinusitis has two or more the following symptoms, such as nasal congestion, nasal discharge (anterior/posterior nasal drip), facial tenderness or facial pain, and a decreased sense of smell.4 the most common risk factor is allergies. while others are asthma, pollution and smoke exposure, immune defi ciency, and septal deviation.5 sinusitis and chronic sinusitis are the most common public health problems worldwide.4 on 107 million people who suff er from chronic sinusitis in mainland china in 2015 showed that chronic sinusitis is common among people with certain medical conditions, including allergic rhinitis, asthma, chronic obstructive pulmonary disease, and gout. the prevalence of men (8.79%) is higher than women (7.28%). the independent risk factors for chronic sinusitis were active smokers and passive smokers. therefore, it is necessary to develop health promotion related to chronic sinusitis, especially in developing countries.5 according to the 2007 national health interview survey data, sinusitis is one of the ten most diagnosed diseases in the united states.6 in europe, about 10.9% of people have symptoms of chronic sinusitis.7 in canada, 5% of the general population suff ers from chronic sinusitis.8 in indonesia, based on data from the ministry of health of the republic of indonesia in 2003, there were 102,817 sinus patients undergoing outpatient treatment, while nasal and sinus disease was ranked 25th out of 50 major disease patterns. a study by amelia et al.7 in 2017 showed 73 patients with chronic sinusitis for one year at dr. mohammad hoesin palembang.7 the aceh provincial health profi le noted that sinusitis was ranked 11th out of the 20 most diseases for outpatients in aceh provincial hospitals in 2012 with 8,183 cases.9 a study by husni and pradista10 in 2012 at the dr. zainoel abidin hospital, banda aceh, indonesia showed that there were 33 suff erers of chronic sinusitis from october to december 2010.10 in establishing the diagnosis of chronic sinusitis, an objective examination is necessary because the symptoms that appeared could be non-specifi c.4 the essential examinations for sinusitis are anterior rhinoscopy, nasoendoscopy, and radiological imaging. radiological imaging involved paranasal sinuses x-ray, paranasal sinuses computed tomography (ct) scan, and magnetic resonance imaging (mri). the radiologic examination is often necessary to confirm chronic sinusitis.11 however, the ct scan of the paranasal sinuses is the gold standard in confi rming the diagnosis of chronic sinusitis.12 mucosal abnormalities, sinus ostium obstruction, anatomic variations, and nasal polyps can be depicted well by ct scan.3 however, the disadvantages of ct scan are its relatively high cost and the large radiation dose.13 this study was conducted at the regional general hospital dr. zainoel abidin (rsudza) banda aceh, a referral hospital in aceh. there has never been such a similar study before. based on the description above, we are interested to learn more about the characteristics of patients with chronic sinusitis based on ct scan images without contrast at the ent-head and neck surgery polyclinic, rsud dr. zainoel abidin, banda aceh, indonesia. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 57teuku husni t.r, et al.: characteristics of chronic sinusitis based on non-contrast ct scan materials and methods this descriptive study was conducted using retrospective data from medical records, describing age, gender, location of the aff ected sinus, number of aff ected sinuses, presence of nasal polyps, and anatomical variations based on non-contrast ct scan. this study was located at the regional general hospital dr. zainoel abidin banda aceh, precisely at the ent-head and neck surgery polyclinic and radiology installation. this study was held from may to december 2020, with data collection time from 23 september to 13 october, 2020. the population in this study were adults with symptoms of chronic sinusitis. the patients were treated at the ent-head and neck surgery polyclinic, rsud dr. zainoel abidin banda aceh in 2019. the sample in this study was patients with chronic sinusitis who met the inclusion and exclusion criteria. the sampling method of this study was using a non-probability side method or the consecutive sampling method. univariate analysis was used to obtain the frequency distribution and the percentage of the variables studied. results and discussion this study was conducted at the ent-head and neck surgery polyclinic and the radiology installation of rsud dr. zainoel abidin banda aceh, in september and october 2020. the number of outpatients who had symptoms of chronic sinusitis and went to the ent-head and neck surgery polyclinic rsudza in 2019 amounted to 146 people; however, there were 35 samples that could not be used because they did not meet the inclusion criteria, so that the total sample in this study amounted to 111 people with the following characteristics as shown in table 1. based on table 1, the majority of the respondents were aged 30-39 years among 42 people (37.8%). the results of this study are in accordance with the study conducted by julyanti that most chronic sinusitis occurs at the age of 31-40 years in a sample of 30 people (25.2%).17 a study by sogebi18 found that the age group of 31-45 years was often aff ected by chronic sinusitis using a sample of 48 people (33.6%).18 moreover, a study by pirzadeh et al.19 found chronic sinusitis mostly occurred in the age group of 30-39 years in a sample of 25 people (30.1%)19 adults are more involved in outdoor activities and more at risk of exposure to the allergens or pollution that may cause or exacerbate chronic sinusitis.20 table 2. characteristics of chronic sinusitis by gender gender n % male 52 46.8 female 59 53.2 total 111 100 based on table 2, 59 female patients (53.2%) had chronic sinusitis in line with the study by trihastuti et al.21 of 38 women (60.32%), compared to 25 men (39.68%).21 in a sample of 42 people, aritonang22 also found that more women (52.5%) suff er from chronic sinusitis.22 a study by pirzadeh et al.19 of 49 subjects (women 55.4%, men 44.6%)19 found women are more likely to have a high level of concern for health, thus, women visit health services more often.14 women were also more susceptible to infection and obstruction due to the small size of the sinus ostium.23 however, amelia et al.7 found that of 73 people, chronic sinusitis was more commonly found in men (43, 58.9%) than women (30, 41.1%) with the ratio of male and female with chronic sinusitis 1.4:1.7 men have more smoking habit and are more often exposed to pollution than women.23 table 1. characteristics of patients with chronic sinusitis by age age (year) n % 20-29 24 21.6 30-39 42 37.8 40-49 23 20.7 50-59 22 19.8 total 111 100 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 58 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 55–61 based on the non-contrast ct scan, the characteristics of patients with chronic sinusitis showed the location of the aff ected sinus, the number of aff ected sinuses, the presence of nasal polyps, and anatomical variations. the characteristics of patients with chronic sinusitis based on a non-contrast ct scan shown in table 3. table 3. distribution of aff ected sinuses location of the aff ected sinus yes no n % n % sinus frontalis 32 28.8 79 71.,2 sinus ethmoidalis 51 45.9 60 54.1 sinus maksilaris 110 99.1 1 0.9 sinus sphenoidalis 26 23.4 85 76.6 based on table 3, it was found that the location of the sinus that was most affected was the maxillary sinus in a sample of 110 people (99.1%). fadda and aversa explain that the maxillary sinus is the sinus that is most often involved in chronic sinusitis.15 kurniasih and ratnawati24 also found that the maxillary sinus was the most common sinus in a sample of 106 people (86.89%).24 enema job also remarked that the maxillary sinus was the most frequently involved sinus in a sample of 49 people (81.7%), followed by the ethmoid sinus in f 41 people (68.3%), frontal sinus in f 24 people (40%), and the least was the sphenoid sinus in 12 people (20%).25 the maxillary sinuses have an ostium that is located higher than the sinus base, thus the maxillary sinus drainage depends on the ciliary function. if an infection occurs, it will impair the ciliary function and interfere with sinus drainage which will eventually lead to chronic sinusitis.26 the inferior wall of the maxillary sinus is also adjacent to the roots of the 1st and 2nd molars, which can cause minor elevations or spots. protruding along the maxillary sinus. the anatomic relationship of the maxillary molars to the maxillary sinus may facilitate the development of periapical or periodontal odontogenic infection within the maxillary sinus. maxillary sinus may facilitate the development of periapical or periodontal odontogenic infection within the maxillary sinus.27 table 4. distribution of the number of sinuses aff ected sinuses aff ected n % single sinusitis 58 52.3 multisinusitis 35 31.5 pansinusitis 18 16.2 total 111 100 table 4 shows the number of sinuses aff ected was single sinusitis, which was 58 people (52.3%). makusidi found that single sinusitis often occurred in 86 people (58.9%).28 nova sitinjak also found 104 cases of single sinusitis (63.8%).29 however, multazar et al.30 stated that chronic sinusitis was more common in multiple sinuses (multisinusitis) in a sample of 22 people (88%). this may be related to the osteomental complex (kom) as the fi nal route of drainage from the frontal sinus, maxillary sinus, and ethmoidal sinus. thus, if there are some disturbances in kom, such as infl ammation or edema, this will allow the occurrence of chronic sinusitis in several sinuses (multisinusitis).16,30 table 5. distribution of nasal polyps nasal polyps n % chronic sinusitis with nasal polyps 22 19.8 chronic sinusitis without nasal polyps 89 80.2 total 111 100 31 32 with nasal polyps.32 however, rowe33 also stated that nasal polyps are only involved in 15-20% of patients.33 chronic sinusitis without nasal polyps is more common than chronic sinusitis with nasal polyps. chronic sinusitis without nasal polyps is characterized by edema and cho et al. also stated that chronic sinusitis without nasal polyps is more common than chronic sinusitis based on table 5, it was found that chronic sinusitis without nasal polyps was more common than chronic sinusitis with nasal polyps in a sample of 89 people (80.2%). this is in accordance with benjamin et al who also found that 507 people (82%) had chronic sinusitis without nasal polyps while 111 people (18%) had chronic sinusitis with nasal polyps. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 59teuku husni t.r, et al.: characteristics of chronic sinusitis based on non-contrast ct scan infl ammation of the sinuses which can be caused by several factors, such as allergies, irritation, and infection, while chronic sinusitis with nasal polyps is characterized by a soft mass formed from the mucous membrane in the nasal cavity called nasal polyps. these polyps can become large enough to block the sinuses and cause sinusitis symptoms.34 the infl ammatory reaction in chronic sinusitis without nasal polyps is th1 and th2 mediated, whereas chronic sinusitis with nasal polyps is th2 dominant, which is characterized by high tissue eosinophilia. it is generally accompanied by an increase in tissue mast cells, innate lymphoid cells, immunoglobulin e, and th2 cytokines.35 chronic sinusitis without nasal polyps shows basal membrane thickening, goblet cell hyperplasia, subepithelial edema, and mononuclear cell infi ltration. meanwhile, chronic sinusitis with nasal polyps shows epithelial damage, edema, and a reduced number of blood vessels and glands.31 table 6. distribution of anatomical variations anatomical variation yes no n % n % septal deviation 25 22,5 86 77.5 konka bulosa 3 2.7 108 97.3 konka media paradoks 0 0 0 0 haller cell 0 0 0 0 agger nasi cell 0 0 0 0 onodi cell 0 0 0 0 table 6 shows that the most anatomical variation was the septal deviation among 25 people (22.5%). this is in accordance with shivakumar et al, that the most common anatomical variation was the septal deviation among 98 people (71%).36 moreover, ratnawati37 stated that septal deviation was the most common anatomical variation among 24 people (77%).37 aramani et al.38 also found that 40 people (74.1%) had a deviated septum and 18 people (33.3%) were chronic sinusitis patients.39 septal deviation is an anatomical variation that is often found and one of the predisposing factors for chronic sinusitis. the presence of nasal septal deviation increased airflow around the osteomental complex (kom), which can result in disruption of the mucociliary clearance process.5,40 ajmal41 found that c-shaped deviation was the type that caused the most chronic sinusitis of 62.5% of 150 patients with chronic sinusitis, while the s-shaped deviation can cause pansinusitis because the s-shaped deviation can block the fl ow of air in both noses.41 while concha bullosa is pneumatization that occurs in the concha media and can narrow the semilunar hiatus and block the infundibular drainage, resulting in sinusitis.42 the bullous conchae are small with a vertical height of less than 50% of the total median conchae as measured on a coronal ct scan. while the bullous conchae are said to be large if the vertical height is more than 50% with an increase in the volume of the media conchae. do santos43 found large bullous turbinates were more than small bullous cones in a sample of 222 people (80%).43 the paradoxical middle turbinate is a condition when the median turbinate bends laterally and can lead to narrowing of the com and chronic sinusitis.38 haller cells or infraorbital ethmoid cells are located between the maxillary and orbital sinuses. it can increase the risk of orbital injury during ethmoidectomy. haller cells potentially narrow the maxillary sinus ostium or ethmoid infundibulum which can obstruct the ostium.16 agger nasi cells are the most anterior ethmoidal cells that can narrow the frontal recess and block the frontonasal duct causing sinusitis.40 onodi cells are the rarest anatomical variation and can extend to the sphenoid sinus and surround the optic nerve. it is necessary to be careful in performing functional endoscopic sinus surgery.39 conclussions patients with chronic sinusitis mostly are aged 30-39 years and primarily women. based on a ct scan without contrast, the most aff ected sinus is the maxillary sinus. the number of sinuses aff ected in chronic sinusitis is single sinusitis. chronic sinusitis without nasal polyps is more common than chronic sinusitis with nasal polyps. the most anatomical variation is nasal septal ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 60 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 55–61 deviation. the number of patients with chronic sinusitis based on ct scan images without contrast in 2019 was 111 patients. acknowledgement the authors would like to thank fakultas kedokteran universitas syiah kuala, all staff members of ent-head and neck surgery polyclinic, rsud dr. zainoel abidin, banda aceh, and all staff members of radiology installation, rsud dr. zainoel abidin, banda aceh. conflict of interest the authors declare that there is no confl ict of interest. references 1. mustafa m, patawari p, shimmi sc, hussain ss. acute and chronic rhinosinusitis, pathophysiology and treatment. international journal of pharmaceutical science invention issn. 2015;4(2):30–6. 2. anon jb. upper respiratory infections. american journal of medicine. 2010;123(4 suppl.):16-s25. 3. rosenfeld rm, piccirillo jf, chandrasekhar ss, brook i, ashok kumar k, kramper m, et al. clinical practice guideline (update): adult sinusitis. otolaryngology head and neck surgery (united states). 2015; 152:1s39. 4. bachert c, pawankar r, zhang l, bunnag c, fokkens wj, hamilos dl, et al. icon: chronic 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orli. 2012. 31. benjamin mr, stevens ww, li n, bose s, grammer lc, kern rc, et al. clinical characteristics of patients with chronic rhinosinusitis without nasal polyps in an academic setting. journal of allergy and clinical immunology: in practice. 2019 mar 1;7(3):1010–6. 32. cho sh, kim dw, gevaert p. chronic rhinosinusitis without nasal polyps. journal of allergy and clinical immunology: in practice. 2016 jul 1;4(4):575–82. 33. rowe sm, hoover w, solomon gm, sorscher ej. cystic fibrosis. in: murray and nadel’s textbook of respiratory medicine. elsevier; 2016. p. 822-852.e17. 34. melbourne ent group. information for patients, families and carers chronic rhino-sinusitis (crs) chronic rhino-sinusitis overview. 2020. 35. laidlaw tm, buchheit km. biologics in chronic rhinosinusitis with nasal polyposis. vol. 124, annals of allergy, asthma and immunology. american college of allergy, asthma and immunology; 2020. p. 326–32. 36. senniappan s, raja k, tomy al, kumar cs, panicker am, radhakrishnan s. study of anatomical variations of ostiomeatal complex in chronic rhinosinusitis patients. international journal of otorhinolaryngology and head and neck surgery. 2018 aug 25;4(5):1281. 37. ratnawati lm, putu yupindra pradiptha i. anatomic variation of ct scan in chronic rhinosinusitis patients in sanglah provincial general hospital. biomedical and pharmacology journal. 2019;12(4):2083–6. 38. aramani a, karadi rn, kumar s. a study of anatomical variations of osteomeatal complex in chronic rhinosinusitis patients-ct findings. journal of clinical and diagnostic research. 2014;8(10): kc01–4. 39. tiwari r, goyal r. study of anatomical variations on ct in chronic sinusitis. indian journal of otolaryngology and head and neck surgery. 2014;67(1):18–20. 40. gupta ak, gupta b, gupta n, tripathi n. computerized tomography of paranasal sinuses: a roadmap to endoscopic surgery. clinical rhinology. 2012;5(1):1– 10. 41. ajmal m, usman n. relation between chronic sinusitis and deviated nasal septum. iosr journal of dental and medical sciences (iosr-jdms) e-issn. 2017;16(5):42–5. 42. wardani rs, wardhana a, mangunkusumo e, wulani v, senior ba. radiological anatomy analysis of uncinate process, concha bullosa, and deviated septum in chronic rhinosinusitis. vol. 47. 2017. 43. do santos zounon a, bidossessi vodouhe u, agai j-b, balde d, adjanohoun s, adjibabi w, et al. vignikinyehouessi. large cocha. 44. bullosa is a risk factor for chronic sinusitis: case control study. in: international journal of otorhinolaryngology [internet].2019. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 � 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. 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. 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 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 identifiers, comorbidities, and outcomes among covid-19 confirmed patients in banda aceh, indonesia budi yanti1,2* , t. zulfikar1,2, devi efrina1, rudi agustika1 1department of pulmonology and medical respirology, school of medicine, universitas syiah kuala, banda aceh, indonesia 2zainoel abidin teaching hospital, banda aceh, indonesia received: january 6th, 2022; revised: april 18th, 2022; accepted: july 8th, 2022 abstract coronavirus disease 2019 (covid-19) is a highly contagious disease with an increasing number of infections in indonesia. however, hypertension and diabetes are chronic diseases with high incidence in aceh, there is still limited information regarding the demographics and clinical data of covid-19 patients. this study aims to explain the clinical characteristics, comorbidities, and outcomes of covid-19 patients. a retrospective method was used to locate data from the medical record of covid-19 patients that were admitted to the hospital between june-october 2020. the characteristics demographics, clinical data on admission, and outcomes were extracted from the medical record. in order to determine the comorbid relationship, the chi-square test was used for the laboratory tests and clinical outcomes. a total of 120 patients were included, and more than half were male 80 (60%) with 41-60 years of age at most (51.2%). most of the patients had comorbid diabetes mellitus (40.5%), hypertension (28.9%), and chronic lung disease (8.3%). furthermore, most covid-19 was severe degrees 56 (46.3%). the patients with recovery are 92 (76.0%) and only 29 (24.0%) died. the neutrophilia, and comorbid had no relationship with the clinical outcome of covid-19 (p>0.05). the lymphopenia and degree of severity had relationship with clinical outcome (p> 0.05). diabetes melitus and hypertension are the most common comorbid reported in the covid-19 patients. the inflammation markers, such as lymphocytes, can be used as an early warning to increase awareness in treating patients with severe disease. keywords: clinical identifier; comorbidities; covid19; outcomes abstrak coronavirus disease 2019 (covid-19) merupakan penyakit yang sangat menular dengan jumlah infeksi yang terus meningkat di indonesia. hipertensi dan diabetes merupakan penyakit kronis dengan insiden yang tinggi di aceh, masih terbatasnya informasi mengenai demografi dan data klinis pasien covid-19. penelitian ini bertujuan untuk menjelaskan karakteristik klinis, penyakit penyerta, dan outcome pasien covid-19. metode retrospektif digunakan untuk mencari data dari rekam medis pasien covid-19 yang dirawat di rumah sakit antara juni-oktober 2020. karakteristik demografi, data klinis saat masuk, dan hasil diambil dari rekam medis. untuk menentukan hubungan komorbiditas, uji chi-square digunakan untuk uji laboratorium dan hasil klinis. sebanyak 120 pasien dilibatkan, dan lebih dari setengahnya adalah laki-laki 80 (60%) dengan usia paling banyak 41-60 tahun (51,2%). sebagian besar pasien memiliki penyakit penyerta diabetes mellitus (40,5%), hipertensi (28,9%), dan penyakit paru kronis (8,3%). selanjutnya, sebagian besar covid-19 adalah derajat berat 56 (46,3%). pasien yang sembuh sebanyak 92 (76,0%) dan yang meninggal hanya 29 (24,0%). neutrofilia, dan komorbiditas tidak memiliki hubungan dengan hasil klinis covid-19 (p >0,05). * corresponding author: byantipulmonologis@unsyiah.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0003-2932-0764 77 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: 76–82 limfopenia dan derajat keparahan memiliki hubungan dengan luaran klinis (p>0,05). diabetes mellitus dan hipertensi adalah penyakit penyerta yang paling umum dilaporkan pada pasien covid-19. penanda peradangan, seperti limfosit, dapat digunakan sebagai peringatan dini untuk meningkatkan kesadaran dalam merawat pasien dengan penyakit parah. kata kunci: covid19; komorbiditas; outcome; penanda klinis how to cite: yanti, b., zulfikar, t., efrina, d., agustika, r. clinical identifiers, comorbidities, and outcomes among covid-19 confirmed patients in banda aceh, indonesia. indonesian journal of tropical and infectious disease. 10(2). 76–82. aug. 2022. introduction coronavirus disease 2019 (covid-19) is a contagious disease caused by sars cov2 with increasing morbidity and mortality.1 indonesia is one of the countries affected by sars cov2.2 clinical symptoms appear varied, asymptomatically, mild and very severe symptoms. these trigger the difficulty of disease control in the community.3 due to the large variety of clinical manifestations of covid-19, and the real-time reversetranscription polymerase chain reaction (rrt-pcr) examination as the gold standard for covid-19 is not always available in various places. therefore, several studies are needed to describe the results of laboratory tests as a guide to determine the severity of the disease, prognosis, and clinical outcome. so, the laboratory examination results are valuable knowledge during the covid-19 pandemic.4 covid-19 patients with comorbidities such as hypertension and diabetes have a poorer prognosis, higher morbidity and mortality, and longer icu stay.5 diabetes mellitus was discovered to be higher in people with obesity, and hypertension was strongly associated with diabetes mellitus in indonesia.6 and even the prevalence of smoking, high blood pressure, and obesity in men are increasing in indonesia.7 although there have been many studies describing the clinical characteristics of patients in china and other countries,8,9,10 a little description of the clinical characteristics of covid-19 patients in indonesia is very significant. a retrospective study in china showed that men with a mean age of 56 were mostly infected, while men in italy with a mean age of 67.5 years were more infected.11 subsequently, there is significant differences between indonesia and other countries based on population demographics, comorbid and clinical outcomes of patients. therefore, this study aims to report on the clinical characteristics, comorbidities, and outcomes of covid-19 patients in banda aceh, indonesia. materials and methods setting study this is an observational analytical study with a retrospective method using medical data from the medical records of confirmed covid-19 patients. furthermore, this study was conducted at the dr. zainoel abidin general hospital banda aceh from october 2021. this study was admitted by institutional review board of the school of medicine, universitas syiah kuala, banda aceh (377/ea/fk-rsudza/2021) and national ethics commission the ministry of health of the republic of indonesia (#1171012p. data were collected from the medical records of patients diagnosed with covid-19 confirmed by positive reverse transcriptase pcr examination from nasal swabs, which were analyzed molecularly in the hospital virology unit according to who.12 variable definitions this study collected demographic data (gender, age, and occupation), and clinical data (clinical symptoms, comorbidities, chest x-rays, laboratory tests). basic demographic, comorbid and clinical symptoms are fully 78 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 budi yanti, et al. clinical identifiers, comorbidities, and outcomes among covid-19 documented in the medical record. the definition of clinical outcome is a patient that has completed his treatment in the hospital, either recovered or died. the severity of the disease is divided only into four categories based on the who guidelines, namely mild, moderate, and severe, and critically ill or very severe.13 statistic analysis exploratory statistical analysis was performed to determine characteristic variables of potential patients, clinical symptoms, laboratory results, and clinical outcomes. the association between comorbid, laboratory results and clinical outcomes was assessed using the chi-square test. for statistical analysis, lymphopenia when the lymphocyte percentage is < 20, absolute lymphocyte count (alc) decreases when < 1500, neutrophil lymphocyte ratio (nlr) decsreases when < 13.5, and increases > 13.5. neutrophils percentage decrease when the neutrophil percentage is < 55 and increases > 70. the significance for all data analyzed was α = 0.05. all statistical analyzes were performed using spss (statistical package for social sciences) for windows version 25.0 (ibm spss inc., usa). results and discussion this study collected medical record data for 120 confirmed covid-19 patients that received diagnostic examinations and treatment. the majority were male 80 (66%) with the age of 41-60 years at most (51.2%) and 31.4% of the patients were over 60 years old. most of the patients had comorbid diabetes mellitus (40.5%) and hypertension (28.9%), only 8.3% patient had chronic lung disease. immunodeficiency cases such as human immunodeficiency virus (hiv), systemic lupus erythematosus (sle), and congenital abnormalities like congenital pulmonary anomaly and cardiac malformation were absent. fever is the most common clinical symptom found in treated 89 patients (73.6%). almost all chest x-rays have bilateral pneumonia (95.9%). moderate covid-19 was mostly reported in 32 patients (26.4%) and severe degrees in 56 (46.3%) patients. all patients were well treated, and most of the patients treated out of the hospital in stable condition (recovery) were 209 (87.1%) and only 31 (12.9%) patients died. (table 1). table 1. baseline characteristics characteristic demographic n % age < 20 years 0 0.0 20 40 years 21 17.4 41 60 years 62 51.2 >60 years 38 31.4 gender male 80 66 female 41 34 comorbid hypertension yes 35 28.9 no 86 71.1 diabetes mellitus yes 49 40.5 no 72 59.5 chronic lung disease yes 10 8.3 no 111 91.7 fever yes 89 73.6 no 32 26.4 cough yes 96 79.3 no 25 20.7 shortness of breath yes 79 65.3 no 42 34.7 anosmia yes 15 12.4 no 106 87.6 chest radiograph pneumonia bilateral 116 95.9 without pneumonia 5 4.1 sore throat yes 30 24.8 no 91 75.2 headache yes 8 6.6 no 113 93.4 anorexia yes 76 62.8 no 45 37.2 degree of severity mild 5 4.1 moderate 32 26.4 severe 56 46.3 very severe 28 23.2 outcome 28 23.1 recover 92 76.0 died 29 24.0 absolute lymphocyte count (alc) normal 39 32.2 decrease 82 67.8 netrophyl lymphocyte ratio (nlr) high 24 19.8 low 97 80.2 total 121 100.0 79 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: 76–82 a number of studies showed that the incidence of sars-cov and sars-cov-2 infection is higher in men than in women.14,15 based on the history of the influenza epidemic, there is a variable risk of gender, where men are more susceptible to infection than women.16 men generally had worse clinical outcomes and higher mortality rates in the sars and mers epidemics.14 likewise, a number of studies showed that they are at greater risk of being infected with covid-19 and most are hospitalized.17 some of the mechanisms that put men at a higher risk of contracting the disease than women are gender hormones and gene xrelated activity, which play a role in modulating innate and adaptive immune responses to viral infections.18 the main route of sars-cov-2 infection is via the ace2 receptor, and therefore the biological differences in the angiotensin-converting enzyme 2 (ace2) receptor also play a role, with men shown to have more ace2 expression in the circulation and lungs than women.17 this is consistent with the findings that most men infected with covid-19 received treatment in the hospital. diabetes is one of the top causes of morbidity and mortality, and relationship with infection has long been allowed. infections such as pneumonia are generally seen in type 2 diabetes mellitus (t2dm) people. china and italy reported that older patients with diabetes were at higher risk for more serious covid-19 and mortality.19,20 patients with comorbid cardiovascular disease are at higher risk of severe symptoms when infected with sars-cov-2. hypertension is a major risk factor related with poor clinical outcomes in covid-19.21 according to this study, it was shown that diabetes mellitus and hypertension was the most common comorbid disease but it was not significantly related to the clinical outcome (p <0.05). this happens because the prevalence of diabetes mellitus and hypertension is quite high in indonesia and half of the hypertensive patient are unaware of the dangers of this comorbid.7 it is not unexpected that immunity and metabolism have coevolved in such proximity. cellular stressors in diabetes mellitus, such as endoplasmic reticulum stress, oxidative stress, and others, might exacerbate inflammatory responses. in covid-19, when sars-cov-2 infects diabetic patients with the aforementioned cellular stressors, the decreased immune response may result in significant lung and other pathology and frequently results in mortality.22 the worse outcomes in covid-19 patients can be partially attributed to hypertension, which plays a significant role in the control of raas, inflammation, immunological responses, and the gastrointestinal tract. because of this, patients who have both hypertension and sars-cov-2 infection may suffer a double blow.23 most patients admitted to the hospital had a complaints of, coughing 96 (79.3%), and shortness of breath 79 (65.3%), and only 8 patients had a headache. the laboratory examinations results at the hospital showed that the mean levels of leukocytes were 11.276 x 103/ul, lymphocytes percentage 14.6, neutrophils percentage 81.2, nlr 6.7, and alc 1375 (table 2). 80 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 budi yanti, et al. clinical identifiers, comorbidities, and outcomes among covid-19 table 2. clinical and laboratory findings on admission parameter min. max. mean sd systolic (mmhg) 14.00 194.00 130.4628 22.32974 diastolic (mmhg) 48.00 275.00 79.9669 20.60014 heart rate (x/i) 71 130 95.13 11.272 respiratory rate (x/i) 18 40 25.51 4.384 sao2 without o2 (%) 40 99 87.69 9.930 leukocytes x 103/ul ( normal range 4.0-10.0) 1100 34400 11276.03 6576.904 lymphocyte percentage, normal range 25-40 2 47 14.60 12.128 neutrophyl lymphocyte ratio (nlr) .00 47.50 6.7660 9.58457 neutrofil percentage, normal range 50-70 26.00 126.00 81.2231 15.17591 absolute lymphocyte count (alc) 77.00 7082.00 1375.4380 1109.49531 systolic (mmhg) 14.00 194.00 130.4628 22.32974 diastolic (mmhg) 48.00 275.00 79.9669 20.60014 a meta-analysis of patients with covid19 had a fever as the most common initial symptom (88.8%), dry cough (68%), and fatigue (33%). other symptoms reported were productive cough (28.5%), shortness of breath (17%), muscle aches (14.4%), sore throat (11.4%), and headache (10.2%). during the first week of the virus phase when the body becomes infected, fever is a manifestation of the body's immune response to virus replication.24 in line with the findings, it shows that cough and fever are the clinical symptoms most complained of by covid-19 patients. the results show that most cases of covid19 are hospitalized in severe cases and the majority return to home in stable condition. this is most likely due to increased health worker awareness about signs, symptoms, early diagnosis, and identification of disease more quickly to reduce the severity of the disease that may occur.25 therefore, this had an impact on the clinical outcome as most of the patients treated were able to return home in a stable condition. neutrophylia and comorbid had no significant relationship with the clinical outcome of patients (p >0.05), and there is a significant relationship between lymphocyte and the degree of severity with clinical outcome (p <0.05). (table 3). table 3. the relationship of clinical characteristics and outcomes clinical outcome t o ta l p died recover hypertension no 17 19.8% 69 80.2% 86 0,09 yes 12 34.3% 23 65.7% 35 diabetes mellitus no 13 18.1% 59 81.9% 72 0.065 yes 16 32.7% 33 67.3% 49 ppok no 28 25.2% 83 74.8% 111 0.280 yes 1 10.0% 9 90.0% 10 alc decreased 23 28.0% 59 72.0% 82 0,127 normal 6 15.4% 33 84.6% 39 nlr low 22 22.7% 75 77.3% 97 0,505 high 7 29.2% 17 70.8% 24 degree of severity mild 0 0.0% 5 100.0% 5 0,000 moderete 0 0.0% 32 100.0% 32 severe 12 21.4% 44 78.6% 56 very severe 17 60.7% 11 39.3% 28 neutrofil decrease 0 0.0% 9 100.0% 09 0,101 normal 1 10.0% 9 90.0% 10 increase 28 27.5% 74 72.5% 102 limfosit decrease 28 30.8% 63 69.2% 91 0.002 normal 1 3.3% 29 96.7% 30 total 29 24,0% 92 76,0% 121 based on the laboratory tests in this study, sars cov2 infection affected the mean level of leukocytes, lymphocytes, and neutrophils (table 2). therefore, there is a significant relationship between the decrease in lymphocyte levels and the patient's clinical outcome. our findings confirm the potential use of lymphocytes in disease severity in covid-19. as is well known, sars cov2 primarily acts on t lymphocytes and further disrupts the stability of neutrophils in the immune system.26 a meta-analysis showed that lymphopenia and neutropenia are associated with poor clinical outcomes in covid-19. 81 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: 76–82 lymphopenia can cause interference with adaptive immunity and cytokine storms that trigger acute respiratory distress syndrome (ards).27 furthermore, low absolute lymphocyte levels can be used as a characteristic marker of diagnosis and describe the prognosis of the disease.11 lymphopenia and neutrophylia observed can be the cause of the poor clinical outcome in covid-19 because there is a disruption in the balance of the immune system in response to viral infection, leading to hyperinflammation and death. because of the defensins and neutrophil elastase (ne) that are generated after excessive neutrophil activation during sars cov2 infection, blood arteries may become more permeable. additionally, net formation may actively harm vascular tissue or significantly contribute to the activation of endothelial cells aggravating the inflammatory circuit and activating alveolar macrophages for clearance.28 conclusions diabetes melitus and hypertension are the most common comorbid reported in the covid-19 patients. furthermore, lymphocyte can be used as markers of disease severity that influence the clinical profile of patients with covid-19. majority of the patients return home with a stable condition, most likely because health worker awareness is quite good regarding this disease. acknowledgement the authors are grateful to zainoel abidin general hospital banda aceh for providing medical record data and also 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jul;146(1):89–100. 27. henry b, cheruiyot i, vikse j, mutua v, kipkorir v, benoit j, et al. lymphopenia and neutrophilia at admission predicts severity and mortality in patients with covid-19: a meta-analysis. acta biomed. 2020 sep;91(3):e2020008–e2020008. 28. reusch n, de domenico e, bonaguro l, schulteschrepping j, baßler k, schultze jl, et al. neutrophils in covid-19. front immunol. 2021;12(march):1–9. 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 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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 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. 124 vol. 6 no. 5 may–august 2017 research report integrating the roles of stakeholders in preventing the hiv/aids transmission in east java, indonesia toetik koesbardiati1, sri endah kinasih1, siti mas’udah2a 1 department of anthropology, faculty of social and political sciences, universitas airlangga 2 department of sociology, faculty of social and political sciences, universitas airlangga a corresponding author: siti.masudah@fisip.unair.ac.id abstract hiv/aids prevention is very important and absolutely necessary. hiv transmission is now entering a fairly alarming level, in which people with hiv/aids in certain subpopulations are emerging. special steps and resources are thus needed to cope with the condition. there are some phenomena potentially encourage hiv transmissions, such as the increasingly common free sex, homosexuality, the use of unsafe and unsterile syringes in narcotics consumption, commercial sex workers and various high-risk sexual activities. one of the crucial concerns that arises when sending prostitutes back to their hometowns without any coordinated and holistic mechanism is that the prostitutes may cause the spreading of hiv/aids in their hometowns. the research objective is to provide the material (input) how the prostitutes themselves may cause the spreading of hiv/aids. the research employed descriptive method with a qualitative approach. the results showed that the implementation and the role division in the closure have been highly coordinated and holistic. the leading sector in the role division is the social welfare epartment of the government in surabaya. in terms of health aspects for the former prostitutes sent back to their hometowns, there has been no policies related to medical screening designed to identify the disease early. screening is very important for early diagnosis during the post-closure phase. the screening mechanism is that the provincial health department has to optimize the monitoring, coordination, cooperation, agreements and partnerships with stakeholders such as the local health department and the national/provincial/distric aids commission, ngos that are concerned with the problems of hiv-aids, international organizations, professional organizations, community leaders, religious leaders and universities. keywords: policy, prostitute reintegration, prostitution, hiv/aids countermeasure abstrak penanggulangan hiv/aids sangat penting dan mutlak dilakukan. saat ini, penularan hiv/aids telah memasuki fase yang cukup krusial. munculnya penderita hiv/ids pada komunitas tertentu memerlukan upaya khusus karena penularan hiv/aids dapat terjadi melalui berbagai cara. satu hal penting yang dikhawatirkan saat pemulangan psk kembali ke daerah asal tanpa adanya mekanisme yang terkoordinasi dan holistik adalah bahwa psk tersebut justru dapat menyebarkan hiv/aids di daerah asal mereka. penelitian ini mengkaji mekanisme reintegrasi psk ke daerah asal yang telah dilakukan dan peran dari masing-masing dinas pemerintah ketika penutupan lokalisasi serta menjembatani kebijakan penanggulangan hiv/aids dengan kebijakan penutupan lokalisasi dalam kerangka sistem kesehatan daerah. tujuan dari penelitian ini untuk bahan (input) penerapan dalam hal penanggulangan hiv/aids pasca penutupan lokalisasi. penelitian ini menggunakan tipe penelitian deskriptif dengan pendekatan kualitatif. hasil penelitian ini menunjukkan bahwa pelaksanaan pembagian peran pra dan saat penutupan enam lokalisasi di surabaya di masing-masing dinas yang ditunjuk oleh pemerintah kota surabaya sangat terkoordinasi dan holistik. dalam pembagian peran ini sebagai leading sectornya adalah dinas sosial kota surabaya. terkait dengan aspek kesehatan bagi psk pada saat reintegrasi ke daerah asal, belum adanya kebijakan terkait dengan medical screening. screening dirancang untuk mengidentifikasi suatu penyakit secara dini, supaya ada intervensi dari dinas kesehatan provinsi, yang bertanggungjawab dalam penyebaran penyakit hiv/aids. screening ini sangat dibutuhkan dan penting untuk diagnosis lebih awal bagi psk pada saat reintegrasi ke daerah asal maupun pasca penutupan lokalisasi dengan melakukan 125koesbardiati, et al.: integrating the roles of stakeholders in preventing the hiv/aids transmission monitoring, koordinasi, kerjasama, kesepakatan-kesepakatan dan kemitraan dengan stakeholder seperti dinas kesehatan dan komisi aids nasional/provinsi/kabupaten, lsm yang peduli dengan masalah hiv-aids, organisasi internasional, organisasi profesi, tokoh masyarakat, pemuka agama dan perguruan tinggi. kata kunci: kebijakan, reintegrasi pekerja seks komersial (psk), prostitusi, penanggulangan hiv-aids introduction hiv/aids prevention is very important and absolutely necessary. hiv transmission is now entering a fairly alarming level, in which people with hiv/aids in certain subpopulations are emerging. special steps and resources are thus needed to cope with the condition. there are some phenomena potentially encourage hiv transmissions, such as the increasingly common free sex, homosexuality, the use of unsafe and unsterile syringes in narcotics consumption, commercial sex workers and various highrisk sexual activities. the policy of closing red-light districts in surabaya, indonesia seems unable to resolve the issues of hiv/aids spreading due to the fact that risky sexual activities are still ongoing. the impacts of the policy implementation include uncontrolled transmission and spreading of hiv/aids from illegal prostitutions (illegal relocation), uncontrollable by government policy. hiv/aids countermeasure is heavily dependent on government policies in both national and local levels to reach the goal. the policy must be included in the health system prevailing in indonesia. it has to be admitted that the conflicting policies above will greatly influence the policies and programs related to hiv/aids. policies and programs on hiv/aids that will be developed cannot be separated from the debate on the red-light district closure. it is already mentioned that the decrease of hiv/ aids cases is one of the main targets of the millennium development goals (mdgs). thus, the research is expected to contribute to decrease aids-related deaths. several studies about hiv have been conducted in other countries. in africa, diagnosis of hiv caused grief, fear, anxiety, and despair.1,2 ugandan women were in denial, afraid, and felt isolated upon knowing their hiv status,3 while congolese women were ravaged and tormented over risk of dying and left their children orphaned; they puzzled why the infection had happened to them.4 a number of zimbabwean lived secret lives characterised by constant concern of relevation.5 furthermore, hiv studies in patrilineal zimbabwe have predictably centered on female prostitutes as dangerous disseminators of,6 the risk of vertical transmission to their children,7 and women as caregivers for orphans or people living with hiv (plhiv).8 in addition, gona (2015) reports that zimbabwean women area unit are at a high risk for hiv infection yet usually aren’t the main focus of inquiry unless they’re participants in controlled trials. in rural areas of mexico, it has become progressively apparent that heterosexual transmission of hiv could be a growing problem.9 compared with the united states, mexico continues to possess lower incidence rates of hiv and aids.10 traditionally, aids has been a disease of enormous urban areas and, till recently, the incidence of hiv infection in remote and rural regions of mexico has been low. 9 researchers have contributed valuable data associated with the spread of hiv in migrant employees, particularly focusing on communities on the u.s.-mexico border.11–13 that migrant workers from mexico working in the united states need culturally appropriate hiv education, access to hiv testing, and access to hiv care. studies have shown a transparent association between poverty and health status.14,15 furthermore, the implications of hiv infection on the individual and also the resultant interventions required, as well as treatment, are well studied and understood.16 basavaraj (2010) stressed that it ought to benoted that for a well-rehabilitated and trained plhiv with social and economic support, having access to quality health care makes a difference not solely in his/her personal life, but also for society in general.17 the framework suggests numerous support areas to help mitigate the impacts of hiv on commercial enterprise security.18 prostitutes and indigenous populations are found to be among the most vulnerable.19 as seen in different countries, indigenous girls area unit overrepresented in statistics for new hiv infections, with the risk of dissemination for sex employees.20 the prostitutes often resorted to drug abuse, a risk behavior that, in conjunction with social violence, made them a lot more suspectible to hiv and other sexually transmited infections (stis).21 strong relationships are found between sex work and drug abuse, with an exaggerated risk of getting hiv and other stis, in addition to being victims of sexual and physical abuse.22,23 there’s a desire, therefore, to put culturally accepted interventions in place and to aim them at the women as well as their clients, whereas taking the issues featured by indigenous prostitutes under consideration.20,24 there is little analysis that explores how to prevent the hiv aids transmission related to sending prostitutes back to their hometowns. this study will observe the reintegration mechanisms of former prostitutes to their hometowns and to create a standardized medical screening policy to reduce hiv/aids transmission to fulfill the tri zero programs in indonesia, which are zero new hiv infections, zero aidsrelated death, and zero discrimination. this study examines how the reintegration mechanisms of former prostitutes 126 indonesian journal of tropical and infectious disease, vol. 6 no. 5 may–august 2017: 124–130 to their hometowns is conducted; what is the role of each agency; what the officers have done on the closure of redlight districts and how to relate the hiv/aids prevention policy with the policy of the red-light districts closure within the framework of the local health system. material and method to obtain empirical data and information, this research applied descriptive research with qualitative approach. several stages were applied, including: (1) determining research locations by purposive, resulting in dupak bangunsari, tambakasri, moroseneng, klakah rejo, jarak and dolly in surabaya, east java, indonesia. there are several considerations leading to the selection of six red-light districts in surabaya for the research. the considerations are: first, east java ranks second for hiv infection prevalence in indonesia. second, these six redlight districts are considered to be the “hotspot” red-light districts in surabaya which had operated for years. third, these six red-light districts are subjects of the east java governor policy for immediate closure. fourth, these six red-light districts were considered by east java governor as the cause of increase of hiv/aids incidence in surabaya. (2) combining several techniques of data collections, including: (a) observation and (b) in-depth interview. (3) selecting the informants for this study that consists of individuals who had the knowledge and experience of the problems studied, (4) grouping and identifying the collected data by theme and then analyzing them to answer the research questions. result and discussion former prostitutes reintegration mechanism to their hometowns the closure process of the red-light districts had actually been done by the surabaya city government in 2002, but it had the success rate of only 10% in reducing the number of prostitutes in surabaya. this was due to the absence of actions and better coordination between surabaya social affair department, surabaya health department, and the people empowerment department as well as government officials in the red-light districts either in the levels of sub-district, urban village, rukun warga (local non-formal officials) and also in the neighborhood. based on both governor’s letters addressed to the mayors and head of regents throughout east java, surabaya city government then immediately performed the closure. the red-light districts closure was completed using two approaches. the first approach was by persuasive approach, meaning that the mayor invited the prostitutes, pimps, community leaders and religious leaders from six red-light districts in surabaya in an event of ramadan iftar dinner. in the event, moral messages were delivered based on religious norms highlighting the fact that becoming prostitutes and the existence of red-light districts in surabaya were great sins for both the prostitutes and the mayor herself as the leader in surabaya. the mayor then called on the consciousness of the prostitutes, pimps and the people that relied their lives on the red-light districts to quickly realize that their actions were big sins. in this event, a dialogue was held between the prostitutes, pimps, community leaders and religious leaders from six red-light districts in surabaya. the dialogue covered problem identification and needs analysis. indeed, some parties in the dialogue admitted that they did not want to be prostitutes and pimps, and their concern about the children in the areas around the red-light districts that could be affected. however, there were also many opposing remarks including the new prostitutes’ contract with their pimps, meaning they were recruited by pimps and therefore indebted to the pimps. the debt must be paid off by serving “guests” or “costumers” where approximately 30% to 40% of the income goes to the pimps. this is how the prostitutes repaid their debts. should the first approach not be successful, the second approach was taken which was by repressive coercion. it is the utmost necessary to prevent and control prostitution and woman trafficking by closing of red-light districts. in the closure, surabaya city government used strategic effort of gradually closing the red-light districts. 1) the plan started from dupak bangunsari, tambakasri/kremil, klakah rejo and sememi (moroseneng) and last jarak and dolly complex; 2) providing skills training for prostitutes according to their interests, and providing venture capital; 3) sending prostitutes from outside surabaya back to their hometowns while keep coaching and mentoring in a continuous and sustainable manner; and 4) transforming the red-light district sites into business hubs with opportunities for the surrounding community. before returning the prostitutes, spiritual guidance and vocational training were given to the prostitutes with the expectation that they could become more independent and accepted by the society. there were role divisions in the administration of the compensation fund. before the prostitutes received compensation fund, officials from surabaya social department would verify the data by matching the names and address in cooperation with the heads of rt and rw (local non-formal officials), urban heads of urban village and head of sub-districts in the red-light districts. in the data verification, the prostitutes must meet two requirements. the first requirement, the prostitutes were required to show their id or work permit from the villages. the second requirement, they must fill out a statement which explained that the prostitutes come from certain brothel houses and they promise not to return to be a prostitute anymore. the surabaya social service could not disburse the financial compensation if these two conditions were not met. in addition, the prostitutes would have voluntarily joined hiv counselling and test with local community 127koesbardiati, et al.: integrating the roles of stakeholders in preventing the hiv/aids transmission health centers in collaboration with surabaya health department and rt & rw in the red-light districts. if there were prostitutes affected by hiv-aids disease, surabaya health department will submit the case to the provincial health department for further monitoring and coordination with the local health department in the hometowns of the former-prostitutes. the local health department in the prostitutes’ prostitutes hometowns were expected to monitor and provide guidance for hiv/aids testing in the community health centers in the prostitutes’ hometowns. the role of each government department and their coordination in the red-light district closure in reality, the program of red-light district closure was holistic and well coordinated to meet the targets of the governor’s decree. all agencies were always linked to each other in running the programs. most of these programs did not overlap among institutions. regarding the postclosure hiv/aids countermeasure, the provincial health department took the role as the leading sector. in addition, the leading sector in hiv/aids countermeasure in the east java, provincial health department also monitored and coordinated with the local health department at the former prostitutes’ hometowns, whether there were prostitutes infected with hiv by conducting voluntary counselling and testing (vct) sessions. what are the forms of coordination and monitoring by the local health department in the former prostitutes’ hometowns? this is an issue related to the spreading of hiv/aids in east java. as stated by surabaya social department, after the closure of red-light districts, the provincial health department would step in as the leading sector. if there were prostitutes affected by hiv/aids found in the red-light districts in surabaya, the information would be submitted to the provincial health department to follow it up with monitoring and coordination with the local health department of the former prostitutes’ hometowns. in terms of local budgets, some of the prostitutes in surabaya red-light districts are from outside surabaya. if this group were in fact infected with hiv and needed therapy, they would use the budget of surabaya city and thus surabaya city government would be burdened with this problem. regarding the prostitutes affected by hiv/aids returning to their hometowns, the same case happened. the local health department in the hometowns also faced similar issue. this is confirmed by the chairman of embun foundation’s statement, explaining that prostitutes would rather check their venereal diseases and hiv/aids to hospitals or community health centers in surabaya. the reason is that they trust them more than the ngos that deal with the local health departments. these prostitutes were familiar with the medical staff there, resulting in more confidence, better understanding and empathy. in addition, they were more familiar with the procedures when they checked themselves at the hospitals. this made the diagnosis and management easier, more understanding and more importantly, their secrets were kept discreet. on the other hand, however, the prostitutes were often not the citizens of surabaya, meaning that the one who should be responsible on funding these problems of venereal disease and hiv-aids inspections were the health departments of the prostitutes’ hometown. the hometown local health departments of the former prostitutes seemed to be less prepared to accept this responsibility. once these former prostitutes were sent back to their hometowns and were identified with hiv/aids disease, there seemed to be lack of attention without any follow up nor monitoring. there was no further guidance. this might be due to the human resource incapability concerning hiv/aids or the inability to budget the hiv/ aids monitoring. the provincial health department as the leading sector seemed to have less than the maximum effort in monitoring and coordinating this problem with the local health departments of the former prostitutes’ hometown. worse still, the local health departments of the former prostitutes’ hometowns were not focused nor had any clear direction to support the need in the local health department level. as a result, the community health centers around the red-light districts in surabaya still often received visits from former prostitutes who still lived in the area as well as those who had been sent back to their hometowns. from all the explanation above, it seems that the provincial health department as the leading sector has not been optimal in monitoring, coordinating and mutually supporting the local health departments in the former prostitutes’ hometowns. the lack of monitoring and coordination between the provincial health department and the local health departments had resulted the inaccurate surveillance data and thus it was difficult to intervene. hence, the target was not reached. data surveillance is intended to strictly control the spreading of the infectious disease of hiv/aids, so that patients can immediately be isolated and control measures can be taken as early as possible. thus, the need to plan programs is expected to have a successful result. from these programs, the disease countermeasures are then taken. if the target is not reached, the epidemic of sexually transmitted infections and hiv/ aids cannot be controlled. in this case, the provincial health department should cooperate and have agreements with local health department on the former prostitutes’ hometowns. the issue of red-light districts closure was not an easy one. prostitutes were experiencing extremely complex problems. prior to being returned to their hometowns, former prostitutes were equipped with training to make culinary, sewing, handicrafts (mats, brooches), introduced to washing machine and manage budget spending. these former prostitutes and their families are also equipped with the knowledge and skills to survive in their hometowns. in fact, some principle findings are found. one of the factors driving women to become prostitutes is that they came from poor families of farm labors, farmers, construction workers, small merchants, housewives, or being the family member of a prostitute. the result of this 128 indonesian journal of tropical and infectious disease, vol. 6 no. 5 may–august 2017: 124–130 study is similar to the one found in bolivia that sex work has become a way of financial support.25 the next factor is low education. prostitutes generally have low education background. likewise, the trafficking victims had less education in average. they only finished elementary school or do not even graduate. this is why they are easily deceived, seduced, and lured by pimps or brokers to become prostitutes. although skill trainings based on their interests and talents has been provided, the question lies on whether in a short time these former prostitutes would be able to survive. they come from poor families with poor education. will they be able to survive with the existing conditions of poverty? there have been trainings for prostitutes before the red-light district closure such as cooking and salon business. nevertheless, the government had made no effort to create jobs for these prostitutes. in the end prostitute remains a prostitute. this would have an impact on the spreading of hiv/aids. the effects of the red-light district closure were very complex. therefore, partnerships with stakeholders to interpret strategic plans into field implementation were highly required. red-light district closure program should always be directed to build and maintain partnerships which involved all stakeholders comprising government agencies, community organizations, non-governmental organizations, international organizations, professional organizations, academics and universities, and the public in general. the partnership is made to build understanding and a shared commitment to develop any necessary efforts in hiv/aids countermeasure. to make the process and objectives of the red-light district closure program successful, surabaya social service took the role as the leading sector in the pre-closure and closure phases and the provincial health department as the leading sector in the post-closure need to consider the partnership as a goal and the foundation to be applied in every implementation of hiv/aids countermeasure efforts by all stakeholders in surabaya and east java. surabaya social service has conducted partnership with rt, rw, urban village chiefs in the red-light districts both in terms of closure socialization, prostitute database collection and verification, and also assessment. nevertheless, it has not made any partnership with ngos, international organizations, professional organizations and colleges. this was confirmed by the statement of the head of hotline surabaya surya that no partnership was made by surabaya social service. other ngos that are concerned with the prevention of hiv/aids such as obor foundation was not involved as well. likewise, after the closure, it seemed that no partnership was made by the provincial health department as the leading sector. as the agency to coordinate and monitor the local health department when prostitutes infected with hiv aids were found, the provincial health department seemingly made no coordination and monitoring. the former prostitutes could have become prostitutes in other brothel with a different format. in general, the local health service had relatively limited human resources and budget. therefore, the concept of partnership was the most likely alternative for local governments (in this case involving the local health department) to develop with non-governmental organizations, international organizations, professional organizations, community leaders, religious leaders and colleges. it was almost difficult to expect partnership without the involvement of all stakeholders. some of the obstacles including: the inability of local governments to offer various forms of partnership and the concept being less feasible in conducting partnership which was offered by the local government in the prostitutes’ hometowns. m e d i c a l s c r e e n i n g s t a n d a r d i z a t i o n a s h i v a i d s countermeasure efforts for former prostitute reintegration at their hometowns although at the time of red-light district closure vct had been conducted by surabaya health department in cooperation with community health centers in six regions of red-light districts in surabaya, it was still necessary to conduct vct in the post-closure. it is the post-closure that the most important moment in hiv/aids countermeasure. being the leading sector after the closure, it was up to the provincial health department to maximize the monitoring, coordination, cooperation, and partnership agreements with stakeholders all the way to the local levels. stakeholders in this study were the local health department, national/ provincial/local aids commissions, and ngos that were concerned with the problem of hiv-aids, international organizations, professional organizations, community leaders, religious leaders and colleges. the monitoring, coordination, cooperation, and partnership agreements with the stakeholders should be right on target to gain an effective and efficient policy (fulfilling mdg target to reduce the number of people with hiv/aids until at least below 0.5 percent). for further details, the reintegration mechanisms of former prostitutes to their hometowns as an effort to countermeasure hiv/aids for the former prostitutes needs to be standardized. the provincial health department must have a national strategic plan to prevent, control, and cope with aids. this strategic plan can be in the form of counseling, prevention, nurturing, monitoring and controlling hiv/aids. provincial health department needed to make an agreement with provincial/local aids countermeasure commission (kpa) to support the policy of hiv/aids prevention and controlling on the red-light district closure program. the socio-cultural barriers and potentials (supports) in the policy model of medical screening standardization to countermeasure hiv/aids in the reintegration of prostitutes to their hometowns was highly dependent on the actors. the actors here means people, groups, organizations or networks that were capable of taking decisions and actions to cooperate in establishing and maintaining a specific rule/structure system. conversely, this could also be conflicting due to different interests for example, 129koesbardiati, et al.: integrating the roles of stakeholders in preventing the hiv/aids transmission whether or not to accept government programs related to both the prevention of hiv/aids. the actors would then interact in forms of cooperation, competition, contradiction, use of coercive force and so on within the social system. the policy process is the means of how policy was initiated, developed or formulated, negotiated, communicated, implemented and evaluated. there are two steps in the process of formulating policies that determines the preferred choice of a policy. at both of these stages, policy makers should ideally understand the situation in details to make implementable decisions. actors in policy model of medical screening standardization to reduce the number of people with hiv/ aids during the prostitute reintegration process to their hometowns included ngos which were concerned about the protection of women and children and hiv/aids spreading, colleges, community organizations, religious organizations, village/district government, counties, and local health departments. these agencies were the actors in formulizing policies on hiv/aids if these agencies have hiv/aids countermeasure policy, it could be said that they support indonesia towards the tri zero condition. these actors are the supporters of the social system that had certain social movements to cooperate in building and maintaining specific rule system. conversely, if these actors did not have any policy, on hiv/aids prevention or treatment, it could be said that they became the inhibitor in building a standardized medical screening. these actors can dispute with each other due to their different interests. this interest can be in the form of selfish sector interests. the provincial health department program may be obstructed/cannot be done, if no partnership was made with colleges, ngos, religious organizations and community organizations including the village as government agency partners in realizing the closure. the goal of the partnership is the realization of red district closure and hiv/aids prevention. if this partnership were conducted by the provincial health department in running the program, there would always be linkages with other stakeholders. provincial health department is not always capable of providing and implementing its own program. therefore, it would require a partnership to support the government to implement the program of red-light district closure. the participation of stakeholders as partners would provide a variety of programs that can be implemented better. the participation of stakeholder partners is expected by the government in terms of role division, access granting with the principle of equality and mutual benefit. in reality, however, this partnership is not always followed by a good coordination between the stakeholders and the red-light district closure had no clear focus nor a clear direction with overlaps so that they did not support each other. if the east java provincial health department were to make partnership with stakeholders, the principle of equality and mutual benefit should be implemented to realize the success of the closure and also the hiv/aids countermeasure. conclusion the role distribution implementation of the pre-closure and closure stages of six red-light districts in surabaya in the appointed departments within the surabaya city government has been highly coordinated and holistically implemented. in the role distribution, the surabaya social service plays the role of the leading sector. during the post-closure phase, there are still role divisions in each service agencies where the east java health department plays the role of the leading sector. the east java health department is appointed by surabaya city government in accordance with hiv/aids prevention. however, the coordination with the east java health department is apparently non-effective. in terms of the health aspects for the former prostitutes during their reintegration to their hometowns, there has been no previous policies related to medical screening which is designed for early detection of the disease and this lack of intervention from the east java health department may affect the spread of hiv/aids. screening is very important for early diagnosis for former prostitutes at the time of reintegration into their hometowns and the red-light districts post-closure phase. the screening mechanism is that the provincial health department has to optimize the monitoring, coordination, cooperation, agreements and partnerships with stakeholders such as the local health department and the national/provincial/distric aids commission, ngos that are 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the mexico-us border region. curr opin psychiatry. 2010 may; 23(3): 215–20. 24. lawan um, abubakar s, ahmed a. risk perceptions, prevention and treatment seeking for sexually transmitted infections and hiv/aids among female sex workers in kano, nigeria. afr j reprod health. 2012; 16(1): 61–7. 25. lópez entrambasaguas om, granero-molina j, hernández-padilla j, fernández-sola c. understanding sociocultural factors contributing to hiv risk among ayoreo bolivian sex workers. j assoc nurses aids care. 2015 nov; 26(6): 781–93. 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 review article prolonged use of protective masks induced facial skin injury in primary healthcare workers during covid-19 pandemic: a systematic review alvian mohamad yapanto1* , aulia rahma isnaeni1 , khairani ayu lestari1 , agung bagus sista satyarsa2 1faculty of medicine, universitas yarsi, jakarta, indonesia 2faculty of medicine, universitas udayana, bali, indonesia received: june 15th, 2022; revised: august 12th, 2022; accepted: november 23rd, 2022 abstract covid-19 transmission necessitates health workers to use personal protective equipment (ppe), especially protective masks when delivering medical services. long-term use of protective masks might cause facial skin injuries. our study aims to provide a systematic review to explore the phenomenon and incidence of protective masks induced facial skin injuries in primary healthcare workers. this systematic review was created by obtaining articles from the pubmed database and the cochrane library from 2020 to 2021, using the keywords "face skin injury," "wearing protective masks for a long time," and "wearing protective masks and facial skin disorders." inclusion criteria were studies that fully report the phenomenon of wearing protective masks and the incidence of facial skin injuries. one hundred and sixty-eight studies were obtained, but only 14 articles matched the inclusion criteria with more than 10,430 participants from different countries that covered various characteristics of facial skin injuries in primary healthcare workers. the findings obtained dominant characteristics of health workers who experienced facial skin injuries: women, n95 masks, and daily n95 coverage for more than 6 hours (p<0.05). facial skin injuries are often seen after using protective face masks, as it is used for an extended period as part of a defensive effort during work. therefore, measures that protect health workers from covid-19 and prevent health workers from potential injuries of protective masks must be taken into account. keywords: covid-19; facial skin injury; long duration; primary health workers; protective masks abstrak penularan covid-19 mengharuskan tenaga kesehatan untuk menggunakan alat pelindung diri (apd), khususnya masker pelindung saat memberikan pelayanan medis. penggunaan masker pelindung jangka panjang dapat menyebabkan cedera kulit wajah. penelitian kami bertujuan untuk memberikan tinjauan sistematis untuk mengeksplorasi fenomena dan kejadian cedera kulit wajah terkait penggunaan masker pelindung pada tenaga kesehatan primer. tinjauan sistematis ini dibuat dengan memperoleh artikel dari database pubmed dan perpustakaan cochrane dari tahun 2020 hingga 2021, menggunakan kata kunci "face skin injury," "wearing protective masks for a long time," dan "wearing protective masks and facial skin disorders". kriteria inklusi adalah penelitian yang secara lengkap melaporkan fenomena pemakaian masker pelindung dengan kejadian luka pada kulit wajah. 168 penelitian diperoleh, tetapi hanya 14 artikel yang sesuai dengan kriteria inklusi dengan lebih dari 10.430 peserta dari berbagai negara yang mencakup berbagai karakteristik cedera kulit wajah pada petugas kesehatan primer. temuan didapatkan karakteristik dominan tenaga kesehatan yang mengalami cedera kulit wajah: perempuan, masker n95, dan cakupan n95 harian lebih dari 6 jam (p<0,05). cedera kulit wajah sering terlihat setelah menggunakan masker pelindung, karena digunakan dalam * corresponding author: alvian.mohamad@students.yarsi.ac.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-2781-8107 https://orcid.org/0000-0003-2774-0167 https://orcid.org/0000-0003-2480-1083 https://orcid.org/0000-0002-2153-2271 199 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license alvian mohamad yapanto, et al. prolonged use of protective masks induced facial skin injury waktu lama sebagai bagian dari upaya defensif selama bekerja. oleh karena itu, langkah-langkah yang melindungi petugas kesehatan dari covid-19 dan mencegah petugas kesehatan dari potensi cedera masker pelindung harus diperhitungkan. kata kunci: covid-19; cedera kulit wajah; durasi panjang; masker pelindung; petugas kesehatan layanan primer how to cite: yapanto, a. m., isnaeni, a. r., lestari, k. a., satyarsa, a. b. s. prolonged use of protective masks induced facial skin injury in primary healthcare workers during covid-19 pandemic: a systematic review. indonesian journal of tropical and infectious disease. 10(3). 198–204. dec. 2022. introduction coronavirus disease (covid-19) is an ongoing global threat requiring the public to abate its transmission by improving personal and communal hygiene practices.1,2 personal protective equipment (ppe) is essential for health workers as they are more at risk of contracting covid-19.3-5 although wearing ppe, especially protective masks, is mandatory to prevent covid-19 infection, its long-term use increases the temperature, which leads to sebum excretion. moreover, the pressure and friction from the protective masks can cause contact dermatitis (injuries of facial skin), seborrheic dermatitis, and acne vulgaris. the most frequent side effect of ppe is pressurebased wounds induced by n95 masks, such as the indentation of the mask on the bridge of the nose of health workers.5 this systematic review will provide a comprehensive overview of the available literature regarding the side effects of the long-term use of protective masks. our main objective is to understand the extent of facial skin injury induced by protective maskwearing among primary healthcare workers during the pandemic of covid-19. methods study design this was a systematic review of facial skin injury induced by protective masks during the covid-19 pandemic. in conducting the literature search and reviewing the article, we adhered to prisma guidelines.4 pubmed and cochrane library were the primary databases to search for articles published from january 2020 to november 2021. the literature search process used the boolean operator "and" or "or" using the keywords "face skin injury," "wearing protective masks for a long time," and "wearing protective masks and facial skin disorders." study selection articles were selected from the databases based on inclusion and exclusion criteria. the article's inclusion process followed several criteria, such as 1) studies reporting the significance of protective masks induced facial skin injury during the pandemic of covid-19; 2) age > 18 years old; 3) medical staff who wore level 2 or 3 ppe while working at the frontline against covid-19, regardless of gender. exclusion criteria included review articles written in languages other than english, conference abstracts, nonhuman research, and studies that did not evaluate the outcome measures. two independent reviewers selected the articles and extracted the key findings. disagreements between the two authors were resolved by reaching a consensus aided by the third reviewer. the full literature search and selection process followed the prisma guideline. study quality assessing the quality of evidence within a systematic review is as important as analyzing the data. selecting an appropriate tool to help analyze strength of evidence and embedded biases within each paper was also essential. therefore, the author used joanna briggs institute (jbi) that provides robust 200 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: 189–204 checklists for the appraisal and assessment of most studies. data extraction and analysis key findings were independently extracted, starting by noting baseline characteristics and outcomes from included articles. extracted data contained first author name, year of publication, study design, age range, diagnosis, sample size, and results. all data results are presented and described descriptively in tabular form. results and discussion the querying process returned 168 studies, with 167 originating from online databases (pubmed and cochrane library) and one article sourced from an organic search. a total of 123 studies were obtained after removing duplicates using computer software (citation manager). upon screening the title and abstract, 17 studies were eligible for further assessment. however, 3 studies did not satisfy the inclusion criteria, 14 of which were still included in the qualitative analysis (systematic study).5–18 figure 1 summarizes the literature search process as indicated by the prisma guideline. figure 1. flowchart prisma from all included articles, 10,430 respondents participated in several observational studies. the median age of respondents was 35 years, with most respondents being women (available in table 1). the dominant health workers are nurses with the most use of n95 while handling patients during a pandemic. in addition, the working time of health workers in each study was between 4–12 hours. table 1. characteristics of studies author, year study design age gender sample size jiang et al., 20205 multicenter observational study 35 years (median) male (12.7%) female (87.3%) 4308 battista et al., 20216 observational study 35.0 ±11.7 years male (33.1%) female (66.9%) 381 abiakam et. al., 20217 prospective study 45 years (median) male (12.0%) female (88.0%) 307 ippolito et al., 20218 a cross-sectional survey 40 years (median) male (49%) female (51%) 2711 han et al., 20219 a cross-sectional study 37.5±10.83 years male (10.0%) female (90.0%) 20 choi et al., 202110 multicenter observational study 35.50±14.45 years male (34.85%) female (65.15%) 330 uthayakumar et al., 202111 rapid report 34 years (median); range 23-60 female : male (4:1) 67 purushothaman et al., 202112 cross-sectional 25.843 years (mean) range 20-48 male (28.4%) female (71.6%) 250 techasatian et al., 202013 prospective cross-sectional 32 (25-41) years (median (iqr)) range 18-87 years male (26.7%) female (73.3%) 833 singh et al., 202014 survey study 32.78±14.51 years male (59.7%) female (40.3%) 43 coelho et al., 202015 cross-sectional 34.08 (8.9) (mean(sd)) male (16.4%) female (83.6%) 1106 yuan et al., 202016 cross-sectional n/a male : female (1:2) 129 shanshal et al., 202017 cross-sectional observational n/a male (36%) female (64%) 276 christopher et al., 202018 cross-sectional 26.94±7.23 years male (33%) female (67%) 200 201 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license alvian mohamad yapanto, et al. prolonged use of protective masks induced facial skin injury personal protective equipment (ppe) is one piece of equipment used by health workers to prevent nosocomial infections and protect patients from the possibility of infection, starting from the patient entering and receiving healthcare and medical action until the patient returns from the hospital.19–22 the scientific summary released by the world health organization (who) reported the presence of sars-cov-2 ribonucleic acid (rna) in air samples taken from under the patient's bed and windows. both areas would have minimal direct contact with patients or health care. researchers also found that 66.7% of air samples taken from hospital hallways contained viral.23,24 the world health organization (who) recommendation is that surgical masks should be sufficient when treating covid-19 patients, and n95 or papr respirators should be used only in the case of aerosol-generating procedures. the cdc insists that n95 respirators be used by all medical professionals who contact covid-19 patients. based on this, if there are difficulties in procurement or vacancies for n95 masks, surgical masks are allowed to make contact with covid-19 patients and to protect, face shields can be used. several studies state no clinically significant evidence of a difference in safety between surgical masks and n95.5,8,20 table 2. unique publications identified author, year ppe and duration outcome quality of study (score) jiang et al., 20205 level 3 ppe, protective masks >4 hours the device-related pressure injury (drpi) was prevalent among healthcare workers wearing ppe against covid19. the risk factors for facial skin injury (p<0.05) were male, wearing level 3 ppe, longer wearing time > 4 hours and sweating. high (8) battista et al., 20216 surgical mask, cotton mask, n95, combination surgical + ffp2/3, <1 hours until > 12 hours most affected individuals were healthcare workers wearing n95 respirator masks for more than six h/d (p<0.05) moderate (6) abiakam et al., 20217 ppe (ffp3), eye protection, gloves, gown >8 hours the adverse skin reactions (facial skin injury) had a significant association with the average daily time of ppe usage during > 8 hours (p<0.05) moderate (7) ippolito et al., 20218 mask (surgical, n95, ffp3, papr), gown, >6 hours 59% of the participants had significant pressure injury on the face area after using an n95 mask in icu for> 6 hours (p<0.05) high (8) han et al., 20219 kf94 respirator dan medical mask 4 hours, 8 hours, dan 14 hours skin injury significantly differed between rpe-covered and uncovered areas after 4 and 8 hours (p<0.05). low (2) choi et al., 202110 n95/kf94/kf80, surgical, cotton ≥6 hours daily use of n95 masks significantly increases the incidence of new contact dermatitis. the duration of wearing ppe >6 hours/day and masks made of cotton significantly increased the incidence of acne and wounds around the face. health workers had a higher incidence of facial skin injuries (p<0.05). moderate (6) uthayakumar et al., 202111 protective masks n95 > 6 hours ppe marked an increase in the impact of facial skin injury; 70% reported a significant adverse effect on their work or study (p<0.05) low (4) purushothaman et al., 202112 n95 + surgical mask, > 4 hour/day excessive sweating around the mouth after used protective mask was 67.6%, resulting in poorer adherence and increased risk of infection in the face area (p<0.05). moderate (7) techasatian et al., 202013 n95 masks, surgical mask, 4 to 8 hours/day 1,92% facial skin injury among 4-8 hours (48.9%) after used protective mask was a significant value in statistics (p<0.05) high (8) singh et al., 202014 n95 masks, face shields, and goggles average 8.76 hours goggles and n95 masks were the most common culprit agent among all ppe, causing skin injuries. the most commonly noted dermatoses were irritant contact dermatitis in the face (p<0.05). moderate (7) coelho et al. 202015 cap, gloves, apron, n95 mask, surgical mask, pff2 mask, face protector, and glasses >6 hours the number of pressure injuries related to personal protective equipment was high (an average of 2.4 injuries per professional). working and wearing personal protective equipment for more than six hours a day was one of the significant factors (p<0.05). high (8) 202 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: 189–204 yuan et al. 202016 n95 mask, goggles, gloves, face mask, gown, and medical protective clothing > 8 hours a total of 122 (94.57%) healthcare professionals experienced discomfort while wearing l3ppe, including varying degrees of face skin injuries, respiratory difficulties, heat stress, dizziness and nausea. moderate (6) shanshal et al. 202017 n95 mask, goggles, gloves, face mask, gown, and medical protective clothing > 8 hours 51% had pressure injury in the facial skin after prolonging (> 8 hours) using ppe, especially in the woman, and 82.5% had facial skin injury (p<0.05) moderate (6) christopher et al. 202018 level 1-3 ppe, protective masks ≥7 hours/day the level of ppe worn and duration of ppe worn daily was factors considerably associated with adverse skin reactions to ppe. low (4) fsi=facial skin injury, manifested in several clinical features, such as dryness, itching, erythema, acne, indentation, and pressure ulcer. further evidence suggested n95 respirator as protective mask causes more severe facial injuries than the kn95 respirator.5 applying polyester tape layering and emollient effectively prevented severe injuries, especially on the cheekbones, chin, nasal bridge and behind the ears.25–29 n95 masks cause skin injury because the material is thick and stiff, causing greater pressure on the skin.9 also, many studies have reported differences in risk between n95 masks and kn95 masks, as observed in our results. the difference in risk is interesting, given that n95 and kn95 masks provide relatively the same level of protection. more recent tests have also shown that n95 and kn95 are quite effective at filtering respiratory particulates, especially those protective mask used by healthcare professionals in treating patients with covid-19. besides that, interestingly, the kn95 mask is not as thick and stiff as the n95, so it is more comfortable to use for a longer period.7,8,30,31 the quality of the study and the bias assessment of the cross-sectional studies was done using the newcastle ottawa scale (nos), as presented in table 2. the overall quality of evidence was moderate-high quality.4,23 our findings recommend using an alternative to kn95 masks instead of n95 in primary care for patients with covid-19. they can promote using wound dressings and emollients to protect facial skin after carrying out services with ppe for > 4–6 hours. in particular, healthcare facilities are expected to provide supplies of protective facial mask and emollients to prevent facial injuries that use ppe too often and for a long time.32,33 previous investigations have yielded similar conclusions, although this study is one of the few to report the phenomenon of facial injuries due to prolonged use of protective masks. these results can be considered, and recommendations can be used in indonesia wisely. however, much remains to be learned about the covid-19 pandemic on the welfare and safety of health workers in primary health care. future studies should explore minimal treatment and prevention options for healthcare workers who suffer these injuries so that services during the pandemic are maximized.34,35 summary facial skin injuries are often seen after using protective masks, as it is used for an extended period of defensive effort during work. the current state of the evidence suggests that some protective face mask have their respective advantages and optimal usage duration. therefore, measures that protect health workers from covid-19 and prevent health workers from potential injuries from protective facial masks must be considered. the choice and duration of protective mask usage must be adjusted according to their working environment. acknowledgement we sincerely acknowledge the help provided by our lecturer in medical faculty of universitas yarsi for the professional guidance and valuable support for this research. 203 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa 4.0 international license alvian mohamad yapanto, et al. prolonged use of protective masks induced facial skin injury conflict of interest the authors 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l. evaluation of n95 respirators, modified snorkel masks and low‐cost powered air‐purifying respirators: a prospective observational cohort study in healthcare workers. anaesthesia. 2021;76(5):617-622. 32. pacis m, azor-ocampo a, burnett e, tanasapphaisal c, coleman b. prophylactic dressings for maintaining skin integrity of healthcare workers when using n95 respirators while preventing contamination due to the novel coronavirus: a quality improvement project. journal of wound, ostomy, and continence nursing. 2020;47(6):551. 33. guschel s, chmiel k, rosenstein j. use of thin dressings under n95 respirators: exploring their effect on quantitative fit testing results to guide hospital practice during the covid-19 pandemic. wound management & prevention. 2020;66(11):13-17. 34. smart h, opinion fb, darwich i, elnawasany ma, kodange c. preventing facial pressure injury for health care providers adhering to covid-19 personal protective equipment requirements. advances in skin & wound care. 2020. 35. cabbarzade c. a practical way to prevent nose and cheek damage due to the use of n95 masks in the covid-19 pandemic. aesthetic surgery journal. 2020;40(10):np608-10. 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 the activities on prevention of malaria and filariasis vector bites among indonesian society: a nationwide disease prevention survey mutiara widawati* , mara ipa , endang puji astuti , tri wahono , yuneu yuliasih research organization for health, national research and innovation agency (brin) indonesia received: may 23rd, 2022; revised: july 7th, 2022; accepted: july, 12th, 2022 abstract vector borne diseases are diseases that cause many problems. these diseases are spread by mosquitoes as the vectors. they transmit parasites to humans through their bites. the people who live in indonesia have several characteristics that make them vulnerable to these diseases. therefore, it is necessary to explore these characteristics in order to gain better prevention promotional targeting strategy. this study aims to determine the factors that can influence mosquito bite prevention behaviour in indonesian society. the data from a nationwide survey research were used with a cross sectional design conducted once in every five years. the riskesdas was conducted from april to may 2018 in all districts in indonesia. the influencing factors observed were including experience of exposure to vector borne diseases (malaria or filariasis), gender, age group, education level and area of residence. this study conducted a multivariate test using logistic regression analysis to determine the factors that influence mosquito bite prevention behaviour. the results demonstrated that the factors of experience of exposure to vector borne diseases, gender, age group, education level and area of residence could determine the mosquitoes bite prevention behaviour in indonesian society. respondents who have experience of being exposed to malaria or filaria, under 60 years old, women, college graduates, and rural communities are more likely to prevent mosquito bites, therefore they could be empowered in promoting public awareness towards mosquito bites prevention. keywords: filaria; malaria; mosquitoe; prevention; sociodemographic abstrak penyakit tular vektor merupakan penyakit yang menimbulkan banyak masalah. penyakit ini disebarkan oleh nyamuk sebagai vektornya. ini menularkan parasit ke manusia melalui gigitannya. masyarakat yang tinggal di indonesia memiliki beberapa karakteristik yang membuat mereka rentan terhadap penyakit tersebut. oleh karena itu, perlu dijajaki ciri-ciri tersebut guna mendapatkan strategi penargetan promosi pencegahan yang lebih baik. penelitian ini bertujuan untuk mengetahui faktor-faktor yang dapat mempengaruhi perilaku pencegahan gigitan nyamuk pada masyarakat indonesia. penelitian ini menggunakan data dari penelitian survei nasional dengan desain cross sectional yang dilakukan setiap lima tahun sekali. penelitian ini menggunakan data penelitian kesehatan dasar yang dilakukan pada bulan april hingga mei 2018 di seluruh kabupaten di indonesia. faktorfaktor yang digunakan dalam penelitian ini meliputi pengalaman pajanan penyakit tular vektor (malaria atau filariasis), jenis kelamin, kelompok umur, tingkat pendidikan dan daerah tempat tinggal. penelitian ini melakukan uji multivariat dengan menggunakan analisis regresi logistik untuk mengetahui faktor-faktor yang mempengaruhi perilaku pencegahan gigitan nyamuk. hasil penelitian menunjukkan bahwa pengalaman pajanan penyakit tular vektor, jenis kelamin, kelompok umur, tingkat pendidikan dan daerah tempat tinggal merupakan faktor yang dapat menentukan perilaku pencegahan gigitan nyamuk pada masyarakat indonesia. * corresponding author: mutiara.widawati@brin.go.id https://e-journal.unair.ac.id/ijtid/ https://orcid.org/0000-0002-1649-4688 https://orcid.org/0000-0002-4831-6536 https://orcid.org/0000-0003-4172-9300 https://orcid.org/0000-0002-6563-6953 https://orcid.org/0000-0003-0050-9520 105 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: 104–112 responden yang memiliki pengalaman terkena malaria atau filaria, di bawah 60 tahun, perempuan, lulusan perguruan tinggi, dan masyarakat pedesaan lebih mungkin untuk mencegah gigitan nyamuk, sehingga mereka dapat diberdayakan dalam meningkatkan kesadaran masyarakat terhadap pencegahan gigitan nyamuk. kata kunci: filaria; malaria; nyamuk; pencegahan; sosiodemografi how to cite: widawati m., ipa, m., astuti, e. p., wahono, t., yuliasih, y. the activities on prevention of malaria and filariasis vector bites among indonesian society: a nationwide disease prevention survey. indonesian journal of tropical and infectious disease. 10(2). 104–112. aug. 2022. introduction vector borne diseases are diseases that cause many problems in the world. malaria in 2018 has caused 405,000 deaths globally.1 lymphatic filariasis is also a health problem globally, especially in many tropical and subtropical countries. untreated lymphatic filariasis can lead to elephantiasis and hydrocele that cause significant social and economic burdens in a person's life.1,2 indonesia is declared as one of the malaria and filariasis endemic countries.3,4 indonesian society have several characteristics that vulnerable to diseases like malaria and filariasis. the characteristics are including tropical climate, population size, high migration rate, socio-economic imbalance, and regional government autonomy.2,5 diseases such as malaria and filariasis are spread by mosquitoes vectors. mosquitoes transmit parasites plasmodium or microfilariae to humans through their bites. therefore, it is important for the community to make efforts to prevent mosquito bites. several studies have shown that action to prevent mosquito bites by individuals were including the use of mosquito nets, repellents, mosquito coils, electric racquets, and electric repellents. repellents and mosquito coils work by hiding human odors from mosquitoes. electric racquets and mosquito nets are used for preventing mosquitoes from landing on humans. these efforts have proven useful in reducing mosquito bites.6,7 indonesian people’s behaviour is still not reliable enough to prevent bites from infectious disease vectors. despite the fact that eradicating mosquito nests is the main effort that can be done to prevent the infection of diseases such as dengue haemorrhagic fever, a disease that is one of the highlights in indonesia, the results of riskesdas’ report that indonesians are not very active in eradicating mosquito nests in their environment.4 nevertheless, research on the relationship between various factors and efforts to prevent mosquito bites in indonesia is still limited. research on the behaviour of mosquito are lacked. a research related to malaria transmission prevention efforts was found carried out only in the eastern part of indonesia.8 no article that focused on the specific factors that can influence the overall mosquito bite prevention behaviour of the overall indonesian people was found. therefore, a study on the influencing factors in preventing mosquito bite among indonesian society has been conducted that may contribute to build a recommendation policy to decrease the spread of mosquitoes-transmitted diseases, such as malaria and filariasis. materials and methods location of the study this is a national study conducted at all city/village in 34 provinces in indonesia. data and analysis the data used in this study was the secondary data of basic health research (riset kesehatan dasar or riskesdas) a survey research that has been conducted and developed since 2007, and was continued in 2010, 2013, and 2018.9–12 the questionnaire used was little different in each year. this includes the development of questions from 2013 to 2018. this research aims to describe 106 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 mutiara widawati, et al. the activities on prevention of malaria the indicators of indonesian public health situations that are used as the basis for policymaking at the national, provincial and district levels. these indicators include access to health services, environmental health, housing conditions, economy, infectious and non-infectious diseases, financial health, maternal and child health, and immunization. this research aims to describe the basic health status of the indonesian population, therefore the sample is taken from the population, which means that all indonesian citizens (265 million people) were taken into account (bps 2018) inform consent the survey was conducted by the health research and development institute of the indonesian ministry of health (badan litbangkes kemenkes). the survey was conducted by trained enumerators. each enumerator was trained to use the questionnaire and communicate with the respondent. enumerators are also trained to convey respondents' rights and obtain permission to collect data from respondents. each enumerator visited each selected household, accompanied by the village head and local health workers. each household, spouse or their elder person was asked to sign the inform consent to participate in this survey before starting the interview. children under 15 years old were interviewed accompanied by their parents/guardians. each respondent was informed about the research, and their option to stop the interview at any time without coercion. respondents who refused, gave up, decided to stop being interviewed and were not willing to be re-interviewed were excluded from the sample of this study. data related to individual identities were removed from the data subset for further analysis. areas with difficult access, natural disasters and conflicts were excluded in this survey. samples and variables the sample frame used in riskesdas 2018 was the 2018 socio-economic survey (susenas) samples of 300,000 households from 30,000 census blocks (bs). census blocks were selected using the probability proportional to size (pps) method with systematic random sampling in each city/village per district/city.4 riskesdas 2018 conducted a survey to a total sample of 295,720 households with a total of 1,091,528 household members (individuals) in 34 provinces of indonesia. data collection used a paper-based structured questionnaire which was asked to all household members. the riskesdas 2018 questionnaire consisted of a household questionnaire and an individual questionnaire. specifically for this study, the questionnaire data used were individual data only. the data used in this study was only the 2018 riskesdas data in the prevention of disease transmission due to mosquito bites (ministry of health research and development agency 2019). the independent variables studied included experience of exposure to malaria or filaria, age group, gender, recent education level, and area of residence. the dependent variable was including the use of repellents/materials to prevent mosquito bites, mosquito nets, and electric mosquito repellent device (example: electric mosquito racket) or not. the data were categorized into 2 categories, "yes" for respondents who prevented mosquito bites and "no" for respondents who did not use mosquito bite prevention at all. the first independent variable was the variable related to the experience of being exposed to malaria or filariasis, respondents who have been positive for malaria/have been tested for malaria/ have been given filarial medicine/have been positive for filaria were classified as having been exposed to vectorborne disease information. the second independent variable is the age group variable. this variable is categorized into 4 categories ranging from the age group 0-20 years old, 21-40 years old, 41-60 years old to older than 60 years old. gender variables include men and women, and the last variable, the area of residence which consisted of urban and rural areas. another 107 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: 104–112 independent variable was the level of education which was divided into 7 categories including never attended school, did not graduate from elementary school, graduated from elementary school, graduated from middle school, graduated from high school, graduated from diploma degree and graduated from college. data analysis this study conducted a multivariate test using logistic regression analysis to determine the factors that influence the behaviour in preventing mosquito bites. multivariate analysis was performed using logistic regression with a backward wald method to determine the relationship of the response variable (mosquito bite prevention behaviour) and each explanatory variable (exposure experience, age group, education level, gender, and regional characteristics). the relationship between variables was described by the value of the odd ratio (or) along with the value of the confidence interval (95% ci). the analysis was carried out to find the most optimal equation model. the difference in or was >10% between the previous model and the model after the variable was removed then used to determine whether the variable can be included in the final model or not. all data were analysed using spss version 15 (ibm inc., chicago, il.usa). ethic statement riskesdas 2018 has received ethical approval from the national ethics commission (ethics commission of the ministry of health research and development agency) number lb.02.01/2/ ke.024/2018. results and discussion this research was conducted to measure people's behaviour in preventing mosquito bites by several factors among indonesian society. the data of various factors was significantly associated with the mosquito bite prevention behaviour as p-values were <0.05 as shown in table 1. the experience of exposure the experience of exposure to vector borne disease was the factor that significantly influenced prevention efforts. respondents who had never been exposed to malaria or filaria were less likely to make an effort in preventing mosquito bites compared to respondents who had been exposed (0.609 times) (ci = 0.601-0.616; p <0.001). age the age group factors influenced prevention efforts. respondents below 20 years old were more likely to undertaken a bite prevention effort compared to those over 60 years old (1,261 times) (ci = 1,240-1,283; p <0.001). respondents between 21-40 years old also have a higher probability in taking mosquito bite prevention than respondents over 60 years old (1,385 times) (ci = 1,3611,411; p <0.001). in the 40-61 years old age group, more people did bite prevention compared to the respondents who were over 60 years old (1,037 times) (ci = 1,000-1,076; p <0.001). gender apart from experience of exposure to disease and age group, gender also has a significant role in influencing prevention efforts. male respondents were less likely to take prevention compared to female respondents (0.906 times) (ci = 0.897-0.915; p <0.001). education education was also a factor influencing prevention efforts. respondents with education level of ‘never attended school' were more likely to take precautions than respondents who graduated from college education level (1,164 times) (ci = 1,132-1,196; p <0.001). respondents who did not complete elementary school were also more likely to take precautions than respondents who graduated 108 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 mutiara widawati, et al. the activities on prevention of malaria from college (1,303 times) (ci = 1,271-1,336; p <0.001). respondents with elementary education level were more likely to take precautions than respondents who graduated from college (1,221 times) (ci=1,192-1,250; p <0.001). respondents who graduated from middle school also showed a tendency to take precautions compared to respondents who graduated from college (1,137 times) (ci = 1,110-1,165; p <0.001). respondents with a high school education level also showed a tendency to take precautions compared to respondents who graduated from college (1,066 times) (ci = 1,042-1,091; p <0.001) based on table 1 it can also be seen that respondents who graduated from diploma education were more likely to do prevention compared to respondents who graduated from college (1,037 times) (ci= 1,000-1,076; p= 0.053). regional the regional factors in table 1 shows that respondents in urban areas were less likely to take bite prevention efforts compared to respondents from rural areas (0.607 times) (ci = 0.601-0.613; p <0.001). the findings this study’s findings show that the percentage of respondents who did not do bite prevention was higher than who did it. these findings are in line with studies elsewhere.16 several studies in indonesia stated that inconvenience in practicing mosquito bite prevention is the main reason why many indonesians do not make efforts to prevent mosquito bites. for example, mosquito nets were reported to cause people to feel stiflingly hot.13 the reason for the inconvenient feeling was also reported by research related to mosquito repellent.14,15 besides the feeling of inconvenient there were other factors that might affect bite prevention practice. this current study revealed that experience of exposure to vector borne diseases (malaria or filariasis), gender, age group, education level, and area of residence were associated with mosquito bite prevention behaviour in indonesian society. experience of exposure to disease can affect a person's belief and knowledge of a disease. the experience of each individual's exposure to disease is closely linked to the public and private sectors. cooperation and participation or community involvement between the public (government) and private sectors are very important in order to create continual promotional messages related to bite prevention.16 table 1. the estimated association between mosquito bite prevention behaviour with various factors by logistic regression. factors prevention behaviour or 95%ci p-value yes (%) no (%) vector diseases experience no 20.2 54.2 0.609 0.601-0.616 <0.001 yes 4.4 21.1 1 age group 0-20 years old 9.2 30,1 1.261 1.240-1.283 <0.001 21-40 years old 6.7 20.7 1.385 1.361-1.411 <0.001 41-60 years old 6.2 18.4 1.296 1.273-1.319 <0.001 >60 years old 2.6 6.1 1 https://paperpile.com/c/wbnhcz/hc55 109 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: 104–112 gender men 12.5 36.5 0.906 0.897-0.915 <0.001 women 12.2 38.9 1 level of education never attended school 2.1 6.3 1.164 1.132-1.196 <0.001 did not finish elementary school 5.1 17.4 1.303 1.271-1.336 <0.001 elementary school graduate 5.7 18.7 1.221 1.192-1.250 <0.001 middle school graduate 4.2 12.6 1.137 1.110-1.165 <0.001 high school graduate 5.8 14.9 1.066 1.042-1.091 <0.001 diploma graduate 0.7 1.7 1.037 1.000-1.076 0.053 college graduate 1.4 3.3 1 area urban 13.2 29.1 0.607 0.601-0.613 <0.001 rural 11.5 46.3 1 in this study, people with exposure experience were the people who had their blood drawn to be tested for malaria/people who were positive for malaria/people who were given filariasis prevention drugs/ people diagnosed with filariasis by health workers. therefore, the delivery of health promotion presented by health workers when they were taking blood for malaria test or administering filariasis drugs is very important to encourage these individuals to take efforts in mosquito bites prevention. for respondents who have been exposed to vector-borne diseases, suffering from the disease is likely lead to behavioural changes in preventive measures to avoid the same infection. action theory states that the individual takes an action based on the experience, perception, understanding or emerges from certain situations, as well as the existence of a stimulus object.16 individuals' behaviour could also be influenced by their experience, acceptance/understanding of information, existing traditions and religion.17 the results of the study in table 1 show that the frequency of mosquito bites prevention is higher in respondents who have been exposed to vector-borne diseases than respondents who have never been exposed to it. the positive effect of the suffering experience has also been reported respondents who have been infected with malaria gave them a strong urge to recover.18 the results of this study indicate that women are more likely to make efforts to prevent mosquito bites than men. this in line with the health belief model theory that behaviour can be influenced by one's gender.19 this is probably because the indonesian women are commonly doing household chores, such as cleaning the house and therefore, mosquito control at home mostly done by housewife. better practice scores among women might be affected by their role and their sense of responsibility in taking care of their family and household needs.20 the same results were also reported bby vannayong 21 that female respondents were more willing to carry out activities to prevent mosquito bites, such as cleaning water reservoirs. the result of this study is also consistent with a study in ecuador, which reported that ecuadorian women had better 110 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 mutiara widawati, et al. the activities on prevention of malaria knowledge and doing better measures in disease prevention than men.22 a person's educational background might affect their job. people with college graduate backgrounds are more likely to have active jobs. a person's job status can inhibit or encourage a person's actions to live healthy, therefore a job status is defined as a predisposing factor.23 another possibility is that people at this level are reluctant to do the bite prevention since they feel that they do not have time to do it. a high level of activity makes someone who has malaria symptoms reluctant to take prevention action because of their busy schedule18. the results of analysis are similar to the results of wong's research 24 that dengue prevention behaviour is mostly carried out by respondents who do not work or workers who do not need skills (unskilled workers). unusual finding showed that the respondents from urban areas had significantly lower probability in doing bite prevention than those who live in rural areas. the high availability of vector’s habitat in rural areas might cause this finding. the malaria and filaria vector mosquitoes prefer the outdoor areas. water is an important factor in the life cycle of these mosquitoes, mosquitoes leave their larvae which then develop into adult mosquitoes. the anopheles mosquito, which acts as a vector for malaria, is commonly found in rice fields, brackish water, swamps, and mountainous areas. mosquitoes can live in clear water and come into direct contact with the ground. meanwhile, the culex mosquito, which is the vector for filariasis, can be found living in polluted water, such as ditches, rivers full of garbage, and standing water, but can also be found living in clear water.25 rural communities are more likely to have larger yards than urban communities. the size of the yards can sometimes cause rural communities to be more active in controlling the mosquito breeding places. when the rainy season comes, the leaves will filled with water and become a mosquitoes breeding place. the lush plants also affects the mosquito population because mosquitoes prefer to perch.25 plants will block sunlight and make the place even more humid and suitable for resting place.26 this increases the risk of being bitten by mosquitoes, which may make the village community more active in carrying out mosquito bite prevention activities. limitations of the study in practice, this study was inseparable from several limitations. the education variable in this study did not have an even number of respondents at each level, the higher education variable tended to be less than the other respondents. therefore, interpretation must be carried out with caution. in addition, this study did not include occupational factors as one of the explanatory factors, therefore further studies are needed to show a direct association between work and mosquito bite prevention practices. we conceptualized the general picture by taking into account urban and rural disparities based on a study conducted by wilson 27 and asingizwe.28 communities living in less developed areas or rural areas might have a lack of access to improved housing, essential health services, effective and timely diagnosis and treatment that might contribute to the higher risk of the transmission of malaria. studies also have shown that financial problems have been a significant challenge in delivering malaria prevention and treatment program in rural communities. other disparities from riskesdas can be seen in other publications.29 conclusions respondents who have experience of being exposed to malaria or filaria, under 60 years old, women, college graduates, and rural communities are more likely to perform mosquito bites prevention, therefore they could be empowered in promoting public 111 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: 104–112 awareness towards mosquito bites prevention. 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 the authors acknowledge the support received from indonesian ministry of health, particularly thank to the director of the national insitute of health research and development, ministry of health of indonesia. we are grateful for the invaluable participation of the indonesian citizen in riskesdas survey. special thanks are also given to the research team and all stakeholders who participated in and contributed to this study. 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. world health organization. world malaria report 2018. 2019. 2 babu bv, swain bk, rath k. impact of chronic lymphatic filariasis on quantity and quality of productive work among weavers in an endemic village from india. trop med int heal 2006. doi:doi:10.1111/j.1365-3156.2006. 01617.x. 3 sitohang v, sariwati e, fajariyani sb, et al. malaria elimination in indonesia: halfway there. lancet glob heal 2018. doi:doi:10.1016/s2214109x(18)30198-0. 4 badan litbangkes kemenkes. the 2018 indonesia basic health survey (riskesdas). jakarta, 2019. 5 supali t, djuardi y, lomiga a, et al. comparison of the impact of annual and semiannual mass drug administration on lymphatic filariasis prevalence in flores island, indonesia. am j trop med hyg 2019. 6 afify a, betz jf, riabinina o, c l, potter cj. commonly used insect repellents hide human odors from anopheles mosquitoes. curr biol 2019; 29: 3669–3680. 7 hogarh jn, agyekum tp, bempah ck, et al. health risks and benefits of the use of mosquito coils as malaria prevention and control strategy. malar j 2018; 17: 265. 8 hermawan a, hananto m. faktor sosiodemografi dan perilaku pencegahan gigitan nyamuk terhadap perilaku pemberantasan sarang nyamuk di indonesia: analisis lanjut 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activities on prevention of malaria 19 fishbein m, ajzen i. belief, attitude, intention, and behavior: an introduction to theory and research. 1977. 20 pujiyanti a, triratnawati a. the knowledge and experience of dengue mosquitoes among housewives. makara j heal res 2011; 15. doi:doi:10.7454/msk.v15i1.792. 21 vannavong n, seidu r, stenström t-a, dada n, hj o. effects of socio-demographic characteristics and household water management on aedes aegypti production in suburban and rural villages in laos and thailand. parasit vectors 2017. 22 cabezas m, fornasini m, dardenne n, borja t, albert a. a cross-sectional study to assess knowledge about hiv/aids transmission and prevention measures in company workers in ecuador. bmc public health 2013; 13: 139. 23 gorsky ra. resource reviews: health promotion planning: an educational and environmental approach, step by step to substance abuse prevention: the planning guide to school-based programs. am j heal promot 1991; 5. doi:doi:10.4278/0890-11715.6.414. 24 wong lp, shakir smm, atefi n, abubakar s. factors affecting dengue prevention practices: nationwide survey of the malaysian public. plos one 2015; 10. doi:doi:10.1371/journal.pone.0122890. 25 agustina e. fauna nyamuk vektor tular penyakit dan tempat perindukannya di kawasan kampus uin ar-raniry. pros biot 2018; 3. 26 marina r, azhar k, lasut d, et al. faktor lingkungan dan perilaku pemberantasan sarang nyamuk terhadap status transmisi demam berdarah dengue di kecamatan mustikajaya, kota bekasi. vektora j vektor dan reserv penyakit 2020; 12: 53–60. 27 wilson ml, krogstad dj, arinaitwe e, arevaloherrera m, l c, ferreira mu et al. urban malaria: understanding its epidemiology, ecology, and transmission across seven diverse icemr network sites. am j trop med hyg 2019; 93. 28 asingizwe d, poortvliet pm, koenraadt cjm, van vliet ajh, ingabire cm, mutesa l et al. role of individual perceptions in the consistent use of malaria preventive measures: mixed methods evidence from rural rwanda. malar j 2019; 18: 270. 29 mara i, widawati m, laksono ad, kusrini i, dhewantara pw. variation of preventive practices and its association with malaria infection in eastern indonesia: findings from community-based survey. plos one 2020; 15. doi:doi:10.1371/journal.pone.0232909. 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 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 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 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 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 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ijtid@itd.unair.ac.id 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. 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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. �� 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 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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 ijtid vol 3 no 2 april juni 2012.indd 76 vol. 3. no. 2 april–juni 2012 analysis of hiv subtypes and clinical staging of hiv disease/aids in east java yulia sari ismail1, soetjipto2, eddy bagus wasito3, nasronudin4 1 alumny doctoral-study program of medical science, graduate school, universitas airlangga 2 biochemistry department, medical faculty, universitas airlangga 3 microbiology department, medical faculty, universitas airlangga 4 institute of tropical disease, universitas airlangga abstract human immunodeficiency virus type 1 (hiv-1) known to cause acquired immune deficiency syndrome (aids) disease are divided into several subtypes (a, b, c, d, f, g, h, j, k) and circulating recombinant form (crf). different characteristics of subtype of the virus and its interaction with the host can affect the severity of the disease. this study was to analyze hiv-1 subtypes circulating in hiv/aids patients from the east java region descriptively and to analyze its relationship with clinical stadiums of hiv/aids. information from this research was expected to complement the data of mocular epidemiology of hiv in indonesia. this study utilited blood plasma from patients who had been tested to be hiv positive who sected treatment to or were reffered to the intermediate care unit of infectious disease (upipi) dr. soetomo hospital surabaya from various area representing the east java regions. plasma was separated from blood samples by centrifugation for use in the the molecular biology examination including rna extraction, nested pcr using specific primer for hiv gp120 env gene region, dna purifying, dna sequencing, and homology and phylogenetic analysis. based on the nucleotide sequence of the hiv gp120 env gene, it was found that the most dominant subtypes in east java were in one group of circulating recombinant form (crf) that is crf01_ae, crf33_01b and crf34_01b which was also found in southeast asia. in the phylogenetic tree, most of hiv samples (30 samples) are in the same branch with crf01_ae, crf33_01b and crf34_01b, except for one sample (hiv40) which is in the same branch with subtype b. hiv subtypes are associated with clinical stadiums (disease severity) since samples from different stages of hiv disease have the same subtype. keywords: hiv, aids, molecular biology examination, subtype, clinical stadium abstrak latar belakang: virus hiv pada manusia (hiv-1) diketahui menyebabkan acquired immune deficiency syndrome (aids) terbagi menjadi beberapa subtipe (a, b, c, d, f, g, h, j, k) dan circulating recombinant form (crf). karakteristik yang berbeda dari subtipe virus dan interaksi dengan host dapat mempengaruhi keparahan penyakit. tujuan: penelitian ini dilakukan untuk menganalisis hiv-1 subtipe secara deskriptif, yang beredar di kalangan pasien hiv / aids dari wilayah jawa timur dan untuk untuk menganalisis hubungan dengan stadion klinis hiv / aids. informasi dari penelitian ini diharapkan dapat melengkapi data epidemiologi mocular hiv di indonesia. metode: penelitian ini menggunakan plasma darah dari pasien yang telah diuji untuk menjadi hiv positif yang berobat ke atau dirujuk ke unit perawatan menengah penyakit infeksi (upipi) dr soetomo surabaya dari berbagai daerah yang mewakili wilayah jawa timur. plasma dipisahkan dari sampel darah melalui sentrifugasi untuk digunakan dalam pemeriksaan biologi molekuler termasuk ekstraksi rna, proses pcr dengan menggunakan primer spesifik untuk wilayah hiv gp120 gen env, pemurnian dna, sekuensing dna, dan analisis homologi dan filogenetik. hasil: berdasarkan urutan nukleotida dari gen gp120 hiv env, ditemukan bahwa subtipe yang paling dominan di jawa timur berada dalam satu kelompok circulating recombinant form (crf) yang crf01_ae, crf33_01b dan crf34_01b yang juga ditemukan di asia tenggara. pada pohon filogenetik, sebagian besar sampel hiv (30 sampel) berada di cabang yang sama dengan crf01_ae, crf33_01b dan crf34_01b, kecuali satu sampel (hiv40) adalah di cabang yang sama dengan subtipe b. hiv subtipe dikaitkan dengan stadion klinis (keparahan penyakit ) karena sampel dari berbagai tahap penyakit hiv memiliki subtipe yang sama. kata kunci: hiv, aids, pemeriksaan biologi molekuler, subtipe, stadium klinis research report 77ismail, et al.: analysis of hiv subtypes and clinical staging of hiv disease / aids introduction aids (acquired immune deficiency syndrome) is one of the most feared diseases in the world today. a disease that causes decreased immunity of a person is caused by germs hiv (human immunodeficiency virus). epidemiological situation of hiv/aids in the world is continued to worry about. the prevalence of aids cases in east java is 9.80 per 100,000 populations, by 3540 of the cumulative number of cases in the province. east java is now ranked as the number of cases and the spread of hiv/aids, rising from third to second place in the dki jakarta (moh, 2010). it is known that there are two types of hiv, namely hiv-1 and hiv-2. the main cause of aids in the world today is the majority of hiv-1. this species is divided into three groups: group m (main), group o (outlier) and group n (new/non-m, non-o). group m is widespread and is the most common cause of hiv/aids epidemic worldwide. group m is divided into several subtypes, which until now has been recognized several subtypes, namely a1, a2, a3, a4, b, c, d, f1, f2, g, h, j, and k.1 between a subtype with other subtypes can form the so-called recombinant crf (circulating recombinant form) and, until now, 43 crf has been found.2 differences in the characteristics of subtypes of the virus and its interactions with the human host may influence the severity of the disease. some studies proposed that the hiv subtype variation associated with the clinical stage of disease. a research in senegal, against sex workers who were infected with subtype a, c, d and g, found that the development of aids increased in patients infected with non-subtype a.3 in thailand, the study of survival of patients infected with crf01_ae showed shorter time since hiv-1 infection to death compared to western populations.4 several other studies in africa also state that certain subtypes that increased the severity of disease in patients with hiv/aids than other subtypes. on the basis of the high prevalence of aids in the community caused by hiv, the required examination of dna subtypes can be done in order to make it a more efficient so that aids prevention and eradication of disease can be more successful. it is necessary for the proper diagnosis of aids patients, and then examined the subtype of virus infected for business/further precautions. information on the rate of subtype differences in clinical stage of hiv/aids is important for proper testing of hiv vaccines aimed at slowing the progression of disease severity and in the management of hiv infected individuals. this genetic information will provide a strong addition to the standard data to determine the epidemiological pattern of the spread of the virus. molecular epidemiology supports classical epidemiology in terms of import sources to confirm the virus known, a virus subtype that is obtained by a known virus subtypeps circulating in a country. this research was expected to contribute data about the types of hiv subtypes in east java, so that the chain of transmission of hiv/aids can be controlled both at the national, regional and global levels. subtypes of hiv virus can also be reported to the who to complement the hiv virus molecular epidemiologic data that already exist in the who. the data of this study can also be used as a consideration for further research in an attempt to perform virus isolation, and manufacture of candidate hiv vaccine for aids is more in line with the subtypes of hiv virus in indonesia and the clinical stage. hiv genome consisting of genes that encode the viral structural proteins are among the major genes gag, pol and env. env sequence variation is high enough. various groups and different hiv subtypes have been characterized genetically by sequences of the env gene. thus, env is the main target area for studying subtype associated with the epidemiology, as it can provide information on all circulating subtypes in a given geographical area.5 the present study used the env gene of hiv-1 gp120 as a target due to its high regional variability (v) and constant region (c). this study aims to determine the descriptive subtypes of hiv-1 circulating in patients with hiv/aids from east java and to study its relationship with clinical stage of hiv disease/aids. in particular, the purpose of this study are as follows: 1. analyzing the most predominant hiv subtype in east java. 2. analyzing kinship (phylogenetic analysis) hiv in east java. 3. analyzing the relationship between clinical stage of hiv infection/aids with hiv subtype in east java. materials and methods because the target to be assessed in this study is the type of hiv virus, blood samples were taken only from patients who had been hiv positive for the virus and went to the intermediate care unit of infectious diseases (upipi) hospital dr. soetomo and had not received antiretroviral therapy. patients were randomly selected from different regions of origin to represent some of the areas in east java. blood sampling performed by medical personnel hospital dr. soetomo was trained. ± 3 ml of blood sample was collected in edta tubes vacutube (to prevent freezing). then, blood samples were taken into a cool box which had been given the icepack/ice cubes to the laboratory hepatitis/aids institute for tropical diseases (tropical disease center/tdc) within 6 hours maximum after taking them. in the laboratory, the sample tubes were centrifuged to separate the plasma from the blood. plasma obtained was transferred into a 2 ml microtube and stored at temperatures –80° c until use. collection of blood samples was carried on until the minimum amount of sufficient, appropriate, statistical calculations. this ultimately obtained blood sample from 46 patients. in addition to taking blood sample, we also collected other data from the patient such as age, gender, origin, stage of disease, cd4 count, history of other diseases, infection group, and so forth. furthermore, hiv viral rna extraction 78 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 76−82 from blood plasma was collected using a reagent qiaamp viral mini kit from qiagen. in this study, the gp120 env gene was the target. for the hiv, rna should be made of dna by the reverse process trancription. by using the onestep rt-pcr reagents from qiagen, we could the reverse trancription and pcr amplification in one step. the process of the first round of pcr (first round pcr) was used, namely hiv-specific primers ed5 and ed12 forward reverse.6,7 from the first round, the pcr amplicon size obtained was 1200 bp. to increase the specificity of the gene sought, and also because the size of the amplicon is large enough so that the concern will complicate the process of dna sequencing, the nested pcr was performed. on the first round of pcr products, pcr was performed with the second round pcr reagents from promega gotaq green pcr using primers as mastermix es7x forward and reverse e1256,7 to produce amplicon size 300 bp ~. pcr process was performed many times for the optimization of annealing temperature to find the right. therefore, the result of the pcr product was good. sometimes, the process of pcr has also repeated on the samples that gave negative results. to view the results pcr was performed on pcr products electrophoresis sample of 2% agarose gel, and then observed using uv transiluminator (short wave = 254 nm). picture of dna bands on the gel was photographed using a digital camera. before performing the dna sequencing, the pcr product had to be purified first. the process of purification was performed using reagents qiaquick purification kit from qiagen. if the sample results of the second round pcr gave a positive result, then the pcr product was used for the sequencing process, but when the second round pcr was negative, it was used for sequencing the first round pcr products. sometimes there were also bands of dna samples that did not look positive or too thin in the first round pcr, but they would appear after the second round pcr. before the purified pcr products of positive/ clear tape on-electrophoresis then what? after that, under long-wave uv light (365 nm) the gel containing the target dna band was cut, then the gel was diluted with buffers contained in the kit, qiaquick dna purification obtained was pure. purified dna was then at-labeled by using the pcr primer es7x specifically for labeling. after obtaining a label, we precipitated dna sequencing according to standard procedures, then inserted into the dna sequencing machines ready abi prism 310 genetic analyzer for sequence traced. the result of this nucleotide sequencing of electroferogram was a diagram showing the peaks representing the nucleotide. sometimes, there was a sample that produced a good picture electroferogram with clear peaks, but there were also samples of which the electroferogram was not good. in some resequencing process the samples needed to be treated to get a good electroferogram. of the 46 samples, 31 samples that produced a pretty good electroferogram were obtained. the other samples have a negative pcr result was that it was impossible to continue the process of sequencing, and some sample results of electroferogram are not good. samples that had been successful in hiv-sequencing and homology analysis were treated to make the filogenetic tree. table 1. characteristic epidemiology and clinical of the subjects co infected hiv in east java characteristic number (people) percentage (%) gender male (age 19-54 years; mean: 34.39) female (age 25-56 years; mean: 34.25) from surabaya madura gresik sidoarjo pasuruan madiun bojonegoro banyuwangi kediri tuban lumajang risk factor penasun homoseksual heteroseksual stage of disease stadium i stadium ii stadium iii stadium iv 30 16 25 4 4 2 2 2 2 2 1 1 1 3 2 41 7 0 29 10 65.22 34.78 54.35 8.70 8.70 4.35 4.35 4.35 4.35 4.35 2.17 2.17 2.17 6.52 4.35 89.13 15.22 0 63.04 21.74 79ismail, et al.: analysis of hiv subtypes and clinical staging of hiv disease / aids results and discussion in this study, several samples of 46 patients with positive acquired human immunodeficiency virus infection (hiv) were obtained. these patients referred to hospitals for treatment or dr. soetomo surabaya from some areas in east java. epidemiological and clinical characteristics of the study subjects can be seen in table 1. the 46 patients who were positively infected by hiv in this study, consisted of 30 men (65.22%) and 16 women (34.78%). the ages of the patients ranged from 19 to 56 years old with the average of 34.39 years old. the ages of the male patients range of 19 to 54 years old, with the average of 34.47 years old. meanwhile, the ages of the female patiens ranged from 25 up to 56 years old, with the average of 34.25 years. the subjects in the study came from several areas in east java and were referred to hospitals for treatment or to dr. soetomo hospital surabaya from some areas in east java as shown in table 1. patients were randomly selected from different regions of origins to represent some of the areas in east java. patients from surabaya, amounted to 25 people (54,35%), dominated the whole subjects of the study. other subjects came from including that include madura (4 people), gresik (4 people), sidoarjo (2 people), makati (2 people), madison (2 people), bojonegoro (2 people), banyuwangi (2 people), karachi (1 person), tuban (1 person) and lumajang (1 person). transmission of hiv/aids can occur through various methods of disease transmission, or so-called risk factors, namely injecting drug users (idus), heterosexual behavior or illicit sex, homosexual sex, from pregnant mothers to the fetus, blood transfusion, and other unknown causes. based on the data obtained in this study, the most dominant risk factor was heterosexuality amounted to 41 cases (89.13%), while the other risk factors are injecting drug users (only 3 cases) and homosexuality (only 2 cases) (table 1). according to the report issued by the national aids commission (nac) in the international symposium in padalarang, west java on october 21, 2011, the behavior or heterosexual sex is now the main culprit in the spread of hiv/aids in indonesia. in 2006, the trend of transmission of hiv/aids in indonesia was dominated by the use of a syringe while a 54.42% of contributor to hiv/aids cases were skil unreported, and contributet to 38.5%. conditions to the contrary were the place in 2011 where injecting drug users risk factor decreased to 16.3%, while the heterosexual risk factors reached 76.3%. this means that the majority of hiv/aids in indonesia is transmitted through casual heterosexual, sex that apparently also happeneds in east java. in table 1, it can also be seen that clinical characteristics of hiv/aids are the subjects of this study. patients with clinical stage i-stage amounted to only 7 people, patients with stage iii amounted to 29 people, and stage iv patients amounted to 10 people. in the present study we found no patients with stage ii. it appears that hiv/ aids patients in hospitals dr. soetomo surabaya are still dominated by advanced stage patients rather than early stage patients. this is probably still due the lack of awareness or the courage to see her early on, that makes them go to hospitals when the condition of the disease is severe. the government, the business, and all the parties need to resolve this issue, for example, by doing a counseling and free examinations for the whole society. because what needs to be assessed in this study are the type of the hiv virus, the blood samples were taken only from patients who had been hiv positive for the virus had gone to the intermediate care unit of infectious diseases (upipi) hospital dr. soetomo and had not received any antiretroviral therapy. the overall patients who become the subjects of this study were examined for antibodies to hiv using a standard procedure for patients in hospitals upipi dr. soetomo, namely by using three kinds of antibody rapid test kit: oncoprobe, sd triline and hiv 1/2. the use of the three types of inspections was intended to avoid mistakes in making the diagnosis, since the diagnosis of hiv infection is a diagnosis that widely affects not only the patients but also the surrounding environment and also controls efforts undertaken by the government. hiv dna detection using pcr technology is one choice of method for diagnosis of hiv infection under situations in which the detection of antibodies gives negative and still questionable results.8 for the pcr in this study we used pairs of primers that had been used and published in international journals.6,7 the 46 samples from hiv-positive patients were infected with hiv. the pcr of the env gene coding for hiv gp120 protein obtained positive pcr results amounted to 34 samples. in the examination of the samples with antibodies to hiv positive and hiv pcr positive, the patients’ bodies still contained hiv rna that they still had the potential to transmit the hiv virus. the pcr process was conducted many times to seek optimization annealing temperature (annealing) until the right one, could be found. therefore, the resulting pcr product appeared to be good. sometimes, the process of pcr was also repeated on samples that gave negative results. in the sample that was pcr negative results with the use of pairs in this study, the possibility of change / mutation in the nucleotide sequence point of attachment of the primary, so the primary cannot be attached to the result that the negative pcr results. primer pairs in this study is that when the primer pair used pcr amplification of nucleotides and can provide a positive, after sequencing, the nucleotide sequence obtained will be used to determine the hiv subtype. to find hiv subtypes, the nucleotide sequence obtained in this study was then compared with the nucleotide sequences that had been published. hiv dna pcr result was further purified by amplification and sequencing using the abi-310 sequencer engine. the result of this sequencing of the electroferogram was a diagram showing the peaks representing the nucleotide. in this study, sometimes, there was a sample that produced a good electroferogram with clear peaks, but 80 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 76−82 there were also samples of electroferogram which appeared not very good. in some re-sequencing, the samples needed to be treated to get a good electroferogram. the 34 samples that produced positive pcr result obtained 31 samples of electroferogram that were pretty good. the other samples had a negative pcr result that it was impossible to continue the process of sequencing, and 3 samples were positive for pcr-sequencing, the electroferograms which were not good or not readable. from the results obtained by sequencing, and by molecular analysis to determine the hiv genotyping and nucleotide, the sequence homology was obtained. to find hiv subtypes, the nucleotide sequence of sequencing results obtained in this study was compared with other hiv subtypes of nucleotide sequences that have been published.9,10,11 then analyzed to make a phylogenetic tree. nucleotide sequence of sequencing results obtained from samples of people with hiv/aids was used to detect genetic variations or mutations in hiv dna pcr results in this study. it has been argued that the identification of hiv-1 that differs in hiv env causes are grouped into: m, n and o. group m is the most frequently encountered and is divided into nine subtypes based on the whole genome which are geographically distinct9,10,11 namely subtypes a, b, c, d, f, g, h , j and k. hiv subtype is further subdivided into subtype, which includes a1, a2, f1 and f2.10 the argument states that the different hiv subtypes may also differ in the effects of transmission (transmission), the emergence of drug resistance and the disease perogresifitas. it has also been argued that subtypes b (found in north america figure 1. tree neighbor-joining phylogenetic models of gp120 env sequences of hiv samples and reference 81ismail, et al.: analysis of hiv subtypes and clinical staging of hiv disease / aids and europe), a and d (africa), c (africa and asia) are the most prevalent ones. these subtypes form branches in a tree depicting the genetic ancestry of the group m of hiv-1. co-infection with different subtypes leads to the increased circulating recombinant forms (crfs). in 2000, a global analysis of prevalent subtypes was made stating that 47.2% of infections worldwide were subtype c, 26.7% were subtype a/crf02_ag, 12.3% were subtype b, 5.3% were subtype d, 3.2% were crf_ae, and the remaining 5.3% consisted of other subtypes as well as crfs.12 most research focused on hiv-1 subtype b, while others focud on the few other subtypes.13 the hiv nucleotide sequences obtained were analyzed and ready (as many as 31 of the 34 sequences that had been expected). we also conducted a molecular phylogenetic analysis of hiv nucleotide and phylogenetic tree drawn by a computer program clone manager version 6 6:00, with 128 nucleotides with various subtypes of hiv-references that had been published previously (http://www.hiv.lanl. gov). the results obtained, turned out to be hiv from hiv/aids patients in this study. as many as 30 samples in one group were circulating recombinant forms (crfs), especially crf01_ae, crf33_01b, and crf34_01b originating from thailand and malaysia. for the first sample, the sample was located in one group hiv40 branching with subtype b. the result of the molecular analysis as the result of 31 hiv sequencing of this study homology in the form of multiple nucleotide alignment was 300 nucleotides long. the results of the molecular analysis which were intended to determine the hiv subtype in the form of a phylogenetic tree of nucleotide along with the length of 300 nucleotides (gp120 env gene v3 region) consisted of 31 samples, and the results of this study of hiv subtypes (a, b, c, d, f, g, h, i , j and k) and a variety of crf have been published. the form of a phylogenetic tree of hiv subtypes are shown in figure 1. 31 samples were successfully determined. 5 samples were from stage i patients. 19 samples were from stage iii patients and 7 samples were from stage iv patients. almost all the samples had the same subtype and only one sample had a different subtype that it can be connected between the degree or the stage of disease infecting the hiv subtypes. from the results of statistical tests that were obtained between hiv subtype and the clinical stage, there was no significant relationship (exact p = 1.000 by contingency coefficient = 0.144). thus, in this study it has not been proven whether the subtypes of hiv influence disease severity. the design of this study was cross sectional so that what the readers can do is analyze the relationship between the subtype of hiv with the degree of disease in a single observation. a longitudinal study in europe by and another one in thailand by stated that there was no difference of disease progression among hiv-1 subtypes that were different. however, the study of in his studies in africa had non-b subtypes that were often found in patients with severe degree of disease (stage iii and iv). conclusion the results of this study was based on nucleotide sequences of the env gene of hiv gp120 it can also be concluded that the most predominant hiv subtype in east java in one group circulating recombinant forms (crfs) that crf01_ae, crf3x_01b, and crf34_01b which were also found in various countries of southeast asia. in the phylogenetic tree, 30 hiv samples in a branching kinship with the subtype crf01_ae, crf34_01b, and crf33_01b, while the hiv-1 samples are in a branching hiv40 kinship with subtype b. in addition, hiv subtype is not associated with clinical stages (disease severity) while samples from different stages of hiv disease have the same dominant subtype. references 1. taylor bs, me sobieszczyk, fe mccutchan, sm hammer. 2008. the challenge of hiv-1 subtype diversity. new england journal of medicine 358; 15: 1590–602. 2. lihana rw, sa khamadi, rm lwembe, jg kinyua, jk muriuki, nj lagat, et al. 2009. hiv-1 subtype and viral tropism determination for evaluating antiretroviral therapy options: an analysis of archived kenyan blood samples. bmc infectious disease 9: 215. 3. kanki pj, dj hamel, jl sankale, et al. 1999. human immunodeficiency virus type 1 subtypes differ in disease progression. journal of infecious disease. 179: 68–73. 4. costello c, ke nelson, v suriyanon, et al. 2005. hiv-1 subtype e progression among norther thai couple: traditional and non-traditional predictors of survival. international journal of epidemiology. 34: 577–84. 5. pieniazek d, j baggs, dj hu, gm matar, am abdelnoor, je mokhbat, m uwaydah, et al. 1998. introduction of hiv-2 and multiple hiv-1 subtypes to lebanon. emerg infect dis. 4: 649–56. 6. foley b, e donegan, n silitonga, fs wignall, mp busch, el delwart. 2001. importation of multiple hiv type 1 strains into west papua, indonesia (irian jaya). aids research and human retroviruses. vol.17, no.17, pp. 1655–9. 7. delwart el, b herring, ag rodrigo, ji mullins. 1995. genetic subtyping of human immunodeficiency virus using a heteroduplex mobility assay. genome research. 4: s202–16. 8. panteleeff dd, g john, r nduati, d mbori-ngacha, b richardson, et al. 1999. rapid method for screening dried blood samples on filter paper for hiv type 1 dna. journal of clinical microbiology. 37(2): 350–3. 9. robertson dl, b hahn, p sharp. 1995. recombination in aids viruses. j mol evol. 40(3): 249–59. 10. antunes r, s figueiredo, is ba’rtolo, m pinheiro, l rosado, et al. 2003. plasma samples from a pediatric population predominantly infected with hiv type 1 subtype g and bg recombinant forms. journal of clinical microbiology. 41(7): 3361–7. 11. khamdi sa, rw lihana, s osman, j mwangi, j muriuki, et al. 2009. genetic diversity of hiv type 1 along the coastal strip of kenya. aids research and human retroviruses. 25(9): 919–23. 12. osmanov s, c pattou, n walker, b schwarlander, j esparza and the who-unaids network for hiv isolation and characterization. 2002. estimated global distribution and regional spread of hiv-1 genetic subtypes in the year 2002. j acquir immune defic syndr. 29: 184–90. 13. perrin l, l kaiser, s yerly. 2003. travel and the spread of hiv-1 genetic variants. lancet infect dis. 3(1): 22–7. 14. alaeus a, lidman k, bjorkman a, giesecke j, albert j. 1999. similar rate of disease progression among individuals infected with hiv-1 genetic subtypes a-d. aids. 13(8): 901–7. 82 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 76−82 15. amornkul pn, tansuphasawadikul s, limpakajanarat k, likanonsakul s, young n, et al. 1999. clinical disease associated with subtype b’ and e infection among 2014 patients in thailand. aids. 13(14): 1963–9. 16. departemen kesehatan ri. 2010. laporan triwulan i statistik kasus hiv/aids di indonesia. ditjen ppm & pl. 17. jacobs gb, c de beer, je fincham, v adams, ma dhansay, et al. 2006. serotyping and genotyping of hiv-1 infection in residents of kayelitsha, cape town, south africa. journal of medical virology. 78: 1529–36. 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. 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 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 ................................................................................................................. 66–76 404 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 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, 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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. vol. 10 no. 1 january–april 2022 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ * corresponding author: yunika.trisnawati.s.kep-2020@fkm.unair.ac.id original article an overview of covid-19 patients in rsud bhakti dharma husada surabaya from september 2020 to june 2021 yunika trisnawati1,2*, firman suryadi rahman3 1prevention and control infections committee of rsud bhakti dharma husada surabaya, indonesia 2master program of epidemiology, faculty of public health, universitas airlangga, surabaya, indonesia 3doctoral program of public health, faculty of public health, universitas airlangga, surabaya, indonesia received: 24th august 2021; revised: 8th september 2021; accepted: 2nd december 2021 abstract the covid-19 pandemic has been lasting more than a year. until now, research on the analysis of an overview of covid-19patients has not been carried out at rsud bhakti dharma husada surabaya. this study aims to describe the covid-19 cases in rsud bhakti dharma husada surabaya about the gender of patients, highest number of patients =, the most recovered patient, the highest death rate occurred, and case fatality rate (cfr). this study is a descriptive observational study with a case series approach. the data used in this study were covid-19 data from the application of online hospital ditjen yankes from september 2020 to june 2021. the majority of covid-19 cases occured in women (53.04 %). the covid-19 patients mostly came to the hospital in june 2021, about 241. the most recovered patients in oktober were 255 patients. the highest death rates occurred in june 2021 ware 47 patients. case fatality rate (cfr) is at 5.79 % because in june 2021 the health facilities were full, and cause patients did not get help quickly. many patients have been forced to self-isolate at home so that they have worsened and fi nally died. most covid-19 patients who were treated at the rsud bhakti dharma husada surabaya from 2020 to june 2021 occurred in women and the most patients who were admitted was in june 2021. keywords: descriptive, overview, patient, covid-19, hospital abstrak pandemi covid-19 telah berlangsung lebih dari satu tahun. penelitian tentang analisis gambaran pasien covid-19 di rsud bhakti dharma husada surabaya hingga saat ini belum dilakukan. penelitian ini bertujuan menggambarkan kasus covid-19 yang ada di rsu bhakti dharma husada surabaya tentang jenis kelamin pasien, pasien yang paling banyak masuk rumah sakit, pasien yang paling banyak sembuh, angka kematian pasien paling tinggi dan case fatality rate (cfr). penelitian ini merupakan penelitian deskriptif observasional dengan pendekatan case series. sumber data pada penelitian ini adalah data ovid-19 terjadi pada perempuan (53.04%). pasien covid-19 paling banyak masuk pada bulan juni 2021 sejumlah 241. pasien paling banyak sembuh ada di bulan oktober yaitu 255 pasien terjadi angka kematian paling tinggi di bulan juni 2021 sebanyak 47 pasien. case fatality rate (cfr) berada di angka 5.79 % sebab di bulan juni 2021 fasilitas kesehatan penuh, sehingga pasien tidak segera mendapatkan perawatan. banyak pasien yang terpaksa isolasi mandiri di rumah sehingga kondisinya semakin parah dan akhirnya meninggal dunia. pasienovid-19 yang dirawat di rsud bhakti dharma husada surabaya pada bulan september 2020-juni 2021 paling banyak terjadi pada perempuan dan pasien paling banyak masuk pada bulan juni 2021. kata kunci: deskrptif, gambaran, pasien,covid d-19, rumah sakit ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 43yunika trisnawati, et al.: an overview of covid-19 patients in rsud bhakti dharma husada surabaya how to cite: trisnawati, y., rahman, f. s. the overview of covid-19 patients in rsud bhakti dharma husada surabaya from september 2020 to june 2021. indonesian journal of tropical and infectious disease, 10(1), p. 42–47, apr. 2022. introduction covid-19 is a communicable disease fi rstly reported as novel coronavirus is as a caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2).ˡ since declared a pandemic by who starting on march 11, 2020, until now the covid-19 pandemic is still ongoing.2 after indonesia reported the first case on march 2, 2020, covid-19 cases in indonesia at the end of december 2020 had reached 743,198 people. 611,097 patients were recovered and 22,138 patients died. cases are increasing and spreading rapidly throughout indonesia, including surabaya. as of july 15, 2021, the surabaya city covid-19 task force reported 32,297 confi rmed covid 19 cases with 1,433 deaths.3,6 rsud bhakti dharma husada is one of the covid-19 referral hospitals with a capacity of 164 beds for covid-19 patients. sirs.kemkes. go.id first version has been used to collect data on covid-19 patients in hospitals from march 2020 to august 2020. in the fi rst version, the data collection is name, email, phone number, address, gender, age, date admission, patient status, date of discharge, discharge status, nik, type of patient (suspect, confirmation), diagnosis, and laboratory examination. since september 2020, sirs.kemkes.go.id the second version has been used where data collection is in the form of daily data for triage er patients, daily data for patients admitted, daily data for patients treated with comorbidities, daily data for patients treated without comorbidities, and daily data for patients discharged.4 this study aims to provide an analysis of the description of covidid-19 patients at the bhakti dharma husada hospital surabaya as an input in handling covid-19 cases in the city of surabaya especially the bhakti dharma husada hospital surabaya. materials and methods materials this research was an observational descriptive study with a case series approach. the source of data in this study is secondary data taken from the online hospital application of the directorate general of health and health version 2 (two) where the data started from september 2020 until the data collection for this study ended in june 2021. this study describes the incidence of covid-19 with a case approach, epidemiology by person, and time. the variables studied in this study were gender, admitted patients, recovered patients, and deceased patients at bhakti dharma husada hospital surabaya. the case fatality rate (cfr) variable is the result of the division between the number of confirmed covid-19 deaths in a certain period and the number of confirmed covid-19 cases in that period multiplied by 100% (who criteria). results and discussion in august 2020, rsud bhakti dharma husada had treated 554 confirmed covid-19 patients (figure 1). figure 1. coronavirus cases march – august 2020 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 44 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 42–47 the numbers of covid-19 patients who entered the bhakti dharma husada hospital in the period, marchaugust 2020 were the most in july namely 167 patients, and the lowest were in march with three patients. the gender of covid-19 patients who entered the bhakti dharma husada hospital in the period september 2020 june 2021 the most were female, namely 436 patients (53.04%). males gender was 386 patients (46.96%) as shown in table 1. the total numbers of covid-19 patients who entered the bhakti dharma husada hospital in the period september 2020 june 2021 were the most in june as many as 241 patients, and the lowest in march 2021 with 21 patients. the most recovered patients were in october about 255 patients, and the lowest in april 2021 with 15 patients. the highest death rate occurred in june 2021 namely 47 patients, the lowest in april and may 2021 namely 0 (zero) as shown in fugre 2. table 1. distribution of covid-19 cases based on people at bhakti dharma husada hospital september 2020-june 2021 cases by people cases (month-year) amount sept oct nov dec jan feb mar apr may jun 2020 2020 2020 2020 2021 2021 2021 2021 2021 2021 gender n % male 44 27 19 64 45 25 7 9 12 133 386 46.96 female 59 34 40 60 66 23 14 14 18 108 436 53.04 total 103 62 59 124 111 48 21 23 30 241 822 100 figure 2. distribution of covid-19 cases based on time at bhakti dharma husada hospital surabaya september 2020-june 2021 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 45yunika trisnawati, et al.: an overview of covid-19 patients in rsud bhakti dharma husada surabaya table 2. distribution of deaths of covid-19 patients at bhakti dharma husada hospital surabaya september 2020-june 2021 no month confirmed died patient confirmed patient cfr(%) 1 september 2020 2 197 1.02 2 october 2020 1 154 0.65 3 november 2020 14 187 7.49 4 december 2020 14 344 4.07 5 january 2021 28 350 8.00 6 february 2021 9 162 5.56 7 march 2021 5 96 5.21 8 april 2021 0 80 0.00 9 may 2021 0 57 0.00 10 june 2021 47 444 10.59 amount 120 2017 5.79 according to the revised ministry of health covid-19 guidelines, covid-19 deaths for surveillance purposes are confirmed/probable covid-19 cases that have died. the case fatality rate (cfr) of the bhakti dharma husada hospital in the period september 2020 june 2021 was 5.79 % as shown in table 2. covid-19 case pattern based on gender the gender of covid-19 patients who entered the bhakti dharma husada hospital in the period september 2020 june 2021 mostly were female, namely 436 patients (53.04%). it is also in line with who that the percentage of infection distribution in males is greater than in females (51% vs 47%) with some variations across age groups. based on the data from 77, 000 deaths in the case-based reporting database (nearly 30% of all known deaths), there appear to be higher numbers of deaths (45,000 or 58%) in men. geographical variations in infection rates and deaths among women and men of diff erent age groups are probable; however, available data come from relatively few countries and are, therefore, skewed. consequently, any interpretation of the gender differences across age groups and countries must be made with great caution. these limitations underline the urgent need for better and completed reporting of data by sex and age, as a minimum, for better identifi cation and understand the key differences and disparities to inform a more effective covid19 response. evidence from past epidemics, such as the sars coronavirus outbreak in 2002−2003, shows that men and women are likely to have both different susceptibilities to the virus and different vulnerabilities to the infection as a result of both sexand gender-related factors. data (on persons tested, the severity of the disease, hospitalization rates, discharge [recovery], and health worker status) that are disaggregated at a minimum by sex and age – as well as by other stratifies such as socioeconomic status, ethnicity, sexual orientation, gender identity, refugee status, etc., where feasible – could help in identifying and addressing health inequities related to covid19.27 covid-19 case pattern based on time according to who science in 5 on covid19, some factors are contributing to increased transmission around the world. the first are these variants of concern, including the delta variant which rapidly takes off and spreads between people more efficiently than even the alpha variant that was first detected around december to january 2021. the second factor is that we have increased social mixing and increased ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 46 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 42–47 social mobility, which increases the number of contacts that individuals have. the third factor is the relaxation or the inappropriate use of public health and social measures. proven public health and social measures we know prevent infections, reduce the spread of somebody who is infected with the virus to others, and save lives. and the fourth factor is the uneven and inequitable distribution of vaccines.9 covid-19 case fatality pattern the results of the study show that more covid-19 deaths occurred in june 2021 with 47 patients as shown in table 2. one of the causes of the high number of cases of death is influenced by the increasing number of active cases in june 2021. this is because the health facilities were full, causing patients not to get help quickly. many patients have been forced to self-isolate at home so that they have worsened and have been admitted to the hospital in severe conditions. the results showed that, from september 2020-june 2021, the majority of covidd-19 cases occurred in june 2021, most patients recovered in october 2020 and most patients died in june 2021 as shown in figure 2. this study shows that the largest increase in cases and death rates of covid-19 patients occurred in june 2021 where this occurred .throughout indonesia and the world 3,5,6 covid-19 case fatality pattern according to table 2, the total mortality of confirmed patients who died was 120 people (cfr 5.79%). age, occupation (entrepreneur and farmer/trader), contact history, symptoms (fever, dyspnea, cough, lethargic, and cold), and comorbidities (diabetes, copd, hypertension, cancer, heart disease, neurological disorders, and immune disorders) were risk factors of covid19 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 covid19 who have risk factors. in another article, the mean case fatality rate for adults aged under 60 is estimated to be less than 0.2%, compared with 9.3% in those aged over 80. even if comorbidities increased mortality risk by five times, the risk would remain lower for younger people than for most older adults.11 conclusions the majority of covid-19 patients treated at the bhakti dharma husada hospital from september 2020 to june 2021 were female ; 436 (53,04 %), the covid-19 patients mostly came to the hospital in june 2021, about 241. the most recovered patients were in october namely 255 patients. the highest death rate occurred in june 2021 namely 47 patients. case fatality rate (cfr) is at 5.79 % because in june 2021 the health facilities were full, and cause patients did not get help quickly. many patients have been forced to self-isolate at home so that they have worsened and fi nally died. the urgent need for better and completed reporting of data by sex and age, as a minimum, for better identifi cation and understand the key diff erences and disparities to inform a more eff ective covid-19 response. assessment of the history of vaccine is very important. based on what we know so far, vaccines are proving eff ective against existing variants, especially at preventing severe disease, hospitalization and death. acknowledgement the authors are grateful for the cooperation of the head and all staff of rsud bhakti dharma husada, and to all local authorities that facilitated this study. conflict of interest the authors declare that they have no conflict of interest. ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 47yunika trisnawati, et al.: an overview of covid-19 patients in rsud bhakti dharma husada surabaya references 1. centers for disease control and prevention (cdc). novel coronavirus (2019-ncov) [internet]. 2020. 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annu. rev. public health. 2007 apr 21;28:33-54. 23. leung k, wu jt, leung gm. effects of adjusting public health, travel, and social measures during the roll-out of covid-19 vaccination: a modelling study. the lancet public health. 2021 aug 11. 24. gan cc, dwirahmadi f. how can the public be better protected against covid-19?. jurnal berkala epidemiologi. 2020 may 31;8(2):97-9. 25. gille f, brall c. public trust: caught between hype and need. international journal of public health.2020.65(3), 233–234. 26. aldrich dp, oum s, sawada y, editors. resilience and recovery in asian disasters: community ties, market mechanisms, and governance. tokyo: springer japan; 2015. 27. who, gender and covid-19 : 2020 [cited 2021 august 20]. available from: https://apps.who.int/iris/ bitstream/handle/10665/332080/who-2019-ncov advocacy_brief-gender-2020.1-eng.pd 4. the health ministry of indonesia.hospital management information system. ditjen yankes.2021 http://sirs. kemkes.go.id/versi/ [cited 2021 july 15]. 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 ijtid vol 3 no 2 april juni 2012.indd 92 vol. 3. no. 2 april–juni 2012 evaluation on the effect of antiretroviral drugs on cd4 t-cell and the increment of body weight among hiv-aids patients in surabaya edith frederika1, irine normalina1, nasronudin, rury mega1 1 institute of tropical disease, universitas airlangga abstract antiretroviral drug discovery has encouraged a revolution in the care of people living with hiv, although it has not been able to cure diseases and to increase the challenge in terms of drug side effects. side effects of antiretroviral drugs are fairly common occurrences in hiv patients and generally occurr within the first three months after initiation of antiretroviral therapy, although long-term side effects are also often found afterwards. this study aims to evaluate the number of cd4 t-cells in patients with aids before and after getting on arv therapy, the side effects arising during the taking of arvs are related to the increment of body weight among the hivaids patients. subjects were then narrowed down from 25 to 12 due to the incomplete data. the results showed that the top three most side effects which often occur in people with aids are appetite loss (20.0%), nausea (17.8%), and diarrhoea (15.6%). meanwhile, about 58% of the subjects experienced increment of their body weight, and 42% were losing weight due to the side effects of the arv therapy. among those who lost their body weight, 50% were in the productive ages between 21–30 years old. the present study shows that combination antiretroviral therapy gives good results to the increased number of cd4 t-cells in patients living with hiv, as shown by the tendency of an increment in the number of cd4 t-cells in patients who received antiretroviral therapy. however, around 42% of those patients were losing weight because of the side effects of the therapy. therefore, the importance of giving specific nutrient to overcome with the weight loss is needed to be given to the patients hiv instead of only giving the arv treatment. keywords: antiretroviral drug side effects, cd4 t-cell, weight loss, weight gain, nutrition abstrak latar belakang: penemuan obat antiretroviral mendorong sebuah revolusi dalam perawatan orang yang hidup dengan hiv, meskipun belum mampu menyembuhkan penyakit dan meningkatkan tantangan dalam hal efek samping obat. efek samping dari obat antiretroviral adalah kejadian yang cukup umum pada pasien hiv dan umumnya terjadi dalam tiga bulan pertama setelah memulai terapi antiretroviral, meskipun jangka panjang efek samping sering juga ditemukan setelah itu. tujuan: untuk mengevaluasi jumlah cd4 t-sel pada pasien dengan aids sebelum dan setelah mendapatkan terapi arv, efek samping yang timbul selama arv dikonsumsi dan dikaitkan dengan peningkatan berat badan di antara pasien hiv-aids tersebut. subyek kemudian dipersempit dari 25 orang menjadi 12 orang karena data yang kurang lengkap. hasil: penelitian menunjukkan bahwa terdapat 3 efek samping tertbanyak yang paling yang sering terjadi pada penderita aids antara lain kehilangan nafsu makan (20,0%), mual (17,8%), dan diare (15,6%). sementara itu, sekitar 58% dari subyek memiliki peningkatan berat badan, dan 42% yang kehilangan berat badan karena efek samping dari terapi arv. di antara mereka yang kehilangan berat badan, 50% berada di usia produktif antara 21 30 tahun. dalam penelitian ini menunjukkan bahwa kombinasi terapi antiretroviral memberikan hasil yang baik bagi peningkatan jumlah cd4 t-sel pada pasien yang hidup dengan hiv, seperti yang ditunjukkan oleh kecenderungan kenaikan dalam jumlah cd4 t-sel pada pasien yang menerima terapi antiretroviral. namun, sekitar 42% dari pasien yang kehilangan berat badan karena efek samping dari terapi tersebut. oleh karena itu, pentingnya memberikan nutrisi yang spesifik untuk mengatasi dengan penurunan berat badan yang perlu diberikan kepada pasien hiv bukan hanya memberikan pengobatan arv saja. kata kunci: efek samping obat antiretroviral, cd4 t-sel, penurunan berat badan, berat badan, nutrisi case report 93frederika, et al.: evaluation on the effect of antiretroviral drugs introduction the human immunodeficiency virus (hiv) is one of the most serious, deadly diseases in human history. hiv causes a condition called acquired immunodeficiency syndrome, commonly known as aids. hiv destroys type of defence cell in the body’s immune system called cd4 helper lymphocyte. a healthy body has cd4 helper lymphocytes cells (cd4¯ t cells) which helps the immune system to function normally and fight off certain kinds of infections by acting as a messenger to other types of immune system cells, telling them to become active and fight against an invading germ. when hiv destroys these lymphocytes, the immune system becomes weak and the people get more serious infection than that of normal people. hiv attaches to these cd4¯ t cells. the virus then infects the cells and uses them as a place to multiply. while doing so, the virus destroys the ability of the infected cells to do their job in the immune system. the body therefore loses the ability to fight many infections. once the number of cd4¯ t cells per microliter (μl) of blood drops below 200, the cellular immunity is lost. acute hiv infection usually progress es overtime to clinical latent hiv infection and then early symptomatic hiv infection and later to aids which is identified on the basis of the amount of cd4¯ t cells remaining in the blood.1 several symptoms of hiv infection and aids may not appear for as long as 10 years or more. people with hiv may not notice any signs that they have the virus. however, doctors nowadays can diagnose someone with aids when that person’s blood lacks a number of cd4¯ t cells which are required to fight the infections. doctors can also diagnose aids if the person has signs of specific illness or diseases that may occur to people with hiv infection, such as fatigue or extreme weakness, rapid weight loss, frequent fever, heavy sweating, swollen lymph glands, chronic diarrhoea, coughing, or even minor infections such as skin rashes.2 aims of study the aim of this study is to evaluate the effect of antiretroviral (arv) drugs that are given to the hiv-aids patients. the antiretroviral (arv) drugs may give effect on the cd4 t-cells and also cause some specific symptoms which lead to neither increment nor deterioration of body weight among the patients. materials and method twenty five patients with hiv positive from a private clinic in surabaya were involved in the study. the treatment for hiv infection was given with high active antiretroviral (arv) therapy which slowed down the progression of the diseases, including preventatives and active treatment of opportunistic infections. current antiretroviral (arv) treatment options according to the who recommendations are combinations consisting of at least three medications belonging to at least two types of antiretroviral agents: an nnrti (nonnucleoside reverse transcriptase inhibitor) and two nrtis (nucleoside analogue reverse transcriptase inhibitors). the typical nrtis include zidoyudine (azt) or tenofovir (tdf) and lamiyudine (3tc) or emtricitabine (ftc). the arv combination may reduce the resistance of hiv replication. therefore the opportunistic infection could be avoided. however, the side effects of the drug therapy may occur within 3 months after the consumption of arv. some medication can upset the stomach which may lead to a weight loss. nevertheless the arv therapy is a way to increase the body weight which may be affected due to the infection.3 each patient was given antiretroviral (arv) drugs with different combinations namely azt+3tc+nvp, azt+3tc+efv, and dat+3tc+efv. conversely, due to the lack of the patient data, the sample of this study reduced to 12 people. among these patients, we only evaluated the side effects of the drugs without differentiatin the types of drug combination associating with their body weight. results and discussion this research shown the evaluation on the effect of arv drug on cd4 t-cell before and after the therapy, and the increment of body weight of the hiv-aids patients, and also shows the side effects that occur during the arv consumption. there were 25 samples in total; however it was reduced to 12 samples due to the incomplete data. among 12 samples, the combination of arv therapy gave a quite good result on the increasing amount of the cd4 t-cell as shown in the graphic below. 0 100 200 300 400 500 af dm sg bd as rc x y ay an stp scp 35 428 394 12 229 7 19 162 59 1 130 176104 257 76.5 14 79.5 1 206.5 44 91 10 79.5 101 before after amount of cd4 t-cell before and after arv therapy graphic 1. the amount of cd4 t-cell before and after arv therapy based on the graphic, some results show the reduction of the cd4 t-cells. these conditions were caused by the 94 indonesian journal of tropical and infectious disease, vol. 3. no. 2 april–june 2012: 92−95 secondary infections which consequenced to the purpose of the therapy. even though the arv therapy may give some side effects however it has been proven to improve the amount of the cd4 t-cells continuously. the 12 samples in this research were vandus age. most of the patients were in productive ages, between 21 to 30 years old (in category). it can be seen in the table below. table 1. age category no age n percentage (%) 1 < 20 years old 0 2 21 – 30 years old 6 50.0 3 31 – 40 years old 3 25.0 4 41 – 50 years old 1 8.3 5 51 – 60 years old 0 6 > 61 years old 2 16.7 half of the samples were in the ages between 21–30 years old, which were consided productive ages. comparisons with the increment the of body weight as a result of the arv therapy are show in the graphic below. graphic 2. the increment of body weight within age category graphic 3. increment of body weight the total samples, approximately 58.3% patients had gained weight. however, around 41.7% lost their weight. this increment of body weight might have associated with the arv therapy, nevertheless there were still patients whose body weight were deteriorated. the side effects of the arv therapy may have possibly caused the weight loss. based on the information acquired, 10 side effects occurred among the hiv-aids patients during the arv therapy within 1 to 3 years. each patient had the most frequent side effects that they experienced within the 10 side effects that occured. the side effects are shown in the table below: table 2. the most frequent side effect of arv therapy no side effect freq. percentage (%) 1 nausea 8 17.8 2 fatigue 3 6.7 3 sleeping problem 7 15.6 4 lose appetite 9 20.0 5 muscle pain 1 2.2 6 diarrhoea 7 15.6 7 skin rashes 4 8.9 8 breathless 1 2.2 9 fever 2 4.4 10 headache 3 6.7 the top 3 most common side effects which occur among the hiv-aids patients are appetite loss (20.0%), nausea (17.8%), and diarrhoea (15.6%). the appetite loss side effect among the hiv-aids patients due to the arv therapy, may have leaded to the reduction of their body weight, as well as the other side effects such nausea and diarrhoea. some of arv medications can upset stomach, this is why the side effects arise and the patients loose their weight.4 with respect to the dietary advice for hiv-aids patient, some evidence has shown a benefit of micronutrient supplements,5 dietary intake of macronutrients at rda (recommended dietary allowance) levels by hiv-infected adults is recommended by the world health organization.6 furthermore, who states that several studies indicate that supplementation of vitamin a, zinc, and iron can reduce adverse effects in hiv-positive adults to help improve body weight. there are some nutrition guidelines for hiv-aids patients. the basic thing is to eat more because extra muscle weight will help to fight hiv. balance diet containing macronutrients are essential.7 protein helps build and maintain muscle.8 the good sources of protein came from meat, fish, beans, nuts, and seeds. carbohydrates are the best macronutrient that give energy, which came from grains, cereals, vegetables, and fruits. carbohydrates are also a good source of fibre. an other micronutrient is fat, which is needed in an appropriate amount. monounsaturated fats are considered good fats if they are found in nuts, seeds, olive oil, and fish. to maintain a balance diet, a moderate exercise will help our body to transform the food into muscles. the easiest way is to include exercises into our daily activities (walking or cycling). moreover, taking supplements can also help to maintain our body weight and get the vitamins and the minerals we need.9 last but not least, enough liquid is necessary. extra water can reduce the side effects of 95frederika, et al.: evaluation on the effect of antiretroviral drugs medications and also helps to concurrence the diarrhoea problem. however, such drinks as tea, coffee, carbonated drinks, chocolate or even alcohol should be avoided since these drinks may actually associate with body liquid loss. conclusion antiretroviral drugs (arv) have been proven to increase the amount of cd4 t-cells which may help our immune system to function normally and fight off certain kinds of infections. these cd4 t-cells acted as the messenger to other types of immune system cells, telling them to become active and fight against an invading germ. however, the side effects of the arv therapy among hiv patients are some symptoms related to the appetite which may cause deterioration to body weight. these symptoms are (1) appetite loss, (2) nausea, and (3) diarrhoea, which then lead to a weight loss. to address the problem, supplementation of vitamin a, zinc, and iron can reduce adverse effects in hiv-positive adults to help them improve their body weight, maintain their body weight and get the vitamins and the minerals they need. moreover, a balance diet must contain macronutrients that are essential such as high carbohydrates (to give energy), and protein (to help building muscles). extra water can also help to reduce the side effects of medications and to overcome diarrhoea problems. furthermore, to maintain a balance diet, it is recommended thats patients keep being active by doing some light exercise such as walking or cycling, in order to retain their body fitness, to gain appetite, as well as to preserve their body muscles. references 1. paton n. i. et al., 2006, “the impact of malnutrition on survival and the cd4 count response in hiv-infected patients starting antiretroviral therapy”, hiv medicine 7(5). 2. who, 2010, “antiretroviral therapy of hiv infection in infants and children in resource-limited settings: towards universal access” 3. koethe, john r md. et al., 2010, ‘association between weight gain and clinical outcomes among malnourished adults initiating antiretroviral therapy in lusaka, zambia’ jaids 53(4) 507–13. 4. tang, a. m. et al., 2002, “weight loss and survival in hiv-positive patients in the era of highly active antiretroviral therapy”, jaids 31(2). 5. friis, h, 2006, “micronutrient interventions and hiv infection: a review of current evidence”, tropical medicine & international health 11(12). 6. who, 2005, “micronutrients and hiv infection: a review of current evidence”. 7. nerad j. et al., 2003, “general nutrition management in patients infected with human immunodeficiency virus”, clinical infectious diseases 36(suppl 2). 8. dudgeon, w. d., 2006, “counteracting muscle wasting in hivinfected individuals”, hiv medicine 7(5). 9. hendricks, k. m. et al., 2007, “dietary supplement use and nutrient intake in hiv-infected persons”, aids reader 17(4). 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 55 vol. 1. no. 2 may–august 2010 research report serotype and clinical performance of dengue virus infection on the year 2009 soegeng soegijanto1,2,3,4, widodo darmowandowo1,2, amor p. ginting1,2 and atsushi yamanaka5 1 department of child health dr. soetomo hospital surabaya 2 medical faculty of airlangga university surabaya 3 head of dengue virus infection researcher team 4 institute of tropical disease center airlangga university 5 kobe university graduate school of medicine abstract dengue hemorrhagic fever is one of the important health problem in indonesia, mortality rate is becoming decrease but many dengue shock syndrome cases is very difficult to be help. previous study showed 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. there are four serotype of dengue virus infection can cause primary and secondary infection. the aim of this research is to know the relationship between clinical performance of dengue virus infection and serotype dengue virus and also to know the role of primary and secondary infection and age of dengue virus cases. a prospective analytic observational study, which was conducted in dr. soetomo hospital since january 2009. rt-pcr was used to attempt to identify the infecting serotype from dengue virus isolated using vero cell. antibody responses were measured by elisa and clinical manifestation were measured with the who criteria 1997. dengue serotype identification by rt-pcr was 70 patients. virus types were den-2 65(92.8%), den-1 3(4.2%), and den-3 2(2.8%). patients with den-1 genotype iv were more trend severe disease dss and unusual infection. commanly usually secondary exposure cause more severe clinical manifestation than primary exposure (p = 0.035) but in this study found that all of den-1 genotype iv, primary or secondary infection to show severe clinical manifestation of dengue virus infection. we can conclude that den-2 was the most dominant serotype in dr. soetomo hospital. on primary and secondary infection, den-1 genotype iv showing more severe than den-2 and den-3. key words: serotype, clinical performance, dengue virus infection introduction 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 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. 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 finded 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. there are four serotype of dengue virus infection which can cause primary and secondary infection. clinical performance of secondary infection to show more severe clinical manifestation than primary infection of dengue virus. the role of serotype and age of cases will influence the severe clinical manifestation of dengue virus infection. based on of the clinical and bio medical problem that mention above the researcher want to identify serotype dengue virus infection that circulating in surabaya; to know the relationship between clinical performance of dengue virus infection and serotype dengue virus and also to know the role of primary and secondary infection and 56 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 55-59 age patient/age virus of dengue virus cases. the study had been done in dr. soetomo hospital on 2009. 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 become in patient at 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. table 1. age distribution of dengue virus infection clinical performance & diagnostic age (year) df dhf dss unusual total 1–4 9 10 5 0 24 5–14 22 16 5 3 46 total 31 26 10 3 70 table 2. sex distribution of dengue virus infection clinical performance & diagnostic sex df dhf dss unusual total boy 15 8 5 2 30 girl 16 18 5 1 40 total 31 26 10 3 70 age and sex distribution of 70 cases dengue virus infection showed that: school age children especially girl were found more prevalence suffering from dhf than pre school age (see table 1 & 2) table 3. distribution of serotype dengue virus based on age serotype of dengue virus age (year) den 1 den 2 den 3 den 4 total 1–4 2* 20 2 0 24 5–14 1 45 0 0 46 total 3 65 2 0 70 serotype den2 was dominant than den1 & den3 (see table 3) had exposured 40 cases of school age children which clinical performance of df 14 cases, dhf 19 cases, dss 7 cases. all of them showed secondary immune response and 5 cases of school age children and 20 cases pre school age showed primary immune respon (see table 7). table 4. distribution of immune response of dengue virus infection based on age immune response of dengue virus infection age (year) primary secondary total 1–4 11 (45,8%) 13 (54,2%) 24 (100%) 5–14 15 (32,6%) 31 (67,4%) 46 (100%) total 26 (37,1%) 44 (62,9%) 70 (100%) this table 4, give information that the school age children showed more higher cases suffering from secondary of dengue virus infection. table 5. 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) 57soegijanto et al.: serotype and clinical performance of dengue virus infection table 6. distribution of serotype on 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) 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 6). serotype den 1 was usually mild case but this study 1 case showed a severe clinical performance as dss and identified as primary infection (see table5). the second case was identified as secondary dengue virus infection and the third case was an unusual case which showed secondary of dengue virus infection (see table 7). based on yamanaka this serotype den 1 might be have genotype iv or mention as den 1 genotype iv. table 7. 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 discussion this study found that 65.7% cases of dengue virus infection were on school age group children and more higher than pre-school age group (34.3%). it correlated with previous study based on explanation that many mosquito of aedes aegypti and albocpitus were found surrounding the school where the pupil got education every day and playing football, baseball where the mosquito can bite one or more pupil especially if the sanitation and hygiene of school were not routine controlled for the population of larva. in pre-school age group children showed primary infection of dengue virus 45.8% were more found and showed a clinical performance of dhf. however the secondary infection more found in school age group children (67.4%). the study in asia & latin america found there were a trend to be increase dengue virus infected cases in older than younger. the outbreak of dhf cases in bangladesh on 2000 found that all of dhf cases death had age more than 5 years old. in school age group children, there were secondary infection of dengue virus with clinical performance of dengue fever (67.4%). e ong (2008) study in singapore has showed there were increasing secondary infection in children with age 1–5 years (0.77%) and age 6–10 years (6.7%). and had a high risk of dengue virus infection in school age group children. den 2 serotype were dominant and then followed by den 1 & den 3. in this study den 1 genotype iv has showed a clinical performance of dss (dhf grade iii & iv) with primary infection and unusual case of dhf with showed primary & secondary infection. study in thailand, den 2 serotype showed more severe of clinical performance and it was influenced by serotype and had a special strain. den 2 in south east asia had showed more severe than den 2 in latin america. den 2 in africa were very rare causing health problem in human being. study in singapore 2008 found serotype den 2 was dominant and followed by serotype den 1 & den 3. our study in surabaya 2009 had found serotype den 1 genotype iv, this showed a severe clinical performance as dengue shock syndrome and unusual dhf cases with primary and secondary dengue virus infection. some studies in thailand showed serotype den 2 more severe clinical performance; it was influenced by dominant serotype and special strain of dengue virus. in south east asia serotype den 2 were dominant than latin america and in africa. in africa serotype den 2 was rarely found and did not make health problem in human being. leit meyer 1999 found that the differences dengue virus virulence based on structure protein e, prm, ns1 & ns3. protein e was a first antigen that influencing to entry and attachment endosom by fusion method and making virion. the differences of amino acid structure at the specific area can cause changing of antigen for attachment can cause replication of virus. rico herse 1990 showed the distribution of den 1 based on protein e & ns1 was found in: genotype i america & africa; genotype ii srilanka; genotype iii japan; genotype iv south east asia, australia and mexico; genotype v taiwan and thailand. manoa 2004 found the differences of clinical performance serotype den 1, such as den 1 genotype v virus can show 58 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 55-59 a clinical performance of dengue fever and den 1 genotype iv showed a clinical performance of dhf & dss in some cases cannot be help and death. some of them showed as primary infection dengue virus. this den 1 genotype iv were also found in indonesia. this phenomena also has founded in this study. halstead hypothesis give information that secondary infection dengue virus can cause a severe clinical performance of dengue virus infection, it maybe due to ade for promoting dengue virus to entry in many target cell of organ making children become a case of dengue virus infection with a severe clinical performance. in this study, serotype den 1 genotype iv can promote a severe clinical performance of dss & unusual dhf infection whatever with primary or secondary infection of dengue virus. laili 2004, den 1 genotype iv one of genotype dengue virus that had been found in indonesia and had shown a severe clinical performance. the other den 1 with difference genotype had shown a mild clinical performance. however secondary infection of dengue virus showed more severe clinical performance than primary infection dengue virus. conclusion 1. dengue virus infection in dr. soetomo hospital surabaya has more cases with den 2 following by den 1 and den 3 2. serotype dengue virus influence to clinical performance of dengue virus infection especially den 1 genotype iv can cause severe clinical performance significant. 3. secondary infection of dengue virus showed more severe clinical performance than primary infection dengue virus based on statistical analysis. however in this study one case of den 1 genotype iv with primary infection tend to have more severe clinical performance of dengue virus infection and this case could not be handled. recommendation 1. the future study focusing on genotype and the differences of structure dengue virus which to learn the pathogenesis of dengue virus infection. 2. study should be done in many cities in indonesia. reference alison i, janet m, 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py, huang jh, 2004. current advances in dengue diagnosis. clinl diagn lab immunol 11:642–650. soegijanto s, 1999. masalah penyakit dbd di indonesia. dalam: soegijanto s. demam berdarah dengue. surabaya: airlangga university press, hlm 5-9. university press, hlm 5-9. sriprom m, pongsumpun p, yoksan s, et al, 2003. dengue haemorrhagic fever in thailand, 1998–2003: primary or secondary infection. dengue bulletin 27:36–46. takol c, siripen k, sukathida u, 2007. dengue virus (denv) antibodydependent enhancement of infection upregulates the production of anti-inflamatory cytokines. j virol 88:365–375. vaughn dw, green s, kalayanarooj s, et al,2000. dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. j infect dis 181:2–9. veeradej vw, endy tp, samakoses r, et al, 2003. transplacentallytransplacentally transferred maternal-infant antibodies to dengue virus. am j trop med hyg 69:123–128. ijtid vol 1 no 2 may-aug 2010.3.pdf ijtid vol 1 no 2 may-aug 2010.4.pdf ijtid vol 1 no 2 may-aug 2010.5.pdf ijtid vol 1 no 2 may-aug 2010.6.pdf ijtid vol 1 no 2 may-aug 2010.7.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 vol. 10 no. 1 january–april 2022 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ review article clostridium diffi cile infection (cdi) by hypervirulent bi/nap1/027 strain: a comprehensive review of toxigenicity, pathogenesis, risk factors, and preventative measures ni nyoman sri budayanti1, i gusti putu suka aryana2*, ni luh putu harta wedari3 1clinical microbiology department, faculty of medicine, universitas udayana, universitas udayana hospital, bali, indonesia 2geriatric division, internal medicine department, faculty of medicine, universitas udayana, sanglah general hospital, bali, indonesia 3clinical microbiology specialist program, faculty of medicine, universitas udayana, sanglah general hospital, bali, indonesia received: 29th october 2021; revised: 17th january 2022; accepted: 26th january 2022 abstract clostridium diffi cile is an anaerobic bacil gram-positive bacteria, able to form spores and toxin, that is transmitted among humans through the fecal–oral route. clostridium diffi cile infection (cdi), a typical nosocomial infection has been contributed to a signifi cant proportion of morbidity and mortality among in-patients with a case-fatality rate of 14% within 30 days after diagnosis. profound culture and toxin examination for c. diffi cile are still minimal in many hospitals in various asian countries. consequently, c. diffi cile reports in asia remain rare. highly virulent form of c. diffi cile caused greater fatality and epidemics severity. elderly age, hospitalization, exposure to antibiotics e.g., cephalosporins, fl uoroquinolones, clindamycin, and penicillin contributed as main risk factors. hypervirulent strain bi/nap1/027 demonstrated to carry cdtloc gene locus encodes cd196 adp-ribosyltransferase (cdt) or known as binary toxin. virulence factors are tcda, tcdb, cdta cdtb in which hypersporulation and mutation of tcd gene by hypervirulent strain led to toxin hyperexpression. early cases detection, building management team to evaluate patient positive with all c. diffi cile toxins, hand hygiene improvement, continuation of contact precautions after diarrhea resolution, audit of infection control, and restriction of antimicrobials should be implemented as preventative measures. focus measures also should emphasize on development of vaccine of c. diffi cile to boost immune state of elderly people. this review aims to describe severity of disease caused by hypervirulent bi/nap1/027 c. diffi cile strain, its mechanism or pathogenesis, risk factors, current treatment options available, along with proposed preventative measures and infection control. keywords: clostridium diffi cile infection (cdi), hypervirulent strain, bi/nap1/027 abstrak clostridium diffi cile adalah bakteri basil gram positif anaerobik, pembentuk spora dan toksin, yang ditularkan di antara manusia melalui rute fekal-oral. clostridium diffi cile infection (cdi), sebuah tipikal infeksi nosokomial telah berkontribusi pada proporsi yang signifi kan terhadap morbiditas dan mortalitas di antara pasien rawat inap dengan tingkat fatalitas kasus 14% dalam waktu 30 hari setelah diagnosis. kultur dan pemeriksaan toksin c. diffi cile masih minim di banyak rumah sakit di berbagai negara asia. akibatnya, laporan c. diffi cile di asia masih jarang. epidemi kematian dan keparahan yang lebih besar dari cdi disebabkan oleh c. diffi cile yang hipervirulen. faktor risiko utama adalah usia lanjut, rawat inap, paparan antibiotik misalnya sefalosporin, fl uoroquinolones, klindamisin, dan penisilin. strain hipervirulen bi/nap1/027 terbukti membawa lokus gen cdtloc yang mengkode cd196 adp-ribosyltransferase (cdt) atau dikenal sebagai toksin biner. faktor virulensi yaitu tcda, tcdb, cdta cdtb; strain hipervirulen mampu melakukan hipersporulasi dan mutasi gen tcd yang menyebabkan hiperekspresi toksin. tindakan pencegahan dapat dilakukan dengan deteksi dini kasus, pembentukan tim manajemen untuk mengevaluasi pasien yang positif semua toksin c. diffi cile, peningkatan kebersihan tangan, kelanjutan tindakan pencegahan kontak setelah resolusi diare, audit pengendalian infeksi, dan pembatasan antimikroba. fokus upaya juga sebaiknya ditekankan pada pengembangan vaksin c. diffi cile untuk meningkatkan * corresponding author: ptsuka_aryana@unud.ac.id ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 28 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 27–41 status kekebalan pada individu berusia lanjut. tinjauan ini bertujuan untuk menggambarkan tingkat keparahan penyakit yang disebabkan oleh strain c. diffi cile bi/nap1/027 hipervirulen, mekanisme atau patogenesisnya, faktor risiko, pilihan pengobatan yang tersedia, serta tindakan pencegahan dan pengendalian infeksi. kata kunci: clostridium diffi cile infection (cdi), strain hipervirulen, bi/nap1/027 how to cite: budayanti, n. n. s., aryana, i. g. p. s., wedari, n. l. p. h., clostridium diffi cile infection (cdi) by hypervirulent bi/nap1/027 strain: a comprehensive review of toxigenicity, pathogenesis, risk factors, and preventative measures. indonesian journal of tropical and infectious disease, 10(1), p. 27–41, apr. 2022. introduction clostridium difficile infection (cdi) has been known as a typical nosocomial infection and contributes to a signifi cant proportion of morbidity and mortality among in-patients with a case-fatality rate of 14% within 30 days after diagnosis.1 c. diffi cile gives rise to numerous infections varying from mild diarrhea to pseudomembranous colitis (pmc), mainly in elderly patients with antibiotic treatment. in addition, high healthcare costs related to cdi increase the fi nancial burden of government on health expenditure. it was recorded that half a million infections were attributed to cdi in the united states in 2011 with an incidence rate of 8.75 cases/1,000 adult admissions in 2009.2,3 a literature study by collins et al.4 found few data of cdi cases. the study found that a study in japan only reported on the ribotyping result of c. diffi cile without any information on cdi prevalence or incidence in japan; cdi incidence increased from 1.7/1,000 to 2.7/1,000 adults in korea, and 17.1/10,000 inpatients in shanghai were attributed to cdi. meanwhile, about 44% and 14% of colitis positive patients were positively diagnosed with the c. diffi cile toxin in philippine and malaysia, respectively.4 a more recent study showed that cdi prevalence was 9.2% in thailand.5 there are only a few reports about cdi incidence or prevalence in indonesia. a study reported that there were eight types of c. diffi cile strains presenting in healthy people,6 while another study showed that the prevalence of c. diffi cile (toxin a) was 1.3% in community and hospital in jakarta.7 the last report originated from central java showing the prevalence of cdi to be 20.6% by 2017.8 profoundly extensive culture and toxin examination for c.diffi cile are still minimal in many hospitals in various asian countries. consequently, c.difficile reports in asia remain rare. in the current study held in malaysia, assays determining toxin a/b from 175 stool samples collected from patients with antibiotic-associated diarrhea have been performed in tertiary hospital in north-eastern suburb; 24 of them (13.7%) tested positive for toxin, where the age most of infected patients is >50 years.9-11 however, no ribotyping or any other molecular test have been done in regard to isolates of malaysian c. diffi cile. similar to malaysia, cdi cases reporting in indonesia is uncommon. it has been found 1.3% test results of stool sample reveal the etiology of diarrhea in indonesia children was c. diffi cile. furthermore, only enzyme immunoassay of toxin a was conducted; therefore, the c. diffi cile true prevalence may have been substantially greater. molecular study of eight isolates collected from indonesia established fi ve of the results identifi ed as toxinotype viii and ribotype 017, assembled into epidemic strains of international 017. two of them are a+b+ toxinotype 0, and one remaining a-b+ isolate was identifi ed as toxinotype xvi binary toxin.12-14 some risk factors including advanced age, antibiotic exposure, and hospitalization are highly associated with cdi.15 regulation of antibiotic usage in asian countries is considered to be poor. there has been a review in southeast asian countries which depicted 47% of pneumonia cases as not receiving proper antibiotic whereas 54% of patients with diarrhea were receiving antibiotic unnecessarily, with 40% of underdose antibiotics prescribed. the advanced age individuals with recent antibiotic treatment are at ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 29ni nyoman sri budayanti, et al.: clostridium difficile infection (cdi) by hypervirulent the highest risk for cdi as they lack of benefi cial gut microbiota and have low immunity due to age and other comorbidities.16-20 this group is excessively aff ected and has the highest mortality rate due to cdi with 2% of risk increase every year after 18 years old of age. a report described around one in ten deaths due to cdi in advanced age people in the usa in 2010. there were no data of cdi on advanced age people in indonesia, which could be due to lack of surveillance on cdi cases followed by limited laboratory facility in the hospital capable of diagnosing cdi. besides, recurrence (relapse/reinfection) and death cases due to cdi in advanced age people would be higher because of improper treatment.21 severe form of c. diffi cile infection (cdi) is caused by hypervirulent strain identifi ed as type 1 of north american pulsed fi eld, type b1 class from analysis of restriction-endonuclease, ribotype 027 as presented by pcr. hypervirulent strain leads nationwide cdi outbreaks in european countries, canada, and the united states (u.s.). first outbreak report of type 027 cdi was in canada where the worst infected was quebec in 2005. in the u.s., type 027 cdi aff ected 38 states. meanwhile based on european centre for disease prevention and control, there were infections in 16 countries due to type 027 cdi. hypervirulent strain of toxinotype iii nurture tcda and tcdb toxin genes, possess deletion of 18-bp in tcdc of toxin regulatory gene, and deletion at area 117. it leads to premature stop codon and frameshift, causing tcdc protein truncation. the rising case of type 027 virulence associated with more excessive toxin production can be attributed to lack control of regulatory tcdc.19-21a cohort study estimated that around 40% of cdi cases were community-acquired cdi (ca-cdi). cacdi occurs in younger age people, less severe symptoms, shorter hospital stay, lower recurrence rate and no deaths have been reported attributable to ca-cdi. besides, cdi is also exacerbated by the discovery of hypervirulent strains and antibiotics resistant to quinolones, gatifl oxacin instead of levofl oxacin.17-19 the emerging of ca-cdi will be a risk factor for domestic and foreign tourists who visit bali. since 2000, greater fatality and severity epidemics of cdi have been caused by a highly virulent form of c. diffi cile. bi/nap1/027 strains have spread widely and robustly over past 10 years and have been associated to cdi epidemics. the prevalent ribotypes in the middle east are 140, 126, 078, 046, 014, 002, and 001, meanwhile the more prevalent ribotypes in asia are 018, 017, 014, 002, and 001. in north america and europe, ribotypes 078, 027, 020, 014, and 001 have been the uppermost strains.22-24 ribotype 027 has been found to possess reduced susceptibility to chloramphenicol, imipenem, clindamycin, moxifl oxacin, rifampicin, and metronidazole. these characteristics implicate to more severe presentation of disease, high morbidity and mortality rates due to antimicrobial resistance juxtaposed to other strains. spores of ribotype 027 expand more robustly and easily in hospital as they able to resist disinfectants, cleaning, and hospital surroundings. observational study on patients with diarrhea in veteran aff airs medical center, u.s. demonstrated around 22% of them were positive of bi/nap1/027 strain.19-24 this literature review aims to describe severity of disease caused by hypervirulent bi/nap1/027 c. diffi cile strain, its mechanism or pathogenesis, risk factors, current treatment options available, along with proposed preventative measures and infection control.22-24 clostridium diffi cile infection (cdi) clostridium diffi cile is an anaerobic grampositive bacillus bacterium, able to form spore and toxin, transmitted in humans by fecal–oral pathway. in the u.s., c. diffi cile is the most frequent nosocomial pathogen reported. a surveillance study of 2011 found 453,000 cdi cases with 29,000 associated deaths, wherein around a quarter of those were communityacquired. nosocomial c. difficile infection quadruples hospitalizations cost causing rise of expenditures by about $1.5 billion in the u.s. yearly. it was recorded that half a million infections were attributed to cdi in the united states in 2011 with an incidence rate of 8.75 cases/1,000 adult admissions in 2009. in hong ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 30 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 27–41 kong, there were more than fi fteen thousand cdi cases from 2006 to 2014 in which most were identifi ed as a nosocomial infection. a nationwide study in korea revealed cdi total incidence was 2.7 cases/1,000 adult admissions in 2008. cdi is also known for its propensity to recurrence among 35% of patients with antibiotic therapy and more than a half of recurrences of cdi are identifi ed as relapse (relapse or reinfection).25 due to cdi, approximately $1.1 billion are utilized in healthcare cost annually in the usa, while about €3 million is associated with healthcare costs in europe. compared to reports from countries across europe and the usa, the prevalence of cdi in asia is not fully known. in korea, survey across 17 tertiary hospitals, from 2004 until 2008, found cdi incidence cases soared from 1.7/1,000 to 2.7/1,000 adults. community-acquired cdi (ca-cdi) proportion over total cdi cases in a hospital in busan was 7.1%, meanwhile 59.4% of cdi cases at a seoul hospital’s emergency department were ca-cdi. based on a comprehensive study in shanghai, china from march 2007 until april 2008, overall cdi incidence was 17.1/10,000 admissions; mild cdi because of younger mean age (62.8 years) compared to 63% patients were ≥65 years in a comprehensive european study. in addition, a survey across 13 asia-pacifi c countries demonstrated the proportion of cdi associated to healthcare facility was 53.6% and ca-cdi was 16.5%. cdi case reports in indonesia remain uncommon. c. diffi cile was identifi ed in 1.3% stool samples of indonesian children. however these data were not enough to reflect global prevalence in asia. furthermore cdi prevalence data of elderly are still unavailable to date.22-26 cdi mostly occurs in advanced age people, which is possibly explained by some of the risk factors, including frequent exposure to healthcare, age-related changes in physiology, increasing antibiotics usage, changes in the composition of gut fl ora, and increased comorbidities. frequent exposure to healthcare increases the opportunity of contacting with environments contaminated with endospores of c. diffi cile and frequent utilization of antimicrobials. carriers of c. diffi cile, both asymptomatic and symptomatic, could contain spores on their skin and discard those into the environment. age-related physiologic changes also increase the risk of cdi, particularly changes in the immune system. the development and recurrence of cdi have been associated with the ability to generate immune responses, and the ability to produce antibodies against toxin may aff ect the progress of colonization and active infection. aging is accompanied by immune senescence – a degeneration of the immune system related to advanced age – and it has been associated with a diminishing adaptive immune system.27-29 c. diffi cile has the ability to do colonization in large intestine, then releasing exotoxins protein (tcda, tcdb) leading to colitis in people with risk factors. figure 1 depicts tcda and tcdb arbitrate c. difficile diarrhea, causing rho family members’ inactivation, rho gtpases (guanosine triphospatases). this is followed by neutrophilic colitis, colonocyte death, functional loss of intestinal barrier, and death of colonocytes. expression of clinical cdi disease is exerted by host immune responses and strain of c. diffi cile. a dramatic increase of severe cdi in hospitals was initially reported in the beginning of 2000s. cdc (centers for disease control and prevention) depicted isolates were group bi of restriction endonuclease, nap1 (gel electrophoresis of north american), and pcr (polymerase chain reaction) 027; therefore, as bi/nap1/027. this strain’s characteristics are high level resistance of fl uoroquinolones, robust production of toxin, effi cient rate of sporulation, and signifi cantly high mortality compared to less virulent c. diffi cile.28,29 bi/nap1/027 strain fi rstly originated in north america and western europe, but currently it spreads to various settings of hospitals across the globe.30,31 even though hospital-acquired cdi has been the majority, ca-cdi has been increasing signifi cantly and contributes to a third of new cdi cases. ca-cdi happens when onset of disease begins within 12 weeks in individuals who did not stay overnight in hospitals or other healthcare facility. ca-cdi could occur in younger patients, who have unclear antibiotics ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 31ni nyoman sri budayanti, et al.: clostridium difficile infection (cdi) by hypervirulent exposure and unknown risk factors. therefore, ca-cdi acquisition main modes are currently in investigation. ca-cdi associated morbidity and mortality remain lower compared to hospitalacquired cdi. nonetheless, 40% of ca-cdi patients need hospitalization and relapse rates are similar to ha-cdi.32 acid suppression infl uence to cdi remains unclear. theoretically, gastric acid suppression allows more vegetative organisms to reach colon. however, c. diffi cile produces spores resistant to acid ph.33,34 hypervirulent bi/nap1/027 c. diffi cile strain risk factors substantial risk factors of cdi by bi/ nap1/027 strain are namely hospitalization, elderly age, and exposure to antibiotics, e.g., cephalosporins and fl uoroquinolones. particular fl uoroquinolones identifi ed being risk factors are ciprofl oxacin, gatifl oxacin, moxifl oxacin, figure 1. progress from asymptomatic colonization to c. diffi cile infection (cdi)22-29 and levofl oxacin, presumed as consequences of fl uoroquinolones-resistance in endemic strain. almost all antibiotics of cephalosporin class are resistant to all c. diffi cile types and have been incriminated as signifi cant risk factor in hospitals where endemic strain exists, as well as its usage for surgical prophylaxis. consumption of agents to lower stomach acid production, e.g., proton pump inhibitors (ppi), and type 2 blockers of histamine have been recognized inconsistently as cdi risk factors in hospital with predominance of endemic strain. besides resistance to current fl uoroquinolones, other specifi c factors of bi/ nap1/027 strain dissemination as well as severe cdi caused by this hypervirulent strain remain speculative and need to be the substance of thorough study or research.32-34 administration to almost all groups of antimicrobials has been delineated to cause cdi, even though cephalosporins, clindamycin, ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 32 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 27–41 penicillin, and present fluoroquinolones are notably reported as common culprits. numerous isolates of c. diffi cile were found containing elements of mobile genetic, markers of antibiotic resistance in chromosomes, and mutations of genetic conferring resistance into rifamycins, chloramphenicol, tetracyclines, streptogramin, lincosamide, macrolide, and fl uoroquinolones. patients also progress to cdi disease following antibiotics therapy which leads to susceptibility of c. diffi cile strain infection. presumably, cdi occurs due to suppression of normal microbiota in intestine for extended periods after discontinuation of antibiotics; therefore, allowing sustained opportunity for colonization and infection of c. diffi cile. isolates resistance to clindamycin, erythromycin frequently associated to epidemics and outbreaks. furthermore, individuals administered by clindamycin possess a remarkable high-rise cdi frequency caused by clindamycin resistant. resistance is commonly associated with erm(b) presence encoding methyltransferase and ground in tn5398 conjugative transposon.35 current exploration of bi/nap1/027 isolates has exemplifi ed re-emergence of erythromycinresistant bi/nap1/027 in european countries, the u.s., and canada. currently, therapy with fl uoroquinolones is identifi ed as bi/nap1/027 c. diffi cile infection risk factors, and there is a proposed association between therapy with fluoroquinolones and emergence of bi/nap1/027 strain. even though all previous isolates of bi/nap1/027 are susceptible to gatifl oxacin, moxifl oxacin, and fl uoroquinolones, yet resistant to levofl oxacin and ciprofl oxacin, almost all current isolates were resistant to all fl uoroquinolone antibiotics. inhibition of dna replication by fl uoroquinolones is as a result of its binding to dna gyrase or topoisomerase ii, or topoisomerase iv. resistance to fl uoroquinolones in c. diffi cile is associated with particular mutations in gyra and gyrb, that encode dna gyrase. fluoroquinolones are broadspectrum antibiotics which act on gram-negative and gram-positive bacteria and are able to decrease normal fl ora in intestine, hence broad use of fl uoroquinolones in hospitals fosters spreading of bi/nap1/027 c. diffi cile strain.32-36 h y p e rv i r u l e n t b i / n a p 1 / 0 2 7 c . difficile strain toxigenicity and pathogenesis h y p e r v i r u l e n t s t r a i n b i / n a p 1 / 0 2 7 i s demonstrated to carry cdtloc gene locus and encodes cd196 adp-ribosyltransferase (cdt) or known as binary toxin. hypervirulent c. diffi cile is able to produce tcda and tcdb, similar with non-027 ribotypes throughout gene locus of paloc. cdt was initially isolated by popoff et al.37 the toxin contains two distinct toxin components separately, namely cdta and cdtb. cdta, adp-ribosyltransferase enzyme acts on actin modifying which leads to depolymerization and destruction of actin cytoskeleton inside gut; meanwhile cdtb hitches to gut cells and stimulates cdta uptake. destruction by cdt accommodates bacteria adherence and surges toxin a and b uptake.38-40 furthermore, hypervirulent strain contains bp frameshift deletion on tcdc gene, nucleotide 117, and functions as negative regulator of toxin a and toxin b. tcdc mutation causes toxins hyperexpression. warny et al.58 demonstrated bi/nap1/027 as able to produce 16 times of toxin a and 23 times of toxin b approximately compared to control strain. one research postulated increasing sporulation by hypervirulent strain possibly has association with robust cdi spreading. nevertheless, previous research demonstrated controverted results in regard to disease severity by hypervirulent strain. a retrospective study by bauer et al.41 concluded hypervirulent strain bi/nap1/027 as associated with declined odds of disease severity ratio (or): 0.35, 95% confi dence interval (ci) 0.13 0.93) and did not increase mortality in hospitalized patients (or: 1.02, 95% ci 0.53 1.96), or (or: 1.16, 95% ci 0.36 3.77) of recurrence rate. meanwhile, some other studies (cohort, casecontrol, and cross-sectional) did not demonstrate worse prognoses compared to other strains as shown in table 1.41 ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 33ni nyoman sri budayanti, et al.: clostridium difficile infection (cdi) by hypervirulent table 1. virulence factors of hypervirulent bi/nap1/027 c. diffi cile strain41 virulence factors mechanism tcda or enterotoxin a (toxin a) destruction of actin within target cells causes infi ltration of neutrophil, infl ammation, and epithelial cells necrosis. tcdb or cytotoxin b (toxin b) destruction of epithelial cells tight junctions causes increasing permeability of vascular, and hemorrhage. cdta toxin modifi es the action with adp-ribosylation leads to depolymerization of actin and damage of cytoskeleton assists bacteria adherence to epithelial cells of gut. cdtb toxin facilitates cdta toxin uptake into epithelial lining of gut. hypersporulation increases bacteria reproduction and spreading. mutation of tcd gene increases assembly of toxin a and toxin b by down-regulation of feedback inhibitor necessitate in diminishing toxin production. based on sirard et al. (2011), even though hypervirulent strain bi/nap1/027 is able to assemble more toxins, they construct spores in fewer numbers and have not always been associated with severe condition of disease.42 in contrast, a cohort study by rao et al.43 demonstrated association between hypervirulent strain ribotype 027 with severe cdi disease (or: 1.73, 95% ci 1.03 2.89; p = 0.037) and higher mortality rate (or: 2.02, 95% ci 1.19 3.43; p = 0.009) juxtaposed to other ribotypes.43 study by see et al.44 demonstrated similar results by using nap1 strain, where analysis of multivariate regression depicted increased severity of cdi (or: 1.66, 95% ci: 1.90 2.54) and higher mortality (or: 2.12, 95% ci: 1.22 3.68).44 furthermore, a study in quebec showed the hypervirulent strain ribotype 027 is associated with disease severity, twice more severe frequently in contrast to other strains. nevertheless, basic reasons of these contradictory results were un-measured confounding factors, setting of study, detection methods of c. diffi cile, size of sample, population of study, and design of study. therefore, the generalization of the study results has to be examined profoundly. therefore, given these contrary fi ndings, healthcare workers or providers advised to center their attention on infection treatment based on clinical reasoning and infection marker related to severe infection, as well as episodes of diarrhea, dehydration signs, albumin level, creatinine level, white blood cell (wbc) count, underlying comorbidities, and immunocompromised condition.45,46 mechanism of endemic strain displacement with hypervirulent ribotype 027 c. diffi cile strain transmission of pathogen occur via fecaloral route with new infections emerge by bacterial spore consumption. c. diffi cile spores are resistance to desiccation and able to persist for about 5 months on hard or solid surface. merrigan et al.45 examined spore accumulation in regard to growth cycle of bacteria with results demonstrating that hypervirulent strains have the ability to sporulate faster and causing signifi cant more spore accumulation per total volume compared to non-hypervirulent strains.45 increase sporulation rate could elucidate the uncommon soaring recurrence correlated to hypervirulent strains, 4-fold according to marsh et al.20,46, as patients tend to transmit the contamination to local surroundings, then re-infect themselves subsequently. subsequently after dormant bacterial consumption and ingestion, germination of c. diffi cile spore occurs as exposure to combination of bile salts and l-glycine. vegetative phase of c. difficile happen as colonization of host’s gastrointestinal tract. even tough colonization is required to cause the disease, most of infected people prevail asymptomatic. cdi manifestations are arbitrated by production of cytotoxic toxins to large intestine epithelial tissue lead the way of immense colon infl ammation and epithelial cell obstruction of the host.46 study by pepin et al.47 and hubert et al.48 demonstrated doubling rate of severe disease as emergence of ribotype 027 in canada. hypervirulent strains associated ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 34 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 27–41 with higher rates of symptomatic disease presumed to be result of increased production of toxin or due to intensifi ed variant clostridial toxins.48 there are three probable mechanisms postulated in accordance to transitions from endemic strains to hypervirulent strains: (1) the more infectious strains are hypervirulent; (2) symptomatic condition with higher rate is caused by hypervirulent strain; (3) outcompete in host’s gut can be done by hypervirulent strain.49 throughout stochastic simulation, c. diffi cile hypervirulent strain invasion to human population cherished endemic strain was investigated. reasoning of some previous models aims to establish infection control strategy in hospital and surroundings. nevertheless, c. diffi cile has been recognized prominently as a global community pathogen, in preference to just segment of healthcare associated pathogen. in addition, present study has demonstrated major source of infection is community. nonetheless, in some conditions if community not the primary source, infections suff ered by high-risk group in healthcare environment. underlying cause of diff erence between endemic strains and hypervirulent strains prevail undetermined regardless of current atypical strains constitute predominant infections in community surroundings. therefore, the consequence of three distinct pathways of intensifi ed were examined namely increasing pathogen infectiousness, increasing rate of colonization to symptomatic disease, and ability of endemic strains displacement by hypervirulent strains in colonized gut.49,52 instinctively, parameters govern these distinct mechanisms have positive correlation to possibility of establishment invading strains in community. nevertheless, comparison of these parameters’ infl uence on invasion rate and prevalence of resultant equipoise yielded different patterns of epidemiology. in accordance to classic epidemiological comprehension, the rate in which an establish pathogen spreading within susceptible individuals is strongly dependent on coeffi cient of transmission, as modelled by increasing the hypervirulent strain infectiousness. simulation demonstrated increased infectious strains tend to establish further, spread robustly, and reach equilibrium to increased prevalence in community. probability of successful established invasion and current steady circumstances of prevalence has been less influenced by rising colonized percentage on clinical disease experience. if individuals colonized by endemic strains were prone to hypervirulent strains, a weaker correlation was constructed with probability of establishment, and no comprehensible correlation was discerned with equilibrium prevalence outcome. spreading of novel strain is substantially independent to endemicity of resident strain when gut is colonized by resident strain as uncolonized gut readily.49-53 clinical reports over the past 15 years have demonstrated substantial increase of disease rate in accordance to prominent and robust switch in dominance of c. diffi cile strain. isolates from pcr-ribotyping in montreal hospital depicted nap1/ribotype 027 were not found in 2000 and 2001. nonetheless, nap1/ribotype 027 constituted more than 75% isolates collected during the outbreak in 2003 until 2004. increasing prevalence of cdi disease has corresponded to ribotype 027 dominance in many countries across the world, comprising england with its peak in 2007-2008, european countries, and north america.49,50 tying to epidemiological model with present analysis results, apparently hypervirulent strains’ ability in displacing endemic strains from readily colonized host’s gut is the slightest mechanism facilitates ribotype 027 dominance, resulting in more severe diarrhea and longer recovery period. in spite of investigating a wide range of parameter values, from resistance of colonization to susceptibility counterpart in uncolonized individuals, novel strain is unsuccessful to reproduce heightened level of prevalence associated with emerging hypervirulent strains. it does not invalidate the probability of more competitive hypervirulent strains compared to typical strains within host. however, it still suggests this mechanism is not a pivotal role for successful invasion and hypervirulent strain of ribotype 027 clonal dominance. importantly, the present study depicted strains’ competition inside host’s gut is not essential for abrupt prevailing strains switching; all surrogate mechanisms ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 35ni nyoman sri budayanti, et al.: clostridium difficile infection (cdi) by hypervirulent of hypervirulent distinctly illustrated previous dominant strains are not merely added on invasion of subsequent new strain, yet excluded throughout exploitative competition.53,54 a n t i b i o t i c r e s i s t a n c e o f hypervirulent bi/nap1/027 c. diffi cile strain investigation of bi/nap1/027 cdi cases in panama showed high resistance to several antibiotics: rifampin, ciprofl oxacin, levofl oxacin, moxifloxacin, and clindamycin, yet remain susceptible to vancomycin and metronidazole. study tested for several antibiotic susceptibility for ribotype 027 and non-027 ribotype in canada with fi ndings 92.2% resistance of ribotype 027 to moxifl oxacin as opposed to 11.2% of other strains. correspondingly, ribotype 027 strains (78.2%) showed resistance to ceftriaxone compared to other strains (15.7%). ribotype 027 was greater than 4-fold higher of minimum inhibitory concentration (mic) compared to metronidazole (4 μg/ml vs 1 μg/ml). in addition, ribotype 027 strain demonstrated 2-fold higher mic of fidaxomicin (1 μg/ml vs 2 μg/ml). nevertheless, resistance for vancomycin and clindamycin was akin both in group of bi/ nap1/027 and other strains. erythromycin resistance is associated with mutation of methylase genes in ribosome, meanwhile fl uoroquinolones resistance is caused by mutation of dna gyrase. resistance to fi daxomicin and rifamycin group is linked to methylation of ribonucleic acid (rna) polymerase. in addition, resistance to linezolid is caused by genes of lincosamide and phenicol. study in several hospitals in mexico demonstrated numerous ribotype 027 isolates possesses decreased susceptibility to fi daxomicin even though this antibiotic is unavailable in mexico and patients had been unexposed to it. basis for bi/nap1/027 strain treatment is antibiotics. presently, specifi c guidelines of the infectious diseases society of america (idsa) remain unavailable to bi/nap1/027 strain.55-58 consequently, based on overall cdi treatment guidelines, infection by bi/nap1/027 strain treatment has been proposed as in table 2. table 2. suggestive antibiotic treatment for bi/nap1/027 strain55 1st line treatment alternative treatment initial non-severe infection vancomycin per oral (p.o.), 125 mg, 4 times daily, 10 days fidaxomicin p.o., 200 mg, twice per day, 10 days. if unavailable, take metronidazole, 500 mg, three times per day, 10 days 1st non-severe recurrency vancomycin p.o., 125 mg, 4 times per day, 10 days oral fi daxomicin, 200 mg, twice per day, 10 days 2nd non-severe recurrency vancomycin p.o. tapering off : 125 mg, 4 times, 7 until 10 days; 125 mg twice per day, 7 days; 125 mg once daily, 7 days; 125 mg per three days, 14 days fidaxomicin p.o., 200 mg, twice per day, 10 days, or transplantation of fecal microbiota later non-severe relapse transplantation of fecal microbiota vancomycin p.o. tapering off with probiotics, fi daxomicin, intravenous immune globulin (ivig) severe disease vancomycin p.o. 125 mg, f4 times daily; rise to 500 mg, 4 times per day. this can be applied only if there is no improvement within 24 48 hours, or associated side eff ects, e.g., ileus, renal failure if patient cannot tolerate vancomycin p.o, fi daxomicin is antibiotic of choice ileus plus intravenous metronidazole 500 mg, every 8 hours to fi daxomicin or oral vancomycin, consultation to general surgery should be considered ivig, intra colonic vancomycin ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 36 indonesian journal of tropical and infectious disease, vol. 10 no. 1 january–april 2022: 27–41 hypervirulent bi/nap1/027 c. diffi cile strain preventative measures and control bi/nap1//027 is well-known to cause outbreaks in hospital, and some reports have represented eff orts and measures in regard to outbreak control. muto56 depicted combined measures to control outbreak in the university of pittsburgh as a bundle of encompassed education; increment of early detection in regard to cdi requires nurses to make an order of toxin texting, and email notifi cation to alert physicians who treat high risk patients, and establish a management team to evaluate patients tested positive for all c. diffi cile toxins. expansion of infection control action comprises environmental cleansing with bleach, replacing alcohol hand rubs with water and soap to improve hand hygiene for cdi patients, continuation of contact precautions following diarrhea resolution, audit of infection control, and restriction of targeted antimicrobials.56 even though particular eff ect of each measures was diffi cult to ascertain, investigators delineated a 78% decrease of cdi incidence and severe cdi proportion. furthermore, only 13% of c. diffi cile isolates were bi/nap1/027 strain by 2005, compared to 51% among clinical isolates in 2001.57-60 in regard to numerous hospitals outbreak in quebec, the canadian government allocated $20 million to upgrade infection control measures in 12 hospitals; whereas in pittsburgh, various approaches were implemented, comprising domestic measures intensifi cation with thorough environmental cleaning of aff ected areas and toilets by applying bleach, cleaning all rooms on a subdivision or section if number of nosocomial occurrence exceeded within three weeks; equipment dedication; applying hand washing rather than alcohol rubs; prompt finding of cdi case by daily enhancement of toxin testing frequency in clinical laboratory; prompt empirical treatment and contact precaution practice subsequent to second diarrhea stool; move patients from 4-bed ward if possible; and education to decrease administration of cephalosporin and fluoroquinolone. consequently, incidence of cdi in these hospitals declined from 22.5 to 12.4 per 1000 admittance as a result of applying these preventative measures, but incidence rates did not reach pre-outbreak extent.61-65 one hospital in quebec implemented antimicrobial stewardship when no effectivity was shown in decrease of cdi incidence after executing infection control measures. this unrestrained strategy leaned on education and commentary or assessment from pharmaceutical parties and hence attained administration reduction to 54% of total antibiotic and 23% of targeted antibiotic. simultaneously, with diminishment in antibiotic consumption, cdi incidence has seen a 60% drop. targeted antibiotics encompassed second and third generation of cephalosporin, macrolides, clindamycin, and ciprofl oxacin. drop in ciprofl oxacin usage has been accompanied by increase. in other places, administration of moxifl oxacinwas used as an agent incriminated as high-risk antimicrobial agent.64,65 some factors contribute a signifi cant role of therapy by fluoroquinolones in epidemics era, encompassing enhance resistance of bi/ nap1/027 strains to group of fl uoroquinolones, juxtaposed to historical isolates not associated to epidemic isolates, expanded consumption of fl uoroquinolones, along with high ascribable risk in regard to fl uoroquinolones of this outbreak. however, considering assorted outcomes of certain fl uoroquinolones restriction, un-assessed hypothesis that could be a “class effect,” subsequently all fluoroquinolones restriction will be a specifi c potential control course of action in hospital with outbreak caused by bi/nap1/027 strain. various measures have been implemented in outbreak control of cdi, especially bi/nap1/027 which poses a remarkable challenge.66-68 coalescence of elderly patients, continual use of antibiotics, contamination of hospital environment with spores are all ideal circumstances of cdi outbreaks, high rate or number of morbidity and mortality. even though infection control measures, such as environmental cleaning, isolation, and hand hygiene, will persist as keystones course of action to prevent c. diffi cile exposure in hospital, methods to reduce disease ijtid, p-issn 2085-1103, e-issn 2356-0991 open access under cc-by-nc-sa share alike 4.0 37ni nyoman sri budayanti, et al.: clostridium difficile infection (cdi) by hypervirulent risk following c. diffi cile infection or ingestion have to be reckoned. nevertheless, decreasing antibiotics use remains absolutely an important approach to reduce cdi risk. these measures have commenced in various hospitals, yet there is still considerable extent to improve antimicrobial stewardship. methods to neutralize antibiotic disruption of microbiota colonization should be incorporated with biotherapeutic methods, e.g., nontoxigenic c. diffi cile strain administration which has demonstrated to be eff ective in hamster model. focus measures also should emphasize on development of vaccine of c. difficile to boost immune state of elderly people. passive immune methods such as monoclonal antibody to enhance immune response to toxin a and b seem to be eff ective in early stage of clinical trials. nevertheless, even though current focus is on bi/nap1/027 c. diffi cile strain, new upcoming epidemic strains are going to emerge in the foreseeable future.69-72 conclusions greater fatality and severity epidemics of cdi have been caused by highly virulent form of c. diffi cile of bi/nap1/027 that spread widely and robustly over decades. main risk factors are elderly age, hospitalization, and exposure to antibiotics, e.g., cephalosporins, fl uoroquinolones, clindamycin, and penicillin. virulence factors are tcda, tcdb, cdta cdtb; hypervirulence is prone to hypersporulation and mutation of tcd gene leads to toxin hyperexpression. preventative measures can be done by early cases detection, building a management team to evaluate patient positive with all c. diffi cile toxins, hand hygiene improvement, continuation of contact precautions after diarrhea resolution, audit of infection control, restriction of antimicrobials, and development of a vaccine of c. diffi cile. acknowledgement we would like to acknowledge clinical microbiology department and geriatric division, internal medicine department, faculty of medicine, universitas udayana to support this literature review. conflict of interest no competing interests have been associated from construction process until publication of the manuscript. no fi nancial support from any parties or organization was obtained regarding the manuscript submitted. references 1. kotila sm, mentula s, ollgren j, virolainenjulkunen a, lyytikäinen o. communityand healthcare-associated clostridium diffi cile infections, finland, 2008−2013. emerging infectious disease journal. 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2356-0991 open access under cc-by-nc-sa share alike 4.0 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. 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; 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 80 105 vol. 6 no. 5 may–august 2017 research report an evaluation study of enzyme-linked immunosorbent assay (elisa) using recombinant protein gra1 for detection of igg antibodies againts toxoplasma gondii infections nina difla muflikhah1a, wayan tunas artama2 1 faculty of dentistry, institut ilmu kesehatan bhakti wiyata, kediri 2 faculty of veterinary medicine, universitas gadjah mada, yogyakarta a corresponding author: ninadifla@gmail.com abstract toxoplasmosis is an infectious disease caused by toxoplasma gondii, an intracellular protozoan parasite that live inside the cells of the reticulo-endothelial and parenchymal cells of human and animals (mammals and birds). some cases of toxoplasmosis usually have no symptoms, but in any cases caused severe symptoms, such as hydrocephalus, microcephalus, intracranial calcification, retinal damage, brain abscess, mental retardation, lymphadenopathy, and others. its severe symptoms usually showed a long time after first exposure, except symptoms showed by congenital transmission caused by infected mother. early diagnosis is important to prevent the illness but methods for toxoplasmosis screening are still too expensive for developing country. enzyme-linked immunosorbent assay (elisa) allow the testing of a large number samples within short time frame and based on antibody or antigen detection. this study aimed to know the sensitivity and specificity of recombinat protein gra1 as antigen using elisa methods. we tested the sensitivity and spesificity of gra1 protein as antigen in elisa methods to diagnose toxoplasmosis and compared with elisa kit commercial. reliable laboratory testing is important to detect toxoplasma gondii infection, and focused to improving the low cost and easy-to-use diagnostic instrument. seventy sera collected and tested using both indirect elisa, commercial elisa kit and gra1 protein coated as antigen. fourty eight and fifty one samples showed positive igg antibody result of elisa-gra1 and elisa kit. negative sample tested by elisa-gra1 was 22 samples and 19 sample tested by elisa kit. the sensitivity and specificity of gra1-based on elisa were 100% and 86.36%, positive prediction value (ppv) was 94.11%. these data indicate that the recombinant protein gra1 is a highly immunogenic protein in human toxoplasmosis and become a promising marker for the screening of toxoplasmosis. keywords: toxoplasmosis, recombinant protein gra1, elisa, sensitivity, specificity abstrak toksoplasmosis merupakan penyakit menular yang disebabkan oleh protozoa parasit intrasel spesies toxoplasma gondii, yang hidup di dalam sel endotelial dan perenkim manusia dan hewan (mamlia dan aves). sebagian besar kasus toksoplasmosis tidak menunjukkan gejala, namun pada beberapa kasus menyebabkan kondisi yang kronis seperti hidrosefalus, mikrosefalus, kalsifikasi intrakranial, kerusakan retina, abses otak, keterbelakangan mental, limfadenopati, dan gejala lainnya. gejala tersebut akan tampak beberapa waktu setelah infeksi pertama, kecuali pada kasus kongenital toksoplasmosis dimana bayi terlahir dalam kondisi cacat akibat infeksi pada ibu pada trisemester kedua. diagnosis toksoplasmosis merupakan kunci utama dalam pencegahan munculnya gejala namun prosedur diagnosis masih tergolong cukup mahal di negara berkembang. salah satu metode diagnosis toksoplasmosis adalah dengan metode enzyme-linked immunosorbent assay (elisa) yang dapat melakukan pemeriksaan untuk sampel dengan jumlah besar dalam waktu singkat berdasarkan reaksi antigen dan antibodi. bentuk upaya pengembangan instrumen diagnosis elisa adalah penggunaan protein rekombinan sebagai antigen, seperti protein gra1. penelitian ini bertujuan untuk mengetahui nilai sensitivitas dan spesifitas protein rekombinan gra1 sebagai antigen menggunakan metode elisa. uji sensitivitas dan spesifitas protein gra1 sebagai antigen dilakukan dengan membandingkan dengan elisa kit yang telah dijual bebas pada 70 sampel. sebanyak 48 dan 51 sampel serum menunjukkan 106 indonesian journal of tropical and infectious disease, vol. 6 no. 5 may–august 2017: 105–108 hasil positif toksoplasmosis yang diuji menggunakan elisa-gra1 dan elisa kit. sebanyak 22 sampel menunjukkan hasil negatif toksoplasmosis menggunakan metode elisa-gra1, sedangkan 19 sampel negatif menggunakan elisa kit. nilai sensitivitas gra1 sebagai antigen adalah 100%, spesifitas sebesar 86,36%, dan nilai prediksi positif sebesar 94,11%. berdasarkan data tersebut, protein rekombinan gra1 diketahui memiliki aktivitas immunogenik yang tinggi pada penderita toksoplasmosis dan dapat digunakan sebagai penanda untuk diagnosis toksoplasmosis sehingga pengembangan alat diagnosis dengan biaya rendah dapat dilakukan. kata kunci: toksoplasmosis, protein rekombinan gra1, metode elisa, sensitivitas, spesifisitas introduction toxoplasmosis is disease caused by infection of obligate protozoan parasite, called toxoplasma gondii. human can infected by t. gondii in many ways such as, congenital, consumptions habits (raw meat, raw vegetables), activity with soil/meat without protection, blood tranfussion, organ transplantation, and etc. oocyst become infective stage when passed out from definitive host and contaminated with water sources, soil, and plants. most of toxoplasmosis are asymptomatic but can be serious problems in immunocompromised patients and newborns with congenital toxoplasmosis. infection can cause encephalitis in immunocompormised hosts, chorioretinitis in immunocompetent hosts or serious congenital disease in developing fetus if pregnant women become infected for the first time during pregnancy.1 more than 60% world population are toxoplasmosis, and 90% of it asymptomatic even they have tg antibodies. it depends on the individual immune responses to prevent the symptoms.2 detection for toxoplasmosis usually using serological methods, such as dye test (dt), modified agglutination test (mat), enzyme-linked immunosorbent assays (elisa), immunosorbent agglutination assay (isaga), indirect fluorescent antibody test (ifat) and indirect haemagglutination assays (iha) to detect tg antibody. demonstrating the parasite in tissue can be done by culture of parasite (in vivo and in vitro) and detection of spesific nucleic acid using dna probe, pcr and l-amp methods. enzyme-linked immunosorbent assays (elisa) is a popular and commercially available easier methods for clinical detection of toxoplasmosis. commercial elisa kits use antigen from native tachyzoites which grown in mice or tissue culture and probably contain varying amounts of extraparasitic material.3 limitations of the tachyzoite antigen for serologic tests can be serious problems, another antigens should become an alternative test, such as using purified recombinant antigens which expressed by tachyzoites and bradyzoites. however, the whole tachyzoite native antigen test is difficult to standarize and in some cases produce false positive reactions.4 tachyzoites is not the only component which could activated the immune response to produce antibody, expression of excretedsecreted antigen from bradyzoites can induces antibody production and igg specific tg always exist in infected host lifetime. gra1 has been identified as excreted-secreted antigen (esa) in tachyzoites and crossreactive with bradyzoites.5 it located in dense granule of both tachyzoites and bradizoites, and used as a marker of secretory organelles of tg. it always secreted in lumen and potentially can be identify in body fluid of infected host. gra1 induces humoral and cellular immune responses in the chronic phase of the infection in mice and humans and increasing production of antibody and ifn-γ. gra1 epitopes present in mhc class i molecules during infection and induces specific ctls.6–8 gra1 was secreted into the lumen of the parasitophorus vacuola as a soluble protein and associated with the membranous tubular network peripherally.9 gra1 needed for secretion of 3 secretory organelles of tg and became marker of dense granule proteins.10 vaccination using gra1 protein show the activity of cd8+ t-cells against parasite-infected cells and a gra1-transfected cell line.7 the costs for serologic testing in developed countries are not prohibitively high. however, in developing countries there is alternative low-cost test with the same sensitivity and specificity. the costs for the development of instrument depend on the efficient production of recombinant antigens.11 previous study, in same project, have developed an efficient system for the production and purification of gra1 proteins and have been tested for immunogenic activity. based on the ability of gra1 to stimulated immune response, we tried its ability as antigens to develop the diagnostic tools. sensitivity and specificity of gra1 as antigens in elisa methods will compared with commercial elisa kit to detect tg-igg antibodies. material and method seventy human sera were obtained from previous study in central java population and approved by ethical committee of faculty of medicine universitas gadjah mada for research in human subject.12 sera were tested using elisa methods and separated for both elisa kit test and elisa-gra1 coated protein as antigen. preparation of recombinant protein gra1 consist of isolation, characterization, cloning, expression, and purification of gra1 protein. isolation, characterization, cloning, and expression of gra1 protein were worked by previous researcher in same project13,14 and culture of e.coli inserted gra1 protein were stored in 40c until we 107muflikhah and artama: an evaluation study of enzyme-linked immunosorbent assay table 1. sensitivity and specificity of gra1 recombinant protein as antigens for toxoplasmosis detections. no. elisa kit positive of tg igg antibody negative of tg igg antibody total no. elisa-gra1 positive of tg igg antibody 48 3 51 negative of tg igg antibody 0 19 19 total 48 22 70 used to this study. we were re-culture the recombinant e coli and isolated gra1 protein used sonication to break the bacterial membrane. gra1 protein purified using ni-ted profino coloumn chromatography and electrophoresis to confirmed the result. protein recombinant gra1 was diluted to the optimized concentration of 2 µl in 200 µl biorad protein assay (pba) and 798 µl h2o then optical density checked by using spectrophotometer with wavelenght 595 nm. gra1 protein level measured following this formula; (optical density+0.057)/0.0465. optical density was found 0.02885 and counted using that formula showed gra1 protein level was 0.5602 µg/µl. counting of protein level was needed to calculated the volume of the protein which is incubated as antigen and must reach 5 µg/100 µl. each microwell was added by 50 µl of protein solution and coated overnight at 370c using coating buffer (na2co3 and nahco3). the processes were done step by step, consist of blocking process, samples additions, conjugates (antihuman igg alkaline phosphatase), substrate (pnpp), and stop solution. washing solution was added three times after each processes to removed all unbounded particles. blocking buffer (pbs-bsa 1% ph 7.00) was added and incubated at 370c in hour. human sera and antihuman igg alkaline phosphatase (conjugate) were diluted 10 times and 5000 times, respectively, and incubated. p-nitrophenyl phosphate as substrate was diluted in substrate buffer 1mg/ ml (diethanolamin and mgcl2) and 150 µl was added into each well then incubated 30 minutes. reaction was stopped immediatelly with addition of stop solution containing hcl. the quantitative result was measured by elisa reader to detect optical density (od). cut off value was counted by mean of negative control od. effectiveness of gra1 as promising-antigen was evaluated by elisa kit commercial (genway biotech) coated with native tachyzoites. procedure of elisa kit which used were followed the manufacture instruction. the kit consist of dilution buffer, washing buffer, negative and positive control, 4 type of calibrator to differentiate negative, low and high positive antibodies concentration, and stop solution. result and discussion serodiagnostic using recombinant proteins of tg evaluated by indirect elisa and compared with commercial elisa kit. sensitivity and specificity were measured to detect the effectiveness of gra1 proteins as antigens. as shown in table 1, there were 51 positive and 19 negative samples by elisa-gra1, while there were 48 positive and 22 negative samples tested by elisa kit. commercial elisa kit usually using native tachyzoites antigens coated in microplate and distributed worldwide to diagnosis of toxoplasma gondii infections. this tools is a high-cost instrument among laboratories and not always accurate because often produces false positive reactions.4 while toxoplasmosis diagnostic is an important test for human in every social-economic status, developing a lowcost diagnostic tools really important to support the health status and epidemiological screening of infected disease in populations. the results of this study explicitly show that gra1 antigen is suitable for detecting serum antibody to tg infections and clearly distinguished mean of optical density (od) and 95% of confidence interval (ci), the method is able to differentiate seropositive and seronegative of tg-igg sera. there were 51 positive and 19 negative samples tested by elisa-gra1, while 48 positive and 22 negative samples tested by elisa kit (genway biotech). all observed sensitivity and specificity estimates greater than 80%. sensitivity of gra1 is 100% and specificity reach 86.36%. based on a sensitivity and specificity of 80%, the observed sample size was sufficient to estimate good sensitivity and specificity as diagnostic tools. dense granule proteins function is to manage modification of the parasitophorus vacuola and intake nutrition from cytoplasm of infected cell.5,15–18 this protein was needed by tachyzoites for continuing their development in infected cell and replicate inside of parasitophorous vacuolar membrane.18 most of dense granules proteins secreted in parasitophorous vacuoles and increase following the number of parasite infections. a molecule became potential antigen if it have weigh over 1 kd, complex structure, and a stabil molecules.19 gra1 proteins have immunogenic and antigenic activity.6,7,20 vercammen et al. (2000) was reported the result of gra1 vaccination induce humoral immune response in mice and produce igg antibodies. naturally, gra1 induces secretion of igg antibodies spesific to gra1 and could be detected using serologic assay. however, there is a significant advantage in the preparation of recombinant proteins over the preparation of crude tg proteins. recombinant tg proteins could be produced economically and in large quantities by e. coli culture, but crude tg antigens must be extracted from tg 108 indonesian journal of tropical and infectious disease, vol. 6 no. 5 may–august 2017: 105–108 within the animal model. these crude extracts contain large amounts of proteins and other macromolecules, and most of them can influence the results of the test.21 purified recombinant proteins could be an alternative substances to detect serum antibodies and will allow better standardization of the immunoassays.21–23 furthermore, a combination of recombinant antigens may enhance the sensitivity of an antibody-based assay. several previous studies have found that recombinant antigens improve the serological diagnosis of tg infection.22,24–26 moreover, recombinant antigens have the potential to be used in the creation of new instrument that differentiate recently acquired infections from those acquired in the more distant past. conclusion our study showed high sensitivity and specificity of gra1 recombinant protein as antigens for detections of toxoplasmosis using enzyme-linked immunosorbent assays. gra1 recombinant proteins became promising antigens performed accurate result and should be develope as alternative tools for tg antibodies detection in toxoplasmosis suspect. acknowledgement there is no conflict of interest to declare. this study was supported by the grant given by the ministry of research and technology, republic of indonesia. gra1 recombinant proteins was kindly obtained from prof. drh. wayan tunas artama, ph.d. the authors would like to acknowledge toxoplasmosis team for their generous help through the project for providing gra1 recombinant proteins. references 1. montoya jg, liesenfeld o. toxoplasmosis. lancet (london, england). 2004 jun 12;363(9425):1965–76. 2. sibley ld. toxoplasma gondii: perfecting an intracellular life style. traffic. 2003 sep;4(9):581–6. 3. aubert d, maine gt, villena i, hunt jc, howard l, sheu m, et al. recombinant antigens to detect toxoplasma gondii-specific immunoglobulin g and immunoglobulin m in human sera by enzyme immunoassay. j clin microbiol. 2000 mar;38(3):1144–50. 4. hassi a, muller wa, aspock h. an identical epitope inpneumocystis carinii andtoxoplasma gondii causing serological cross reactions. parasitol res. 1991;77(4):351–2. 5. cesbron-delauw m-f, lecordier l, mercier c. role of secretory dense granule organelles in the pathogenesis of toxoplasmosis. in: toxoplasma gondii. berlin, heidelberg: springer berlin heidelberg; 1996. p. 59–65. 6. vercammen m, scorza t, huygen k, de braekeleer j, diet r, jacobs d, et al. dna vaccination with genes encoding toxoplasma gondii antigens gra1, gra7, and rop2 induces partially protective immunity against lethal challenge in mice. infect immun. 2000 jan;68(1):38–45. 7. scorza t, d’souza s, laloup m, dewit j, de braekeleer j, verschueren h, et al. a gra1 dna vaccine primes cytolytic cd8(+) t cells to control acute toxoplasma gondii infection. infect immun. 2003 jan;71(1):309–16. 8. sulistyaningsih e, moeljopawiro s, subandono j, artama wt. cloning of cdna encoding gra1 protein of tachyzoite toxoplasma gondii local isolate. indones j biotechnol. 2005;10(1):763–7. 9. s i b l e y l d , m o r d u e d g , s u c , r o b b e n p m , h o w e d k . genetic approaches to studying virulence and pathogenesis in toxoplasma gondii. philos trans r soc lond b biol sci. 2002 jan 29;357(1417):81–8. 10. carruthers vb. host cell invasion by the opportunistic pathogen toxoplasma gondii. acta trop. 2002 feb;81(2):111–22. 11. hiszczynska-sawicka e, kur j, pietkiewicz h, holec l, gsior a, myjak p. efficient production of the toxoplasma gondii gra6, p35 and sag2 recombinant antigens and their applications in the serodiagnosis of toxoplasmosis. acta parasitológica. 2005;50(3):249– 54. 12. retmanasari a, widartono bs, wijayanti ma, artama wt. analisis spasial dan faktor risiko toksoplasmosis di jawa tengah bagian selatan. universitas gadjah mada; 2015. 13. utami ws. isolasi, karakterisasi dan uji diagnostik protein rekombinan gra-1 takizoit toxoplasma gondii isolat lokal. universitas gadjah mada; 2009. 14. widayanti e. subkloning dan over ekspresi gen penyandi protein gra-1 takizoit toxoplasma gondii isolat lokal. universitas gadjah mada; 2008. 15. wu x-n, lin j, lin x, chen j, chen z-l, lin j-y. multicomponent dna vaccine-encoding toxoplasma gondii gra1 and sag1 primes: anti-toxoplasma immune response in mice. parasitol res. 2012 nov 27;111(5):2001–9. 16. black mw, boothroyd jc. lytic cycle of toxoplasma gondii. microbiol mol biol rev. 2000 sep;64(3):607–23. 17. miller cm, boulter nr, ikin rj, smith nc. the immunobiology of the innate response to toxoplasma gondii. int j parasitol. 2009 jan;39(1):23–39. 18. nam h-w. gra proteins of toxoplasma gondii: maintenance of hostparasite interactions across the parasitophorous vacuolar membrane. korean j parasitol. 2009 oct;47 suppl:s29-37. 19. tizard i. veterinary immunology. 9th ed. philadelphia (pennsylvania): saunders; 2012. 20. döşkaya m, kalantari-dehaghi m, walsh cm, hiszczyńska-sawicka e, davies dh, felgner pl, et al. gra1 protein vaccine confers better immune response compared to codon-optimized gra1 dna vaccine. vaccine. 2007 feb 26;25(10):1824–37. 21. holec-gasior l. toxoplasma gondii recombinant antigens as tools for serodiagnosis of human toxoplasmosis: current status of studies. clin vaccine immunol. 2013 sep;20(9):1343–51. 22. subekti dt. study of antigenicity and immunogenicity gra1 protein from toxoplasma gondii. indones bull anim vet sci. 2014 apr 30;23(3). 23. wang z, ge w, huang s-y, li j, zhu x-q, liu q. evaluation of recombinant granule antigens gra1 and gra7 for serodiagnosis of toxoplasma gondii infection in dogs. bmc vet res. 2014 apr 30;10(1):158. 24. zhou h, min j, zhao q, gu q, cong h, li y, et al. protective immune response against toxoplasma gondii elicited by a recombinant dna vaccine with a novel genetic adjuvant. vaccine. 2012 feb 27;30(10):1800–6. 25. ferrandiz j, mercier c, wallon m, picot s, cesbron-delauw m-f, peyron f. limited value of assays using detection of immunoglobulin g antibodies to the two recombinant dense granule antigens, gra1 and gra6 nt of toxoplasma gondii, for distinguishing between acute and chronic infections in pregnant women. clin vaccine immunol. 2004 nov 1;11(6):1016–21. 26. gedik y, gülçe i̇z s, can h, değirmenci döşkaya a, i̇smet deliloğlu gürhan s, gürüz y, et al. immunogenic multistage recombinant protein vaccine confers partial protection against experimental toxoplasmosis mimicking natural infection in murine model. trials vaccinol. 2016;5:15–23. 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 80 152 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. 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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. 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 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. 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 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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. 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: budayantinns@unud.ac.id research article antimicrobial resistance profi le of mdr & non-mdr meropenemresistant pseudomonas aeruginosa isolates of patients in intensive care unit of tertiary hospital imaculata sonia vidaryo lameng1, ni nyoman sri budayanti1,2*, luh inta prilandari2, agus indra adhiputra1 1clinical microbiology study program, faculty of medicine, udayana university sanglah hospital, denpasar, bali 2clinical microbiology departement, faculty of medicine, udayana university – sanglah hospital, denpasar, bali received: 18th september 2021; revised:19th october 2021; accepted: 13th december 2021 abstract pseudomonas aeruginosa is one of the gram-negative bacteria that frequently causes infection of patients in the intensive care unit (icu) which is easily resistant to antimicrobial drugs. patients infected with carbapenem-resistant p. aeruginosa are predicted to have a poor prognosis. this study aims to know the resistance profi le of meropenem-resistant p. aeruginosa of patients in the icu. the results of this study can be used as a measure on the success of antimicrobial resistance control, infection control programs and become a reference for empirical therapy in the icu. this study used descriptive research and was carried out at the clinical microbiology laboratory of sanglah hospital denpasar for three years, from 2018 to 2020. the results showed 38 of the 93 isolates of p. aeruginosa in the icu were resistant to meropenem and were derived from sputum and urine. the percentage of meropenem-resistant p. aeruginosa isolates was higher in the multi-drug-resistant group and mostly came from sputum specimens. in 2018, non-mdr meropenem-resistant p. aeruginosa isolates was that 100% sensitive to all other antibiotics used to treat p. aeruginosa infections, including; ceftazidime, cefepime, ciprofl oxacin, gentamicin, amikacin, and piperacillin-tazobactam. in 2019 no meropenem-resistant p. aeruginosa isolates were found. in 2020, its sensitivity to antibiotics ceftazidime and piperacillin-tazobactam was 20.0%, ciprofl oxacin 60.0% and to antibiotics gentamicin and amikacin 100%. mdr meropenem-resistant p. aeruginosa isolates in 2018 were still sensitive to ceftazidime (15.4%) and amikacin (69.2%) antibiotics, while in 2019 they were only sensitive to amikacin (37.5%). in 2020, p. aeruginosa isolates were sensitive to the antibiotics ceftazidime and cefepime (11.1%), piperacillin-tazobactam (22.2%), and amikacin (88.9%). amikacin may be the choice of treatment for mdr meropenem-resistant p. aeruginosa. keywords: resistance profi le; pseudomonas aeruginosa; icu; meropenem; resistance abstrak pseudomonas aeruginosa merupakan salah satu bakteri gram negatif penyebab infeksi pada pasien di intensive care unit (icu) yang mudah resisten. pasien terinfeksi p. aeruginosa yang resistan karbapenem diindikasikan memiliki prognosis yang buruk. pengendalian infeksi dan menjadi acuan pemberian terapi empiris di icu. penelitian ini menggunakan metode penelitian deskriptif dan dilakukan di instalasi mikrobiologi klinik rumah sakit sanglah denpasar selama tiga tahun, dari 2018 hingga 2020. hasil penelitian menunjukan 38 dari 93 isolat p. aeruginosa di icu resistan terhadap meropenem dan berasal dari spesimen sputum dan urine. presentasi isolat p. aeruginosa yang resistan meropenem lebih tinggi pada kelompok multi-drug resistan dan sebagian besar berasal dari spesimen sputum. pada tahun 2018, isolat p. aeruginosa non-mdr yang resistan meropenem, 100% sensitif terhadap semua antibiotik lainnya yang digunakan untuk terapi infeksi p. aeruginosa, antara lain ; ceftazidime, cefepime, ciprofl oxacin, gentamicin, amikacin, dan piperacillin-tazobaktam. pada tahun 2019 tidak ditemukan isolat p. aeruginosa non-mdr resistan meropenem. pada tahun 2020, sensitifi tasnya terhadap antibiotik ceftazidime dan piperacillin-tazobactam 20,0%, ciprofl oxacin 60,0% dan terhadap antibiotik gentamicin serta amikacin 100%. isolat p. aeruginosa mdr resistan meropenem pada tahun 2018 masih sensitif terhadap antibiotik ceftazidime (15,4%) dan amikacin (69,2%), sedangkan pada tahun 2019 hanya sensitif terhadap antibiotik amikacin (37,5%). pada ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 tahun 2020, isolat p. aeruginosa sensitif terhadap antibiotik ceftazidime dan cefepime (11,1%), piperacillin-tazobactam (22,2%), serta amikacin (88,9%). amikacin dapat menjadi pilihan terapi p. aeruginosa mdr resistan meropenem. kata kunci: profi l resistansi; pseudomonas aeruginosa; icu; meropenem; resistansi how to cite: lameng, i.s.v, budayanti, n.n.s, prilandari, l.i, adhiputra, a.i. antimicrobial resistance profi le of mdr & non-mdr meropenem-resistant pseudomonas aeruginosa isolates of patients in intensive care unit of tertiary hospital. indonesian journal of tropical and infectious disease, 9(3) introduction pseudomonas aeruginosa is one of the gramnegative bacteria that is often found as a contaminant in hospitals.1,2 this bacterium can be an opportunistic pathogen causing nosocomial infections in the blood, lungs, and other body parts after surgery, especially in immunocompromised patients, patient received appropriate medical procedure, invasive, surgical wound or burns.2,3 nosocomial infections are estimated to occur annually in 1.75 million hospitalized patients worldwide and result in 175,000 deaths.4 p. aeruginosa accounts for 10%20% of hospital acquired infections in europe.5 the national healthcare safety network (nhsn) reported, p. aeruginosa as the third most common gram-negative bacteria causing nosocomial infections during 2011-2014.6,7 research by ribeiro et al. from january 2010 to december 2013 found that p. aeruginosa was the second most common bacterium in the icu sao paulo hospital brazil (14.5%), of which 48.7% was multi-resistant drug organism.8 at sanglah hospital itself in the second half of 2020, p. aeruginosa ranked third highest bacteria that cause infection in the intensive care unit (icu) and high care unit (hcu).9 patients admitted to the icu have a fi ve to ten times higher risk of developing p. aeruginosa infection compared to patients admitted to other inpatients.10 high frequency infection in the icu is associated with a decrease in the patient’s immunity due to the disease and the use of invasive devices such as catheters, nasogastric tubes, and ventilators.3,10 p. aeruginosa has quorum sensing ability which is associated with the occurrence of biofi lms on invasive medical devices in patients in the icu.1,2 the spread of p. aeruginosa infection through person-to-person contact is also more prone to occur in the icu due to several factors a patient is combined in one relatively small room.10 in the management of infection therapy, the selection of empiric antibiotics in the icu is not easy.4 according to performance standard for antimicrobial susceptibility testing on clinical and laboratory standard institute (2021), p. aeruginosa sensitive to antibiotics, beta-lactam combinations such as piperacillin tazobactam, 3rd generation cephalosporins especially ceftazidime, 4th generation cephalosporins (cefepime), aminoglycosides (gentamicin, amikacin), monobactams (aztreonam), carbapenems (except ertapenem) and fl uoroquinolones (ciprofl oxacin).11 the aztreonam group often becomes resistant.1 treatment of infectious diseases caused by p. aeruginosa becomes difficult because p. aeruginosa is easily resistant to various types of antibiotics. the prevalence of p. aeruginosa resistance to antibiotics is higher in icu patient isolates compared to non icu patients.10,12 the irresponsible use of an antibiotic widely, repeatedly, and over a long period of time can lead to the emergence of antibiotic resistance.10 the increase in treatment costs in cases of infection due to resistant bacteria is caused by various factors, including; patients get longer treatment, longer hospital stays, more intensive attention from health professionals such as doctors and nurses, or the use of newer antibiotics. newer antibiotics generally cost more than older antibiotics. the potential for increased costs in overcoming cases of infection by resistant bacteria needs attention because it can increase the fi nancial burden that must be borne by the state in the era of implementing the national health insurance program (jkn). 14 carbapenem is a type of beta-lactam antibiotic which has a broad spectrum of antibacterial imaculata sonia vidaryo lameng, et al.: antimicrobial resistance profile of mdr & non-mdr 153 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 activity.14 carbapenems such as meropenem and imipenem are potential antimicrobial agents that also used to treat multi-drug resistant pseudomonas aeruginosa (mdrpa) infections.14,15 increasing resistance to carbapenem antibiotics is one of the phenomena that must be watched out for at this time. patients infected with carbapenem-resistant p. aeruginosa are indicated to have a poor prognosis.16 this study aims to know the resistance profi le of meropenem-resistant p. aeruginosa in the icu. the results of this study can be used as a measure on the implementation of antimicrobial resistance control, infection control programs and become a reference for empirical therapy in the icu. material and methods this study used a descriptive research and was conducted at the clinical microbiology laboratory of sanglah hospital denpasar for three years, from 2018 to 2020. sanglah hospital denpasar is a tertiary referral hospital and the main health care center for the eastern part of indonesia with facilities of 710 beds. the sample was clinical isolates of meropenemresistant p. aeruginosa from patients admitted to icu. all type of specimens were included in this study. identifi cation and antimicrobial susceptibility testing were conducted using the vitek 2 automated system with gn card for identifi cation and ast gn 93 for antimicrobial susceptibility testing, according to the 2020 clinical laboratory standard institute (clsi) standard.11,17 data of antibiotic susceptibility test were collected and resistance profi le was analyzed. meropenem-resistant p. aeruginosa is a p. aeruginosa isolate with a minimum inhibitory concentration of ≥ 8 g/ml based on a dosage regimen of 1 gram every 8 hours.11 the antibiotics assessed in this study were the antibiotics of choice for p. aeruginosa that were available and included in the sanglah hospital formulary, including piperacillin tazobactam, ceftazidime, cefepime, gentamicin, amikacin, and ciprofl oxacin.18 multidrug-resistant (mdr) is a condition in which bacteria are resistant to at least one type of antibiotic from 3 antibiotic groups.13 mdr p. aeruginosa is a p. aeruginosa isolate that is resistant to at least one of three or more classes of antibiotics of choice for this bacterium, including: quinolones (ciprofl oxacin), extended-spectrum cephalosporins (ceftazidime, cefepime), penicillin (piperacillin tazobactam), aminoglycosides (gentamicin or amikacin) and carbapenems (meropenem).11,13 the exclusion criteria were incomplete data on meropenemresistant p. aeruginosa isolates from the icu including the results of antibiotic sensitivity tests as well as data from other treatment rooms including the covid-19 special icu. results this study observe the sensitivity pattern of meropenem-resistant p. aeruginosa in 3 consecutive years, from 2018 to 2020. in general, from 2018 to 2020, 93 p. aeruginosa bacteria were isolated in the icu sanglah hospital denpasar. the number of non-multidrug-resistant (nonmdr) p. aeruginosa isolates in the icu showed an increasing number but the mdr isolates showed a decreasing number (figure 1). figure 1. pseudomonas aeruginosa in icu during the period of 2018-2020 most of the p. aeruginosa isolates came from sputum specimens, both non-mdr and mdr, followed by urine and blood specimens. mdr p. aeruginosa isolates in 2018, 100% came from sputum specimens, while in 2019 and 2020, specimens came from sputum and urine specimens (table i). indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 152–159 firda typewritten text 154 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 table i. pseudomonas aeruginosa in icu during the period of 2018-2020 based on specimen specimen 2018 (%) 2019 (%) 2020 (%) non-mdr mdr non-mdr mdr non-mdr mdr sputum 9(81.8) 15(100) 19(100) 9(81.8) 25(89.3) 8(88.9) blood 2(18.2) 0(0) 0(0) 0(0) 0(0) 0(0) urine 0(0) 0(0) 0(0) 2(18.2) 3(10.7) 1(11.1) total 11(100) 15(100) 19(100) 11(100) 28(100) 9(100) of the 93 isolates of p. aeruginosa in the icu, 38 (40.9%) isolates had developed resistance to meropenem. the meropenem-resistant isolates were obtained from sputum and urine specimens. there were no meropenem-resistant isolates from blood. in 2018, 16 isolates were found from sputum, of which 3 of 9 (33.3%) were non-mdr and 13 of 15 (86.7%) mdr. in 2019, 8 of 11 mdr isolates were resistant against meropenem, which was 66.7% in sputum and 100% in urine. in the non-mdr group, no meropenem-resistant isolates were found. in 2020, the number of meropenemresistant isolates were 14 isolates, 5 non-mdr isolates came from sputum (20.0%) and 9 isolates from mdr, of which 8 isolates from sputum (100%) and 1 isolate from urine (100%). the percentage of meropenem-resistant p. aeruginosa isolates was higher in the mdr group (figure 2). based on table ii, in 2018, non-mdr meropenem-resistant p. aeruginosa isolates was 100% sensitive to all other antibiotics used to treat p. aeruginosa infections, including; ceftazidime, cefepime, ciprofl oxacin, gentamicin, amikacin, and piperacillin-tazobactam. in 2019 no meropenem-resistant p. aeruginosa isolates were found. in 2020, the percentage of sensitivity of other antibiotics used to treat p. aeruginosa infection decreased dramatically compared to 2018, especially ceftazidime and piperacillintazobactam (20.0%), followed by ciprofl oxacin (60.0%) while the sensitivity to gentamicin and amikacin was still 100%. mdr meropenemresistant p. aeruginosa isolates in 2018 were still sensitive to ceftazidime (15.4%) and amikacin (69.2%). in 2019, mdr meropenem-resistant isolates of p. aeruginosa were only 37.5% sensitive to amikacin. the percentage was decreasing compared to 2018 and other antibiotics were already resistant. however, in 2020 the table ii. results of antibiotic susceptibility test of meropenem-resistant p. aeruginosa isolates in 2018-2020 no antibiotics non-mdr(%s) mdr(%s) 2018(n=3) 2019(n=0) 2020(n=5) 2018(n=13) 2019(n=8) 2020(n=9) 1 ceftazidime 3/3 (100) 0/0 1/5 (20.0) 2/13 (15.4) 0/8 (0.0) 1/9 (11.1) 2 cefepime 3/3 (100) 0/0 1/5 (100) 0/13 (0.0) 0/8 (0.0) 1/9 (11.1) 3 ciprofl oxacin 3/3 (100) 0/0 3/5 (60.0) 0/13 (0.0) 0/8 (0.0) 0/9 (0.0) 4 gentamicin 3/3 (100) 0/0 5/5 (100) 0/13 (0.0) 0/8 (0.0) 0/9 (0.0) 5 amikacin 3/3 (100) 0/0 5/5 (100) 9/13 (69.2) 3/8 (37.5) 8/9 (88.9) 6 piperacilin tazobactam 3/3 (100) 0/0 1/5 (20.0) 0/13 (0.0) 0/8 (0.0) 2/9 (22.2) figure 2. meropenem-resistant pseudomonas aeruginosa in icu (n=38 isolates) imaculata sonia vidaryo lameng, et al.: antimicrobial resistance profile of mdr & non-mdr 155 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 sensitivity of meropenem-resistant p. aeruginosa isolates to antibiotics improved, including to the ceftazidime and cefepime (11.1%), piperacillintazobactam (22.2%), and amikacin (88.9%). the sensitivity pattern of mdr meropenemresistant p. aeruginosa isolates (table 3) showed that there was a pattern of mdr meropenemresistant p. aeruginosa isolates that were also resistant to all antibiotics in the hospital. this pattern always exists every year with a fl uctuating percentage and a signifi cant decline in 2021. the pattern of mdr meropenem-resistant p. aeruginosa isolates sensitive to ceftazidime and amikacin antibiotics was only found in 2018. in 2021, the pattern of antibiotic sensitivity was more diverse. most of them were resistant to various antibiotics, but each of these sensitivity patterns was still sensitive to amikacin antibiotics and one isolate of mdr p. aeruginosa was meropenemresistant, besides being sensitive to amikacin, they were also sensitive to the ceftazidime, cefepime, piperacilin, tazobactam antibiotics. the nonmdr meropenem-resistant p. aeruginosa isolate in 2018, the sensitivity pattern of 100% showed sensitivity to all anti-pseudomonal antibiotics table iii. resistance profi le of mdr and non-mdr meropenem-resistant p. aeruginosa isolates c ef ta zi di m e c ef ep im e p ip er az ili n ta zo ba ct am c ip ro fl o xa ci n g en ta m ic in a m ik ac in 2018 2019 2020 no meropenem-resistant pseudomonas aeruginosa mdr(%) mdr(%) mdr(%) 1 s r r r r s 2/13 (15.4) 0/8 (0.0) 0/9 (0.0) 2 r r r r r s 5/13 (58.8) 3/8(37.5) 6/9 (66.7) 3 r r r r r r 4/13 (30.8) 5/8 (62.5) 1/9 (11.1) 4 r r s r r s 0/13 (0.0) 0/8 (0.0) 1/9 (11.1) 5 s s s r r s 0/13 (0.0) 0/8 (0.0) 1/9 (11.1) no meropenem-resistant pseudomonas aeruginosa non-mdr (%) non-mdr (%) non-mdr (%) 1 s s s s s s 3/3 (100) 0/0 1/5 (20.0) 2 r r r r s s 0/3 (0.0) 0/0 2/5 (40.0) 3 r r r s s s 0/3 (0.0) 0/0 2/5 (40.0) available at sanglah hospital denpasar. however, in 2020, the pattern of sensitivity to antibiotics varied because some antibiotics were already resistant. discussion pseudomonas aeruginosa is one of the environmental bacteria that is often found in hospitals.1,2 these bacteria can be opportunistic pathogens that cause nosocomial infections, including pneumonia, urinary tract infections, sepsis, osteomyelitis and skin infections including wounds and burns.2 p. aeruginosa can grow in a variety of environmental conditions. incidence of infection and resistance is common in the icu. the bacteria found were often resistant to antibiotics.10,19 based on data from the antibiogram of sanglah hospital for the julydecember 2020 period, p. aeruginosa was the third highest bacteria in the icu and hcu.9 the high rate of frequency of p. aeruginosa in the icu is related to the decreased immunity itself, as well as the use of invasive devices such as catheters, nasogastric tubes and ventilators.3,10 indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 152–159 firda typewritten text 156 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 according to the performance standard for antimicrobial susceptibility testing at the 2020 edition of the clinical and laboratory standard institute, p. aeruginosa is sensitive to beta lactam combination antibiotics such as piperacillin tazobactam, 3rd generation cephalosporins especially ceftazidime, 4th g e n e r a t i o n c e p h a l o s p o r i n ( c e f e p i m e ) , aminoglycosides (gentamicin, amikacin), monobactam (aztreonam), carbapenems (except ertapenem) and fl uoroquinolones.11 the aztreonam group often becomes resistant.1 widespread, repeated, and long-term use of an antibacterial agent can lead to the emergence of antibacterial resistance.15 p. aeruginosa is intrinsically resistant to several antibiotics and has the ability to rapidly generate resistance to new antimicrobials. p. aeruginosa was the fi rst bacterium to show an mdr phenotype.8 carbapenem antibiotics have become important in clinical management.8,20 carbapenem-resistant p. aeruginosa infections are common.21 in february 2017, the world health organization made a priority list of pathogenic bacteria in the development of new antibiotics. carbapenem-resistant p. aeruginosa is second ranked in the group of top priority (critical) bacteria because of its high resistance to most antibiotics including carbapenems and thirdgeneration cephalosporins which are the best choices in the treatment of mdr bacteria.22 in the united states, 10%–20 % of clinical isolates of p. aeruginosa identifi ed in health facilities, resistant to at least one carbapenem group antibiotic.23 p. aeruginosa became meropenem-resistant due to upregulation of the effl ux pump.24 in this study, the percentage of meropenemresistant p. aeruginosa isolates was higher in the multidrug-resistant group and most of them came from sputum specimens. most of the sputum specimens collected in this study were from endotracheal tube secretions. p. aeruginosa has the ability of a bacterial cell-cell communication mechanism, known as quorum sensing (qs) which plays a role in gene expression and biofi lm formation. the results of this study also support research in new york by walter et al., that during july-october 2015 carbapenem-resistant p. aeruginosa was most commonly found in sputum specimens followed by urine.18 research by asempa te et al., in 2017-2018 also showed that 89% meropenem-resistant p. aeruginosa was found in respiratory specimens.25 the sensitivity of meropenem-resistant p. aeruginosa isolates varied to various antibiotics. research carried out by vitkauskienė a et al., in 2003 and 2008, isolates of p. aeruginosa that were resistant to carbapenems were more often resistant to ciprofl oxacin and gentamicin than isolates sensitive to carbapenems. in 2008, isolates that were carbapenem-resistant were also more frequently resistant to ceftazidime, cefepime, aztreonam, piperacillin, and amikacin.26 results from the study by asempa te et al. in july-october 2017 showed that most of the carbapenem-resistant p. aeruginosa isolates had lower resistance to ceftazidime.25 research by garcinuno et al. the data from 2009-2013 found that most of the meropenem-resistant p. aeruginosa isolates were also resistant to fl uoroquinolones. administration of amikacin therapy resulted in a more than threefold reduction in the risk of resistance.27 overuse of fl uoroquinolones in the treatment of p. aeruginosa infections increased bacterial resistance to fluoroquinolones in recent years. resistance to fl uoroquinolones is mainly due to: (1) point mutations in the dna gyrase (gyra and gyrb) and topoisomerase iv (parc and pare) genes, (2) the presence of transferable plasmid-mediated quinolone resistance (pmqr), and (3) mutations in genes that regulate efflux expression and decreased expression of outer membrane porins.28 from the results of this study, in 2018 non-mdr meropenem-resistant p. aeruginosa isolates were still sensitive to all antibiotics but in 2020 most of the sensitivity patterns showed sensitivity to gentamicin and amikacin. although the mdr meropenem-resistant isolates of p. aeruginosa showed less sensitivity to various antibiotics, in 2020 the percentage of sensitivity to antibiotics except ceftazidime increased. sanglah hospital published guidelines for the use of prophylactic and therapeutic antibiotics in 2019, ceftazidime is included in the watch group of antibiotics. ceftazidime not recommended as empiric imaculata sonia vidaryo lameng, et al.: antimicrobial resistance profile of mdr & non-mdr 157 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 antibiotic therapy but should be based on the results of bacterial culture (defi nitive therapy) to p. aeruginosa, so its use restricted, in which allows the sensitivity p. aeruginosa to the ceftazidime increased in sanglah hospital. this can be seen in the pattern of sensitivity, where although there is a pattern that is already resistant to all antibiotics every year, the percentage shows a fl uctuating picture and signifi cantly decreases in 2020. in addition, there were various other sensitivity patterns that showed mdr meropenem-resistant p. aeruginosa isolates were still sensitive to amikacin. the results of this study support the statement of baseti et al. in 2018 which stated that all antipseudomonal antibiotics except amikacin were associated with the emergence of resistance in p. aeruginosa. aminoglycosides modifying enzymes (ame) inactivate aminoglycosides by attaching acetyl, phosphate or adenyl groups to the amino and hydroxyl substituents on the antibiotic molecule. this modifi cation signifi cantly reduced the affi nity of the aminoglycoside for the target of the 30s ribosomal subunit and blocks the activity of the aminoglycoside. however, compared to other aminoglycosides, amikacin is usually a poor substrate for this enzyme and is known to provide better antibiotic activity against p. aeruginosa.28 the results of this study also support the study carried out by khan f et al. in 2012-2013 regarding the prevalence and susceptibility pattern of multi drug resistant clinical isolates of pseudomonas aeruginosa in karachi and research by anggraini d et al., regarding the prevalence and sensitivity pattern of multidrug resistant antimicrobial pseudomonas aeruginosa in arifin achmad pekanbaru hospital in 2015 that amikacin is a therapeutic option for mdr p. aeruginosa.29,30 conclusion the resistance profi le of meropenem-resistant p. aeruginosa in the icu varies. meropenemresistant p. aeruginosa isolates that were nonmdr for 3 years were still mostly sensitive to gentamicin and amikacin, while in multi-drug resistant isolates, amikacin was the choice of treatment. acknowledgement the authors are grateful for cooperation of: 1. head and all staff clinical microbiology of sanglah hospital. 2. antimicrobials resistance control sub committee of sanglah hospital. conflict of interest references 1. ryan kj, ahmad n, alspaugh ja, drew wl, reller m. sherris medical microbiology. 7th ed. new york: mcgraw hill education; 2014. 2. talaro kp, chess b. foundation microbiology. 10th ed. new york: mcgraw-hill education; 2018. 3. centers for disease control and prevention. pseudomonas aeruginosa in healthcare settings [internet]. centers for disease control and prevention; 2019 [cited 29 april 2021]. available from: https://www.cdc.gov/hai/ organisms/pseudomonas.html 4. guggenbichler jp, assadian o, boeswald m, kramer a. incidence and clinical implication of nosocomial infections associated with implantable biomaterials catheters, ventilator-associated pneumonia, urinary tract infections. gms krankenhhyg interdiszip. 2011;6(1). doi: 10.3205/dgkh000175 5. ramos gp, rocha jl, tuon ff. seasonal humidity may infl uence pseudomonas aeruginosa hospital-acquired infection rates. int j infect dis. 2013;17(9):e757-e761. doi: 10.1016/j.ijid.2013.03.002 6. weiner lm, webb ak, limbago b, dudeck ma, patel j, kallen aj, et al. antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the national healthcare safety network at the centers for disease control and prevention, 2011-2014. infect control hosp epidemiol. 2016;37(11):1288-1301. doi: 10.1017/ice.2016.174. 7. centers for disease control and prevention. gramnegative bacteria infections in healthcare settings [internet]. centers for disease control and prevention; 2013. [cited 29 april 2021]. available from: https:// www.cdc.gov/hai/organisms/gram-negative-bacteria. html 8. ribeiro ácds, crozatti mtl, silva aad, macedo rs, machado amo, silva ata. pseudomonas aeruginosa in the icu: prevalence, resistance profi le, and antimicrobial consumption. rev soc bras med trop. 2019;53:e20180498. oi: 10.1590/0037-86820498-2018. the authors declare that they have no confl ict of interest. indonesian journal of tropical and infectious disease, vol. 9 no. 3 september–december 2021: 152–159 firda typewritten text 158 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 9. rsup sanglah. pola kepekaan mikroorganisme rsup sanglah periode juli-desember 2020: pola kepekaan mi kroorganisme ruang icu dan hcu rsup sanglah denpasar juli desember 2020. denpasar: rsup sanglah; 2020. 10. dharmayanti igam, sukrama idm, karakteristik b a k t e r i p s e u d o m o n a s a e r u g i n o s a d a n p o l a kepekaannya terhadap bakteri di intensive care unit (icu) rsup sanglah pada bulan november 2014 – januari 2015. e-jurnal medika. 2019;8(4). issn 23031395. available at: <https://ojs.unud.ac.id/index.php/ eum/article/view/50011> 11. clsi. performance standards for antimicrobial susceptibility testing. 31st ed. clsi guideline m100. wayne, pa: clinical and laboratory standards institute; 2021. 12. yusuf e, van herendael b, verbrugghe w, ieven m, goovaerts e, bergs k, et al. emergence of antimicrobial resistance to pseudomonas aeruginosa in the intensive care unit: association with the duration of antibiotic exposure and mode of administration. ann intensive care. 2017 dec;7(1):72. doi: 10.1186/s13613-017-0296-z. 13. magiorakos ap, srinivasan a, carey rb, carmeli y, falagas me, giske cg, et al. multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. clin microbiol infect. 2012 mar;18(3):268-81. doi: 10.1111/ j.1469-0691.2011.03570.x. 14. halim sv, yulia r, penggunaan antibakteri golongan carbapenem pada pasien dewasa rawat inap sebuah rumah sakit swasta di surabaya. jurnal farmasi klinik indonesia, desember. 2017;6(4). doi: http://10.15416/ ijcp.2017.6.4.267 15. fusté e, jiménez ll, segura c, gainza e, vinuesa t, viñas m. carbapenem resistance mechanisms of multidrug-resistant pseudomonas aeruginosa. journal medical microbiology. 2013 sep;62(pt 9):1317-1325. doi: 10.1099/jmm.0.058354-0 16. deni j, pangalila fjv. hubungan keberhasilan terapi pneumonia nosokomial resistan pseudomonas aeruginosa dan acinetobacter baumannii dengan dosis karbapenem di icu rs royal taruma periode 2012-2017. tarumanagara medical journal. 2019;2(1): 65-76. doi: http://dx.doi.org/10.24912/tmj.v2i1.5865 17. garcia ls (ed). clinical microbiology procedures handbook, 3rd edition [internet]. american society for microbiology press; 2010. 18. rsup sanglah. formularium edisi xiii rumah sakit umum pusat sanglah tahun 2020-2021: rsup sanglah; 2021. 19. lee j, zhang l. the hierarchy quorum sensing network in pseudomonas aeruginosa. protein & cell. 2015;6(1):2641. doi: 10.1007/s13238-014-0100-x. 20. walters ms, grass je, bulens sn, hancock eb, phipps ec, muleta d, et al.. carbapenem-resistant pseudomonas aeruginosa at us emerging infections program sites, 2015. emerg infect dis. 2019 jul;25(7):1281-1288.doi: 10.3201/eid2507.181200 21. zilberberg md, shorr af. 2013. secular trends in gram-negative resistance among urinary tract infection hospitalizations in the united states, 2000– 2009. infect control hosp epidemiol 34:940–946. doi:10.1086/671740. 22. world health organization. who publishes list of bacteria for which new antibiotics are urgently needed [internet]. world health organization; 2017. [cited 1 juni 2021]. available from: https://www.who.int/news/ item/27-02-2017-who-publishes-list-of-bacteria-forwhich-new-antibiotics-are-urgently-needed 23. huband md, castanheira m, flamm rk, farrell dj, jones rn, sader hs. in vitro activity of ceftazidimeavibactam against contemporary pseudomonas aeruginosa isolates from u.s. medical centers by census region, 2014. antimicrob agents chemother 2016; 60:2537–41. doi: 10.1128/aac.02252-16 24. yohei d. treatment options for carbapenem-resistant gram-negative bacterial infections. clinical infectious diseases, volume 69. issue supplement_7[internet]. 1 december 2019. pages s565–s57. https://doi. org/10.1093/cid/ciz830 25. asempa te, nicolau dp, kuti jl. carbapenemnonsusceptible pseudomonas aeruginosa isolates from intensive care units in the united states: a potential role for new β-lactam combination agents. j clin microbiol. 2019;57(8):e00535-19. doi: 10.1128/ jcm.00535-19 26. vitkauskienė a, skrodenienė e, jomantienė d, macas a, sakalauskas r. changes in the dependence of pseudomonas aeruginosa o serogroup strains and their resistance to antibiotics in a university hospital during a 5-year period. medicina (kaunas, lithuania). 2 0 11 ; 4 7 ( 7 ) : 3 6 1 3 6 7 . h t t p s : / / d o i . o rg / 1 0 . 3 3 9 0 / medicina47070051 27. garcinuño p, santibañez m, gimeno l, sánchezbautista a, coy j, sánchez-paya j, boix v, et al. empirical monotherapy with meropenem or combination therapy: the microbiological point of view. eur j clin microbiol infect dis. 2016 nov;35(11):18511855. doi: 10.1007/s10096-016-2737-2. 28. bassetti m, vena a, croxatto a, righi e, guery b. how to manage pseudomonas aeruginosa infections. drugs context. 2018 may 29;7:212527. doi: 10.7573/ dic.212527 29. khan f, khan a, kazmi su. prevalence and susceptibility pattern of multi drug resistant clinical isolates of pseudomonas aeruginosa in karachi. pak j med sci. 2014;30(5):951-954. doi: 10.12669/ pjms.305.5400 30. anggraini d, yulindra ug, savira m, djojosugito fa, hidayat n. prevalensi dan pola sensitivitas antimikroba multidrug resistant pseudomonas aeruginosa di rsud arifin achmad. majalah kedokteran bandung. 2018; 5(1), 6-12. doi: https:// doi.org/10.15395/mkb.v50n1.1150 imaculata sonia vidaryo lameng, et al.: antimicrobial resistance profile of mdr & non-mdr 159 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. 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. 15 vol. 7 no. 1 january–april 2018 c o m b i n a t i o n a n t i f u n g a l t h e r a p y f o r onychomycosis nur khamidah1a, evy ervianti1 1 dermato-venereology departement, school of medicine universitas airlangga, dr. soetomo hospital surabaya a corresponding author: midul_mbem@yahoo.com abstract onychomycosis is a fungal infection of the nail unit including the nail matrix, the nail bed and the nail plate by both dermatophyte and non-dermatophyte agents. it is disturbs not only cosmetic disfigurement, but also it may have an impact on patients’ emotional, social and occupational functioning, finally affecting the overall quality of life. the incidence rate tends to increase, management of onychomycosis is still challenging. important problems regarding antifungal monotherapy have experienced many failures and recurrences. in general, pharmacological approaches for onychomycosis can be topical or oral antifungal. antifungal monotherapies often lead to failure treatment, also high incidence of recurrence. one strategy for this problem is a combination antifungal therapy. in vitro studies show the synergistic effect of using combination two antifungals (both oral antifungal or combination topical and oral antifungal), hence it is mycologically or clinically expected to increase the success rate of onychomycosis therapy. this review tries to evaluate the previous study exploring the effectiveness of antifungal combination therapies on onychomycosis. two oral antifungals usually used are terbinafine as fungicidal agent and itraconazole as fungistatic agent. there is combination between topical and oral antifungal such as itraconazole or terbinafine with amorolfine or ciclopirox, also other combination like griseofulvin and amorolfone or tioconazole. all the combination therapies show better result than monotherapy alone, but it is still difficult to conclude whether antifungal combinations in onychomycosis will increase effectiveness due to variations in therapeutic duration, result definition, and statistical evaluation on existing studies. further research is required with longer duration of observation, uniform patient criteria and definition of success, random control and blinding to minimize bias. keywords: antifungal, combination, therapy, onychomycosis, effectiveness abstrak onikomikosis adalah infeksi jamur pada bagian kuku baik pada matriks kuku, bantalan kuku, maupun lempeng kuku, oleh agen dermatofit maupun non-dermatofit. onikomikosis adalah penyakit yang sangat mengganggu bukan hanya karena masalah kosmetik, namun juga dampaknya pada faktor emosional, sosial, dan pekerjaan, yang selanjutnya akan mempengaruhi kualitas hidup pasien. angka kejadiannya cenderung meningkat, sedangkan penatalaksanaannya masih merupakan tantangan. masalah penting oleh karena dengan antijamur monoterapi banyak mengalami kegagalan dan kekambuhan. secara umum, pendekatan strategi farmakologi antijamur dapat berupa terapi topikal atau oral. antijamur monoterapi sering menunjukkan kegagalan, dan kejadian rekurensi yang tinggi. salah satu strategi untuk mengatasi masalah ini adalah dengan terapi kombinasi antijamur. penelitian in vitro menunjukkan efek sinergisme penggunaan kombinasi dua buah antijamur (dua buah antijamur oral atau kombinasi antijamur topikal dan oral), sehingga secara klinis dan mikologi diharapkan dapat meningkatkan keberhasilan terapi onikomikosis. telaah pustaka ini bertujuan mengkaji penelitian sebelumnya yang menunjukkan efektivitas kombinasi antijamur pada onikomikosis. dua buah antijamur oral yang biasa digunakan adalah terbinafin sebagai agen fungisidal dan itrakonazol sebagai agen fungistatik. untuk kombinasi oral dan topikal berupa terbinafin atau itrakonazol dengan amorolfin atau siklopiroks, dengan kombinasi lain seperti griseofulvin dengan amorolfin atau tiokonazol. seluruh kombinasi terapi ini menunjukkan hasil yang lebih baik dibandingkan monoterapi, namun sayangnya masih sulit menarik simpulan dari penelitian yang ada apakah kombinasi antijamur pada onikomikosis akan meningkatkan efektivitas oleh literature review 16 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 15–20 karena adanya variasi durasi terapi, variasi definisi hasil, evaluasi statistik pada penelitian-penelitian yang ada. dibutuhkan penelitian lebih lanjut dengan durasi pengamatan yang panjang, kriteria pasien dan definisi keberhasilan yang sama, kontrol acak dan blinding untuk meminimalisir bias. kata kunci: antijamur, kombinasi, terapi, onikomikosis, efektivitas introduction onychomycosis is a progressive fungal infection of the nails and surrounding tissue, characterized by thickening and/or nail color change, as well as the separation of the nail from the nail bed. onychomycosis can occur either on the nail bed, plate, matrix, or a combination of the parts of the nail.1–4 onychomycosis accounts for about 30% of all superficial fungal infections and 50% of all nail abnormalities. the incidence of onychomycosis tends to increase due to increasing geriatric population and cases of immunocompromised patients such as diabetes, peripheral arterial disease, immunosuppressed conditions such as hiv infection and consumption of immunosuppressant agents. onychomycosis in children is rare, usually it comes from indirect environment contamination of trauma or dystrophic nails.5,6 in addition there are other factors influencing as climate, prolonged use of occlusive footwear, repeated trauma to the nails, and genetic predisposition.2,3,7–9 onychomycosis can cause cosmetic problem, pain, discomfort, and affect the emotional or psychosocial aspects of the patient, thus requiring optimal and total therapy. the therapy remains a challenge for dermatologists, due to the duration of therapy (in accordance with the slow growth of the nail plate), poor patient compliance, and often takeing long to observe the success of therapy.1 the failure rate of onychomycosis therapy reaches 20%, along with a high recurrence rate of 10-53%.3,6,8,9 some factors that also contribute to the unsuccessful therapy are patient’s susceptibility, pattern of resistant fungal growth, the presence of fungal dormant spores on the nail, low bioavailability of the drug, and lack of drug penetration into the nail.1,3 therefore, many methods are introduced to overcome problems in onychomycosis therapy, including a combination of two oral antifungals, or combination of both oral and topical antifungals.4 this review will try to discuss the combination of two oral antifungal or topical and oral combination to improve therapeutic efficacy of infection in onychomycosis cases. definition onychomycosis onychomycosis is a fungal infection of the nails by both dermatophytes and non-dermatophytes such as yeast and mold. ninety percent of onychomycosis cases occur in the toes and most cases of onychomycosis of the fingers are caused by dermatophytes. the most common dermatophyte species are trichophyton rubrum, trichophyton mentagrophytes var. digitale or epidermophyton floccosum. infection by other filamentous fungus or mold such as scopulariopsis bravicaulis, aspergillus spp., fusarium spp., acremonium spp., alternaria spp., can act as primary pathogen, secondary pathogens, as well as contaminants. yeasts such as candida albicans, candida parapsilosis are the third leading cause of fungal infections of the fingernails and usually arise when there are certain predisposing factors such as immunosuppression and diabetes.2,3,10 clinically, onychomycosis can be differentiated into several subtypes: distal and lateral subungual onychomycosis (dlso), white superficial onychomycosis (wso), proximal subungual onychomycosis (pso), endonyx onychomycosis (eo), total dystrophic onychomycosis (tdo), mixed onychomycosis, candida onychomycosis.2,7,8,11 predilection on toe nails is more frequent (4-10 times) than fingernails, usually affecting multiple fingers and often accompanied with tinea plantar pedis. onychomycosis of the toes is usually more difficult to treat, because the growth of toenails is slower than the hands, and limited blood flows in the area, especially in the elderly.6,9 diagnosis of onychomycosis is made based on the clinical and mycological examinations by microscopic and culture examinations. microscopic identification is considered enough to support determination of therapy, but the culture remains important as the gold standard to determine the causative species of onychomycosis.3 there are several other investigation methods, including histopathology, onychoscopy, stripe dermatophytes, fluorescent microscopy, raman spectroscopy, and polymerase chain reaction (pcr).2,11 topical and systemic antifungal therapy the purpose of onychomycosis therapy is eradication of fungus and restoration of the nail to be normal as before or complete clearance, which is defined as mycologically clear (including both negative direct microscopy and negative culture) and clinically clear (as disappearance of all lesions or residual disease of no more than 10% of the original total suffering surface).12 an ideal antifungal agent for the treatment of onychomycosis are favorable nail kinetics (ability to diffuse through the nail bed and be incorporated into the nail matrix), high mycologic and clinical cure rate, low incidence of relapse and effectiveness as short term therapy, low incidence of side effects, few drug interactions and cost effectiveness.12 in general there are two main pharmacological strategies, oral and topical.8 17khamidah and ervianti: combination antifungal therapy for onychomycosis the role of monotherapy of topical antifungal is only effective in wso (except in transverse or striata infections), early dlso (except for longitudinal lines) with nail plate involvement <80% and lack of associated lunula, or contraindications to systemic antifungal use.13,14 some topical therapies used are azoles (ketoconazoles, clotrimazole, miconazole, sulconazole, oxiconazole and econazole); whitfield’s ointment, potassium permanganate, amorolfine nail lacquer, ciclopirox olamine, allylamines (naftifine, terbinafine); organic acids (salicylic acid, phytex paint and undecenoates), halogenated phenolic esters (haloprogin); thiocarbamate derivatives (tolnaftate); and polyenes (nystatin). amorolfine and ciclopirox are widely used than other topical agent8,12 lacquer is a form of drug which can maintain proper concentration of the active substance on the nail surface, and it just need to be applied once or twice weekly, which is of great convenience. and cosmetic nail varnishes would not affect the antifungal efficacy and can be applied concomitantly with amorolfine in the treatment of onychomycosis.15 amorolfine is a group of morpholine which is a synthetic antifungal drug, with fungistatic and fungicide activity and broad spectrum. amorolfine works by inhibiting the enzyme of 14 delta reductase, delta 8 and delta 7 isomerase in ergosterol biosynthesis pathways, and fungicidal against c. albicans and t. mentagrophytes. amorolfine is available in the form of 5% nail polish and applied to affected nails 1 or 2 times a week for 6-12 months, after removing as many infected areas as possible from the nail plate. amorolfine persists for 14 days after complete therapy.8,11 ciclopirox is a hydroxypiridone derivative with extensive antifungal activity against t. rubrum, s. brevicaulis, and candida spp. ciclopirox will inhibit metaldependent enzymatic processes, including nutrient uptake, cellular energy production, and toxic intracellular peroxide degradation. ciclopirox available in the form of nail polish 8% concentration, used once a day for more than 48 weeks. recommended duration for treatment with ciclopirox is 24 weeks for fingernails and 48 weeks for toenails. no studies has directly compared the effectiveness of amorolfine and ciclopirox but clinical improvement with ciclopirox is usually lower.1,8,11 systemic antifungal therapy gives higher effectiveness than topical route. the oral antifungal therapy that has been widely used for the therapy of onychomycosis includes itraconazole, terbinafine, fluconazole, griseofulvin and ketoconazole. itraconazole and terbinafine appear to be the best systemic drugs for the therapy of onychomycosis due to their reservoir effects in the nails.16 terbinafine works by inhibiting squalene epoxidase, which is very important for ergosterol biosynthesis to be an integral component to the cell wall of the fungus, which are directly fungicidal. recommended dosage of terbinafine is 250 mg/day for 6 weeks for fingernails and 12-16 weeks for toenails infections. for pulsed therapy some studies use a dosage of 250 mg/day for a week per month (3 months duration) or 500 mg/day for a week per month (3 months duration). terbinafine is lipophilic and well distributed on the skin and nails. terbinafine can be detected on the nail after one week from the start of therapy and persist for up to 6 months after complete therapy, due to the long half-life of the drug. terbinafine has potent and extensive fungicidal activity against dermatophytes, especially t. rubrum, t. mentagrophytes, but has low fungistatic activity against candida spp., compared with azole derivatives. oral terbinafine is generally well tolerated. surveillance studies show the most common side effects are on the gastrointestinal tract (49%) such as nausea, diarrhea, or taste disorders, and dermatological reactions such as rash, pruritus, urticaria, eczema, liver side effects are low. terbinafine is not recommended for onychomycosis in children cases. oral terbinafine has minimal drug interactions, especially with drugs that metabolized by the p450 2d6 isoenzym cytochrome.8,11 itraconazole fights against fungi from dermatophytes, yeasts, and molds. the minimum of inhibitory concentration (mic) is 10 times greater than terbinafine, but not as active as terbinafine against dermatophytes. in general itraconazole is fungistatic but can become fungicidal by increasing the concentration to 10 times of mic. the mechanism of action of itraconazole is similar to other azole derivatives, by inhibiting the p450 cytochrome oxidase mediating the ergosterol synthesis that is required for fungal cell wall synthesis. itraconazole is absorbed optimally along with food and acidic ph. it is lipophilic and metabolized in the liver by the p450 cytochrome 3a4 system, which increases the risk of interaction with other drugs metabolized through this pathway. itraconazole dose that can be administered is 200 mg/day for 12 weeks continuously or 400 mg/day pulse therapy for 1 week per month. fingernails onychomycosis is recommended to be used two periods pulsation therapy and toe nails given three periods of pulse therapy. similar to terbinafine, itraconazole has rapid penetration into the nail and can be detected one week from the start of administration, and persists in the nail up to 6-9 months after the therapy is stopped. the most common side effects of itraconazole are headache, and gastrointestinal disorders. the side effects will be lower when given with pulse therapy. abnormalities of liver function occur in 1.9% of patients treated with itraconazole with pulsation method and 3% at a daily dosage. hepatitis is common in continuous therapy usually after 4 weeks of therapy. monitor liver function is recommended in patients with previous liver disorders, who receive continuous therapy for more than one month, and use other hepatotoxic drugs. itraconazole is a contraindication to patients with congestive heart disease because of the increased risk of negative inotropes. itraconazole may prolong the qt interval and co-administration of other drugs may increase the qt interval.8,17 fluconazole is less effective than terbinafine and itraconazole in the treatment of onychomycosis, but it is a choice when patients do not tolerate other oral antifungal agents. the minimal dosage of fluconazole should be 150 18 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 15–20 mg/week for at least 6 months. fluconazole can still be detected on toe nails 6 months after the therapy is stopped. fluconazole can fight dermatophytes and candida. although there is no license for the use of fluconazole for cases of onychomycosis, the study is still conducted with emphasis on the use of dosage of 450 mg once a week. this may be possible given the pharmacokinetic ability, and with weekly dosages will be able to improve adherence and decrease the cost of therapy. there have been several studies comparing the effectiveness of fluconazole with itraconazole and terbinafine, but the results show that fluconazole is less effective than both drugs. the most common side effects of fluconazole are headache, skin rash, gastrointestinal disorders, and insomnia. fluconazole is a lower inhibitor of p450 cytochrome compared to itraconazole, resulting in little interaction with other drugs.8,9,16 griseofulvin and ketoconazole were the first group of systemic antifungals but no longer used for the treatment of onychomycosis because of some reasons such as long duration of therapy, low clinical and mycologic cure rates, high probability of relapse within 2 years and potentially significant side effects. high risk of hepatotoxicity in longterm use of ketoconazole makes its use limited in the usa and europe.8,13 griseofulvin is a fungistatic agent which works by inhibiting nucleic acid synthesis, stopping cell division, and inhibiting fungal cell wall synthesis. griseofulvin is the only licensed antifungal agent for children with onychomycosis, recommended dosage for age 1 month and above is 10 mg/ kg body weight/day, along with fatty foods to increase absorption and support bioavailability. for adults, the recommended dosage is 500-1000 mg/day for 6-9 months for fingernail infection and 12-18 months for toenail infection. contraindications for griseofulvin are pregnancy and men who plan to have children 6 months after therapy because according to an experimental study in mice, it had significant effects to the sperm. a study comparing the use of griseofulvin, terbinafine, and itraconazole showed a lower improvement for griseofulvin. griseofulvin has several limitations including low effectiveness, long duration of therapy, greater drug interaction risk, while on the other side there are new antifungal agents with higher availability. for these reasons, the use of griseofulvin is no longer recommended for onychomycosis cases.8,11 antifungal combination therapy a l t h o u g h m o n o t h e r a p y e x h i b i t e d s h o r t t e r m effectiveness, the substantial proportion of patients does not show complete recovery forever. the use of antifungal combinations and synergistic exploitation has been performed in the field of mycology. combination therapy is one of the ways to improve the rate of healing and overall improvement of onychomycosis cases completely.8,12,13 the combination of two antifungals can provide several advantages such as improving the effectiveness and speed of therapeutic improvements, enhancing the broader antifungal spectrum, better resistance suppression, tolerability and patient safety. this advantage is often attributed to the synergistic effects of drugs. in this context, synergism can be defined as the use of a combination of two antifungals will have a better effect than the use of both drugs individually.4 combined regimens may be sequentially or paralleled. sequential therapy means that the combination is given separately. the first drug is given for a certain duration and then followed by second drug afterwards. parallel therapy means simultaneous combination of both drugs. there is no election guideline when sequential therapy and parallel therapy are used. parallel therapy is recommended for the patients who are expected to experience treatment failure, for example in patients with onychomycosis with diabetes. sequential therapy is recommended for patients with minimal response to therapy, such as patients with positive culture after 3-6 months of therapy.4,13 two oral antifungal combination therapy onychomycosis caused by dermatophytes, molds, and yeasts shows differential susceptibility to the oral systemic treatments. this differential susceptibility to antifungals suggests that a combined therapy of oral treatments might be more effective against mixed infection or resistant fungi.18 there were two studies about oral antifungal combination itraconazole and terbinafine in the management of onychomycosis by dermatophytes in toenails. the first study is showed the continued combination therapy (sequential), while the other study showed a parallel combination. sequential therapy with itraconazole and terbinafine for 12-16 weeks showed a higher cure rate than the terbinafine pulse therapy alone. combination therapy of itraconazole and terbinafine paralel in short duration (6 weeks) showed results that were comparable with sequential therapy terbinafine or itraconazole pulse therapy for 12 weeks. these data are indicated the advantages of combination therapy depending on the length of the therapy period.18 on the other hand, the prospective study by gupta in april 1996 until december 2004 compared the four treatment groups: the first group was itraconazole 200 mg/ day for weeks 1 to 4 and terbinafine 250 mg/day for weeks 3 to 6 (2-week overlap of itraconazole and terbinafine); second group was continuous terbinafine 250 mg/day for 12 weeks; third group with intermittent terbinafine (250 mg/day for 4 weeks on, 4 weeks off, 4 weeks on); forth group with pulsed itraconazole (one pulse 200 mg twice daily for 7 days on, 21 days off) for three pulses. mycological recurrence showed 57% in the first group, 32% in the second group, 36% in the third group, and 59% in the fourth group.19 19khamidah and ervianti: combination antifungal therapy for onychomycosis combination oral and topical antifungal the combination of oral and topical antifungal therapy will provide the increas penetration effect on the infected tissue, which when given separately will not accumulate at effective concentrations. rapid oral therapy accumulates on the nail bed, whereas effective topical therapy penetrates the nail plate and the lateral borders but not the deeper layers of the nail, therefore in combination therapy there will be two-way penetration of the nail plate by topical agents and on the nail bed by oral agents and prevent reinfection. combination therapy with oral and topical antifungals has been shown to lead to a marked improvement of mycological and clinical outcomes, may be more cost effective, reduce duration, also minimize the side effect of systemic treatment.3,4,18 systemic treatment that is widely used for combination treatment are terbinafine and itraconazole, while topical agent usually used are amorolfine and ciclopirox, eventhough some studies also reported another combintion agent. in vitro studies have shown that the use of itraconazole and amorolfine combinations in some dermatophyte and non-dermatophyte strains will show synergistic and additive effects, with no antagonistic effects. the explanation of the synergism effect of these two drugs is not known clearly.18 study by lecha in 2001 used itraconazole 200 mg/ day for 12 weeks and in combination with 5% amorolfine once-weekly for 24 weeks. combination therapy is showed a higher cure rate (93.9%) than monotherapy (69%).20 another study from rigopoulos in 2003 revealed that higher complete cure of patient with combination amorolfine 5% (weekly for 6 months) and 400 mg itraconazole (daily for 1 week on/3 weeks off for 2 months), than itraconazole alone (93% and 81%). the subjects of this study were onychomycosis patients >50% surface area involvement.21 the effectiveness combination of terbinafine and amorolfine are demonstrated in 2001 by baran study with 147 patients with severe onychomycosis in the nail. terbinafine and amorolfin for 12 weeks are showed better healing (72.3%) than monotherapy terbinafine (37.5%). randomized controlled studies on onychomycosis dermatophytic therapy in the nail matrix are showed that 5% amorolfine in combination with oral terbinafine 250 mg per day for 3 months were more effective than the terbinafine monotherapy at the same time.22 avner in 2005 used 250 mg terbinafine daily for 4 months alone and compared it with 250 mg terbinafine daily for 4 months combination with 8% ciclopirox daily for 9 months. this study is showed higher complete cure rate in combination therapy (68%) than terbinafine alone (50%). mycological cure also higher in combination group (88%), while 65% in monotherapy group.23 another evaluation is performed by gupta in 2005 with treatment groups was similar to the previous studies, but with fewer patients over longer periods, resulting in greater mycological outcomes in combination groups than in single terbinafine. the results are obtained were 66.7% (14/21) in the first group (terbinafine 250 mg/day (4 weeks of therapy 4 weeks stop 4 weeks of therapy) ciclopirox combination 8% daily for 48 weeks), 70.4% (19/27) in the second group (terbinafine for 12 weeks ciclopirox combination 8% daily for 48 weeks), and 56.0% (14/25) in the third group (terbinafine 250 mg/day for 12 weeks). the results of this study are showed that p value is not significant.24 jaiswal used three arm groups: first with 250 mg terbinafine twice daily for 1 week/month for 4 months; second group terbinafine with addition 5% amorolfine weekly for 4 months; and the third group terbinafine combination with 8% ciclopirox daily for 4 months. the result is showed that mycological cure for first group was 82.6%, while second group 70%, and third group 83.3%.25 baran in 2007 is compared 250 mg terbinafine daily for three months with addition 5% amorolfine weekly for 15 months, and is showed higher complete cure in combination group than terbinafine alone.26 even though griseofulvin no longer used in treatment onychomycosis, the combination of griseofulvin with amorolfine showed a greater effectiveness is compared to griseofulvin with placebo. research using 233 samples are showed mycological improvement in 67% of patients with combination therapy and 45.3% for griseofulvin monotherapy group. positive culture was found in 7.4% of patients with combination therapy, while single griseofulvin was 34.7%. the clinical cure for combination therapy groups was twice as high as the number of patients with griseofulvin monotherapy.4 one gram of griseofulvin in daily combination of tioconazole 28% solution was being compared to one gram griseofulvin and placebo for 1 year in the bilateral onychomycosis patients by trichophyton rubrum. each patient received combination therapy on one side of his leg and a placebo for the other side. clinical and mycological improvement in combination therapy was 69%, while in griseofulvin and placebo 41%.4 some literatures are said that topical treatment administration as additional therapy any systemic antifungal agent, should continue for at least 1 year and, if necessary, up to 18 months which could produce a better clinical outcome that reflects true nail pathology.27–30 summary the combination of antifungal agents with different pharmacological effects is thought to improve the success of the therapy. combination therapy will exploit the strength of each antifungal agent to achieve simultaneous treatment success. in addition, by working mechanisms of two different drugs, it improves the effectiveness and speed of therapeutic improvement, enhanced broader antifungal spectrum, better resistance suppression, tolerability and patient safety. 20 indonesian journal of tropical and infectious disease, vol. 7 no. 1 january–april 2018: 15–20 in vitro studies had been proven that the use of an antifungal combination has a synergistic effect, which will improve the effectiveness of therapy compared to monotherapy. however, direct clinical trials in several studies have shown mixed results, making it difficult to draw conclusions. references 1. vora d, bharti b, solanki p, kothari a, meher k. a study to compare efficacy of various oral antifungals (fluconazole, terbinafine, itraconazole) in treatment of onychomycosis. j res med dent sci. 2014;2(4):49. 2. piraccini b, alessandrini a. onychomycosis: a review. j fungi. 2015 mar 27;1(3):30–43. 3. evans eg. the rationale for combination therapy. br j dermatol. 2001 oct;145 suppl:9–13. 4. olafsson jh, sigurgeirsson b, baran r. combination therapy for onychomycosis. br j dermatol. 2003 sep;149 suppl:15–8. 5. gupta ak, daigle d, foley ka. network meta-analysis of onychomycosis treatments. ski appendage disord. 2015 sep; 1(2):74–81. 6. tabara k, szewczyk ae, bienias w, wojciechowska a, pastuszka m, oszukowska m, et al. amorolfine vs. ciclopirox – lacquers for the treatment of onychomycosis. adv dermatology allergol. 2015;1:40–5. 7. kb, ss, wi, lm r, sw, ee. dermatomikosis superfisialis pedoman dokter dan mahasiswa kedokteran. jakarta: badan penerbit fakultas kedokteran universitas indonesia; 2013. 86-89 p. 8. ameen m, lear jt, madan v, mohd mustapa mf, richardson m. british association of dermatologists’ guidelines for the management of onychomycosis 2014. br j dermatol. 2014 nov;171(5):937–58. 9. córdoba-fernández a, távara-vidalón p, mandredi-márquez mj. therapeutic approach for toenail onychomycosis: literature review and cost-effectiveness analysis. foot ankle online j 7. 2014; 2(3):3–10. 10. lipner sr, scher rk. prognostic factors in onychomycosis treatment. infect dis ther. 2015;3(1):1–6. 11. wolff k, la g, si k. fitzpatrick ’ s dermatology in general medicine. seventh edition. two. 2009;17(2):149–50. 12. ayanlowo o, oladele ro. onychomycosis: updates and management challenges. a review. niger postgrad med j. 2014 jun; 21(2): 185–91. 13. vlahovic tc. onychomycosis: evaluation, treatment options, managing recurrence, and patient outcomes. clin podiatr med surg. 2016 jul;33(3):305–18. 14. gupta ak, daigle d, foley ka. topical therapy for toenail onychomycosis: an evidence-based review. am j clin dermatol. 2014 dec;15(6):489–502. 15. sigurgeirsson b, olafsson jh, steinsson jt, kerrouche n, sidou f. efficacy of amorolfine nail lacquer for the prophylaxis of onychomycosis over 3 years. j eur acad dermatol venereol. 2010 aug;24(8):910–5. 16. shenoy mm, shenoy ms. fungal nail disease (onychomycosis); challenges and solutions. arch med heal sci. 2014;2(1):48. 17. nenoff p, krüger c, paasch u, ginter-hanselmayer g. mycology an update part 3: dermatomycoses: topical and systemic therapy. j dtsch dermatol ges. 2015 may;13(5):387–410; quiz 411. 18. gupta ak, paquet m. improved efficacy in onychomycosis therapy. clin dermatol. 31(5):555–63. 19. gupta ak, cooper ea, paquet m. recurrences of dermatophyte toenail onychomycosis during long-term follow-up after successful treatments with monoand combined therapy of terbinafine and itraconazole. j cutan med surg. 17(3):201–6. 20. lecha m, alsina m, torres rodríguez jm, de erenchun fr, mirada a, rossi ab. an open-label, multicenter study of the combination of amorolfine nail lacquer and oral itraconazole compared with oral itraconazole alone in the treatment of severe toenail onychomycosis. curr ther res. 2002 jun;63(6):366–79. 21. rigopoulos d, katoulis ac, ioannides d, georgala s, kalogeromitros d, bolbasis i, et al. a randomized trial of amorolfine 5% solution nail lacquer in association with itraconazole pulse therapy compared with itraconazole alone in the treatment of candida fingernail onychomycosis. br j dermatol. 2003 jul;149(1):151–6. 22. baran r, feuilhade m, combernale p, datry a, goettmann s, pietrini p, et al. a randomized trial of amorolfine 5% solution nail lacquer combined with oral terbinafine compared with terbinafine alone in the treatment of dermatophytic toenail onychomycoses affecting the matrix region. br j dermatol. 2000 jun;142(6):1177–83. 23. avner s, nir n, henri t. combination of oral terbinafine and topical ciclopirox compared to oral terbinafine for the treatment of onychomycosis. j dermatolog treat. 2005;16(5–6):327–30. 24. gupta ak, onychomycosis combination therapy study group. ciclopirox topical solution, 8% combined with oral terbinafine to treat onychomycosis: a randomized, evaluator-blinded study. j drugs dermatol. 4(4):481–5. 25. jaiswal a, sharma rp, garg ap. an open randomized comparative study to test the efficacy and safety of oral terbinafine pulse as a monotherapy and in combination with topical ciclopirox olamine 8% or topical amorolfine hydrochloride 5% in the treatment of onychomycosis. indian j dermatol venereol leprol. 73(6):393–6. 26. baran r, sigurgeirsson b, de berker d, kaufmann r, lecha m, faergemann j, et al. a multicentre, randomized, controlled study of the efficacy, safety and cost-effectiveness of a combination therapy with amorolfine nail lacquer and oral terbinafine compared with oral terbinafine alone for the treatment of onychomycosis with matrix invol. br j dermatol. 2007 jul;157(1):149–57. 27. ghannoum ma, long l, isham n, bulgheroni a, setaro m, caserini m, et al. ability of hydroxypropyl chitosan nail lacquer to protect against dermatophyte nail infection. antimicrob agents chemother. 2015 apr;59(4):1844–8. 28. ghannoum m, isham n, catalano v. a second look at efficacy criteria for onychomycosis: clinical and mycological cure. br j dermatol. 2014 jan;170(1):182–7. 29. scher rk, tavakkol a, sigurgeirsson b, hay rj, joseph ws, tosti a, et al. onychomycosis: diagnosis and definition of cure. j am acad dermatol. 2007 jun;56(6):939–44. 30. ghannoum m, sevin k, sarkany m. amorolfine 5% nail lacquer exhibits potent antifungal activity compared to three acid-based devices indicated for the treatment of onychomycosis: an in vitro nail penetration assay. dermatol ther (heidelb). 2016 mar; 6(1): 69–75. 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 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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 136 vol. 5. no. 5 may–august 2015 the effect of gendarussin a isolates of justicia gendarussa burm.f. leaf in reverse transcriptase inhibition of hiv type i in vitro bambang prajogo ew1)., prihartini widiyanti2.4), nasronudin2,3),bimo aksono2,5) 1 department of pharmacognosy and phytochemicals, faculty of pharmacy, universitas airlangga, surabaya, indonesia 2 institute of tropical diseases, universitas airlangga, surabaya, indonesia 3 faculty of medicine universitas airlangga, surabaya, indonesia 4 faculty of science and technology, universitas airlangga, surabaya, indonesia 5 faculty of veterinary, universitas airlangga, surabaya, indonesia abstract screening has been done to a few extracts from the leaves justicia gendarussa burm.f to see the growth rate of the virus from the blood plasma of hiv patients at dr soetomo hospital. it is known that j. gendarussa leaf extract inhibits hiv type 1 reverse transcriptase. in addition, its main content is gendarussin a, besides gendarussin b, jgf1, jgf2 and jgf3, which have just identified. at the beginning, extraction and fractionation were performed with 3 models that highlight the absolute methanol, 70% methanol and 70% ethanol with the release of alkaloids. furthermore, samples of each fraction were incubated in plasma of hiv patients with a titer of 3.6 106 copies for 1 h in concentrations of 1.64 ppm, 4.1 ppm, 8.2 ppm, 16.4 ppm and 41.0 ppm. after incubation, examination was performed by using nucli sens a machine, which is a combination of pcr and elisa, thus avoiding direct contact with the highly pathogenic virus. the result showed that the activity sequence from the most potential to the weak, among others, was 1.64 ppm > 4.1 ppm > 8.2 ppm > 16.4 ppm > 41.0 ppm, each with barriers value of 0.62 106, 1.4 106, 1.6 106, 2.4 106 and 5.2 106 cells/ml. in conclusion, highest anti-hiv activity comes from the concentration of gendarussin a isolate at 1.64 ppm. furthermore, after linear regression of y = -3.063 x + 81.37 was done, the ic50 of 10.24 ppm was obtained. keywords:, justicia gendarussa, gendarussin a,reverse transcriptase, inhibition, anti hiv abstrak penelitian telah dilakukan pada beberapa ekstrak daun justicia gendarussa burm.f untuk melihat angka pertumbuhan virus plasma darah pasien hiv di rumah sakit dr. soetomo. telah diketahui bahwa ekstrak daun j. gendarussa menghambat hiv tipe 1 reverse trancipitase. selain itu, kandungan utama dari j. gendarussa adalah gendarussin a disamping gendarussin b, jgf1, jgf2, dan jgf3 yang telah diidentifikasi. pada awalnya, ekstraksi dan fraksinasi ditunjukkan oleh 3 model yang ditandai oleh absolute methanol, 70% methanol dan 70% ethanol dengan melepaskan alkoid. selanjutnya, sebagian dari masing-masing sampel diinkubasi ke dalam plasma hiv pasien hiv dengan titer 3.6 106 sebanyak masing-masing 1h dengan konsentrasi 1.64 ppm, 4.1 ppm, 8.2 ppm, 16.4 ppm dan 41.0 ppm. setelah inkubasi, pengujian ditunjukkan menggunakan mesin nucli sens yang dikombinasikan dengan pcr dan elisa untuk menghindari kontak langsung dengan virus yang memiliki resiko pathogen tinggi. hasil menunjukkan bahwa rangkaian aktivitas tersebut dari yang paling berpotensi tinggi ke yang paling berpotensi rendah diantaranya adalah 1.64 ppm > 4.1 ppm > 8.2 ppm > 16.4 ppm > 41.0 ppm dengan masing-masing nilai penghalang 0.62 106, 1.4 106, 1.6 106, 2.4 106 and 5.2 106 cells/ml. kesimpulannya. aktivitas anti-hiv tertinggi diperoleh dari konsentrasi gendarussin a yang dipisahkan pada 1.64 ppm. selanjutnya, setelah regresi linier y = -3.063 x + 81.37 selesai, diperoleh ic50 of 10.24 ppm. kata kunci: justicia gendarussa, gendarussin a, reverse transcriptase, inhibisi, anti-hiv 137prajogo, et al.: the effect of gendarussin a isolates of justicia gendarussa burm.f. leaf introduction the transmission of hiv-aids in indonesia continues to widen, particularly in the group of young and productive individuals. data from the ministry of health showed that up to june 2008 there were approximately 6782 individuals aged 20-29 years who suffered from aids. the number of people with aids in this age group is the highest compared to other groups, the second highest were those with age 30-39 years, comprising only 3539 people. the young generation is as if being chased by this deadly disease, and, unfortunately, the spread of this disease is like an iceberg phenomenon. the problem that comes to the surface is actually just a piece of the reality in the field. integrated biological and behavior survey (survey terpadu biologi dan perilaku, stbp) regarding hiv prevalence in indonesia in 2007 showed that about 43-56% percent of drugs (narcotics, psychotropic substances and additives) users or injection drug users in four cities, medan, jakarta, bandung and surabaya has been infected with hiv.1 hiv-aids becomes the fifth leading cause of death in population aged 25-44 years in the united states. at global level, 25 million people have died in vain since the epidemics of this infectious disease and 40.3 million people worldwide are currently living with hiv-aids. hiv causes aids, the virus attacks immune system and stay in the body that can spur the onset of infection and cancer. generally, bacteria, yeast, and viruses seem not to become a serious illness when the immune system in healthy condition. this may be different and will be fatal in people who suffer from aids. hiv is found in saliva, tears, nervous tissue and spinal fluid, blood, semen (seminal fluid in ejaculate), vaginal fluid and breast milk. but only through blood, semen, vaginal secretions and breast milk generally these infections can be transmitted.2 aids begins with hiv infection. hiv-infected person may be no symptoms for 10 years or more, but remain infected and can transmit infection to others. meanwhile, if the infection is not detected or without treatment, immune system gradually weakens, and aids develops. generally, symptoms may present as flu with fever, rash, sure throat, sweats, chills, swollen lymph nodes, weakness and weight loss. hiv infection is associated with decreased cd4 cells, a type of immune cells called “t cell” or “helper cell”. indications of a viral infection is when the number of “cd4 cells” below 350 cells/ml, and, specifically in hiv infection, if cd4 cell count is below 50 cells/ml. furthermore, for monitoring hiv patients the number of cd4 cells, known as hiv-rna, is used.3 traditional medicine contributes much to the discovery of new compounds that has anti-hiv activity. there are the plants that have proteins that can inhibit hiv reverse transcription in vitro.4 some isolated single chain ribosom inactivating protein (scrip) showed the power of the antiviral action of dna and rna viruses. for example, map30 and tap 29 are scrip protein isolated from momordica charantia seeds and tubers of trichosanthes kirilowii. both materials can inhibit the replication of hiv-1 infected cells and also the activity of inhibitors of hiv-1 virus associated with reverse transcriptase.5water extracts and 80% ethanol extracts of the plants andrographs paniculata, justicia gendarussa, vitex trifolia and tinospora crispa have the activity of inhibitors of hiv-1 reverse transcriptase.6 natural materials, particularly the class of polyphenol, have anti-hiv activity, whose action can work, for example, by inhibiting hiv cycle: (1). virus adsorption, (2) virus-cell fusion (3) reverse transcription, (4) integration, (5) proteolytic cleavage, (6) glycosylation and (7) assembly/release.7 justicia gendarussa is a plant often used by indonesian people as medicine, either as a drug for internal and external use. as a male contraceptive, research has been carried out on its biological and pharmacological activity, even reaching preclinical and clinical trials phase i and ii.8,9,10 from phytochemical studies, the major component of gendarussin a has been identified, in addition to minor components of other flavonoids. there is also alkaloid content bhagya. furthermore, the anti-hiv screening showed that 70% etoh extracts justicia gendarussa in 100 ppm can reduce hiv growth inhibition (viral load) to 1.47 105 copies/ml after incubation of 60 minutes. it is even more potential if gendarussin a isolates are used.10due to the high potential of gendarussin a isolates and the needs of reconfirmation with different concentrations, it was necessary to carry out reisolation, which is quite difficult, even it has already standardized method. this effort is aimed to gain the stabil gendarusi a level in extracts. due to humanity consideration, this research is very important, since until now there is no potential anti-hiv drugs originating from indonesia that have been subjected to clinical trials. therefore, this study examined the ability of the isolates of gendarussin a, a polyphenol compound from justicia gendarussa leaves, as the activity inhibitor of hiv-1 reverse transcriptase. material and methods justicia gendarussa leaf powder were obtained from pacet mojokerto, methanol pa, methanol pro hplc, gendarussin a isolate, jgf1 isolate, jgf2 isolate, jgf2 isolate, and silica gel f254. some equipments such as percolator, hplc shimadzu lc10ad, coloumn waters novapack c18, easyq hiv-1 v2.0 worksheet nuclisens magnetic extraction, rotavapor buchi r-114, rotavapor buchi r-153. sample extraction and isolation justicia gendarussa extract were obtained by maceration of 1.5 kg of powdered gendarussa leaves which was soaked with 3 l. methanol for 24 hours with stirring. extraction was repeated 2 times with the same method and solvent volume. gendarussin a isolate was obtained by several times preparative hplc running with a 200 μl per injection. 138 indonesian journal of tropical and infectious disease, vol. 5. no. 5 may–august 2015: 136-141 furthermore, the collections of isolates were subjected to lyophilization with freeze dryer until constant weight was obtained. identification of isolate preparation amorphous-shaped gendarussin a isolate was identified and compared with standard gendarussin a isolates through hplc analysis, 1h-13c nmr, chemical reactions and physical characterization that could be done. inhibitions to hiv-1 replication were tested in several concentrations from 5, 10, 20, 40, 80, 100 ppm (each in triplicate). determination of ic50 of gendarussin a isolate obtained gendarussin a isolates was diluted in methanol with various concentrations, which could be done by making more than six points around 10 ppm, and analyzed by probit which would reveal the regression equation, so that the chart pattern would also be obtained figure 1. scheme of flavonoids isolation would reveal the regression equation, so that the chart pattern would also be obtained between the concentration and the viral load (hiv), and from the extrapolation we would have the ic50. extraction with meoh partition with hexan / h2o extraction with n-buoh / h2o ods c.c.(meoh/h2o = 3/7 1/0) sio2 c.c.c.(chcl3/meoh = 1/0 0/1) ods hplc (meoh/0.1% hcooh aq = 40% figure 1. scheme of flavonoids isolation justicia gendarussa burm f (leaf powder) methanol extract 6,18/32 hexan extract 1.5 residue etoac extract 790mg residue n-buoh extract 777 mg h2o compound 1a 16.1 mg (0.23%) compound 1b 410 mg (0.07%) compound 4 2.7 mg (0.04%) compound 5 3.1 mg (0.05%) between the concentration and the viral load (hiv), and from the extrapolation we would have the ic50. hiv monitoring procedure easyq hiv-1 v2.0 worksheet nuclisens magnetic extraction was used and it has some steps such as lysis, binding,washing and dilution. in lysis step, lysis buffer tube was centrifugated for 10 seconds at 1500 g, and then 0.1 ml or 0.5 ml or 1.0 ml sample were added into it. the mixed solution was vortexed and was incubated for 10 minutes at room temperature. then premix solution was prepared by entering 550 cal diluent into grain-shaped cal and 550 ul salica solution was added and was vortexed maximum 20 minutes, premix solution must be added to the sample. in binding step, premix solution was vortexed and 10 µl premix solution was added. the solution then was vortexed and was incubated for 10 minutes at room temperature 139prajogo, et al.: the effect of gendarussin a isolates of justicia gendarussa burm.f. leaf figure 2. alkaloid isolation scheme extraction with meoh partition with etoac / 3% tartaric acid solution sat. na2co3 aq (ph 9) partitioned with chcl3/h2o extraction with n-buoh / h2o sio2 c.c.c(chcl3/meoh = 1/0 0/1) ods hplc (meoh/0.1% hcooh aq = 2/3) figure 2. alkaloid isolation scheme justicia gendarussa burm f (leaf powder) methanol extraction (14/32) etoac extract 5.0g residue chcl3 extract 58.4 mg residue n-buoh extract 2.1 mg h2o compound 2a 0.6 mg (0.004%) compound 2b 0.4 mg (0.003%) compound 3 0.3 mg (0.002%) without homogenization (without mixing). in washing step, the lysis buffer tube was vortexed for 2 minutes at 1500g and then was discarded the supernatant.400 µl wash buffer 1 (transparent) was added, homogenized and transfered this solution into 1.5 ml tubes and was washed for 30 seconds on the menu step 1 on the nuclisens mini mag (magnetic on). after that the supernatant was discarded. 400 µl wash buffer 1 (transparent) (magnet off) was added step 4 and 5 was repeated. 500 µl wash buffer 2 (red●) (magnet off) was added and was repeated step 4 and 5. after that step 7 was repeated.500 µl wash buffer 3 (yellow●) (magnet off) was added and was washed for 15 seconds on the menu step 1 at nuclisens min mag (magnet on). all supernatant/liquid (magnet on) was discarded. in dilution step, 25 µl elution buffer (yellow●) was added and was incubated for 5 min, 60°c in thermoshaker (speed 1400 rpm). after that tube was placed in magnetic rack with open tube, and 15 µl extracted samples was moved to 8 tube strips in the amplification area or store at a certain temperature. results and discussion in some extraction isolation models, the type of solvent, methanol and ethanol, and alkaloid-free or not extract were influenced the quantity of fractions which were obtained. the largest amount is in the polar fraction from the methanol and ethanol in various concentrations. whereas, in non-polar solvents, like n-hexane and chloroform, it was found in small amounts. it is known that the main content in the polar fraction is gendarussin a, which is a type of flavonoid glycosides.11 the process of isolation in butanol fraction revealed isolates jgf1 (2 mg), jgf2 (4.2 g) and jgf3 (1.3 mg), while from chloroform fraction we obtained jga1 alkaloids (0.96 mg). from the physicochemical analysis, it was found that jgf1, jgf2 and jgf3 are derivatives of apigenin as that in gendarussin a and has the same molecular weight of 534.14. they are different only in ribose, xylose and arabinose sugar structures. then, in jga1 alkaloid is also observed. it is the derivative of benzyl 140 indonesian journal of tropical and infectious disease, vol. 5. no. 5 may–august 2015: 136-141 amino alcohol bound to carboxylic acid on both sides of the core. of the various isolates, such as gendarussin a, jgf1, jgf2 and jgf3 with various concentrations, antihiv test was done using nucli sens machine (table 1). from the results of incubation of isolates tested against human plasma hiv for 60 minutes in vitro, it was shown that the gendarussin a isolate provided the strongest activity (3.6 106) at 793 ppm compared to other isolates. in various concentrations, it was observable that anti-hiv activity is determined by the viral load. determination of anti-hiv activity can be seen with inverse proportion between viral load and cd4 count. this indicated that the effect is positive when viral load is lower than the negative control (patients’ titer ) or cd4 count increases compared to a negative control. if gendarussin a isolate provides good effect compared to other isolates, it means that those isolates contain apegenin glycoside compounds with xylose and arabinose sugar. in tested sample 70% ethanol fraction contained 1.4% gendarussin a, as determined by hplc method. in previous clinical trials, it was found that bioavailability test in plasma or blood serum detected gendarussin a metabolite, which also appeared in ejaculate and urine. 12 thus, this in vitro test can then be used as a model of direct interaction with the virus and it has been known previously that the virus growth inhibition is due to the inhibition of transcriptase enzyme function of hiv type 1, whose function is to replicate itself. the certainty of viral death can be tested through the identification of proteins, since the virus is highly pathogenic. in the hiv viral load measurement results, 200μl sample after 60 minutes incubation is showing the result sample gendarussin a with concentration 793ppm, jgf1, jgf2, jgf3 show the viral load value 3.1×10 6; 6.4×106; 3.1×106 and 8.1×106 subsequently. this effects of flavanoid compounds in hiv sample could be seen in the figure 4. the concentration of gendarussin a showed the tendency to be increased as high as the viral load result. the data show in figure 5. the isolates of gendarussin a, jgf1, jgf2 and jgf 3 of the leaf j. gendarussa at concentrations 793, 2000, 4200, and 1300 ppm produces viral load 3.1×106 ; 8.1×106 ; 6.4×106 and 3.1×106 copies/ml. the inhibition concentration 50% (ic50) of gendarussin a is 235.3 ppm. figure 3. alkaloid and flavonoid compounds in justicia gendarussa leaves 534.14. they are different only in ribose, xylose and arabinose sugar structures. then, in jga1 alkaloid is also observed. it is the derivative of benzyl amino alcohol bound to carboxylic acid on both sides of the core. of the various isolates, such as gendarussin a, jgf1, jgf2 and jgf3 with various concentrations, anti-hiv test was done using nucli sens machine (table 1). from the results of incubation of isolates tested against human plasma hiv for 60 minutes in vitro, it was shown that the gendarussin a isolate provided the strongest activity (3.6 106) at 793 ppm compared to other isolates. in various concentrations, it was observable that anti-hiv activity is determined by the viral load. determination of anti-hiv activity can be seen with inverse proportion between viral load and cd4 count. this indicated that the effect is positive when viral load is lower than the negative control (patients' titer ) or cd4 count increases compared to a negative control. if gendarussin a isolate provides good effect compared to other isolates, it means that those isolates contain apegenin glycoside compounds with xylose and arabinose sugar. in tested sample 70% ethanol fraction contained 1.4% gendarussin a, as determined by hplc method. in previous clinical trials, it was found that bioavailability test in plasma or blood serum detected gendarussin a metabolite, which also appeared in ejaculate and urine. 12 thus, this in vitro test can then be used as a model of direct interaction with the virus and it has been known previously that the virus growth inhibition is due to the inhibition of transcriptase enzyme function of hiv type 1, whose function is to replicate itself. the certainty of viral death can be tested through the identification of proteins, since the virus is highly pathogenic. figure 3. alkaloid and flavonoid compounds in justicia gendarussa leaves figure 4. diagram of the effects of flavonoid compounds in the plasma of hiv in vitro in the hiv viral load measurement results, 200µl sample after 60 minutes incubation is showing the result sample gendarussin a with concentration 793ppm, jgf1, jgf2, jgf3 show the viral load value 3.1 106; 6.4 106; 3.1 106 and 8.1 106 subsequently. this effects of flavanoid compounds in hiv sample could be seen in the figure 4. figure 4. diagram of the effects of flavonoid compounds in the plasma of hiv in vitro the concentration of gendarussin a showed the tendency to be increased as high as the viral load result. the data show in figure 5 below. (photo) figure 5: the effect of gendarussin a on human plasma in patients with hiv-1 the isolates of gendarussin a, jgf1, jgf2 and jgf 3 of the leaf j. gendarussa at concentrations 793, 2000, 4200, and 1300 ppm produces viral load 3.1 106 ; 8.1 106 ; 6.4 106 figure 5. the effect of gendarussin a on human plasma in patients with hiv-1 in the hiv viral load measurement results, 200µl sample after 60 minutes incubation is showing the result sample gendarussin a with concentration 793ppm, jgf1, jgf2, jgf3 show the viral load value 3.1 106; 6.4 106; 3.1 106 and 8.1 106 subsequently. this effects of flavanoid compounds in hiv sample could be seen in the figure 4. figure 4. diagram of the effects of flavonoid compounds in the plasma of hiv in vitro the concentration of gendarussin a showed the tendency to be increased as high as the viral load result. the data show in figure 5 below. (photo) figure 5: the effect of gendarussin a on human plasma in patients with hiv-1 the isolates of gendarussin a, jgf1, jgf2 and jgf 3 of the leaf j. gendarussa at concentrations 793, 2000, 4200, and 1300 ppm produces viral load 3.1 106 ; 8.1 106 ; 6.4 106 141prajogo, et al.: the effect of gendarussin a isolates of justicia gendarussa burm.f. leaf in conclusion, this fact is referred to the protency and strength of gendarussin a and other active compound of jucticia gendarussa leaf in inhibition of hiv replication. conclussion in conclusion, this fact is referred to the protency and strength of gendarussin a and other active compound of jucticia gendarussa leaf in inhibition of hiv replication. acknowledgements great gratitude to director of the directorate general of higher education, ministry of national education, jakarta for the research funding. references 1. health profile: indonesia. united states agency for international development (march 2008). accessed august 25, 2008. 2. joann o’toole, lauren robertson, susan walters schmid, https:// www.atrainceu.com/course/washington-hiv-aids-4-units-136 3. yu lm, easterbrook pj, marshall t, 1997. relationship between cd4 countand cd4% in hiv infected people. international jof epidemiology, vol 26 no 6, great britain. 4. ng, tb, huang b, fong,wp., yeung., h.w., 1997. anti-human immunodeficiency virus(anti-hiv) natural products with special emphasis on hiv reverse trancriptase inhibitors. life science 61, 933-949. 5. lee-huang, s.,huang, p.l., nara, p.l, chen, h.c., kung, h.f., huang, p., huang, h.i,, huang, p.l., 1990. map 30 a new inhibitor of hiv-1 infection and replication, febs letters 272, 12-18. 6. woradulanyapinij, w., soonthornchareonnon, n., wiwat, c., 2005. in vitro hiv type 1 reverse transcriptase inhibitory activities ofthai medicinal plants and canna indica l. rhizomes. journal of ethnopharmacology 101, pp. 84-89. 7. andrae-marobela, kerstin; ghislain, fotso wabo; okatch, harriet; majinda, runner r. t, 2013. polyphenols: a diverse class of multitarget anti-hiv-1 agents current drug metabolism;may2013, vol. 14 issue 4, p392 8. prajogo bew, noor ch, hudi, aucky h, dian, mustaina, kasmijati, anggraeni, radjaram, 2008. pengaruh ekstak etanol 70 % pada pria fertile (uji klinik fase i). fakultas farmasi unair-bkkbn pusat. 9. prajogo bew, noor ch, hudi, aucky h, dian, mustaina, flouresia, anggraeni, radjaram, 2009. pengaruh ekstak etanol 70 % pada pria pasangan usia subur (pus) (uji klinik fase ii). fakulas farmasi unair-bkkbn pusat. 10. prajogo bew, nasronudin, noor ch dan neny p, 2009. aktivitas penghambatan reverse transcriptase hiv tipe i tanaman obat justicia gendarussa burm.f. lppm unair. 11. izzah z, prajogo bew, radjaram a, 2010. studi stabilitas kimia gendarussin a dalam sediaan granul fraksi air daun justicia gendarussa burm f, majalah farmasi airlangga, vol.8 no.1, april 2010 12. nianhang chen, lian wen, henry lau, sekhar surapaneni, and gondi kumar, 2012. pharmacokinetics, metabolism and excretion of [14c]-lenalidomide following oral administration in healthy male subjects cancer chemother pharmacol. 2012 mar; 69(3): 789–797. published online 2011 oct 29. doi: 10.1007/s00280-011-1760-3cid: pmc3286592 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 151 404 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 �� vol. 2. no. 1 january–march 2011 profile of community acquired pneumonia in children at soetomo hospital surabaya in �00�–�00� retno asih setyoningrum, landia setiawati department of child health medical school, airlangga university dr. soetomo hospital surabaya indonesia abstract background: community acquired pneumonia (cap) is one of the most important health problem affecting children all over the world. clinical findings, laboratory and radiological examination of cap may largely vary from mild to severe. objective: to report profile of cap in children hospitalized at soetomo hospital surabaya in 2007–2008 methods: this research was a retrospective study. data of children with primary diagnosis of cap in 2007–2008 were obtained from medical records of the department of child health soetomo hospital surabaya. the diagnosis cap was based on who criteria (pneumonia clinical syndrome). the clinical features of illness, laboratory and radiological examination were recorded and presented descriptively. results: during the study period, 438 patients were diagnosed as cap. more than half (83.4%) patients aged 3 months– 3 year. beside cough and tachypnea, most common symptom and signs were chest indrawing (76.2%) and fever (23.8%). leucocytosis (39.6%). bacteria was found in 8.2%. accompanying diseases (i.e congenital heart disease, neurological and gastroenterological disorders) were found in 36.4%. one hundred fifty seven patients (35.8%) had malnutrition. patchy infiltrate was found in 80.8% chest x-ray examination. mortality was found in 4.3%. conclusions: community acquired pneumonia in children still count as a major problem at soetomo hospital surabaya. key words: children, community acquired pneumonia, clinical features of illness introduction community acquired pneumonia (cap) is one of the most important health problem affecting children all over the world. clinical findings, laboratory and radiological examination of cap may largely vary from mild to severe. the term “community-acquired pneumonia” (cap) refers to pneumonia in a previously healthy person who acquired the infection outside a hospital.a,2 the world health organization has defined pneumonia solely on the basis of clinical findings obtained by visual inspection and timing of the respiratory rate. the cause of cap is often difficult to establish. the most effective methods are often invansive and cannot always be justified and serological diagnosis is too late to be of any therapeutic use. despite the progress made in the diagnosis of pneumonia, it takes a few days to identify the causative microorganism in the blood or sputum samples and the etiology of half of all patients with cap remains uncertain. physicians need reliable data on the relative prevalence of different etiological agents in the patients area of residence, in addition to the clinical, laboratory and radiological findings in order to initiate antibiotic treatment empirically. the relative frequency of etiological agents varies among different geographical areas. the profile of community acquired pneumonia in children at soetomo hospital surabaya is not known the present study was undertaken to determine the profile of cap in children hospitalized at soetomo hospital surabaya in 2006. material and methods this research was a retrospective study. data of children with diagnosis of cap in january 2007–december 2008 were obtained from medical records of the department of child health soetomo hospital surabaya. the diagnosis cap was based on who criteria (peumonia clinical ��setyoningrum and setiawati: profile of community acquired pneumonia syndrome). the clinical features of illness, laboratory and radiological examination were recorded and presented descriptively. no patients had received the pneumococcal conjugate or polysaccharide vaccines. the radiologist assigned standardized and mutually exclusive diagnoses that included normal, patchy infiltrate, lobar consolidation and pleural effusion. results during the study period, 438 patients were diagnosed as cap. more than half (83.4%) patients aged 3–36 month. beside cough and tachypnea, most common symptom and signs were chest indrawing (76.2%), and fever (23.8%). leucocytosis (39.6%). bacteria was found in 8.2%. accompanying diseases (i.e congenital heart disease, neurological and gastroenterological disorders) were found in 36.4%. one hundred fifty seven patients (35.8%) had malnutrition. patchy infiltrate was found in 80.8% chest x-ray examination. mortality was found in 4.3%. table 1. profile of patients with cap characteristic no percentage sex, male 273 62.3 age at diagnosis < 3 month 52 11.8 3–36 month 365 83.4 > 36 months 21 4.8 clinical characteristic chest indrawing fever 334 104 76.3 23.7 nutritional status well nourished moderate nourished severe nourished 281 150 7 64.2 34.2 1.6 presence of accompanying disease 120 52.9 chest x-ray. normal lobar consolidation pleural effusion patchy infiltrat 60 21 3 354 13.7 4.8 0.7 80.8 figure 1. mortality of cap discussion during the study period, 438 patients were diagnosed as cap. we showed that children with aged 3 months– 3 year had the greatest degree of cap, indicating that the infant have at most as many epidoses of pneumonia as older children. the clinical features all patients who diagnosed as cap in this study were cough and tachypnea, based on the who criteria. the other clinical features that were most strongly associated with pneumonia were chest indrawing (76.3%), and fever (23.7%). pneumonia should be suspected if tachypnea occurs in a patient younger than two years with a temperature higher than 38° c (100,4° f). measurement of tachypnea requires a full one-minute count while the child is quiet. the world health organization’s age-spesific criteria for tachypnea are the most widely used: a respiratory rate of more than 50 breaths per minute in infants two to 12 months of age; more than 40 breaths per minute in children one to five years of age; and more than 30 breaths per minute in children older than five years.2 accompanying diseases (i.e congenital heart disease, neurological and gastroenterological disorders) were found in 36.4%. one hundred fifty seven patients (35.8%) had malnutrition. patchy infiltrate was found in 80.8% chest x-ray examination. complications of cap such as respiratory failure occurred in 8.8% cases and sepsis in 10.5%, leading to mortality of 4.3%. the mortality from pneumonia is high particularly in patients with associated co-morbid conditions. severe cap requiring intensive care unit (icu) admission, spread of radiographic infiltrates and �� indonesian journal of tropical and infectious disease, vol. 2. no. 1 january–march 2011: 12-14 previous treatment with immunosupressive drugs have all been associated a poor outcome.3,4 the mortality in our study was 4.3%. analysis with student t test, malnutrition (p = 0,036) and accompanying diseases (p = 0,029) have significant correlation with the mortality of cap. conclusions community acquired pneumonia in children still count as a major problem at soetomo hospital surabaya. references 1. ostapchuk m, roberts dm, haddy r. community–acquired pneumonia in infants and children. am fam physisician 2004; 70: 899–908. 2. world health organization. essential drugs and medicines policy. drugs used in bacterial infections. accesed online february 27, 2008, at: http://www.who.int/medicines/library/bacterial 3. bansai s, kashsyap, pai ls and goel a. clinical and bacteriological profile of community acquired pneumonia in shimia, himachal pradesh. indian j chest dis allied sci 2004; 46: 17–22. 4. lakhanpaul m, atkinson m, stephenson t. community acquired pneumonia in children: a clinical update. arch dis child ed pract 2004; 89: 29–34. 5. palafox m, guiscafré h, reyes h, muñoz o, martinez h. diagnostic value of tachypnoea in pneumonia defined radiologically. arch. dis. child. 2000; 82: 41–5. 6. vallès x, marcos a, pinart m, piñer r, marco, mensa jm, et al. hospitalized community-acquired pneumoniae due to streptococcus pneumonia: has resistance to antibiotics decreased? chest 2006; 130; 800–6. 7. owell s, kupronis ba, zell er, stat m,hay dks. mortality fromowell s, kupronis ba, zell er, stat m,hay dks. mortality frommortality from pneumonia in children in the united sstates 1939 through 1996..the new engl j of med 2008; 342: 1399–1407. 8. hazir t, yasir bin nisar, qazi sa, khan sf, raza m, zameer s, et al. chest radiography in children aged 2-59 months diagnosed with non-severe pneumonia as defined by world health organization: descriptive multicentre study in pakistan. bmj 2006; 333: 629. 9. victora cg, fuchs sc, flores jac, fonseca w, kirkwood b. risk factors for pneumonia among children in a brazilian metropolitan area pediatrics 1994; 93: 977–985. 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/ 87 vol. 7 no. 3 september–december 2018 determinant factors of drop out (do) among multi drugs resistance tuberculosis (mdr tb) patients at jakarta province in 2011 to 2015 sitti farihatun1, putri bungsu2a 1 graduate student of epidemiology department, public health faculty, universitas indonesia 2 lecturer of epidemiology department, public health faculty, universitas indonesia a corresponding author: putri.bungsu10@ui.ac.id abstract the prevalence of drop out (do) among multi drugs resistant tuberculosis (mdr tb) patients increases every year in jakarta province. the latest data of 2016 contains 367 drug resistant tb patients and 78 patients (21.2%) were do. this study was aimed to analyze the determinant factors of drop out (do) among mdr tb patients in jakarta province between 2011 to 2015 based on risk factors of age, sex, hiv status, sputum test, type of patient, number of previous treatments and number of drugs resistance. this study was used secondary data that source from cohort registration e-tb manager from dki jakarta health office with total 516 samples. the design study was an observational cross sectional quantitative study. do is a condition of patients who have been treated and drop out of treatment for 2 consecutive months or more. the crude prevalence of do among mdr tb patients was 44.6%. trend of do among mdr tb was increased since 2011 to 2015. there was a further increase more than 10% in every year. the proportion of do among mdr tb in jakarta was more than 64 years old (63.6%), male (47.3%), patients with status hiv negative (44.9%), patients that never or ever consumed drugs less than 1 month (61.2%), and patients with >2 drugs resistance (45.7%). the results of this study indicated that proportion of do among mdr tb patients at jakarta province in 2011-2015 was high. therefore, it is necessary efforts that can decrease do cases among mdr tb patients. this study was expected to be a reference for jakarta province health office in implement p2tb program implementation and reach target precisely. keywords: drop out, mdr, tuberculosis, jakarta, determinant factors abstrak prevalensi drop out/ putus obat (do) pada pasien tuberkulosis multi drugs resistant (tb mdr) terus meningkat setiap tahunnya di provinsi dki jakarta. data terakhir di tahun 2016 tercatat sebanyak 367 pasien tb mdr dan 78 pasien (21.2%) berstatus do. penelitian ini bertujuan untuk menganalisis faktor determinan kejadian drop out (do) pada pasien tb mdr di provinsi dki jakarta pada tahun 2011 sampai 2015 berdasarkan faktor risiko umur, jenis kelamin, status hiv, hasil test sputum, tipe pasien, riwayat pengobatan tb sebelumnya (jumlah), dan jumlah resistansi obat. data yang digunakan adalah data sekunder yang bersumber dari data register kohort e-tb manager dengan jumlah sampel sebanyak 516 sampel. desain penelitian ini adalah studi kuantitatif observational cross sectional. do ada studi ini adalah kondisi pasien yang telah diobati dan putus pengobatan selama 2 bulan berturut-turut atau lebih. prevalensi do pasien tb mdr pada penelitian ini yaitu 44.6% yang merupakan prevalensi kasar. tren kejadian do pada penelitian ini cenderung mengalami peningkatan dari tahun 2011 hingga 2015 dan prevalensi do terus melebihi angka 10% setiap tahunnya. proporsi do pada pasien tb mdr di provinsi dki jakarta tahun 2011-2015 banyak terjadi pada pasien dengan usia >64 (63.6%), jenis kelamin laki-laki (47.3%), status hiv negatif (44.9%), pasien yang belum pernah atau pernah menelan obat namun kurang <1 bulan (58.8%), dan paling banyak pada pasien dengan jumlah resistansi >2 obat (45.7%). hasil penelitian ini menunjukkan bahwa proporsi kasus do pada pasien tb mdr di provinsi dki jakarta tahun 2011-2015 masih tinggi. oleh karena itu, perlu adanya research report 88 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 87–92 introduction multi drug resistant tuberculosis (mdr tb) is a condition in which mycobacterium tuberculosis resistant to the type of first-line drugs treatment such as rifampicin and isoniazid simultaneously, with or without being followed by other first-line drugs resistance treatment.1 the number of incidences of mdr tb in the world by 2014 was estimated to be 300.000 new cases of mdr tb and by 2014 increased to 480.000 new cases of mdr tb.2 in indonesia, the number of confirmed cases of mdr tb discovery was likely to increase from 2009 to 2015.3 the spread of mdr tb cases is mostly found in the provinces of jakarta, west java, and east java.3 mdr tb takes 20-26 months of treatment, expensive requires, more complex treatment, and more side effects of drug sensitive tb, making it difficult to be controlled.1 the bmain factor in tb treatment failure was treatment termination before the trial period was completed; it’s called as drop out (do) cases.4 do is a condition of patients who have been treated and drop out from treatment for 2 consecutive months or more.1 in indonesia, the rate of do among patients with drug resistant tb in 2009 was 10.7%. unfortunately, this number increased in 2013 to 28.7%.5 do rates among tb patients exceeding 10% can result in a high proportion of future retreatment cases.4 in jakarta province, the number of drug resistant tb patients based on e-tb manager surveillance data in jakarta provincial health office 2011-2016 was tended to increase. while the rate of do among drug resistant tb patients from 2011 to 2016 was very volatile, but the do number of drug resistant tb patients was always more than 10% annually. the latest data of 2016 contained 367 drug resistant tb patients and 78 patients (21.2%) were do. the purpose of this study was to analyze the determinant factors of drop out (do) among mdr tb patients in jakarta province between 2011 to 2015. material and method t h e d e s i g n s t u d y w a s a n o b s e r v a t i o n a l c r o s s s e c t i o n a l q u a n t i t a t i v e s t u d y . i n d e p e n d e n t variables include predisposing factors (age, sex, and hiv status), and enabling factors (duration of treatment, adherence, previous tb treatment history, and number of drugs resistances). dependent variable was do among mdr tb patients. the definition of do in this study was mdr tb patients who didn’t come for treatment and didn’t take the medication for 2 consecutive months or more before the end of treatment. inclusion criteria of this study include were patients with pulmonary mdr tb, age >15 years, domiciled in jakarta province, pulmonary mdr tb patients have been evaluated the final results of treatment, and pulmonary mdr tb patients that data were recorded fully in e-tb manager of jakarta provincial health office. exclusion criteria were pulmonary mdr tb patients who died and failed treatment related to discontinued treatment or requires a permanent change in mdr tb treatment; mixture of two or more mdr drugs. this study was using secondary data (e-tb manager) from jakarta provincial health office with total 516 samples. the analysis data used chi-square through stata 12 software. this study has obtained permission from the owner of the data jakarta provincial health office) with letter number 1713/sdk/xi/2017. in addition, this study also has passed ethical approval from ethical committee of public health faculty, universitas indonesia reg number: 08/un2.f10/ ppm.00.02/2018. name of respondents/patients withheld by using id for privacy of respondents. result and discussion firstly, the total of eligible sample who include in this study was 602 samples. then, there were 86 samples have been excluded which was consist of 71 of patients died and 15 of failed treatment. so, the percentage of participant rate was 85.7%. table 1 was showed the percentage of multidrugs tuberculosis patients in this study. table 1 was showed the results of this study indicated the prevalence of do among patients with mdr tb at dki jakarta province in 2011-2015 was 44.6%. figure 1 was described about the prevalence of mdr tb patients at dki jakarta province in 2011-2015 increased from 2011 to 2014, then it was decreased by 2015 to 24.6%. while the prevalence of mdr tb patients at dki jakarta province in 2011-2015 tends to increased and >10% each year. the highest prevalence of do occured in 2015 (53.50%) and the lowest in 2011 (16.7%). upaya untuk dapat mengurangi jumlah kasus do pada pasien tb mdr. diharapkan penelitian ini dapat menjadi acuan bagi dinas kesehatan provinsi dki jakarta dalam menjalankan program p2tb yang lebih baik dan tepat sasaran. kata kunci: drop out, mdr, tuberkulosis, jakarta, faktor determinan table 1. description of prevalence of do among mdr tb patients patient status n % not do (recover and treatment complete) 286 55.4 do 230 44.6 total 516 100 89farihatun dan bungsu: determinant factors of drop out (do) in some previous studies, the prevalence of do among mdr tb patients was lower than the prevalence of do obtained in this study (44.6%) (figure 1).6 the prevalence of mdr tb patients with do in previous studies in russia was showed 12% prevalence of do mdr tb patients.6 studies which conducted in lima city, peru showed as much as 10%.7 studies which conducted in kwazulu-natal, south africa showed the prevalence of do in patients with mdr by 21%.8 a study in uzbekistan which showed a large prevalence of do in patients with mdr tb by 20%.9 it was indicated that the prevalence of do in mdr tb patients might be influenced by other factors such as geography, environmental, and behavioral variations.9 from previous similar studies in indonesia, the findings of prevalence results in this study reached 41.1% higher than previous studies.10 a study that has been done at persahabatan hospital in 2010 was showed the prevalence of do in patients with mdr tb was 34.5%.10 in githrif’s study (2016) was mentioned that the prevalence of do in patients with mdr tb at gresik in 2011-2015 was 33.7%.11 the high prevalence rate of do which find in this study can be attributed to the limitations of this study. in this study, a considerable number of patients couldn’t be included in the study because they did not met the inclusion criteria and the exclusion criteria, so the proportion of mdr tb patients at dki jakarta province in 2011-2015 was high. table 2 was showed correlation between predisposing factors and do among mdr tb patients. the results showed that the age group of 45-64 years had significant association with do of mdr tb patients with p = 0.042 and do risk in mdr tb patients increased with age of mdr tb patients. while gender and hiv status had no significant relationship with do among mdr tb patients. this study is in line with lalor’s research, et al. in 2013 that indicating an association between age and incidence of do in patients with mdr tb (p value 0.022), which patients aged >45 years had a risk of 1.71 (95% ci: 1.072.73) for experienced do compared to patients aged ≤45 years of mdr.9 other previous study was showed that age had an effect on decreasing the success of mdr tb treatment and was statistically significant (or = 0.955; 95% ci = 0.921-0.991; p = 0.014).12 the older age of mdr tb sufferers were more likely to have a risk of drop out or have a tendency to experience irregularity in taking medication because the older age needs additional support to access tb treatment.13 the proportion of sex-based dos is more prevalent in males than in females. however, they did not show much different proportions. the statistical test was showed no significant relationship between male and female. it was found that men had a risk of do 1.151 times (95% ci: 0.715-1.852) than women. previous studies was showed that sex were not statistically correlated with do among mdr tb patients with or values of 1.6 (95% ci: 0.83.0).7,14 in addition, studies were also showed that there was no significant association between gender and do figure 1. t r e n d o f p r e v a l e n c e o f d o a m o n g m d r tb patients table 2. the correlation between predisposing factors and do among mdr tb patients variable predisposing factors patient status pr (ci 95%) p valuenot do n (%) not do n (%) age (years) 15-24 25-44 45-64 > 64 23 (69.7%) 105 (63.6%) 46 (48.9%) 4 (40%) 10 (30.3%) 60 (36.4%) 48 (51.1%) 6 (60%) ref 1.314 (0.586 – 2.947) 2.4 (1.031 – 5.589) 3.45 (0.96 – 14.958) ref 0.507 0.042* 0.098 gender female male 69 (61.1%) 109 (57.7%) 44 (38.9%) 80 (42.3%) 1.151 (0.715 – 1.852) 0.646 hiv status negative positive unknown 134 (56.8%) 4 (80%) 40 (65.6%) 102 (43.2%) 1 (20%) 21 (34.4%) ref 0.328 (0.036 – 2.983) 0.69 (0.383 – 1.241) ref 0.323 0.215 90 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 87–92 incidence in mdr tb patients, it was found that male mdr tb patients had a risk of 1.4 times (95% ci: 0.96-2.05) to have do compared with female mdr tb patients (p value 0.083).9 regarding to the research from indonesia, this study is in line with several previous which were research which were that showed there was no sex relationship with the incidence of mdr patient tb patients.11,15,16 in contrast, previous study is showed brust’s research, et al. in 2010, a significant association between sex with do in mdr tb patients, which men had 1.9 times greater risk (95% ci 1.2-3.1) to have do than women.8 other study was suggested that women were more likely to seek health care and tend to be more adherent to treatment with dots compared with men (wu et al., 2013). different opinions were also obtained from studies in africa, bangladesh, and syria which was stated that married women tend to have to ask their husbands for permission to come to health services for tb treatment (ibrahim et al., 2014). the proportion of do patients with mdr tb in patients with hiv negative status was higher than the proportion of mdr patients with hiv positive status. the result of statistical test was showed that there was no significant relationship between hiv status and do with mdr tb patients with p value = 1.00 (p > 0.05). the results of this study were consistent with the study in nigeria in 2011-2012 which shows that hiv status has no statistically significant association with do in mdr tb patients.17 similar research in sagamu, nigeria by daniel, et al. and hasker’s research, et al. was showed similar results that hiv status had no significant relationship with the occurrence of do in mdr tb patients.18,19 in contrast, in kwazulu-natal, south africa in 2000-2003 was showing that patients with hiv-positive status (tb-hiv co-infection) have risk factors that can increase do of mdr tb by 2 times (95% ci: 1.3-3.1). this might be happened because of the research was done in south africa which had a high prevalence of hiv cases. the absence of any association in this study can occur due to the large number of mdr tb patients whose hiv status is unknown. in addition, data on tb-hiv coinfection may also have unreported data on e-tb manager so that the proportion of hiv-positive tb patients was low (under reporting). the results of sputum examination is an early indication to be able to know the presence of bta. the status of this bta examination may reflect the extent of lesions in the lung. 3+ bacilli on initial pre-treatment can be used as a predictor for difficult conversion after two months of treatment. the longer the time required for conversion will be the longer the initial treatment. this may affect the patient in treatment and may cause the patient to drop out (nwokeukwu & awujo, 2013). the proportion of do in mdr tb patients between patients was exposed to smear positive with smear negative did not show much different numbers. the proportion of do in mdr tb patients was more prevalent in patients exposed to smear negative than the proportion of do in smear positive patients. the result of statistical test was showed that patients who had sputum smear + at the beginning of treatment had a risk of having positive smearpositive mdr tb patients at 0.836 (95% ci: 0.504–1.385) risk of drop out compared to negative smear-negative mdr tb patients. this study was showed similar results with the previous research which is showed no significant relationship between sputum examination result and do incidence, the value of crude or 1.15 (95% ci: 0.7-1.89) and adjusted or 1.02 (95% ci: 0.43-1.30) (table 3).18 in contrast, the results of alobu’s research, et al. was showed a significant relationship between sputum examination results and the incidence of do in mdr tb patients, which the value of crude or 2.1 (95% ci: 1.4-3.1) and adjusted or table 3. distribution of do among mdr tb patients in 2011-2015 based on enabling factors variable enabling factors patient status pr (ci 95%) p valueno do n (%) do n(%) sputum test negatif positif 48 (55.8%) 130 (60.2%) 38 (44.2%) 86 (39.8%) 0.836 (0.504 – 1.385) 0.571 type of patient new recurent defaulter failed in category 1 failed in category 2 others (unclear) 3 (50%) 58 (58.6%) 22 (55%) 41 (63.1%) 49 (61.2%) 5 (41.7%) 3 (50%) 41 (41.4%) 18 (45%) 24 (36.9%) 31 (38.8%) 7 (58.3%) ref 0.707 (0.136 – 3.679) 0.818 (0.147 – 4.557) 0.585 (0.109 – 3.134) 0.633 (0.12 – 3.335) 1.4 (0.195 – 10.032) ref 0.680 0.819 0.532 0.589 0.738 number of previous treatment ≤2 times of treatment >2 times of treatment 150 (60%) 28 (53.8%) 100 (40%) 24 (46.2%) 1.286 (0.705 – 2.345) 0.506 number of drugs 2 drugs 3 drugs ≥4 drugs 48 (57.8%) 55 (55.6%) 75 (62.5%) 35 (42.2%) 44 (44.4%) 45 (37.5%) ref 1.097 (0.609 – 1.977) 0.823 (0.465 – 1.457) ref 0.758 0.504 91farihatun dan bungsu: determinant factors of drop out (do) 2.3 (95% ci: 1.5–3.6).17 the others study was showed a significant relationship between sputum and do results, but the or values which were obtained in this study resulted in protective pr, with or values of 0.57 (95% ci: 0.33 0.97) and adjusted or 0.42 (0.024 0.75).19 the statistical results were showed that there was no relationship between the types of patients with do among mdr tb patients. it was found that the “other” type (unclear history) of patient group had a risk of 1.4 times (95% ci: 0.195-10.032) for drop out compared to the new patient type group. patients who were failed in treatment of second category had 0.633 (95% ci: 0.12-3.335) times of risk to have do compared with new patients. patients who were failed at first category had 0.585 times (95% ci: 0.109-3.134) of risk having do experience compared with new patients. defaulter patients had a risk of 0.818 times (95% ci: 0.147-4.557) to have do compared with new patients. recurrent patients had a risk of 0.707 times (95% ci: 0.136-3.679) to have do compared with new patients. this research is in line with some previous research which indicate that the type of patient did not have statistical correlation with do in mdr tb patients.18,17,20 in contrast, previous study santha study, et al. (2002) and lalor, et al. (2013) were showed that the type of patient had a significant association with do in mdr tb patients. in the santha study, et al (2002) were showed re-treatment patients had or 2.5 (95% ci: 1.5-4.3) of having do compared with the new patient type and adjusted or 2.8 times (95% ci : 1.6-4.9).9,21 research from lalor, et al. (2013) was showed that the type of patient defaulter had a risk of 2.10 (95% ci: 1.02-4.37) for do compared with the new patient type and adjusted or 2.38 (95% ci: 1.09-5.24), patients failed in category 2 had a risk of 0.57 times (95% ci: 0.35 to 0.93) to have do compared with the new patient type and adjusted or 0.85 (95% ci: 0.49-1.49). the proportion of do patients with mdr tb in patients in the previous treatment group group was >2 times more than patients with previous treatment amount ≤2 times. the results of statistical tests showed that there was no statistically significant relationship between the amount of previous treatment with do in mdr tb patients. it was found that patients with previous treatment >times had a risk of 1.286 (95% ci: 0.705-2.345) for drop out compared to patients with previous treatment ≤2 times. in fact, most mdr tb patients in indonesia are patients with a history of previous tb treatment. treatment which performed previously treated petients with first-line oat, requires treatment with second-line oat, where secondline oat is more complicated in its management, and second-line oats have more and more severe side effects than first-line oat, thus allowing mdr tb patients to drop out.1 this study was showed similar results with previous study which was showed there was no relationship between the number of previous treatments and the incidence of do (p value = 1.0).20 this study is also in line with the research of franke, et al. (2008) indicating no significant association between the number of previous treatments and the incidence of do in mdr tb patients. patients with previous treatment amounts >2 treatments had a risk of 1.2 times (95% ci: 0.70-2.05) for dropouts compared to patients with previous treatment ≤2 times of treatment. researchers have not found previous research which results indicating that there was a significant relationship between the number of previous treatments and the incidence of do in mdr tb patients, but based on the results of research franke, et al. in 2008, the value of or >1 means that the number of previous treatments is a risk factor for the incidence of do in patients with mdr tb, so the researchers included this number of previous treatment factors to be investigated as independent variables of research and variables are substantially important.7 the amount of oat resistance is divided into resistant to 2 drugs, 3 drugs and ≥4 drugs. the proportion of do in patients with drug resistant mdr 3 was higher than the proportion of do in the group of other oat resistance levels. the result of statistical test was showed that there was no relationship between the amount of oat resistance with do patient of mdr tb. the results of this study was indicated that the patient group of the ≥4 drug resistance group had a risk of 0.823 times (95% ci: 0.465-1.457) for drop out compared to the 2-drug resistance group. the researchers also have not found previous research which their results related to the variable amount of oat resistance indicating a statistically significant relationship between the amount of oat resistance and the incidence of do in patients with mdr tb, but the researcher still incorporates this amount of oat resistance factor to be studied as an important independent variable of research. from the results of this study it is known that the number of drug resistance has a risk of 1.097 times (95% ci: 0.6091.977) for drop out compared to the 2-drug resistance group. conclusion determinant factors that associated with do in mdr tb patients in dki jakarta province 2011-2015 is aged 4564 years old. while the others variables are not proved by statistically to have association with do among mdr tb in this study. the age group that significantly associated with the incidence of do in mdr tb patients was 45-64 years who had a risk of 2.4 times (95% ci: 1.031-5.589) for do compared to the 15-24 age group. acknowledgement the authors thank to jakarta province health office especially vector and zoonotic contagious diseases section, diseases control and prevention department who gave us permission for using tb surveillance data. 92 indonesian journal of tropical and infectious disease, vol. 7 no. 3 september–december 2018: 87–92 references 1. magister manajemen ut. buku panduan manajemen terpadu pengendalian tuberkulosis resistan obat. in jakarta: ministry of health ri; 2014. 2. world health organization. global tuberculosis report. 2016. 3. ministry of health ri. toss tb: temukan tb obati sampai sembuh. 2016. 4. departemen kesehatan r. pedoman nasional pengendalian tuberkulosis. j kesehat masy. 2011;2011. 5. efek i, obat s. berita meso. 2007;25(2):6184. 6. shin ss, pasechnikov ad, gelmanova iy, peremitin gg, strelis ak, mishustin s, et al. treatment outcomes in an integrated civilian and prison mdr-tb treatment program in russia. int j tuberc lung dis. 2006 apr;10(4):402–8. 7. franke mf, appleton sc, bayona j, arteaga f, palacios e, llaro k, et al. risk factors and mortality associated with default from multidrug-resistant tuberculosis treatment. clin infect dis. 2008 jun 15;46(12):1844–51. 8. brust jcm, gandhi nr, carrara h, osburn g, padayatchi n. high treatment failure and default rates for patients with multidrug-resistant tuberculosis in kwazulu-natal, south africa, 2000-2003. int j tuberc lung dis. 2010 apr;14(4):413–9. 9. lalor mk, greig j, allamuratova s, althomsons s, tigay z, khaemraev a, et al. risk factors associated with default from multi and extensively drug-resistant tuberculosis treatment, uzbekistan: a retrospective cohort analysis. plos one. 2013;8(11):e78364. 10. sri mm, nawas; a, soetoyo; dk. pengamatan pasien tuberkulosis paru dengan multidrug resistant (tb-mdr) di poliklinik paru rsup persahabatan. j respirologi indones. 2010;30(2):1 of 13. 11. universitas airlangga. analisis faktor yang mempengaruhi drop out dalam pengobatan tb mdr di kabupaten gresik. 2016. 12. widyasrini er, probandari an. factors affecting the success of multi drug resistance ( mdr-tb ) tuberculosis treatment in residential surakarta. 2015;45–57. 13. wu j, liu w, he l, huang f, chen j, cui p, et al. sputum microbiota associated with new, recurrent and treatment failure tuberculosis. plos one. 2013;8(12):e83445. 14. holtz th, lancaster j, laserson kf, wells cd, thorpe l, weyer k. risk factors associated with default from multidrug-resistant tuberculosis treatment, south africa, 1999-2001. int j tuberc lung dis. 2006 jun;10(6):649–55. 15. reviono et al. multidrug resistant tuberculosis (mdr-tb): tinjauan epidemiologi dan faktor risiko efek samping obat anti tuberkulosis multidrug resistant tuberculosis (mdr-tb): epidemiologic review and adverse events risk factors of anti tuberculosis drugs. mkb. 2014;46(4):189–96. 16. fauziyah n. faktor yang berhubungan dengan drop out pengobatan pada penderita tb paru di balai pengobatan penyakit paru-paru ( bp4 ) salatiga. univ negeri semarang. 2010; 17. alobu i, oshi sn, oshi dc, ukwaja kn. risk factors of treatment default and death among tuberculosis patients in a resource-limited setting. asian pac j trop med. 2014 dec;7(12):977–84. 18. daniel oj, oladapo ot, alausa ok. default from tuberculosis treatment programme in sagamu, nigeria. niger j med. 15(1):63–7. 19. hasker e, khodjikhanov m, usarova s, asamidinov u, yuldashova u, van der werf mj, et al. default from tuberculosis treatment in tashkent, uzbekistan; who are these defaulters and why do they default? bmc infect dis. 2008 jul 22;8:97. 20. kartika. analisis faktor-faktor yang berhubungan dengan default penderita tuberkulosis paru di rsud budhi asih jakarta tahun 2008. 2009; 21. santha t, garg r, frieden tr, chandrasekaran v, subramani r, gopi pg, et al. risk factors associated with default, failure and death among tuberculosis patients treated in a dots programme in tiruvallur district, south india, 2000. int j tuberc lung dis. 2002 sep;6(9):780–8. 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. 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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 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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. 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 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[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. 50 vol. 7 no. 2 may–august 2018 research report t h e e f f e c t i v e n e s s o f h e r b a l m o s qu i t o c o i l s “morizena” against aedes aegypti death rina priastini susilowati1a, win darmanto2, nanik siti aminah3 1 department of biology, faculty of medicine, universitas krsten krida wacana 2 departement of biology, faculty of science and technology, universitas airlangga 3 departement of chemistry, faculty of science and technology, universitas airlangga a corresponding author: rina.priastini@ukrida.ac.id abstract aedes aegypti is a mosquito vector of dengue hemorrhagic fever (dhf) which is one of endemic diseases in indonesia. until now the way of handling is to use synthetic insecticides. the mosquito coil is the most widely used insecticide by the community because it is cheap and easy to use. however, its continuous use can cause resistance to mosquitoes and disrupt the health of its users. the purpose of this study was to find alternative insecticide chemicals that are safer for use derived from indonesian plants. morizena is a natural mosquito coil which is a mixture of 40% of permot leaf extract (passiflora foetida), 40% chrysanthemum extract (chrysanthemum cinerariaefolium), 20% essential oil of citronella (cymbopogon nardus). the herbal mosquito coil “morizena” is as effective to kill ae. aegypti as the use of transflutrin synthetic chemical mosquito coil. exposure to herbal mosquito repellent “morizena” on ae. aegypti for 8 hours / day with a concentration of 500 ppm (p1), 1000 ppm (p2), 2000 ppm (p3), 3000 ppm (p4), 4000 ppm (p5) and transflutrin 2500 ppm (k1) synthetic fuel mosquito positive control group, and group without exposure to mosquito coils (k0) as negative controls. animal models which are used in this experiment are 25 tail of ae. aegypti mosquitos for each treatment with 4 replications. the experimental design which are used was a probit test to calculate lc50 and lc90. the results of herbal mosquito coils “morizena” test given for 8 hours/day was yielded mortality of ae. aegypti by 92% for a concentration of 3000 ppm (p4) and 100% to a concentration of 4000 ppm (p5) and 100% for test to synthetic mosquito coils transfluthrin 2500 ppm (k1). ae. aegypti lc50 and lc90 value for treatment of exposure to herbal mosquito coils “morizena” are 999 ppm and 2977 ppm. treatment of herbal mosquito coils “morizena” with graded doses up to 4000 ppm and synthetic mosquito coils transfluthrin 2500 ppm was caused an increase in the enzyme acetylcholinesterase activity of ae. aegypti. based on the ae. aegypti lc90 value is 2977 ppm. the effective dose of herbal mosquito coils “morizena” to kill ae. aegypti is 2977 ppm. keywords: herbal mosquito coils, ae. aegypti, morizena, dengue abstrak aedes aegypti adalah nyamuk vector penyakit demam berdarah dengue (dbd) yang merupakan salah satu penyakit endemic di indonesia. hingga saat ini cara penanggulangannya adalah menggunakan insektisida sintetik. obat nyamuk bakar adalah jenis insektisida yang paling banyak digunakan oleh masyarakat karena harganya murah dan mudah digunakan. namun penggunaannya yang terus menerus dapat menyebabkan resistensi pada nyamuk dan mengganggu kesehatan penggunanya. tujuan penelitian ini adalah menemukan alternative bahan kimia insektisida yang lebih aman untuk digunakan yang berasal dari tanaman indonesia. morizena adalah obat nyamuk bakar berbahan alami yang merupakan campuran dari 40% ekstrak daun permot (passiflora foetida), 40% ekstrak bunga krisan (chrysanthemum cinerariaefolium), 20% minyak atsiri daun-batang sereh (cymbopogon nardus). obat nyamuk bakar herbal “morizena” sama efektifnya untuk membunuh aedes aegypti seperti penggunaan obat nyamuk bakar berbahan kimia sintetik transflutrin. paparan obat nyamuk bakar herbal “morizena” pada ae. aegypti selama 8 jam/hari dengan konsentrasi 500 ppm (p1), 1000 ppm (p2), 2000 ppm (p3), 3000 ppm (p4), 4000 ppm (p5) dan paparan obat nyamuk bakar sintetik transflutrin 2500 ppm (k1) sebagai kelompok kontrol positif, dan kelompok tanpa paparan obat nyamuk bakar (k0) sebagai kontrol negatif. hewan coba yang digunakan adalah nyamuk ae. aegypti dewasa sebanyak 25 ekor untuk setiap perlakuan dengan 4 ulangan. rancangan percobaan yang digunakan adalah uji probit untuk menghitung lc50 dan lc90. hasil penelitian yang diperoleh dari paparan obat nyamuk bakar herbal “morizena” selama 8 jam/ 51susilowati, et al.: the effectiveness of herbal mosquito coils hari adalah mortalitas ae. aegypti sebesar 92% pada konsentrasi 3000 ppm (p4) dan mortalitas 100% pada konsentrasi 4000 ppm (p5) dan 100% untuk obat nyamuk bakar sintetik transfluthrin 2500 ppm (k1). nilai lc50 and lc90 pada kelompok paparan obat nyamuk bakar herbal “morizena” adalah 999 ppm dan 2977 ppm. paparan obat nyamuk bakar herbal “morizena” dengan dosis bertingkat hingga 4000 ppm dan obat nyamuk bakar sintetik transflutrin 2500 ppm menyebabkan peningkatan aktivitas asetilkolinesterase ae. aegypti. kesimpulannya adalah nilai ae. aegypti lc90 sebesar 2977 ppm, berarti dosis efektif obat nyamuk bakar herbal “morizena” yang dapat membunuh ae. aegypti adalah 2977 ppm. kata kunci: obat nyamuk bakar herbal, ae. aegypti, morizena, dengue introduction dengue hemorrhagic fever (dhf) is an infectious disease caused by the dengue virus and is transmitted through the bite of ae. aegypti mosquito as the main factor.1 the number of districts/cities in indonesia infected by dhf in 2008 about 355 districts/cities (71.72%), in 2009 about 384 districts/cities (77.28%), in 2010 about 400 districts/ cities (80.48%) and in 2011 about 374 districts/cities (76.5%). the number of patients with dhf are reached 65432 cases, about 596 of them dies. in 2014, 100347 cases of dengue were reported, and within the case, 907 people were dead, compared to the year 2013 as many as 112511 cases.2 it can be seen from these data that the dhf prevention efforts in indonesia has not been optimized yet because the number of cases are increasing every year. one of the methods to break the chain of mosquitoes is by using insecticides to control the vector. this is because the synthetic insecticides are effective, practical, efficacious and economically more profitable. however, because the use of synthetic insecticides continuously will cause environmental pollution, the death of a wide variety of other living things and cause mosquitoes to become resistant, it can even cause mutations of genes in this species. synthetic insecticides are bio active, contain chemicals which are difficult to be degraded in nature so the residue can contaminate the environment and can degrade the quality of the environment.3 in addition, the presence of synthetic insecticides in the food chain can cause the death of some other living things and eventually will disrupt the ecosystem stability. although before it is produced, synthetic insecticides have undergone very rigorous testing requirements regarding its safety. because synthetic insecticide is bio active and a toxic substance, it will always create harmful effect to humans and environment. regarding to the side effects which caused by synthetic insecticides, it is necessary to find alternative materials which not only more environmentally friendly, but also effective in controlling mosquito population. the use of bioinsecticides as a substitute for synthetic insecticides is expected to reduce the problem of environmental pollution. this is due to the use of bioinsecticides does not cause environmental pollution because it contains material that is easily and rapidly degraded in nature and do not harm the environment, both animal and human. it has been also stated by quarcoo et al. in 2014 that the bioinsecticides has superior properties as biodegradable and harmless to humans and the environment.4 one of the chemical controls is mosquito coils. mosquito coils are a material which produces insecticide fumes and it is widely used to reduce mosquito bites and reduce the density of mosquitoes either by the researchers and the community. mosquito coils are easy to use, effective and cheap.5 therefore; this study is aimed to make mosquito coils made from plants called “morizena”. herbal mosquito coils “morizena” is made from a mixture of plant extracts of passiflora foetida leaf, chrysanthemum cinerariaefolium flower extract and essential oil of cymbopogon nardus leaf-stem. one of the medicinal plants which is assumed contains active ingredients that can be used as a bioinsecticides is passiflora foetida. passiflora foetida is a trailing plant from indonesia which grows wildly between other plants and can be easily found in garden or trail on the wall and because of that it usually burned or thrown away.6-8 based on the previous research conduct by susilowati in 2013 on how passiflora foetida leaf extract affect the mortality of aedes aegypti larvae in a various concentration start from 500 ppm, 1000 ppm, 1500 ppm to 2000 ppm. the results from the linear regression equation can achieve lc50 from passiflora foetida leaf extract precise concentration of 1068 ppm.9 however, after tested with the rate of concentration 500 ppm, 1000 ppm and 2000 ppm, it has not been able to reach mortality rate of ae. aegypti more than 90%.10 other plant that has been previously used as popular insect repellent active ingredients are pyrethrins found in chrysanthemum cinerariaefolium flower seeds. kardinan in 2000 was reported that one of the member of asteraceae family is chrysanthemum cinerariaefolium which contains bioactive compounds, pyrethrin.11 these compounds are toxic contact that works as a neurotoxin to insects and can inhibit egg lying and egg hatching of insects. furthermore, pyrethrin is a component that can kill mosquitoes and have low toxic levels for humans and other mammals and does not leave residue and safe for the environment.12 the leaves and stem extract of lemongrass (cymbopogon nardus) has an active substance of citronellal, citronellol, and geraniol which can be used as mosquito repellent. citronellal oil can also be used as an anti-bacterial, antifungal, anti-viral, cytotoxic, anti aflatoxigenic, perfume, food seasoning, the aroma of tea, and insect repellent 52 indonesian journal of tropical and infectious disease, vol. 7 no. 2 may–august 2018: 50–55 because it contains citronellal, citronellol, and geraniol. citronellal substance has the characteristics of contact toxin. as a contact toxin, citronellal can cause death due to continuous fluid loss so that the mosquito is dehydrated.13 according to sakulku et al. in 2009 and fradin in 1998 among the essential oils that exist, citronellal oil is the most effective which can serve as a repellent for 2 hours.14,15 citronellal oil showed good efficacy against 44 ae. aegypti with the concentration ranging from 0.05% to 15% (w/v) or in combination with other natural materials or commercial products of insect repellent. to get more than 90% of ae. aegypti, by referring to some previous research results on bioinsecticides, herbal mosquito coils morizena is made by mixing a few active ingredient: passiflora foetida leaf extract containing harmaline, harmine and ermanin, chrysanthemum cinerariaefolium flower seed extract containing pyrethrins and the essential oil of cymbopogon nardus leavesstems with active ingredient citronellal using the mixed composition 40% passiflora foetida leaf extract, 40% chrysanthemum cinerariaefolium flower seed extract and 20% of essential oil of cymbopogon nardus leaves and stems. material and method herbal mosquito coils “morizena” after several stages of research to determine the percentage of the mixture, then herbal mosquito coils “morizena” made by mixing a few active ingredient: 40% passiflora foetida leaf extract with ethanol, 40% chrysanthemum cinerariaefolium seed flower extract with ethanol and 20% of essential oil of cymbopogon nardus leaves-stems. adhesive materials for herbal mosquito coils “morizena” are starch and honey, filler of mosquito coils is from the residue of passiflora foetida leaf extract and the powder of coconut shell. experimental animals experimental animals used were adult mosquitoes ae. aegypti. ae. aegypti eggs that will be bread for two generations to get a pure line for adult mosquitoes obtained from the veterinary faculty of bogor agricultural institute. the number of mosquitoes used was 25 each treatment with 4 replications. adult mosquitoes were used ± 14 days after instar iv phase. treatment dose in this research, the treatment dose for ae. aegypti are grouped as follows: negative control k0 (without treatment of mosquito coils), positive control k1 (synthetic mosquito coils transfluthrin 2500 ppm), p1 (herbal mosquito coils “morizena” 500 ppm), p2 (herbal mosquito coils “morizena” 1000 ppm), p3 (herbal mosquito coils “morizena” 2000 ppm), p4 (herbal mosquito coils “morizena” 3000 ppm) and p5 (herbal mosquito coils “morizena” 4000 ppm). each group was given the treatment for 8 hours/day. the treatment begins by burning mosquito coils at 8:00 to 16:00 in each mosquito cage with a multilevel dosage. the bioassaytest the bioassay testing was used ae. aegypti aged 3–5 days that had previously been fed with sugar water, put in 12x12x12 cm3 cage with 25 mosquitoes in each cage. the number of cage is matched up to 2 control groups and 5 treatment groups. mosquito coils were placed next to the cage from 17.00 until 01.00. they were observed in the 5th, 10th, 15th, 30th, 45th, 60th, 120th, 180th, 240th, 300th, 360th, 420th, and 480th minute, by counted the number of mosquitoes which pass out and dead, and the calculated the percentage of the death. temperature, ph and air relative humidity during the test period is also measured and recorded. mosquito knockdown rate recorded in 0.5 minutes at intervals of 1 minute up to 10 minutes for the ae. aegypti. the criteria for knockdown mosquitoes are when they are not be able to keep their balance and not be able to fly any longer.16 efficacy criteria is taken based on the time of paralysis of the mosquitoes tested (calculated from the data corrected by the mortality and paralysis of tested mosquitoes) in the control group. the acetylcholinesterase activity test the analysis of acetylcholinesterase activity is performed based on the method from ellman et al. in 1961 which applied on the ae. aegypti larvae.17 however, for this research, the method is modified so it can be applied to the adult ae. aegypti. in a test tube containing potassium phosphate buffer 1.95 ml, with 0.1 m ph 7.5, added 200 µl homogenate of ae. aegypti, 150 µl dtnb 0.0011 m in phosphate buffer and 100 µl test insecticide solution in water and emulsifier alkylaryl polyglycol ether 400 mg/l 0.1%. the insecticide suspension control mixture was replaced with buffer solution. after perfectly whipped and left for 10 minutes, then in each test tube added 100 µl of acetylcholine iodide 0.0105 m in phosphate buffer. the test was conducted on six concentrations of mosquito coils. blank mixture contains the same components as tested mixture, except homogenates enzyme source was replaced by phosphate buffer. the reaction was left for 30 minutes, and then light absorption of the solution in each tube was measured at a wavelength of 412 nm using a spectrophotometer. acetylcholinesterase activity expressed in molar substrate hydrolyzed per minute per mg. data analysis data were analyzed by linear regression to the death of a mosquito mortality rate, lc50 and lc90 using probit test.18 one way anova test is used to determine the effect of mosquito coils in the activity of the acetylcholinesterase ae. aegypti. 53susilowati, et al.: the effectiveness of herbal mosquito coils result and discussion the bioassay test of ae. aegypti toxicity test or bioassay is aimed to determine the killing power of each concentration which tested against ae. aegypti. toxicity test was conducted on mosquitoes by using herbal mosquito coils “morizena” from the level of 500 ppm, 1000 ppm, 2000 ppm, 3000 ppm to 4000 ppm (p1-p5), and also using synthetic mosquito coils transfluthrin 2500 ppm (k1). determination tests are conducted on ae. aegypti every minute for 8 hours/day reflects the increasing number of deaths (mortality) due to the use of herbal mosquito coils “morizena” (p1-p5) and synthetic mosquito coils transflutrin 2500 ppm (k1), the result can be seen in figure 1. result and discussion the bioassay test of ae. aegypti toxicity test or bioassay is aimed to determine the killing power of each concentration which tested against ae. aegypti. toxicity test was conducted on mosquitoes by using herbal mosquito coils “morizena” from the level of 500 ppm, 1000 ppm, 2000 ppm, 3000 ppm to 4000 ppm (p1-p5), and also using synthetic mosquito coils transfluthrin 2500 ppm (k1). determination tests are conducted on ae. aegypti every minute for 8 hours/day reflects the increasing number of deaths (mortality) due to the use of herbal mosquito coils “morizena” (p1-p5) and synthetic mosquito coils transflutrin 2500 ppm (k1), the result can be seen in figure 1. figure 1.graph the number of deaths ae. aegypti on treatment of herbal mosquito coils “morizena” graded dose and synthetic mosquito coils transfluthrin 2500 ppm after 8 hours of observation from one-way anova test results are showed that the probability value 0,000, means that there is a significant difference (p<0.05) of all treatment groups to the number of deaths (mortality) of ae. aegypti. furthermore, to compare the effects of each dose herbal mosquito coils “morizena” (p1-p5), synthetic mosquito coils transflutrin 2500 ppm (k1) with negative control (without treatment of mosquito coils) on the number of deaths of ae. aegypti used least significant difference test (lsd). the least significant difference test results showed that the number of deaths of ae. aegypti without exposure to mosquito coils (k0) was significantly different with group exposed with synthetic mosquito coils transfluthrin 2500 ppm (k1) and all groups exposed with herbal mosquito coils morizena graded doses (p1-p5). meanwhile, among the group of synthetic mosquito coils transfluthrin 2500 ppm (k1) was not significantly different with herbal mosquito coils “morizena” 3000 ppm (p4) and 4000 ppm (p5). this study has been showed that administration of synthetic mosquito coils transfluthrin 2500 ppm (k1) and a group of herbal mosquito coils “morizena” 3000 ppm (p4) and4000 ppm (p5) caused the death of ae. aegypti more than 90%. probit test is commonly used in toxicology to determine the relative toxicity of chemicals, most common outcome of a dose-response experiment in which probit test is used is the lc50. the probit test resulted regression equation y = 1.8372x + 0.0159 (y is the mortality of ae. aegypti and x is the dose of herbal mosquito coils “morizena” with graded dose). from the regression equation the value of the effective dose of herbal mosquito coils “morizena” that causes mortality of ae. aegypti by 50% (lc50) is 999 ppm and by 90% (lc90) is 2977 ppm. based on the results of this research it can be concluded that herbal mosquito coils figure 1. graph the number of deaths ae. aegypti on treatment of herbal mosquito coils “morizena” graded dose and synthetic mosquito coils transfluthrin 2500 ppm after 8 hours of observation. from one-way anova test results are showed that the probability value 0,000, means that there is a significant difference (p < 0.05) of all treatment groups to the number of deaths (mortality) of ae. aegypti. furthermore, to compare the effects of each dose herbal mosquito coils “morizena” (p1-p5), synthetic mosquito coils transflutrin 2500 ppm (k1) with negative control (without treatment of mosquito coils) on the number of deaths of ae. aegypti used least significant difference test (lsd). the least significant difference test showed was results that the number of deaths of ae. aegypti without exposure to mosquito coils (k0) was significantly different with group exposed with synthetic mosquito coils transfluthrin 2500 ppm (k1) and all groups exposed with herbal mosquito coils morizena graded doses (p1-p5). meanwhile, among the group of synthetic mosquito coils transfluthrin 2500 ppm (k1) was not significantly different with herbal mosquito coils “morizena” 3000 ppm (p4) and 4000 ppm (p5). this study has been showed that administration of synthetic mosquito coils transfluthrin 2500 ppm (k1) and a group of herbal mosquito coils “morizena” 3000 ppm (p4) and4000 ppm (p5) caused the death of ae. aegypti more than 90%. probit test is commonly used in toxicology to determine the relative toxicity of chemicals, most common outcome of a dose-response experiment in which probit test is used is the lc50. the probit test resulted regression equation y = 1.8372x + 0.0159 (y is the mortality of ae. aegypti and x is the dose of herbal mosquito coils “morizena” with graded dose). from the regression equation the value of the effective dose of herbal mosquito coils “morizena” that causes mortality of ae. aegypti by 50% (lc50) is 999 ppm and by 90% (lc90) is 2977 ppm. based on the results of this research it can be concluded that herbal mosquito coils “morizena” is more effective and environmentally friendly compared to synthetic mosquito coils transfluthrin 2500 ppm because the exposure of herbal mosquito coils “morizena” up to a dose of 4000 ppm can cause 100% death of ae. aegypti. ae. aegypti mosquito lc90 is 2977 ppm, which means the effective dose of herbal mosquito coils “morizena” to kill ae. aegypti is 2297 ppm and the effective dose of synthetic mosquito coils transfluthrin 2500 ppm. the activity of the acetylcholinesterase after ae. aegypti mosquitos which were used in this research dead, they were tested for the activity of the acetylcholinesterase. each of ae. aegypti mosquitos were homogenized in the appropriate buffer solution at a temperature of 4oc and 10 g supernatan is used to determine the activity of acetylcholinesterase. the activity of acetylcholinesterase was calculated in the 1st, 4th, 8th hour after the ae. aegypti dead. measurement of the acetylcholinesterase activity in the ae. aegypti conducted using a modified method of ellman et al. in 1961.17 acetylcholinesterase activity measurement results were expressed in molar substrate hydrolyzed per minute per mg protein as shown in figure 2. “morizena” is more effective and environmentally friendly compared to synthetic mosquito coils transfluthrin 2500 ppm because the exposure of herbal mosquito coils “morizena” up to a dose of 4000 ppm can cause 100 % death of ae. aegypti. ae. aegypti mosquito lc90 is 2977 ppm, which means the effective dose of herbal mosquito coils “morizena” to kill ae. aegypti is 2297 ppm and the effective dose of synthetic mosquito coils transfluthrin 2500 ppm. the activity of the acetylcholinesterase after ae. aegypti mosquitos which were used in this research dead, they were tested for the activity of the acetylcholinesterase. each of ae. aegypti mosquitos were homogenized in the appropriate buffer solution at a temperature of 4oc and 10 g supernatan is used to determine the activity of acetylcholinesterase. the activity of acetylcholinesterase was calculated in the 1st, 4th, 8th hour after the ae. aegypti dead. measurement of the acetylcholinesterase activity in the ae. aegypti conducted using a modified method of ellman et al. in 1961.17 acetylcholinesterase activity measurement results were expressed in molar substrate hydrolyzed per minute per mg protein as shown in figure 2. figure 2. acetylcholinesterase enzyme activity graph of ae. aegypti on herbal mosquito coils “morizena” graded dose and synthetic mosquito coils transfluthrin 2500 ppm on the hour to 1, 4 and 8 after the ae. aegypti die the analysis of variants of acetylcholinesterase activity in ae. aegypti which exposed with herbal mosquito coils “morizena” graded dose (p1-p5) for 8 hours/day are showed that the probability value is 0.000. this means that there are significant differences of treatment effect of herbal mosquito coils “morizena” graded dose (p1-p5), positive control group which was treated with synthetic mosquito coils transfluthrin 2500 ppm (k1) and negative control group without treatment of mosquito coils on the activity of the acetylcholinesterase ae. aegypti. then the least significant difference test was performed to compare the significance of treatment effects of herbal mosquito coils “morizena” graded dose (p1-p5), synthetic mosquito coils transfluthrin 2500 ppm (k1) and negative control group without treatment of mosquito coils on the activity of the acetylcholinesterase in ae. aegypti. the least significant difference test results were showed that each concentration levels of herbal mosquito coils “morizena” (p1-p5), synthetic mosquito coils transfluthrin 2500 ppm (k1) and without treatment of mosquito coils (k0) provide a significant difference between each concentration. an attempt to control the population of ae. aegypti is by spraying chemical insecticides, which can cause adverse effects to the environment, among others, the effects of the emergence of resistance in vectors and cause environmental pollution by chemical insecticides present in figure 2. acetylcholinesterase enzyme activity graph of ae. aegypti on herbal mosquito coils “morizena” graded dose and synthetic mosquito coils transfluthrin 2500 ppm on the hour to 1, 4 and 8 after the ae. aegypti die. the analysis of variants of acetylcholinesterase activity in ae. aegypti which exposed with herbal mosquito coils “morizena” graded dose (p1-p5) for 8 hours/day are showed that the probability value is 0.000. this means that there are significant differences of treatment effect of herbal mosquito coils “morizena” graded dose (p1-p5), 54 indonesian journal of tropical and infectious disease, vol. 7 no. 2 may–august 2018: 50–55 positive control group which was treated with synthetic mosquito coils transfluthrin 2500 ppm (k1) and negative control group without treatment of mosquito coils on the activity of the acetylcholinesterase ae. aegypti. then the least significant difference test was performed to compare the significance of treatment effects of herbal mosquito coils “morizena” graded dose (p1-p5), synthetic mosquito coils transfluthrin 2500 ppm (k1) and negative control group without treatment of mosquito coils on the activity of the acetylcholinesterase in ae. aegypti. the least significant difference test results were showed that each concentration levels of herbal mosquito coils “morizena” (p1-p5), synthetic mosquito coils transfluthrin 2500 ppm (k1) and without treatment of mosquito coils (k0) provide a significant difference between each concentration. an attempt to control the population of ae. aegypti is by spraying chemical insecticides, which can cause adverse effects to the environment, among others, the effects of the emergence of resistance in vectors and cause environmental pollution by chemical insecticides present in the form of a spray, mosquito coils, mats and others. the use of synthetic chemical insecticides with materials or insecticides to anticipate the negative effects that are more environmentally friendly which called biological insecticides (bioinsecticides) is required. therefore, this research is tried to find new alternative of herbal mosquito coils “morizena” which is mixture of permot leaf (passiflora foetida), chrysantemum cineriaefolium flower seed and leaf-stem lemongrass (cymbopogon nardus). toxicity test with the statistical approach is measured through lc50 and lc90 concentration of herbal mosquito coils “morizena” to kill 50% of ae. aegypti, the test used was probit test.18 probit test analysis of ae. aegypti death after 8 hours/day of exposure showed that lc50 is 999 ppm and lc90 is 2977 ppm. based on the toxicity test, the level of concentration applied is the main factors that affect the mortality of ae. aegypti tested. it can be proved with the higher the concentration of treatment the greater the number of dead mosquitoes, with effective dose of herbal mosquito coils “morizena” is 2977 ppm. heterogenity seen in the probit test is due to the variation of the concentration of mosquito coils used in toxicity testing on ae. aegypti. as hartati in 2000 was stated that the power of each concentration of insecticide mosquito coils can affect the rate of oxygen consumption and metamorphosis of ae. aegypti.19 killing power of biological insecticides (bioinsecticides) is because there is a toxic substance that is toxic to the stomach and contact toxin of the bodied animals. in addition, the use of bioinsecticide is not intended to put synthetic insecticides aside, but only as an alternative way with the aim that people should not rely on synthetic insecticides. another goal is that the use of synthetic insecticides can be minimized so that the environmental damage can be reduced. besides that, the use of bioinsecticides will help the development of agroindustries.20 tarumingkeng in 1989 was stated that the first step in observing the effects of poisoning is to observe the physical response behavior of the tested animals.21 the response is the basis for the classification of toxic materials. furthermore, the symptoms shown due to the influence of the treatment given will show four stages of the animal’s response include excitation, convulsions, paralysis and death.22 the stage of adult mosquitoes were stated by who in 2009 that the symptoms which has been showed as a result of the effect of insecticides which can stiffen the body of the mosquito. the insecticides attack the nervous system which was indicated by the mosquito’s inability to fly, paralysis and knockdown for several times and eventually will die.24 death was caused by toxin in the digestive system (spiracles) that causes the mosquito dead. generally, a neurotoxin causing four stages of symptoms, namely: excitation, convulsions, paralysis and death. these stages are not necessarily complete and only a few that can be observed. the time between the toxin application and the appearance of first phase symptoms is called the latent period. latent period is often found in the stomach toxin application. excitation phase is often preceded by anxiety. at this anxiety stage, the mosquito is showed movements such as cleaning the body such as the antenna or the other body parts. the poisoning process raises signs such rapid movements, restless leg raise, rolling and head twitching. at the end of the excitation mosquitoes will lose balance and ataxia and finally falls.21 from this research, lc90 which can cause death of ae. aegypti is 2977 ppm. from these results, it can be stated that herbal mosquito coils “morizena” which were tested against ae. aegypti, have been showed high toxic effects (poison) so that it can be classified as a bioinsecticide. according to who in 2006, the insecticides is said to be good if it shows high level of concentration with the shorter amount of time.24 the result is showed that the herbal mosquito coils “morizena” with graded doses up to 3000 ppm capable in causing ae. aegypti high mortality rate (more than 90%). from observation after exposure of herbal mosquito coils “morizena”, the mortality of ae. aegypti with the concentration of 3000 ppm (p4) and synthetic mosquito coils transfluthrin 2500 ppm (k1) occur simultaneously in a short time. however, ae. aegypti at the concentration of 500 ppm and 1000 ppm, 2000 ppm were only paralyzed and then were recovered. this is because the amount of toxins in herbal mosquito coils “morizena” less than 1000 ppm were not clinical enough to ae. aegypti.25,26 in this case the target, which is ae. aegypti mosquito, will die when inhaling bioinsecticides in this case is the herbal mosquito coils “morizena” that contains toxic toxins in sufficient quantities (mixture of harmaline harmine, ermanine, pyrethrin and citronellol). as for the contact toxin due to direct contact with the insecticide through the skin (epidermal tissue). pyrethrin compounds contained in extracts of chrysanthemum flower seeds and citronellol 55susilowati, et al.: the effectiveness of herbal mosquito coils of the leaves and stems lemongrass essential oil has a mechanism that inhibits the acetylcholinesterase, resulting in phosphorylation of the amino acid serine at the center of related synaptic enzyme. the toxicity symptoms appear due to accumulation of acetylcholine which causes central nervous system disorders, seizures, respiratory abilities obstruction and death. mortality occurs in ae. aegypti due to the ability of the active ingredient of herbal mosquito coils “morizena” for instance harmaline, harmine and ermanine on permot leaf extract, pyrethrin on chrysanthemum flower seeds extract and citronellol in essential oils of lemongrass leaves and stems can interrupt the flow of na+ (sodium) in the nerve cells and neurotransmitters (chemical transmitter) in the synapse.26 harmaline, harmine, ermanine and pyrethrin in nerves will extend na+ ions to flow into the membrane by slowing or blocking the channel closure. if harmaline and pyrethrin slow down the channel closure, the nerve will be depolarized in a long time, so there will be many na+ ions entering the membrane. this will cause symptoms of seizures and trembling. harmaline, harmine, ermanine and pyrethrins are also able to block the closure of the channel, this situation will cause excess membrane ion na+ eventually become inactive nerves. inactivity is occurring because the nerve is too positive and difficult to repolarize (back to its original state). symptoms that will appear is paralysis.26 pyrethrin presence at the synapse will interfere the transmitter chemicals or neurotransmitter which is acetylcholine.26,27 pyrethrin will increase acetylcholine and inhibit the enzyme from breaking down the acetylcholine. acetylcholine function is to provide permeability properties at postsynaptic membranes that cause the displacement na+ ions, causing depolarization. acetylcholines will be hydrolyzed by the acetylcholinesterase, which is present in large amounts at the synapse.25 with pyrethrin, enzymes cannot break down the chemical transmitter acetylcholine so that acetylcholine will continue to increase as the result of the membrane excess positive ions. in these circumstances mosquitoes will paralyze and will eventually die.27 problems of safe environmental pollution for mosquito coils morizena can be done by the method of reducing the concentration of carbon dioxide released through the smoke. conclusion exposure of herbal mosquito coils “morizena” with graded doses can increase the activity of the acetylcholinesterase enzyme in ae. aegypti and cause death of mosquito. references 1. s s. masalah vector demam berdarah dengue (dbd) dan pengendaliannya di indonesia. buletin jendela epidemiologi. 2. kementerian kesehatan ri. profil data kesehatan indonesia tahun 2015. jakarta: kementerian kesehatan ri; 2015. 3. rl m, whl. introduction to insect pest management. 3rd ed. new york: john wiley and sons; 2011. 4. f q, c b, pesticides tn. agricultural and biological sciences: pesticides – toxic aspects. environ hum heal. 2014; 5. rozendaal ja. vector control, method for use by individuals and communities. geneva : world health organization; 1997. 6. k h. tumbuhan berguna indonesia. jakarta: badan penelitian dan pengembangan kehutanan, departemen kehutanan; 1987. 7. hmh w. tanaman berkhasiat obat di indonesia. jakarta: penerbit pustaka kartini; 1995. 8. s s. chemical ecology and function of alkaloids. 2014. 9. rp s. efektivitas ekstrak daun permot (passiflora foetida) terhadap mortalitas larva nyamuk aedes aegypti. laporan penelitian. jakarta: fakultas kedokteran, universitas kristen krida wacana; 2013. 10. rp s. daya bunuh obat nyamuk bakar berbahan ekstrak daun permot (passiflora foetida) terhadap nyamuk aedes aegypti. jakarta: fakultas kedokteran, universitas kristen krida wacana; 2014. 11. a. k. piretrum (chrysanthemun cinerariaefolium trev): bahan insektisida nabati potensial. j litbang pertan. 2000;19(4):122–8. 12. novizan. membuat dan memanfaatkan pestisida ramah lingkungan. jakarta : penerbit agromedia pustaka; 2002. 13. h p. potret insektisida nabati pengendali nyamuk. inside. 2007;11(2). 14. sakulku u, nuchuchua o, uawongyart n, puttipipatkhachorn s, soottitantawat a, ruktanonchai u. characterization and mosquito repellent activity of citronella oil nanoemulsion. int j pharm. 2009 may 8;372(1–2):105–11. 15. fradin ms. mosquitoes and mosquito repellents: a clinician’s guide. ann intern med. 1998 jun 1;128(11):931. 16. adanan cr, zairi j, ng kh. efficacy and sublethal effects of mosquito mats on aedes aegypti and culex quinquefasciatus (diptera: culicidae). proc fifth int conf urban pests. 2005. 17. ellman gl, courtney kd, andres v, featherstone rm. a new and rapid colorimetric determination of acetylcholinesterase activity. biochem pharmacol. 1961 jul;7(2):88–95. 18. dj f. probit analisis. 3rd ed. london: cambridge university press; 2009. 19. w h. uji kepekaan larva aedes egypti linn terhadap ekstrak biji srikaya. semarang: universitas diponegoro; 2000. 20. n e-w. botanical pesticides and their mode of action. gesunde pflanz. 2013;65:125–49. 21. rc t. insektisida : sifat, mekanisme kerja dan dampak penggunaannya. jakarta: universitas kristen krida wacana; 1992. 22. brown ae, ph d, ingianni e, assistant p. no. 41: mode of action of structural pest control chemicals. 2013;(41). 23. world health organization. dengue and dengue haemorrhagic fever. fact sheet. 2009; 24. haouas d, halima-kamel m ben, habib m, hamouda b, supérieur i, chott a de. insecticidal activity of flower and leaf extracts from chrysanthemum species against tribolium confusum. tunis j plant prot. 2008;3(2):87–94. 25. winslow l. the effects of pyrethrins and pyrethroids on human physiology. 2002. 26. dr s. characterization of insect acetylcholinesterase enzyme: dmso-mediated allosteric effects, inhibitor pharmacological profile and role in the neurotoxicity of insect repellents. usa : university of florida; 2012. 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. 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 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, 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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. 156 vol. 5. no. 6 september–december 2015 c o m p a r a t i v e s t u d y o f f i l a r i a l d e t e c t i o n b y microscopic examination and serological assay utilizing bmr1 and bmxsp recombinant antigens for evaluation of filariasis elimination program at kampung sawah and pamulang, south tangerang district, banten, indonesia silvia f. nasution1 1 faculty of medicine and health sciences, uin syarif hidayatullah jakarta a silvia.nasution@gmail.com abstract south tangerang district is one of the endemic areas for filariasis; and based on an evaluation study in 2008-2009 which covered several subdistricts, the prevalence of microfilaria was between 1–2.4%. nevertheless, the evaluation by serological assay has never been reported. a cross-sectional study was conducted to detect the microfilaremia and anti-filarial igg4 antibody status in kp sawah and pamulang subdistricts. cluster sampling was performed in kp sawah by collecting finger-prick blood (fpb) and venous blood samples from inhabitants who lived with and nearby the four elephantiasis subjects in the area. the fpb were only collected in pamulang area by consecutive sampling method. the detection method included microscopic evaluation of fpb and serological detection using recombinant antigens bmr1 and bmsxp by elisa and lateral flow rapid tests. symptomatic patients who had 2nd and 3rd degree of elephantiasis were clinically determined in 10% (4/40) subjects. among those with elephantiasis, 2 were positive serologically but their microscopic results were all negative (40/40). meanwhile, the microscopic result for 107 subjects from pamulang were all negative. the results of the rapid tests showed that 15% (6/40) of the positive cases were detected by brugia rapid and 27.5% (11/40) by panlf. meanwhile, the elisa showed that 20% (8/40) of the cases were positive with bmsxp, whereas only 2.5% or 1/40 sample was found to be positive with bmr1. even though the sensitivity of the rapid test was lower when compared to microscopic examination for these samples, the assay showed good specificity ranging from 72.5 to 97.5%. the optical density (od) values of elisa has ranged between 0.3–3.045. key words: microfilaremia, bmr1, bmsxp, brugia rapid test, panlf abstrak kabupaten tangerang selatan merupakan salah satu wilayah endemik filariasis; dan berdasarkan studi evaluasi tahun 2008-2009 yang mencakup beberapa kecamatan dengan prevalensi antara 1–2.4%. namun demikian, belum ada laporan tentang hasil evaluasi secara serologi. studi potong lintang dilakukan untuk mendeteksi status mikrofilaremi dan keberadaan antibodi anti-filaria igg4 di kecamatan kp sawah dan pamulang. pengambilan sampel dilakukan secara cluster sampling dengan sampel darah jari (sdj) dan sampel darah vena dari penduduk yang tinggal di sekitar empat penderita elefantiasis di wilayah kp sawah. sedangkan di wilayah pamulang hanya dilakukan pengambilan darah jari dengan metode consecutive sampling. metode deteksi dilakukan secara mikroskopis terhadap sdj dan secara serologi dengan menggunakan rekombinan antigen bmr1 dan bmsxp dengan cara elisa dan tes cepat brugia rapid. penderita simptomatik yang terdeteksi elefantiasis berjumlah 10% (4/40) diketahui dengan status limfedema ekstremitas derajat 2 dan 3. diantara penderita elefantiasis tersebut, 2 orang terdeteksi positif secara serologis, namun hasil mikroskopisnya negatif (40/40). sementara itu, hasil mikroskopis dari 107 sdj di wilayah pamulang seluruhnya negatif. hasil tes cepat menunjukkan 15% (6/40) positif terhadap brugia rapid dan 27.5% (11/40) positif terhadap panlf. hasil elisa pada sampel penelitian ini menunjukkan research report 157nasution: comparative study of filarial detection by microscopic examination and serological assay 20% (8/40) positif terhadap bmsxp, namun hanya 2.5% (1/40) yang positif terhadap bmr1. meskipun nilai sensitifitas tes cepat lebih rendah dibandingkan mikroskopis pada sampel penelitian ini, namun nilai spesifisitasnya tinggi yang berkisar antara 72.5 to 97.5%. nilai optical density (od) dari hasil elisa berkisar antara 0.3–3.045. kata kunci: microfilaremia, bmr1, bmsxp, brugia rapid test, panlf introduction lymphatic filariasis is targeted for the global elimination program initiated by who and the program is expected to be successful by 2020. an epidemiological data maps out that until 2008, there are 316 regencies/ municipalities out of 471 regencies/municipalities in indonesia which have been declared as the endemic areas of filariasis.1 the south tangerang regency is one of endemic area for filariasis with a prevalence of microfilaria ranges between 1–2.4% covering several subdistricts as mentioned by an evaluation in 2008.2 the health department of south tangerang district in the same period found that the prevalence of filariasis in ciputat subdistrict has reached 1.6% with 8 patients has clinically suffered from lymphedema or elephantiasis in 2002; while in other subdistricts including pondok aren, setu and pamulang, the prevalence are 1.8%, 1%, and 2.4%, respectively. an area is defined to be endemic for filariasis when the microfilaria rate has 1% of prevalence.2 a previous study to evaluate microfilaremia and antigenemia status, which was conducted in kp sawah, ciputat, south tangerang district in 2012, showed that 5% subjects were positive for microfilaria and 27.5% subjects had positive results for igg4 antifilarial antibody using rapid test.2 there are some factors that may affect the success of filariasis elimination program, i.e. accurate diagnosis and evaluation on the success of continued diagnostic work-up and treatment.3 mass drug administration for filariasis in south tangerang district, has been performed annually and been evaluated microfilaremia by using finger-prick blood (fpb) since 2002. nevertheless, an evaluation by serological assay to detect antigenemia in blood vein has never been reported. the present cross-sectional study was conducted to identify the microfilaremia and antibody anti-filarial igg4 status in kp sawah (ciputat) and pamulang areas. the method included microscopic evaluation for fpb and serological detection using recombinant antigens bmr1 and bmsxp1 for blood vein samples of inhabitants living in kp sawah area. diagnostic tests were also performed to identify the sensitivity and specificity of both antigens in detecting the presence of antibody anti-filarial igg4 in the blood. material and methods a cross-sectional study was designed to conduct filariasis evaluation by observational, questionairy, and laboratory methods. diagnostic tests were performed to detect the presence of microfilaria by microscopic and igg4 antibody antifilarial by rapid test and elisa. samples were collected using cluster sampling technique in kp sawah by obtaining samples from some inhabitants who lived nearby the 4 patients who had been diagnosed with elephantiasis in the area. finger-prick blood (fpb) was also collected in west pamulang area by consecutive sampling; however, the local health department advised that the blood vein samples should not be collected at the time. samples were collected at night (10 pm – 2 am.) as the microfilaria activity in peripheral blood reaches its peak in those hours. microscopic examinations were performed at the parasitology laboratory faculty of medicine and health sciences, syarif hidayatullah state islamic university on 40 samples obtained from kp sawah and 107 samples obtained from west pamulang. the fpb were prepared into thick-blood smear slide and subsequently stained using giemsa staining (merck©) before they were examined under microscope. the volume of blood for microscopic was 1-2 drop(s) of peripheral blood. the calculation of microfilaria found in fpb was performed using the following formulation: mf density (mfd) = total number of microfilariae found in the sample × ×50* total number of slides with positive mf * 50 is the correctional factor for blood volume of 20 μl; while for different blood volume, the correctional factor is also different.4 subsequently, the vein blood was examined using recombinant antigens bmr1 and bmsxp to detect antibody anti-filarial of igg4 in blood circulation. the serological examination was performed at the laboratory of institute for moleculer medicine (informm) in university science malaysia (usm) using both rapid test and elisa. the measurement of rapid test was done using recombinant antigens (bmr1 and bmsxp) and the results were characterized by the development of 2-3 strips (bands) indicating positive results or the presence antibody antifilarial of igg4 in sample serum. the instrument used for detecting the presence of brugia sp infection is brugia rapid test; while for detecting w. bancrofti, the ict 158 indonesian journal of tropical and infectious disease, vol. 5. no. 6 september–december 2015: 156-160 comparison figure 1. method of diagnostic stages to detect lymphatic filariasis table 1. results of diagnostic test for samples from kp. sawah and pamulang index detail from kp sawah total result kp sawah result from pamulang microscopic examina tion:  microfilaremic  amicrofilaremic rapid test  positive panlf  positive brugia rapid  positive brugia rapid & panlf  negative elisa:  positive anti bmxsp1 (od: 0.3–3.045 or strong positive)  positive anti bmr1 (od: 0.645 or strong positive)  positive w. bancrofti and brugia s  negative 0 100% (40/40) 27.5% (11/40) 15% (6/40) 15% (6/40) 60% (24/40) 20% (8/40) 2.5% (1/40) 2.5% (1/40) 77.5% (31/40) rapid test:  positive: 27.5%  negative 72.5% elisa:  positive: 20%  negative: 80% amicrofilaremic: 100% (107/107) no serological test was performed bancrofti or pan lf was utilized. (reszon diagnostik international. bhd, malaysia). the measurement of igg4 antibody antifilarial level using elisa technique was also performed according to the standard procedure at the laboratory of informm-usm (penang, malaysia) as mentioned by rahmah et al (2001a). each well of the elisa plate was coated with 100 μl of recombinant antigen bmr1/bmsxp in 20 μg/ml nahco3 buffer (ph 9.6). conjugates containing monoclonal anti-human igg4hrp (horseredish peroxidase-clb netherland) were inserted in each well of the plate as much as 1: 4500 in pbs. additional substrate of abts (boehringer mannheim, germany) was given for each well after washing and the plate was subsequently covered with aluminium foil and incubated for ½ hour.5 the result of reaction was read using elisa spectrophotometer (dynatech, usa) at 410 nm wavelength. the measurement results were presented in optical density (od) with a cut-off point of 0.300.5 serum sample with od ≥ 0.300 was categorized as sample with positive igg4 and those with od < 0.300 was considered negative. result and discussion evaluation on the success of filariasis elimination program was supported by instruments of assay, which had high sensitivity and specificity in detecting the presence of specific infection of filarial species in filariasis endemic area.4 the use of recombinant antigens of bmr1 and bmsxp by utilizing rapid test and elisa had higher sensitivity and specificity compared to using microscopic examination. 159nasution: comparative study of filarial detection by microscopic examination and serological assay it can detect and differentiate filarial infection caused by wuchereria bancrofti and brugia sp, both for individuals with amicrofilaremia and those with microfilaremia. the recombinant antigen is to confirm microscopic result, which have some limitations, i.e. the sensitivity depends on blood volume and parasite periodicity; therefore, it is less sensitive, particularly for individual with symptomatic amicrofilaremia.6 several study report by using microscopic in some endemic filariasis area in south tangerang and banten province has found negative result in all slides of fpb. however, serological examination has never been reported for these areas. an evaluation on filariasis elimination program in the last 5th-annual period in 2013 at ciputat and pamulang area reveal the following results: symptomatic subjects who had 2nd and 3rd degree of elephantiasis at kp sawah in the present study were 4/40 subjects or 10% of all subjects in the study. among the respondents who had elephantiasis, there were only 2 subjects who had positive serological results for w. bancrofti filariasis, but their microscopic results were all negative; therefore, the inhabitants in the study site could be categorized into: a. asymptomatic microfilaremic patient: 0 b. asymptomatic amicrofilaremic patients: 90% (36/40) c. symptomatic microfilaremic patients: 0 d. symptomatic amicrofilaremic patients: 10% (4/40) meanwhile, the microscopic result for 107 subjects from pamulang were all negative, and there was no blood vein collection nor serological test was performed at the time. total for microscopic result of 147 samples from the two areas are negative. the results of the rapid tests showed that 15% (6/40) of the positive cases were detected by brugia rapid and 27.5% (11/40) by panlf. this is not surprising since both recombinants antigens can detect both kinds of filariasis, however bmsxp has greater diagnostic sensitive for bancroftian filariasis while bmr1 is more sensitive in detecting brugian filariasis. noordin, 2003 has reported that bmsxp antigen showed 91% sensitivity using serum of w. bancroftiinfected individuals and 39% sensitivity using serum from brugian filariasis patients. meanwhile, the elisa showed that 20% (8/40) of the cases were positive with bmsxp, whereas only 2.5% or 1/40 sample was found to be positive with bmr1. these results indicated that the study site is endemic for bancroftian filariasis and this idea is supported by the clinical manifestations. the optical density (od) values ranged between 0.3–3.045. even though the sensitivity of the elisa test was lower when compared to microscopic examination, the assay showed good specificity ranging from 72.5 to 97.5%. the serological diagnostic test can also detect and differentiate infection specifically between wuchereria bancrofti and brugia sp since there is a recombinant filarial antigen of bmr1 and bmsxp1 coated on the rapid test as well as for the elisa. the results of serological test, which were mostly positive with recombinant antigen of bmsxp, indicates that the study site was endemic for bancrofti and this idea is supported by the clinical manifestations, which revealed the presence of 2nd and 3rd degree of elephantiasis. however, positive result of brugia found by elisa in one single sample of asymptomatic subject and in 6 samples of panlf rapid test has indication of possibility for potential transmission of brugria filariasis in the area. appropriate results may become a reference point for evaluation of filariasis program, whether the program is successful or not in the endemic area. it will affect the future policy of filariasis program that should be taken into consideration by the local health department, i.e. whether they will continue the filariasis program or whether it should be stopped. the process of stopping mda for filariasis is illustrated in the figure below. conclusion serological detection using antigen bmr1 and bmsxp for inhabitants in kp sawah and pamulang area shows that infection of filaria w. bancrofti and brugia sp is remained endemic. eventhough sensitivity of the elisa test was lower when compared to microscopic examination, the assay showed good specificity ranging from 72.5 to 97.5% for the presence of w. bancrofti and brugia filaria with titer of igg4 antifilarial antibody ranging between 0.3–3.045. acknowledgement i would like to express my honor and gratitude for all the contribution and coordination to: a. research center (pusat penelitian) uin syarif hidayatullah jakarta b. prof rahmah noordin, phd. vice deputy informm– usm, penang, malaysia c. head of health department of south tangerang district and staff p2m subdit filariasis d. head of upt puskesmas kp sawah and staff e. head of district health laboratory (labkesda) south tangerang f. all participants from kp sawah, ciputat and pamulang barat for their samples contribution 160 indonesian journal of tropical and infectious disease, vol. 5. no. 6 september–december 2015: 156-160 references 1. buletin jendela epidemiologi, 2010. volume 1, juli. 2. nasution sf and ekawati e. 2013. prevalensi mikrofilaria dan respons antibodi antifilaria igg4 pada tahun keempat program pengobatan masal di wilayah endemik filariasis kp. sawah ciputat, tangerang selatan. jurnal biologi lingkungan,; vol. 6, no. 2, p. 113–119. 3. rahmah n., et al. 2004. homologs of the brugia malayi diagnostic antigen bmr1 are present in other filarial parasites but induce different humoral immuneresponses. filarial journal, 3:10. 4. who. 2005. handbook filariasis. jakarta, indonesia. 5. rahmah n., et al. 2003. multicentre laboratory evaluation of brugia rapid dipstick test for detection of brugian filariasis.tropical medicine and international health, volume 8 no 10 pp. 895–900. 6. lammie pj. et al. 2004. recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis – a multicenter trial. filaria journal, 3:9. 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. 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 80 122 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 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and malang regions of east java kurnia desiandura1, nunuk dyah retno lastuti2a, lucia tri suwanti2,4, didik handijatno3 1 faculty of veterinary medicine, universitas airlangga 2 department of parasitology veteriner, faculty of veterinary medicine, universitas airlangga 3 department of microbiology veteriner, faculty of veterinary medicine, universitas airlangga 4 institute of tropical disease, universitas airlangga a coresponding author: nunuk_dyah@yahoo.com abstract scabies is a zoonotic skin disease caused by sarcoptes scabiei mites. as an emerging/re-emerging parasitic disease, scabies represents a significant global threat to both human and animal health. numerous cases of scabies in indonesia have been reported, which support research on the prevalence of s. scabiei. however, most such studies have involved conventional morphological studies, with limited molecular diagnostic studies. the purpose of the present study was the genetic characterization of s. scabiei var. cuniculi in domestic rabbits to generate baseline genotypic data. s. scabiei var. cuniculi was isolated and identified from scabies-infected rabbits from the surabaya and malang regions of east java. molecular identification was performed using polymerase chain reaction (pcr) using specific primers targeting the cox1 gene. we performed cox1 pcr using rabbit isolates of s. scabiei from indonesia. to the best of our knowledge, no such study had been reported previously. this study was performed in the laboratory of veterinary parasitology, faculty of veterinary medicine and the tropical disease diagnostic center laboratory, universitas airlangga. the results with agarose gel electrophoresis revealed a 289 bp pcr product amplified from the dna of s. scabiei isolates from both surabaya and malang in accordance with the expected cox1 amplicon size, that indicated a single band 289 bp in length, demonstrating specific detection of s. scabiei var. cuniculi from surabaya and malang using cox1 primers. the results were consistent with the calculated amplicon size based on primer positions within the cox1 locus, with the forward primer spanning nucleotides 61–94, and the reverse primer spanning nucleotides 331–350 ( 350 − 61 = 289 bp). pcr genotyping of the isolates yielded an identical nucleotide length of 289 bp. further studies are required to sequence the amplified fragments for homology assessment. keywords: sarcoptes scabiei, scabies, pcr, 289 bp, zoonosis abstrak scabies disebabkan oleh tungau sarcoptes scabiei adalah tungau penyebab penyakit kulit yang bersifat zoonosis. scabies sebagai penyakit parasite yang muncul kembali dan mengancam kesehatan manusia maupun hewan di dunia. kasus scabies yang tinggi pada beberapa hewan domestic seperti kelinci di indonesia hanya didukung dengan hasil penelitian prevalensi s.scabiei, namun hanya bedasarkan tinjauan gejala klinis dan morphologi, sedangkan penelitian berbasis molecular seperti karakterisasi genetic dari tungau s.scabiei masih terbatas. tujuan dari penelitian ini untuk mendeteksi karakterisasi genetik tungau sarcoptes scabiei var.cuniculi dari kelinci domestik sebagai data dasar informasi genetik. penelitian ini dilakukan isolasi dan identifikasi s.scabiei var.cuniculi dari kelinci yang menunjukkan gejala scabies dari daerah surabaya dan malang, selanjutnya dilakukan ektraksi dna dari isolate s.scabiei kelinci indonesia, untuk diproses dengan polymerase chain reaction (pcr) menggunakan primer spesifik dengan target gen cox1 289 bp. studi ini dilakukan di laboratorium parasitology veteriner, fakultas kedokteran hewan dan laboratorium pusat diagnostik penyakit tropik, universitas airlangga. hasil studi genetic s.scabiei isolate lokal indonesia untuk menambah ilmu pengetahuan karena belum ada hasil studi yang dilaporkan sebelumnya. hasil electrophoresis tungau s.scabiei var.cuniculi dari kelinci asal surabaya dan malang menunjukkan panjang nukleotida sebesar 289 bp sesuai dengan target. hasilnya konsisten dengan ukuran amplikon yang 151desiandura, et al.: molecular identification of sarcoptes scabiei dihitung berdasarkan posisi primer di dalam lokus cox1, dengan primer ke depan mencakup nukleotida 61-94, dan primer terbalik mencakup nukleotida 331-350 (350 61 = 289 bp). genotipe pcr dari isolat lokal menghasilkan panjang nukleotida identik 289 bp. diperlukan penelitian lebih lanjut untuk mengurutkan fragmen yang diperkuat untuk penilaian homologi. kata kunci: sarcoptes scabiei, scabies, pcr, 289 bp, zoonosis introduction scabies or mange is an infectious zoonotic skin disease caused by the mite sarcoptes scabiei and is considered an important disease in both humans and animals. it is estimated that more than 300 million people are infected each year.1,2 the sarcoptes mite is an obligate parasite of the skin; the mites burrow into the stratum corneum complete their life cycle starting from the egg to the adult stage.2–4 s. scabiei infection may trigger multiple reactions including allergic reactions, inflammation, innate immune reactions, and activation of immune components in the skin.5 significant clinical symptoms of scabies include thickening of skin, crust formation, alopecia involving the eyes, ears, mouth, legs, and itching accompanied by the formation of red spots (rash). occasionally, 3-cm long lines or grooves are formed on the skin, which lead to crust, papule, or vesicle formation.6–8 scabies mainly occurs in areas with high poverty rates and low nutritional status and is transmitted via direct contact with infected humans or animals.9 numerous cases of scabies in indonesia have been reported, which support research on the prevalence of s. scabiei. however, most such studies have involved conventional morphological studies, with limited molecular diagnostic studies.10,11 esther et al. (2015) reported pcr based detection of genotypic differences between s. scabiei from different hosts including pigs, rabbits, foxes, jackals, and porcupines in comparison with the genbank database using three different genes, including cytochrome c oxidase 1 (cox1) with a 467 bp amplicon and glutathione-stransferase class 1 (gst1) with a 670 bp amplicon. the highest percentage identity of database-reported sequences, at 99%, was with s. scabiei from rabbits, obtained using cox1 pcr. another study reported an s. scabiei cox1 amplicon of 250 bp in an isolate from hong kong.11 the purpose of this study was the genetic characterization of s. scabiei var. cuniculi from domestic rabbits to generate baseline genotypic data. we performed cox1 pcr using rabbit isolates of s. scabiei from indonesia. to the best of our knowledge, no such study had been reported previously. material and method s. scabiei mites (adults, nymphs, and larvae) were isolated from domestic rabbits from two different regions in east java, including four rabbits from surabaya and two from malang. domestic rabbits showed clinical signs of scabies such as thickening of skin, crust formation, and alopecia involving the eyes, ears, mouth, and legs. mites were morphologically identified using identification keys.6,7 this study was performed in the laboratory of veterinary parasitology , faculty of veterinary medicine and the tropical disease diagnostic center laboratory, universitas airlangga. pcr based detection of s. scabiei in rabbits dna extraction was performed using mini kit qiaamp dna (qiagen, hilden, germany), following the manufacturer’s protocol as follows: 20 µl of qiagen protease was added to a microcentrifuge tube with a 200 µl suspension of s. scabiei; 180 µl of buffer tissue lysis (atl buffer) was then added and the tube was centrifuged at 8,000 rpm for 3 min, vortexed for 15 s, and incubated at 56°c for 24 h. further, 200 µl of buffer lysis (al buffer) was added to the tube and vortexed for 15 s; 200 µl of 96% ethanol was added to the tube, vortexed for 15 s, and centrifuged at 8,000 rpm for 1 min to pellet dna. next, 500 µl of buffer washing 1 (aw1 buffer) was added to the pellet and centrifuged at 8000 rpm for 1 min; 500 µl of aw2 buffer was then added to the pellet and the tube was centrifuged at 13,000 rpm for 3 min, at 13,000 rpm for 1 min, and 50 µl of elution buffer (ae buffer) was added to the final dna pellet; following an incubation at 15–25°c for 1 min, the contents were centrifuged at 8,000 rpm for 1 min to collect dna.12 next, pcr to amplify a 289 bp region of the cox1 gene was performed using the forward primer 5’tcttaggggctggatttagtatg-3’ and the reverse primer 5’-agttcctctaccagttccac-3’. pcr was performed in an automatic thermocycler (biorad) with an initial denaturation step at 95°c for 5 min, and 35 cycles of 94°c for 1 min, 55°c for 1 min, and 72°c for 1 min, followed by a final extension step at 72°c for 5 min.13 the pcr reaction product was then resolved using 2% agarose gel electrophoresis. result and discussion s. scabiei var. cuniculi mites were isolated from rabbits that showed clinical signs of scabies including thickening of the skin, crust formation, alopecia involving the eyes, ears, mouth, and legs. morphological appearance of mites is spherical, transparent, and oval.6,7 the dorsal surface of their body contains fine-grooved lines equipped with a plastron, conical scales, and spines. they have four pairs 152 indonesian journal of tropical and infectious disease, vol. 6 no. 6 september–december 2017: 150–153 of short legs; two pairs of legs on the anterior (notothorax) with a sucker and ambulacra, and two pairs of posterior legs (notogaster). the mouth consists of a chelicera, capitulum, and hypostome 6 (figure 1). agarose gel electrophoresis revealed a 289 bp pcr product amplified from the dna of s. scabiei isolates from both surabaya and malang in accordance with the expected cox1 amplicon size. pcr reactions from both samples showed a band between the 200 bp and 300 bp molecular weight markers. the results were consistent with the calculated amplicon size based on primer positions within the cox1 locus, with the forward primer spanning nucleotides 61–94, and the reverse primer spanning nucleotides 331–350 ( 350 − 61 = 289 bp). electrophoresis results are shown in figure 2 and figure 3. phylogenetic studies on s. scabiei from rabbits using the cox1 sequence are limited; wong et al. (2005) reported that the cox1 amplicon size using s. scabiei var. cuniculi isolated from hong kong was 250 bp, while that using s. scabiei var. cuniculi isolated from indonesia, specifically malang and surabaya, was 289 bp. zhao et al. (2015) reported that it is unclear whether the same species of sarcoptes mites parasitize humans and animals, and data on genetic diversity of these mite populations in humans is scarce.14 in a study conducted in china to identify s. hominis and s. canis, genomic dna was extracted from ten individual mites (five isolated from patients with scabies and five from dogs with mange) for amplification of its2 rdna, 16s mtdna, and cox1 fragment sequences. future studies assessing the sequence of pcr products from s. scabiei are required to determine the level of homology among s. scabiei var. cuniculi originating from different countries, which can aid the development of subunit vaccines. scabies poses a significant threat to public health and causes economic loss because of high prevalence and the lack of efficient prevention measures. dissatisfaction with use of insecticides is increasing because of the emergence of resistant parasites, environmental pollution, and consumer rejection of livestock products containing pesticide residues.15 vaccination is the best available alternative because it causes least damage to the environment and is safe for consumer use, and potentially more effective than scabicide and inexpensive; however, several obstacles hinder large scale vaccine production. therefore, several studies have been aimed at developing molecular subunit vaccines using recombinant dna cloned from s. scabiei.16–19 8000 rpm for 1 min to pellet dna. next, 500 µl of buffer washing 1 (aw1 buffer) was added to the pellet and centrifuged at 8000 rpm for 1 min; 500 µl of aw2 buffer was then added to the pellet and the tube was centrifuged at 13000 rpm for 3 min, at 13000 rpm for 1 min, and 50 µl of elution buffer (ae buffer) was added to the final dna pellet; following an incubation at 15–25 °c for 1 min, the contents were centrifuged at 8000 rpm for 1 min to collect dna.12 next, pcr to amplify a 289 bp region of the cox1 gene was performed using the forward primer 5'tcttaggggctggatttagtatg-3' and the reverse primer 5'agttcctctaccagttccac-3'. pcr was performed in an automatic thermocycler (biorad) with an initial denaturation step at 95 °c for 5 min, and 35 cycles of 94 °c for 1 min, 55 °c for 1 min, and 72 °c for 1 min, followed by a final extension step at 72 °c for 5 min.13 the pcr reaction product was then resolved using 2% agarose gel electrophoresis. result and discussion s. scabiei var. cuniculi mites were isolated from rabbits that showed clinical signs of scabies including thickening of the skin, crust formation, alopecia involving the eyes, ears, mouth, and legs. morphological appearance of mites is spherical, transparent, and oval.6,7 the dorsal surface of their body contains fine-grooved lines equipped with a plastron, conical scales, and spines. they have four pairs of short legs; two pairs of legs on the anterior (notothorax) with a sucker and ambulacra, and two pairs of posterior legs (notogaster). the mouth consists of a chelicera, capitulum, and hypostome 6 (figure 1). figure 1. sarcoptes scabiei ventral view (olympus optilab camera microscope, magnification: 400×) agarose gel electrophoresis revealed a 289 bp pcr product amplified from the dna of s. scabiei isolates from both surabaya and malang in accordance with the expected cox1 amplicon size. pcr reactions from both samples showed a band between the 200 bp and 300 bp molecular weight markers. the results were consistent with the calculated amplicon size based on primer positions within the cox1 locus, with the forward primer spanning nucleotides 61–94, and the reverse primer spanning nucleotides 331–350 ( 350 − 61 = 289 bp). electrophoresis results are shown in figure 2 and figure 3. hypostome legs bristle figure 1. sarcoptes scabiei ventral view (olympus optilab camera microscope, magnification: 400×) figure 2. agarose gel electrophoresis of the pcr product from s. scabiei var. cuniculi isolated from surabaya figure 3. agarose gel electrophoresis of the pcr product from s. scabiei var. cuniculi isolated from malang phylogenetic studies on s. scabiei from rabbits using the cox1 sequence are limited; wong et al. (2005) reported that the cox1 amplicon size using s. scabiei var. cuniculi isolated from hong kong was 250 bp, while that using s. scabiei var. cuniculi isolated from indonesia, specifically malang and surabaya, was 289 bp. zhao et al. (2015) reported that it is unclear whether the same species of sarcoptes mites parasitize humans and animals, and data on genetic diversity of these mite populations in humans is scarce.14 in a study conducted in china to identify s. hominis and s. canis, genomic dna was extracted from ten individual mites (five isolated from patients with scabies and five from dogs with mange) for amplification of its2 rdna, 16s mtdna, and 300bp 200bp 100bp 289bp m s 300bp 200bp 100bp 289bp s information: m = marker s = sample information: m = marker s = sample figure 2. agarose gel electrophoresis of the pcr product from s. scabiei var. cuniculi isolated from surabaya figure 2. agarose gel electrophoresis of the pcr product from s. scabiei var. cuniculi isolated from surabaya figure 3. agarose gel electrophoresis of the pcr product from s. scabiei var. cuniculi isolated from malang phylogenetic studies on s. scabiei from rabbits using the cox1 sequence are limited; wong et al. (2005) reported that the cox1 amplicon size using s. scabiei var. cuniculi isolated from hong kong was 250 bp, while that using s. scabiei var. cuniculi isolated from indonesia, specifically malang and surabaya, was 289 bp. zhao et al. (2015) reported that it is unclear whether the same species of sarcoptes mites parasitize humans and animals, and data on genetic diversity of these mite populations in humans is scarce.14 in a study conducted in china to identify s. hominis and s. canis, genomic dna was extracted from ten individual mites (five isolated from patients with scabies and five from dogs with mange) for amplification of its2 rdna, 16s mtdna, and 300bp 200bp 100bp 289bp m s 300bp 200bp 100bp 289bp s information: m = marker s = sample figure 3. agarose gel electrophoresis of the pcr product from s. scabiei var. cuniculi isolated from malang 153desiandura, et al.: molecular identification of sarcoptes scabiei conclusion sarcoptes scabiei isolated from rabbits that were infected with scabies was detected using cox1 pcr with the forward primer 5’-tcttaggggctggtattagtatg-3’ and the reverse primer 5’-agttcctctaccagttccac-3’, yielding the expected amplicon size of 289 bp. references 1. arlian lg, vyszenski-moher dl, pole mj. survival of adults and developmental stages ofsarcoptes scabiei var.canis when off the host. exp appl acarol. 1989 apr;6(3):181–7. 2. morgan ms, rider sd, arlian lg. identification of antigenic sarcoptes scabiei proteins for use in a diagnostic test and of nonantigenic proteins that may be immunomodulatory. vinetz jm, editor. plos negl trop dis. 2017 jun 12;11(6):e0005669. 3. walton sf, currie bj. problems in diagnosing scabies, a global disease in human and animal populations. clin microbiol rev. 2007 apr 1;20(2):268–79. 4. lastuti nd. specific antigenic protein 57.3 kda of sarcoptes scabiei var. caprae as material candidate of scabies diagnostic kit for goat and toll-like receptor mediated immune responses. universitas airlangga; 5. erster o, roth a, pozzi ps, bouznach a, shkap v. first detection of sarcoptes scabiei from domesticated pig (sus scrofa) and genetic characterization of s. scabiei from pet, farm and wild hosts in israel. exp appl acarol. 2015 aug 23;66(4):605–12. 6. soulsby ejl. helminths, arthropods and protozoa of domesticated animals. 6th ed. london: e.l.b.s.; 1978. 7. lastuti nd. exploration of whole proteins sarcoptes scabiei var. cuniculi cause of rabbit’s scabies. media kedokteran hewan. 2008;80–5. 8. veteriner bp. masalah skabies pada hewan dan manusia serta penanggulangannya. 1998;28–34. 9. wardhana ah, manurung j. skabies : tantangan penyakit zoonosis masa kini . dan masa datang. ivartazoa. 2002;vol. 16 no(30): 40–52. 10. choy jl, currie b, arlian l, mcbroom j, kemp dj, bonson a, et al. genetically distinct dog-derived and human-derived sarcoptes scabiei in scabies-endemic communities in northern australia. am j trop med hyg. 1999 oct 1;61(4):542–7. 11. wong ssy, poon rws, chau s, wong scy, to kkw, cheng vcc, et al. development of conventional and real-time quantitative pcr assays for diagnosis and monitoring of scabies. gilligan ph, editor. j clin microbiol. 2015 jul;53(7):2095–102. 12. reed r, holmes d, weyers j, jones a. practical skills in biomolecular sciences (2nd edition). second. prentice hall; 2003. 237-319 p. 13. rantam fa. methods of immunology. surabaya: airlangga university press; 2003. 149-155 p. 14. andriantsoanirina v, ariey f, izri a, bernigaud c, fang f, charrel r, et al. sarcoptes scabiei mites in humans are distributed into three genetically distinct clades. clin microbiol infect. 2015 dec;21(12):1107–14. 15. tarigan s. vaksin skabies dibutuhkan namun sulit diwujudkan. wartazoa. 2007;17(30):38–45. 16. rider sd, morgan ms, arlian lg. draft genome of the scabies mite. parasit vectors. 2015 dec 10;8(1):585. 17. arlian lg, morgan ms, rider sd. sarcoptes scabiei: genomics to proteomics to biology. parasit vectors. 2016 dec 1;9(1):380. 18. zhang r, jise q, zheng w, ren y, nong x, wu x, et al. characterization and evaluation of a sarcoptes scabiei allergen as a candidate vaccine. parasites and vectors. 2012;5(1):1–9. 19. casais r, granda v, balseiro a, del cerro a, dalton kp, gonzález r, et al. vaccination of rabbits with immunodominant antigens from sarcoptes scabiei induced high levels of humoral responses and pro-inflammatory cytokines but confers limited protection. parasit vectors. 2016 dec 8;9(1):435. 63 vol. 7 no. 4 january-april 2019 research report l e p r o s y a n d h u m a n i m m u n o d e f i c i e n c y v i r u s coinfection: a rare case eva lydiawati1, chukmol sirithida2, sou vannda2, hak vortey2, heng ratana2, m. yulianto listiawan1, indropo agusni1, evy ervianti1 1 department of dermatology and venereology, faculty of medicine, universitas airlangga/dr. soetomo general hospital surabaya, indonesia 2 department of dermatology and venereology, faculty of medicine, university of health sciences, phnom penh, cambodia a corresponding author: evalydiawati@gmail.com abstract leprosy, or morbus hansen, is a chronic infectious disease which caused by mycobacterium leprae. it is associated with inflammation that may damage the skin and peripheral nerves. leprosy remains an important public health problem in southeast asia, america, and africa. it has been speculated that, as with tuberculosis, human immunodeficiency virus (hiv) infection may exacerbate leprosy lesions and/or lead to increase susceptibility to leprosy. we are reported the case of leprosy and hiv coinfection and reveals its clinical manifestation. a 34-year-old female came to outpatient clinic complaining of redness plaque on her face of 2-months duration. it was also accompanied with thick sensation without itchy or burning sensation. we found thick erythematous plaque with sharp margin and hypoesthesia on her face and body. there were no madarosis, saddle nose, lagophthalmos and sign of neuritis. the slit-skin smear revealed bi 1+ globi and mi 2%. from laboratory examination we found igm anti pgl-1 titer was 1265 u/ml and igg anti pgl-1 was 834 u/ml. the similar lesion of leprosy was found on her both of ear lobe and legs by using histological examination. the detection of hiv antibody was positive with cd4 count on 325 cells/μl. we treat her with multidrug treatment (mdt) for multibacillary leprosy along with anti-retroviral therapy or art consist of tenofovir, lamivudine, and efavirenz. after 6-months follow-up we are observed no progression of the lesions though the slit-skin smear become negative. m. leprae does not seem to accelerate the decline of immune function when associated with hiv infection. hiv infection does not seem to affect the clinical classification and progression of leprosy. the treatment of the hiv-leprosy coinfected patient consists of the combination of arts and anti-leprosy agents. those treatment gives the good result in the bacteriological state of the patient. keywords: leprosy, hansen disease, hiv co-infection, leprosy-hiv, mh. abstrak kusta, atau morbus hansen, merupakan penyakit inflamasi kronik yang disebabkan oleh mycobacterium leprae, terkait dengan inflamasi yang merusak kulit dan saraf perifer. kusta tetap menjadi masalah kesehatan masyarakat di asia tenggara, amerika, dan afrika. koinfeksi dengan hiv memiliki pengaruh besar terhadap perkembangan penyakit, terutama penyakit mikobakterial. telah diduga sebelumnya, seperti halnya tuberkulosis, infeksi human immunodeficiency virus (hiv) menyebabkan eksaserbasi lesi kusta dan/atau meningkatkan kerentanan terhadap kusta. kami melaporkan kasus koinfeksi kusta dan hiv dan menunjukkan manifestasi klinisnya. seorang wanita 34 tahun datang ke klinik rawat jalan mengeluhkan bercak kemerahan sejak 2 bulan. hal ini disertai pula dengan sensasi tebal tetapi tanpa gatal atau rasa terbakar. tidak disertai demam atau benjolan. dari pemeriksaan ditemukan plak eritematosa berbatas jelas dan hipoestesia pada wajah dan badan. tidak ditemukan madarosis, hidung pelana, lagoftalmos, atau tanda neuritis. pemeriksaan basil tahan asam menunjukkan bi 1+ globi dan mi 2%. pemeriksaan laboratorium darah menunjukkan dalam batas normal, titer ig m anti pgl-1 1265 u/ml dan igg anti pgl-1 834 u/ml. kedua pemeriksaan histologis dari cuping telinga dan tungkai menunjukkan gambaran menyerupai kusta. deteksi antibody hiv positif dengan hitung cd4 325 cells/μl. pasien diterapi dengan multidrug treatment (mdt) untuk leprosy tipe multibasiler dan anti-retroviral therapy atau art yang terdiri dari tenofovir, lamivudine, and efavirenz. setelah 6 bulan terapi dapat kami amati bahwa tidak ditemukan perkembangan bermakna dari lesi meskipun pemeriksaan basil tahan asam menjadi negatif. kasus ini merupakan koinfeksi kusta-hiv. m. lepra tampaknya tidak 64 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 63–68 mempercepat penurunan fungsi imun terkait infeksi hiv. infeksi hiv juga tidak mempengaruhi klasifikasi klinis dan perkembangan kusta. terapi pasien koinfeksi kusta-hiv terdiri dari kombinasi art dan agen anti-kusta mdt. terapi tersebut memberikan perbaikan hasil bakteriologis yang cukup baik untuk pasien. kata kunci: kusta, penyakit hansen, koinfeksi hiv, kusta-hiv, mdt introduction leprosy, or hansen disease (hd), is a chronic infectious disease which caused by mycobacterium leprae which is associated with inflammation that may damage the skin and peripheral nerves.1 despite the claim by the world health organization (who) that it would no longer be a public health problem after the year 2000, leprosy is far from being eliminated, with more than 200,000 new cases being reported yearly during the past 5 years. leprosy remains an important public health problem in southeast asia, america and africa.2,3 human immunodeficiency virus (hiv) infection prevalence rates are high in many countries where leprosy is still endemic.2,4,5 in 2008, 121 countries were reported a total of 249,007 new leprosy cases to who. most endemic countries for leprosy also have a high hiv prevalence, increasing the possibility of hiv-leprosy coinfection. although the number of coinfected patients has not been estimated yet, the increasing geographic overlap of these two diseases will result in increasing number of person being dually infected.6 meanwhile, there are few number of case reports of leprosy that have association with hiv infection.4,6 a few studies have tried to evaluate reasons for this rare coexistence. tissue cell-mediated immune response against m. leprae is known to be preserved even though the peripheral blood lymphocyte count was reduced in concurrent leprosy and hiv-infected patients.6 thus probably, there are less reports of leprosy in association with hiv. the present case of leprosy in an hiv-infected person is herewith reported for its rarity. this case report is aimed to describe the different manifestation of leprosy and hiv coinfection. the understanding about the existence of coinfection should be remember and it bring also the obligation to follow standardized guideline treatment. case a 34-year-old female came to the dermatology outpatient clinic of dr. soetomo general hospital surabaya on december 13th 2017. she came with chief complaint of redness plaque on her face. she is complained about it since about 2 months before admission. firstly, she got this lesion on her face with small size, by the time then this lesion became larger. this symptom was also accompanied with thick sensation over the red area but without itchy or burning sensation. she had no fever before. she went to several general practitioners and was diagnosed with atopic dermatitis. she got some medications but there were no significant differences before and after taking those treatments. after several weeks back then, there were some erythematous and blackish macule that was spread on her extremities. because of feeling afraid of this condition, she sought any help to dermatology and venereology outpatient clinic of dr. soetomo general hospital and was diagnosed as leprosy. about one month after she got treatment from there, she had a chronic diarrhea and had a low fluid intake until she became severe dehydration. because of this condition she was hospitalized in other hospital and did some general examination which is one of those examination panel was hiv rapid testing. those laboratory data revealed that she got hiv infection. she was started on antiretroviral therapy (art) one month later. the patient was married with a man since about 10 years ago. she is refused to have a sexual intercourse before she was married. she is claimed that her husband was the one and only sexual partner of her. her husband was a worker on the building construction project. the history of sexual activity of her husband was unknown. the patient is denied of the same disease before and her husband was not having the same symptoms. there was no history of consuming drugs before the lesions appeared, drug hypersensitivity, blood transfusion, injection drug user, or drug abuser. history of fever, headache, malaise, and weight loss were denied. the physical examination of general state was all within normal limit. blood pressure was 100/70 mmhg, pulse rate was 84 times per minute, respiratory rate was 18 times per minute and body temperature was 36,40 c. from head and neck, there were no signs of anemia, cyanosis, icterus, or dyspneu. from thorax examination, heart and lungs were normal. from abdomen, liver and spleen were not palpable. from her upper and lower extremities there were no edema and warm on palpation. there was no enlargement of the cervical, axillar, inguinal and genital lymph nodes. dermatological examination on her right face especially on the periorbital region discovered the thick erythematous plaques with sharp margin, some are covered with white fine scales and hypoaesthetic (fig. 1 a). no madarosis of the eyebrows or eyelashes was observed. there were no saddle nose or diffuse infiltrate on the face, and lagophthalmos. there was also multiple erythematous macule that sharply marginated accompanied with erythematous papules that varied in size about 0,5-1 cm on her trunk, upper, and lower extremities (fig. 1 b-e). there was no thickened peripheral nerves on the left and right ulnar nerves and did not accompanied with tenderness on palpation. in addition, peripheral neurological symptoms, including motoric, sensory and autonomic nerve disturbance were not detected based on a neurological assessment that included light 65lydiawati, et al.: leprosy and human immunodeficiency figure 1. the clinical manifestation of the patient on the first examination on right facial region, there was erythematous plaque with sharp margin (picture a); the other manifestation of the patient on the trunk and extremities region, there were multiple erythematous and hyperpigmented macules that varied in size (picture b-e) touch, pin–prick test, thermal sensory test, manual muscle strength test and monofilament test. we found that acid-fast bacilli was detected by the slit – skin smear test of the ear lobes and lesion (bacterial index: 1+ globi; morphological index: 2%). the laboratory examination on december 13th 2017 was revealed: haemoglobin was 14,0 g/dl, white blood count 8.090/mm3, thrombocyte 302.000/l, and hematocrite 41,2%. detection of hiv antibody (3 methods) on january 2018 was positive with cd4 count on 525 cells/μl. serologic test by detecting antiphenolic glycolipid i (anti pgl-1) antibody was positive by the score of igm = 1265 (cutt off = 605 u/ml) and igg = 834 (cutt off = 630 u/ml). histological examination of the ear lobe skin was revealed atrophy and short-flattening of rete ridge on the upper epidermis, there were some group of hystiocyte or foam cell on superficial to deep dermis. no specific microorganisms were identified by fite – faraco staining. the conclusion of that biopsy was borderline leprosy. the picture of this examination can be clearly seen on the figure 2a. because of our suspicion on several diagnosis of the lesions on her trunk and extremities, we did the biopsy examination on that location too. the skin biopsy on extremity was revealed atropy and short-flatening of rete ridges on epidermis, some epitheloid cells which form granuloma, some lymphocyte and eosinophil infiltration on the dermis. there was no bacteria were observed on fite– faraco staining. the conclusion was similar to the lesion of borderline tuberculoid leprosy. in those two examination we did not see any differences in the manifestation of the disease according to the histologic examination. the picture of this examination can also be clearly seen on the figure 2b. figure 2. the histologic examination of ear lobe (picture a) revealed atrophy of epidermis with short-flattening of rete ridges, we found group of histiocyte or foam cell on superficial to deep dermis and datia langhans cell, there were no bacteria observed and the conclusion was borderline leprosy; the other histologic examination on the extremity (picture b) revealed a slight different that we found epithelioid cells that form granuloma and some lymphocyte and eosinophil infiltration, we conclude the result as borderline tuberculoid leprosy 66 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 63–68 figure 3. the skin lesion on her face at the first come (picture a), 3rd month of mdt (picture b); and after 6th month of mdt (picture c). we found no progression of the lesions. based on these findings, from physical and laboratory examination, the diagnosis of multibacillary, borderline lepromatous (bl) leprosy with hiv coinfection was established. there was no sign of the leprosy reaction at this time. the patient were observed for the period of time to observe the amendment of her condition. according to the world health organization (who) classification, she was classified as having multibacillary leprosy and got multidrug treatment of leprosy (mdt). those regiment consisted of rifampicin 600 mg monthly, clofazimine 300 mg once a month and 50 mg daily, and dapsone 100 mg daily for 12 months which is the who recommended for multibacillary leprosy. she was also initiated on first-line antiretroviral therapy (art) regimen including tenofovir, lamivudine, and efavirenz. six months since initiating mdt for leprosy, the patient remained stable without new lesions or neurological deficits. however, there were no progression of the lesions even though she has been treated for 6 months. the progression of the disease was clearly described in table 1 and the lesions can be seen on figure 3 to 5 discussion leprosy is one of a deliberately progresssive infectious disease caused by mycobacterium leprae. it is a disease which primarily affects the skin and peripheral nerve, and in highly bacillated state, any internal organ except central nervous system can be affected too. the damage to peripheral nerves results in sensory and motor impairment which characterized by dreadful abnormalities and debilities.7-9 talhari et al. were proposed the classification for leprosy associated with hiv infection. this classification recognizes true leprosy-hiv coinfection, opportunistic leprosy disease, and leprosy related to art.8 recently, it was suggested that even though leprosy–hiv coinfection does not manifest homogenously across affected populations, immunological features seem to be shared by certain subgroups. in this context, a clinical classification of m. leprae and hiv/ figure 5. the lesions on her back were the same between her first come (picture a) and after 6th month of mdt (picture b) table 1. progression of the disease figure 4. the skin lesion on her forearms at the first come (picture a), 3rd month of mdt (picture b); and after 6th month of mdt (picture c). we still found no progression of the lesions. aids-coinfected patients including in the following criterias.2 the first criteria are m. leprae–hiv true coinfection. this group consists of hiv positive individuals who do not fulfill aids criteria and are not under haart. the patients have similarity to immunocompetent subjects.2 the next criteria are opportunistic leprosy disease. this criteria consist of aids patients who do not 67lydiawati, et al.: leprosy and human immunodeficiency receive haart, presenting usually with multibacillary leprosy. this group would include individuals manifesting leprosy as an opportunistic mycobacteriosis, as expected in immunosuppressed individuals.2 the last criteria are haart-related leprosy. this criteria include aids patients presenting all clinical forms of leprosy related or not to iris. combined haart and mdt may cause upgrading shift within the leprosy clinical spectrum, as may be revealed by long-term follow-up.2 according to those criterias, we could define the leprosy and hiv in this case as m. leprae–hiv true coinfection. this case illustrates clinical manifestations of leprosy that was not worsen by hiv infection, although it slowly progressed during the follow-up. it is a well-known fact that in tuberculosis (tb) and hiv coinfected patients, tb and hiv infection itself contributes to the progression of each other.10-12 active tb infection in hiv-infected patients is associated with increased immunodeficiency and mortality in those patients.13-15 it has been hypothesized that hiv infection may exacerbate leprosy lesions and/or lead to increased susceptibility to leprosy. this condition was thought to be like in tb and hiv infection. however, there is less evidence to support this hypothesis. in the contrary, there were several studies that have found that in leprosy and hiv coinfected patients, each disease progresses independently.6,16 a few studies were performed to evaluate the reasons for this rare co-existence. tissue cell-mediated immune response against m. leprae is known to be preserved even though the peripheral blood lymphocyte count was reduced in concurrent leprosy and hiv-infected patients.6 the deficiency in cell-mediated immunity (cmi) is specific to the m. leprae antigens and has nothing to do with the decreased peripheral cd4 count of hiv.17 thus probably, there are less reports of leprosy in association with hiv. mycobacterium leprae does not seem to accelerate the decline of immune function when associated with hiv infection. this condition was different with the fact which often happens in tuberculosis coinfection.18,19 reactional states may occur more frequently in individuals with hiv coinfection. however, there are still many conflicting data regarding increased reaction frequency in this group.16 as noted in this patient, hiv infection did not seem to affect the clinical classification and progression of leprosy. as we found in a study by pereira et al. that the clinical, immunologic, histopathology, and virology features among 22 hiv-leprosy coinfected brazilian patients indicate that each disease is progressed as in single infection.20 despite overall hiv-associated immunosuppression, cell-mediated immune responses to m. leprae are well preserved at the site of the disease.20,21 based on our experience as we found in our patient, the disease was progressed slowly, and the lesions did not alter morphologically over a period of 6 months follow up. this suggests that the pathogenesis of leprosy in this patient was unaffected by her immunodeficiency. this finding was similar to the result of the study that was mentioned above.20 initiation of haart has been associated with immune reconstitution and inflammatory syndrome (iris) in various situations. iris in leprosy may trigger potential adverse effects, such as leprosy acute inflammatory episodes.7,10,22 this usually leads to a worsening of the initial lesion characterized by erythema and tenderness in the setting of rising cd4 count and falling viral load. these reactions are more common in patients with low cd4 counts especially during the initial 3 months of initiation of art. typically, as the immune system further recovers, the lesions become tuberculoid or paucibacillary as opposed to lepromatous. in our patient, there was no change in the appearance of the skin lesions after starting haart with no evident virological suppression and immune reconstitution with the latter. more so, there were no neurological deficits noted even after 6-months therapy of mdt and haart. the follow-up of this case after 6 months of mdt for leprosy combined with haart was revealed negativity of skin slit smear. although it has no significant different in the clinical manifestation, but the progression in skin slit smear indicates the cure of leprosy in this patient. moreover, we still continue the mdt regiment for 12 months for multibacillary leprosy based on the who’s recommended treatment regimens for multibacillary leprosy. the therapy for leprosy with hiv coinfection is still the same with leprosy without coinfection. hiv infection might affect the efficacy of multidrug therapy for leprosy. the hiv positive patients are potentially taking longer to be treated or experiencing a higher relapse rate of leprosy. but some published data were suggested that leprosy-hiv coinfected patients respond equally well to multidrug therapy without the need for prolonged treatment.16 relapses are rare after multidrug therapy. it counts about 1 per 1000 person-years for tuberculoid patients and 0-20.4 per 1000 person-years for multibacillary patients.16,23 conclusion based on the available data, we can conclude that leprosy and hiv coinfection has three different criteria. one of them is the true coinfection, such in this case, is the diseases that progress independently. in general, the therapy for this patient is the same as the disease was separately. those treatment includes standard who-mdt in conjunction with haart according to the patient’s clinical state. the influence of hiv infection on cell-mediated immune responses to m. leprae in the hivinfected patient needs more exploration. leprosy and hiv coinfection is an evolving situation with ongoing discoveries and further research needs. acknowledgement the authors would like to express their genuine thanks to the dermatovenereology ward and outpatient’s clinic 68 indonesian journal of tropical and infectious disease, vol. 7 no. 4 january–april 2019: 63–68 of dr. soetomo general hospital surabaya and patient who participated in this study. references 1. region ea, region m. global leprosy situation. week epid rec 2012; (august 2012): 2011–2015. 2. massone c, talhari c, ribeiro-rodrigues r, sindeaux rhm, mira mt, talhari s, et al. leprosy and hiv coinfection: a critical approach. expert rev anti infect ther 2011; 9(6): 701–10. 3. global leprosy situation. wkly epidemiol 2010; rec. 85(35): 337 48. 4. naafs b. leprosy and hiv: an analysis. hansen int 2000; 25(1): 63–6. 5. naafs b. some observations from the past year. hansen int 2004; 29: 51–6. 6. lockwood dnj, lambert sm. human immunodeficiency virus and leprosy: an update. dermatol clin 2011; 29(1): 125–128. 7. kwobah cm, wools-kaloustian kk, gitau jn, siika am. human immunodeficiency virus and leprosy coinfection: challenges in resource-limited setups. case rep med 2012; 2012: 2–3. 8. talhari c, mira mt, massone c, braga a, chrusciak‐talhari a, santos, m, et al. leprosy and hiv coinfection: a clinical, pathological, immunological, and therapeutic study of a cohort from a brazilian referral center for infectious diseases. j infect dis 2010; 202(3): 345–354. 9. kumar b, kar hk. ial textbook of leprosy, the health sciences publisher. london, 2nd edition. 2017. p 343–47. 10. pires caa, de miranda mfr, bittencourt m, de js, de brito ac, xavier mb. comparison between histopathologic features of leprosy in reaction lesions in hiv coinfected and non-coinfected patients. ana bras dermatol 2015; 90(1): 27–34. 11. rahman s, gudetta b, fink j. compartmentalization of immune responses in human tuberculosis: few cd8+ effector t cells but elevated levels of foxp2+ regulatory t cells in the granulomatous lesions. am j pathol 2009; 174: 2211–24. 12. ribeiro-rodriguez r, hirsch cs, boom wh. a role for cd4+ cd25+ t cells in regulation of the immune response during human tuberculosis. clin exp emmunol 2006; 144: 25–34. 13. toossi z, mayanja-kizza h, hirsch cs. impact of tuberculosis (tb) on hiv-1 activity in dually infected patients. am j clin exp immunol 2001; vol. 123, no. 2: 233–8. 14. falvo jv, ranjbar s, jasenosky ld, goldfeld ae. arc of a vicious circle: pathways activated by mycobacterium tuberculosis that target the hiv-1 long terminal. am j respir cell mol biol 2011; vol. 45, no. 6: 1116–24. 15. cdc. hiv testing and treatment among tuberculosis patients—kenya 2006–2009. morb mortal wkly rep (mmwr) 2010; vol. 59, no. 46: 1514–7. 16. ustianowski ap, lawn sd, lockwood dn. interactions between hiv infection and leprosy: a paradox. lancet infect dis 2006; 6(6): 350–60. 17. gupta tsc, sinha pk, murthy vs, kumari gs. leprosy in an hiv-infected person. indian j sex transm dis 2007; 28 (2): 100–2. 18. whalen c, horsburgh cr, hom d, lahart c, simberkoff m, ellner j. accelerated course of human immunodeficiency virus infection after tuberculosis. am j respir crit case med 1995; 151: 129–35. 19. whalen cc, nsubuga p, okwera a, johnson jl, hom dl, michael nl, et al. impact of pulmonary tuberculosis on survival of hivinfected adults: a prospective epidemiologic study in uganda. aids 2000; 14: 1219–28. 20. pereira gas, stefani mma, araujofilho ja, souza lcs, stefani gp, martelli smt. human immunodeficiency virus type 1 (hiv-1) and mycobacterium leprae coinfection: hiv-1 subtypes and clinical, immunologic, and histopathologic profiles in a brazilian cohort. am j trop med hyg 2004; 71(5): 679–84. 21. deps p, lucas s, porro am, maeda sm, tomimori j, guidella c, et al. clinical and histological features of leprosy and human immunodeficiency virus coinfection in brazil. clin exp dermatol 2013; 38: 470–7. 22. sarno en, illarramendi x, nery ja, sales am, gutierrez-galhardo mc, penna ml, et al. hiv-m. leprae interaction: can haart modify the course of leprosy? public health rep 2008; 123: 206–12. 23. britton wj, lockwood dnj. leprosy. lancet 2004; 363: 1209–19. 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. 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. �� 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. 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 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 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. 82 vol. 1. no. 2 may–august 2010 identification of streptomyces sp-mws1 producing antibacterial compounds wiwin retnowati department of medical microbiology, airlangga university school of medicine surabaya, indonesia institute of tropical disease airlangga university abstract an actinomycete, designated streptomyces sp-mws1, was isolated from mangrove ecosystem soil in the eastern coast of surabaya. this organism was capable of producing a series of antibiotics that strongly inhibit the growth of gram-positive and gram-negative bacteria. furthermore, culture morphological and physiological characteristics of the isolated strain, streptomyces sp-mws1 were compared to other reference strains belong to streptomyces species. the analysis of nucleotide sequence of the 16s rdna indicated similarity binary 98% with streptomyces species. key words: streptomyces, mangrove, antibiotic, 16s rdna introduction the”strike back” of pathogens has revitalized the research for new drugs (lemonick, 1994; jaroff, 1994). novel antibiotics are required to counter drug-resistant bacteria, fungi, and viruses. only about 10% of the estimated total number of microbial species are knownthere is an extensive and diverse resource that can be tapped for useful products, such as antibiotics, and processes, such as novel mechanisms of action (bull et al., 1992). in this respect, natural antibiotics (particularly those from the genus actinomycetes, the most abundant microbial source of antimicrobial compounds (miyadoh, 1993) are as important as those, which are derived from chemical modification of existing antibiotics. in our screening program for bioactive compounds, an actinomycete (which we designated streptomyces spmws1) was isolated from mangrove ecosystem soil in the eastern coast of surabaya. this actinomycete is capable of producing antibiotics that strongly inhibit the growth of gram-positive and gram-negative bacteria. we present the identification of streptomyces sp-mws1 through a study of its biological properties. material and methods microorganisms and culture conditions streptomyces sp-mws1 was isolated from mangrove ecosystem soil in the eastern coast of surabaya. isolation and enumeration of actinomycetes colonies performed by soil dilution plate technique using isp-4 agar medium. one gram of soil was suspended into test tube containing 9 ml steril phosphat buffer ph 7.3 solution and heated at 50° c for 10 min. different dilutions, 10–3, 10–5, 10–7 of the suspension were plated onto agar medium. the plates were incubated for 7 to 10 days at 28° c. selected colonies were transfered from mixed culture of the plates onto respective agar plates and incubated onto at 28° c for other 7 days. after incubation, typically pigmented, dry, powdery colonies were selected from mixed plate culture and maintained on fresh medium to get pure cultures (shirling and gottlieb, 1966). plates containing pure cultures were stored at 4° c until further examination. the letter in an isolates name designate what location is came from. cultural and morphological characteri�ation cultural characteristics of streptomyces sp-mws1 were determined according to the international streptomyces research report 83retnowati: identification of streptomyces sp-mwsi project (isp) (shirling and gottlieb, 1966). the general criteria used for streptomyces spp. identifications are morphology, production of diffusible pigments, utilization of various carbon sources and antimicrobial activity (arment et al., 2004; simon et al., 1999). strain was maintained as spore suspensions in 20% glycerol at –80° c and/or as agar plugs cut from actively growing plates and stored at –80° c. morphology was examined by light microscope (model se; nicon) by using the methode of shirling and gottlieb (1966). active purified isolate of actinomycetes were identified up to the species level by comparing their morphology of spore bearing hyphae with entire spore chain and structure of spore chain with the actinomycetes morphologics, as described in bergey’s manual. this was done by using cover-slip method cross (1989) in which individual cultures were transferred to the base of cover slips burried in isp-4 medium. carbon utilization was determined on plates containing nutrien agar medium to which separately-sterilized carbon sources were added to a final concentration 1%. the plates were incubated at 28° c and growth was noticed after 7 days. carbohydrate utili�ation utilization of carbohydrates was investigated with a basal carbon nutrient medium (pridham and gottlieb, 1948, waksman, 1967). methods and media used for physiological tests were as described by waksman (1967) luedemann and brodsky (1964). the cutural broth was tested for its antimicrobial activity using the cup or the paper disc diffusion methods (wu, 1984). total dna isolation streptomyces sp-mws1 was inoculated in 25 ml of the isp broth medium and incubated at 28° c with agitation speed 200 rpm overnight. after that genomic dna of the strain was isolated as described by pospiech and newmann (1995). the collected pellets were left to dry and dissolved in a suitable volume (100 ml) of te buffer (100 mm nacl, 1 mm edta, 100 mm tris-hcl, ph 8) or deionized water and storage at –20° c. 16s rdna sequencing the 16s rdna analysis for this isolate was done by extracting dna using a qiagen dneasy plant mini kit according to the standard protocols, and made ready for dna amplification. the 16s rdna gene was then amplified by pcr using the lyticase enzyme and the following pairs of primers : 9f (forwards: 5’-gagtttgatcctggc cag3’) and 1541 r (reverse:5’-aaggaggtgatccagcc3’) (zhang, et al., 2003). the pcr reaction mixture (50 ml) contained pcr beads 2 ml from each primer 9f and 1541 r and 10 µl template dna up to finale volume 50 ml reached by distilled water. amplification was performed with an initial denaturation step of 3 min at 94° c and then 35 cycles of (60 sec denaturation at 95° c, 60 sec at 60° c for pimer annealing and 60 sec at 72° c for primer extension) and kept at 72° c for 10 min to complete extension. electrophoresis of the pcr products was carried out on 1% agarose gel containing ethidium bromide (0.5 mg/ml–1), to ensure that fragment of the the correct size had been amplified and detected by gel documentation system. agarose gel analysis of the amplified pcr products showed that the amplified dna has size of about 1600 bp. pcr product were purified before sequencing using the magnetic water (ampure) and magnetic plate procedure according to the standard protocol. purified pcr product was sequenced using big dye terminator v3.1 cycle sequencing ready reaction kit original. sequencing was performed in a total final of 10 ml. products were then analized using a dna sequencer (abi prism 3100). phylogenetic analysis of streptomyces nukleotide sequences were compared with those maintained in the genbank database through ncbi blast. for phylogenetic analysis, sequences were aligned with those of reference strains with the program bioedit version 7.0.4.01. the phylogenetic tree was derived from distance matrices using neighbor-joining method. results and discussion cultural and morphological characteristics the characterization of streptomyces species is mainly based on the aerial, substrate mycelia color, soluble pigment production, the shape and ornamentation of spore surface. other additional testes are also considered to ascertain species classification of a new isolate. streptomyces sp-mws1 grew on isp-4 media. the abundance and the color of aerial mycelium depended on the medium composition and the age of the culture. the results indicated of streptomyces sp-mws1 that the white aerial mass color and soluble pigment produced on isp-4 media. for streptomyces sp-mws1, it was observed that the aerial hyphae bears spores of no spiral type as shown in figure 1. figure 1. morphology of spore-bearing aerial hyphae of streptomyces sp-mws1 after 14 days cultivation on isp-4 agar medium at 28° c showing sporechain no spiral (400×) 84 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 82-85 carbohydrate utili�ation the utilization of various carbohydrates by streptomyces sp-mws1 suggests a very narrow pattern of carbon source assimilation. amylum and glucose were utilized well, and l-arabinose, d-xylose, d-mannitol, lactose, saccaharose were not utilized. antimicrobial activities streptomyces sp-mws1 showed a broad antimicrobial spectrum against gram-positive and gram-negative bacteria (table 1). many such broad-spectrum antibiotics have been produced by streptomyces sp. (korzybski et al, 1967). table 1. antimicrobial activities of antibiotics streptomyces mws-1 indicatorsa diameter zone of inhibition (mm) streptomycin (control) streptomyces sp.mws-1 bacillus subtilis 20,6 17,9 staphylococcus aureus atcc 25923 14,4 21,9 pseudomonas aeruginosa atcc 7853 16,0 24,9 e. coli atcc 25922 12,9 22,4 salmonella typhimurium 13,4 25,7 a bacteria were incubated on nutrient agar medium at 37° c for 24 hours comparison with the known strain the characteristics of streptomyces sp-mws1 were compared with published descriptions of various streptomyces species (shirling and gottlieb, 1968). in general, the taxonomic classification and identification of streptomycetes is based on morphological and physiological test, thus molecular methods (16s rdna gene sequence analysis) could represent an improvement (rintala et al., 2001). nowdays, the detection and clasification of streptomyces are now most commonly performed by molecular approaches based on selective pcr amplification (locatelli et al., 2002). many approaches have been tried to aid in the classification of streptomyces isolates to the genus, species, and strain levels. genetic methods are more rapid and convenient than classification methods based on phenotypic characteristics (park et al., 2006). pcr amplification experimental analysis of the pcr amplification performance the forward primer 9f in conjunction with the reverse primer 1541r was conducted under the reaction conditions described. the primer pairs 9f/1541r amplified a fragment of the expected size from dna isolated from positive control strain streptomyces griseus atcc 10971. the specificity of the primers was further examined by pcr amplification using dna isolated from streptomyces spmws1. the specificity of the pcr is affected by multiple factors, such as the primers, the properties of the gene regions flanking the target site, the annealing temperature in the pcr reaction and the reaction conditions. phylogenetic taxonomy of streptomyces the alignment of the nucleotide sequences (1430 bp) of streptomyces sp.mws-1 was done through matching with the 16s rdna reported genes sequences in the gene bank. the database of ncbi blast available at (www. nvbi.nlm.nig.gov) was used to compare the isolated strains with sequence of the reference species of streptomyces contained in genomic database bank. the results exhibited similarity level 98% with 17 strains from the uncultured known streptomyces species. the 16s rdna nucleotide sequence of streptomyce sp-mws1 consisted of 1430 bp and the gc-content was 70%. these results were in accordance to many authors who mentioned that, the gccontent of streptomyces dna is 60–78%. (anderson ad wellington. 2001). figure 2. the phylogenetic position of streptomyces sp-mws1 among neighboring method showing 16s rdna tree of the phylogenic similarity (%) comparing with the sequences of other known streptomyces species modern streptomyces identification systems are based on the 16s rdna sequence data, which have provided invaluable information about streptomyces systematic and then have been used to identify several newly isolated streptomyces (hyo et al., 2006). for data analysis, the phylogenetic tree in fig. 2 was derived from distance matrices using neighbor-joining method (saitou and nei, 1987). the majority of sequences clustered into groups in the phylogenetic analysis. however, the results indicate the presence of several different type of streptomyces 16srdna sequences in buildings, suggesting higher diversity with several species. these strains together with 85retnowati: identification of streptomyces sp-mwsi sequences from uncultured known streptomyces species which were differed for them in the some morphological characters; carbon utilization and active secondary metabolites production, where the different detection limits of the methods make presence/absence comparisons difficult (suutari et al., 2002). conclusion streptomyces sp-mws1 producing antibacterial compounds isolated from the soil of mangrove ecosystem in the eastern coast of surabaya indicated similarity binary 98% with sreptomyces species. acknowledgement this work was supported in part by hibah bersaing dirjen dikti, contract no: 318/sp3/pp/dp2m/2007. references anderson, a.s., wellington, e.m.h. 2001. the taxonomy of streptomyces and related genera. int. j. syst. evol. microbiol. 57: 797–814. armen, e.a., arthur, a.h., anichka, s.h., rafael, a.a., andranik, a.v., ashot, a.s. 2004. isolation, purification and physiological characterization of water-soluble bacillus thuringiensis melanin. pigment cell res. 18:130–135. bull, a.t., m. goodfellow, j.h. slater. 1992. biodiversity as a source of innovation in biotechnology. annu. rev. microbial. 46: 19–252. cross, t. 1989. growth and examination of actinomycetes some guidelines. in bergey’s manual of systematic bacteriology. williams and wilkins company. baltimore. 4: 2340–2343. hyo, j.k., l. sung, k.h. byung. 2006. streptomyces cheonanensis sp.nov., a novel streptomycete with antifungal activity. in. syst. evol. microbial. 56: 471–475. jaroff, l. 1994. counterattack : how drugmakers are fighting back. time. 37: 46. korzybski, t., z. kowszyk-gindifer, w. kurylowicz. (eds). 1967. antibiotics, origin, nature, and properties. translated by paryski, e. pergamon press. oxford, pp. 450–715. lemonick, m.d. 1994. the killers all arround. time 37: 40–47. luedemann,g.m., b.c. brodsky. 1964. taxonomy of gentamicinproducing micromonospora. antimicrob. agents chemother. 1963: 1116–124. locatelli, l., s. tarnawski, j. hamelin, p. rossi, m. aragno, n. fromin. 2002. specific pcr amplification for the genus pseudomonas targeting the 3 half of 16s rdna and the whole 16s-23s rdna spacer. syst. appl. microbiol. 25: 220–227. miyadoh,s. 1993. research on antibiotic screening in japan over the last decade: a producing microorganism approach. actinomycetologica. 7: 100–106. park, h.s., j. john, i.i kilbane. 2006. rapid detection and high resolution discrimination of the genus streptomyces based on 16s-23s rdna spacer region and denaturating gradient gel electrophoresis. j. ind. microbiol. biotechnol. 33: 289–297. pospiech, a., newmann, b. 1995. a versality quick –prep of genomic dna from gram positive bacteria. trends genet. 11: 217–218. pridham, t.g. d. gottlieb. 1948. the utilization of carbon compounds by some actinomycetales as in aid for species determination. j.bacteriol. 56: 107–114. rintala, h., a. nvalainen, e. ronka, m. suutari. 2001. pcr primers targeting the 16s rrna gene for the specific detection of streptomyces. molecular and celullar probes. 15: 337–347. shimon, d.g., r. m., petinate, martins, r.r. rosalie, coelho, l. nazareth, meirelles, h. marta, a.v. branquinha, beatriz. 1999. influence of growth medium in protease and pigment production in streptomyces cyzneus. mem. inst. oswwaldo. cruz. 94(2): 173–177. saitou, n. and m. nei. 1987. the neighbor-joining method : a new method for reconstructing phylogenetic trees. mol. bio. evol. 4: 406–425. shirling, e.b., d. gottlieb,. 1966. methods for characterization of streptomyces species. int. j. syst. bacteriol. 16: 313–340. shirling, e.b., d. gottlieb,. 1968. cooperative description of type culture of streptomyces. ii. species description from first and second study. int. j.syst. bacteriol. 18: 279–392. shirling, e.b., d. gottlieb. 1972. cooperative description of type culture of streptomyces. v. additional description. int. j.syst. bacteriol. 22: 265–394. suutari, m., u. lignell, a. hyvarinen, a. nevalainen. 2002. media for cultivation of indoor streptomycetes. j. microbial. methods. 51: 411–416. waksman, s.a. (ed). 1967. the actinomycetes. a summary of current knowledge. ronald press co., new york. wu, r.y. 1984. studies on the streptomyces sc4. ii. taxonomic and biological characteristics of streptomyces strain sc4. both. bull. acad. sin. 25: 111–123. zhang, q., w.l. jun, x.c. long. 2003. streptomyces yunnanensis sp.nov., a mesophile from soils in yunnan, china. int. j. sys. evol. microbiology. vol. 53. 217–221. ijtid vol 1 no 2 may-aug 2010.30.pdf ijtid vol 1 no 2 may-aug 2010.31.pdf ijtid vol 1 no 2 may-aug 2010.32.pdf ijtid vol 1 no 2 may-aug 2010.33.pdf 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. 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. 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 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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. 93 vol. 7 no. 5 may-august 2019 research report combined target site vgsc mutations play a primary role in pyrethroid resistant phenotypes of aedes aegypti as dengue vector from palu city, central sulawesi purwaningsih1, sitti rahmah umniyati2a, budi mulyaningsih2 1 post graduate student of tropical medicine program, faculty of medicine, public health and nursing, universitas gadjah mada, yogyakarta. 2 department of parasitology, faculty of medicine, public health and nursing, universitas gadjah mada, yogyakarta. a coresponding author: sitti-rahmah@ugm.ac.id abstract it has been reported that aedes aegypti mosquitoes in palu city had been resistant to cypermethrin insecticide but the resistance mechanism is not well known. this study aimed to determine the resistance status of ae. aegypti to cypermethrin and whether the mutation of voltage-gated sodium channel (vgsc) was associated with pyretroid resistance in high and low dengue endemic areas in palu city. aedes aegypti collected from each village was reared to adult and assayed for susceptibility test to cypermethrin using the cdc bottle bioassay method. pcr primers of aascf1 and aascr4 were used for screening of iis6 vgsc gene mutation. pcr primers of aascf7 and aascr7 were used for screening of iiis6 vgsc gene mutation. for an identification of mutation sites were sequenced and aligned to gene bank (access no. ab914689 and ab914690) for iis6 vgsc and gene bank (access no. ab914687 and ab914688) for iiis6 vgsc gene mutation. the susceptibility status of ae. aegypti to cypermethrin was resistant in high dengue endemic areas and moderately resistant in low dengue endemic areas. it was found double point mutation at s989p and v1016g in ae. aegypti from high and low dengue endemic areas in palu city and there was a single point mutation only in high dengue endemic area at target site v1016g. aedes aegypti from both high and low dengue endemic areas were resistant to cyperpethrinn and the two alleles had a major role in the occurrence of cypermethrin resistance in palu city. keywords: aedes aegypti, resistance, pyrethroid, vgsc gene mutation abstrak telah dilaporkan bahwa nyamuk aedes aegypti di kota palu telah resisten terhadap insektisida sipermetrin, tetapi mekanisme resistensinya belum diketahui dengan baik. penelitian ini bertujuan untuk mengetahui status resistensi ae. aegypti terhadap sipermetrin dan untuk menentukan apakah mutasi voltage gated sodium channel (vgsc) dikaitkan dengan resistensi piretroid di daerah endemis dengue yang tinggi dan rendah di kota palu. aedes aegypti yang dikumpulkan dari masing-masing desa dipelihara sampai dewasa dan diuji untuk uji kerentanan terhadap sipermetrin menggunakan metode cdc botol bioassay. primer pcr aascf1 dan aascr4 digunakan untuk skrining mutasi gen iis6 vgsc. primer pcr aascf7 dan aascr7 digunakan untuk skrining gen iiis6 vgsc. untuk identifikasi lokasi mutasi disekuensing dan disejajarkan dengan bank gene (akses no. ab914689 dan ab914690) untuk iis6 vgsc dan gene bank (akses no. ab914687 dan ab914688) untuk mutasi gen iiis6 vgsc. status kerentanan ae. aegypti terhadap sipermetrin resisten di daerah endemik dengue tinggi dan resisten sedang di daerah endemik dengue rendah. ditemukan titik ganda mutasi pada s989p dan v1016g ae. aegypti dari daerah endemik dengue tinggi dan rendah di kota palu, dan ada satu titik mutasi hanya di daerah endemik dengue tinggi pada target site v1016g. aedes aegypti dari daerah endemik dengue tinggi dan rendah resisten terhadap sipermetrin, dan kedua alel memiliki peran besar dalam terjadinya resistensi sipermetrin di kota palu. kata kunci: aedes aegypti, resisttensi, pyrethroid, mutasi gen vgsc 94 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 93–98 introduction aedes aegypti is the most efficient vector for arboviruses because it is highly anthropophilic, frequently bites, and thrives in close proximity to humans.1 indonesia is a hyperendemic area with the spread of cases in both urban and rural areas.2,3 dhf often causes outbreaks in several districts/cities in indonesia. the number of dengue cases always increase every year. in 2016, palu city had the highest number of dhf cases in central sulawesi province, 2 people were died from balaroa village. balaroa village is categorized as high dengue endemic area while siranindi village is categorized as low dengue endemic area of dhf. almost all primary health facilities in palu city had problems with dhf every year, and there are reported cases of death.4 various dhf prevention policies and strategies have been programmed. the government of palu city issued regional regulation no. 2, 2016 about concerning the control of dhf as proof of the seriousness in dhf control efforts, but the results are still not optimal until now.4 aedes aegypti resistance to insecticides is a global phenomenon. resistance is inherited and a single obstacle to the success of chemical vector control.5 the using of insecticides from the community is a factor that triggers resistance.6 the health office of palu city used malathion insecticides for vector control since its establishment in 1997, but the using of malathion was stopped in 2013. the using of cypermethrin insecticide began in 2014.4 the latest information from several areas in palu city is that adult mosquitoes of ae. aegypti have been resistant to cypermethrin 0.05% based on the conventional method of impragnated paper.7 resistance mechanism of ae. aegypti to cypermethrin insecticide has not been identified using this method. there are several methods to determine the resistance mechanism, including biochemical testing to detect the mechanism of metabolic resistance and molecular tests to determine the target site resistance mechanism to insecticides. an example of the target site resistance mechanism to pyrethroids is known as knockdown resistant (kdr)/voltage gated sodium channel (vgsc) gene. the molecular method can determine mutations in the vgsc gene due to selection pressure of insecticides in the organochlorine and pyrethroid groups.8 this study was aimed to determine the resistance status of ae. aegypti to cypermethrin and to determine whether the mutation of the voltage-gated sodium channel (vgsc) gene was associated with pyrethroid resistance in high and low dengue endemic areas in palu city. materials and methods the observational study with cross-sectional analytical design9 was approved by the medical and health research ethics committee of faculty of medicine of universitas gadjah mada number ke /fk/262/ec/2017. one hundred and twenty ovitraps were installed in high and low dengue endemic area (balaroa and siranindi villages). the coordinates of sampling locations are recorded using gps (global positioning system). the ovitraps were installed for 3-4 days and the ovistrips were carefully dried, labeled and inserted in clear plastics and labeled to be stored. the mosquito eggs were colonized in the insectarium of department of parasitology of the faculty medicine, public health and nursing, universitas gadjah mada, yogyakarta. the eggs hatch into larvae were kept until they became pupae. they were taken with a pipette and kept in a cage. pupae developed into adult mosquitoes after 2 days. adult mosquitoes were fed 10% sugar solution which was absorbed in cotton. the temperature of test room was 27 + 2°c, humidity was 75 + 10% and photoperiod consisted of 12 hours of light: 12 hours of darkness.10 the adult mosquitoes were identified to determine ae. aegypti mosquito and were colonized to the f1 generation mosquito cdc bottle bioassay each testing was involved 125 female adults of ae. aegypti f1 generation, aged 3-5 days. female mosquitoes were fed only with 10% sugar solution the day before testing. the test used 1 control bottle, and 4 test bottles. each bottle was labeled (4 test bottles, 1 control bottle). the test bottle was filled with 1ml of cypermethrin 10μg/ ml solution and the control bottle was filled with 1ml of acetone, then the bottle was tightly closed and the solution was coated on the wall, bottom and bottle cap. bottles were dried at room temperature for 24 hours. using an aspirator 25 mosquitoes were introduced into each test bottle and control bottle. a number of knockdown and or alive mosquitoes were recorded every 5 minutes during the diagnostic time of 30 minutes of exposure. observation was continued until all are dead or up to 2 hours. mortality was corrected with abbott’s formula if the mortality at 2 hours in the control bottle is between 3% and 10%. the result should be discarded if the mortality in the control bottle > 10%. mosquitoes were moved to an insecticide-free recovery cage and were administered with 10% sugar for 24 hours and recorded the number of dead mosquitoes. the resistance status was classified in three categories according to who as follows: 98–100% mortality at the recommended diagnostic time indicates susceptibility; 80–97% mortality at the recommended diagnostic time suggests the possibility of resistance that needs to be confirmed; <80% mortality at the recommended diagnostic time suggests resistance.11 the molecular test the isolation of genomic dna was done individually using genomic dna mini kit geneaidtm cat no. gb100. lot no.jm02202 according to the manufacturer’s 95purwaningsih, et al.: combined target site vgsc mutations play a primary role instructions.12 pcr primers aascf1 (aga caa tgt gga tcg ctt cc) and aascr4 (gga cgc aat ctg gct tgtta) were used for screening of iis6 vgsc gene mutation. pcr primers of aascf7 (gag aac tcg ccg atg aac tt) and aascr7 (gac gac gaa atc gaa cag gt) were used for screening of iiis6 vgsc gene mutations.13 the pcr mixtures contained 15 μl of mix pcr (go taq® green master mix. 2x), 11 μl of ddh2o (nuclease-free water lot. 0000123190. promega), 2 μl of r & f primers (20 μm), 2 μl of the dna template in a total volume of 30μl. pcr was performed under the following conditions: initial denaturation at 94°c for 3 min; 35 cycles each of 94°c for 15s, 55°c for 30s, and 72°c for 30s; and a final elongation step at 72°c for 10 min.13 the dna amplification results were separated according to the size of the base pair by electrophoresis technique. electrophoresis technique used 2% agarose gel which added 1 μl gel red. the gel was inserted in chamber electrophoresisis which already contained solution buffer 50x tae to cover surface gel. product pcr was taken up 7 μl and inserted on gel well. standard molecules were used 100 kb ladder marker. power supply was run with potential difference of 100 volts for approximately 45 minutes. observation of the dna bands was done below uv light on gel doc. the electrophoresis results were read on the target band and documented. the samples which showed bands of target dna were sent to pt. genetica science indonesia. samples would be sent to 1st base laboratories singapore for sequencing. for an identification of mutation sites were sequenced and aligned to gene bank (access no. ab914689 and ab914690) for iis6 vgsc gene mutation (s989p, i1011m/v, v1016g/i) and aligned to gene bank (access no. ab914687 and ab914688) for iiis6 vgsc gene mutation (t1520i and f1534c)13 using mega version 7.0.18 and bio edit version 7.2.6. results and discussion result of cdc bottle bioassay it was shown the susceptibility status of ae. aegypti to cypermethrin based on cdc bottle bioassays in high and low dengue endemic areas. the result of statistical analysis through bivariate test using independent t-test, obtained that the result the susceptibility status based on mortality rate between high and low dengue endemic area were significant differences with p value = 0.000 (p <0,05). it was indicated that there was knockdown resistance (kdr) of ae. aegypti mosquitoes from high and low dengue endemic areas, because there were reduction of mortality, about 26% dan 19,5% after 24 hours in recovery cages (table 1). result of molecular assays and sequencing analysis the amplification results of iis6 and iiis6 vgsc gene were visualized with 2% agarose gel electrophoresis and read under uv obtained specific band, 619 bp and 748 bp respectively (figure 1 and figure 3). the pcr product was sequenced to determine the mutation of iis6 vgsc gene (figure 2). the point mutation of s989p at iis6 site occurred because one of nucleotide base changed from thymine to citocin at codon tcc → ccc caused the amino acid changed from serine to proline. figure 2 showed, there were double point mutation in target site s898p and v1016g and there was single point mutation in target site v1016g. the pcr product was sequenced to determine the mutation of iiis6 vgsc gene (figure 4). table 1. the result of cdc bottle bioassay of ae. aegypti to cypermethrin 10 μg/bottle (diagnostic dose) location (villages) generation mortality (%) category 30 minutes 2 hours holding 24 hours 24 jam high endemic dengue area (balaroa) test bottle-1 f1 65 98 66 resistant test bottle-2 f1 59 100 82 resistant test bottle-3 f1 66 99 71 resistant average high endemic 63,33 99 73 resistant control bottle f1 0 0 0 low endemic dengue area (siranindi) test bottle-1 f1 91,2 95,2 75,2 moderate resistant test bottle-2 f1 89,6 95,2 80,8 moderate resistant test bottle-3 f1 90,4 97,6 73,6 moderate resistant average low endemic 90,4 96 76,5 moderate resistant control bottle f1 0 0 0 laboratory strain f1057 99 100 100 susceptible 96 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 93–98 discussion it was shown that the susceptibility status of ae. aegypti to cypermethrin insecticide was resistant in high endemic dengue area and moderate resistant in low endemic dengue area. differences in susceptibility status between regions can occur because they are influenced by differences in knowledge, education, control efforts and frequency of insecticides used for health and agricultural purposes.14 cypermethrin insecticide began to be used for the benefit of the program in palu city, in 2014. insect resistance to insecticides generally occurs after 2-20 years of use. the use of insecticides on a large scale, continuously for a long period of time and high frequency can cause a decrease in susceptibility to mosquitoes targeted.15 the process of the occurrence of vector resistance to certain insecticides is influenced by multiple factors, namely genetic (presence of specific gene frequencies), operational (insecticide type and application) and biological (size and characteristics of vector populations).16 the different of susceptibility status in these two regions due to operational factors, namely vector control through fogging from the program until now is still being used. fogging is carried out when there is a dhf case report. dengue endemic areas have a higher fogging frequency compared to low dengue endemic areas. another factor that triggers a decrease in the susceptibility status of ae. aegypti mosquitoes from these two villages were the use of household insecticides by the local community. some household insecticides such as aerosol formulations and other formulations were used in balaroa and siranindi villages, 28.2% and 36.95% respectively. the active ingredients used were malation and other active ingredients such as propoxur (bendiocarb) which can cause multiple resistance. aedes aegypti were still undergoing selection pressure on organophosphate insecticides in palu city, but selection pressure was higher for cypermethrin insecticides because of the use of the program and the effect of exposure from household insecticides. the data of research through structured interviews showed that household insecticides in balaroa and siranindi villages were equal to 76.67%. most household insecticides were pyrethroids. the percentage of household insecticides which was quite high affects the susceptibility status to cypermethrin insecticides in both regions. another factor that influences the increase in resistance status is the ability of mosquitoes to adapt and evolve well. mosquitoes have a high reproductive speed and a short generation period so that mosquitoes are susceptible to genetic mutations.17 this is evidenced by research in malaysia which shows an increase in ae. aegypti resistance to permethrin is 5-18 times after five generations.18,19. figure 1. visualization of iis6 vgsc gen amplification of ae. aegypti from balaroa (1-6) and siranindi (7-10), m (100 lader dna marker), k(negative control, without vgsc gene dna), k+ (positive control of vgsc gene) figure 2. result sequensing aligned with gen bank access ab914689 and ab914690 indicated mutation of iis6 vgsc gene of ae. aegypti target site serin (tcc) 989 prolin (ccc) and valin (gta) 1016 glycin (gga) (mega version of 7.0.18 and bio edit version of 7.2.6) figure 3. visualization of iiis6 vgsc gene amplification of ae. aegypti from balaroa (1-6) and siranindi (7-10), m (100 lader dna marker), k(negative control, without vgsc gene dna), k + (positive control of vgsc gene) figure 4. results of sequencing aligned with gene bank access ab914687 and ab914688. there weren’t change ttc (phenilalanine) to cystein (tgc) (mega version of 7.0.18 and bio edit version of 7.2.6) 97purwaningsih, et al.: combined target site vgsc mutations play a primary role figure 5. iis6 vgsc double point mutation target site s989p and v1016g associated to resistance to pyrethroid from ae. aegypti were mostly detected in this study aedes aegypti resistance against cypermethrin insecticide occurs in west venezuela,20 and several regions in central java (semarang, grobogan, purbalingga and kendal).21 the resistance of ae. aegypti against deltamethrin is also reported in central java (semarang, jepara, blora, salatiga, surakarta, tegal, magelang and purwokerto).22 the mechanism of resistance to pyrethroid insecticides can be detected molecularly. this study indicated that there was a target site mutation in the vgsc gene. the target site mutation in vgsc gene regarding resistance to pyrethroids suggests that there is an ongoing resistance mechanism. detection of vgsc gene mutations can directly assess the transformation of target cells which are the target of insecticides. gene mutation causes conformational changes in the sodium channel because it can not be opened by insecticide molecules. mutations like this can only be detected by molecular methods. the basic principle of molecular detection of resistance in vectors is identify genes. the results showed that most of the samples of ae. aegypti from balaroa and siranindi villages, palu city experienced double-point mutations (two-point mutations simultaneously) at s989p, and v1016g in iis5-s6 and iis6 vgsc genes respectively (figure 5). the results of susceptibility test gave a very specific description of phenotypic resistance events and were supported by mutations found in ae. aegypti mosquito in vgsc gene (genotypic resistance). a valine to glycine transversion in domain ii of the vgsc (v1016g) is associated with resistance to type i and ii pyrethroids, such as permethrin and deltamethrin, respectively.23the v1016g mutation is often found with a serine to proline mutation (s989p) in domain ii. they have also been found in several other regions of asia. mutations were reported at these points in thailand, myanmar, vietnam, taiwan and indonesia.24 in this study, the only one sample experienced a single point mutation at the v1016g target site in balaroa village. it was also reported in klaten, central java.23 the results of study by rajatileka25 and srisawat26 found a point mutation at target site v1016g in vgsc gene of ae. aegypti that was associated with pyrethroid synthetic resistance in thailand. the v1016g allele seems limited in southeast asia, but recently the v1016g allele was found in mecca.27 different substitutions of v1016i are found in ae. aegypti populations from brazil v1016i.28,29 the v1016i allele was distributed in south and north america (alvarez et al., 2013)20, but the v1016i allele was also found in palembang-indonesia (ghiffari et al., 2013).30 transformation in valine to glycine iis6 vgsc (v1016g) were associated with resistance to type i and ii pyrethroids, such as permethrin and deltamethrin.23 pyrethroids mainly affect the peripheral and central nervous system in insects by binding to the vgsc target site in the nerve membrane. some of the advantages of insecticides from this group include low levels of toxicity to humans and mammals in general and easily decompose in the soil (martins et al., 2009).28 pyrethroid is divided into 2 types based on chemical structure & the effects, such as pyrethroid type i and type ii. pyrethroid type ii contains parts of α-cyano-3phenoxybenzyl alcohol such as cypermethrin, sifulthrin, deltamethrin, fenvalerate, esfenvalerate and lamellalhalrin (ishak et al, 2015).31 it was reported by al nazawi27 that the ae. aegypti sample was resistant to deltamethrin. the strain from mecca experienced a point mutation simultaneously s989p and v1016g. point mutations found simultaneously were also reported in ae. aegypti populations from latin america in different substitutions and alleles, i1011v and v1016i (plernsub et al., 2016).32 mutation is a marker for monitoring resistance (ishak et al., 2015).31 according to widyastuti et al. (2015)33 that vgsc gene mutations in several positions can occur simultaneously in one individual mosquito and the possible effect will be greater on mosquito resistance properties. aedes aegypti v1016g strains (occurring with and without s989p) and f1534c mutations are common and widespread throughout asia. the g1016 allele was known to be associated with resistance to type i and ii pyrethroids. the c1534 allele was mainly associated with resistance to pyrethroid type i and known as recessive alleles (ghiffari et al., 2013.30 f1534 allele of this study are similar to those conducted by stenhouse.24 conclusion aedes aegypti from high and low dengue endemic areas were resistant to cyperpethrinn, and the two alleles (v1016g and s989p) had a major role in the occurrence of cypermethrin resistance in palu city. acknowledgement the author would like to express sincerely gratitude the health office of central sulawesi province for funding this research and faculty of medicine, public health and nursing, universitas gadjah mada yogyakarta for providing an opportunity to do this study in laboratory of department of parasitology and integrated research laboratory. 98 indonesian journal of tropical and infectious disease, vol. 7 no. 5 may-august 2019: 93–98 references 1. who. dengue guidelines for diagnosis, treatment, prevention and control. geneva, switzerland: who; 2009. 2. world health organization. comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. south-east asia: who, 2011. 3. world health organization. national guidelines for clinical management of dengue fever. india: who, 2015. 4. dinas kesehatan kota palu. 2017. pengendalian penyakit demam berdarah dengue. peraturan daerah (perda) kota palu no. 2 tahun 2016. available from: https://peraturan.bpk.go.id/home/ details/20800. 5. kementrian kesehatan republik indonesia. pedoman penggunaan insektisida (pestisida) dalam pengendalian vektor. jakarta: direktorat jendral pengendalian dan penyehatan lingkungan, 2012. available from:http://perpus.b2p2vrp.litbang.depkes.go.id/opac/index. php?p=show_detail&id=1107&keywords= 6. yuliani ts, triwidodo h, mudikdjo k, panjaitan, nk, sjafrida manuwoto s. pestisida rumah tangga untuk pengendalian hama permukiman pada rumah tangga. jpsl, 2011;2 (1): 73-83. 7. jastal. pemetaan status resistensi insektisida di indonesia tahun 2015. laporan penelitian. balai litbangkes donggala, 2015. available from: https://docplayer.info/44333679-laporan-tahunan-tahun-balaipenelitian-dan-pengembangan-pengendalian-penyakit-bersumberbinatang-donggala.html 8. smith lb, kasai s, scott jg. pyrethroid resistance in aedes aegypti and aedes albopictus: important mosquito vectors of human diseases. pestic. biochem. physiol. 2016. 133: 1-12. 9. sastroasmoro s dan ismail s. dasar-dasar metodologi penelitian klinis. 5th ed. jakarta: cv. sagung seto. 2014. 104-110. 10. world health organization. monitoring and managing insecticide resistance in aedes mosquito populations. genewa: who, 2016. available from:https://www.who.int/csr/resources/publications/zika/ insecticide-resistance/en/ 11. center for desease control and prevention gudline for evaluating insecticide resistance in vector using the cdc bottle bioassay. usa: cdc, 2010. 1-83. 12. anonymus. genomic dna mini kit (blood/cultured cell). geneaid www.geneaid.com available from: geneaid www.geneaid.com. http://www.geneaid.com/products/genomic-dna-purification/dnaextraction-kit-blood-cultured-cell-miniprep. 13. kawada h, oo szm, thaung s, kawashima e, maung ynm, thu hm, et al. 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(4): 39. 22. sunaryo, ikawati b, rahmawati., widiastuti d. status resistensi vektor demam berdarah dengue (aedes aegypti) terhadap malation 0,8% dan permethrin 0,25% di provinsi jawa tengah. ekologi kesehatan. 2014. vol. 13 no. 2. 23. ikawati b, sunaryo,widiastuti d. peta status kerentanan aedes aegypti (linn.) terhadap insektisida cypermethrin dan malation di jawa tengah. aspirator. 2015. 7(1): 23-8. 24. stenhouse sa, plernsub s, yanola j, lumjuan n, dantrakool a, choochote w, somboon p. detection of the v1016g mutation in the voltage-gated sodium channel gene of aedes aegypti (diptera: culicidae) by allele-specific pcr assay, and its distribution and effect on deltamethrin resistance in thailand. parasites & vectors. 2013.6: 253. 25. rajatileka s, william cb, saavedra kr, yuwadee t, chamnarn a, mccalla pj, and hilary r. development and application of a simple colorimetric assay reveals widespread distribution of sodium shannel mutations in thai populations of aedes aegypti. acta tropica. 2008. 108: 54–7. 26. srisawat r, komalamisra n, eshita y, zheng m, ono k, itoh tq, matsumoto a, petmitr s, and rongsriyam y. point mutations in domain ii of the voltage-gated sodium channel gene in deltamethrinresistant aedes aegypti (diptera: culicidae). appl. entomol. zool. 2010; 45. 275–282. 27. al nazawi am, aqili j, alzahrani m, mccall pj, and weetman d. combined target site (kdr) mutations play a primary role in highly pyrethroid resistant phenotypes of aedes aegypti from saudi arabia. parasites & vectors. 2017;10:161. 28. martins aj, lima jb, peixoto aa, and valle d. frequency of val1016ile mutation in the voltage-gated sodium channel gene of aedes aegypti brazilian populations. trop med int health. 2009.14(11): 1351-5, issn 1365-3156. 29. lima ep, paiva mhs, de araújo ap, da silva évg, da silva um., de oliveira a.e.g. santana, c.n.barbosa, c.c.p. neto, m.o.f goulart, c.s. wilding, ayres ln, and santos mavm. insecticide resistance in aedes aegypti populations from ceará, brazil. parasites & vectors. 2011. 4(5):1-12. 30. ghiffari a, fatimi h, dan anwar c. deteksi resistensi insektisida sintetik piretroid pada aedes aegypti (l.) strain palembang menggunakan teknik polymerase chain reaction. aspirator. 2013. 5 (2): 37-44. 31. ishak ih, jaal z, ranson h, and wondji cs. contrasting patterns of insecticide resistance and knockdown resistance (kdr) in the dengue vectors aedes aegypti and aedes albopictus from malaysia. parasites & vectors. 2015. 8:181. 32. plernsub s, saingamsook j, yanola j, lumjuan n, tippawangkoso p,sukontason k, walton c, and somboon p. additive effect of knockdown resistance mutations, s989p, v1016g and f1534c,in a heterozygous genotype conferring pyrethroid resistance in aedes aegypti in thailand. parasites & vectors, 2016; 9:417. 33. widyastuti d, sunaryo, pramestuti n, sari tf, wijayanti n. deteksi mutasi v1016g pada gen voltage-gated sodium channel pada populasi aedes aegypti (diptera: culicidae) di kabupaten klaten, jawa tengah dengan metode allele-specific pcr. vektora. 2015. 7 (2): 65-70. 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 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. 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 105 vol. 1. no. 3 september–december 2010 research report hepatitis c virus infection in hemodialysis patients: comparison of the surabaya dialysis center and juntendo university hospital dialysis centre djoko santoso1, pranawa1, moh. yogiantoro1, widodo1, a. wardana1, n. mardiana1, c. irwanadi1, soewanto1, ichiyu shou2, kunimi maeda2, chieko hamada2, mitsumine fukui2, satoshi horikoshi2 and yasuhiko tomino2 1 division of nephrology – hypertension, department of internal medicine, dr.soetomo hospital, faculty of medicine, airlangga university – surabaya, indonesia 2 division of nephrology, department of internal medicine, juntendo university faculty of medicine, tokyo, japan abstract hepatitis c virus infection is highly prevalence in chronic hemodialysis (hd) patients. the present study will compare prevalence of hcv positive population in difference countries where there are great contrasts in and diversity of care available to patients who have end stage renal disease. all serum samples of the 100 patients were tested for hcv antibodies, using third-generation enzyme immunoassay. the prevalence of anti-hcv was correlated with a history of blood transfusion and with duration of hemodialysis. hcv prevalences were 88% of surabaya group and 6% of juntendo group, respectively. in surabaya group, prevalence of hcv positive was high and the risk factors are not only those of the juntendo group, but also a combination of poor living conditions, frequent blood transfusions, and lack of adherence. much needs to be studied about the role of universal screening and effective techniques for primary prevention in surabaya group key words: epidemiology, hemodialysis, hepatitis c virus, risk factor introduction patients with end stage renal disease (esrd) are at higher risk of acquiring hcv infection.[1,2] published prevalence data for hcv infection among hemodialysis (hd) patients in various countries in consistently higher than in healthy populations,[3] ranging from 2 to 6% in northwestern europe to more than 20% in japan and over 60% in saudi arabia.[4,5,6] these variations seem not only to reflect local prevalence of hcv but also to suggest that aspects of the dialytic process may expose patients to an increased risk of developing hcv. this means the geographical region of the study population, methods used for detection of hepatitis c and the study design lead to varied results, as it was recently suggested in uk.[7} even though general epidemiological information has been obtained for hd populations that are increasing worldwide, a considerable regional variability has been reported.[8,9,10,11] the present study will compare prevalence of hcv positive population in difference countries where there are great contrasts in and diversity of care available to patients who have end stage renal disease. using demographic and laboratory databases, the present incidence of hcv positive hemodialysis patients, environmental conditions, and availability of sophisticated dialysis programes and care for these patients or the lack of treatment facilities will be compared. subjects and methods health conditions and patients there are two groups, dr. soetomo hospital dialysis center, surabaya, indonesia and juntendo university hospital dialysis center, tokyo, japan. surabaya group: fifty hemodialysis patients (41 males and 9 females). informed consent was obtained from each patient enrolled in this study. their mean age was 48.7±12.7 years (range 15–74 years). the causal diseases were divided into diabetes melitus by 24% and other diseases by 76%. the mean duration of hd treatment was 37.45 ± 33.46 106 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 105-109 months (range 7 months to 120 months) (table 1). the patients were treated with standard hd for 4 hours twice weekly, for almost hd patients were applied acetate buffer, cuprophane dialyzer which were reused. standard heparin (multi dose ampules) was used to prevent coagulation . there was no special area for hepatitis positive patients. erythropoietin treatment was rare because the surabaya group is economically disadvantaged with almost all patients coming from the low income population with poor sanitary conditions (table 1). hepatitis prevalence in the normal population of the region is very high. in surabaya, there is a reservoir of hcv infected persons who can transmit the infection to others and who are at risk for hcv related chronic disease. during the time of unknown hcv transmission within the unit, the frequent sharing of facilities over a prolonged period resulted in accumulated risk. surabaya patients frequently received multiple blood products. history family related hcv infections was not examined. juntendo group: there were 50 hemodialysis patients (33 males and 17 females),with a mean age 65.4 ± 14.7 years (range 30 to 89 years). the length of time on dialysis treatment was 110 3 ± 61.65 months (range 32.5 months to 249 months, median of 90.9 months). all patients were on hd thrice weekly. the duration of the dialysis procedure ranged from 4 to 5 hours per session. the blood flow was between 200 and 250 ml/min and dialysate flow was 500 ml/min. almost all hd patients received bicarbonate buffer and recombinant human erythropoietin. underlying renal diseases were: diabetes (34%) and other diseases (66%). information on duration of hemodialysis, and number of blood transfusions was obtained from medical records (table 1). the unit concerned provides two dialysis shifts daily. dialysis machines are not moved, and wherever possible, patients occupy the same dialysis station. a few specified machines were used to dialysed all known hcv positive patients and a few patients with hepatitis b were dialysed within the unit. dialysis was carried out using nipro machines in all patients. between dialysis sessions, machine sterilization was carried out according to manufactured recomandations. the sterilization procedures were based on the instructions of the manufacturers. application of universal precautions to prevent staff members from facilitating transmission of hcv between patients and also to limit the risk of contracting hcv themselves were carried out according to standard infection control practices. all hd patients and staff members were regularly tested for hepatitis b and c. almost all patients did not any receive blood. dialyzers used were either cuprophan or polysulfone. disposable equipment was used for dialysis and dialysers were not reused. single use heparin vials were used to administer bolus and continuous heparin infusions. informed consent was also obtained from each patient enrolled in this study. laboratory tests all serum samples of the 100 patients were tested for hcv antibodies using the inno-test ab iii enzyme immunoassay (eia) for the presence of antibodies to hcv. all tests were carried out and interpreted strictly in accordance with the manufacturer's instructions. the liver tests, which included determinations of aspartate aminotransferase and alanine aminotransferase, were performed using the hitachi analyzer (boehringermanheim, germany). statistical analysis. epidemiological data are presented as means ± sd and percentages of the mean. further statistical analysis of risk factors for hcv infection (duration of hemodialysis, and number of blood transfusions) was also performed. table 1. characteristics of anti-hcv-positive in two groups patient characteristics surabaya group juntendo group p anti hcvanti hcv + anti hcvanti hcv + sex male female 41 9 33 17 hemoglobin concentration (gr/dl) 7.34 + 1.66 10.2 + 0.77 <0.001 age (years) 48.7 + 12.7 65.4 + 14.7 <0.001 standard hemodialysis for 4 hours twice/week thrice/week dialyzer reused disposable median duration of hd treatment (months) 37.45± 33.46 110 3± 61.65 <0.001 underlying renal diseases dm/non-dm 24% / 76% 34% / 66% standard infection control practices ± (incomplete) + (complete) erythropoeitin treatment not any/rare + 107santoso et al.: hepatitis c virus infection in hemodialysis patients results serum sample were collected from a total of 100 dialysis patients (surabaya and juntendo groups).the collected sera were subjected to serological tests. of those tested for hepatitis c virus antibodies by third generation elisa, 44 were positive (88%) in the surabaya group and 3 were positive (6%) in the juntendo group. statistically, there was significant differences between the two groups. the surabaya group had a significantly higher prevalence than that in juntendo group (p < 0.0001)(fig. 1). figure 1. prevalence of anti hcv positive in hemodialysis patients, surabaya: juntendo university hospital dialysis center. time on hemodialysis in the surabaya patients was 37.45 ± months less than that in the juntendo group (110.3 + 61.7 months) with a significant difference (table.1). the surabaya hcv antibody positive patients showed a significantly lower mean level of hemoglobin (7.34 ± 1.66 gr/dl than the juntendo group, whose hb values were 10.2 ± 0.77 gr/dl) (p < 0.0001) (table 1). many surabaya antibody positive patients one to multiple blood transfusions but no juntendo patients received any transfusions (table 2). table 2. relationship between parameters and prevalences of anti-hcv positive patients. parameter surabaya juntendo anti-hcv positive anti-hcv negative anti-hcv positive anti-hcv negative sex male female 36 (81.81%) 8 (18.19%) 5 (83.33%) 1 (16.67%) 2 (66.66%) 1 (33.34%) 31 (65.95%) 16 (34.05%) elevated alt ast 5 5 1 2 0 0 0 0 data obtained from this study demonstrated that prevalence of anti hcv antibodies in the surabaya group was much higher than in those in the juntendo group, which may be attributed to several risk factors, including blood transfusion, duration of dialysis, and a lack of access to dialysis treatment due to limited health care resources. the prevalence rate of positive anti-hcv antibody in surabaya group was 88%, with a positive correlation between anti-hcv positive cases and longer duration on dialysis (p = 0.0001 ) and blood transfusion (p = 0.001), suggesting the presence of clear regional differences within populations in the incidence of hcv antibody. the relationship between hcv infection and transfusion in the surabaya subgroup is more complex since subgroup i patients (time on dialysis < 1 year) and subgroup ii patients (> 3 years) have the same frequency and those in subgroup iii have a higher frequency than those with intermediate values (table 3). hcv positive patients attributed to transfusion were observed in patients on dialysis with both short and long times. discussion prevalence of anti-hcv antibodies among patients on dialysis is consistently higher than in healthy populations,[3] suggesting that dialysis patients may be at higher risk of acquiring hcv infection.[1,2] in different countries, prevalence of this disease among dialysis patients shows wide variations.[12,13,14] studies performed in a selected group of dialysis centers showed that the prevalence of table 3. relationship between the duration of hd and the prevalence of anti hcv positive among hd patients in surabaya and juntendo parameter surabaya juntendo anti-hcv positive (n = 44) antihcv negative (n = 6) anti-hcv positive (n = 3) anti-hcv negative (n = 47) duration of hemodialysis (months) <12 12–48 >48 8 (18.18%) 8 (18.18%) 28 (63.64%) 5 (83.3%) 1 (16.7%) 0 (0%) 0 (0%) 0 (0%) 3 (100%) 0 (0%) 6 (12.77%) 41 (87.23%) duration on hemodialysis (months) 42.04±34.06 9.7±2.5 148.5±70.5 108.7±62.5 tabel 4. relationship between hd patients previously with blood transfussion, with no blood transfussion and prevalence anti hcv positive in surabaya centre hd patients anti hcv (+) anti hcv (–) previously with blood transfussion 36 (81,82%) 6 (100%) 42 (84%) previously with no blood transfussion 8(18,18%) 0 (0%) 8 (16%) total 44 6 50 prevalence anti hcv + among hd patients: 42/50 = 84% prevalence anti hcv + among hd with blood transfussion: 36/43 = 81,81% prevalence anti hcv + among hd with no blood transfussion : 8/43 = 18,18% 108 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 105-109 hcv infections among hemodialysis (hd) patients, is much higher than that among healthy donors,[3] ranging from 2 to 6% in northwestern europe to more than 20% in japan and over 60% in saudi arabia.[4,5,6] many factors are involved in these variations which may explain the differences in prevalence of anti-hcv positivity among the dialysis units.[5] the increased requirement for blood transfusion in the dialysis population[10,15,16,17,18] and duration of hemodialysis are risk factors for hcv infection (independent of previous transfusions), transmission within the dialysis unit,[17] the mode of transmission is through environmental contamination.[19,20] data obtained from this study demonstrated that prevalence of anti hcv antibodies in the surabaya group was very different from than in the juntendo group. the low prevalence of hcv infection among the juntendo group has to be contrasted with the 88% prevalence of infection found in the surabaya group. such the high variations have also been reported by other authors.[5] using these estimates and assuming that the incidence is related to many factors for developing hcv infection in the population, this study proposes a several mechanisms explaining the difference in prevalence in the two groups. the reason for this difference may be explained by the unique condition in the two countries epidemiologically, the different prevalence of hcv infections in hd patients in both countries, suggests an increase in the number of patients at higher risk for the development of hcv infection and such regional variability has been reported.[21] in fact the hepatitis c prevalence in the general population of the region is higher,[22] suggesting infection from a common source. a dramatically higher prevalence is anti hcv positive hd patients in the surabaya group has been found, which has made it a significant health burden in the surabaya group. it is well known that administration of blood products is the main risk factor for developing hepatitis c.[15] in indonesia including surabaya, blood and blood products are important factors in the transmission of hcv infection, because hepatitis c virus is still the major cause of posttransfusion hepatitis. therefore, multiple blood transfusions seemed to be an important risk factor for hd patients in the acquisition of hcv infection in the surabaya group, as recently stressed by other authors.[23] the fact that these patients had never received erythropoeitin suggest that the need for blood transfusion has still continued to correct renal anemia in hemodialysis patients. the introduction of erythropoietin and screening of blood products for anti hcv infection can be highly effective in preventing transmission of hcv infection. in the present study of the juntendo group, patients had almost no hcv infection (only 6%) and the number of transfusions in dialysis patients was much lower. in contrast to the juntendo group with availability of a sophisticated dialysis program, the surabaya group had a prevalence of hcv positive patients of more than 87%. the nonrandom distribution of hcv infection in the surabaya group indicated that local factors may play a role in the epidemiology of hcv. the reason for this phenomenon is not entirely clear, but there are several proposed mechanisms to explain the high prevalence of hcv infection in the surabaya group. undoubtedly, the use of blood products was a major contributory factor, with this mode of transmission is now largely historical in japan, especially in the juntendo university hospital dialysis centre. this condition is supported by evidence of the prevalence rate varied between 0 and 53% on a multicentre study in 11 centres in japan.[22] in the present study, antihcv positive hd patients received significantly more units of blood products than anti-hcv negative patients. frequent bood transfusions (multiple blood transfusions) over a prolonged period may result in an accumulated risk and the present study found a positive correlation between blood transfusions and the risk of infection by hcv, as recently stressed by other authors.[24] the major risk factor is longer duration of dialysis. in surabaya group, the risk factors are not only those of the juntendo group, but also a combination of poor living conditions, frequent blood transfusions, and lack of adherence to universal infection precautions including poor infection control practices, sharing of instruments or medication, nurses not regularly wearing gloves, spread through blood spillage, and presence of other levels of hygienic standards that make dialysis prone to hepatitis c viral infection. the such high prevalence is a burden on the health care system especially for the surabaya group. hcv is major health problem in hd patients in the surabaya centre. identifiable risk factors may be longer duration of dialysis, blood transfusion, and the lack of adherence to universal infection precautions. the major difference in prevalence of anti-hcv antibody in the surabaya and juntendo groups illustraties the diversity of care available to patients in developing and developed countries. references 1. pereira bjg, hepatitis c virus infection in dialysis: a continuing problem, arificial organs 23: 51–60,1999. 2. estaben ji, estaben r, viladomiou l et al. hepatitis c virus antibodyhepatitis c virus antibody among risk groups in spain. lancet 1989; 334: 294–296. 3. mcintyre pg, mccruden ea, dow bc et al. hepatitis c virus infection in renal dialysis patients in glasgow. nephrol dial transplant 1994; 9: 291–295. 4. schneeberger pm, keur i, vliet w, hoek k, boswijk h, loon am, dijk wc, kauffmann rh, quint w, doorn lj. hepatitis c virus infection in dialysis centers in the netherlands: a national survey by serological and molecular methods. j of clin microbiology. 1998, 36: 1711–1715. 5. huraib s, al-rashed r, aldrees a, aljefry m, arif m and al-faleh fa. high prevalence of and risk factors for hepatitis c in hemodialysis patients in saudi arabia: a need for new dialysis strategies. ndt 1995, 10: 470–4. 6. fujiyama s, kawano s, sato s, shimada h, matsushita k, ikezaki n, nakano t, sato t. 1995. changes in prevalence of anti-hcvchanges in prevalence of anti-hcv antibodies associated with preventive measures among hemodialysis patients and dialysis staff. hepato-gastroenterology 42: 162–165. 7. mclaughlin kj, cameron so, good t, mccruden e, ferguson jc, davidson f, simmonds p, mactier ra, mcmillan ma. nosocomial 109santoso et al.: hepatitis c virus infection in hemodialysis patients transmission of hepatitis c virus within a british dialysis centre. nephrol dial transplant 1997, 12: 304–309. 8. hinrichsen h, leimenstoll g, stegen g et al. prevalence and riskprevalence and risk factors of hepatitis c virus infection in haemodialysis patients: multicentre study in 2796 patients.gut 2002, 51: 429–433. 9. morikawa t, nakata k, hamasaki k, et al. prevalence andprevalence and characterization of hepatitis c virus in hemodialysis patients. intern med 1999; 38: 626–31. 10. knudsen f, wantzin p, rasmussen k, et al. hepatitis c in dialysishepatitis c in dialysis patients: relationship to blood transfusions, dialysis and liver disease. kidney int 1993; 43: 1353–6. 11. sandhu j, preiksaitis jk, campbell pm, et al. hepatitis c prevalence and risk factors in the northern alberta dialysis population. am j epidemiol 1999; 150: 58–66. (abstract ) 12. dusheiko g, schmilovitz-weiss h, brown d et al. hepatitis c virus genotypes: an investigation of type-specific differences in geographic origin and disease. hepatology 1994; 19: 13–18. 13. tokita h, okamoto h, iizuka h et al. hepatitis c virus variants fromhepatitis c virus variants from jakarta, indonesia classifiable into novel genotypes in the second (2e and 2f), tenth (10a) and eleventh (11a) genetic groups. j gen virol 1996; 77: 293–301. 14. bosmans jl, nouwen ej, behets g et al. prevalence and clinicalprevalence and clinical expression of hcv-genotypes in haemodialysis patients of two geographically remote countries: belgium and saudi arabia. clin nephrol 1997; 47: 256–262. 15. medin c, allander t, roll m, et al. seroconversion to hepatitis c virus in dialysis patients. a restropective and prospective study. nephron 1993; 65: 40–5. 16. jadoul m, cornu c, van ypersele de strihou c, and ucl collaboratory group. incidence and risk factors for hepatitis c seroconversion in hemodialysis: a prospective study. kidney int 1993; 44: 1322–6. 17. fabrizi f, martin p, dixit v, et al. acquisition of hepatitis c virus inacquisition of hepatitis c virus in hemodialysis patients: a prospective study by branched dna signal amplification assay. am j kidney dis 1998; 31: 647–54. 18. katsoulidou a, paraskevis d, kalapothaki v, et al. molecularmolecular epidemiology of a hepatitis c outbreak in a haemodialysis unit. multicentre haemodialysis cohort study on viral hepatitis. nephrol dial transplant 1999; 14: 1188–94. 19. muller g, zabaletta me, arminio a et al. risk factors for dialysis-risk factors for dialysisassociated hepatitis c in venezula. kidney int 1992; 41: 1055–8. 20. yoshida cft, takahashi c, gaspar amc, schatzmyr hg, ruzany f. hepatitis c virus in chronic hemodialysis patients with non-a, non-b hepatitis. nephron 1992; 60: 150–3. 21. ponz e, campistol jm, bruguera m et al. hepatitis c infection among kidney transplant recipients. kidney int 1991; 40: 748–51. 22. oguchi h, miyasaka m, tokunaga s et al. hepatitis virus infection (hbv and hcv) in eleven japanese hemodialysis units. clin nephrol 1992; 38: 36–43. 23. shusterman n, singer i. infectious hepatitis in dialysis patients. am j kidney dis 1987; 447–55 24. hruby z, sliwinski j, molin i, zalewska m, knysz b, czyz w, steciwko a, bogucki j, gladysz a. high prevalence of antibodies to hepatitis c virus in three haemodialysis centers in south-western poland. nephrol dial transplant 1993, 8: 740–43. ijtid vol 1 no 3 sep-dec 2010.3.pdf ijtid vol 1 no 3 sep-dec 2010.4.pdf ijtid vol 1 no 3 sep-dec 2010.5.pdf ijtid vol 1 no 3 sep-dec 2010.6.pdf ijtid vol 1 no 3 sep-dec 2010.7.pdf 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 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ijtid@itd.unair.ac.id 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 80 133 vol. 1. no. 3 september–december 2010 the etio-pathogenesis of periodontal disease dewi nurul mustaqimah abstract the etiology of polymicrobial disease such as periodontitis is likely to be more complex than suggested by the traditional paradigm of disease involving a single virulent organism which up to now has been believed. this review limits its discussion to the other subgingival microbiota which is not yet cultivable, however it is suggested be implicated with the severity of periodontal disease. the intricate interactions between viruses and bacteria within periodontal pockets as a co-infection process reveal its role in the etiopathogenesis of periodontal disease. also archaea domain participate in syntrophic relationship with the microbiota life members in the subgingival crevice, promote colonization by special bacterial group during periodontitis. it is clear that periodontal diseases are not monoinfections. key words: polymicrobial disease; co-infection process; syntrophic relationship; severe periodontal disease; etio-pathogenesis mechanism literature review introduction it has been known that few subgingival microbiota associated with periodontal infections. isolating and growing putative periodontal pathogens on artificial media in the laboratory has led to recognition of a widely accepted group of cultivable periodontal pathogens. in the current genomic area of exploration, it is now possible to detect and study the ‘not yet cultivable� components of the subgingival microbiota. accordingly, the list of putative pathogens is growing, and now includes a very diverse group of organisms in both the bacteria and archaea domains. it is becoming increasingly apparent that periodontal infections are caused by a much more diverse microbiota than merely gram-negative anaerobes.[1] therefore, members of the oral microbiota that are in the ‘not-yet-cultivated� group, approximately 50%, were not included as etiological candidates.[2] archaea have been found in the mouth of patients with periodontitis. it establishes correlations between the presence of disease and the presence of archaeal dna, the severity of periodontal disease and the relative abundance of archaeal dna in subgingival plaque, and between disease resolution and diminished archaeal dna abundance.[3] it has now been established that human viruses are part of the micro-ecosystem of the oral cavity. evidence supports a co-infection in which the development and progression of periodontal disease is associated with infection by certain human viruses in conjunction with an increase in opportunistic pathogenic bacteria in subgingival microbiota.[1] this review limits its discussion to the other subgingival microbiota which is ‘not-yet-cultivable�, however it is suggesting be implicated with the severity of periodontal disease, and the etiopathogenesis of it. periodontitis as an infectious disease periodontitis represents a specific inflammatory response to microbial residents of the subgingival biofilm. there is considerable variability in terms of clinical manifestation and disease progression rates. this variability may be attributed to differences in the composition of the subgingival microbial flora.[4] in the case of periodontal infections, this is not a simple task, because disease onset and progression is not related merely to the presence of a specific microorganism, but most importantly, to the imbalance between levels and proportions of periodontal pathogens and beneficial species in different sites of the mouth.[5] oral microbiota is an enormously complex and dynamic entity that is profoundly affected by perpetually changing local environments and host-mediated selective pressures. these microorganisms live in hard-to-study biofilms 134 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 133-137 comprising organized polymicrobial communities that are elegantly adapted to thriving and surviving in the multiple micro-ecosystem of the oral cavity.[2] the consistent finding that putative periodontal pathogens are often found in periodontally healthy people for long periods without doing anyharm, supports the idea that these microorganisms are part of the normal oral microbiota as commensal opportunistic pathogens. periodontal disease occurs when there is a disruption in the host-microbe homeostasis associated with health.[2] microbiological culture and culture-independent molecular studies have identified more than 1,200 bacterial species and 19,000 phylotypes in the oral cavity.[6] only a limited number of bacterial species, from more than 500 identified in the subgingival biofilm, which have been associated with periodontitis.[7] but fewer than 20 species are considered to be major periodontal pathogens.[6] although only a few dozen types of microorganisms have been strongly implicated as etiological agents for periodontitis, they do not act independently of their neighbors. the disease-producing abilities of periodontal pathogens are clearly modified by their interactions with other members of the biofilm community. in some instances, their virulence is enhanced, whereas in other cases it is inhibited by the influence of neighboring microorganisms within the biofilm.[2] bilichodmath et al. (2009)[8] reported that the etiopathogenesis of periodontal disease is a complex process, involving the multifarious interaction between microbial and host factors, a variety of disease-modulating environmental factors, and a minor extent on yeasts and parasites. bacterial etiology alone has not been able to substantiate various aspects such as 1) rapid periodontal tissue breakdown with minimal plaque, 2) phase of disease activity and quiescence, 3) site specificity in periodontal disease, 4) progression to advanced periodontal destruction which occurs in a fraction of a given population. an interesting finding from culture-independent studies is the association of microorganisms from the archaea domain with periodontal disease. their association with periodontitis is quite striking as they appear in progressively greater numbers in the subgingival microbiota as a function of disease severity (i.e. probing depth and clinical attachment loss).[2] it has now been established that human viruses (hvs) are part of the micro-ecosystem of the oral cavity. evidence from a variety of sources supports a co-infection hypothesis in which development and progression of periodontal disease is associated with dual infection by certain human viruses (e.g. epstein-barr virus /e-bv and cytomegalovirus/cmv) in conjunction with an increase in opportunistic pathogenic bacteria residing in the endogenous subgingival microbiota.[1] subgingival microbiota most of the destruction found in all cases of periodontitis is mediated by host inflammatory and immunological responses to subgingival biofilms.[1] these infections are caused by an extremely diverse consortium of microorganisms that are part of the endogenous microbiota of most people. the pathogens do not act alone but are part of complex microbial communities called biofilms. biofilms are populations of microorganisms that are concentrated on a surface and surrounded by an extracellular slime matrix of microbial origin known as the glycocalyx.[2] it is well established that the biochemical and metabolic properties of free-floating or planktonic microorganisms are quite different when these same microbes become part of an adherent biofilm community. for example, some bacteria appear to sense when they touch a solid surface. this contact triggers activation of genes that facilitate irreversible attachment of the bacteria to the solid surface. on the other hand, genes for certain virulence factors are up-regulated in some bacteria once they become members of a biofilm. this is important from an etiological perspective.[2] an important feature of microbial communities in biofilm is increased synthesis of quorum-sensing molecules that promote coordinated reactions of the entire biofilm to external stimuli. indeed, biofilm tend to behave like multicellular organisms when challenged with significant environmental changes.[2] finally, horizontal gene transfer (hgt) between similar and dissimilar microorganisms promotes the preservation of genes that enhance survival in highly organized and competitive biofilm ecosystems. it has been observed that hgt events play an important role in the adaptation of microbes to new environments.[2,9] it has been detected that archaea might be linked to virulence as donors of virulence-promoting genes to pathogenic bacteria through the process of hgt.[9] the development of 16s rrna phylogenetic methodology has widened the scope of detectable microorganisms to include uncultivable organisms that may play significant, as yet undefined, roles in pathogenesis. members of the domain archaea are found in greater abundance in dental plaque from sites with periodontal disease than in plaque from non-diseased sites. it can also be detected in root-canal samples.[10] archaea have been isolated from the human oral cavity, gut, colon, and vagina;[3,9] but have not been established as causes of human disease.[3] it has been detected that interactions between viruses and bacteria within periodontal pockets is as a process of etiopathogenesis of periodontal diseases.[8] armitage et al. (2010)[1] wrote that human viruses are part of the micro-ecosystem of the oral cavity of periodontitis patients. even slots (2010)[6] stated that healthy periodontal site of 135mustaqimah: the etio-pathogenesis of periodontal disease periodontitis patients may harbor more herpesviruses than healthy periodontal site of individuals with a generally healthy periodontium. co-infection by viruses periodontal health is associated with median genome detection rates of 8% for e-bv and for cmv. healthy periimplant site have demonstrated an absence of cmv. the observation of few or no herpesvirus genomes in the healthy periodontium is in accordance with a herpesvirus (hv) infection of periodontal inflammatory cells. herpesvirusinfected periodontal healthy and gingivitis sites typically harbor the viruses in a nontranscriptional state. [6] bilichodmath et al. (2009)[8] also wrote that herpesviruses are usually present in the body in inactive state. herpesviruses may cause direct cytopathic effects on keratinocytes, fibroblasts, endothelial cells and inflammatory cells. some researchers have detected cmv in monocytes/macrophages and t lymphocytes, e-bv 1 in b lymphocytes, and herpes simplex viruses (hsv) in monocytes/macrophages and t lymphocytes.[8] absence of herpesviral infection or viral reactivation may clarify why some individuals carry periodontopathic bacteria while still maintaining periodontal health or minimal disease. herpesvirus reactivation may occur spontaneously or as a result of various types of impairment of the host immune defense, including hiv infection, pregnancy, hormonal changes, and psychosocial and physical stress. however, bacterial enzymes or other inflammation-inducing products have probably also the potential to activate periodontal herpesviruses. factors that activate herpesviruses are also recognized risk indicators of periodontitis.[11] an active herpesvirus infection initiates periodontal tissue breakdown and that host immune response against the herpesvirus infection are an important component of the etiopathogeny of the disease.[6] this infection would further diminish the resistance of periodontal tissues, thereby allowing subgingival upgrowth of periodontal pathogenic bacteria.[11] syntrophic relationship like bacteria, archaea are widely distributed and have been isolated from the human oral cavity, root-canal space, human gut, colon, and vagina.[9,10,11] they resemble bacteria in their shapes and various cell structures, but they differ immensely in the chemical composition of their structures.[9] thus physically resemble bacteria but have different nucleotide sequences in their 16s rrna genes and are therefore not in the bacteria domain. archaea is a member of microbial life (among eukarya, bacteria and archaea domain). archaea is the only group in which pathogens have not yet been demonstrated.[2] methanobrevibacter oralis is the most commonly found archaean associated with periodontal disease.[2] members of genus methanobrevibacter are strict anaerobes.[3] this methane-producing microorganism is the dominant oral methanogens.[2,12] although a cause-and-effect relationship has not been shown, archaea have never been found in the subgingival microbiota of periodontally healthy individuals or at healthy sites in patients with periodontitis. although m. oralis has been cultured in the laboratory, it is difficult to grow and is not isolated during routine microbiological assessments. it has been shown that m. oralis antigens induce the production of specific igg antibody by periodontitis patients who harbor the microorganism as part of their subgingival microbiota.[2] there is some evidence that subgingival methanogens ‘outcompete� sulfate-reducing bacteria (srb) and acetogenic bacteria for available h2 in the local environment. these three groups of hydrogenotrophic microorganisms play an important role in the overall subgingival ecology by regulating the levels of h2 and thereby affecting the levels of secondary fermenting periodontal pathogens.[2] such knowledge could provide basic information on the role of h2 consumption in the regulation of the periodontal biofilm ecosystem (i.e. interspecies hydrogen transfer as a possible driving force to promote proliferation of fermenting pathogens).[12] the co-occurrence of two (or all three) groups indicates h2 levels to be sufficiently high in periodontal plaque to allow partitioning of h2 consumption or alternative metabolic strategies of srb and/or acetogens. this could include the use of electron donors others than h2 or the fermentation of short-chain fatty acids with possible production of h2. in the latter case, a mutual relationship with methanogens rather than competition could be possible. here antagonistic interactions, and hence competition among h2 utilizers, seems to prevail. the srb should outcompete methanogens for the substrate h2 if sulfate is not a limiting factor. [12] a recent study provided evidence for the possibility of an opposite order of competitivity (i.e., methanogens outcompeting srb) in the human colon.[9,12] methanogenic archaea has no direct pathogenic effects but contribute to the overall pathogenicity of subgingival biofilms by syntrophic interactions with other microbes. such interactions are those that promote or otherwise affect the pathogenicity of neighboring microbes.[2] archaea have not been established as causes of human disease,[3] because no virulence genes have been identified in archaea.[9] as archaea has been found to be capable of colonizing in the human host as the normal flora, however, no virulence genes have been identified in archaea till now. recent studies have led to identification of the possibility of a probable role of archaea in causing virulence. horizontal gene transfer (hgt) has been thought of as a mode to acquire novel virulence genes in pathogens. recent sequencing of bacterial and archaeal genomes has shown that inter-domain transfer is common. since archaea are 136 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 133-137 not directly involved in causing a disease but there are similarities between pathogenic bacterial genome and archaeal genome, archaea might be linked to virulence as donors of virulence-promoting genes to pathogenic bacteria through the process of lateral gene transfer. there are some evidence that hgt has taken place from archaea to bacterial and may have contributed to virulence in bacteria.[9] discussion periodontal disease is a polymicrobial anaerobic infection that, besides possibly leading to loss of the involved teeth (if untreated), is considered to constitute a risk factor contributing to the development of lifethreatening systemic diseases, such as endocarditis, atherosclerosis, and stroke, as well as being cocausative for preterm birth. the list of oral microorganisms involved in periodontal disease is large, consisting of several hundred species, of which approximately 50% represent as-yetuncultivable phylotypes.[12] no single species has been identified so far as the ultimate causative agent. instead, the disease is likely to be a result of the activities of different and varying microbial complexes.[12] the periodontal putative pathogens are not limited to gram-negative anaerobic bacteria, but also a large number of gram-positive bacteria. even non-bacterial microbes from the archaea domain and also humanviruses may have an etiological role. for example bacteria in the tm7 division which are gram-positive and now have yet been cultured in the laboratory. this 1025 clone of tm7 has recently been associated with periodontal disease as well as with active inflammatory bowel disease.[2] human viruses have been established as part of the micro-ecosystem of the oral cavity of periodontitis patients. some evidence supports co-infection with herpesviruses results in a local increase in proinflammatory cytokines that subsequently disrupts the homeostatic balance between the resident periodontal microbiota and the host. members of the subgingival microbiota that thrive under inflammatory conditions (e.g. poprphyromonas gingivalis /pg, tannerella forsythia /tf, treponema spp.) proliferate and contribute to the development and progression of periodontitis.[1,11] profound hormonal changes at the onset of puberty may re-activate a periodontal cmv infection, resulting in suppression of antibacterial immune defenses and overgrowth of exogenous-like bacteria, sech as aggregatibacter actinomycetemcomitans (aa).[6] this aa has long been known as periodontal putative pathogen. the aa and pg have ability to invade and proliferate in human gingival tissues. aggressive types of periodontitis also exhibit a close relationship with hvs. herpesviruses can multiply in gingival tissue and tend to reach higher copy counts in gingival tissue than in subgingival sites. in localized aggressive periodontitis, cmv and pg seemed to act synergistically to influence the risk for both the occurrence and the extent of the disease. antibodies against the hvs were predominantly of the iga isotype in the gingival crevice fluid, and of the igg isotype in serum.[6] the recognition that periodontitis is a multifactorial disease involving hvs, bacteria and host reactions may explain why aggressive periodontitis is relatively uncommon in most populations despite a high prevalence of individuals harboring both herpes-viruses and bacterial pathogens. it might be that periodontal tissue breakdown is because of simultaneous occurrence of a number of infections disease events as stated by kamma and slots (2003).[11] those events including 1) adequate hv load (gingivitis level) in periodontal sites, 2) activation of hvs in the periodontium, 3) inadequate protective antiviral cytotoxic t-lymphocyte response, 4) presence of specific periodontal pathogenic bacteria, and 5) inadequate protective antibacterial antibody response. archaea are widely distributed in human body. the ubiquity of hydrogenotrophs in periodontal pockets[2,9,12] shows the association of archaea domain with periodontitis is quite striking as they appear in progressively greater numbers in the subginival microbiota as a function of disease severity (i.e. probing depth and clinical attachment loss).[2] the role of h2 consumption in the regulation of the periodontal biofilm ecosystem is as interspecies hydrogen transfer for a possible force to promote proliferation of fermenting pathogens.[12] as the high level of fermenting pathogens shows the high h2 production, and methanogenic archaea indirectly promote periodontal disease in some patients by serving as a hydrogen sink. lepp et al. (2004)[3] and mirza et al. (2010)[9] reported hgt between archaea and pathogenic bacteria. gene virulence transfers with direction from archaea to bacteria. it has been shown from studies of deep periodontal pockets with high-virulence pathogenic group compared to deep periodontal pockets with non-virulence pathogenic group. the first group has severe destructive periodontal disease and abundant of methanogenic archaea. hence methanogenic archaea indirectly promote severe destructive periodontal disease. treponema species are capable of homoacetogenesis, a hydrogen-consuming process. the relative abundance of treponemal rdna was significantly lower in sites with archaeal rdna than in sites without archaeal rdna. this is suggesting that some treponema species may compete with methanogens. methanogens and treponemes may serve as alternative syntrophic partners with other members of the subgingival biofilm community.[3,9,12] this condition supported by armitage (2010)[2] which was stated that in sites with high levels of methanogenic archaea have low levels of treponema spp, and vise versa. armitage (2010)[2] stated that subgingival methanogens outcompete sulfate-reducing bacteria (srb). the competition is in h2 consumption. this clearly shows that treponema spp also hydrogen-utilizing organisms. it is concluded that archaea transfer the gene laterally to pathogens putative periodontal and may have contributed 137mustaqimah: the etio-pathogenesis of periodontal disease to virulence in bacteria. the infected inflammatory cells by herpesviruses expressed no clinical signs because these viruses still in inactive state. these infected cells enter the periodontal tissues because an event of gingival inflammation induced by highly virulence pathogenic bacteriae. these events trigger the active herpesviruses infection, and would further diminish the resistance of periodontal tissues, thereby allowing subgingival upgrowth of periodontal pathogenic bacteriae. interaction among h2 consumers and h2 producers in plaque biofilms may be as important as those in other anaerobic environments for overall functioning of this disease-associated microbial ecosystem. the role of archaea as a reservoir of a variety of metabolic innovations for bacteria. references 1. armitage gc, cullinan mp, seymour gj. comparative biology of chronic and aggressive periodontitis: introduction. periodontol 2000 2010; 53: 7–11. 2. armitage gc. comparison of the microbiological features of chronic and aggressive periodontitis. periodontol 2000 2010; 53: 70–88. 3. lepp pw, brinig mm, ouverney cc, palm k, armitage gc, relman da. methanogenic archaea and human periodontal disease. proc natl acad sci 2004; 101(16): 6176–81. 4. bartold pm, cantley md, haynes dr. mechanisms and control of pathologic bone loss in periodontitis. periodontol 2000 2010; 53: 55–69. 5. faveri m, figueiredo lc, duarte pm, mestnik mj, mayer mpa, feres m. microbiological profile of untreated subjects with localized aggressive periodontitis. j clin periodontol 2009; 36: 739–49. 6. slots j. human viruses in periodontitis. periodontol 2000 2010; 53: 89–110. 7. vernal r, leon r, silva a, van winkelhoff aj, garcia-sanz ja, sanz m. differential cytokine expression by human dendritic cells in response to different porphyromonas gingivalis capsular serotypes. j clin periodontol 2009; 36: 823–9. 8. bilichodmath s, mangalekar sb, sharma dcg, prabhakar ak, reddy sb, kalburgi nb, et al. herpesviruses in chronic and aggressive periodontitis patients in an indian population. j oral sci 2009; 51: 79–86. 9. mirza hb, anwar m, bokhari sh. in silico identification of potential horizontal gene transfer events between archaea and pathogenic bacteria. j bioinform seq anal 2010; 2(3): 36–41. 10. vickerman mm, brossard ka, funk db, jesionowski am,gill sr.phylogenetic analysis of bacterial and archaeal species in symptomatic and asymptomatic endodontic infections. j med microbiol 2007; 56: 110–8. 11. kamma jj, slots j. herpesviral-bacterial interaction in aggressive periodontitis. j clin periodontol 2003; 30: 420–6. 12. vianna me, holtgraewe s, seyfarth i, conrads g, horz hp. quantitative analysis of three hydrogenotrophic microbial groups, methanogenic archaea, sulfate-reducing bacteria, and acetogenic bacteria, within plaque biofilms associated with human periodontal diseases. j bacteriol 2008; 190(10): 3779–85. ijtid vol 1 no 3 sep-dec 2010.31.pdf ijtid vol 1 no 3 sep-dec 2010.32.pdf ijtid vol 1 no 3 sep-dec 2010.33.pdf ijtid vol 1 no 3 sep-dec 2010.34.pdf ijtid vol 1 no 3 sep-dec 2010.35.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 80 137 vol. 7 no. 6 september-december 2019 prevalence, intensity and risk factors of soil transmitted helminths infections among elementary school students in ngis village, karangasem district, bali sang ayu arta suryantari1, agung bagus sista satyarsa1, i gusti ngurah bagus rai mulya hartawan1, i kadek yana parastuta1 and i made sudarmaja2a 1 faculty of medicine of udayana university, denpasar, bali, indonesia 2 department of parasitology, faculty of medicine of udayana university, denpasar, bali, indonesia a corresponding author : made_sudarmaja@unud.ac.id abstract soil transmitted helminths (sth) infection is one of health issues in indonesia based on social and environment problems. it is classified as neglected disease. the indonesian government already has sth eradication program, but it is not supported by evaluation and monitoring program. the purpose of this study is to determine the prevalence and relation of each risk factors related to sth infections in elementary school in ngis village, karangasem regency, bali. the study was done by analytical description using cross sectional study. samples were selected from population based on inclusion and exclusion criteria. primary data about suspected risk factors were collected using questionnaire. diagnosis was established using kato-katz modification method. data were analyzed using chi-square with confidence interval 95% or p value ≤0.05 categorized as significant. 138 students was enrolled in this study, the median age is 9 (6-13) years. the prevalence of sth infections is 10.1% with 78.6% is single infection of trichuris trichiura and 21.4% mixed infections. the proportion of sth infections in males (64,3%) is higher than female (35,7%) but it is statistically non significantly different. sth infections have significant relationship with some risk factors such as unwashed hand after defecation, unwashed hand after playing with soil, barefoot, uncut nails and dewormed. the highest risk factor of sth infections in ngis village is not having available and proper latrine. (or=33.9; 95%ci=5.749-199.769). the prevalence of sth infection is quite high with mild to moderate intensity and risk factors namely low hygiene and limited latrines. the implementation of monitoring and evaluation can be an effort to control risk factors and stop the sth transmission chain. keywords: elementary school, intensity, kato-katz modification, risk factors, soil transmitted helminths (sth). abstrak soil transmitted helminths (sth) adalah salah satu masalah kesehatan di indonesia berdasarkan aspek sosial dan lingkungan yang digolongkan sebagai penyakit terabaikan (neglected disease). pemerintah saat ini telah melaksanakan program eradikasi, namun tidak didukung dengan tahap evaluasi dan monitoring (monev). penelitian ini bertujuan untuk menentukan prevalensi dan hubungan antara faktor risiko dengan kejadian infeksi sth pada siswa sekolah dasar di desa ngis, karangasem, bali. penelitian ini dilakukan dengan metode deskriptif analitik menggunakan studi cross-sectional. sampel dipilih dari populasi berdasarkan kriteria inklusi dan eksklusi. data primer mengenai faktor-faktor risiko yang dicurigai dikumpulkan dengan menggunakan kuesioner tervalidasi. diagnosis ditegakkan menggunakan metode kato-katz modifikasi. analisis data menggunakan chi-square dengan tingkat kepercayaan 95% atau dikategorikan sebagai signifikan apabila nilai p ≤0,05. sebanyak 138 siswa berpartisipasi dengan median usia 9 (6-13) tahun. prevalensi kejadian infeksi sth yakni 10,1%, dengan 78,6% infeksi trichuris trichiura dan 21,4% infeksi campuran. infeksi sth dominan terjadi pada laki-laki (64,3%) daripada perempuan (35,7%) tetapi secara statistik tidak bermakna. kejadian infeksi sth memiliki hubungan yang signifikan dengan faktor risiko seperti; tidak mencuci tangan setelah buang air besar, tidak mencuci tangan setelah bermain tanah, tidak memakai alas kaki, tidak memotong kuku dan minum obat cacing secara rutin. faktor risiko tertinggi kejadian infeksi sth pada research report 138 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 137–143 siswa di desa ngis adalah tidak adanya ketersediaan jamban (or=33,9; ik%95=5,749-199,769). prevalensi infeksi sth tergolong cukup tinggi dengan intensitas ringan – sedang dan faktor risiko yaitu rendahnya higienitas dan keterbatasan jamban. pelaksanaan monev dapat menjadi upaya untuk mengontrol faktor risiko dan menghentikan rantai transmisi sth. kata kunci: faktor risiko, infeksi sth, intensitas, kato-katz modifikasi, sekolah dasar. introduction soil transmitted helminths (sth) infection is one of the health issues in indonesia that has environment and social basis.1 inadequate sanitation, poor economic conditions, and suitable climatic conditions for worm growth support the high prevalence of helminthiasis in indonesia. sth infection is classified as neglected disease which is defined as an infection that is rarely noticed and chronic without causing obvious clinical symptoms. the impact of the infection is usually noticable in long term such as malnutrition, growth and developmental disorder, and cognitive impairment in children.2 more than two billion of the world’s population is estimated to be infected with sth. approximately 300 million of them are people with severe infections with 150 thousand cases of death due to sth infection occur every year. most infections were caused by ascaris lumbricoides of 1.2 billion, trichuris trichiura of 795 million, and necator americanus and ancylostoma duodenale as many as 740 million cases.3 the prevalence of sth infection, especially in indonesia, is still high with most infections caused by ascaris lumbricoides.4 as many as 60% to 80% of indonesia’s population is infected by sth,1 the prevalence is even higher in certain regions.1,3,5 primary school age is a high-risk group to be infected with sth.5 this is due to poor immunity and lack of awareness to live clean and healthily.6 especially in bali, the prevalence of sth infections in rural areas is still high. in the village of telaga, the prevalence of intestinal worm infections was reached 68.41% of 93 public elementary school students of telaga i and 83.87% of 72 elementary school students in telaga ii. the most prevalent infection was ascaris lumbricoides (49.65%).7 factors which cause high sth infection are poor sanitation, such as the habit of unwashed hand before eating and after defecation (defecation), uncut nails, snacking in unhygenic places, not having a decent toilet and difficult to access clean water.1-3 the impact of sth infection is quite serious, therefore an effective and efficient control strategy is needed. the world health organization (who)8 was recommended routine deworming as a major morbidity control strategy in countries with a high prevalence of helminthiasis.8 the program has already been implemented in indonesia, especially in the province of bali which still has a high prevalence of sth infection. some areas in karangasem regency still have a high incidence of sth infection even though the government has implemented a worm eradication program in the form of routine deworming in every elementary school. therefore, to increase the effectiveness of helminthiasis, valid data is needed regarding the incidence of helminthiasis, and education on the prevention of intestinal worm infections and administration of helminthic drugs to infected students is necessary.5-8 based on the exposition above, this study is to find out the prevalence and risk factors which contribute to the incidence of sth infection in elementary school children in ngis village, karangasem district, bali. material and methods this research is a descriptive analysis study with crosssectional study design. this research was conducted in three elementary schools in ngis village, manggis subdistrict, karangasem regency, bali, namely sdn 1 ngis, sdn 2 ngis, and sdn 3 ngis. the study was conducted on july 28 to august 27, 2017. the target population of this study is school-age children (6-12 years). reachable population of this study were all elementary school students in ngis village totaling 157 people. reachable populations are selected in the period of july 2017. the sampling process was not done randomly (non probability sampling) with total sampling technique. samples were selected from the population based on the inclusion criteria and exclusion criteria as follows. inclusion criteria were students who were willing to become respondents, aged 6 to 12 years, filled out validated questionnaires and collected feces. exclusion criteria were students who moved school or did not approve inform consents. research instrument research instruments in the form of tools and materials used in the study are distilled water, glycerin solution, 3% malachite green solution, physiological nacl or 2% eosin, object glass, kato-katz modification kit, 10-15 ml plastic pot (faecal pot), filter wire, toothpicks, plastic sticks, cellophane tape, wipes, waterproof markers, scissors, rubber gloves, microscopes, and questionnaires. kato-katz modification method stool pots are distributed a day before feces collection. before being given stool pots, students were given a 139suryantari, et al.: prevalence, intensity and risk factors validated questionnaire regarding worm disease risk factors. personal data on validated questionnaires were adjusted according to data on faecal pots. the amount of stool that is put into a pot is about 100 mg (as big as a marble or thumb). the procedure used in this research is the kato-katz modification method (see figure 1). this method is used to assess the degree of infection. the degree of infection established by who is defined as the number of worm eggs per gram of feces (epg). the degree category of ascaris lumbricoides infection is mild (1-4,999 epg), moderate (5000-49,999 epg) and severe (> 50,000 epg). category of degree of trichuris trichiura infection is mild (1-999 epg), moderate (1000-9.999 epg) and severe (> 10,000 epg).8 a b c d e f figure 1. kato-katz modification method.9 a) label the glass slide with the sample number and take the faecal sample from container b) place a small amount of the faecal sample on a paper c) press the faecal with wirenet to filter the debris d) place a hollow carton on slide glass then fill with faecal e) replace the cartoon with cellophane which has been soaked overnight in methylene blue glycerol solution and press the top slide firmly to spread the stool f) let the slide dries about 20-30 minutes before reading on microscope. data analysis techniques were carried out using spss software. the data that has been obtained is analyzed descriptively and analytically. first, univariate analysis is carried out, is shows data in proportion in the form of respondent characteristics presented in tables and graphs. second, continued bivariate analysis was carried out to determine the relationship between sth-infected students and risk factors analyzed using chi-square test in crosstab. odd ratio (or) analysis was carried out to determine risk factors that affect the occurrence of sth infection in elementary students in ngis village with a confidence interval (ci) of 95%. the results of bivariate analysis are presented through tables and graphs. result and discussion based on the data obtained (table 1), there were 138 students who participated in this study with 14 people (10.1%) having sth infection. male students (64.3%) have a higher proportion of infections compared to female students (35.7%). third grade students (19%) and five (13.8%) had the highest proportion of students who had sth infection. there were eleven students experiencing a single type of trichuris trichiura infection (78.6%) and the other three experienced mixed infections with ascaris lumbricoides and trichuris trichiura (21.4%). of the eleven students who had a single trichuris trichiura infection, ten students had a mild infection (90.9%) and one student had a moderate infection (9.1%). of the three people who had mixed infections with ascaris lumbricoides and trichuris trichiura, two students had a mild infection (66.7%) and one person had a moderate infection (33.3%). of all students who were infected with sth, there were no students who had severe infections. table 1. the characteristics of respondents. characteristics sth infection (n=138) positive (n=14) negative (n=124) total (n=138) age (median) 9 (6-13) 9 (6-13) 138 gender, n (%) male 9 (12,5) 63 (87,5) 72 (100,0) female 5 (7,6) 61 (92,4) 66 (100,0) grade level, n (%) grade 1 grade 2 grade 3 grade 4 grade 5 grade 6 1 (5,0) 2 (9,1) 4 (19,0) 2 (9,1) 4 (13,8) 1 (4,2) 19 (95,0) 20 (90,9) 17 (81,0) 20 (90,9) 25 (86,2) 23 (95,8) 20 (100,0) 22 (100,0) 21 (100,0) 22 (100,0) 29 (100,0) 24 (100,0) type of infection, n (%) single infection t. trichiura 11 (78,6) mixed infections a. lumbricoides and t. trichiura 3 (21,4) sth infection intensity, n (%) single infection trichuris trichiura mild moderate severe 10 (90,9) 1 (9,1) mixed infections ascaris lumbricoides and trichuris trichiura mild moderate severe 2 (66,7) 1 (33,3) 140 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 137–143 table 2. sth infection risk factors in elementary students in ngis village, karangasem, bali. sth infection positive n (%) negative n (%) p pr 95%ci gender male 9 (12,5) 63 (87,5) 0,339 0,57 0,182-1,809 female 5 (7,6) 61 (92,4) often playing with soil yes 3 (8,1) 34 (91,9) 0,631 1,38 0,364-5,270 no 11 (10,9) 90 (89,1) dewormed yes 9 (7,6) 110 (92,4) 0,012* 0,23 0,067-0,781 no 5 (26,3) 14 (73,7) unwashed hands after defecation yes 2 (66,7) 1 (33,3) 0,001** 20,50 1,730-242,98 no 12 (8,9) 123 (91,1) unwashed hands after playing with soil yes 6 (42,6) 7 (53,8) 0,000** 12,54 3,401-46,210 no 8 (6,4) 117 (93,6) barefoot yes 3 (37,5) 5 (62,5) 0,008** 6,491 1,365-30,856 no 11 (8,5) 119 (91,5) uncut nails regularly yes 3 (17,6) 14 (82,4) 0,274 2,143 0,538-8,625 no 11 (9,1) 110 (90,1) unavailable latrine yes 5 (71,4) 2 (28,6) 0,000** 33,89 5,749-199,77 no 9 (6,9) 122 (93,1) explanation: * p< 0,05, ** p< 0,01 children are one of the groups that is vulnerable to infection due to poor self-protection efforts, both in terms of the body’s immunity and adequate knowledge about hygiene.10 village children in bali often spend their time playing with the soil and this causes them to be easily infected by bacteria or parasites.11-14 the incidence of sth infection in children has brought the attention of the government. the indonesian government program currently wants to reduce the incidence of helminthiasis in children, both through counseling and deworming for six months on a regular basis, targeting primary school children in remote villages. in this study, researchers were found the prevalence of sth infection in elementary school children is 10.1%. this result is lower than the results obtained by siregar11 which is 25.7% and damayanti12 in baturiti, tabanan is 38.57%. in 2017, the nation-wide prevalence is in the range of 28.9%. this difference is caused by several factors such as time of study, geographical location, culture, social and economic conditions.11,14-16 based on the data in table 2, the results of statistical tests found that the proportion of male sex is higher than that of women experiencing sth infection. gender has no risk for the incidence of sth infection. there was a low proportion of elementary school students who were infected with sth with frequent playing behavior (8.1%). students who often play with soil are not at risk for the incidence of sth infection. there were 66.7% of students with sth infection who did not wash their hands after defecation. unwashed hands after defecation has a significant relationship to the incidence of sth infection (p <0.05). furthermore, unwashed hands after defecation can increase the risk of sth infection by twenty times with a confidence interval of 1,730-242,98. it was also found that the proportion of students infected with sth who did not wash their hands after playing with soil was 42.6%. unwashed hands after playing the soil can cause sth infection (p <0.05). this can put the student at risk for by twelve times higher. sth infection is also caused by being barefoot. the proportion of students who are barefoot with the incidence of sth infection is greater than those who have are not barefoot. this is supported by a significant relationship between being barefoot with the incidence of sth infection (p <0.05). students who are barefoot is 141suryantari, et al.: prevalence, intensity and risk factors at risk of sth infection by six times. the proportion of students infected with sth by uncut nails is greater than those that cut nails regularly. uncut nails is not a risk factor that affects the incidence of sth infection in elementary students in ngis village. there were 5 (71.4%) students who were infected with sth with unavailable latrine. the availability of toilet is one of the efforts made to protect against sth infection. this is proven through the research results which states the absence of latrines can put students at risk of sth infection by 33 times higher. a significant relationship between sth infection and unavailable latrines in elementary students in ngis village (p <0.05) was also found. there was a low proportion of sth infected elementary school students who took deworming drugs regularly. dewormed regularly is a protective effort against the occurrence of sth infection. this is proven by a significant relationship between taking deworming drugs regularly with no sth infection (p <0.05). based on some of these risk factors, there are risk factors that influence the incidence of sth infection in students in ngis village, including unwashed their hands after defecation, unwashed their hands after playing with soil, being barefoot, unavailable latrines, and dewormed regularly. in this study, researchers were found that elementary school children had previously gone through dewormed as part of government program in eradicating the incidence of sth infection in the area. this was conveyed by the head of community health center (puskesmas) ii manggis during the interview session, who stated the administration of deworming drugs was implemented as a district government program to eradicate the incidence of sth infection in elementary school children. as for the program implementation, the administration of worm medicine (albendasol dose 1x400mg) once in six months was done as an effort to eradicate or reduce the incidence of sth infection in children. the administration of these drugs is carried out routinely and is carried out directly, meaning the administration of deworming drugs was done in the classroom to avoid students who do not take deworming drugs (program in 2016). this program has been carried out twice in december 2016 and in july 2017. however, the implementation of this program only provides deworming drugs directly to children, without educating them on basic health hygiene practices, such as washing hands with soap after playing. in addition, there is a limitation to this program, namely the lack of monitoring and evaluation during program implementation. it also becomes an obstacle in determining whether the program is implemented properly or not. based on the results of our study, it was found that only 10.1% of children infected with sth. it is below the nation-wide prevalence of sth infection in indonesia by 25.7%, thus this program had an effect on decreasing the incidence of sth infection in children.17,18 furthermore, the intensity or degree of infection of children who have sth infection is mild to moderate according to the results of calculations with the kato-katz modification method.15 this program can be continued to reduce the incidence of sth infection in children especially in karangasem district, bali. in this study, it was found that children who had sth infection lacks knowledge about self hygiene. it was proven that the hygiene of children from the habit of hand washing and playing outside the house was known to be significant in causing the incidence of sth infection. another risk factor which caused the occurrence of sth infection is hygiene after defecation, because children often forget to wash their hands after defecation. infection is transmitted through dirty hand nails which mediate the entry of worm eggs into the body. in addition, the habit of unwashed hands after defecation can be a strong risk factor in children having sth infection. it was found that playing with soil often is not a risk factor for sth infection in elementary students in ngis village. these results are differ from the research results by samad who found contamination of ascaris lumbricoides eggs in children who enjoy playing with soil.19 it is also inversely proportional to juhairiyah’s findings which was stated that the habit of playing with soil will increase the risk of sth infection.20 these results may be influenced by children who no longer play with soil as often. it was also found that the proportion of students infected with sth who did not wash their hands after playing the soil was 42.6%. not washing hands after playing the soil can increase risk of being infected with sth by twelve times in elementary students. these results are in line with the research results obtained by wiryadana et al.14 in addition, which researcher also was found that children who are barefoot when they are outside the house was also significantly associated with the incidence of sth infection. being barefoot can cause the occurrence of ascaris lumbricoides infection. in line with kartini’s21 research which was stated that is a relationship between children who were barefoot against sth infection, but in wiryadana et al.14 study, no significant results were found.14,21 researcher was also found that children who did not have latrines had a significant association with the incidence of sth infection. however researcher was found the protective value of children who have latrines. supported by the research results of wiryadana et al.,14 children who have latrines are not infected with sth due to the inability of worms to develop in the latrine whereas on the ground it can develop and re-infect.14 this study was found that there were still children who did not have latrines at home. this is the biggest risk factor for the incidence of sth infection. countermeasures are needed in the form of latrines for families who do not have latrines as an effort to reduce the incidence of sth infection.14,20,21,22 in this study, researchers were also found that elementary school children had been dewormed before. researchers were found that taking deworming drugs had a protective relationship to sth infection. this is supported by the results of research conducted by the researcher, which found that dewormed regularly can prevent students from 142 indonesian journal of tropical and infectious disease, vol. 7 no. 6 sept-dec 2019: 137–143 getting sth infection.14 therefore, regular administration of deworming drugs can be a prophylactic effort to prevent children from having sth infection. however, there were some children who had mild sth infection even though they had been dewormed. it is also part of the who program to eradicate sth infections.21 the sth eradication program should be implemented as the government’s priority program. the development of this program will be good if proper monitoring and evaluation can be carried out. in eradicating sth infection, information and health education is needed for both parents and children as well as all components of society. collaboration and good performance in this program will make the target of eradicating sth a success.14,20-22 the limitations of this study are the distance between the location of the research and the examination laboratory. due to the unavailability of the epidemiological data from bali and indonesia, this study was conducted with a crosssectional design. at present, it is not possible to carry out studies with a more comprehensive design. thus, this study was used a cross-sectional design which could not assess the risk factors but limited only to the incidence of sth. furthermore, the researchers collect only one specimen in each child which may be different from the worm egg count in that child later on. conclusion based on the results of the study and the discussion above, it can be concluded that the prevalence of sth infection in elementary students in ngis village is 10.1%. intensity of sth infection occurs with mild to moderate. risk factors that cause the incidence of sth infection in students in ngis village are unwashed hands after defecation, unwashed hands after playing with soil, being barefoot, unavailable latrines, and not dewormed regularly. researcher was found a decrease in the prevalence of sth infection compared to the national prevalence rate. the knowledge in prevention and attitude of elementary school students are fairly good but efforts are needed to improve hygiene in order to prevent the onset of infection. monitoring and evaluation efforts from the community health center (puskesmas) are also needed to maximize the efforts to eradicate sth infection. acknowledgements the researcher would like to thank and the katokatz modification method. researcher also thank i made marta pradnyana and i komang tri as the chairman of the research and community service (peniti 2017), one of the program of the of the medical student association (hmku) of fk unud, as well as the rest of the committee members who helped during the research. researcher also thank the laboratory staff at the laboratory of parasitology fk unud who have helped and guided the researchers in the examining the worm eggs using modified kato-katz method. references 1. pullan rl, smith jl, jasrasaria r, brooker sj. global numbers of infection and disease burden of soil transmitted helminth infections in 2010. parasites & vectors. 2014 dec;7(1):37. 2. zerdo z, yohanes t, tariku b. soil-transmitted helminth reinfection and associated risk factors among school-age children in chencha district, southern ethiopia: a cross-sectional study. journal of parasitology research. 2016;2016. 3. idris oa, wintola oa, afolayan aj. helminthiases; prevalence, transmission, host-parasite interactions, resistance to common synthetic drugs and treatment. heliyon. 2019 jan 1;5(1):e01161. 4. ganguly s, barkataki s, karmakar s, sanga p, boopathi k, kanagasabai k, et al. high prevalence of soil-transmitted helminth infections among primary school children, uttar pradesh, india, 2015. infectious diseases of poverty. 2017;6(1). 5. yuwono n, husada d, basuki s. prevalence of soil-transmitted helminthiasis among elementary children in sorong district, west papua. indonesian journal of tropical and infectious disease. 2019 feb 22;7(4):86-91. 6. simarmata n, sembiring t, ali m. nutritional status of soiltransmitted helminthiasis-infected and uninfected children. paediatrica indonesiana. 2015;55(3):136. 7. devi np, sudarmaja im, swastika k. prevalensi infeksi soil transmitted helmith di sekolah dasar negeri 1 padangbulia kecamatan sukasada kabupaten buleleng, bali-indonesia. intisari sains medis. 2018;9(3): 59-61. 8. who. soil-transmitted helmithiases. in: sustaining the drive to overcome global impact of neglected tropical diseases: second who report on neglected tropical disease. geneva: who; 2013: 106-111. 9. miércoles. técnica de kato katz para la cuantificación de huevos de helmintos [online]. laboratorio de parasitología 2009 [cited 2018 jun 28]. available from: http://parasitosintestinalesguatemala.blogspot. com/2009/09/comentarios.hhtm. 10. montresor a. helminth control in school-age children. geneva: world health organization; 2011. 11. siregar m. kejadian infeksi sth dan gambaran kepemilikan jamban dan air bersih pada anak usia sekolah dasar di yayasan nanda dian nusantara 2011 [thesis]. jakarta: universitas islam negeri syarif hidayatullah; 2012. 12. damayanti pa. pengobatan dan penilaian status gizi anak sdn 1 luwus, baturiti yang menderita cacingan (soil-transmitted helminthiasis). buletin udayana mengabdi. 2009;12(1). 13. diarthini nl, swastika ik, ariwati l, isyaputri r, 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 oct 31;7(3):57-61. 14. wiryadana k, putra i, rahayu p, pradnyana m, adelaida m, sudarmaja i. risk factors of soil-transmitted helminth infection among elementary school students. paediatrica indonesiana. 2018;57(6):295. 15. endris m, tekeste z, lemma w, kassu a. comparison of the kato-katz, wet mount, and formol-ether concentration diagnostic techniques for intestinal helminth infections in ethiopia. isrn parasitology. 2012 oct 22;2013. 16. ginandjar p, saraswati l, martini . soil-transmitted helminth infection in elementary school children: an integrated environment and behavior case study in bandungan sub-district, semarang district. advanced science letters. 2017;23(4):3565-3568. 143suryantari, et al.: prevalence, intensity and risk factors 17. elmi, sembiring t, dewiyani bs, hamid ed, pasaribu s, lubis cp. status gizi dan infestasi cacing usus pada anak sekolah dasar. bagian ilmu kesehatan anak fk usu. [cited 2018 june]. available from:www.repository.usu.ac.id. 18. hackenberger d, palijan g, lončarić ž, jovanović glavaš o, hackenberger b. influence of soil temperature and moisture on biochemical biomarkers in earthworm and microbial activity after exposure to propiconazole and chlorantraniliprole. ecotoxicology and environmental safety. 2018;148:480-489. 19. marlina l, widjaja j. hubungan pendidikan formal, pengetahuan ibu dan sosial ekonomi terhadap infeksi soil transmitted helminths pada anak sekolah dasar di kecamatan seluma timur kabupaten seluma bengkulu. indonesian journal of health ecology. 2012;11(1). 20. juhairiyah j, annida a, indriyati l. gambaran faktor resiko kecacingan pada anak sekolah dasar di kota banjarmasin. jurnal vektor penyakit. 2016;9(1):21-27. 21. kartini s. kejadian kecacingan pada siswa sekolah dasar negeri kecamatan rumbai pesisir pekanbaru. jurnal kesehatan komunitas. 2016;3(2):53. 22. blum aj, hotez pj. global” worming”: climate change and its projected general impact on human helminth infections. plos neglected tropical diseases. 2018 jul 19;12(7):e0006370-. 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. 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 ijtid vol 8 no 2 may-agustus 2020_newfromsarah.indd vol. 8 no. 2 may–august 2020 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 case report recurrent giant condylomata acuminata caused by human papilloma virus in hiv with homosexual male 1emy kusumaningsih, 2lita setyowatie 1department of dermatology and venereology, faculty of medicine, universitas brawijaya, malang, east java, indonesia 2dr. saiful anwar regional, general hospital, malang, east java, indonesia received: 21st december 2018; revised: 15th october 2019; accepted: 11th february 2020 abstract perianal giant condylomata acuminate (gca) is a rare clinical condition associated with low-risk human papillomavirus (hpv) type 6 and 11 infections. human immunodefi ciency virus (hiv) infection is one of the risk factors for gca, that can increase the condylomata acuminate incidence and spread caused by hpv. a 28-year-old man came with a caulifl owerlike mass complaint in his perianal and anal since 2 months ago. the patient did not complain of pain or itching on the mass but often bled when defecating. the patient is a male who has sex with men (msm) and often changes partners. he has been diagnosed with hiv since 11 months ago and regularly taking anti-retroviral drugs, efavirenz 600 mg daily. he was also diagnosed having lung tuberculosis at the same time, got 6 months treatment and was declared cured. the venereological examination of the perianal and anal region revealed erythematous and grayish stem-shaped vegetation and papules, verrucous surface, multiple, well defi ned, with 3 x 1.5 x 2 cm in size. a positive act of white examination was obtained. blood tests revealed cd+4 230 cells /μl. polymerase chain reaction (pcr) examination for hpv obtained hpv types 6 and 11 infections. histopathologic examination revealed acanthosis, papillomatosis, and hyperkeratotic epidermis and koilocytotic cells. the patient was treated with electrodesiccation three times but obtained mass in anal getting bigger with a size of 6 x 3 x 3 cm. therefore, he agreed to be referred to the surgical department with an extensive surgical excision plan. screening of gca using pcr is not a routine examination but pcr has high sensitivity and specifi city for determining the type of hpv, is useful for determining gca prognosis and therapy, and is recommended for malignant and possible gca recurrence detection. keywords: giant condylomata acuminate, hpv, recurrent, hiv, msm abstrak perianal giant condylomata acuminata merupakan kondisi klinis yang jarang dan dihubungkan dengan infeksi rekuren human papillomavirus (hpv) low-risk tipe 6 dan 11. infeksi human immunodefi ciency virus (hiv) merupakan salah satu faktor risiko gca, yang dapat meningkatkan risiko kejadian kondilomata akuminata dan penyebaran yang disebabkan oleh hpv. laki-laki 28 tahun datang dengan keluhan benjolan seperti bunga kol di anus dan sekitar anus sejak 2 bulan yang lalu. pasien tidak mengeluhkan nyeri maupun gatal pada benjolan tersebut, namun sering berdarah saat buang air besar. pasien berhubungan seksual dengan sesama jenis dan sering berganti pasangan. pasien telah didiagnosis hiv sejak 11 bulan yang lalu dan rutin minum anti-retroviral, efavirenz 600 mg setiap hari.pasien juga didiagnosis menderita tuberculosis paru pada saat yang bersamaan, mendapatkan 6 bulan terapi dan dinyatakan sembuh. pemeriksaan venereologis pada regio perianal dan anal didapatkan vegetasi bertangkai serta papul-nodul eritematous dan keabuabuan, permukaan verukosa, multipel, batas tegas, ukuran 3 x 1,5 x 2 cm. pemeriksaan acetowhite positif. pemeriksaan darah cd+4 230 sel/μl. pemeriksaan polymerase chain reaction (pcr) untuk hpv, didapatkan hasil hpv tipe 6 dan 11. pemeriksaan histopatologis didapatkan epidermis akantosis, papilomatosis, hyperkeratosis dan sel-sel koilositosis. pasien diterapi dengan elektrodesikasi sebanyak 3x namun didapatkan benjolan semakin membesar dengan ukuran 6 x 3 x 3 cm sehingga pasien setuju dirujuk ke bagian bedah dengan rencana wide surgical excision. skrining gca dengan menggunakan pcr bukanlah pemeriksaan yang * corresponding author : emy.kusumaningsih@gmail.com 132 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: 131–136 rutin dilakukan, namun pemeriksaan pcr ini mempunyai sensitivitas dan spesifi tas tinggi untuk menentukan tipe hpv yang berguna untuk menentukan prognosis serta terapi gca dan disarankan untuk deteksi keganasan serta deteksi kemungkinan rekurensi gca. kata kunci: giant condylomata acuminata, hpv, rekurensi, hiv, lsl how to cite: kusumaningsih, emy., & setyowatie, lita. (2020). recurrent giant condylomata acuminata caused by human papilloma virus in hiv with homosexual male. indonesian journal of tropical and infectious disease, 8(2), 1–8. introduction the incidence of anogenital condylomata acuminata (ca) has increased in the past decades and is, to date, the most common sexually transmitted disease in western countries. condylomata acuminata is correlated with lowrisk human papillomavirus (hpv) type 6 and 11 infections, whereas high-risk hpv type 16 is frequently present in anogenital malignant lesions.1 perianal giant condyloma acuminatum (gca) is a rare clinical condition related to hpv infection and characterized by a circumferential, exophytic, caulifl ower-like mass with an irregular warty surface localized in the anal region.1 the giant form of this disease has a rare event rate, no more than 0.1% genital warts. most of the incidence attacks middle-aged men, with a male-to-female ratio at 3:1.2 risk factors for gca include anoreceptive intercourse, human immunodeficiency virus (hiv), and immunosuppression.3 human immunodefi ciency virus infection is a predisposition that increases the ca incidence and spread caused by hpv.4 many examination techniques are used to detect hpv infection. to mention one is polymerase chain reaction (pcr) technique. by pcr, it is now possible to amplify enzymatically specifi c target deoxyribonucleic acid (dna) sequences to higher levels so that they are now readily detectable by additional methods to detect the type of hpv.5 detection and subsequent hpv types have a profound role in assessing the prognosis and therapy of genital lesions and evaluation of effi cacy therapy.5 classifi cation of hpv infection types is important for the identifi cation of patients at risk of developing malignant transformation and for the detection recurrence rates of gca.5,17 a case of giant condylomata acuminata caused by hpv types 6 and 11, identifi ed by pcr techniques in a 28-year-old male patient with hiv-infected who had sex with men (msm). case report a man, 28 years old, came to dermatovenereology’s outpatient clinic of saiful anwar regional general hospital (rssa) malang with a complaint of caulifl ower-like mass on his anal and perianal since 2 months ago. it initially appeared as a small bump that got bigger in both anal and perianal, and some reddish and some brownishgray in color. there was no itching or pain in the bumps. the caulifl ower-like mass was rapidly enlarged. three days before his visit, the patient felt diffi cult to defecate due to the mass getting bigger and bled after defecation, accompanied by an unpleasant odor. the patient had a history of similar complaints 2.5 years ago, initially obtained small bumps around the anal, enlarged within a year. the bump in the anal was also getting bigger, and the patient complained often of bleeding after defecation. he checked to the private hospital and was diagnosed as “giant condylomata acuminate.” he was referred to the surgical department in rssa and performed surgery in august 2017 (6 months ago). the complaint reappeared 4 months later. the patient had been diagnosed with hiv since march 2017, and routinely taking antiretroviral (arv), efavirenz 600 mg daily, from internal department’s outpatient clinic of a private hospital in malang. he was also diagnosed 134 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: 131–136 at the follow-up, fi ve weeks after the patient back from abroad, mass in the anal region grew larger by 6 x 3 x 3 cm (figure 2). therefore, the patient agreed to be referred to the surgical department with an extensive surgical excision plan. discussion giant condylomata acuminata (gca) is a slow-growing, large, caulifl ower-like tumor with locally destructive behavior that typically appears in the anogenital region.3,6,8 originally described as a penile lesion by buschke in 1896 and lӧwenstein in 1925, it is a genital infection caused by human papillomavirus (hpv) types 6 and 11.7 the fi rst description of anorectal gca was by dawson et al. in 1965. giant condylomata acuminata is a rare lesion tending to present in the fi fth decade with a 2.7:1 male: female ratio. for patients under 50 years old, this ratio increases to 3.5:1.3,8 in some cases, series of these lesions have a high recurrence rate of between 18% and 67%, with an overall mortality rate of 21%.9 according to some literature, gca is a low-grade and welldiff erentiated squamous cell carcinoma. giant condylomata acuminata or verrucose carcinoma should be considered as a diff erential diagnosis in lesions larger than 1 cm.7 risk factors of gca include anoreceptive intercourse, hiv and immunosuppression. the prevalence of hpv infection in the anal is very high, around 57% in men with human immunodefi ciency virus (hiv)-negative who have sex with men (msm); and among people, with hiv-positive infections, the incidence rate is about 60 times higher than in the general male population.9 in this case report, the patient is experiencing an msm for approximately 8.5 years, acted as a “bottom” and rarely used condoms. there were lesions in the form of stemmed vegetation with the largest size of 3 x 1.5 x 2 cm in the perianal and anal region. the patient was also diagnosed with hiv-positive and took arv daily. the anal disease is a common disease in patients with hiv infection, especially in msm patients.10 anal hpv infection and anal intraepithelial neoplasia (ain) are more common in hiv-positive compared to hiv-negative msm.11 recurrent anal condylomata are stronger with hiv and cd+4 lymphocytopenia compared to hpv persistence indicating that people with hiv-negative can clear the virus more easily.12 presumably, there is a complex interaction between hiv, hpv and local mucosal immune mechanisms. hiv increases hpv transcription and resets hpv e7 which affects cellular diff erentiation, leading to higher amounts of hpv dna in the tissue.9 furthermore, hpv causes a decrease in the number of local macrophages, langerhans and cd+4 cells and decreases local cytokine production, which results in impaired local immunity control against hpv infection.9 since hiv appears to increase hpv replication, one would expect that antiretroviral therapy initiation with future suppression of hiv viral load should lead to a decrease in the amount of hpv in the infected mucosa, followed by clinical improvement. it has been reported that a paradoxical case illustrates the impairment of gca as a consequence of immune reconstitution syndrome after arv, in patients with low cd+4 counts at the beginning of treatment (50 / mm3) .13 a study of hiv positive women showed that antiretroviral drugs could reduce the incidence of genital warts and vulvar intraepithelial neoplasia and this eff ect was mediated through an increase in cd+4 count and hiv viral load reduction .14 histologically, gca appears to be similar to condyloma, but grows both upward and downward and indicates a local invasion.15 in a limited biopsy, the pathologist may only see hyperkeratotic benign epithelium, but the fully developed lesions exhibit an exophytic and endophytic growth pattern.15 knowledge of hpv is obtained through several examinations such as cytological examination, histopathology, immunohistochemistry, molecular hybridization, and pcr.16 polymerase chain reaction techniques have high sensitivity and specificity. they can be used to amplify and sequence dna viral processes and to determine the type of hpv that is defi ned as dna sequence homology. pcr 133emy kusumaningsih, et al.: recurrent giant condylomata acuminata caused by human papilloma virus copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 with pulmonary tuberculosis (tb) at the same time, received complete tb treatment for up to 6 months and was declared cured in september 2017. the patient has had sex with men (msm) since his age of 17 years. the patient acts as a “bottom”. he claimed to have had a pair of 7 men known through social media applications. the patient and his couple rarely use condoms during intercourse. the last time he had sex was around 2.5 years ago. currently, the patient works as an entrepreneur. a general examination of the patient showed mild illness. vital signs were within normal limits. venereological examination of the corpus penis, glans penis, ostium urethra external, and scrotum was within normal limits. preputium has been circumcised. the perianal and anal region revealed stemmed vegetation and erythematous to grayish papules, verrucous surfaces, multiple, well defi ned, varying in size with the largest size at 3 x 1.5 x 2 cm (figure 1). acetowhite test using 5% acetic acid revealed the mass changed becoming paler. blood and urinalysis examination revealed normal limits, while cd+4 was 230 sel/μl. hpv dna genotyping was performed using the pcr method, as tissue was taken from warts in the anal region. it found that the mass was due to types 6 and 11hpv infection. histopathologic examination taken from mucocutaneous lesions in the perianal region, f o u n d : a c a n t h o s i s , p a p i l l o m a t o s i s , a n d hyperkerathosisepidermis. there were also koilocytosis cells, whereas in the dermis layer there was no abnormality. no malignancy was found in the tissues. the conclusion was a condylomata acuminata. having diagnosed as giant condylomata acuminata, the patient was treated with electrodesiccation on genital warts in the perianal. meanwhile, in anal warts due to extensive bleeding, electrodesiccation was done gradually. he was educated to routinely treat wounds and maintain hygiene. the evaluation was done every two weeks. in the second week, evaluation for the rest of the electrodesiccation had dried up. after three times electrodesiccation, the mass in the anal region was getting bigger and bled easily with a size of 4 x 2.5 x 2.5 cm. since the patient went abroad, the electrodesiccation was postponed. figure 1. anal and perianal region revealed caulifl ower-like mass. figure 2. follow up of the 5th week, mass in the anal area grew larger by 6 x 3 x 3 cm. 135emy kusumaningsih, et al.: recurrent giant condylomata acuminata caused by human papilloma virus copyright © 2020, ijtid, p-issn 2085-1103, e-issn 2356-0991 examination requires only 10 copies of hpv.16 because information on the type of hpv is clinically useful for prognosis and treatment of condyloma, molecular epidemiology of hpv using the pcr method has been widely used. clinical classifi cation of hpv types is important for identifying patients at risk of developing malignant transformation and detection risk of gca recurrence.7 the result of the pcr examination of the patient showed that his gca was caused by multiple infections, namely types 6 and 11 hpv. cong x et al. (2015)17 conducted a study of hpv type correlation and clinical features in patients with ca in china and found out that multiple hpv infection results in the formation of largersize ofca (gca) and associated with higher recurrence rates, and extended disease course.17 this corresponds to a patient’s history that 6 months ago the patient had undergone surgery at rssa for his gca in the anal region and then started growing again 4 months later. the patient, in this case, was then referred to the surgical department for wide surgical excision. the treatment choice for the management of gca is considered a wide surgical excision.18 surgical excision alone has been shown to result in a disease-free state in up to 46% of cases.18,19 oral and topical chemotherapeutic modalities can be used as an adjuvant, to surgery. some factors that need to be taken into account during treatment choice include the size and thickness of the lesion, anatomic site, associated hpv subtype, and immune status.18,19,20,21 the direct-applied modalities that are targeted to remove warts locally do not destroy all the very small or subclinical lesions in the surrounding healthy-looking skin and this may be the cause of recurrence.20,21,22 the polymerase chain reaction was not a routine examination for gca. nevertheless, hivinfected men with anal condylomatous lesions were at high risk of having high-grade squamous intraepithelial lesions and harboring multiple hpv infections involving high-risk hpv types in the canal anal in comparison to hiv-infected men without condylomata. these data emphasize the importance of screening and follow-up of condylomata in the anal canal in hiv-infected men. one of the screenings is using pcr to determine the type of hpv.23,24,25 conclusions the 28-year-old male patient, msm, has been reported with recurrent giant condylomata acuminata and hiv positive. the patient was then referred to the digestive surgical department for wide surgical excision. recurrent gca in this patient may root in his immunosuppression condition due to hiv infection, multiple infections of some hpv types, or previous operations that were not completely clean. polymerase chain reaction genotyping of hpv dna obtained types 6 and 11hpv. screening of gca using pcr is not a routine examination but it is very important to determine prognosis, therapy and possible of gca recurrence. acknowledgment special thanks to dermatology and venereology departement medical faculty, universitas brawijaya, malang. conflict of interest there is no confl ict of interest of this study. references 1. guttadauro a, chiarelli m, macchini d, frassani s, maternini m, bertolini a, gabrielli f. circumferential anal giant condyloma acuminatum: a new surgical approach. diseases of the colon & rectum. 2015; 58(4): e49–52. 2. akhavizadegan h. electrocautery resection, shaving with a scalpel, and podophyllin: a combination therapy for giant condyloma acuminatum. the world journal of men’s health. 2015; 33(1): 39–41. 3. de toma g, cavallaro g, bitonti a, polistena a, onesti mg, scuderi n. surgical management of perianal giant condyloma acuminatum (buschke-löwenstein tumor). european surgical research. 2006; 38(4): 418–22. 4. murtiastutik d. kelainan infeksi menular seksual pada infeksi hiv. dalam: barakbah j, lumintang h, 136 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: 131–136 martodihardjo s, editors. buku ajar infeksi menular seksual. surabaya: airlangga university press; 2008. h. 260–268. 5. mills a, balasubramaniam r, longacre t, kong c, pinsky b. laboratory-developed l1 sequencing and type-specifi c, real-time polymerase chain reaction for the detection and typing of human papillomaviruses in formalin-fixed, paraffin-embedded tissues. archives of pathology & laboratory medicine. 2013; 137(1): 50–54. 6. chao mw, gibbs p. squamous cell carcinoma arising in a giant 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immune defi ciency syndromes. 2012; 59(3): 259–265. 12. arany i, evans t, tyring sk. tissue-specifi c hpv expression and downregulation of local immune responses in condylomas from hiv seropositive individuals. sexually transmitted infections. 1998; 74(5): 349–53. 13. moussa r, stephenson i, fisk p, dhar j, nicholson kg, wiselka mj. buschke–loewenstein lesion: another possible manifestation of immune restoration infl ammatory syndrome?. aids. 2004; 18(8): 1221–3. 14. massad ls, silverberg mj, springer g, minkoff h, hessol n, palefsky jm, strickler hd, levine am, sacks hs, moxley m, watts dh. eff ect of antiretroviral therapy on the incidence of genital warts and vulvar neoplasia among women with the human immunodefi ciency virus. american journal of obstetrics & gynecology. 2004; 190(5): 1241–8. 15. martin jm, molina i, monteagudo c, marti n, lopez v, jorda e. buschke-lowenstein tumor. journal of dermatological case reports. 2008; 2(4): 60. 16. koutsky la, kiviat nb. genital human papillomavirus. in: holmes kk, sparling pf, lemon sm, stamm we, piot p, wasserheit jn, editors. sexually transmitted disease. 3rd ed. new york: mc graw-hills; 1999. p. 347–59 17. cong x, sun r, zhang x, wang y, wang l, yu y. correlation of human papillomavirus types with clinical features of patients with condyloma acuminatum in china. international journal of dermatology. 2016; 55(7): 775–80. 18. lilungulu a, mpondo bc, mlwati a, matovelo d, kihunrwa a, gumodoka b. giant condyloma acuminatum of vulva in an hiv-infected woman. case reports in infectious diseases. 2017; 2017. 19. mistrangelo m, cornaglia s, pizzio m, rimonda r, gavello g, dal conte i, mussa a. immunostimulation to reduce recurrence after surgery for anal condyloma acuminata: a prospective randomized controlled trial. colorectal disease. 2010; 12(8): 799–803. 20. silvera rj, smith ck, swedish ka, goldstone se. anal condyloma treatment and recurrence in hivnegative men who have sex with men. diseases of the colon & rectum. 2014; 57(6): 752–61. 21. leszczyszyn j, lebski i, lysenko l, hirnle l, gerber h. anal warts (condylomata acuminata)-current issues and treatment modalities. adv clin exp med. 2014; 23(2): 307–11. 22. ockenfels hm. therapeutic management of cutaneous and genital warts. jddg: journal der deutschen dermatologischen gesellschaft. 2016 sep; 14(9): 892–9. 23. thomas r, steben m, greenwald z, stutz m, rodier c, deangelis f, rampakakis e. recurrence of human papillomavirus external genital wart infection among high-risk adults in montréal, canada. sexually transmitted diseases. 2017 nov 1; 44(11): 700–6. 24. darwich l, cañadas mp, videla s, coll j, piñol m, cobarsi p, molina-lópez ra, vela s, garcía-cuyás f, llatjos m, sirera g. condylomata, cytological abnormalities and human papillomavirus infection in the anal canal in hiv-infected men. hiv medicine. 2012 oct 1; 13(9): 549–57 25. zou h, tabrizi sn, grulich ae, hocking js, bradshaw cs, cornall am, morrow a, prestage g, law mg, garland sm, chen my. site-specifi c human papillomavirus infection in adolescent men who have sex with men (hyper): an observational cohort study. the lancet infectious diseases. 2015 jan 1; 15(1): 65–73. 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. 1 january–april 2021 available online at ijtid website: https://e-journal.unair.ac.id/ijtid/ ijtid, p-issn 2085-1103, e-issn 2356-0991 original article diagnosis based on detection of cxcl10 in urine as biomarker for the determining diagnosis of active lung tuberculosis i gede yogi prema ananda1, ni made mertaniasih2*, soedarsono3, deby kusumaningrum2 1faculty of medicine universitas airlangga, surabaya indonesia 2 department of medical microbiology, faculty of medicine universitas airlangga, surabaya indonesia 3 department of pulmonology and respiratory medicine, faculty of medicine, universitas airlangga, surabaya indonesia received: 24th september 2020; revised: 4th february 2019; accepted: 9th february 2021 abstract tuberculosis diagnosis is an important component in decreasing tb incidence and prevalence. because of the difficulty to collect sputum in some cases, urine specimens are used as it is easier to garner. one of the biomarkers in urine that can be used to diagnose pulmonary tb is ip-10, which can be represented by the cxcl10 gene. the study aims to determine the accuracy of diagnosis based on detection of the cxcl10 gene in urine as a biomarker for the patients with suspected pulmonary tb in dr. soetomo hospital in surabaya from november 2019 until march 2020. thus, this is an observative laboratory research with a cross-sectional study. cxcl10 gene was examined using pcr for 36 urine samples, and then, the data, together with the medical records of clinical manifestations of pulmonary tb, genexpert mtb /rif, blood count, and thorax radiograph, were processed using ibm spss statistics 26. the results of the genexpert mtb/rif and thorax radiograph criteria show positive results of pulmonary tb, which were 44.4% and 69.4% respectively. cxcl10 gene was not found in all urine of healthy people (negative), while 2.8% (1/36 samples) positive cxcl10 gene was found in a patient with positive genexpert, also with negative clinical manifestations and urine culture. in this study, the accuracy of diagnosis based on detection of the cxcl10 gene in urine for diagnosis of active pulmonary tb was 2.8%. future research is needed to improve the methods, among them are bigger size of urine samples and clearer medical history of patients. abstrak diagnosis tuberkulosis merupakan komponen penting dalam menurunkan insiden dan prevalensi tb. karena sulitnya mengumpulkan dahak pada beberapa kasus, spesimen urin digunakan karena lebih mudah didapatkan. salah satu biomarker dalam urin yang dapat digunakan untuk mendiagnosis tb paru adalah ip-10 dengan cara mendeteksi keberadaan gen cxcl10. penelitian ini bertujuan untuk mengetahui akurasi diagnosis berdasarkan deteksi gen cxcl10 dalam urin sebagai biomarker untuk diagnosis pasien tb paru di rsud dr. soetomo surabaya dari november 2019 hingga maret 2020. oleh karena itu, penelitian ini termasuk penelitian laboratorium observatif dengan studi cross-sectional. pemeriksaan gen cxcl10 dilakukan menggunakan pcr, kemudian data, bersama dengan hasil rekam medis manifestasi klinis tb paru, genexpert mtb/rif, menghitung darah, dan rontgen dada, diolah menggunakan ibm spss statistics 26. hasil kriteria genexpert mtb/rif dan rontgen dada menunjukkan hasil positif masing-masing 44,4% dan 69,4% tb paru. semua urine orang sehat menunjukkan hasil gen cxcl10 negatif, didapatkan hasil sebesar 2,8% gen cxcl10 positif dalam urin pasien dengan genexpert positif dengan manifestasi klinis dan kultur urin negatif. dalam penelitian ini akurasi diagnosis berdasarkan deteksi gen cxcl10 dalam urin untuk diagnosis tb paru aktif adalah 2,8%. penelitian lebih lanjut dibutuhkan untuk meningkatkan metode yang diguna digunakan, terutama agar menggunakan lebih banyak sampel urin dan riwayat pasien yang lebih jelas. * corresponding author: nmademertaniasih@gmail.com open acces under cc-by-nc-sa share alike 4.0 keywords: tuberculosis; cxcl10; biomarker; urine; diagnosis ijtid, p-issn 2085-1103, e-issn 2356-0991 58 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 57–65 kata kunci: tuberkulosis; cxcl10; biomarker; urin; diagnosis how to cite: ananda, igyp., mertaniasih, nm., soedarsono., kusumaningrum, d.diagnosis based on detection of cxcl10 in urine as biomarker for the determining diagnosis of active lung tuberculosis. indonesian journal of tropical and infectious disease, 9(1), 57–65. introduction tuberculosis (tb) is a pulmonary infectious disease caused by mycobacterium tuberculosis and one of the top 10 causes of death as well as the number one cause of death from infection in the world. in 2017, 1.3 million people died from tb, and 10 million people were infected with tb.1 again, in 2018, as many as 1.3 million people died from tb and 10 million people were infected.2 indonesia is one of the 20 countries with the most tb cases, with 845 thousand people infected and 563 thousand diagnosed, and 98 thousand of them died from tb in 2018. indonesia is included in high burden countries (hbc), countries with a high burden of tb based on 3 indicators, namely tb, hiv-tb coinfection, and mdr-tb. as indonesia is included in all indicators, tb becomes one of the main health problems in indonesia.2 in indonesia, the detected and reported tb cases were 53% in 2017.1 and then, it increased to 67% in 2018.2 of the unreported cases, 29% were detected but not reported and 18% were not detected at all. java and bali are the regions with the highest number of unreported tb cases, which is at 42%. puskesmas as primary care in indonesia has 15% of unreported cases, relatively lower than cases not reported by hospitals, which reaches 65%, and the highest by a combination of general practitioners, clinics, and laboratory practices was 96%.1 indonesia targets to increase tb disease control by decreasing the number of people with tb disease from 293 people per 100,000 population in 2013 to 245 people per 100,000 population in 2019 (4). indonesia also targets tb elimination by 2035 and tb-free indonesia by 2050.5 diagnosis is an important component in achieving the target of reducing tb incidence and prevalence. diagnosis of pulmonary tuberculosis begins with clinical criteria, chronic cough symptoms for more than 2 weeks, accompanied by fever, night sweats, and weight loss. for a country with a high tb prevalence such as indonesia, all patients with suspected pulmonary tb clinical criteria are immediately diagnosed with pulmonary tb disease. the problem is that not a ll patients showing symptoms of chronic cough proved to be acid resistant basil (afb) positive, and vice versa. data shows that 10-25% of patients with positive smear do not show symptom s of cough.3 the most common laboratory microscopic examinations are the sputum smear using the ziehl-neelsen staining technique and the genexpert mtb/rif molecular rapid test.3 both of these methods have disadvantages, that it is difficult for the patient to pass sputum, and consequently, there have not been enough sputum specimens collected for examination. a method of examination using specimens that is easier to collect, such as urine, is needed. it is easier to ask the patient to urinate than to expel phlegm. besides, urine collection is noninvasive, not too risky for the medical personnel involved, and requires no special equipment or expertise. one of the biomarkers in urine that can be used for diagnosis of pulmonary tb is ip-10, or interferon-gamma(ifn-γ)-inducible protein of 10 kda, which is represented by the cxcl10 gene, a pro-inflammatory chemokine released by exposed cells with antigens and cause activated t lymphocytes to move toward g open access under cc-by-nc-sa share alike 4.0 results i gede yogi prema ananda, et al.: diagnosis based on detection of cxcl10 in urine 59 ijtid, p-issn 2085-1103, e-issn 2356-0991 the site of inflammation. inflammation in active pulmonary tb spreads inflammatory cells lymphogenously and haematogenously throughout the body, including the kidneys, to be excreted together with urine. urinary ip-10 levels are significantly elevated in patients with pulmonary disease, be it tb or other infections. the level of ip-10 in the urine of pulmonary tb patients who were examined at the onset of the disease was higher than in patients who had recovered.12 (cannas et al., 2010). ip-10 levels increase in patients with active pulmonary tb and decrease when tb treatment is complete.19 this study aims to analyze the accuracy of the diagnosis based on the detection of the cxcl10 gene in the urine of patients with suspected pulmonary tb. the results of this study are expected to determine the accuracy of the cxcl10 gene detection method in urine as a laboratory tool for diagnosis of pulmonary tb. materials and methods this research was a laboratory observational study with a cross-sectional study design using primary data of the results of the cxcl10 gene examination using pcr and secondary data of medical records unit in dr. soetomo hospital. medical records include clinical manifestations of pulmonary tuberculosis, molecular rapid test of genexpert mtb/rif (cepheid, canada), laboratory examinations, complete blood count, physical examination, and radiological photos of the thorax. the research was conducted in the period of november 2019 march 2020. urine samples were collected from 36 pulmonary tb adult patients. laboratory procedure for urine examination was urine processing using centrifugation, dna extraction using te buffer with boiling, and pcr optimization. the primer for pcr were 5’-ttcctgcaagccaattttgtcc3’ for forward and 5’for the reverse. gcagctgatttggtgaccat-3’ 3 urine cu urine culture based on standard solid medium culture method, 200 µl sediment of urine processing, was inoculated in middlebrook 7h10. the accuracy was determined by detecting the cxcl10 gene, which represented ip-10 protein in the urine of active pulmonary tb patients, and nucleic acid amplification tests (naats) method using pcr. the results was determined as positive if the band measured was 305 basepair (bp) and it matched with the primer set. the collected data were then processed with ibm spss statistics 26. after processing the data, the next step was to analyze the data whether the existing research hypothesis were to be accepted or rejected. data analysis was used to describe, understand, and explain the relationship between the variables studied. based on the study, there were 36 samples of patients with suspected pulmonary tb consisting of 20 (55.6%) men and 16 (44.4%) women. most samples were found in the age range of 20-29 years old, i.e., 10 people (27.8%). the complete findings are: 4 people aged 10-19 (11.1%), 1 person aged 30-39 (2.8%), 6 people aged 40-49 (16.7%), 3 people aged 50-59 (8.3%), 8 people aged 60-69 (22.2%), and 4 people aged 70-79 (11, 1%). the results shows that there were 33.3% of patients with low bmi, 2.8% of patients with high bmi, and 63.9% of patients with normal bmi. the majority of the research samples came from surabaya with 20 people (55.6%), while 16 people (44.4%) came from outside surabaya. the majority of the sample, 15 people (41.7%) of the 36, did not work, 10 of them were private workers (28.7%), 6 were students or students (16.7%), 3 were farmers (8.3%), while for merchants and regional senators (dprd) were with the same number, each consisting of 1 person (2.8%) as can be seen in table 1. open acces under cc-by-nc-sa share alike 4.0 ijtid, p-issn 2085-1103, e-issn 2356-0991 60 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 57–65 characteristics total percentage gender male 20 55,6% female 16 44,4% total 36 100% age 10-19 4 11,1% 20-29 10 27,8% 30-39 1 2,8% 40-49 6 16,7% 50-59 3 8,3% 60-69 8 22,2% 70-79 4 11,1% total 36 100% body mass index normal 23 63,9% underweight 12 33,3% overweight 1 2,8% region surabaya 20 55,6% outside surabaya 16 44,4% total 36 100% job student 6 16,7% private worker 10 27,8% farmer 3 8,3% merchant 1 2,8% dprd 1 2,8% no job 15 41,7% total 36 100% the comparison of the results of cxcl10 gene detection in urine with clinical manifestations has a specificity of 100%, a sensitivity of 6.6%, and an accuracy of 61.1% as can be seen in table 2. table 2. comparison of the results of the cxcl10 gene detection in urine with clinical manifestations detection of the cxcl10 gene in urine clinical manifestation total positive negative positive total 1 0 1 % 2,80% 0,00% 2,80% negative total 14 21 35 % 38,90% 58,30% 97,20% total total 15 21 36 % 41,70% 58,30% 100,00% the comparison of the results of the cxcl10 gene detection in urine with physical examination has a specificity of 97.2% and sensitivity of 2,8% as can be seen in table 3. table 3. comparison of the results of the cxcl10 gene detection in urine with physical examination detection of the cxcl10 gene in urine physical examination total positive negative positive total 0 1 1 % 0,00% 2,80% 2,80% negative total 0 35 35 % 0,00% 97,20% 97,20% total total 0 36 36 % 0,00% 100,00% 100,00% the comparison of the results of the cxcl gene detection in urine with the manifestations of laboratory tests of complete blood has a specificity of 100%, sensitivity of 11%, and accuracy of 77.7% as can be seen in table 4. table 4. comparison of cxcl10 gene detection results in urine with manifestations of complete blood count detection of the cxcl10 gene in urine complete blood count total positive negative positive total 1 0 1 % 2,80% 0,00% 2,80% negative total 8 27 35 % 22,20% 75,00% 97,20% total total 9 27 36 % 25,00% 75,00% 100,00% the comparison of the results of the cxcl gene detection in urine with the radiological results of the chest radiograph has a specificity of 100%, sensitivity of 4% and an accuracy of 33.3% as can be seen in table 5. table 1. frequency distribution of patients with lung tb suspect based on characteristics in the dots clinic of dr. soetomo surabaya in november 2019 march 2020 period open access under cc-by-nc-sa share alike 4.0 i gede yogi prema ananda, et al.: diagnosis based on detection of cxcl10 in urine 61 ijtid, p-issn 2085-1103, e-issn 2356-0991 table 5. comparison of the results of the cxcl gene detection in urine with the results of cadiological chest radiographs detection of the cxcl10 gene in urine chest radiograph total positive negative positive total 1 0 1 % 2,80% 0,00% 2,80% negative total 24 11 35 % 66,70% 30,50% 97,20% total total 25 11 36 % 69,50% 30,50% 100,00% the comparison of the results of the cxcl gene detection in urine with the results of genexpert has a specificity of 100%, a sensitivity of 6.2%, and an accuracy of 58.3% as can be seen in table 6. table 6. comparison of cxcl10 gene detection results in urine with genexpert results detection of the cxcl10 gene in urine genexpert total positive negative positive total 1 0 1 % 2,80% 0,00% 2,80% negative total 15 20 35 % 41,70% 55,60% 97,20% total total 16 20 36 % 44,40% 55,60% 100,00% the comparison of the results of the cxcl gene detection in urine with the results of urine culture has a specificity of 97.2%, a sensitivity of 0%, and an accuracy of 97.2% as can be seen in table 7. table 7. comparison of the results of the cxcl gene detection in urine with the results of urine culture detection of the cxcl10 gene in urine urine culture result total positive negative positive total 0 1 1 % 0,00% 2,80% 2,80% negative total 0 35 35 % 0,00% 97,20% 97,20% total total 0 36 36 % 0,00% 100,00% 100,00% patients (10.8%) with mrsa carrier events as much as zero (0%).18 discussion in this study, the prevalence of mrsa in subjects with stage five ckd were 6/150 (4%) there were no significant differences in the incidence of mrsa carriers in stage five ckd non hd or hd groups. this study shows that mrsa colonization exists in stage five ckd sufferers who have or who have not received hd therapy. pulmonary tuberculosis is a disease caused by mycobacterium tuberculosis. these bacteria are transmitted through droplets that enter the respiratory tract. the clinical symptoms are 3 weeks or more cough with phlegm, hemoptysis, fever, chest pain, weight loss, night sweats, and tightness. the results show that 91.7% of the patients had cough symptoms for 3 weeks or more. this is supported by a research conducted.8 which states that 81% of patients had cough symptoms for 3 weeks or more low bmi is associated with the risk of developing pulmonary tb because it is correlated with malnutrition and susceptibility to infectious diseases. the results show that there were 33.3% of patients with low bmi, 2.8% of patients with high bmi, and 63.9% of patients with normal bmi. research states that low bmi correlates with the risk of being infected with pulmonary tb, but not with extrapulmonary tb.13 research in taiwan also states that low bmi increases the risk of infection and mortality of tb disease.28 another study in korea shows that a high bmi lowers the risk of tb infection, but a very high bmi does not reduce the risk.20 meanwhile, research in china shows that high bmi and obesity are associated with the risk of tb infection, possibly because the excess cell adiposity weakens the immune system.30 the majority of patients showed that vital signs were outside the normal limits, and the results of the study showed that most of the patients had abnormal temperatures, which was in 33.3% of patients. another study states open access under cc-by-nc-sa share alike 4.0 ijtid, p-issn 2085-1103, e-issn 2356-0991 62 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 57–67 that 22.5% of 40 patients experienced increased body temperature as a result of tb disease.24 the vital sign that was most often outside the normal limit was the respiratory rate, with 79.5% of 49 patients showing a respiratory rate that exceeded the normal limit.26 complete blood count can be a parameter for diagnosis, prognosis, or response to pulmonary tb disease treatment.27 the results show that 19.4% of patients had leukocytosis and 5.6% of patients had decreased hemoglobin (hb). tb patients experienced decreased leukocytosis and hb. decreased hemoglobin is one of the hematological problems that often appears in tb patients.23 there were 61.5% of tb patients with anemia, where 43% of them suffered from moderate and severe anemia, and 49% suffered from iron deficiency anemia and anemia of chronic diseases.9 a chest radiograph is an important examination for people with suspected pulmonary tuberculosis but showing negative results of smear examination. it can also be used to determine disease progression and evaluate responses to therapy. radiological photos alone cannot diagnose pulmonary tuberculosis, it is also necessary to combine it with a physical examination and clinical symptoms.3 the results show that there were 69.4% of patients with positive chest radiology results. in the right lung, the most found were infiltrates and infiltrates accompanied by fibrosis, each of 5 people (13.9%), pleural effusions as many as 4 people (11.1%), and cavities accompanied by infiltrates as many as 2 people (5,6 %). in the left lung, the most found was infiltrates as many as 5 people (13.9%) and infiltrates with fibrosis in 3 people (8.3%). the most common features on chest radiological radiographs include 55% consolidation, 26% pleural effusion, and 17% lung collapse (appleton et al., 2017). another study shows that the most commonly seen features were 45% non-specific apex, 33% normal apex, and d 16% apex of the lung with infiltrates. when compared with patients whose culture results were negative, pulmonary tb patients showed 43% of infiltrates at the apex and 14% of cavities.6 pleural effusion was found in 38% of new cases of pulmonary tb patients.10 research conducted in nigeria shows that the use of genexpert for the diagnosis of pulmonary tuberculosis has better results than smear testing.15 the results show that there were 44.4% of patients with positive genexpert results, and 2.8% of patients showed resistance to rifampicin. examination using genexpert is more accurate than occasional sputum examination with higher sensitivity and negative predictive value (npv).29 another study conducted in china shows that examination with genexpert has a sensitivity of 94.6%, specificity of 82.9%, positive predictive value (ppv) 77.3%, and negative predictive value (npv) of 96.1% so that it can be used for examinations that require a shorter time, are simpler, and more efficient.23 research conducted in iran shows that 3.1% of 162 pulmonary tb patients whose sputum test results were positive were also resistant to rifampicin from the results of the genexpert examination.7 another study conducted in ethiopia shows that 1 out of 14 pulmonary tb patients who were bacteriologically positive was also resistant to rifampicin.17 in this study, the results show that none of the patients had positive urine culture results. meanwhile, a study conducted on hiv positive pulmonary tb patients in ethiopia shows that urine culture could help improve detection of the bacterium mycobacterium tuberculosis in hiv positive patients. of the 45 people, there were 14.5% positive culture patients in lowenstein-jensen media, 6% positive culture patients from urine smears, and 24.8% positive patients from rd9-based pcr examinations.14 another study conducted in india shows that 26.1% of the 46 patients with suspected pulmonary tb with positive sputum culture results also showed positive urine cultu open acces under cc-by-nc-sa share alike 4.0 i gede yogi prema ananda, et al.: diagnosis based on detection of cxcl10 in urine 63 ijtid, p-issn 2085-1103, e-issn 2356-0991 determine the duration of clinical symptoms that varied among the patients. overall, the accuracy of the method of diagnosing pulmonary tb based on detection of the cxcl10 gene in urine cannot be measured because of several reasons, i.e., lack of medical history, especially in treatment of anti-tuberculosis drugs, small sample size of active tb patients, and the difficulty to collect urine directly from hospitalized patients. further research in cohort study is needed with more complete clinical variable data, a wider scope, more samples, a real-time pcr method to detect the cxcl10 gene in urine, and the next generation sequencing (ngs) method. the validity of this research can also be increased by using 50 ml of the patients’ first morning urine. culture results.16 the results of this study show that there were 2.8% of patients with positive urine detection of the cxcl10 gene. the detection accuracy of the cxcl10 gene in urine was 2.8% compared with clinical manifestation of pulmonary tb, chest radiograph, and genexpert. active pulmonary tb patients have higher urine levels of the cxcl10 gene than healthy people. the detection of cxcl10 in urine using elisa (quanterix) has a sensitivity of 78% and a specificity of 94%.22 the detection of the cxcl10 gene in serum has a sensitivity of 87.5% and a specificity of 78.9%.21 another study reveals that detection of cxcl10 in serum showed positive results in 87.5% of active pulmonary tb patients, 45.5% of latent tb, and 9.5% in control variables.18 the differences in the accuracy rate between this research and other studies may be caused by some reasons. the study from petrone et al. in 2019.22 was conducted using elisa (quanterix) to measure cxcl10 in urine, while this research was using pcr. studies by nonghanphithak et al. in 2017. 21 and hong et al. in 2012.18 measured cxcl10 in serum of tb patient, while this research measured it in urine to avoid invasive procedures. if the detection of the cxcl10 gene in urine is to be compared with other tests, the highest sensitivity is shown by complete blood laboratory examination, which is 11%. the examination with the highest specificity are shown in the manifestation of clinical symptoms, complete blood laboratory tests, gene genexpert results, and radiological radiographs of the chest, which are 100% respectively. the examination with the highest accuracy is physical examination and urine culture result, which is 97.2%. this study has limitations in that the samples representing low bmi and optimal bmi are not sufficient. bmi is also a determining factor for the production of the cxcl10 gene. also, it was difficult to determine conflict of interest there is no confict of interest regarding this study. acknowledgement the authors would like to thank mrs. agnes, mrs. ria, mr. amin, and mr. agus for the help as lab technicians, to mrs. sri and the other nurses for the help in getting samples, and to all the patients. conclusion in this study, the accuracy of diagnosis based on detection of the cxcl10 gene in urine as a biomarker for diagnosis of active pulmonary tb is 2.8%. future research is needed to improve the methods by increasing urine samples to 50 ml and u sing clear medical history of the patients especially the history of anti-tuberculosis drugs. open acces under cc-by-nc-sa share alike 4.0 ijtid, p-issn 2085-1103, e-issn 2356-0991 64 indonesian journal of tropical and infectious disease, vol. 9 no. 1 january–april 2021: 57–65 13. casha a, scarci m. the link between tuberculosis and body mass index. journal of thoracic disease. 2017;9(3):e301-e303 14. chemeda a, abebe t, ameni g, worku a, mihret a. utility of urine as a clinical specimen for the diagnosis of pulmonary tuberculosis in people living with hiv in addis ababa, ethiopia. journal of clinical tuberculosis and other mycobacterial diseases. 2019;17:100125 15. ejeh e, undiandeye a, akinseye v, okon k, kazeem h, kudi c et al. diagnostic performance of genexpert and ziehl-neelson microscopy in the detection of tuberculosis in benue state, nigeria. alexandria journal of medicine. 2018;54(4):529533 16. gopinath k, singh s. urine as an adjunct specimen for the diagnosis of active pulmonary tuberculosis. international journal of infectious diseases. 2009;13(3):374-379 17. habte d, melese m, hiruy n, gashu z, jerene d, moges f et al. the additional yield of genexpert mtb/rif test in the diagnosis of pulmonary tuberculosis among household contacts of smear positive tb cases. international journal of infectious diseases. 2016;49:179-184 18. hong j, jung g, kim h, kim y, lee h, cho s et al. efficacy of inducible protein 10 as a biomarker for the diagnosis of tuberculosis. international journal of infectious diseases. 2012;16(12):e855-e859 19. kim s, kim j, kim d, kang y, bong s, lee j et al. urine ip-10 as a biomarker of therapeutic response in patients with active pulmonary tuberculosis. bmc infectious diseases. 2018;18(1) 20. kim s, ye s, ha e, chun e. association of body mass index with incident tuberculosis in korea. plos one. 2018;13(4):e0195104 21. nonghanphithak d, reechaipichitkul w, namwat w, naranbhai v, faksri k. chemokines additional to ifn-γ can be used to differentiate among mycobacterium tuberculosis infection possibilities and provide evidence of an early clearance phenotype. tuberculosis. 2017;105:28-34 22. petrone l, bondet v, vanini v, cuzzi g, palmieri f, palucci i et al. first description of agonist and antagonist ip-10 in urine of patients with active tb. international journal of infectious diseases. 2019;78:15-21 23. rohini k, surekha bhat m, srikumar p, mahesh kumar a. assessment of hematological parameters in pulmonary tuberculosis patients. indian journal of clinical biochemistry. 2015;31(3):332-335 references 1. world health organization. global tuberculosis report 2018. geneva: world health organization; 2018 2. world health organization. global tuberculosis report 2019. geneva: world health organization; 2019 3. world health organization. international standards for tuberculosis care. san fransisco: world health organization; 2014 4. kementerian kesehatan republik indonesia. rencana strategis kementerian kesehatan tahun 2015-2019. jakarta: kementerian kesehatan republik indonesia; 2015 5. kementerian kesehatan republik indonesia. peraturan menteri kesehatan republik indonesia nomor 67 tahun 2016 tentang penanggulangan tuberkulosis. jakarta: kementerian kesehatan republik indonesia; 2016 6. appleton s, connell d, singanayagam a, bradley p, pan d, sanderson f et al. evaluation of prediagnosis emergency department presentations in patients with active tuberculosis: the role of chest radiography, risk factors and symptoms. 2021 7. atashi s, izadi b, jalilian s, madani s, farahani a, mohajeri p. evaluation of genexpert mtb/rif for determination of rifampicin resistance among new tuberculosis cases in west and northwest iran. new microbes and new infections. 2017;19:117-120 8. bark c, dietze r, okwera a, quelapio m, thiel b, johnson j. clinical symptoms and microbiological outcomes in tuberculosis treatment trials. tuberculosis. 2011;91(6):601-604 9. barzegari s, afshari m, movahednia m, moosazadeh m. prevalence of anemia among patients with tuberculosis: a systematic review and meta-analysis. indian journal of tuberculosis. 2019;66(2):299-307 10.bhalla a, goyal a, guleria r, gupta a. chest tuberculosis: radiological review and imaging recommendations. indian journal of radiology and imaging. 2015;25(3):213 11. bowman a, jain a, baker b, milano p, terp s, desai s. chest x-ray findings in emergency department patients evaluated for pulmonary tuberculosis: the experience of a large urban academic emergency department. annals of emergency medicine. 2015;66(4):s104 12. cannas a, calvo l, chiacchio t, cuzzi g, vanini v, lauria f et al. ip-10 detection in urine is associated with lung diseases. bmc infectious diseases. 2010;10(1) open acces under cc-by-nc-sa share alike 4.0 i gede yogi prema ananda, et al.: diagnosis based on detection of cxcl10 in urine 65 ijtid, p-issn 2085-1103, e-issn 2356-0991 fachri m, bahrun u et al. hematologic parameters in open acces under cc-by-nc-sa share alike 4.0 26. shao y, peng h, chen c, zhu t, ji m, jiang w et al. evaluation of genexpert mtb/rif for detection of pulmonary tuberculosis at peripheral tuberculosis clinics. microbial pathogenesis. 2017;105:260-263. 27. singla r, raghu b, gupta a, caminero j, sethi p, tayal d et al. risk factors for early mortality in patients with pulmonary tuberculosis admitted to the 25. chandni r., rajan g., udayabhaskaran v. extra pulmonary tuberculosis presenting as fever with massive splenomegaly and pancytopenia. idcases. 2016; 4: 20-22 emergency room. pulmonology. 2021;27(1):35-42 28. wikanningtyas t, hatta m, massi m, pratiwi i, 24. rohini k, surekha bhat m, srikumar p, mahesh kumar a. assessment of hematological parameters in pulmonary tuberculosis patients. indian journal of clinical biochemistry. 2015;31(3):332-335 in pulmonary tuberculosis patients based on the microscopic sputum examination. enfermería clínica. 2020;30:243-246 29. yen y, chuang p, yen m, lin s, chuang p, yuan m et al. association of body mass index with tuberculosis mortality. medicine. 2016;95(1):e2300 30. yeong c, byrne a, cho j, sintchenko v, crighton t, marais b. use of genexpert mtb/rif on a single pooled sputum specimen to exclude pulmonary tuberculosis among hospital inpatients placed in respiratory isolation. international journal of infectious diseases. 2020;92:175-180 31. zhang h, li x, xin h, li h, li m, lu w et al. association of body mass index with the tuberculosis infection: a population-based study among 17796 adults in rural china. scientific reports. 2017;7(1) 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 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 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 122 vol. 1. no. 3 september–december 2010 what is malaria? indah s tantular institute of tropical disease and department of parasitology, airlangga university school of medicine surabaya abstract malaria persists as an undiminished global problem and still is the cause of much human morbidity and mortality. although the disease has been eradicated in many temperate zones, it continues to be endemic throughout much of the tropics and subtropics. many tools for understanding its biology and epidemiology are well developed, with a particular richness of comparative genome sequences. studies of the epidemiology, prevention, and treatment of human malaria have already been influenced by the availability of molecular methods, and analyses of parasite polymorphisms have long had useful and highly informative applications. the molecular epidemiology of malaria is currently undergoing its most substantial revolution as a result of the genomic information and technologies that are available in well-resourced centers. however, great progress in malaria control has been made in some highly endemic countries. vector control is assuming a new importance with the significant reductions in malaria burden achieved using combined malaria control interventions. education of health workers and communities about malaria prevention, diagnosis and treatment is a vital component of effective case management, especially as diagnostic policies change. key words: malaria, plasmodium, anopheles mosquito, parasite detection, malaria control introduction malaria is probably one of the oldest diseases known to mankind that has had profound impact on our history. for centuries it prevented any economic development in vast regions of the earth. it continues to be a huge social, economical and health problem, particularly in the tropical countries. malaria has been and still is the cause of much human morbidity and mortality. although the disease has been eradicated in many temperate zones, it continues to be endemic throughout much of the tropics and subtropics. forty percent of the world's population lives in endemic areas. epidemics have devastated large populations a malaria poses a serious barrier to economic progress in many developing countries. malaria is caused by members of the genus plasmodium. plasmodium species are apicomplexa and exhibit a heteroxenous life cycle involving a vertebrate host and an arthropod vector. vertebrate hosts include: reptiles, birds, rodents, monkeys and humans. plasmodium species are generally host and vector specific in that each species will only infect a limited range of hosts and vectors. four distinct species infected humans: p. falciparum, p. vivax, p. ovale and p malariae. the four human parasite species differ in regards to their morphology, details of their life cycles, and their clinical manifestations. plasmodium falciparum is the most common species in tropical areas and is transmitted primarily during the rainy season. this species is the most dangerous, accounting for half of all clinical cases of malaria and 90 percent of deaths from the disease. plasmodium vivax is the most widely distributed parasite, existing in temperate as well as tropical climates. plasmodium malariae can also be found in temperate and tropical climates but is less common than plasmodium vivax. plasmodium ovale is a relatively rare parasite, restricted to tropical climates and found primarily in eastern africa.[4,8] based on field survey on malaria at flores island, east nusa tenggara province, indonesia, tantular et al found single infections with either plasmodium falciparum or p. vivax were predominantly , mixed infection with p. vivax and p. malariae and 6 triple infections with p. falciparum, p. vivax, p. malariae.[20] plasmodium parasites undergo many stages of development, and their complete life cycle occurs in both humans and mosquitoes. the parasites are transmitted to humans by female mosquitoes of the genus anopheles. about 60 of the 390 species of anopheles mosquito transmit the malaria parasite. of these, only a dozen literature review 123tantular: what is malaria species are important in the transmission of malaria worldwide. usually just one or two species play a role in malaria transmission in a particular region where the disease is prevalent. it is a disease that can be treated in just 48 hours, yet it can cause fatal complications if the diagnosis and treatment are delayed. it is re-emerging as the first infectious killer and it is the number one priority tropical disease of the world health organization. malaria ranks third among the major infectious diseases in causing deaths after pneumococcal acute respiratory infections and tuberculosis. it is expected that by the turn of the century malaria would be the number one infectious killer disease in the world. (trigg and kondrachine, 1998; rosemary, 2010). history of plasmodium parasites malaria is an ancient disease that has plagued humans throughout history. the greek physician hippocrates described malaria in his writings during the 400s bc. throughout history and even today outbreaks of malaria have often been associated with warfare, migrations, and other societal disruptions. more soldiers have been lost to malaria than to bullets in the wars of the 20th century. historians believe that malaria was brought to the western hemisphere by european explorers. however, the exact cause of malaria was not understood until the closing years of the 19th century. in 2002 an international team of scientists deciphered the genome (genetic makeup) of plasmodium falciparum and other malaria parasites. scientists hope to use information gained from researching the parasite�s genome to design more effective antimalaria drugs and vaccines. scientists have also decoded the genome sequence of anopheles gambiae, one of the most common malaria mosquitoes. insecticides that target specific proteins produced by one or more genes in this mosquito�s genome may one day be used to control and possibly eliminate the mosquito from many areas.[1,3] the tropical disease malaria, which results in more than one million deaths annually, is considered one of the most significant infectious diseases worldwide. malaria is caused by protozoan parasites of the genus plasmodium and transmitted by blood-feeding anopheline mosquitoes. malaria infects hundreds of millions of people worldwide and kills an estimated 900,000 a year, taking an especially high toll on children in sub-saharan africa. at present, at least 300,000,000 people are affected by malaria globally, and there are between 1,000,000 and 1,500,000 malaria deaths per year. malaria parasites have been with us since the dawn of time. they probably originated in africa (along with mankind) and fossils of mosquitoes up to 30 million years old show that the vector for malaria was present well before the earliest history.[22] the plasmodium parasites are highly specific, with man as the only vertebrate host and anopheles mosquitoes as the vectors. this specificity of the parasites also points towards a long and adaptive relationship with our species. malaria is generally endemic in the tropics, with extensions into the subtropics. malaria in travellers arriving by air is now an important cause of death in non-malarious areas, and this is not helped by the common ignorance or indifference of travellers to prophylaxis. distribution varies greatly from country to country, and within the counties themselves, as the flight range of the vector from a suitable habitat is fortunately limited to a maximum of 2 miles, not taking account of prevailing wind etc.[4,5] in 1990, 80% of cases were in africa, with the remainder clustered in nine countries: india, brazil, afghanistan, sri-lanka, thailand, indonesia, vietnam, cambodia and china. current data for africa is unavailable. the disease is endemic in 91 countries currently, with small pockets of transmission in a further eight. plasmodium falciparum is the predominant species, with 120,000,000 new cases and up to 1,000,000 deaths per year globally. it is the plasmodium falciparum species which has given rise to the formidable drug resistant strains emerging in asia. in 1989, who declared malaria control to be a global priority due to the worsening situation, and in 1993, the world health assembly urged member states and who to increase control efforts.[5,22] epidemiology malaria is primarily a disease of the tropics and subtropics and is widespread in hot humid regions of africa, asia and south and central america. currently malaria is endemic in more than 100 countries and 40% of the world�s population lives in areas at risk for infection. the level of malaria transmission varies in different regions, countries and also within countries. endemic regions are characterized by warm temperatures and rainfall, both suitable for mosquito breeding, and where populations of human hosts and malaria parasites co-exist. because of the climatic conditions required, seasonal maps can be drawn up that allow prediction of when transmission will be at its highest and where epidemics are likely to occur. such seasonal information should help with the development of malaria control calendars and assist health services in appropriately focusing their control activities, such as drug procurement and anti-vector measures. the map shows areas of the world with different levels of endemicity or transmission intensity.[4,22] malaria is prevalent in sub-saharan africa, as well as other tropical and sub-tropical regions such as central and south america, asia, and the middle east. the geographic distribution of malaria is coordinate with the mosquito vector. as a result, malaria is generally not found in high altitude places. though anopheles mosquitoes can be found in the united states, public health interventions have disrupted parasite transmission. most cases of malaria in the united states are therefore "imported malaria," or malaria 124 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 122-127 acquired by a person traveling from an endemic region to the united states.[21] factors which may precipitate a malaria epidemic fall into two categories: natural (climatic variations, natural disasters), and man-made (conflict and war, agricultural projects, dams, mining, logging). most of these factors modify the physical environment, and increase the capacity of mosquitoes to transmit malaria. some factors also result in massive population movements that expose nonimmune populations to malaria infection the epidemiology of malaria is very important in understanding which populations will be most at risk, so that control programmes can be designed accordingly.[19,22] biology of plasmodium parasites and anopheles mosquitos malaria, a parasitic infection caused by plasmodium spp., requires two hosts: a human and a mosquito. mosquito bites transmit malaria to people. before mosquitoes were identified as the vectors of malaria, people thought the disease was the result of drinking bad water or breathing bad air (in fact, malaria means bad air). even after scientists realized mosquitoes carried malaria, many believed infection resulted from drinking water in which mosquitoes had died. today, scientists know that plasmodium spp., the parasites that cause malaria, require both the mosquito and the vertebrate host to complete their life cycle. when someone has symptoms of malaria, the parasites circulate in the blood stream; at this stage in their life cycle they are found inside red blood cells (erythrocytes). the so-called erythrocytic phase of the malaria parasite culminates with the development of gametocytes male and female malarial parasites. when a mosquito bites a person who has malaria, it sucks both blood and malarial parasites into its stomach. most of these parasites die and are digested with the blood, but if the mosquito is an anopheles sp. mosquito, and there are gametocytes in the ingested blood, the gametocytes develop to gametes. once an anopheles mosquito is infected with malaria, it remains infected for life and can infect a human each time it takes a blood meal. it appears that infected anopheles mosquitoes feed more often than those that are not infected, increasing the potential for the parasite to be passed on.[16] the plasmodium genus of protozoal parasites (mainly p.falciparum, p.vivax, p.ovale, and p.malariae) have a life cycle which is split between a vertebrate host and an insect vector. the plasmodium species, with the exception of p.malariae (which may affect the higher primates) are exclusively parasites of man. the mosquito is always the vector, and is always an anopheline mosquito, although, out of the 380 species of anopheline mosquito, only 60 can transmit malaria. only female mosquitos are involved as the males do not feed on blood.[14,16] transmission of malaria infectious bites each year more than 200 million people develop malaria. more than one million people die from malaria each year. when a mosquito bites, it injects a small amount of fluid. if the mosquito has plasmodium sp. sporozoites in its salivary glands, some of the sporozoites enter the prey bloodstream. sporozoites remain in the blood only minutes to hours before they invade liver cells, where they begin multiplying again. in cameroon, the researchers recorded how often people were bitten by a mosquito that carried malaria parasites. they also examined the number of transmittable parasites in the blood of infected persons. the number of gametocytes per person was season and age dependent. children were found to be by far the most important source of malaria transmission in the area. people who live in areas where malaria is prevalent, can develop a natural immunity that stops the development of the parasite in the mosquito. this prevents the parasite from spreading further. the presence of this immunity, the so-called transmission-reducing activity, is determined using a laboratory test. van der kolk discovered that people who are often infected with malaria could quickly acquire this immunity. he also found that people with higher numbers of gametocytes are more frequently immune.[10,16] malaria parasites are transmitted from the vertebrate host to the mosquito vector by sexual blood stages (gametocytes). when taken up in the bloodmeal by the engorging female mosquito, male gametocytes (microgametocytes) undergo exflagellation, producing up to eight male gametes; a female gametocyte (macrogametocyte) produces only one female gamete, which is fertilized by a single male gamete. the gametocyte sex ratio tends to be female-biased in all species of malaria parasites and several authors have considered that the theory of local mate competition, which successfully explains many other cases of biased sex ratios , determines the gametocyte sex ratio of malaria parasites. according to this theory, when an infection consists of a few parasite clones, whose offspring will mate among themselves, a female-biased sex ratio is favored by natural selection, because it will reduce competition among brothers for mates. however, empirical data are conflicting and the mechanism of gametocyte sex determination in malaria parasites remains unknown. gametocyte sex is not determined by segregation of sex-determining genes or chromosomes, because malaria parasites are haploid in the vertebrate host and a single clone can produce both male and female gametocytes.[2,13] other modes of transmission rarely malaria can spread by the inoculation of blood from an infected person to a healthy person. in this type of malaria, asexual forms are directly inoculated into the 125tantular: what is malaria blood and pre-erythrocytic development of the parasite in the liver does not occur. therefore, this type of malaria has a shorter incubation period and relapses do not occur. 1. blood transfusion (transfusion malaria) this is fairly common in endemic areas. following an attack of malaria, the donor may remain infective for years (1-3 years in p. falciparum, 3-4 years in p. vivax, and 15-50 years in p. malariae. most infections occur in cases of transfusion of blood stored for less than 5 days and it is rare in transfusions of blood stored for more than 2 weeks. the clinical features of transfusion malaria occur earlier and any patient who has received a transfusion three months prior to the febrile illness should be suspected to have malaria. donor blood can be tested with indirect fluorescent antibody test or elisa, and direct examination of the blood for the parasite may not be helpful. in transfusion malaria, pre-erythrocytic schizogony does not occur and hence relapses due to dormant hepatic forms also does not occur. 2. mother to the growing fetus (congenital malaria) intrauterine transmission of infection from mother to child is well documented. placenta becomes heavily infested with the parasites. congenital malaria is more common in first pregnancy, among non immune populations. 3. needle stick injury accidental transmission can occur among drug addicts who share syringes and needles. therapeutic inoculation of malarial parasites, so as to induce fever, was a mode of treatment for neurosyphilis.[1,12] detection of malaria parasites microscopic examination is the primary method of malaria parasite detection and species identification, although problems with this have been recognized for some time have not diminished in recent years. even the most skillful morphological analysis of stained parasites on blood films is not a very reliable basis for determining the identity of a malaria parasite species. difficulties are compounded when infections contain more than one parasite species or when an unusual species is present. although the peripheral blood smear examination that provides the most comprehensive information on a single test format has been the "gold standard" for the diagnosis of malaria, the immunochromatographic tests for the detection of malaria antigens, developed in the past decade, have opened a new and exciting avenue in malaria diagnosis.[13] identification of malaria parasites in peripheral blood samples can now be most reliably performed by analysis of dna, and this, to some extent, transforms the possibilities for diagnosis and epidemiology of malaria. parasite morphology has proved unsuitable for a systematic analysis of the relationships among the different species. other parasitological features, such as data on the course of experimental infections (including periodicity of replication in the blood), also have little reliability for systematic purposes. the use of pcr-based methods to detect malaria parasites in blood samples increases the sensitivity of detection compared with microscopy. qualitative pcr protocols that are robust, sensitive, and species specific have been available since the 1990s, and there are now several quantitative pcr methods that allow estimation of parasitemia levels as well as positivity.[15,17] thick-film microscopy can allow the examination of ~0.1 to 1 µl of blood (50 to 500 high-power fields with ~0.002 µl per field) and, thus, the detection of more than ~10 parasites µl–1. most applications of pcr typically involve amplification of dna template from the equivalent of 1 to 10 µl blood and are thus either slightly more or up to 100 times more sensitive than microscopy. dna template can be prepared from larger volumes of blood to give even higher sensitivity, with detection of ~20 parasites ml–1 being achieved, which is useful in clinical vaccine trials in which the time to first detectable blood-stage parasitemia is the endpoint (singh et al., 1999; mangold et al., 2005). sensitive pcr methods for parasite detection have been used to good effect in epidemiological studies, revealing surprisingly high proportions of individuals that have persistent asymptomatic infections in some populations in areas of endemicity. use of real-time quantitative pcr methods is being evaluated in clinical diagnostic laboratories in countries of endemicity with good resources and reference laboratories in countries with substantial numbers of imported malaria cases. however, there is a limitation to the information provided by any method that samples peripheral blood for estimation of p. falciparum parasitemia, as sequestered mature asexual parasites may sometimes outnumber those in the peripheral blood (roper et al., 1996; singh et al., 1999). signs and symptoms malaria is a febrile illness characterised by fever and related symptoms. however it is very important to remember that malaria is not a simple disease of fever, chills and rigors. in fact, in a malarious area, it can present with such varied and dramatic manifestations that malaria may have to be considered as a differential diagnosis for almost all the clinical problems. malaria is a great imitator and trickster, particularly in areas where it is endemic.[18] all the clinical features of malaria are caused by the erythrocytic schizogony in the blood. the growing parasite progressively consumes and degrades intracellular proteins, principally hemoglobin, resulting in formation of the 'malarial pigment' and hemolysis of the infected red cell. this also alters the transport properties of the red cell membrane, and the red cell becomes more spherical and less deformable. the rupture of red blood cells by merozoites releases certain factors and toxins.[8,21] clinical manifestations of malaria are attributable to the blood stage component of the parasite life cycle. symptoms and symptoms of malaria include non-specific 126 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 122-127 flu-like symptoms, malaise, abdominal pain, anemia, splenomegaly, chills, and fever. the fevers of malaria have a periodic onset that is commisserate with asexual parasite development. this periodic fever, however, is frequently not seen clinically, and therefore is not often helpful in distinguishing malaria from other causes of fever. for example, fevers arising from infection with p. falciparum emerge every 48 hours. this corresponds to the time needed for p. falciparum to proceed from the ring stage to rbc rupture and invasion of other rbcs, a point at which the parasite once again adopts the morphologically-ascribed ring form. anemia results from the rupture of infected erythrocytes, splenomagaly, and vascular sequestration. more severe manifestations of malaria include cerebral malaria, severe anemia, renal failure, respiratory distress, and death. the death rate from falciparum malaria may be as high as 40% in non-immune individuals.[6,12] in an endemic area, malaria often presents with atypical manifestations, such as atypical fever in an endemic area, it is rather unusual to find cases with typical fever pattern. some patients may not have fever at all and may present with other symptoms. many present with fever of various patterns low grade to high grade, with or without chills, intermittent to continuous, or even as cases of prolonged fever. headache headache may be a presenting feature of malaria, with or without fever. it can be unilateral or bilateral. some times the headache could be so intense that it may mimic intracranial infections or intra-cranial space occupying lesions. it may also mimic migraine, sinusitis etc. body ache, back ache and joint pains these symptoms are fairly common in malaria. these can occur even during the prodromal period and at that stage these are generally ignored and diagnosis of malaria is impossible owing to lack of peripheral parasitemia. they are also common accompaniments of the malaria paroxysm. sometimes, malaria may present only with these symptoms, particularly in cases of recurrent malaria. cough cough may be a presenting feature of malaria, particularly p. falciparum infection. patient may have pharyngeal congestion and features of mild bronchitis. patients who have persistent cough and/or fever even after clearance of parasitemia should be evaluated for secondary bacterial pneumonias/ bronchopneumonia and bronchitis. weakness sometimes patients may present with history of weakness, malaise. on examination they may have significant pallor, hypotension, dehydration etc. algid malaria may present like this and the patient may not have fever et all. hepatosplenomegaly patients can present with enlargement of liver and/or spleen, tender or non-tender, with or without fever. rapid enlargement of spleen or liver in malaria can cause acute pain in the abdomen or chest. generally, organomegaly is noticed in the second week of malarial illness. however, in cases of relapse or recrudescence, it may be present earlier. although splenomegaly is a cardinal sign of malaria, absence of splenomegaly does not rule out the possibility of malaria.[9,12,18,21] malaria control malaria continues to be a significant public health issue and a major hindrance to economic growth. sustained control and management of malaria presents significant challenges. effective interventions are available, these include widespread implementation of effective vector control and prompt and effective treatment with act following accurate diagnosis. effective control and treatment of malaria presents enormous logistical challenges. the key to addressing the challenge of reducing the burden of malaria is an integrated approach that combines preventative measures, such as long-lasting insecticide-treated bed nets (llins) and indoor residual spraying (irs), with improved access to effective anti-malarial drugs. however, malaria is a disease that stems from and causes poverty, and many at-risk populations live in extremely destitute, remote areas. poor, rural families are the least likely to have access to these preventative measures that are fundamental to malaria control, and may live kilometres from the nearest healthcare facility. they are also less able to afford treatment once infection has occurred.[3,23] the historic successful eradication of malaria in various parts of the world was achieved primarily by vector control, indicating that renewed efforts in this field, other than the current insecticide-based strategies, should be considered a central aspect of any malaria eradication strategy. however, given the increasing prevalence of mosquito resistance against the currently used chemicals, the need for development of new insecticides has high priority. novel tools for the control of the adult mosquito include entomopathogenic fungi, insect-pathogenic viruses, the introduction of genetically engineered mosquitoes and the sterile insect technique (sit).[19] the development and spread of parasite resistance to certain anti-malarial agents has presented a major barrier to successful disease management in malaria-endemic areas, and has probably contributed to the resurgence of infection and the increase in malaria-related deaths in recent years. resistance to almost all commonly used anti-malarials, notably chloroquine and sulphadoxinepyrimethamine, but also amodiaquine, mefloquine, and quinine, has been observed in the most lethal parasite species, p. falciparum.[19,23] 127tantular: what is malaria reducing malaria transmission by reducing gametocyte carriage with effective drugs can be an important factor in highly endemic areas. some endemic countries are forging a path in malaria control and prevention using combined interventions and have achieved significant reductions in malaria burden. however, factors such as patient access to effective treatments and preventative measures, availability of training programmes and educational materials, and development and spread of resistance to certain antimalarials are hindering progress. addressing the continuing challenges presented by malaria in the years ahead will require responsive strategies such as innovative vector control methods, widespread implementation of biological diagnosis prior to treatment with effective anti-malarials, and close monitoring of local malaria epidemiology to identify areas of resurgence.[4,10,23] references 1. anderson, t. j. c., r. e. l. paul, c. a. donnelly, and k. p. day. 2000. do malaria parasites mate non-randomly in the mosquito midgut? genet. res. 75: 285–96. 2. a. m. talman, o. domarle, f. e. mckenzie, f. ariey, and v. robert, “gametocytogenesis: the puberty of plasmodium falciparum,” malaria journal, vol. 3, article 24, 2004. 3. beck, h. p. 1999. how does molecular epidemiology help to understand malaria? trop. med. int. health 4: 1–3. 4. greenwood bm, fidock da, kyle de, kappe shi, alonso pl, collins fh, duffy pe 2008) malaria: progress, perils, and prospects for eradication. journal of clinicalinvestigation 118: 1266–76. 5. guerra ca, snow rw, hay si: mapping the global extent of malaria in 2005. trends parasitol 2006, 22: 353–358. 6. curtis, h. and n. barnes (1989). biology. new york, worth publishers, inc. 7. g. pradel, “proteins of the malaria parasite sexual stages: expression, function and potential for transmission blocking strategies,” parasitology, 134(14): 1911–1929, 2007. 8. john h. adams, b.a., m.sc., ph.d. "malaria," encyclopedia 2005. http://encarta.msn.com (accessed at november 9, 2010) 9. juckett, g. (1999). malaria prevention in travelers. american family physician. 59(9): 2523–30. 10. m. e. smalley and r. e. sinden, “plasmodium falciparum gametocytes: their longevity and infectivity,” parasitology, 74(1): 1–8, 1977. 11. mangold, k. a., r. u. manson, e. s. koay, l. stephens, m. regner, r. b. thomson, jr., l. r. peterson, and k. l. kaul. 2005. real-time pcr for detection and identification of plasmodium spp. j. clin. microbiol. 43: 2435–40. 12. miller lh, baruch di, marsh k, doumbo ok. the pathogenic basis of malaria. nature. 2002 feb 7; 415(6872): 673–9. 13. r. carter and p. m. graves, in malaria: principles and practice of malariology, w. h. wernsdorfer and i. mcgregor, eds. (churchill livingstone, london, 1988), vol. 1, p. 253. 14. r. e. sinden, “gametocytes and sexual development,” in malaria: parasite biology, pathogenesis, and protection, i. w. sherman, ed., pp. 25–48, asm press, washington, dc, usa, 1998. 15. roper, c., i. m. elhassan, l. hviid, h. giha, w. richardson, h. babiker, g. m. h. satti, t. g. theander, and d. e. arnot. 1996. detection of very low level plasmodium falciparum infections using the nested polymerase chain reaction and a reassessment of the epidemiology of unstable malaria in sudan. am. j. trop. med. hyg. 54: 325–331. 16. rosemary drisdelle, 2010 do mosquito bites cause malaria?: how mosquitoes spread malarial parasites from person to person http://www.suite101.com/content/do-mosquito-bites-cause-malaria (accessed at november 9, 2010). 17. snounou, g., s. viriyakosol, x. p. zhu, w. jarra, l. pinheiro, v. e. do rosario, s. thaithong, and k. n. brown. 1993. high sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. mol. biochem. parasitol. 61: 315–320. 18. snow, r., guerra, c., noor, a., myint, h. & hay, s. (2005) the global distribution of clinical episodes of plasmodium falciparum malaria. nature 434: 214–17. 19. takken w, knols bg. malaria vector control: current and future strategies. trends parasitol. 2009; 25:101–104. doi: 10.1016/ j.pt.2008.12.002. 20. tantular, is, iwai, k, matsuoka,h, kawamoto f, et al, 1999, ‘field trials of rapid test for g6pd deficiency in combination with a rapid diagnosis of malaria�, trop med and international health, 4(4): 245–50. 21. trigg, p. and a. kondrachine (1998). malaria: parasite biology, pathogenesis, and protection. i. sherman. washington, dc, asm press. 22. who world malaria report 2005, who/htm/mal/2005.1102. 23. who guidelines for the treatment of malaria 2006. 24. http://www.who.int/malaria/docs/treatmentguidelines2006.pdf (accessed at november 4, 2010). ijtid vol 1 no 3 sep-dec 2010.20.pdf ijtid vol 1 no 3 sep-dec 2010.21.pdf ijtid vol 1 no 3 sep-dec 2010.22.pdf ijtid vol 1 no 3 sep-dec 2010.23.pdf ijtid vol 1 no 3 sep-dec 2010.24.pdf ijtid vol 1 no 3 sep-dec 2010.25.pdf 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. 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 93 vol. 1. no. 2 may–august 2010 description analysis of human behavior that causes the emergence of hiv/aids infectious diseases in surabaya yayuk susilawati1, nasronudin2, atika3 1 airlangga university school of medicine, department of biochemistry, institute of tropical disease airlangga university 2 airlangga university school of medicine, department of internal medicine, institute of tropical disease airlangga university 3 airlangga university school of medicine, department of public health abstract hiv virus is transmitted to other individuals particularly through sexual contact with infected individuals, narcotic abuse using shared infected needle, maternal-fetal transmission in perinatal period, either during pregnancy, labor, and breastfeeding, or through infected blood donor. the diagnosis of hiv/aids infection is established using laboratory examination with the indication of clinical symptoms or high risk behavior. this descriptive study was intended to describe human behaviors that cause the occurrence of hiv/aids in surabaya. to find the description of the disease, the percentage of total hiv/aids patients according to behavioral risk factors was estimated. total patients in 9 hospitals at each risk factor were divided with total patients in those hospital, multiplied with 100. the description of the disease according to behavioral risk factors in surabaya is as follows: total patients between january and december 2005 was 382 individuals; 204 due to sexual contact (53.40%), 161 due to injected drug use (idu) (42.15%), 6 perinatal cases (1.57%) and 11 with unknown causes (2.88%). from risk factor sexual relationship behavior as many as 204 people, respectively heterosexual 174 people (85.29%), homosexual 17 people (8.33%) and bisexual 13 people (6.37%). further analytical studies are needed to analyze correlation between human behavior and the occurrence of hiv/aids in surabaya. key words: description analysis, high risk behavior, hiv/aids introduction hiv virus infectious to others primarily through: sexual contact with an infected person, the use of narcotic drugs interchangeably with syringe, mother to child transmission in the perinatal period either during pregnancy, childbirth or breastfeeding, or can also be transmitted through blood donation infected.1 behaviors at high risk for transmission of this virus, such as free sex, drug use amongst injecting drug turns, blood donors, perinatal period, health workers, occupational accident, are particularly vulnerable to the development of the hiv virus. east java province which is ranked third highest prevalence of cases of hiv/aids after papua and jakarta, east java under the following namely west java, bali and riau. the six provinces have now signed epidemic levels in concentrated zones that must be addressed.4 number of people with hiv/aids in surabaya, the highest among the 37 districts/municipalities in east java with a total that reached 50% of all people with hiv/aids.10 but no one has ever done research to determine its prevalence in surabaya and how the description of human behaviors that cause the emergence of infectious diseases of hiv/aids in surabaya, therefore we need to do research on this. human immunodeficiency (hiv) is a virus that attacks the human immune system and cause aids (acquired immunodeficiency syndrome). aids is a collection of symptoms caused by diseases of the immune system. progressive damage to the immune system causes people with hiv/aids (odha) is very fragile and easily affected by various diseases. disease that usually is not dangerous even in the long run will cause the patient severe pain and even death. the experts identified two types of hiv virus, hiv-1 and hiv-2. hiv-1 is the major cause of aids in the world, hiv-2 is found mostly in west africa.2,9 hiv-2 cause of death that occurred more slowly than hiv-1.2,7 clinical manifestations of hiv infection can be caused by his own hiv (acute retroviral syndrome, hiv dementia), opportunistic infections or aids-related cancers. travelling with hiv disease is divided into stages based on clinical and cd4 cell count: acute retroviral infection, an asymptomatic period, the early symptoms and the symptoms continued. research report 94 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 93-95 while hiv and aids diagnosis can be established through clinical manifestations and investigation. early diagnosis is established by laboratory examination of clinical symptoms with the instructions or the existence of high-risk behavior. for hiv diagnosis, which is commonly used is the elisa, western blot and pcr. diagnosis of aids is the final stage of hiv infection. patients were expressed in the development of aids when hiv infection further indicates opportunistic infections and life-threatening cancer patients with cd4 count < 200/mm3.11 development of hiv/aids epidemic in indonesia is strongly influenced by the interaction of high-risk groups, among other groups of commercial sex workers, users of narcotic drugs and free sex adherents.3,6 there are not many epidemiological studies conducted on human behavior in the role of infectious diseases caused by hiv/aids in indonesia. while a lot of areas in indonesia with huge potential for development of the hiv virus. individual is an integral part of the social environment in which he lived. individual behavior is psychologically very complex and difficult to learn without a long learning process associated with the form of his life experiences. rotter, a psychologist provides psychological concepts such behavior5: 1) behaviour is an event where individuals living organism as a subject, 2) aspects of behavior always has a direction and goals, and objectives of this getting a big influence of reinforcement conditions. reinforcement is a pleasant situation of the individuals who received social environment or of the results achieved through activities of its behavior. the purpose of this study is to reveal the behaviors that cause the emergence of infectious diseases hiv/aids in surabaya. data from this study are expected to be useful to health stakehoulder as a reference in determining the precautions to reduce the spreading rate or epidemic rate of hiv/aids in surabaya and is also useful as a reference for further study in the future. research method this study was a descriptive study to know the description of human behavior that causes the emergence of hiv/aids infectious diseases in surabaya. the population study was all patients with hiv/aids in surabaya district. the samples are hiv/aids patients who carry out checks in the nine hospitals in surabaya district. region chosen as the research is surabaya city. the choice of location was based because the absence of description data of human behavior causes the disease of hiv/aids in surabaya municipality health office. this research was conducted in the nine hospitals in surabaya municipality area. data needed for research are primary and secondary. the primary data obtained from research instruments such as questionnaires given to patients with hiv/aids who carry out checks in the nine hospitals in surabaya city area. secondary data obtained from the data of hiv/aids are collected by the nine hospitals in the area of surabaya municipality either actively or passively. active data is data obtained actively from activities within the hospital for examination of patients with hiv/aids and activities that serve the system of referral from other health units. while passive data is data obtained from the reports-external (public/community organizations, government agencies, lsm, etc.). to know the description of infectious diseases, hiv/ aids calculate the percentage of hiv/aids according to behavioral risk factors. number of patients at nine hospitals each risk factor divided by the total number of patients at nine hospitals, multiplied by 100%. results and discussion from the research results can be reported that during the month of january 2005–august 2006 there were 382 table 1. distribution of hiv-positive patients based on behavioral risk factors in january 2005–august 2006 in surabaya hospital risk factors totalsexual relationship injection drug user (idu) transfusion perinatal unknown jlh % jlh % jlh % jlh % jlh % jlh % dr. soetomo 157 50.00 151 48.09 0 0 6 1.91 0 0 314 100 navy hospital 37 100.00 0 0 0 0 0 0 0 0 37 100 dr. soewandhi 3 100.00 0 0 0 0 0 0 0 0 3 100 karang tembok 4 80.00 1 20.00 0 0 0 0 0 0 5 100 darmo 0 0 3 60.00 0 0 0 0 2 40.00 5 100 al irsyad 0 0 0 0 0 0 0 0 6 100.00 6 100 budi mulia 2 25.00 5 62.50 0 0 0 0 1 12.50 8 100 dankesda 0 0 0 0 0 0 0 0 2 100.00 2 100 air force hospital 1 50.00 1 50.00 0 0 0 0 0 0 2 100 total 204 53.40 161 42.15 0 0 6 1.57 11 2.88 382 100 95susilawati et al.: description analysis of human behavior 2. from risk factor sexual relationship behavior occupied the first-ranking were heterosexual behaviour (85.29%), the second-ranking was homosexual behaviour (8.33%) and the third-ranking/last was bisexual behaviour (6.37%). recommendation 1. further analytical studies are needed to analyze correlation between human behavior and the occurrence of hiv/aids in surabaya. 2. further studies are needed to identificated hiv virus type that infected patients in surabaya as basic for effort to prevent and therapy. references 1. centers for disease control & prevention (cdc), 2003: how is hiv passed from one person to another?, national center for hiv, std, and tb prevention, divisions of hiv/aids prevention, atlanta, usa, 15 desember 2003. 2. de cock km, brun-vezinet f, soro b, 1991: hiv-1 and hiv-2 infections and aids in west africa, aids, 5 suppl 1: s21–8. 3. dinas informasi dan komunikasi pemda jatim, 2004. jatim terbesardinas informasi dan komunikasi pemda jatim, 2004. jatim terbesar ketiga jumlah penderita hiv/aids, d-infokom-jatim, 22 april 2004. 4. dinas informasi dan komunikasi pemda jatim, 2005. jatim urutan ketiga prevalensi tinggi hiv/aids, d-infokom-jatim, 03 maret 2005. 5. faisal s, mappiare a, 1981. dimensi-dimensi psikologi, usahafaisal s, mappiare a, 1981. dimensi-dimensi psikologi, usaha nasional, surabaya, hal: 225–227. 6. gsianturi, 2002. dicanangkan, gerakan nasional penanggulangan hiv/aids, gizi.net, 25 april 2002. 7. grant ad, djomand g, de cock km, 1997: natural history andgrant ad, djomand g, de cock km, 1997: natural history and spectrum of disease in adults with hiv/aids in africa, aids, 11 suppl b: s43–54. 8. greaves w.w., 1993. epidemiology course study guide, master of public health degree program in general preventive medicine and public health, departement of preventive medicine the medical college of wisconson. 9. rudolf j. kotula md, 2004: hiv/aids: definition and transmission of hiv/aids, private practice in infectious diseases, methodist hospital, omaha, nebraska, university of lowa family practice handbook, fourth edition, chapter 11. 10. sembiring, murphy j., 2004. penderita hiv/aids di jatim surabaya tertinggi, harian surya edisi 26 juni 2004, hal 22. 11. setyono j, 2004. human immunodeficiency virus/acquired immunodeficiency syndrome, mandala of health a scientific journal, januari 2004, vol 1 (1): 41–49. idu, 42.15% 0,00% 1,57% 2.88% 53,40% sexual relationship idu transfusion perinatal unknown figure 1. the percentage of hiv/aids according to behavioral risk factors people with a positive hiv test results, where 204 people (53.40%) were obtained from a sexual relationship, 161 people (42.15%) due to injection drug user (idu), people with children who got hiv from their mothers on perinatal cases, either during pregnancy, childbirth or breast-feeding as many as six people (1.57%) and 11 people has no known cause (2.88%). from risk factor sexual relationship behavior as many as 204 people, respectively heterosexual 174 people (85.29%), homosexual 17 people (8.33%) and bisexual 13 people (6.37%). distribution data amount of hiv/aids patients based on sexual relationship behavioral risk factors can be showed in tabel 2. from the data distribution above showed that risk factors sexual relationship behaviour occupied the topranking were heterosexual behaviour (85.29%) and that was sexual relationship behaviour the most susceptable to spreading of hiv infection. whereas the second-ranking was homosexual behaviour (8.33%) and the third-ranking/ last was bisexual behaviour (6.37%). conclusion and recommendation conclusion 1. description of hiv/aids infectious deseases according to behavior risk factor in surabaya: for month january 2005–august 2006 in surabaya were found hiv/aids patients as many as 382 people. 204 people cause of sexual relationship (53.40%), 161 people due to injection drug user/idu (42.15%), 6 patients perinatal case (1.57%) and the cause unknown 11 people (2.88%). tabel 2. distribution hiv/aids patients with sexual relationship risk factor in month january 2005–august 2006 in surabaya hospital heterosex homosex bisex total jlh % jlh % jlh % jlh % dr.soetomo 142 90.45 15 9.55 0 0 157 100 navy hospital 24 64.86 0 0 13 35.1 37 100 dr.soewandi 2 66.67 1 33.33 0 0 3 100 karang tembok 3 75 1 25 0 0 4 100 budi mulia 2 100 0 0 0 0 2 100 air force hospital 1 100 0 0 0 0 1 100 total 174 85.29 17 8.33 13 6.37 204 100 ijtid vol 1 no 2 may-aug 2010.41.pdf ijtid vol 1 no 2 may-aug 2010.42.pdf ijtid vol 1 no 2 may-aug 2010.43.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 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 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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 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(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. 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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):236 102 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 32 vol. 5. no. 2 may–august 2014 research report heart abnormality classifications using fourier transforms method and neural networks endah purwanti1, amadea kurnia nastiti2, adri supardi3 1 biomedical engineering study program, faculty of science and technology, universitas airlangga, surabaya 2 bachelor of biomedical engineering study program, faculty of science and technology, universitas airlangga, surabaya 3 physics study program, faculty of science and technology, universitas airlangga, surabaya abstract health problems with cardiovascular system disorder are still ranked high globally. one way to detect abnormalities in the cardiovascular system especially in the heart is through the electrocardiogram (ecg) reading. however, reading ecg recording needs experience and expertise, software-based neural networks has designed to help identify any abnormalities of the heart through electrocardiogram digital image. this image is processed using image processing methods to obtain ordinate chart which representing the heart’s electrical potential. feature extraction using fourier transforms which are divided into several numbers of coefficients. as the software input, fourier transforms coefficient have been normalized. output of this software is divided into three classes, namely heart with atrial fibrillation, coronary heart disease and normal. maximum accuracy rate of this software is 95.45%, with the distribution of the fourier transform coefficients 1/8 and number of nodes 5, while minimum accuracy rate of this software at least 68.18% by distribution of the fourier transform coefficients 1/32 and the number of nodes 32. overall result accuracy rate of this software has an average of 86.05% and standard deviation of 7.82. key words: cardiac abnormalities, image processing, electrocardiogram, fourier transforms, artificial neural networks abstrak masalah kesehatan dengan gangguan sistem kardiovaskular masih menduduki peringkat tinggi secara global. salah satu cara untuk mendeteksi kelainan pada sistem kardiovaskular terutama di hati adalah melalui membaca rekaman elektrokardiogram (ekg). namun, membaca rekaman ekg membutuhkan pengalaman dan keahlian, jaringan saraf berbasis software telah dirancang untuk membantu mengidentifikasi kelainan jantung melalui gambar digital elektrokardiogram. gambar ini diproses menggunakan metode pengolahan citra untuk mendapatkan grafik ordinat yang mewakili potensi listrik jantung. ekstraksi fitur menggunakan transformasi fourier yang terbagi menjadi beberapa jumlah koefisien. sebagai input software, transformasi fourier koefisien telah dinormalkan. output dari program ini dibagi menjadi tiga kelas, yaitu jantung dengan fibrilasi atrium, penyakit jantung koroner dan normal. tingkat akurasi maksimum dari software ini adalah 95,45%, dengan distribusi fourier transform koefisien 1/8 dan jumlah node 5, sedangkan tingkat akurasi minimal software ini setidaknya 68,18% dengan distribusi fourier transform koefisien 1/32 dan jumlah node 32. secara keseluruhan hasil tingkat akurasi software ini memiliki rata-rata 86,05 % dan standar deviasi 7.82. kata kunci: abnormalitas jantung, pemrosesan citra, elektrokardiogram, fourier transform, jaringan saraf tiruan introduction health problems with cardiovascular system disorder is still ranked high, according to data from the world health organization (who) reported that approximately 31% of the cause of death globally was cardiovascular disease. variety of prevention and detection of cardiac abnormalities such as by using the device as a diagnostic tool, where the most commonly used is the electrocardiograph (ecg). electrocardiograph (ecg) used to capture and record the potential changes of heart using leads which are placed on the patient’s body at a particular location. ecg results are in a form of image called the electrocardiogram.13 although knowing how the ecg works is relatively easy, 33purwanti e, et al.: heart abnormality classifications using fourier transforms method and neural networks but determining the information on the ecg recording data requires experience and knowledge about heart disease and its symptoms. manual extraction of the essential information on ecg signal is inefficient because of the amount of data that must be observed.16 on the other side, this recent year’s studies using artificial neural network (ann) have been developed. ann is a computational method of artificial intelligence based on human biological neural model of a computer or a machine that can duplicate human intelligence.18 from this phenomenon, one solution to analyze the heart’s electrical signals on the ecg is by using a software based on artificial neural network (ann) into a computational analysis to identify and classify abnormalities of the heart through the scanned ecg records. to reduce the computational load due to the amount of data that needs to be observed, feature extraction with image transformation is used. several studies about the use of scanned ecg records has done by endarko6 by image processing in order to obtain numerical data as an ann input to detect coronary heart disease. at karimah.11 bachrowi2 and asmaria1 research, using a ecg graph ordinate retrieval feature extraction as input to ann. the use of image transformation as feature extraction previously done by kaur10 and sarkaleh15 by using wavelet transform. some of these studies become the basis of this study as an attempt to help identify heart abnormalities. feature extraction done with the discrete fourier transform, and also because the fourier transform can bring in the form of frequency characteristics of image that often appears in the image, which can’t be seen with the eye.5 the study consists of pre-processing, segmentation and morphological operations, feature extraction, and classification of cardiac abnormalities. transformations done at ecg graph to obtain fourier coefficients as an input feature for ann. input patterns were divided into 3 groups, namely normal heart, atrial fibrillation, and coronary heart disease. the whole program is created using matlab. methods data collection research data collections include the acquisition of ecg image that has been diagnosed by a cardiologist manually. from the data collection obtained 87 data; 33 cardiac disorder atrial fibrillation, 13 coronary heart, and 41 normal heart. initial preparation which is cutting the image, with the intention of making the entire cycle on one ecg lead, where in one lead consisted of three ecg cycles. lead which used in this study is data from the lead ii, with the length of 530 pixels. software design broadly the image will be processed by pre-processing, grayscaling, followed by image segmentation with thresholding to obtain binary image, which then continued by morphological image processing. from the resulting image, ordinate value of the image soughted to show the electrical potential value of cardiac which will form the graphic visualization of ecg image. the starting point was taken from the tip of electrocardiogram isoelectric line, with upward deflection is positive and downward deflection is negative. so that obtained pixel value equal to the height of the image pixels on the electrocardiogram. feature extraction in image processing is done by fourier transformation on the value. data were normalized before feature transformation results of are used as networks input data. normalization aims to facilitate the artificial neural networks in the training process, the process of finding the weight, and the testing process. normalization performed on the input features and the target for the network whilst denormalization to the output value of the network to return to its original shape. training process using backpropagation network according to flowchart in figure 1: figure 1. training process flowchart. 34 indonesian journal of tropical and infectious disease, vol. 5. no. 2 may–august 2014: 32–36 result and discussion data processing result a total of 87 data is passing through a series of image processing, include grayscale, segmentation, dilation and erosion morphological operations, and feature extraction. from this amount of data are divided into two, namely 65 training data and 22 testing data. both groups include all of data categories, which are heart with atrial fibrillation disorder, coronary heart disease, and normal heart. testing data used in test validation, this aims to determine the accuracy of the program that has been created. preprocessing this preprocessing includes grayscale using matlab process that aims to transform the rgb image into 8 bit image that has a scale range of 0-255. the rgb image into 8 bit image that has a scale range of 0-255. figure 6 normal heart image after grayscale process figure 2. normal heart image after grayscale process. segmentation the image that has been through a preprocessing segmentation then has the background removed by segmentation. this process causing only the graph remains. segmentation is done by delivering threshold value, so that the image obtained is a binary image which simply made up by black and white color. figure 3. segmentation process result. morphology operations earlier segmentation process causes the binary image in figure 7 produces intermittent graphs in some parts, that it is necessary to conduct morphology operations to rebuild disconnected parts of the graph. operations performed include dilation and erosion. (a) (b) figure 4. morphological image improvement (a) dilation (b) erosion. feature extraction feature extraction process aims to obtain the characteristic features of the image. prepared image is a binary image that has been repaired through morphological operations. initial stages feature extraction is to find the value of each pixel ordinate in the image which represents the potential value of ekg graph. ordinate value search includes the starting point of the graph, which is point 0 on the image y axis. point 0 on y axis adjusted to the isoelectric line of the ecg graph, so its value will be in accordance with the high of pixel graphs. graph visualization derived from the matrix which contains the values obtained from the y axis. next, fourier coefficients calculated from each value, in order to get 530 pieces of fourier transform coefficients which in accordance to the number of pixels at the image length. on this fourier transform results, conducted coefficient part retrieval as an input for artificial neural networks training and testing. coefficient part retrieval aims to reduce the high computational load because of the many data features were trained, and find the smallest number of figure 5. graph visualization length. figure 10 fourier transforms plotting figure 5. fourier transforms plotting 35purwanti e, et al.: heart abnormality classifications using fourier transforms method and neural networks input features that can provide the best results. a complete fourier coefficient parts and number of features for neural network input is presented in table 1. backpropagation network formation backpropagation network training the training process uses 65 data that divided into three classes. the data used for training consisted of 30 normal heart data, 9 coronary heart data and 25 atrial fibrillation data. in this study, the manipulated variable is number of neurons in a hidden layer network. the weights used are the random weights to bias, input and hidden weight. thus in this study weight training which has the best accuracy results for each neuron are used. activation function used is bipolar sigmoid function which ranged (-1,1), because network output target is -1 for atrial fibrillation, 0 for coronary heart and 1 for normal heart. searching the best weight without normalization tends to be more difficult and less efficient because there is a quite far numbers range differences in the fourier coefficients. training accuracy results obtained from neural network training parameter changes can be found in table 2. table 2 shows that the greater number of neurons, the smaller mse generated where this result applies to any number of features. the smaller number of features also led to smaller mse generated. weight searching also tends to be faster on fewer input features. this can be seen in the table above, which generating smaller mse. the use of coefficient parts peaked at 1/16 coefficient, where on the use of 1/32 coefficient, mse results are no smaller from the use of 1/16 coefficient. backpropagation network testing the network testing process uses 22 data which haven’t been used for training. this testing data consists of 10 normal heart data, 4 coronary heart data and 8 atrial fibrillation data. the testing process is done with the same parameter variation as was done during the training process, yet only using the final weights of each variation, to obtain the best results without repeating the training process. backpropagation network test results can be seen in table 3. table 1. fourier coefficient parts and number of features for neural network input coefficient parts number of input features whole coeffients 530 1/2 coeffients 265 1/4 coeffients 133 1/8 coeffients 66 1/16 coeffients 33 1/32 coeffients 16 table 2. training data results neuron coefficient parts mse accuracy 3 1 0.0134 100% 1/2 0.0134 100% 1/4 0.0128 100% 1/8 0.0122 100% 1/16 0.0120 100% 1/32 0.0324 98.46% 5 1 0.0121 100% 1/2 0.0121 100% 1/4 0.0120 100% 1/8 0.0118 100% 1/16 0.0117 100% 1/32 0.0306 98.46% 10 1 0.0119 100% 1/2 0.0118 100% 1/4 0.0118 100% 1/8 0.0118 100% 1/16 0.0117 100% 1/32 0.0119 100% 20 1 0.0118 100% 1/2 0.0118 100% 1/4 0.0118 100% 1/8 0.0117 100% 1/16 0.0117 100% 1/32 0.0119 100% 30 1 0.0118 100% 1/2 0.0118 100% 1/4 0.0117 100% 1/8 0.0117 100% 1/16 0.0117 100% 1/32 0.0118 100% from table 3 can also be seen the influence of the parameter changes; number of neurons on the number of features. the greater the number of features, the greater the number of neurons needed to achieve the highest level of accuracy. rate of accuracy will decrease after reaching the optimal number of neurons. based on the results of accuracy rate in table 3, the highest accuracy rate of this network is 95.45%. this rate is can be found on several variations of fourier coefficient parts and number of neurons. however, if associated with the mse of training results in table 2, and considering the amount of computational load, the highest accuracy rate and optimal parameters found in the 36 indonesian journal of tropical and infectious disease, vol. 5. no. 2 may–august 2014: 32–36 references 1. pratanu, sunoto, 1999, "buku ajar ilmu penyakit dalam", fk ui, jilid 1, edisi ke-3, jakarta 2. schamroth, l., 1990, an introduction to electrocardiography, blackwell science, oxford. 3. waslaluddin s dan wahyudin a, 2010. klasifikasi pola elektrik jantung pada elektrokardiogram (ekg) menggunakan jaringan saraf tiruan berbasis backpropagation, bandung: universitas pendidikan indonesia, 62–65. 4. endarko, et al. 2006. aplikasi pengolahan citra elektrokardiograf dan jaringan saraf tiruan untuk identifikasi penyakit jantung koroner. jurnal fisika dan aplikasinya fmipa its surabaya. pp. 35–38. 5. karimah, fatimul, 2012, implementasi learning vector quantization sebagai alat bantu identifikasi kelainan jantung melalui citra elektrokardiogram, skripsi, fakultas sains dan teknologi univeresitas airlangga surabaya 6. bachrowi t, purwanti e. 2012. deteksi sinyal ecg irama myocardial ischemia dengan jaringan saraf tiruan. program studi teknobiomedik fakultas sains dan teknologi universitas airlangga, surabaya. 32–34. 7. asmaria t, purwanti e. 2012. deteksi dua belas sadapan sinyal elektrokardiogram untuk mengenali kelainan jantung menggunakan jaringan saraf tiruan dengan metode backpropagation. program studi teknobiomedik fakultas sains dan teknologi universitas airlangga, surabaya. 45–56. 8. kaur, jasminder, raina jps, 2012. an intelligent diagnosis system for electrocardiogram (ecg) images using artificial neural network (ann), international journal of electrical, electronics and computer engineering, 1(1): 147–151. 9. sarkaleh mk, shahbahrami a, 2012. classification of ecg arrithmias using discrete wavelet transform and neural networks, international journal of computer science, engineering and applications (ijcsea) vol. 2, no. 1. pp. 123–125. 10. dougherty, geoff. 2009. digital image processing for medical applications, cambridge university press new york. coefficient parts of 1/8 with number of neurons 5, and the final mse 0.0118. lowest accuracy rate obtained on the use of coefficient parts of 1/32 and the number of neurons 30, which is 68.18%. from the overall results in table 3, overall accuracy rate of this software has an average of 86.05% and a standard deviation of 7.82. conclusion image features for neural network software in this study was obtained through image processing, which begins from grayscaling, segmentation, dilation, erosion and followed by ecg signal graphs feature extraction with fourier transforms. this software using artificial neural networks backpropagation, with processed scanned ecg records image as an input. this image converted to a onedimensional time series signal to obtain the value that is equivalent to voltage value on the ecg image which is then transformed with discrete fourier transform. output of this software is a numerical value cardiac abnormalities classification. maximum accuracy rate of this software is 95.45%, with the distribution of the fourier transform coefficients 1/8 and number of nodes 5, while minimum accuracy rate of this software at least 68.18% by distribution of the fourier transform coefficients 1/32 and the number of nodes 32. overall result accuracy rate of this software has an average of 86.05% and standard deviation of 7.82. tabel 3. testing data accuracy fourier coefficient parts 1 1/2 1/4 1/8 1/16 1/32 n eu ro n 3 81.81% 95.45% 81.81% 86.36% 86.36% 72.72% 5 86.36% 90.90% 95.45% 95.45% 81.81% 72.72% 10 90.90% 86.36% 90.90% 90.90% 81.81% 72.72% 20 90.90% 95.45% 86.36% 90.90% 90.90% 77.27% 30 95.45% 90.90% 90.90% 86.36% 81.81% 68.18% 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 69 vol. 1. no. 2 may–august 2010 a patient with suspected diphtheria m. vitanata arfijanto1,2, siti irma mashitah3, prihartini widiyanti2, bramantono1,2 1 division of tropical infectious disease, departemen ilmu penyakit dalam fk unair rsud dr. soetomo surabaya 2 institute of tropical disease airlangga university 3 departement of cardiology fk unair rsu dr. soetomo abstract it was reported that a mature woman, mrs. s, 42 years old with several complaints and symptoms such as fever, swallowing pain weak body, swollen tonsil with beslag, dirty uvula of mouth cavity and tongue, and bullneck. the final diagnosis indicated that the patient was suspected diphtheria, candidiasis oris, sepsis, and pneumonia. the sudden death of the patient was probably caused by myocarditis. keywords: myocarditis, diphtheria, sepsis introduction diphtheria has been a nightmare for thousand years hunting human life and health. it was firstly found in hippocrates era when the first epidemic occurred in the 6th century b.c. (nandi et al, 2003). this disease is still endemic in many developing countries in africa, asia, and south america (shah, 2005). the change of diphtheria epidemiology happens in the entire part of the world. the proportion of adults who are susceptible to diphtheria is mostly discovered in many developing and developed countries (mattos et al., 2003). it was reported that there were 39 cases in east java province in 2006 including 8 cases in surabaya, 7 cases in kabupaten sidoarjo, 4 cases in kabupaten, sumenep, and 4 cases in probolinggo (health department east java, 2006). this classic disease caused by gram-positive bacillus called corynebacterium diphteriae which usually occurs in the upper respiratory tract. it is indicated by the formation of pseudomembrane in the infected place followed by the general symptoms caused by exotoxin produced by the bacillus (acang, 2006). the emergence of immunization program caused relatively low cases of diphtheria as a contagious disease (health department east java, 2006). the progress of the disease is very fast. therefore, the high level of suspicion towards the disease is important to be maintained (mattos et al., 2003). in an acute state, it has case-fatality ratio > 20% if there is no sufficient diagnostic procedure and therapy option. however, the ratio can decrease up to 3% if there is an antitoxin (volzke, 2006). because the disease rarely occurs, many doctors also rarely face the diphtheria case. therefore, it might cause diagnosis failure through clinical examination. not all laboratories regularly perform throat swab culture for c. diphtheriae. it probably increases the case of wrong diagnosis or late diagnosis for diphtheria (bonnet, 1999). if we are late to diagnose and cure the disease, it can increase the probability of death rate up to 20 times higher than its normal death rate. the most dominant factor causing death is myocarditis (acang, 2006). myocarditis diphtheria incidences related to nasopharyngeal diphtheria were 10-20% with death rate up to 50%–60% (kneen et al., 1998; dung et al., 2002). in this case, a patient was reported with suspected diphtheria. she died and it was probably caused by myocarditis. this study focused on its diagnostic problems and management. case a woman, mrs. s, 42 years old, a moslem, a javanese, an office girl in a factory in manukan tandes surabaya, living in bongso wetan pengalangan menganti gresik, came to rsud dr. soetomo through icu (intensive care unit) in april 29, 2009. the major symptom, problem, or complaint was fever or high body temperature. she had experienced the fever since 9 days before she was hospitalized. her body also shook when she got the fever. analgesic medication gave some relief to the case report 70 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 69-76 fever then she started to sweat only for a moment and the temperature rose again. she also complained about high body temperature together with sore throat like a misnomer. and she had nausea without vomiting and her body was felt so weak. since 4 days before hospitalized, she didn’t want to eat because of pain when she widely opened her mouth and tried to swallow. it was accompanied by white spots which looked dirty in her mouth cavity since 3 days before hospitalized. in addition, one day before hospitalized, her neck was getting bigger like a mumps. there was no cold/ flu, cough, dyspnoea, or husky voice, while excrement and urination were normal. from the immunization record, the patient was not sure whether she had been immunized before. she also stated that she used dentures since a month before she was hospitalized. either her family living in the same house with the patient or her neighbors never experienced an illness like her before. in addition, the patient lived near prostitution area. previously, she was a patient in bhakti rahayu hospital suspected diphtheria and hospitalized there for 7 days with therapies as follows: ciprofloxacin infusion 400 mg/12 hours, metoclopramide, paracetamol tablets 3x500 mg. she brought the examination result of diphtheria sample. from the throat swab analysis, granular bacilli were found and through the examination of spore sample, yeast cells were discovered. physical examination in april 29, 2009 (at night) the general condition of the patient was weak, blood pressure 120/80 mmhg, pulse rate 90 bpm regular, respiratory rate 22/60 hz, axillary temperature 36.8° c (after paracetamol intake). through head and neck examinations, there was no pale conjunctiva, icterus, cyanosis, dyspnoea, respiratory stidor, or rhinorrhea from nose. there was a lymph node in neck with the appearance of bullneck. through the examination of mouth cavity, it was found that both tonsils were swelling and looked dirty (beslag/membrane+) it also occurred in uvula, mouth cavity, and tongue. from the chest examination, it indicated that there was no movement from auxiliary breathing muscle; the first and the second heart beats were sole and regular; there was no murmur, gallop sound, or extrasistole; and there was no ronchi or wheezing. abnormality was not found in stomach or gastro-intestinal system examination. through extremity test, there was no edema or wound with normal physiology reflect. laboratory examination in april 29, 2009 blood test showed these following information: hemoglobin 11.6 g/dl, leukocyte 4.100/mm3, glucose 121 mg/dl, creatinine serum 0.5 mg/dl, bun (blood urea nitrogen) 7.9 mg/dl, sgot (serum glutamic-oxaloacetic transaminase) 29 iu/l, albumin 3.1 g/dl. blood gas analysis revealed these data: ph 7.54, pco2 29 mmhg, hco3 – 24.8 mmol/l, be 2.3 mmol/l, so2 97%. supporting or additional examination april 29, 2009 chest x-ray : normal heart and lungs electrocardiography : sinus rhythm 100 beats/minute with normal axis diagnosis : suspected faucial diphtheria with candidiasis oris + sepsis. to support the diagnosis, throat and nose swabs were performed and the patient was taken into an isolated room. therapy : o 2 nasal 3–4 lpm, tutofusin infusion: rd5: pz (21 drops/ minute), high calorie and high protein diet through sonde 6×150 cc, anti-diphtheria serum 40.000 units, ppc (phosphatidylcholine) i n j e c t i o n 2 × 6 0 0 . 0 0 0 i u / i m , nystatin drop, and paracetamol 3×1. monitoring : vital signs, airway obstruction case history treatment day 1 s: fever, sore throat, pain in swallowing, difficulty in eating, slight drinking, nausea without vomiting o: general condition was weak with gcs 456, blood pressure 110/70 mmhg, pulse rate 88 bpm, rr 22/60 hz, axillary temperature 38° c. head: no anemia, icterus, cyanosis, dyspnoea swelling tonsil with beslag, dirty mole on uvulapalate of the tongue thorax: symmetric without retraction of respiratory muscle cor: s1 s2 sole, without murmur pulmo: without wheezing or ronchi abdomen: intestinal noise +, liver and lien were not palpable extremity: warm acral without edema complete urine test: protein +1, epitel cell 6–8 the result of the second throat swab: bacillus gram negative, yeast a: suspected faucial+candidiasis oris+sepsis p: tutofusin injection rd5: pz (21 drops/minute), sonde diet high calorie high protein 6×150 cc, ppc 2×600.000 iu/im, ranitidine injection 2×1 amp iv, ketoconazole 2×1, nystatin drop, and paracetamol 3×1 treatment day 2 s: fever, sore throat, pain in swallowing, slightly eating, finish the milk, sometimes nausea o: general condition was weak with gcs 456, blood pressure 100/80 mmhg, pulse rate 90 bpm regular, rr 20/60 hz, axillary temperature 38° c. head/neck: no anemia, icterus, cyanosis, dyspnoea 71arfijanto et al.: aatient with suspected diphtheria swelling tonsil with beslag, dirty mole on uvula-palate of the tongue still appears thorax: symmetric without retraction of respiratory muscle cor: s1 s2 sole, without murmur pulmo: without wheezing or ronchi abdomen: no abnormality extremity: warm acral without edema the result of throat swab: bacillus gram negative a: suspected diphtheria, candidiasis oris+sepsis p: tutofusin injection rd5:pz (21 drops/minute), sonde diet porridge, ppc 2×600.000 iu/im, ketoconazole 2×1, nystatin drop, and paracetamol 3×1 treatment day 5 s: fever, less sore throat, eat more porridge, no nausea and vomiting o: general condition was weak with gcs 456, blood pressure 110/70 mmhg, pulse rate 100 bpm regular, rr 22/60 hz, axillary temperature 38° c. head/neck: no anemia, icterus, cyanosis, dyspnoea less swelling tonsil with beslag, dirty mole on uvulapalate of the tongue started to be thorax: symmetric without retraction of respiratory muscle cor: s1 s2 sole, without murmur pulmo: without wheezing or ronchi abdomen: no abnormality extremity: warm acral without edema vct result: hiv antibody non reactive a: suspected diphtheria, faucial+candidiasis oris+sepsis p: tutofusin injection rd5:pz (21 drops/minute), sonde diet porridge, ppc 2×600.000 iu/im, ketoconazole 2×1, nystatin drop, and paracetamol 3×1 treatment day 6 s: fever, no appetite, less pain in swallowing, cough with phlegm, no nausea, vomiting, or dyspnoea o: general condition was weak with gcs 456, blood pressure 120/70 mmhg, pulse rate 96 bpm regular, rr 22/60 hz, axillary temperature 39° c. head/neck: no anemia, icterus, cyanosis, dyspnoea less swelling tonsil with beslag, dirty mole on uvulapalate of the tongue started to clean thorax: symmetric without retraction of respiratory muscle cor: s1 s2 sole, without murmur pulmo: without wheezing or ronchi abdomen: no abnormality extremity: warm acral without edema the result of throat swab: bacillus gram negative a: suspected diphtheria, faucial+candidiasis oris+suspected pneumonia+sepsis p: tutofusin injection rd5:pz (21 drops/minute), sonde diet porridge, ppc 2×600.000 iu/im, cefriaxone 2 gr 1×1 iv, ketoconazole 2×1, nystatin drop, & paracetamol 3×1 examination suggestion: a plan for lungs consultation based on the suspected pneumonia was postponed waiting the result of the second thorax rontgen (cito). treatment day 7 s: still fever and cough with phlegm, the patient didn’t want to eat even the swallowing pain decreased, no nausea, vomiting, or dyspnoea o: general condition was weak with gcs 456, blood pressure 120/70 mmhg, pulse rate 106 bpm regular, rr 24/60 hz, axillary temperature 38.5° c. head/neck: no anemia, icterus, cyanosis, dyspnoea, there was tachypnea less swelling tonsil with beslag, dirty mole on uvulapalate of the tongue started to clean thorax: symmetric without retraction of respiratory muscle cor: s1 s2 sole, without murmur pulmo: without wheezing or ronchi abdomen: no abnormality extremity: warm acral without edema a: suspected diphtheria, faucial+candidiasis oris+suspected pneumonia+sepsis p: o2 nasal 3–4 lpm, tutofusin injection rd5:pz (21 drops/minute), sonde diet porridge, ppc 2×600.000 iu/im, cefriaxone 2 gr 1×1 iv, ketoconazole 2×1, nystatin drop, and paracetamol 3×1 examination suggestion: blood culture, thorax rontgen cito (delayed). at 2 p.m. in the 7th day of treatment it was reported that suddenly the patient had apneusis. when the pupils was checked, it had been mydriasis. previously, based on the family statements, she had just finished the meal and she was able to eat half portion of the meal. she asked for a meal because she felt uncomfortable in liver area and felt cold in her body. after finishing the meal, the patient’s family went out from the isolation room for about 15 minutes to wash their hand in the bathroom. however, when they came back to the room, the patient didn’t breathe anymore. final diagnosis: suspected diphtheria, faucial+candidiasis oris+suspected pneumonia+sepsis+suspected myocarditis the result of throat and nose swabs proliferation: before finished the therapy in may 8, 2009 no development of corynebacterium diphtheria. the result of blood culture: finished in may 12, 2009, it was found escheria coli (esbl+) without the development of anaerobe microbes. analysis diphtheria is an acute infection in mucosa of respiratory tracks (tonsil, pharynx, larynx, or nose) sometimes it appears on skin but rarely occurs in other mucosa such as eye, ear, and genital. diphtheria is caused by gram-negative 72 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 69-76 bacillus which is aerobe, no capsule, non-motile, no spore, and produces exotoxin (white, 2003; tiwari, 2008). human is the only reservoir of diphtheria infection (lumio, 2003). the spreading of the disease is through direct contact from respiratory liquid droplet (cough, sneeze, and talk), exudates of patient’s skin lesion or diphtheria carrier, or through indirect contact with contaminated dust, cloth, book, or toy (shah, 2005; acang, 2006). before the development of immunization program, diphtheria was a childhood disease. since 1980, the immunization program had increased the probability of the infection in adults (acang, 2006). because the incidence of diphtheria was rare, the exposure to the bacteria causing the disease was not a common thing to do as well as its repeated exposure. if adult people had not been exposed naturally with diphtheria or administered booster dosage of toxoid diphtheria, the immunity gained in the childhood will decrease in the manhood. therefore, adults are susceptible to diphtheria (nandi et al., 2003). antibody level towards diphtheria is considered protective, if the level ≥ 0.1 iu/ml (full protection) (prosppero et al., 1997; tiwari, 2008). the immunity of the half recovered patients of diphtheria was not adequately developed. therefore, immunization is still needed to be given after the patients recover from diphtheria (prosppero et al., 1997; shah, 2005). the result of a research in turkey stated that the protection upon diphtheria starts to decrease after the age of 30 and achieves its lowest level in the age of 40–49 (cavus et al, 2007). in developing countries where diphtheria is still endemic, the disease characteristics indicate that it has high fatality level, numerous complications, and mostly attacks young people or adults (mattos et al, 2003). some studies proofed that women has higher risk towards the diphtheria infection because they have lower immunity level than men. therefore, women are more likely to be attacked by diphtheria (acang, 2006). the patient was a woman aged 42 years old, born in 1967. the immunization program had just established in 1980. therefore, probably the patient had not been administered by diphtheria immunization. even if she had been infected before, the immunity formed was not adequate to protect her from the second attack of the disease. the diagnosis of diphtheria is based on signs and clinical symptoms supported by laboratory data, test, or confirmation (bishai, 2008). clinical suspicion with a high index is the key of the diagnosis, therefore, antitoxin treatment can be immediately administered (lakkireddy, 2005). diphtheria is not easily and clinically diagnosed. mild cases such as pharyngitis streptococcal and classical form of diphtheria with pseudomembrane in pharynx might not develop, especially in person who had been given an immunization (bonnet, 1999). in one side infection of tonsil, the appearance of edema is often misinterpreted with peritonsillar abscess which tonsillectomy is not necessary to be performed (lumio, 2003). the characteristic of diphtheria pathology because of destructive effect of toxin in epitel cell is the existence of imperfect membrane (patchy) then it becomes thick in the following days called pseudomembrane (lumio, 2003; bishai, 2008). the diagnosis of diphtheria is should not be based on the appearance of pseudomembrane. many studies stated that pseudomembrane was not found in diphtheria patients for about 1/4–3/4 of the entire observed cases. it might never appear any membrane or it has gone in the beginning of the disease attack. although it not always appears, it has a special thick form and following substances: fibrin, broken epitel cell, bacteria, polymorphonuclear cell, grey or white in color and very sticky, if we try to get rid of it, it will leave a wound with bleeding surface (bonet, 1999; lumio, 2003). for clinical purpose, it will be much easier to classify this disease based on the location of lesion anatomy. the classifications of the locations are nasal, faucial (tonsil and pharynx), laryngeal or laryngotracheal, and non-respiratory: cutaneous, conjunctiva, and genital (lumio, 2003; white, 2003). diphtheria occurs after c. diphtheriae enters nose or mouth with incubation period around 2-5 days or even 10 days after the infection (lumio, 2003; bishai, 2008). unspecific complaints or symptoms will occur such as sore throat and fever sometimes with shiver, great pain and difficulty in swallowing food, nausea, vomiting, and headache as a part of faucial diphtheria (lumio, 2003). pseudomembrane initially formed in the tonsil might dilate to uvula, platum mole, oropharynx, nasopharynx, or larynx accompanied by lymph node, pain and oedema called bullneck. nasal diphtheria is usually mild and chronic characterized by nasal discharge which is usually clear or serous then it becomes serosanguineous and bleeding both unilateral and bilateral. larynx diphtheria is characterized by husky voice which gradually becomes heavier and stidor followed by breathe difficulty as the extension of faucial diphtheria. we can see indirectly the swollen epiglottis and subglottis with pseudomembrane through laryngoscopy. cutaneous diphtheria usually appears in foot in a form of pustule or vesicle to chronic ulcer with dirty grey membrane (bonnet, 1999; lumio, 2003; white, 2003). malignant diphtheria has more acute onset. the patients will easily become toxic, high fever, fast pulse, hypotension, and cyanosis. pseudomembrane widens fast together with bullneck state (white, 2003). the patient was diagnosed as suspected faucial diphtheria based on signs and clinical symptoms obtained from anamnesis such as high body temperature, sometimes with shiver followed by sore throat, then continued with difficulty in swallowing food, nausea, vomiting, weak body, swollen neck similar to goiter, and dirty state of mouth cavity. the physical examination showed axial temperature ³38° c, beslag in both tonsils with inflammation which is uncommon for diphtheria. uvula, palatum mole, and tongue looked dirty and there was a bullneck. laboratory diagnosis is used to confirm the infection and used as a supporting data not a replacement for clinical diagnosis (lumio, 2003). one of presumptive diagnosis which can be performed is the use of gram coloring 73arfijanto et al.: aatient with suspected diphtheria (acang, 2006). although c. diphtheriae is described as a positive-gram bacterium, it is easily faded during the coloring procedure and it might appear as negative-gram bacterium (white, 2003). definite diagnose is obtained and based on the discovery of c. diphtheria through cultural examination in selective media such as loffler, tellurite media, and tinsdale agar taken from throat and nose swabs. it is suggested that it is better to take the substances from the membrane or lower membrane if there is any (efstratiou, 1999). this examination is performed before and after the treatment (acang, 2006). however, the culture is often negative (40% cases) or other organism grow in it. the negative culture is especially resulted after the patient consumes antibiotic before coming to the hospital (white, 2003; lakkireddy, 2005). the treatment or cure using antibiotic causes 84%-96% patients showing negative result in the day 2–3 (lumio, 2003). if the result of the culture of the patient suspected diphtheria is negative, isolation of the bacteria from close contact is important to support the diagnosis confirmation (efstratiou, 1999). another test to diagnose the existence of diphtheria microbe is by detecting the presence or the absence of toxin through elek plate test (acang, 2006) but this examination is often misinterpreted. other different method is polymerase chain reaction (pcr) which is simple and fast (5-6 hours) (lumio, 2003). pcr is used to detect gene organizing the production of toxin (dtxr) and dyptheria toxin gene (tox) in a state where diphtheria bacterium organism is not found in culture when antibiotic has been given (tiwari, 2008). the patient brought the result of laboratory examination of throat swab with a negative result. however, the reexamination using gram coloring in rs dr. soetomo indicated that negative-gram bacilli were found with negative diphtheria in culture examination result. the treatment history of the patient was that during her treatment in rs. bhakti rahayu, she had been administered drip antibiotic ciprofloxacin 2×500mg. this condition was similar to several cases in other countries when there were negative results in both throat swab culture and pseudomembrane sample, while the pcr result of diphtheria was positive (lurie et al., 2004). the difference was that mrs. s did not perform the pcr examination. therefore, until her death, she was considered as patient with suspected diphtheria case. other laboratory examinations usually performed for diphtheria are complete blood examination and urinalysis. patients often show the moderate increasing of leukocyte and mild proteinuria (1+ to 2+) might also be found (frasseto, 2008). the result of complete blood examination tended to show leucopenia. it might be caused by sepsis from other infections. it was confirmed by the finding of e-coli (esbl+) and after her death, yeast in throat swab was found. sepsis was a clinical state related to infection with sirs manifestations (body temperature >38° c or <36° c, heart frequency >20/60 hz, respiratory frequency >20/60 hz or paco2 <32 mmhg, leucocytes >12.000/mm 3 or <40.000/mm3 or bacilli >10%) (chen, 2004). other evidences which showed that the patient was in a sepsis state were heart frequency ever reached up to 100/60 hz; body temperature was always >38° c; paco2 29 mmhg; average respiratory rate 22/60 hz daily. through urinalysis, it was found proteinuria +1. as e-coli (esbl+) found in the blood culture, it indicated that there was bacteria infection which produced esbl (extended spectrum beta lactamase). the patient suffered from several infections such as urine tube infection, peritonitis, cholangitis, abscess intra abdominal, ventilatorassociated pneumonia, and central-line associated bacteremia. there were several factors causing the infection and bacteria colony producing esbl: 1. the installation of catheter (artery, central vein, urine tube, gastrostomy or jejunostomy tube, and umbilical catheters); 2. surgery actions (abdominal and emergency laparotomy surgeries); 3. the use of antibiotics (cephalosporin 3rd generation especially ceftazidime, fluoroquinolone, trimethoprim-sulfamethoxazole); 4. previous treatment in nursing home; and 5. the length of treatment in the hospital or icu. e.coli producing esbl is multiresistant so that it increased the tension of the disease (wahjono, 2007). the treatment of diphtheria is divided into two: general and specific or special treatment. the general treatments includes: 1. isolation, 2. bed rest at least 2–3 weeks, 3. soft or liquid food depending on the state of the patient, 4. cleanliness of respiratory track and liquid absorption, and 5. electrocardiography control 2-3 times a week for 4-6 weeks to detect myocarditis earlier. the specific or special treatment aims to: 1. neutralize toxin produced by diphtheria bacilli, and 2. kill diphtheria bacilli producing toxin (acang, 2006). from other source, it stated that the patient should be strictly monitored especially related to heart and respiratory functions. in the patient with a wide pseudomembrane, it is necessary to consult with ear, nose, and throat (ent) specialist or anesthetist. it is recommended for performing tracheostomy or intubation if it is possible (bishai, 2008). c. diphtheriae is susceptible to antibiotics. nowadays, penicillin and eritromicin are recommended by who (world health organization) to cure diphtheria (kneen et al, 1998). antibiotics are administered to the patients until they are able to swallow without feeling pain. procaine penicillin g with dosage of 600.000 unit i.m. is given every 12 hours then followed by peroral drug 500mg every 6 hours up to 14 days until they can swallow (bishai, 2008). the dosage of antibiotics given is based on the location of primary infection, the dilatation of pseudomembrane, and the duration between onset and the intake of antitoxin: 20.000–40.000 units for faucial or cutaneous diphtheria which is less than 48 hours, 40.000–80.000 for faucial or laryngeal diphtheria which the onset is more than 48 hours, and 80.000–100.000 units for malignant diphtheria (white, 2003). 74 indonesian journal of tropical and infectious disease, vol. 1. no. 2 may–august 2010: 69-76 generally, the treatment given to the patient was appropriate with the standard for diphtheria treatment, although the hospital was late to give antitoxin. diphtheria antitoxin was given in the day 4 after the appearances of clinical signs and symptoms with minimal dosage (40.000 units). consultation with other division such as ent division was not performed because the suspicion toward infection dilatation in larynx was not found when husky voice or stidor was not found in the patient. in addition, ekg monitoring was also skipped. complication is the major cause of diphtheria morbidity or mortality. factors contributing the high mortality rate for diphtheria patients are inadequate immunization intake, low socio-economy standard, population density, lateness treatment, and the absence or lateness of antitoxin administration (jayashree et al., 2005). mechanic complication of diphtheria is caused by membrane, while the systemic effect is caused by toxin (shah, 2005). clinical intention should be focused on the obstruction of respiratory track, acute systemic toxicity, and myocarditis-neuritis mediated by the toxin (mattos et al., 2003). pseudomembrane can lose or widen to larynx and tracheobronchial branch so it obstructs the respiratory track (bishai, 2008). mortality in diphtheria case was mostly caused by complications mediated by toxin of c. diphtheriae which created wide damage (such as myocarditis or secondary respiratory failure resulted from peripheral neuropathy, larynx edema, kidney failure, disseminated intravascular coagulation) (dung, 2002). other diphtheria complications are pneumonia, embolic pulmonary, encephalitis, cerebral infarct, acute tubular necrosis (white, 2003; bishai, 2008). pneumonia sepsis or septicemia was also reported as diphtheria complication although the bacteria were rarely isolated from blood. bad state or status of patient’s immunology might be responsible for its manifestation (barakett et al., 1992). the patients was suspected myocarditis complication and pneumonia, while the sepsis was caused by other secondary bacterial infections not from the suspected diphtheria, but from e-coli (esbl+) as shown in blood culture result. the risks of complications in this patient were the absence of immunization, low socio-economy standard (office girl), late administration of antitoxin (in the 4th day after clinical symptoms occurred) with minimal dosage. myocarditis is an inflammation disease in myocardium caused by either infection or non-infection. in the examination post-mortem of myocarditis, it was found 1–9% of myocarditis. therefore, it was suspected as the cause of sudden death (alwi et al, 2006). heart damage or failure in diphtheria case is the major factor of mortality in adults (³40 years old) (lumio et al., 2004; nalmas et al, 2007). myocarditis is often found in patients with inadequate immunization history and late administration of antitoxin (jayashree et al., 2006). complication of heart increases 2–3 times in patients who receive antitoxin > 48 hours from the onset of the disease (white, 2003). diphtheria myocarditis generally occurs in the first 2 weeks of illness (bishai, 2008). the principles of its manifestation are cardiomyopathy dilatation, various types of disritmia and conduction problems. the effects can be asymptomatic and undetected if there is no routine hearth detection (volzke, 2006). sudden death might happen, although general description of heart failure is progressively unclear (fine, 1950). approximately, 50% of diphtheria myocarditis patients developed severe conduction problems related to the fatal outcome. it is because diphtheria toxic is irritable so that tachyarrhythmia might easily happen (dung et al., 2002). electrocardiography is a noninvasive procedure and it has an important role in the measurement of disease severity in various infections. it also might indicate heart complication and give information about prognosis (nalmas et al., 2007). ekg can show myocarditis caused by acute infection when there is only a minimal clinical signs, unclear, or even no sign at all (fine et al., 1950). ekg abnormality was reported from 16.5%–84% diphtheria patients (morgan, 1963). the change of t wave and heart block in the first degree could occur without any clinical sign and develop into severe heart block (mattos, 2003). conduction abnormality causing cardiogenic shock is the most manifestation happens in diphtheria case (jayashree et al., 2006). in a research, to early detect myocarditis diphtheria indicated by lengthen qt interval which causes ventricular arrhythmia, ekg with 12 lead serial can be performed as alternate say in 1st–10th day then continued given once a week after going out from the hospital (kneen et al., 1998). a strict ekg monitoring is an indication to detect heart problem caused by diphtheria toxic, especially in the first week of illness (white, 2003). it can be performed 2–3 times a week for 4–6 weeks to early detect myocarditis (shah, 2005). another monitoring to detect myocarditis is cardiac enzyme examination (acang, 2006), in which there is an increase of cardiac enzyme level (white, 2003; alwi et al., 2006). the risky factors which cause fatal diphtheria myocarditis in this patient were: age > 40 years old (without immunization history), administered by antitoxin > 48 hours after clinical diphtheria onset. she suddenly died in the day 7 of the treatment or in the second week since she complained about fever and sore throat in the previous hospital. the condition in 15 minutes before the death showed that she was in a good condition indicated from the appetite. she could eat the meal more than in the previous days. vital signs recorded in the morning were still good even though the patient in febris and there was an increase of breath frequency. it was probably a myocarditis as a complication caused by diphtheria toxic in heart. unfortunately, during the treatment in isolation room, she was not observed through strict ekg serial as an effort to early detect myocarditis in suspected diphtheria patient. the first ekg examination showed sinus rhythm 100/60 hz with normal axis. moreover, the physical observation of heart diagnostic was not maximal and there was no other 75arfijanto et al.: aatient with suspected diphtheria supporting examination for detecting myocarditis such as re-taking of thorax x-ray, cardiac enzyme, and previous echocardiography. it was also noted that the patient was an adult who epidemiology indicated to have higher risk of diphtheria. the outcome was fatal because it was supported by other mortality factor. summary and conclusion from the description of this case, there are several suggestions given: 1. because the diphtheria toxic can attack many organs, strict observation is needed to control all of the symptoms which occur either from patient’s complaints, physical examination, or other supporting information from the patient (acang, 2006) especially to the one who receive late diagnosis of diphtheria; 2. besides, consultation with other divisions is also needed related to the occurrence of complications (bishai, 2008), so that the patient can be completely treated; 3. if the patient is suspected sepsis state, it is necessary to immediately identify the vector bacteria. nasroudin stated that antibiotics can be administered in the first hour, if there’s an indication, while antibiotic monotheraphy can be given if there is an infection without neutropenia. if there is neutropenia, the combination of antibiotics is needed. definitive antibiotic is given based on reproduction result and sensitivity tests in the first 48–72 hours. reference 1. acang, n., 2006. difteri. dalam: a.w. sudoyo, dkk, ed. buku ajar ilmu penyakit dalam jilid 3 edisi iv. jakarta: pusat penerbitan dep. ipd fkui. pp. 1858–1861. 2. alwi, i. & mukmin, l.h., 2006. miokarditis. dalam: a.w. sudoyo, dkk, ed. buku ajar ilmu penyakit dalam 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decadesprevious history of diphtheria. eur j clin microbiol infect dis, 25, pp. 651–56. 28. wahjono, h., 2007. peran mikrobiologi klinik pada penanganan penyakit infeksi. pidato pengukuhan guru besar mikrobiologi universitas diponegoro, [online]. available at : http://eprints.undip. ac.id/320/1/hendro_wahjono.pdf [diakses 1 september 2009]. 29. white, n.j. & hien t.t., 2003. diphtheria. in: g.c.cook & a.zunla, eds. manson’s tropical diseases 21 ed. london: saunders. pp. 1137–1140. ijtid vol 1 no 2 may-aug 2010.17.pdf ijtid vol 1 no 2 may-aug 2010.18.pdf ijtid vol 1 no 2 may-aug 2010.19.pdf ijtid vol 1 no 2 may-aug 2010.20.pdf ijtid vol 1 no 2 may-aug 2010.21.pdf ijtid vol 1 no 2 may-aug 2010.22.pdf ijtid vol 1 no 2 may-aug 2010.23.pdf ijtid vol 1 no 2 may-aug 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. 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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 4.0 138 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 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tissue herawati1 and jenny sunariani2 1 department of periodontology 2 department of oral biology faculty of dentistry, airlangga university surabaya indonesia abstract tobacco contains thousands of chemical substances which known to be harmful to periodontal tissues. nicotine was considered as the most toxic substances to periodontal tissues. the datas in this review indicate that smoking may have a significant role in the initiation and progression of periodontal destruction. the conclusion of this and the other studies indicate that smokers have a less favorable response to periodontal therapy than non smoker. nicotine is potentially toxic substances that have a detrimental effect on periodontal tissue, by altering the host response or directly damage the cells of normal periodontium. key words: nicotine, risk factor, periodontal disease, vasocontriction, host response literature review introduction smoking had been a human�s habit for almost four centuries. at the first time, columbus brought tobacco to europe which he gets from the indians in america. from europe, smoking was spread throughout the world by portuguese and spanish�s world exploration and became an epidemic throughout the world. but recent studies found that smoking had a detrimental effect to our health.[1] smoking may be considered as a serious health issue as its epidemiological studies had shown that it may have a strong relationship to cancer and may cause physiological disturbances in pulmonary, cardiovascular and gastrointestinal. smoking was also associated to various changes in the oral cavity which was related to oral cancer. in the last two decades, the awareness of smoking effect was increased, especially on its detrimental effect towards periodontal tissue which may be followed by loose teeth.[2] recent researches revealed that smoking might be the most potential risk factor for periodontal disease. tobacco contains thousands of chemical substances e.c. nicotine, tar, and carbonmonoxydes. therefore, smoking and periodontal disease may be considered as public health problems. nicotine nicotine is an alkaloid, colorless, and highly volatile liquid. nicotine change its color to brown after contact with air and smell like tobacco. tobacco containes 1–2% of nicotine.[5] nicotine is an alkaloid derived from dried leaves of nicotiana tabacum and nicotiana rustica. nicotine is a tertiary amine of the pyridine and pyrrolidine rings. nicotine is a weak alkali (pka=8). at physiologic ph, 31% nicotine will not be ionized, therefore may penetrate the cell membrane. nicotine is water-and alcohol-soluble. cigarette smoke is an acidic substances (ph 5.5). in this acidic ph, nicotine is in ionic form and cannot easily penetrate the membrane so nicotine absorption in cheek mucosa occurs only from the smoke.[6] nicotine is easily absorbed from the airway, mucous membranes, and skin. percutaneous nicotine absorption may be poisonous. while smoking, the nicotine is removed from the alveoli into the blood. under acidic condition in alveolus, small particles undergo diffusion through the alveolar membrane or in the form of nicotine salt will removed as uncharged particles. these particles form nicotine which will be attached to the hair on mucosal lining of airway duct.[6] compared to the lipid solubility, ph differences may have stronger influence towards the distribution of nicotine throughout body�s organs and tissues. at physiological ph, small changes in ph may be happened due to non ionized substantial changes in nicotine�s fraction. lipid soluble amine bases such as nicotine was known to 152 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 151-154 have higher levels in tissue than in the blood. nicotine administered intravenously degrades slower in the arterial blood compared to when the nicotine ia absorbed during smoking. in addition, the nicotine in blood lipid fractions will be degraded completely by the liver.[7] inhaled nicotine fraction is metabolized by the lungs. nicotine�s metabolytes are known as konitin and nicotine-n-oxide which is formed from a-carbon oxide and n-oxidation of the pirolidin ring. half-life nicotine elimination obtained from smoking or parenteral injection is 30–60 menit.[8] nicotine and metabolites can be eliminated by the kidneys. the excretion rate of nicotine via urine is dependent upon ph. under alkaline condition, the excretion rate will be decreased. nicotine may also be excreted in breast milk in woman.[1] once it in the body, it might bind nicotinicacethylcholine receptors located in peripheral (neuromuscular junction, adrenal medulla, autonomic ganglia) and central nervous system. peripheral effects, included changes in the endocrine, and alteration in metabolic function of systemic blood vasoconstriction. these effects developed into body�s tolerance.[6] nicotine facilitated platelet adhesion associated with cardiovascular disease and hypertension. nicotine easily penetrated the blood brain barrier (blood-brain barrier) and stimulated the brain and released several neurochemical acetylcholine, beta-endorphin, dopamine, norepinephrine and vasopressin. in the medulla oblongata nicotine directly effect to the respiratory center. here, the nicotine in small doses stimulates breathing activity. but in large doses, nicotine will suppress respiration, and an overdose may cause death.[9] smoking can affect the behavior due to carbon monoxide and nicotine poisoning to the body. nicotine stimulates the production of adrenaline and cortisone. this hormone decreases the serotonin activity which is soothing the brain.[10] when people smoke, the nicotine come into the brain within seconds, which cause someone to feel comfortable. psychological effects of nicotine can cause addiction. an important indicator of physical dependence is the presence of withdrawal. the signs and symptoms of the withdrawal symptoms are dizziness, constipation, diarrhea, drowsiness, fatigue, unable to sleep, unable to concentrate, and depression. the degree of withdrawal symptoms for each person may be different.[7] it might be said that smoking was dangerous, so smoking habit should be stopped. smoking cessation is often as a result of the development of nicotine withdrawal symptoms. nicotine dependence can be treated by behavior and psychological counseling. the other nicotine replacement are nicotine chewing gum and nicotine transdermal. transdermal therapy was proven to be more secure and effective to stop smoking. terapi used for patients who are determined to stop smoking.[1] oral cavity is the first organ exposed to harmful effects of smoking. the alteration in oral cavity may be present as it become the first place to absorp any poisonous substances from smoking. the temperature cigarette on lips is approximately 30° c. while the temperature at the ends of burning cigarette is approximately 900° c.[11] hot smoke continually blowing into the oral cavity acts as a heat stimulus that causes alteration in blood flow and reduces saliva production. as a result the oral cavity becomes dry and anaerobic, thus providing a suitable environment for the anaerobic bacteria in plaque. smokers may have a higher risk of periodontal disease than those who do not smoke. effect of cigarette smoke irritates the gingiva directly and may affect the body through the bloodstream and saliva indirectly.[12] periodontal tissues such as gingiva, periodontal ligament, alveolar bone may be damaged by disruption of the normal function of host response to infection and can stimulate the body to destroy the surrrounding healthy tissues.[13] nicotine may play a role in initiating the periodontal disease because nicotine may be absorbed by soft tissues in oral cavity including the gingiva through the bloodstream and gingival attachment to the tooth surface and roots. nicotine can be found on the surface of the tooth�s root and the continin as it metabolism result may be found in gingival crevicular fluid.[14] periodontitis periodontitis is defined as an inflammatory disease of the supporting tissues of the teeth caused by specific microorganism or groups of specific microorganisms, resulting in progressive destruction of periodontal ligament and alveolar bone with pocket formation, recession, or both. periodontal disease occurs when bacterial toxins and enzymes destroy the supporting tissues of teeth and bone. plaque, attached to the teeth, may form hard deposits called calculus or tar within 48 hours. it is easily happened in smokers.[15] once the calculus has been attached to the teeth, it will not be easily cleaned with regular toothbrushing. scaling is the only available method to remove calculus. calculus which is located under the gums may cause inflammation and infection in periodontal tissue. because the symptoms are absent, very few people may recognize this disease at the early phase. people will usually recognize it in later phase. periodontal disease has some characteristics that can be described clinically. a. gingival inflammation gingival inflammation and the presence of bleeding after a light touches are the early signs of periodontitis. healthy gingiva is charaterized with hard-consistency, live pink coloured, and normal contours. there is no sign of bleeding after probing and the patient has no bleeding complaint when brushing the tooth. the severity of inflammation depends on the status of oral hygiene, lack 153herawati and sunariani: the effects of nicotine on the periodontal tissue of oral hiegene may exhibit gingival infection and bleeding when brushing teeth or sometimes spontaneous bleeding. b. periodontal pocket pocket is a gap between the teeth and gums are interpreted as an increase in the sulcus ginginva be pathological. normal gingival sulcus has a depth of between 2–3 mm. the pocket depth measurement is an important part in periodontitis� diagnosis. some factors may cause the deepening of normal gingival sulcus: 1) coronal movement of the gingival due to gingival inflammation; 2) apical displacement of the gingival attachment; and 3) a combination of both. pocket with a depth of 4 mm is considered as a initial sign of periodontal disease. c. gingival recession gingival recession is the exposure of tooth roots, which is happened together with chronic peridontitis but was not considered as a sign of periodontal disease. if there is a recession, pocket depth measurement is only a partial reflection of the amount of the whole periodontal damage.[16] discussion who developed a community periodontal index of treatment needs (cpitn). this index is received and used by several countries. some researchers conducted a study to compare between smokers and nonsmokers using cpitn reference. it was found that smoking was harmful to health. in oral region it may cause periodontal disease. several studies using cpitn revealed that smoking may harm periodontal health.[17] there was a strong relationship between smoking and periodontal disease, it is expected that the smokers have more routine periodontal treatment. the researchers reported that smokers have a higher cpitn scores than non-smokers. smoking can also change the body�s response to various periodontal therapies. besides, smoking may also play a role in the development of refractory periodontitis.[17] the number of cigarretes consumption determined the severity of periodontal destruction, especially the risk of oral cancer. there were some differences in the severity of periodontal destruction indicated that smoking might worsen host�s response to periodontal therapy. this means that smoking had a negative impact on periodontal treatment.[18] healing following periodontal therapy in smokers might need a longer time because the attachment of connective tissue and collagen fibers was inhibited, so it would disturb healing and tissue regeneration after treatment.[19] nicotine was toxic ingredients contained in cigarettes which was known to have a harmful effect on periodontal tissue, it may alter host�s tissue response or directly damage the cells of the normal periodontium. it could be proven that small amount of nicotine was stored-in and released from fibroblasts in the periodontal tissues.[20] nicotine was known to be able to inhibit cells splitting processes in osteoblast cultures by stimulating osteoblast alkaline phosphate activity in vitro. nicotine might also alter the periodontal cells in vivo. this meaned that nicotine had a tendency to disturb reparative and generative potential of the periodontium.[21] nicotine in cigarettes stimulates the sympathetic ganglia to produce neurotransmitters including katekolamin.[22] it stimulates alpha (a) receptor in blood vessels which then may cause vasoconstriction. vasoconstriction of peripheral blood vessels caused by smoking may also affect the periodontal tissues.[23] nicotine metabolism might cause vasoconstriction and suppress functional activity of polimorphonuclear (pmn) cells and macrophages. the quantity of neutrophils in the peripheral blood increased and penetrated cappilary wall.[24] polimorphonuclear (pmn) are phagocytes which are present in most common location of acute inflammation and have an important role in the defense of the periodontal tissue from bacterial invasion on gingival margin.[15] several studies showed that the effects of nicotine in cigarettes can reduce levels of salivary antibodies (iga) and serum igg antibody, which serve as a defence against porphyromonas intermedia and fusobacterium nucleatum.[25] it becomes clear that periodontium in smokers are prone to be infected with pathogens compared to non-smokers. nicotine in cigarettes could reduce levels of serum igg2. besides, it could also reduce the local oxygen tension and this can cause the growth of anaerobic bacteria. nicotine can also facilitate pathogen adhesion to epithelial cells. deep periodontal pockets may facilitate the growth of pathogenic anaerobic bacteria by providing a low-oxgygen environment.[15] in microbiology, it was hypothesized that the smokers had more plaque bacteria in periodontal disease compared to the non-smokers,. the smokers have more calculus accumulation. calculus may irritate the local tissue and provide a local environment filled with bacteria phatogens.[26] severity of periodontal disease was determined by the amount of cigarettes consumption and the duration of smoking. until now the data measuring the effectiveness of smoking cessation programs in reducing or inhibiting diseases periodontal has not been available yet.[4] in addition, nicotine may suppress the production of pro-inflammatory cytokines interleukin 1 (il-1) and tumor necrosis factor-alpha (tnf-a) which was considered as a key in regulating the host response against microbial infection.[15,27] periodontitis is destructive inflammation which causes loss of periodontal attachment and the supporting alveolar bone. however, the use of nicotine in tobacco cause damage to the collagen tissues, by increasing the production of colagenase.[4] nicotine suppress the growth of gingival fibroblast, and the production of collagen and fibronectin. in addition it also had an impact to leukocytes, by decreasing the migration ability of neutrophils and phagositosis.[20] 154 indonesian journal of tropical and infectious disease, vol. 1. no. 3 september–december 2010: 151-154 as a conclusion from this literature that nicotine may be a cause of periodontal disease. smoking may suppress the immune response and destroy vascular endothelial glands. from this literature study was also showed that smokers had less response to periodontal therapy. references 1. wardjowinoto s. hubungan antara merokok dengan penyakit periodontal. dental journal art. no. 2000; 628; p. 54–57. 2. johnson gk, slach na. impact of tobacco use periodontal status. j dental education. 2001; 65: 306–21. 3. martinez-ganut p, lorca a, magan r. smoking and periodontal disease severity. j clinic periodontal 2003; 22: 743–9. 4. ruslan g. efek merokok terhadap rongga mulut. cermin dunia kedokteran 1996; (113): 41–3. 5. chew cl, et al. long term dissolution of mercury from a non mercury releasing amalgam. clinical preventive dent. 1991; (3): 5–7. 6. goodman, gilman�s. pharmacological basis of therapeutic. 11th ed. mc graw hill co. inc; 2006. p. 213, 559. 7. gora mi. nicotine transdermal systems. the annals of pharmacotherapy 1993; 27: 742–8. 8. darby td, mcname je, van rossum jm. cigarette smoking pharmacokinetic and relationship to smoking behaviour. clinical pharmacokinetic. 1984; 9: 435–49. 9. wallstrom m, sand l, nilsson f, hirsch jm. the long trem effect of nicotine on the oral mucosa. j perio 1999; 94: 417–23. 10. kinane df, radvar m. the effect of smoking on mechanical an antimicrobial periodontal therapy. j perio 1997; 68: 467–72. 11. thomson wm, broadbent jm, welch d beck. cigarette smoking and perodontal disease among 32-years –old. j clinic periodontal 2007; 34: 828–38. 12. lamster ib. smoking as major risk factor for adult periodontitis. j clinic perio 1992; 23: 151–4. 13. barbour se, et al. tobacco and smoking: environmental factors that modify the host respon and impact on periodontal health. crit rev oral biol med 1997; 8: 437–60. 14. dm winn. tobacco use and oral disease. j dental education 2001; 65: 306–12. 15. pejcic a, obradovic r, kesic l, kojovic d. smoking and periodontal disease a review. medical biology 2007; 4(2): 53–9. 16. alamsyah rm. faktor yang mempengaruhi kebiasaan merokok dan hubungan dengan status penyakit peridontitis dikota medan. fkg usu. 2009. 17. kaldahl wb, johnson gk, patil kd, kalkwalf kl. levels of cigarette consumptiom and response to periodontal therapy. j periodontology 1996; 67: 675–81. 18. ryder mi. tobacco use and the periodontal patient. j periodontology 1996; 67: 51–4. 19. power jt. vascular damage from smoking: disease mechanisme at the arterial wall. vasc med 2001; 3: 21–8. 20. zhou j. olson bl windsor lj. nicotine increase the collagen degrading ability of human gingival fibroblasts. j periodontal res 2007; 42: 228–35. 21. esmeralda a, martinez t. root surface condotioning with nicotine or cotine reduces viability and density of fibroblas invitro. j periodontal 2005; 31: 180–6. 22. trauth ja, seidler fj, ali sf. slotkin ta. adolescent nicotine exposure produces immediate and long term cns noradrenergic and dopaminergic fuction brain res. 2001; 892: 269–80. 23. clark ng, hirsch rs. personalized risk factor generalized periodontitis. j clinic pero 1995; 22: 136–45. 24. macfarlane gd, herzberg mc, wolff lf, hardie na, refractory periodontitis associated with abnormal pmn leucocyte phagocytosis and cigarrete smoking j perio 1992; 63: 908–13. 25. ramon mj, calsina g, cheveria jj. effect of smoking on periodontal tissue. 2002. 29: 771–6. 26. marta en. understanding the etiology of periodontitis–an overview of periodontal risk factors. perio 2000. 2004; 32(1): 11–23. 27. kornmann ks, page rc. the host response to microbial challenge in periodontitis. periodontology 2000. 1997; 14: 33–5. ijtid vol 1 no 3 sep-dec 2010.49.pdf ijtid vol 1 no 3 sep-dec 2010.50.pdf ijtid vol 1 no 3 sep-dec 2010.51.pdf ijtid vol 1 no 3 sep-dec 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 80 404 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. 4. no. 4 october–december 2013 update management dengue shock syndrome in pediatric cases soegeng soegijanto1,2, eva chilvia2 1 dengue team of institute tropical disease indonesia 2 collaboration research center emerging re-emerging infections disease, institute of tropical disease, universitas airlangga kobe university japan 3 doctor in charge at rsab soerya hospital sidoarjo indonesia abstract background: since 1968 dengue virus infection has been found in indonesia, especially at surabaya and jakarta city. firstly management of dengue virus infection very difficult to improve, therefore the higher mortality nearly 41,4 % had been found but on the following years in five decades the mortality rates was becoming to decrease until 1,27 % on 2011. aim: to find the new management of dengue shock syndrome to reach the lower fatality rate below 1%. method: until now to manage dengue shock syndrome is very difficult, some cases can be improved but the other lost due to the late coming in the hospital and not involved in criteria diagnosis base on who 1997. to solve this problem who 2009 had made new criteria diagnosis dengue virus infection focusing on early detection of severe dengue virus infection especially dengue shock syndrome. result: on 2011 who had made an integrated criteria diagnosis base on who 2009 and who 1997. these criteria was focusing in update management of dengue shock syndrome in pediatric cases. based on this action, this paper will improve clinical management to reach the lower mortality of dengue shock syndrome in community until cfr < 1%. conclusion: by using integrated criteria of who 2009 and 1997, update management of dengue shock syndrome in pediatric cases, can improve clinical management to reach the lower mortality in community until cfr < 1%. key words: dengue virus infection; criteria diagnosis who; update management, shock, pediatric cases abstrak latar belakang: sejak tahun 1968, virus infeksi demam berdarah telah ditemukan di indonesia, khususnya di kota surabaya dan jakarta. pada awalnya, manajemen virus infeksi demam berdarah ini sangat sulit untuk dikembangkan, maka dari itu, ditemukan tingkat kematian hampir sebesar 41,4%; namun dalam beberapa tahun di 5 abad terakhir, tingkat kematian telah menurun sampai 1,27% di tahun 2011. tujuan: untuk menemukan manajemen baru dari sindrom shock demam berdarah untuk mencapai tingkat kematian yang lebih rendah yaitu dibawah 1%. metode: sampai saat ini pengendalian sindrom shock demam berdarah masih sangat sulit untuk dilakukan, beberapa kasus dapat dikembangankan namun lainnya tidak tertata akibat keterlambatan penanganan di rumah sakit dan tidak masuk dalam kriteria diagnosis berdasarkan pada who 1997. untuk memecahkan masalah ini, who 2009 teolag membuat kriteria diagnosis infeksi virus demam berdarah baru yang berfokus pada deteksi awal pada beberapa infeksi virus demam berdarah khususnya sindrom shock karena demam berdarah. hasil: pada tahun 2011, who telah membuat kriteria diagnosis yang terintegrasi berdasarkan pada who 2009 dan who 1997; kriteria ini berfokus pada manajemen terbaru di sindrom shock karena demam berdarah pada ilmu kedokteran anak. berdasarkan pada tindakan tersebut, penelitian ini akan memotivasi kita untuk mencapai tingkat kematian untuk menurunkan kurs dari 1% menjadi 0%. kesimpulan: dengan menggunakan kriteria dari who 2009 yang telah terintegrasi, pembaharuan manajemen dari sindrom shock akibat demam berdarah diharapkan dapat memotivasi kita untuk mencapai tingkat kematian di masyarakat lebih rendah kurang dari 1%. kata kunci: infeksi virus demam berdarah, kriteria diagnosis who, pembaharuan manajemen, syok, kasus anak-anak research report �0 indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 9–22 introduction dengue is the most rapidly spreading mosquito borne disease in the world. in last 50 years, incidence has increased 30-fold with increasing geographic expansion to new countries and, in the present decade, from urban to rural settings. in indonesia, where more than 35% of the country’s population lives in urban areas, 150,000 cases were reported in 2007 (the highest on record) with over 25,000 cases reported from both jakarta and west java. the case-fatality rate was approximately 1%. reported case in fatality rates for the region approximately 1%, but in india, indonesia and myanmar, focal outbreaks away from the urban areas have reported case-fatality rate of 3–5%. the mechanisms leading to the severe manifestations of dengue virus (denv) infections are still not completely understood but are likely to be multifactorial. the genetic background of the host influences the way that the immune response reacts to denv infection. upon inoculation of denv into the dermis, langerhans cell and keratinocytes will primarily be infected. the virus subsequently spreads via the blood (primary viremia) and infects tissue macrophages in several organs, especially the machrophages in the spleen. the replication efficiency of denv in dendritic cells (dc), monocytes and macrophage, as well as its tropism for and replication efficiency in endothelial cells (ec), bone marrow, stromal cells and liver cells, collectively determine the viral load measured in blood. this viral load represents an important risk factor for development of severe disease. essestially, infection of machrophages, hepatocytes and ec influence the hemostatic and the immune responses to denv. infected cells die predominantly through apoptosis and to a lesser extent trough necrosis. necrosis results in release of toxic products, which activate the coagulation and fibrinolystic systems, depending on the extent of infection of bone marrow stromal cells and the levels of il-6, il-8, il-10 and il-18, hemopoiesis is suppressed, resulting in decrease blood thrombogenicity. platelets interact closely with ec and a normal number of functioning platelets is necessary to maintain vascular stability. a high viral load in blood and possibly viral tropism for ec, severe thrombocytopenia and platelet dysfunction may results in increased vascular permeability and coagulopathy is amplified. in addition, enhancing igg antibodies bind heterologous virus during secondary infection and enhance infection of apcs, thereby contributing to the increased viral load that is in during secondary viremia in some patients. furthermore, a high viral load overstimulates both low and high-avidity cross reactive t cells. in the context of certain hla haplotypes, cross-reactive t cells delay virus clearance, while producing high levels of proinflamatory cytokines and other mediators. ultimately, these high levels of soluble factors, many of which still remain to be identified, induces changes in ec leading to the coagulopathy and plasma leakage characteristic of dss. dengue infection is a systemic and dynamic disease. it has a wide clinical spectrum that includes both severe and non-severe clinical manifestations. after the incubation period, the illness begins abruptly and is followed by the three phases, febrile, critical and recovery. laboratory diagnosis methods for confirming dengue virus infection may involve detection of the virus, viral nucleic acid, antigens or antibodies or a combination of these techniques. after the onset of illness, the virus can be detected in serum, plasma, circulating blood cells and other tissues for 4–5 days. during the early stages of the disease, virus isolation, nucleic acid or antigen detection can be used to diagnose the infection. at the end of the acute phase of infection, serology is the method of choice for diagnosis. until now to manage dengue shock syndrome is very difficult, some cases can be improved but the other lost due to the late coming in the hospital and not involved in criteria diagnosis base on who 1997. to solve this problem, who 2009 had made new criteria diagnosis dengue virus infection focusing on early detection of severe dengue virus infection especially dengue shock syndrome. on 2011 who had made an integrated criteria diagnosis base on who 2009 and who 1997. these criteria was focusing in update management of dengue shock syndrome in pediatric cases. based on this action, this paper will motivate us to reach the lower mortality of dengue shock syndrome in community until cfr < 1%. epidemiology dengue is the most rapidly spreading mosquito-borne viral disease in the world. in the last 50 years, incidence has increased 30-fold with increasing geographic expansion to new countries and, in the present decade, from urban to rural settings (figure 1). the countries of the region have been divided into four distinct climatic zones with different dengue transmission potential. epidemic dengue is a major public health problem in indonesia, myanmar, sri lanka, thailand and timorleste which are in the tropical monsoon and equatorial zone where aedes aegypti is widespread in both urban and rural areas, where multiple virus serotypes are circulating, and where dengue is a leading cause of hospitalization and death in children. cyclic epidemics are increasing in frequency and in-country geographic expansion is occurring in bangladesh, india and maldives – countries in the deciduous dry and wet climatic zone with multiple virus serotypes circulating. over the past four years, epidemic dengue activity has spread to bhutan and nepal in the subhimalayan foothills. reported case fatality rates for the region are approximately 1%, but in india, indonesia and myanmar, focal outbreaks away from the urban areas have reported case-fatality rates of 3–5%. ��soegijanto and chilvia: update management dengue shock syndrome in pediatric cases in indonesia, where more than 35% of the country’s population lives in urban areas, 50,000 cases were reported in 2007 (the highest on record) with over 25,000 cases reported from both jakarta and west java. the case-fatality rate was approximately 1%. criteria for diagnosing dengue (with or without warning signs) and severe dengue are presented in figure 2. it must figure 1. countries/areas at risk of dengue transmission (who, 2008) be kept in mind that even dengue patients without warning signs may develop severe dengue. expert consensus groups in latin america (havana, cuba, 2007), south-east asia (kuala lumpur, malaysia, 2007), and at who he adquarters in geneva, switzerland in 2008 agreed that: “dengue is one disease entity with different clinical presentations and often with unpredictable probable dengue live in /travel to dengue endemic area. fever and 2 of the following criteria: • nausea, vomiting • rash • aches and pains • tourniquet test positive • leukopenia • any warning sign laboratory-confirmed dengue (important when no sign of plasma leakage) signs* • abdominal pain or tenderness • persistent vomiting • clinical fluid accumulation • mucosal bleed • lethargy, restlessness • liver enlargment > 2 cm • laboratory: increase in hct concurrent with rapid decrease in platelet count * (requiring strict observation and medical intervention) 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 organs figure 2. suggested dengue case classification and levels of severity (who, 2009) �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 9–22 clinical evolution and outcome”, the classification into levels of severity has a high potential for being of practical use in the clinicians decision as to where and how intensively the patient should be observed and treated (i.e. triage, which is particularly useful in outbreaks), in more consistent reporting in the national and international surveillance system, and as an end-point measure in dengue vaccine and drug trials. this model for classifying dengue has been suggested by an expert group (geneva, switzerland, 2008) and is currently being tested in 18 countries by comparing its performance in practical settings to the existing who case classification. the process will be finalized in 2010. for practical reasons this guide adapts the distinction between dengue and severe dengue. dengue inflicts a significant health, economic and social burden on the populations of endemic areas. globally the estimated number of disability-adjusted life years (dalys) lost to dengue in 2001 was 528.1 the number of cases reported annually to who ranged from 0.4 to 1.3 million in the decade 1996–2005. as an infectious disease, the number of cases varies substantially figure 3. proposed model for the pathogenesis of df, dhf and dss based on an integrated view of the data presented (see section the integrated view in the text). black arrows, processes leading to the indicated event, colored boxes with white centers, pathological events. each event will ultimately affect the ec or the haemostatic system (purple arrows). (who, 2009) ��soegijanto and chilvia: update management dengue shock syndrome in pediatric cases from year to year. underreporting and misdiagnoses are major obstacles to understanding the full burden of dengue.2 on average, a hospitalized case of dengue cost three times what an ambulatory case costs. combining the ambulatory and hospitalized patients and factoring in the risk of death, the overall cost of a dengue case is us$ 828. merging this number with the average annual number of officially reported dengue cases from the eight countries studied in the period 2001–2005 (532.000 cases) gives a cost of officially reported dengue of us$ 440 million. children are at a higher risk of severe dengue.3 intensive care is required for severely ill patients, including intravenous fluids, blood or plasma transfusion and medicines. dengue afflicts all levels of society but the burden may be higher among the poorest who grow up in communities with inadequate water supply and solid waste infrastructure, and where conditions are most favourable for multiplication of the main vector, ae. aegypti. travellers play an essential role in the global epidemiology of dengue infections, as viraemic travellers carry various dengue serotypes and strains into areas with mosquitoes that can transmit infection.4 pathogenesis the mechanisms leading to the severe manifestations of denv infections are still not completely understood but are likely to be multifactorial (figure 3). the genetic background of the host influences the way that the immune response reacts to denv infection. upon inoculation of denv into the dermis, langerhans cells and keratinocytes will primarily be infected. the virus subsequently spreads via the blood (primary viremia) and infects tissue macrophages in several organs, especially the macrophages in the spleen. the replication efficiency of denv in dc, monocytes, and macrophages, as well as its tropism for and replication efficiency in ec, bone marrow stromal cells, and liver cells, collectively determine the viral load measured in blood. this viral load represents an important risk factor for development of severe disease. essentially, infection of macrophages, hepatocytes, and ec influences the hemostatic and the immune responses to denv. infected cells die predominantly through apoptosis and to a lesser extent through necrosis. necrosis results in release of toxic products, which activate the coagulation and fibrinolytic systems. depending on the extent of infection of bone marrow stromal cells and the levels of il-6, il-8, il-10, and il-18, hemopoiesis is suppressed, resulting in decreased blood thrombogenicity. platelets interact closely with ec, and a normal number of functioning platelets is necessary to maintain vascular stability. a high viral load in blood and possibly viral tropism for ec, severe thrombocytopenia, and platelet dysfunction may result in increased capillary fragility, clinically manifested as petechiae, easy bruising, and gastrointestinal mucosal bleeding, which is characteristic of dhf. at the same time, infection stimulates development of specific antibody and cellular immune responses to denv. when igm antibodies that cross-react with ec, platelets, and plasmin are produced, the loop that results in increased vascular permeability and coagulopathy is amplified. in addition, enhancing igg antibodies bind heterologous virus during secondary infection and enhance infection of apcs, thereby contributing to the increased viral load that is seen during secondary viremia in some patients. furthermore, a high viral load overstimulates both lowand high-avidity cross-reactive t cells. in the context of certain hla haplotypes, cross-reactive t cells delay virus clearance, while producing high levels of proinflammatory cytokines and other mediators. ultimately, these high levels of soluble factors, many of which still remain to be identified, induce changes in ec leading to the coagulopathy and plasma leakage characteristic of dss.5 clinical management and delivery of clinical services dengue infection is a systemic and dynamic disease. it has a wide clinical spectrum that includes both severe and non-severe clinical manifestations.6 after the incubation period, the illness begins abrupt and is followed by the three phases – febrile, critical and recovery (figure 4). for a disease that is complex in its manifestations, management is relatively simple, inexpensive and very effective in saving lives so long as correct and timely interventions are instituted. the key is early recognition and understanding of the clinical problems during the different phases of the disease, leading to a rational approach to case management and a good clinical outcome. activities (triage and management decisions) at the primary and secondary care levels (where patients are first seen and evaluated) are critical in determining the clinical outcome of dengue. a well-managed front-line response not only reduces the number of unnecessary hospital admissions but also saves the lives of dengue patients. early notification of dengue cases seen in primary and secondary care is crucial for identifying outbreaks and initiating an early response differential diagnosis needs to be considered. febrile phase patients typically develop high-grade fever suddenly. this acute febrile phase usually lasts 2–7 days and is often accompanied by facial flushing, skin erythema, generalized body ache, myalgia, arthralgia and headache.6 some patients may have sore throat, injected pharynx and conjunctival injection. anorexia, nausea and vomiting are common. it can be difficult to distinguish dengue clinically from non-dengue febrile diseases in the early febrile phase. a positive tourniquet test in this phase increases the probability of dengue.7,8 in addition, these clinical �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 9–22 features are indistinguishable between severe and nonsevere dengue cases. therefore monitoring for warning signs and other clinical parameters is crucial to recognizing progression to the critical phase. mild haemorrhagic manifestations like petechiae and mucosal membrane bleeding (e.g. nose and gums) may be seen.7,9 massive vaginal bleeding (in women of childbearing age) and gastrointestinal bleeding may occur during this phase but is not common.9 the liver is often enlarged and tender after a few days of fever.7 the earliest abnormality in the full blood count is a progressive decrease in total white cell count, which should alert the physician to a high probability of dengue. critical phase around the time of defervescence, when the temperature drops to 37.5–38° c or less and remains below this level, usually on days 3–7 of illness, an increase in capillary permeability in parallel with increasing haematocrit levels may occur.10,11 this marks the beginning of the critical phase. the period of clinically significant plasma leakage usually lasts 24–48 hours. progressive leukopenia7 followed by a rapid decrease in platelet count usually precedes plasma leakage. at this point patients without an increase in capillary permeability will improve, while those with increased capillary permeability may become worse as a result of lost plasma volume. the degree of plasma leakage varies. pleural effusion and ascites may be clinically detectable depending on the degree of plasma leakage and the volume of fluid therapy. hence chest x-ray and abdominal ultrasound can be useful tools for diagnosis. the degree of increase above the baseline haematocrit often reflects the severity of plasma leakage. shock occurs when a critical volume of plasma is lost through leakage. it is often preceded by warning signs. the body temperature may be subnormal when shock occurs. with prolonged shock, the consequent organ hypoperfusion results in progressive organ impairment, metabolic acidosis and disseminated intravascular coagulation. this in turn leads to severe haemorrhage causing the haematocrit to decrease in severe shock. instead of the leukopenia usually seen during this phase of dengue, the total white cell count may increase in patients with severe bleeding. in addition, severe organ impairment such as severe hepatitis, encephalitis or myocarditis and/or severe bleeding may also develop without obvious plasma leakage or shock.12 those who improve after defervescence are said to have non-severe dengue. some patients progress to the critical phase of plasma leakage without defervescence and, in these patients, changes in the full blood count should be used to guide the onset of the critical phase and plasma leakage. those who deteriorate will manifest with warning signs. this is called dengue with warning signs cases of dengue with warning signs will probably recover with early intravenous rehydration. some cases will deteriorate to severe dengue. recovery phase if the patient survives the 24–48 hour critical phase, a gradual reabsorption of extravascular compartment fluid takes place in the following 48–72 hours. general wellbeing improves, appetite returns, gastrointestinal symptoms abate, haemodynamic status stabilizes and diuresis ensues. some patients may have a rash of “isles of white in the sea of red”.13 some may experience generalized pruritus. bradycardia and electrocardiographic changes are common during this stage. figure 4. the course of dengue illness (who, 2009) ��soegijanto and chilvia: update management dengue shock syndrome in pediatric cases the haematocrit stabilizes or may be lower due to the dilutional effect of reabsorbed fluid. white blood cell count usually starts to rise soon after defervescence but the recovery of platelet count is typically later than that of white blood cell count. severe dengue severe dengue is defined by one or more of the following: (i) plasma leakage that may lead to shock (dengue shock) and/or fluid accumulation, with or without respiratory distress, and/or (ii) severe bleeding, and/or (iii) severe organ impairment. as dengue vascular permeability progresses, hypovolaemia worsens and results in shock. it usually takes place around defervescence, usually on day 4 or 5 (range days 3–7) of illness, preceded by the warning signs. during the initial stage of shock, the compensatory mechanism which maintains a normal systolic blood pressure also produces tachycardia and peripheral vasoconstriction with reduced skin perfusion, resulting in cold extremities and delayed capillary refill time. uniquely, the diastolic pressure rises towards the systolic pressure and the pulse pressure narrows as the peripheral vascular resistance increases. patients in dengue shock often remain conscious and lucid. the inexperienced physician may measure a normal systolic pressure and misjudge the critical state of the patient. finally, there is decompensation and both pressures disappear abruptly. prolonged hypotensive shock and hypoxia may lead to multi-organ failure and an extremely difficult clinical course. the patient is considered to have shock if the pulse pressure (i.e. the difference between the systolic and diastolic pressures) is ≤ 20 mm hg in children or he/she has signs of poor capillary perfusion (cold extremities, delayed capillary refill, or rapid pulse rate). in adults, the pulse pressure of ≤ 20 mm hg may indicate a more severe shock. hypotension is usually associated with prolonged shock which is often complicated by major bleeding. 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. massive bleeding may occur without prolonged shock in instances when acetylsalicylic acid (aspirin), ibuprofen or corticosteroids have been taken. unusual manifestations, including acute liver failure and encephalopathy, may be present, even in the absence of severe plasma leakage or shock. cardiomyopathy and encephalitis are also reported in a few dengue cases. however, most deaths from dengue occur in patients with profound shock, particularly if the situation is complicated by fluid overload. severe dengue should be considered if the patient is from an area of dengue risk presenting with fever of 2–7 days plus any of the following features: 1. there is evidence of plasma leakage, such as: high or progressively rising haematocrit; pleural effusions or ascites; circulatory compromise or shock (tachycardia, cold and clammy extremities, capillary refill time greater than three seconds, weak or undetectable pulse, narrow pulse pressure or, in late shock, unrecordable blood pressure). 2. there is significant bleeding. 3. there is an altered level of consciousness (lethargy or restlessness, coma, convulsions). 4. there is severe gastrointestinal involvement (persistent vomiting, increasing or intense abdominal pain, jaundice). 5. there is severe organ impairment (acute liver failure, acute renal failure, encephalopathy or encephalitis, or other unusual manifestations, cardiomyopathy) or other unusual manifestations. laboratory diagnosis and diagnostic tests dengue virus infection produces a broad spectrum of symptoms, many of which are non-specific. thus, a diagnosis based only on clinical symptoms is unreliable. early laboratory confirmation of clinical diagnosis may be valuable because some patients progress over a short period from mild to severe disease and sometimes to death. early intervention may be life-saving. before day 5 of illness, during the febrile period, dengue infections may be diagnosed by virus isolation in cell culture, by detection of viral rna by nucleic acid amplification tests (naat), or by detection of viral antigens by elisa or rapid tests. virus isolation in cell culture is usually performed only in laboratories with the necessary infrastructure and technical expertise. for virus culture, it is important to keep blood samples cooled or frozen to preserve the viability of the virus during transport from the patient to the laboratory. the isolation and identification of dengue viruses in cell cultures usually takes several days. nucleic acid detection assays with excellent performance characteristics may identify dengue viral rna within 24–48 hours. however, these tests require expensive equipment and reagents and, in order to avoid contamination, tests must table 1. the various clinical problems during the different phases of dengue 1. febrile phase dehydration; high fever may cause neurological disturbances and febrile seizures in young children 2. critical phase shock from plasma leakage; severe haemorrhage; organ impairment 3. recovery phase hypervolaemia (only if intravenous fluid therapy has been excessivea and/or has extended into this period) �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 9–22 observe quality control procedures and must be performed by experienced technicians. ns1 antigen detection kits now becoming commercially available can be used in laboratories with limited equipment and yield results within a few hours. rapid dengue antigen detection tests can be used in field settings and provide results in less than an hour. currently, these assays are not type-specific, are expensive and are under evaluation for diagnostic accuracy and costeffectiveness in multiple settings. table 2 summarizes various dengue diagnostic methods and their costs. after day 5, dengue viruses and antigens disappear from the blood coincident with the appearance of specific antibodies. ns1 antigen may be detected in some patients for a few days after defervescence. dengue serologic tests are more available in dengueendemic countries than are virological tests. specimen transport is not a problem as immunoglobulins are stable at tropical room temperatures. for serology, the time of specimen collection is more flexible than that for virus isolation or rna detection because an antibody response can be measured by comparing a sample collected during the acute stage of illness with samples collected weeks or months later. low levels of a detectable dengue igm response – or the absence of it – in some secondary infections reduces the diagnostic accuracy of igm elisa tests. results of rapid tests may be available within less than one hour. reliance on rapid tests to diagnose dengue infections should be approached with caution, however, since the performance of all commercial tests has not yet been evaluated by reference laboratories.16 a four-fold or greater increase in antibody levels measured by igg elisa or by haemagglutination inhibition (hi) test in paired sera indicates an acute or recent flavivirus infection. however, waiting for the convalescent serum collected at the time of patient discharge is not very useful for diagnosis and clinical management and provides only a retrospective result. differential diagnosis dengue fever can easily be confused with nondengue illnesses, particularly in nonepidemic situations. depending on the geographical origin of the patient, other etiologies – including non-dengue flavivirus infections – should be ruled out. these include yellow fever, japanese encephalitis, st louis encephalitis, zika, and west nile, alphaviruses (such as sinbis and chikungunya), and other causes of fever such as malaria, leptospirosis, typhoid, rickettsial diseases (rickettsia prowazeki, r. mooseri, r. conori, r. rickettsi, orientia tsutsugamushi, coxiella burneti, etc.), measles, enteroviruses, influenza and influenza-like illnesses, haemorrhagic fevers (arenaviridae: junin, etc.; filoviridae: marburg, ebola; bunyaviridae: hantaviruses, crimean-congo haemorrhagic fever, etc.). both the identification of virus/viral rna/viral antigen and the detection of an antibody response are preferable for dengue diagnosis to either approach alone (see table 3). unfortunately, an ideal diagnostic test that permits early and rapid diagnosis, is affordable for different health table 2. summary of operating characteristics and comparative costs of dengue diagnostic methods9 diagnostic methods diagnostic of acute infection time to results specimen time of collection after onset of symptoms facilities cost viral isolation and serotype identification confirmed 1–2 weeks whole blood, serum, tissues 1–5 days mosquito or cell culture facilities, bsl-2/bsl-3° laboratory fluorescence microscope or molecular biology equipment $$$ nucleic acid detection confirmed 1 or 2 days tissues, whole blood, serum, plasma 1–5 days bsl-2 laboratory, equipment for molecular biology $$$ antigen detection not yet determinated confirmed 1 day > 1 day serum tissue for immuno-chemistry 1–6 days na elisa facilities facilities for histology $$ $$$ igmelisa igm rapid test probable 1–2 days 30 minutes serum, plasma, whole blood after 5 days elisa facilities no additional supplies $ igg (paired sera) by elisa, hi or neutralization test confirmed 7 days or more serum, plasma, whole blood acute sera, 1– days, convalescent after 15 days elisa facilities bsl-2 laboratory for neutralization assay $ table 3. interpretation of dengue diagnostic tests adapted from dengue and control (denco) study highly suggestive confirmed one of the following 1. igm + in a single serum sample 2. igg + in a single serum sample with a hi titre of 1280 or greater one of the following: 1. pcr + 2. virus culture + 3. igm seroconversion in paired sera 4. igg seroconversion in paired sera or fourfold igg titer increase in paired sera ��soegijanto and chilvia: update management dengue shock syndrome in pediatric cases systems, is easy to perform, and has a robust performance, is not yet available. recommendations for treatment patients who require emergency treatment and urgent referral when they have severe dengue patients require emergency treatment and urgent referral when they are in the critical phase of disease, i.e. when they have: severe plasma leakage leading to dengue shock and/or fluid accumulation with respiratory distress; severe haemorrhages; severe organ impairment (hepatic damage, renal impairment, cardiomyopathy, encephalopathy or encephalitis). all patients with severe dengue should be admitted to the hospital with access to intensive care facilities and blood transfusion. judicious intravenous fluid resuscitation is the essential and usually sole intervention required. the crystalloid solution should be isotonic and the volume just sufficient to maintain an effective circulation during the period of plasma leakage. plasma losses should be replaced immediately and rapidly with isotonic crystalloid solution or, in the case of hypotensive shock, colloid solutions. if possible, obtain haematocrit levels before and after fluid resuscitation. there should be continued replacement of further plasma losses to maintain effective circulation for 24–48 hours. for overweight or obese patients, the ideal body weight should be used for calculating fluid infusion rates. a group and cross-match should be done for all shock patients. blood transfusion should be given only in cases with suspected/severe bleeding. fluid resuscitation must be clearly separated from simple fluid administration. this is a strategy in which larger volumes of fluids (e.g. 10–20 ml boluses) are administered for a limited period of time under close monitoring to evaluate the patient’s response and to avoid the development of pulmonary oedema. the degree of intravascular volume deficit in dengue shock varies. input is typically much greater than output, and the input/output ratio is of no utility for judging fluid resuscitation needs during this period. the goals of fluid resuscitation include improving central and peripheral circulation (decreasing tachycardia, improving blood pressure, pulse volume, warm and pink extremities, and capillary refill time < 2 seconds) and improving end-organ perfusion i.e. stable conscious level (more alert or less restless), urine output ≥ 0.5 ml/kg/hour, decreasing metabolic acidosis. ringer acetate or koloid (hes or gelatin) bolus 10-20 ml/kg/hour pulse stabil vrine production pulse takikardi blood pressuasytole diastole < 20mmhg urine pvo production ringer acetate ds 7 ml/kg/hour 5 ml/kg/hour 3 ml/kg/hour 24 48 hour expected recoverygood pcv hb pcv blood 10ml/kg/hour & can be repeated as needed better result give plasma 10ml/kg/hour & can be repeated 3 times in 1 day ?? respon pcv not respond pcv figure 5 algorithm for fluid management in compensated shock (who, 2009 with modified) �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 9–22 treatment of shock the action plan for treating patients with compensated shock is as follows (figure 5) 1. start intravenous fluid resuscitation with isotonic crystalloid solutions at 5–10 ml/kg/hour over one hour. then reassess the patient’s condition (vital signs, capillary refill time, haematocrit, urine output). the next steps depend on the situation. 2. if the patient’s condition improves, intravenous fluids should be gradually reduced to 5–7 ml/kg/hr for 1–2 hours, then to 3–5 ml/kg/hr for 2–4 hours, then to 2–3 ml/kg/hr, and then further depending on haemodynamic status, which can be maintained for up to 24–48 hours. 3. if vital signs are still unstable (i.e. shock persists), check the haematocrit the first bolus. if the haematocrit increases or is still high (> 50%), repeat a second bolus of crystalloid solution at 10–20 ml/kg/hr for one hour. after this second bolus, if there is improvement, reduce the rate to 7–10 ml/ kg/hr for 1–2 hours, and then continue to reduce as above. if haematocrit decreases compared to the initial reference haematocrit (<40% in children and adult females, <45% in adult males), this indicates bleeding and the need to cross-match and transfuse blood as soon as possible (see treatment for haemorrhagic complications). 4. further boluses of crystalloid or colloidal solutions may need to be given during the next 24–48 hours. 5. awarness of using ringer lactat solution in dengue virus infection cases can induce severity. 6. one of indicator not using ringer lactat is an increasing liver enzyme, ast and alt with level more than 100200 u/l it is marker of liver damage. 7. therefore we should choose other solution such as ringer acetat or physiology salt. 8. using ringer acetat as fluid therapy in dengue virus infection is better to prevent liver damage than using ringer lactate. patients with hypotensive shock should be managed more vigorously. the action plan for treating patients with hypotensive shock is as follows (figure 6). ringer acetate or colloid (hes 130,140 or gelatin) bolus 10-20 ml/kg/hour, 10-20 minutes syntetic colloid hes 130/0,14 or gelatine pulse stabil urine production glue ringer acetate 7 ml/kg/hour 5 ml/kg/hour 3 ml/kg/hour 24 48 expected recovery puise takikardi blood pressure sistolr – diastole < 20 mmhg urine production pcv hb pcv blood 10ml/kg/hour & can be repeated as needed give plasma 10ml/kg/hour & can be repeated 3 times in 1 day better resultbetter result respon pcv not respon pcv figure 6. algorithm for fluid management in hypotensive shock (who, 2009 with modification) ��soegijanto and chilvia: update management dengue shock syndrome in pediatric cases 1. initiate intravenous fluid resuscitation with crystalloid or colloid solution (if available) at 20 ml/kg as a bolus given over 15 minutes to bring the patient out of shock as quickly as possible. 2. if the patient’s condition improves, give a crystalloid/ colloid infusion of 10 ml/ kg/hr for one hour. then continue with crystalloid infusion and gradually reduce to 5–7 ml/kg/hr for 1–2 hours, then to 3–5 ml/kg/hr for 2–4 hours, and then to 2–3 ml/kg/hr or less, which can be maintained for up to 24–48 hours. 3. if vital signs are still unstable (i.e. shock persists), review the haematocrit obtained before the first bolus. if the haematocrit was low (<40% in children and adult females, <45% in adult males), this indicates bleeding and the need to crossmatch and transfuse blood as soon as possible (see treatment for haemorrhagic complications). 4. if the haematocrit was high compared to the baseline value (if not available, use population baseline), change intravenous fluids to colloid solutions at 10–20 ml/kg as a second bolus over 30 minutes to one hour. after the second bolus, reassess the patient. if the condition improves, reduce the rate to 7–10 ml/kg/hr for 1–2 hours, then change back to crystalloid solution and reduce the rate of infusion as mentioned above. if the condition is still unstable, repeat the haematocrit after the second bolus. 5. if the haematocrit decreases compared to the previous value (<40% in children and adult females, < 45% in adult males), this indicates bleeding and the need to cross-match and transfuse blood as soon as possible (see treatment for haemorrhagic complications). if the haematocrit increases compared to the previous value or remains very high (> 50%), continue colloid solutions at 10–20 ml/kg as a third bolus over one hour. after this dose, reduce the rate to 7–10 ml/kg/hr for 1–2 hours, then change back to crystalloid solution and reduce the rate of infusion as mentioned above when the patient’s condition improves. 6. further boluses of fluids may need to be given during the next 24 hours. the rate and volume of each bolus infusion should be titrated to the clinical response. patients with severe dengue should be admitted to the high-dependency or intensive care area. patients with dengue shock should be frequently monitored until the danger period is over. a detailed fluid balance of all input and output should be maintained. parameters that should be monitored include vital signs and peripheral perfusion (every 15–30 minutes until the patient is out of shock, then 1–2 hourly). in general, the higher the fluid infusion rate, the more frequently the patient should be monitored and reviewed in order to avoid fluid overload while ensuring adequate volume replacement. if resources are available, a patient with severe dengue should have an arterial line placed as soon as practical. the reason for this is that in shock states, estimation of blood pressure using a cuff is commonly inaccurate. the use of an indwelling arterial catheter allows for continuous and reproducible blood pressure measurements and frequent blood sampling on which decisions regarding therapy can be based. monitoring of ecg and pulse oximetry should be available in the intensive care unit. urine output should be checked regularly (hourly till the patient is out of shock, then 1–2 hourly). a continuous bladder catheter enables close monitoring of urine output. an acceptable urine output would be about 0.5 ml/kg/ hour. haematocrit should be monitored (before and after fluid boluses until stable, then 4–6 hourly). in addition, there should be monitoring of arterial or venous blood gases, lactate, total carbon dioxide/bicarbonate (every 30 minutes to one hour until stable, then as indicated), blood glucose (before fluid resuscitation and repeat as indicated), and other organ functions (such as renal profile, liver profile, coagulation profile, before resuscitation and as indicated). changes in the haematocrit are a useful guide to treatment. however, changes must be interpreted in parallel with the haemodynamic status, the clinical response to fluid therapy and the acid-base balance. for instance, a rising or persistently high haematocrit together with unstable vital signs (particularly narrowing of the pulse pressure) indicates active plasma leakage and the need for a further bolus of fluid replacement. however, a rising or persistently high haematocrit together with stable haemodynamic status and adequate urine output does not require extra intravenous fluid. in the latter case, continue to monitor closely and it is likely that the haematocrit will start to fall within the next 24 hours as the plasma leakage stops. a decrease in haematocrit together with unstable vital signs (particularly narrowing of the pulse pressure, tachycardia, metabolic acidosis, poor urine output) indicates major haemorrhage and the need for urgent blood transfusion. yet a decrease in haematocrit together with stable haemodynamic status and adequate urine output indicates haemodilution and/or reabsorption of extravasated fluids, so in this case intravenous fluids must be discontinued immediately to avoid pulmonary oedema. treatment of haemorrhagic complications mucosal bleeding may occur in any patient with dengue but, if the patient remains stable with fluid resuscitation/ replacement, it should be considered as minor. the bleeding usually improves rapidly during the recovery phase. in patients with profound thrombocytopaenia, ensure strict bed rest and protect from trauma to reduce the risk of bleeding. do not give intramuscular injections to avoid haematoma. it should be noted that prophylactic platelet transfusions for severe thrombocytopaenia in otherwise haemodynamically stable patients have not been shown to be effective and are not necessary.14 if major bleeding occurs it is usually from the gastrointestinal tract, and/or vagina in adult females. internal bleeding may not become apparent for many hours until the first black stool is passed. �0 indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 9–22 paptients at risk of major bleeding are those who: 1. have prolonged/refractory shock; 2. have hypotensive shock and renal or liver failure and/or severe and persistent metabolic acidosis; 3. are given non-steroidal anti-inflammatory agents; 4. have pre-existing peptic ulcer disease; 5. are on anticoagulant therapy; 6. have any form of trauma, including intramuscular injection. patients with haemolytic conditions are at risk of acute haemolysis with haemoglobinuria and will require blood transfusion. severe bleeding can be recognized by: 1. persistent and/or severe overt bleeding in the presence of unstable haemodynamic status, regardless of the haematocrit level; 2. a decrease in haematocrit after fluid resuscitation together with unstable haemodynamic status; 3. refractory shock that fails to respond to consecutive fluid resuscitation 40–60 ml/kg; 4. hypotensive shock with low/normal haematocrit before fluid resuscitation; 5. persistent or worsening metabolic acidosis ± a wellmaintained systolic blood pressure, especially in those with severe abdominal tenderness and distension. blood transfusion is life-saving and should be given as soon as severe bleeding is suspected or recognized. however, blood transfusion must be given with care because of the risk of fluid overload. do not wait for the haematocrit to drop too low before deciding on blood transfusion. note that haematocrit of < 30% as a trigger for blood transfusion, as recommended in the surviving sepsis campaign guideline,15 is not applicable to severe dengue. the reason for this is that, in dengue, bleeding usually occurs after a period of prolonged shock that is preceded by plasma leakage. during the plasma leakage the haematocrit increases to relatively high values before the onset of severe bleeding. when bleeding occurs, haematocrit will then drop from this high level. as a result, haematocrit levels may not be as low as in the absence of plasma leakage. the action plan for the treatment of haemorrhagic complications is as follows: • give 5–10 ml/kg of fresh-packed red cells or 10–20 ml/kg of fresh whole blood at an appropriate rate and observe the clinical response. it is important that fresh whole blood or fresh red cells are given. oxygen delivery at tissue level is optimal with high levels of 2,3 di-phosphoglycerate (2,3 dpg). stored blood loses 2,3 dpg, low levels of which impede the oxygen-releasing capacity of haemoglobin, resulting in functional tissue hypoxia. a good clinical response includes improving haemodynamic status and acid-base balance. • consider repeating the blood transfusion if there is further blood loss or no appropriate rise in haematocrit after blood transfusion. there is little evidence to support the practice of transfusing platelet concentrates and/or fresh-frozen plasma for severe bleeding. it is being practised when massive bleeding can not be managed with just fresh whole blood/fresh-packed cells, but it may exacerbate the fluid overload. treatment of complications and other areas of treatment fluid overload fluid overload with large pleural effusions and ascites is a common cause of acute respiratory distress and failure in severe dengue. other causes of respiratory distress include acute pulmonary oedema, severe metabolic acidosis from severe shock, and acute respiratory distress syndrome (ards) (refer to standard textbook of clinical care for further guidance on management). causes of fluid overload are: 1. excessive and/or too rapid intravenous fluids; 2. incorrect use of hypotonic rather than isotonic crystalloid solutions; 3. inappropriate use of large volumes of intravenous fluids in patients with unrecognized severe bleeding; 4. inappropriate transfusion of fresh-frozen plasma, platelet concentrates and cryoprecipitates; 5. continuation of intravenous fluids after plasma leakage has resolved (24–48 hours from defervescence); 6. co-morbid conditions such as congenital or ischaemic heart disease, chronic lung and renal diseases. early clinical features of fluid overload are : 1. respiratory distress, difficulty in breathing; 2. rapid breathing; 3. chest wall in-drawing; 4. wheezing (rather than crepitations); 5. large pleural effusions; 6. tense ascites; 7. increased jugular venous pressure (jvp). late clinical features are: 1. pulmonary oedema (cough with pink or frothy sputum ± crepitations, cyanosis); 2. irreversible shock (heart failure, often in combination with ongoing hypovolaemia). additional investigations are: 1. the chest x-ray which shows cardiomegaly, pleural effusion, upward displacement of the diaphragm by the ascites and varying degrees of “bat’s wings” appearance ± kerley b lines suggestive of fluid overload and pulmonary oedema; 2. ecg to exclude ischaemic changes and arrhythmia; 3. cardiac enzymes. the action plan for the treatment of fluid overload is as follows: 1. oxygen therapy should be given immediately. 2. stopping intravenous fluid therapy during the recovery phase will allow fluid in 3. the pleural and peritoneal cavities to return to the ��soegijanto and chilvia: update management dengue shock syndrome in pediatric cases intravascular compartment. this results in diuresis and resolution of pleural effusion and ascites. recognizing when to decrease or stop intravenous fluids is key to preventing fluid overload. when the following signs are present, intravenous fluids should be discontinued or reduced to the minimum rate necessary to maintain euglycaemia: » signs of cessation of plasma leakage; » stable blood pressure, pulse and peripheral perfusion; » haematocrit decreases in the presence of a good pulse volume; » afebrile for more than 24–48 days (without the use of antipyretics); » resolving bowel/abdominal symptoms; » improving urine output. 4. the management of fluid overload varies according to the phase of the disease and the patient’s haemodynamic status. if the patient has stable haemodynamic status and is out of the critical phase (more than 24–48 hours of defervescence), stop intravenous fluids but continue close monitoring. if necessary, give oral or intravenous furosemide 0.1–0.5 mg/kg/dose once or twice daily, or a continuous infusion of furosemide 0.1 mg/kg/hour. monitor serum potassium and correct the ensuing hypokalaemia. 5. if the patient has stable haemodynamic status but is still within the critical phase, reduce the intravenous fluid accordingly. avoid diuretics during the plasma leakage phase because they may lead to intravascular volume depletion. 6. patients who remain in shock with low or normal haematocrit levels but show signs of fluid overload may have occult haemorrhage. further infusion of large volumes of intravenous fluids will lead only to a poor outcome. careful fresh whole blood transfusion should be initiated as soon as possible. if the patient remains in shock and the haematocrit is elevated, repeated small boluses of a colloid solution may help. other complications of dengue both hyperglycaemia and hypoglycaemia may occur, even in the absence of diabetes mellitus and/ or hypoglycaemic agents. electrolyte and acid-base imbalances are also common observations in severe dengue and are probably related to gastrointestinal losses through vomiting and diarrhoea or to the use of hypotonic solutions for resuscitation and correction of dehydration. hyponatraemia, hypokalaemia, hyperkalaemia, serum calcium imbalances and metabolic acidosis (sodium bicarbonate for metabolic acidosis is not recommended for ph ≥ 7.15) can occur. one should also be alert for coinfections and nosocomial infections. if found cases with dengue shock syndrom with hipotonous heart muscle complication at figure 7. to prevent life threatening hypotension in dss as follow figure 7. flow chart of dengue shock syndrom with hypotonous heart muscle complication(who, 2009) supportive care and adjuvant therapy supportive care and adjuvant therapy may be necessary in severe dengue. this may include: renal replacement therapy, with a preference to continuous veno-venous haemodialysis (cvvh), since peritoneal dialysis has a risk of bleeding; vasopressor and inotropic therapies as temporary measures to prevent life-threatening hypotension in dengue shock and during induction for intubation, while correction of intravascular volume is being vigorously carried out; further treatment of organ impairment, such as severe hepatic involvement or encephalopathy or encephalitis; further treatment of cardiac abnormalities, such as conduction abnormalities, may occur (the latter usually not requiring interventions). in this context there is little or no evidence in favour of the use of steroids and intravenous immunoglobulins, or of recombinant activated factor vii. conclusion by using integrated criteria of who 2009 and 1997, update management of dengue shock syndrome in �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 9–22 pediatric cases, can improve clinical management to reach the lower mortality in community until cfr < 1%. using ringer acetat as fluid therapy in dengue virus infection is better to prevent liver damage than using ringer lactate. references 1. cattand p et al. tropical diseases lacking adequate control measures: dengue, leishmaniasis, and african trypanosomiasis. disease control priorities in developing countries, 2nd ed. new york, ny, oxford university press, 2006 (pp 451–466). 2. suaya ja, shepard ds, beatty me. dengue burden of disease and costs of illness. working paper 3.2 in: report of the scientifc working group meeting on dengue, geneva, 1–5 october 2006. geneva, world health organization, special programme for research and training in tropical diseases, 2007 (pp 35–49) (document tdr/ swg/07).dengue: guidelines for diagnosis, treatment, prevention and control 20. 3. guzman mg. effect of age on outcome of secondary dengue 2 infections. international journal of infectious diseases, 2002, 6(2): 118–124. 4. wilder-smith a, wilson me. sentinel surveillance for dengue: international travelers (unpublished report). 5. byron e.e martina, penelope koraka and albert d. m.e osterhaus erasmus mc, “dengue virus pathogenesis: an integrated view” departement of virology, rotterdam, the netherlands. vol. 22, no. 4: 571–573 6. rigau-perez jg et al. dengue and dengue haemorrhagic fever. lancet, 1998, 352: 971–977. 7. kalayanarooj s et al. early clinical and laboratory indicators of acute dengue illness. journal of infectious diseases, 1997, 176: 313–321. 8. phuong cxt et al. evaluation of the world health organization standard tourniquet test in the diagnosis of dengue infection in vietnam. tropical medicine and international health, 2002, 7: 125–132. 9. balmaseda a et al. assessment of the world health organization scheme for classification of dengue severity in nicaragua. american journal of tropical medicine and hygiene, 2005, 73: 1059–1062. 10. srikiatkhachorn a et al. natural history of plasma leakage in dengue hemorrhagic fever: a serial ultrasonic study. pediatric infectious disease journal, 2007, 26(4): 283–290. 11. nimmannitya s et al. dengue and chikungunya virus infection in man in thailand, 1962–64. observations on hospitalized patients with haemorrhagic fever. american journal of tropical medicine and hygiene, 1969, 18(6): 954–971. 12. martinez-torres e, polanco-anaya ac, pleites-sandoval eb. why and how children with dengue die? revista cubana de medicina tropical, 2008, 60(1): 40–47. 13. nimmannitya s. clinical spectrum and management of dengue haemorrhagic fever. southeast asian journal of tropical medicine and public health, 1987, 18(3): 392–397. 14. lum l et al. preventive transfusion in dengue shock syndrome – is it necessary? journal of pediatrics, 2003, 143: 682–684. 15. dellinger rp, levy mm, carlet jm. surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2008. critical care medicine, 2008, 36: 296–327. 16. who. dengue haemorrhagic fever: diagnosis, treatment, prevention and controlrigau-perez jg et al. dengue and dengue haemorrhagic fever. lancet, 1998, 352: 971–977. kalayanarooj s et al. early clinical and laboratory indicators of acute dengue illness. journal of infectious diseases, 1997, 176: 313–321. 17. hunsperger ea et al. evaluation of commercially available anti– dengue virus immunoglobulin m tests. emerging infectious diseases (serial online), 2009, march (date cited). accessible at http://www. cdc.gov/eid/content/15/3/436.htm 18. 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/ 19. whitehorn j, simmons cp. the pathogenesis of dengue. vaccine. 2011; 29: 7221–8. [pubmed] 20. chuansumrit a, chaiyaratana w, tangnararatchakit k, yoksan s, flamand m, sakuntabhai a. dengue nonstructural protein 1 antigen in the urine as a rapid and convenient diagnostic test during the febrile stage in patients with dengue infection. diagn microbiol infect dis. 2011; 71: 467–9. [pubmed] 21. barniol j, gaczkowski r, barbato ev, da cunha rv, salgado d, martínez e, et al. usefulness and applicability of the revised dengue case classification by disease: multi-centre study in 18 countries. bmc infect dis. 2011; 11: 106. [pmc free article] [pubmed] �� vol. 4. no. 4 october–december 2013 quick diagnosis of japanese encephalitis for new diagnosed emerging disease using pcr technique in surabaya, indonesia muhammad qushai yunifiar matondang1, nasronudin1, eduardus bimo ah1, mari inge l1, aldise mareta nastri1, nur syamsiatul fajar1, lilis mundri jannah1 1 tropical disease diagnostic center (tddc) – institute of tropical disease, universitas airlangga. abstract background: japanese enchepalitis (je) is a viral disease that considered as zoonotic disease, which transmitted through mosquito vectors that had je virus. mainly caused by the mosquito c. tritaeniorhynchus (the most important vector is the mosquito culex, which feeds on cattle in preference to human). je virus disease can also cause disturbances in the central nervous system eg. brain, bone marrow, and meninges which has serious impact on public health. this disease has been reported from japan, korea, taiwan, india, myanmar, thailand, western pacific and southeast asia to indonesia. however, the incidence of this disease in indonesia has not been well known in various animal species or humans. aim: the purpose of this study is to develop rapid diagnostic examinations on patient diagnosed je virus in surabaya by using pcr (polymerase chain reaction). because, je disease can lead to dead-end at the patient if not treated immediately. method: the research methods, extraction method, pcr (1st rt-pcr and 2nd nested pcr) are conducted using japanese encephalitis pcr detection kit. result: the results of the examination showed that 2 out of 17 people (11,765%) are positive with pcr bands 227 bp (basepair). this diagnostic technique to determine and to deal with early onset of the disease. solutions for preventive actions can be started from the termination of the cycle vectors to vaccination measures. conclusion: for his own medical factors given to reduce fever and swelling and reduce the pain. key words: japanese encephalitis, pcr, new emerging disease, preventive, indonesia abstrak latar belakang: japanese encephalitis (je) adalah penyakit virus yang dianggap sebagai penyakit zoonosis , yang ditularkan melalui vektor nyamuk yang memiliki virus je. terutama disebabkan oleh nyamuk culex c. (vektor yang paling penting adalah nyamuk culex, yang menyusu pada sapi dalam preferensi untuk manusia). penyakit virus je juga dapat menyebabkan gangguan pada sistem saraf pusat misalnya: otak, sumsum tulang, dan meninges yang memiliki dampak serius pada kesehatan masyarakat. penyakit ini telah dilaporkan dari jepang, korea, taiwan, india, myanmar, thailand, pasifik barat dan asia tenggara ke indonesia. namun, kejadian penyakit ini di indonesia belum dikenal di berbagai spesies hewan atau manusia. tujuan: tujuan dari penelitian ini adalah untuk mengembangkan pemeriksaan diagnostik cepat pada pasien didiagnosis virus je di surabaya dengan menggunakan pcr (polymerase chain reaction). karena, penyakit je dapat menyebabkan buntu pada pasien jika tidak segera diobati. metode: metode penelitian, metode ekstraksi, pcr (1st rt -pcr dan 2 nested pcr) dilakukan menggunakan japanese ensefalitis pcr deteksi kit . hasil: hasil pemeriksaan menunjukkan bahwa 2 dari 17 orang (11.765%) positif dengan pcr band 227 bp (basepair). teknik diagnostik ini untuk mengetahui dan menangani onset awal disease. solusi untuk tindakan pencegahan dapat dimulai dari penghentian vektor siklus tindakan vaksinasi. kesimpulan: untuk faktor medis sendiri diberikan untuk mengurangi demam dan pembengkakan dan mengurangi rasa sakit . key words: japanese encephalitis, pcr, new emerging disease, pencegahan, indonesia research report ��matondang, et al.: quick diagnosis new emerging disease “japanese encephalitis” introduction japanese encephalitis (je) is a disease can cause brain inflammation in animals and humans which can be transmitted from animals to the human through mosquito bites. this disease has become widespread in parts of east asia such as japan, korea, siberia, china, taiwan, thailand, laos, cambodia, vietnam, philippines, malaysia, indonesia, myanmar, bangladesh, india, sri lanka, and nepal. in indonesia, je case was first reported in 1960. japanese encephalitis virus is part of the flaviviridae family. this virus has the envelope (about 50 nm) with a small lipoprotein that surrounds the nucleocapsid core protein and consists of a single chain of rna. je virus is related to west nile virus and st. louis encephalitis viruses. on the outer layer are formed by (e) protein and act as protective antigen. this helps in the entry of the virus into the cell1. je disease in humans is a way of ending the cycle of transmission (dead end), because viraemia in humans occurs only a few hours which is difficult to spread further to other people. human disease can result in death if not treated properly. wei and gautama, in the same year reported that the most vulnerable age among children infected with je is between 5 to 9 years.23 in asia, with around 50,000 cases and 10,000 deaths per year in children below 15 years of age. the jev has shown a tendency to extend to other geographic regions. case fatality averages 30% and a high percentage of the survivors are left with permanent neuropsychiatric sequelae. je is a disease of public health importance because of its epidemic potential and high fatality rate. in endemic areas, the highest age-specific attack rates occur in children of 3 to 6 years of age. approximately one third of patients die, and half of the survivors suffer severe neuropsychiatric sequelae from the disease.10 the clinical symptoms commonly shown in the case of japanese encephalitis is usually a non specific symptom such as fever, followed by headache, vomiting, and decreased level of consciousness. because the tissue covering the brain and spinal cord become infected and swollen, the patient will usually experience stiffness in the neck and painful. then within two or three days, the patient began to experience the effects of swelling on the brain. these effects may include interference with balance and coordination, paralysis on several groups of muscles, tremors, seizures, and disturbances in consciousness.4 patients also experience dehydration and weight loss. if the patient can survive with the pain, the fever will drop down about day 7, and the symptoms will begin to rise again approximately on day 14. meanwhile there are also people who will continue to have a very high fever and get even worse. in this case, the symptoms will usually be followed by coma and then death occurs within 7–14 days. however, the area also quite a few patients who had recovered but was followed by permanent disability due to brain damage4. some reports suggest that children and teenagers are prone to this disease. in thailand, allegedly 40 of 100,000 children to adolescents aged 5–25 years suffering from this disease. in addition, it was reported also that a lot of je cases occur in rural areas. by all means this case epidemiology in the northern vietnam, northern thailand, korea, japan, taiwan, china, nepal and northern india more common in summer sat. within the area of southern vietnam, southern thailand, indonesia, malaysia, philippines, sri lanka, and southern india, je cases occur sporadically throughout the year. this disease also has been reported to cause behavioral abnormalities. in some children the clinical symptoms that appeared to be a single seizure, followed by a rapid recovery of consciousness. the symptoms of seizures are a common cause shaking on digits or mouth, eye deviation, nystagmus, excess salivation, or irregular respiration.4 in indonesia the first time in the case of je in serological report which occur in humans in 1999 in bali. examination of serum specimens from 12 patients with clinical diagnosis of viral encephalitis, meningitis or dengue hemorrhagic fever (dhf) found two of them positively infected with japanese encephalitis.11 je cases in humans were also reported in some areas, namely in west sumatra, west kalimantan, yogyakarta, central java, east java, west nusa tenggara, east nusa tenggara and papua.5 a recent report there are even reported cases of je virus infection in tourists who holiday in bali. the tourists traveling 3 weeks to java and bali, including vacation stricken rural to rural. last week of march was spent in bali. after returning home, the patient complained of fatigue and 5 days later he fell ill with numbness in both clengan, and can not use a knife and fork while eating. he also vomited and fell to the floor several times, can not stand by itself. when admitted to the hospital on the same night, the patient was febrile (39.18° c), but in general good condition. the next day he became confused and do not understand simple questions or instructions. test results show the conditions that lead to the condition encephalitis je.12 based on the background of the above, the study is to conduct a quick diagnostic on japanese encephalitis using pcr techniques among patients in surabaya, indonesia. some above incident, due to the lack of knowledge about the disease is accompanied by a rapid diagnostic examination for checking the disease and so far as je is a viral disease, then there is no treatment to stop or slow the progression of the virus. treatment can only be done in a way that is symptomatic relieve symptoms seen each patient. the aims of the study are to get a quick diagnose of japanese encephalitis virus using pcr techniques, and to be able to diagnose je virus in surabaya to treat early-infected patients from je disease. the method is by using pcr technique (japanese encephalitis virus detection kit). action is one step vaccination is effective in preventing the disease. generally, vaccines are given to children to adolescents under 17 years usis in je endemic areas. for tourists or travelers visiting endemic areas of je can also take advantage of this vaccine as a precautionary measure. another preventive measure is to efforts to control mosquito populations.13 and factors are the main �� indonesian journal of tropical and infectious disease, vol. 4. no. 4 october–december 2013: 42–45 factors for the prevention of outbreaks of je virus cope in endemic areas. materials and methods the samples are considered as positive when there is a band that emerged in 227 bp. the samples are mainly from whole blood, serum, csf. the samples are extracted. first pcr conducted starting with reverse transcription reaction cycle in 45° c for 30 minutes, then continue with 30 cycles of denaturation in 94° c for 45 second, annealing prosess in 72° c for 60 second, extention phase in 72° c for 60 second, and finally 1 cycle of final extension in 72° c for 5 minutes. second pcr was conducted after the first finish. starting wih pre-denaturation 1 cycle in 94° c for 2 minutes, continue with denaturation 30 cycle in 94° c for 30 second, annealing in 50° c for 30 second, extension in 72° c for 30 second, and final extension 1 cycle in 72° c for 5 minutes. after the pcr process is completed, we continue to electrophoresis with agarose gel for 30 min (110v). result and discussion there are total 17 patients has been examined in institute of tropical disease (itd) universitas airlangga. 2 person was found posistive with je virus. the pcr result showed positive je virus. je virus was first discovered in indonesia proved by ha and hi antibody tests. in the tropics, je virus continues to circulate among mosquitoes, birds and pigs.6 the mechanism of transmission of je virus in humans occurs because of mosquito cx. tritaeniorhynchus were supposed to be zoophilic population becomes heavy or sudden there was an increase of mosquito populations and thus be forced even this mosquito bite humans around him. also, it can also occur because of the number of pigs suffering from viraemia (virus containing je) virus became much so that in nature reserves increased and easily transmitted to humans. age, je vector, culex mosquitoes, ranged between 14–21 days and culex flight distance can reach more than 3 km. culex generally breed in stagnant water overgrown with plants such as rice fields and irrigation channels, shallow ditches or ponds that are not used. in pigs, viraemia occurs during 2–4 days and is followed by the formation of antibodies in the first period of up to 4 weeks. je virus can cross the placenta depends on the gestation and je virus strains. fetal death and mummification can occur when the je infection takes place at 40–60 days gestation. while the je infection after 85 days gestation, abnormalities caused very little. je incubation period in humans ranges from 4 to 14 days.13 japanese enchephalitis virus is quite a new problem in public health, especially in indonesia as the cases still not many but could be pandemic based on the availability of the vectors of disease. precaution may be a good way to be applied to both vectors, source of transmission (pigs), human and living environments. first way is by against the vector (mosquito), by using insecticides to kill adult mosquitoes and larvae and try to remain the hygiene of water in our home. second way is against the source of transmission (pig), by giving vaccination to infantile pigs, and make sure that the case of the pigs are surrounded by wire netting, sprayed insecticides, and should be free of mosquitoes. and the third way is prevention to ourself, by getting vaccination. it is an act that should be done 1 month before the time of transmission and addressed to people who have a high risk for getting the infection. due to the one-month period in an environment of high risk people can take precautions as a whole (the three preventive measures), as well as stop the line of the spread of the virus. alongside, we could also use mosquito repellent during sleep or during activity. conclusion je disease is a viral disease that is zoonotic and disturbing the public, which causes agents and vectors and animal reservoir potential. the existence of je disease itself can be seen with the examinee 17 peoples in surabaya 2 peoples je disease was detected. then made an attempt to overcome these problems. any suggestions with regard to the presence of je control such depth research on je in humans in indonesia in order to know the region spread in indonesia. references 1. erlanger te, weiss s, keiser j, et al. 2009. past, present, and future of japanese encephalitis. emerging infectious diseases 15(1): 1–7 2. wei, l. 2005. disease burden of japanese encephalitis: epidemiologic perspectives. workshop and training surveilans je di rumah sakit, jakarta, 17–19 februari, 2005. 26 him. figure 1. can be seen as well as the positive control samples are at base pair 227 bp which indicates that the samples was positive jev. ��matondang, et al.: quick diagnosis new emerging disease “japanese encephalitis” 3. gautama, k. 2005. pelaksanaan surveilans je di bali . workshop and training surveilans je di rumah sakit. jakarta. 17–19 februari, 2005. 24 him. 4. solomon t, dung nm, kneen r, et al. 2000. japanese encephalitis. j neurol neurosurg psychiatry 68: 405–415 5. ompusunggu s, hills sl, maha ms, et al. 2008. confirmation of japanese eneephalitis as an endémie human disease through sentinel surveillance in indonesia. atn j trop med hyg 79(6): 963–970. 6. harwood, r.f. and m.t. james. 1979. entomology in human and animal health. mc. millan pub. co. inc. new york, toronto, london, 548 pp. 7. simpson, n.i.h., e.t.w. bowen, h.l way, g.s. platt, m.n. hill, s. kamath, tw. lim, p.l f. bendel, and o.h.u. heathcote. 1974. arbovirus infections in sarawdk; october 1968 febf1k1ry 1970: japanese encephalitis virus isolations from mosquitoes. anti. trop. med. parasitol. 68(4): 393–404. 8. benerjee, k., p.k. deshmukh, m.a llkal, and v. dhanda. 1978. transmission of japanese encephalitis virus by culex bitaeniorhyllchus giles. indian j. med. res. 67: 889–893. 9. soedarto. 1992. penyakit-penyakit infeksi di indonesia. widya medika. jakarta. 88–93. 10. sarika tiwari; rishi kumar singh; ruchi tiwari; tapan n. dhole, 2012. japanese encephalitis: a review of the indian perspective. brazilian journal of infectious diseases. vol. 16 no. 6. 11. yoshida m, igarashi a, suwendra p, et al. 1999. the first report on human cases serologically diagnosed as japanese encephalitis in indonesia. southeast asian j trop med public health 30(4): 698–706. 12. stlund mro, kan b, karlsson m, et al. 2001. japanese encephalitis in a swedish tourist after travelling to java and bali. scand j infect dis 36: 512–513. 13. sendow i, bahri s. 2005. perkembangan japanese encephalitis di indonesia. wartazoa 15(3): 111–118. �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. �� 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. 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 67 vol. 5. no. 3 september–december 2014 literature review bacteria caused sepsis biomarkers artaria tjempakasari, nasronudin1,2 1 tropical and infectious disease division department of internal medicine, dr. soetomo general hospital faculty of medicine universitas ailangga, surabaya, indonesia 2 institute of tropical disease universitas airlangga, surabaya, indonesia abstract sepsis is a clinical condition of patients with serious infections that show a systemic inflammatory response, with or without a positive blood culture. sepsis is one of the most frequent causes of death in patients in intensive care units. we are at urgent need for biomarkers and reliable measurements that can be applied to risk stratification of septic patients and that would easily identify those patients at the highest risk of a poor outcome. such markers would be of fundamental importance to decision making for early intervention therapy. pro-inflammatory cytokines such as tumor necrosis factor- (tnf), interleukins-1,-6,-8 (il-1, il-6, il-8) are postulated to play a major role in the pathogenesis of the syndrome. c-reactive protein (crp) and procalcitonin (pct) are among a few biomarkers that incorporated into clinical practice although their precise role in the pathopysiology of sepsis and organ dysfunction still unclear. key words: sepsis, biomarker, inflammatory, c-reactive protein (crp), procalcitonin (pct) abstrak sepsis adalah suatu kondisi klinis penderita dengan infeksi serius yang memperlihatkan respons inflamasi sistemik, dengan atau tanpa kultur darah yang positif. sepsis merupakan salah satu penyebab kematian yang paling sering pada penderita di unit perawatan intensif.. kita berada pada kebutuhan mendesak akan biomarker dan pengukuran yang handal yang dapat diaplikasikan untuk risiko stratifikasi pada pasien septik dan akan diidentifikasi dengan mudah pada pasien dengan risiko tertinggi pada hasil. penanda akan menjadi kepentingan mendasar dalam pengambilan keputusan untuk therapy. pro-inflammatory sitokin seperti nekrosis tumor faktor-α (tnfα), interleukins-1,-6,-8 (il-1, il-6, il-8) merupakan acuan dasar yang berperan penting dalam sindrom patogenesis. c-reactive protein (crp) dan procalcitonin (pct) adalah beberapa biomarker yang dimasukkan ke dalam kegiatan klinis meskipun mereka berperan patifisiologi sepsis dan disfungsi organ masih tak jelas. kata kunci: sepsis, biomarker, inflamasi, c-reactive protein (crp), procalcitonin (pct) introduction sepsis is a clinical condition of patients with serious infections that show a systemic inflammatory response, with or without a positive blood culture.1 the diagnosis of sepsis was referring to the consensus criteria in 1991. these criteria are inviting a lot of dissatisfaction, so that a better approach might be to the stratification system. the new system is based on the piro characterize sepsis predisposition, basic infection, response and organ dysfunction. however this system works well, is very important to identify biomarkers of response profiles that can identify patients at risk of developing into an organ dysfunction.2 sepsis is one of the most frequent causes of death in patients in intensive care units. in america, there are approximately 700,000 patients each year and 210,000 of them died. despite new therapies that support and more potent antibiotics, sepsis remains often causes death in 30–70% of patients with severe sepsis and significantly lowers the quality of life for patients who survived.2 generally, sepsis is a spectrum disorder that is caused by infection by bacteria, viruses, fungi or parasites or toxic products. the spectrum of disorders of sepsis is the result of microbial invasion of the bloodstream or intoxication with early signs of circulatory compromise include tachycardia, tachypnea, peripheral vasodilation and fever 68 indonesian journal of tropical and infectious disease, vol. 5. no.3 september–december 2014: 67–71 (or hypothermia) to circulatory collapse with multiple organ dysfunction and death. several different bioactive molecules have been proposed as a biomarker to assess the degree of patients with sepsis. among them are bacterial products such as endotoxin and bacterial dna, acute phase proteins (protein c, procalcitonin), coagulation factors (fibrin degradation products, antithrombin iii, d-dimer), cellular processes (apoptosis), hormones (cortisol, acth) and cytokines (tnf-, il-1, il-6, il-8, il-10). unfortunately, only a few biomarkers that can be used in clinical practice.2 in this literature review will discuss some of the biomarkers are often used in studies. phatophysiology of sepsis the body’s defense mechanism against bacterial infection is influenced by the structure and bacterial pathogenicity. depending on the structure of the cell walls, the microbes are classified in the class of gram-positive bacteria, gram-negative, and spirokaeta mycobacteria. there are several general overview of the immune response to microbes, namely: defense against microbial-mediated effector mechanisms of innate immunity and acquired immunity, non-specific immune response against microbes play an important role in determining the specific immune response that will take place, the immune system is able to specialize and respond differently to the types of microbes, survival and microbial pathogenicity is strongly influenced by the ability of microbes to evade the host immune system, tissue damage and disease as a consequence of infection is generally caused by the host response to microbes and their products.3 innate defense system of the body is the first line of defense against infection and can be activated when pathogen via natural defense barrier. the body’s defense system includes the humoral elements (the alternative pathway and mannan-binding lectin of the complement system, acute phase proteins and cytokines) and cellular elements (monocytes, macrophages, neutrophils and dendritic cells natural killer cells).4 detection of invading microorganisms mediated by receptors expressed on the surface of innate immune cells. these receptors can recognize structures that are usually found in microbial pathogens.4 lipopolysaccharide (lps) bacteria are the main targets of immune recognition. macromolecules is only found in the outer lipid bilayer that surrounds the walls of gram-negative bacteria. there are two proteins that recognize humoral lps is lps-binding protein and soluble cd14.5 parslow, 2001. cd14/lps complex then interacts with toll-like receptor-4 (tlr4). tlr4 activation causes the transcription of a number of inflammatory genes and the immune response through the mediation mechanism of nuclear factor-b (nf-b).4 gram-positive organisms can also cause sepsis least through two mechanisms: through the production of exotoxins that act as superantigens and through the cell wall components that stimulate the immune cells. superantigens are molecules that are bound to mhc class ii molecules on antigen presenting cell and t cell receptor vb chain to produce large amounts of proinflammatory cytokines. staphylococcus enterotoxin, toxic shock syndrome toxin1 and streptococcal pyrogenic eksotosin are examples of bacterial superantigens. toll-like receptor 2 (tlr2) mediates cellular responses to kill gram-positive bacteria and the structure of the cell wall (peptidoglycan, lipoproteins, lipoteichoic acid and phenolsoluble modulin).4 innate immune defense is another important group of serum proteins called complement pathway. complement can be activated via three routes, all via the c3 complement activation: the classical pathway, the alternative pathway and the lectin pathway.5 with the exception of c3, almost all soluble mediators of innate immunity found in small amounts in normal conditions. this concentration can be increased to 1000 times during a serious infection, which is part of the protective reaction called the acute phase response. in these circumstances, the liver increases the synthesis of more than 30 different serum proteins, called acute phase proteins. some of them are complement factors c3 and b, mbl (mannan-binding lectin), lbp (lps-binding protein), c-reactive protein and amyloid p protein and other coagulation factors such as fibrinogen include, granulocyte colony-stimulating factor anti -oxidants and serum protein -binding metal. acute phase response occurs when hepatocytes associated with cytokines, especially interleukin-6 (il-6), interleukin-1 (il-1) or tumor necrosis factor- (tnf-) are released locally or into the bloodstream by other cells.5 excessive stimulation by proinflammatory cytokines or other mediators may cause systemic damage and endothelial cell dysfunction. endothelial cell activation leads to increased expression of nitric oxyde synthase which causes nitric oxyde and intracell adhesion molecules excessive, stimulates neutrophil chemotaxis and the interaction of endothelial cells.6 bone et al., 1997. split pathophysiology of sepsis into 5 stages:1,7 stage 1: enforcement infection. when the infectious organisms will begin to proliferate produced inflammatory molecules such as lekotrien, complement components, cytokines and antigen-antibody complexes, attract neutrophils to areas of infection, followed by monocytes. stacking leukocytes at sites of inflammation is facilitated by il-8, endothelial cell selectins and cellular adhesion molecules. leukocytes recognize and phagocytize bacteria and fungi teropsonisasi. this process is due to local release of cytokines from macrophages (monocyte tissue). proinflammatory cytokines and other mediators including tnf-, il-1, il-2, il-6, interferon-, platelet-activating factor (paf) and others. the release of these mediators will be balanced by compensating anti-inflammatory response 69artaria tjempakasari and nasronudin: bacteria caused sepsis biomarkers of il4, il-10, il-11, il-13, soluble tnf receptor-, il1ra, tgf-b and other substances. stage 2: early systemic response. in a state of severe infection, proinflammatory cytokines would result in systemic symptoms. the emergence of clinical symptoms shows microenvironment unable to control the infection. proinflammatory cytokines in this process is tnf-, il1, il-6 and interferon-. the reaction of the body heat produced by the release of il-1 that reach the hypothalamus. prostaglandin e2 may also be produced locally in the hypothalamus and increases the set point temperature. stage 3: systemic response. endothelial cell dysfunction is a cause of pathophysiological changes at this stage. as a result of the activity of tnf-, il-1 and other cytokines, endothelial cell phenotype shift toward prothrombotik stage. inflammatory cells and platelets move towards endothelial injury. disturbances in endothelial cell physiology will affect the ability of the endothelium to regulate blood flow. consequently there is an increase in microvascular permeability, fluid transudation, organ dysfunction and shock. stage 4: the reaction of anti-inflammatory compensation. normally, a cascade of proinflammatory mediators followed by a counter-regulatory cytokine that rapidly regulate the secretion of proinflammatory cytokines and clinical manifestations of sepsis. this regulatory cytokines in principle is il-4, il-10, transforming growth factor-b (tgf-b) and other anti-inflammatory molecules. stage 5: the failure of the immune system. this is the final stage, which is seen in some patients. this stage is characterized by the inability of monocytes to respond physiologically, increasing the risk of developing an infection, organ failure and death. biomarker detection biomarkers are any characteristic that can be objectively measured and evaluated as an indicator of biological processes, pathogenic processes, or pharmacologic responses to therapeutic intervention. measurement of existing biomarkers using elisa method, measured by immunoluminometric procalcitonin assay is similar in principle to the elisa.8 protein molecules associated with sepsis is very broad, including cytokines, chemokines, adhesion mediator, soluble receptors and acute phase proteins. protein biomarker research is currently focused primarily on procalcitonin and interleukin some magic bullet as diagnostic biomarkers for infection. the standard method for assessing the use of this diagnostic is characteristic receiver operator curve (roc) by determining the cut point (cut-off) in order to obtain true-positive diagnoses (sensitivity) and false-positive diagnosis.8 tumor necrosis factor- (tnf ) tnf- is a 17-kd polypeptide is expressed in the form bound to the membrane and form secretion. activated macrophages and monocytes, t cells and nk synthesize tnf-. tnf- is secreted bound to the cell surface receptors: type i (55 to kd) or type ii (75-kd). stimulation of type i receptor causes activation of nf-b, induction of il-6, the expression of tissue factor (tf), regulate thrombomodulin (tm) and tm increases catabolism, activation of fibrinolysis, regulation of endothelial cells, induction of nitric acid synthase, neutrophil activation and biological effects other. receptor type ii facilitates tnf- binding to type i receptors and signal transduction.1 tnf- is an early factor in the activation of the body’s response and a series of cytokines released during infection, where the concentration is increased 24 times (828 ng/l) compared to the concentration before infection at 2 h after lps interacts with endotoxin in vivo during the study. however, the use of tnf- as a diagnostic tool is not good, in terms of differentiating inflammation and infection. analysis of the roc curve shows the sensitivity and specificity were weak. difficulty tnf- as a diagnostic tool of sepsis due to an increase in the concentration of bacteria associated with rapid and short half-life of about 17 minutes.8 interleukin-1 (il-1) other proinflammatory cytokines associated with sepsis is the il-1 which include il1 il-1b and il-1 receptor antagonist (il-1ra) in which excessive amounts of il-1b during sepsis.1,8 il-1b interests to provide diagnostic disagreement, between the increase and decrease, so does the same thing has been reported in neonates. instead concentration of il-1ra showed a consistent increase in patients with sepsis with a concentration of 2–31 mg/l (concentration in normal individuals is not detected). roc analysis showed sensitivity of 93% and a specificity of 92% at the time of the diagnosis. but it should be noted that high concentrations have also been reported in patients who underwent thoraco-abdominal aneurysm repair.8 il-1 is the best along with il-8 in terms of predicting the output. this means that the predictive value of these cytokines is better than the prototype clinical prognostic scores were used in the intensive care unit, the acute physiology and chronic health evaluation score (apache ii).2 interleukin-6 (il-6) interleukin-6 (il-6) is a glycoprotein 21-30-kd widely produced by the cells, including monocytes and macrophages, t cells, endothelial cells, fibroblasts and keratinocytes. this molecule is the biggest cause of the acute phase response, causing the growth and differentiation of t cells, nk cell activity and promote the maturation of megakaryocytes. il-6 can inhibit endotoxin -induced tnf- and il-1 and increase the degree of soluble tnf- receptor type i and il-1ra.1 il-6 is a cytokine with important prognostic value in sepsis. although the role of il-6 in this syndrome remains controversial, il-6 cytokine proposed as an important 70 indonesian journal of tropical and infectious disease, vol. 5. no.3 september–december 2014: 67–71 biomarker in sepsis due to the slow kinetic plasma, stable and easily detected in blood samples and correlated well with the intensity of the inflammatory response. persistent increases in levels of il-6 associated with organ failure and death.2,6 such as tnf-, il-6 plays a role in the immune response at the beginning. value for adults in sepsis reported to range from 300–2700 ng/l, above 100 ng/l for sirs. however, there are reports that say that no significant difference between the concentrations in sirs and sepsis, and between sepsis and trauma patients. this contradiction has been confirmed by the lack of sensitivity and specificity based on roc analysis.8,9 interleukin-8 (il-8) interleukin-8 (il-8) is a chemokine, an agency that recruits inflammatory cells to the site of injury. il-8 is synthesized by monocytes, macrophages, neutrophils and endothelial cells. tnf-, il-1b and il-2 stimulates the release of il-8. following stimulation of il-8, also stimulated neutrophil function, promote chemotaxis, adhesion molecule expression and regulation of activity of respiration changes and degranulation.1 among other biomarkers associated with sepsis, il-8 were higher in adult studies, although the main focus is the diagnosis of il-8 in neonatal research. concentrations of il-8 in septic neonates was 94–4335 ng/l compared with 2–42 ng/l in healthy neonates. although in one study said that is not useful for the diagnosis, the majority of studies reported a consistent increase of the concentration of il-8. roc analysis mentioned sensitivity 92 % and specificity of 70%.8 the degree of il-8 correlated with lactic acid, the presence of dic, severe hypoxemia and mortality in patients with severe infection or septic shock (balk, 2004). il-8 together with il-1 cytokines are the best in terms of prediction output.2 c-reactive protein (crp) c-reactive protein is a member of the pentraxin family of proteins decomposed during acute inflammation, causing the immune response to the antigen, activates the complement and enhance the production of monocyte tissue factor. c-reactive protein binds phosphoryl kholin on the surface of bacteria, acts as opsonin for gram-positive bacteria and play a role in the body’s defenses. c-reactive protein also binds low density lipoprotein cholesterol (ldl-c) in vitro, suggesting a direct interaction with atherogenic lipids.9 crp is often used as a marker of bacterial infection, however crp may also be released because of non bacterial stimuli such as state after surgery, autoimmune diseases and rheumatic even on myocardial infarction and malignancy.10 crp is an acute -phase proteins, which are in a state of acute phase plasma levels were varied. crp is an additional biomarker in the diagnosis of sepsis. crp has a plasma half-life that is constant in almost all circumstances. levels in the plasma is determined by the speed of synthesis, which reflects the presence and spread of disease activity. induction of crp requires a minimum of 12–18 hours and crp increased late during sepsis also decline takes several days. crp is not useful to distinguish the evolution of sepsis in severe sepsis and septic shock and septic complications in patients with trauma, the slow period after the trauma of high crp values. patients with sirs also have elevated levels of crp. opinions on the usefulness of crp as a diagnostic tool varies, on the one hand claim to have high value and low on the other side. concentrations were reported in patients with sepsis is between 12–159 mg/l, showing overlap with sirs patients who are between 13–119 mg/l. roc analysis showed low sensitivity and specificity.8,11,12 procalcitonin procalcitonin (pct) is a peptide with 116 amino acids with a sequence that is identical to the prohormone of calcitonin, but pct itself has no activity as a hormone. in normal metabolic conditions, pct only in thyroid gland c cells. in bacterial infection and sepsis, intact pct is found in the blood and more importantly pct levels associated with severe sepsis.12 during severe systemic infection, procalcitonin allegedly generated by the extra thyroid tissue. patients who previously underwent total thyroidectomy procalcitonin still produce at a high level during severe sepsis. procalcitonin for sepsis pathophysiology is unclear.10 in normal physiology pct is a precursor of calcitonin. calcitonin is known to regulate the function of bone and calcium metabolism and inhibits osteoclast resorption. regulation of the release of calcitonin was first influenced by the concentration of ionized calcium in plasma. whether this can be attributed to a condition with hypocalcemia in patients with sepsis, remains unclear.13 serum procalcitonin levels increased during bacterial infections, parasites or fungi with systemic manifestations. in severe viral infections or inflammatory reactions of non-infectious cases, procalcitonin levels are not increased or only a modest increase. in patients without the presence of infection is very low procalcitonin levels (< 0.1 ng/l) or very high (6–53 ng/l) in severe infections. resolution of infection with antibiotic therapy reduce levels of procalcitonin. local bacterial infection and viral infection causes only mild and moderate increase (0,3–1,5 ng/l). that’s why the proposed procalcitonin as an indicator of severe infection or sepsis.12,14 for the record, procalcitonin levels may be elevated in the first days of life in the absence of infection. patients with c-cell carcinoma of the thyroid gland may also be there is an increase in the level of procalcitonin in the absence of underlying infection.14 in vivo studies showed increased lps stimulation after a period of 2–6 hours after injection, with a plateau curve from 8–24 hours. pct as a biomarker measurement is 71artaria tjempakasari and nasronudin: bacteria caused sepsis biomarkers preferred because it has a half-life 22–29 hours and this increase will be long during sepsis. positive and gram negative organisms causing an increase in the concentration of pct in the absence of a significant difference.8 procalcitonin levels increased with increasing degree of inflammatory response in response to infection. when patients were categorized into sirs, sepsis, severe sepsis and septic shock, particularly increased procalcitonin levels in patients with severe sepsis and septic shock. in a recent study, the levels of tnf-, il-6, c-reactive protein and procalcitonin were followed for 14 days after the diagnosis of sepsis. procalcitonin levels were consistently lower in patients compared to survivors who did not over a period of 14 days. while tnf- and il-6 are not consistent and not significantly increased in patients who can not be saved, possibly because of a too high variability from day to day. c-reactive protein is increased in both, patients who survived and did not survive. procalcitonin levels associated with the severity of the inflammatory response to infection, efficient therapy may be adjusted by a decrease in the levels of procalcitonin. instead procalcitonin levels indicated poor prognosis. so, procalcitonin can be used as an important indicator for the severity of infection and prognosis of infection and can determine the wisdom of therapy efficacy measurements.10 castelli et al, reported differences in crp and pct picture. crp concentrations increased immediately during severe organ dysfunction and systemic inflammation, but the value is not increased during stage organ dysfunction gain weight. while increased levels of pct, especially in patients with organ dysfunction, severe sepsis and septic shock.11 summary sepsis has been diagnosed according to the consensus guidelines established in 1991 as an infection in addition to the symptoms of systemic inflammatory response syndrome. it is frequently fatal infectious condition. the incidence continues to increase despite the use of specific antibiotics. we are at urgent need for biomarkers and reliable measurements that can be applied to risk stratification of septic patients and that would easily identify those patients at the highest risk of a poor outcome. such markers would be of fundamental importance to decision making for early intervention therapy. pro-inflammatory cytokines such as tumor necrosis factor- (tnf-), interleukins-1, -6, -8 (il-1, il-6, il-8) are postulated to play a major role in the pathogenesis of the syndrome. c-reactive protein (crp) and procalcitonin (pct) are among a few biomarkers that incorporated into clinical practice although their precise role in the pathopysiology of sepsis and organ dysfunction still unclear. references 1. balk ra, ely ew, goyette re, 2004. the pathophysiology of sepsis. in: sepsis handbook 2nd ed. society of critical care medicine. thomson advanced therapeutics communications and vanderbilt university school of medicine, pp. 24–32. 2. bozza fa, bozza pt. 2005. beyond sepsis pathophysiology with cytokines: what is their value as biomarkers for disease severity? mem inst oswaldo cruz 100 (suppl): 217–221. 3. kresno sb. 2003. respons imun pada infeksi. dalam: imunologi: diagnosis dan prosedur laboratorium. edisi keempat. balai penerbit: fkui. jakarta, hlm. 161–167. 4. bochud py, calandra t. 2003. pathogenesis of sepsis: new concepts and implications for future treatment. bmj 326: 262–266. 5. parslow tg, bainton df. 2001. innate immunity. in: medical immunology 10th ed. editors: parslow tg, stites dp, terr ai, imboden jb. mcgraw-hill companies. new york, pp. 19–40. 6. martins ga, carvalho m, gattass cr. 2003. sepsis: a follow up of cytokine production in different phases of septic patients. international journal of molecular medicine 11: 585–591. 7. bone rc, grodzin cj, balk ra. 1997. sepsis: a new hypothesis for pathogenesis of the disease process. chest 112: 235–243. 8. carrigan sd, scott g, tabrizian m. 2004. toward resolving the challenges of sepsis diagnosis. clinical chemistry 50: 1301– 1314. 9. harbarth s, holeckova k. 2001. diagnostic value of procalcitonin, interleukin-6, and interleukin-8 in critically ill patients admitted with suspected seopsis. american journal of respiratory and critical care medicine 164: 396–402. 10. reinhart k, karzai w. 2000. procalcitonin-a new marker of the systemic inflammatory response to infections. european society of anaesthesiologists refreseher courses. germany, sunday april 2. 11. castelli gp, pognani c, meisner m. 2004. procalcitonin and c-reactive protein during systemic inflammatory response syndrome, sepsis and organ dysfunction. critical care 8: r234-r242. 12. chan yl, tseng cp, tsay pk. 2004. procalcitonin as a marker of bacterial infection in the emergency department: an observational study. critical care 8: r12-r20. 13. landenberg p, schoenfeld y. 2001. new approaches in the diagnosis of sepsis. imaj 3: 439–442. 14. reinhart k, meisner m. 2001. markers of inflammation in sepsis: clinical and therapeutic implications. european society of anaesthesiologists refreseher courses. germany, sunday april 7. 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. �� 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. 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 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[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 � 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. 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(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. 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 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. 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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 142 404 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 manifestations of acute pancreatitis in severe covid-19 patients: is this a coincidence? pradana zaky romadhon1, satriyo dwi suryantoro2*, choirina windradi2, bagus aulia mahdi1, esthiningrum dewi agustin2, krisnina nurul w1, dwiki novendrianto1 1faculty of medicine, universitas airlangga, surabaya, indonesia 2universitas airlangga hospital, surabaya, indonesia received: 27th april 2021; revised: 20th may 2021; accepted: 7th june2021 abstract coronavirus disease-19 (covid-19) adalah penyakit yang disebabkan oleh severe acute acute respiratory coronavirus-2 (sars-cov2) yang berasal dari china, menyebar dengan cepat ke seluruh bagian negara lain yang menyebabkan pandemi dunia. dengan derajat gejala yang bervariasi yang disebabkan oleh covid-19, virus ini menyebabkan kerusakan pada beberapa organ, baik karena efek infl amasi tidak langsung maupun efek sitopatik. data terkait keterlibatan pankreas dalam kasus covid-19 masih belum jelas. seorang laki-laki usia 83 tahun dirawat karena gejala covid-19 berat. dalam perawatan, pasien memberikan gejala dan tanda pankreatitis akut tanpa diketahui faktor resiko yang terkait. pada pemeriksaan didapatkan rt-pcr sars-cov2 positif dari swab nasofaring, amilase lipase yang meningkat serta gambaran ultrasound khas untuk pankreatitis akut. tatalaksana pasien tetap berdasar pada kasus sars-cov2 dengan isolasi, oksigenasi, pemberian anti virus dan suportif. pemberian antibiotik juga didasarkan pada terapi empiris yang kemudian disesuaikan hasil sensitifi tas kultur. skor prognosis pankreatitis menunjukkan risiko kematian pada kasus moderate. pada perjalanan, pasien meninggal karena shock sepsis. prevalensi pankreatitis akut dan tingkat keparahannya perlu diamati. dalam artikel ini, kami menyajikan kasus pankreatitis akut yang terjadi pada covid-19 parah dengan faktor risiko yang tidak diketahui.diagnosis penyebab kasus pankreatitis masih belum jelas tetapi beberapa bukti autopsi kasus infeksi sars-cov2 dengan pankreatitis menyebutkan bahwa infeksi virus ini dapat menyebabkan injuri pada pankreas.. kondisi sepsis dapat diakibatkan infeksi virus sars-cov2 (viral sepsis) atau ko-infeksi bakteri. oleh karena itu, rasionalisasi penggunaan antibiotik juga diperlukan. kasus ini merupakan kasus yang membutuhkan managemen holisitik dan intensif karena kedua kondisi berpotensi dapat memperberat satu sama lain. pengenalan awal kegawatan serta terapi tepat merupakan hal yang penting dapat menunjang kesintasan pasien. keywords: pancreatitis, infectious disease, covid-19, organ damage, sepsis abstract coronavirus disease-19 (covid-19) is a disease caused by severe acute respiratory syndrome coronavirus-2 (sarscov2) came from china, this disease is highly infectious causing rapid spread throughout the world. covid-19 had various of symptoms, manifestation, and also degree to cause multiorgan dysfunctions either due to indirect infl ammatory eff ects or cytopathic eff ects. data regarding the involvement of the pancreas in covid-19 cases is still unclear. an 83-year-old man was being treated for severe covid-19 symptoms. he had received treatment for severe covid-19. unfortunately, during hospitalization, the patient presenteds the symptoms and signs of acute pancreatitis without any known risk factors. physical fi ndings supported the diagnosis criteria for acute pancreatitis. moreover, supporting examination found a positive sars-cov2 rt-pcr from a nasopharyngeal swab, increased amylase lipase and a typical ultrasound image for acute pancreatitis. patient management wasremains based on covid-19 cases witconsisting ofh isolation, oxygenation, antiviral and other supportive medical treatment. antibiotic administration wasis also based on empirical therapy which wasis then adjusted for the results of culture sensitivity. although the etiology diagnosis of this patient was uncertain, we assumed sars-cov2 infection couldan cause injury to the pancreas. * corresponding author: satriyo.dwi.suryantoro@fk.unair.ac.id 80 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 79–84 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 we did observe patient based on clinical and laboratory fi ndings other than that based on ranson’s score the patient wais in poor prognosis. eventually patient died due to septic shock. sepsissis conditions in covid-19 patients could be due to viral sepsis and bacterial co-infection. therefore, a rationalization of the use of antibiotics isis also needed. this case is a case that requires intensive and holistic management because the two conditions can potentially aggravate each other. early recognition of emergency and appropriate therapy is important to support patient survival. kata kunci: pankreatitis, pandemi, covid-19, kerusakan organ, sepsis how to cite: romadhon, pz., suryantoro, sd., windradi, c., mahdi, ba., agustin, ed., nurul, wk., novendrianto, d. (2021). manifestations of acute pancreatitis in severe covid-19 patients: is this a coincidence?. indonesian journal of tropical and infectious disease, 9(2), 79-84 introduction sars-cov2 causes covid-19 infection that until now has become a world pandemic 1-3. coronavirus (cov) is a group of enveloped viruses possibly identified in animals. some cov found in animals may precipitate infectious diseases, such as viral gastroenteritis (tgev), porcine epidemic diarrhea virus (pedv), avian infectious bronchitis virus (ibv), and swine acute diarrhea syndrome coronavirus (sads-cov)4-6. covid-19 also came with extrapulmonary manifestations which involve the role of the ace2 receptor. this may include neurological, renal, hepatic, gastrointestinal, thromboembolic, cardiac, endocrine, and dermatology systems 7, 8. to date, the most common gastrointestinal manifestations of covid-19 are nausea, vomiting, diarrhea, abdominal pain 9-15. however, in our case. the occurrence of acute pancreatitis without an understandable risk factor was the direct injury to the pancreas gland by sarscov2 16. case mr. s, 83 years old, came with tightness 2-3 days ago getting more severe for two days. the patient did not previously complain of having fever and runny nose. there are no complaints of nausea or vomiting. sometimes the patient complains of abdominal pain so that he is merely able to eat half of the usual portion. daily, the patient can still do activities such as bathing, walking, and wearing clothes. however, since then, the patient was unable to carry out his activity. he was weak and only able to lie down and sit with assistance. the patient started having a cough 2 days ago, thus the abdominal tightness was getting worse when coughing. the patient self-checks peripheral oxygen saturation at home and turned out only 72-75% room air. the patient did not previously have diabetes mellitus but had a history of hypertension with heart disease. there is no history of drinking alcohol. the patient takes clopidogrel 75 mg once daily and bisoprolol 2.5 mg once daily. respiratory muscles were found retracted. neither ronchi nor wheezing was heard from the lung examination. the apex beat is dilated, which is in line with cardiomegaly fi gured out on x-ray. initial examination revealed a positive sars-cov2 antigen swab with a chest x-ray showing pneumonia (see figure 1). nasooropharynx polymerase chain reaction (pcr) swab was positive for sars-cov2 with ct 27.5. figure 1. pneumonia covid-19 chest x-ray. 81pradana zaky romadhon, et al.: manifestations of acute pancreatitis in severe covid-19 patients ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 laboratory fi ndings showed hb 14.2 g / dl, white blood count 6530 u / l, urea 69.8; serum creatinine 2.84, d-dimer 8.54. blood gas analysis showed moderate to severe hypoxia with ph 7.43; pco2 24, po2 146, be 17, hco3 16.2, sao2 96. the patient received supportive and symptomatic treatments such as oxygen supplementation, meropenem, nebivolol, codeine, dexamethasone for ten days, fondaparinux, and remdesivir drip for fi ve days. on the third day of treatment, we found that the patient's right upper abdominal pain was severe. unfortunately, our abdominal examination found discoloration of the left fl ank that we suspect as pancreatitis (see figure 3). subsequent laboratory tests showed an increase in amylase number 138 u / l and lipase 200 u / l with normal liver function. serum cholesterol was normal. the infl ammatory markers of crp did not show an increase of 0.7 mg / l while procalcitonin was 0.36 ng / ml. abdominal ultrasound results showed a slightly enlarged pancreas with a very hypoechoic structure (see figure 2). at the current examination, we found a positive ranson prognostic ranson’s score for acute pancreatitis for the variable age 83 years. however, after a 48-hour evaluation, there was a positive ranson criterion for increased hematocrit, urea, and base excess defi cit. therefore, the prognosis of mortality is around 11-15%. figure 2. ultrasonography of pancreatitis. based on the results of the examination, the patient was diagnosed with covid-19 and acute pancreatitis. some of the most common etiologies of pancreatitis are etiologies such as gallstones, alcohol, pancreatitis-causing drugs such as furosemide, thiazide, sulfa, hypertriglyceridemia, other viral infections, autoimmune, posttraumatic were not found in this patient. besides, the use of clopidogrel might considerably be also one of the triggering factors since lai et al. declared that persons actively consume clopidogrel were at 8.46-fold increased odds for acute pancreatitis. 27therefore, however, tthe diagnosis of the cause of acute pancreatitis can be confi rmed by autopsy to detect sars-cov2 in pancreatic cells. but we could not nt performed it because this examination was not done routinely. however, we still manage patients according to the management of acute pancreatitis. at the time of the initial diagnosis of pancreatitis, the patient was fasting and receiving parenteral nutritional support, adequate fl uid administration, also antibiotics according to culture results. the results of the patient’s blood culture showed the growth of staphylococcus sp. then we changed the antibiotic treatment based on the results of the sensitivity test. meanwhile, the patient has received remdesivir as the anti-viral agent. unfortunately, the patient falls into a state of septic shock then the patient died. figure 3. abdominal appearance indicating suspicious acute pancreatitis. 82 indonesian journal of tropical and infectious disease, vol. 9 no. 2 may–august 2021: 79–84 ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 discussion covid-19 often manifests as respiratory complaints, but extrapulmonary ones require extra attention. recent studies show that gastrointestinal symptoms can reach 50% with symptoms such as nausea (17.3%), diarrhea (12.9%), anorexia (12.2%), abdominal pain (5.8%). meanwhile, in a journal written by wang et al, in a case series, nine people had acute pancreatitis along with covid19 infection. liu et al wrote that 17% of the 67 cases of severe covid-19 had acute pancreatitis, although only 7.46% of the pancreatic injury was able to be captured by computed tomography 14, 16. covid-19 utilizes angiotensin-converting enzyme 2 (ace2) as a receptor for the entry of viruses into human cells. ace2 receptors are not only in the lungs but also widely spread in the esophageal, enterocyte, cardiovascular, renal, and pancreatic epithelial cells. surprisingly, the amount of ace2 rna messenger was found more in the pancreas than in the lungs. ace2 expression took both in the exocrine glands of the pancreas and in the islet cells. the spike protein (s) acts as a support for the ace2 receptor. expression of ace2 and transmembrane serine protease 2 (tmprss2) that plays a vital role in the successful fusion of sars-cov2 into human cells, is found in β cells of the pancreas 7. acute pancreatitis is a condition when the pancreas is inflamed. this inflammatory process may be confi ned to the pancreatic or peripancreatic tissue. the causes of this ap vary while severity also is divided to diff erent degrees 12, 17-19. the most common risk factors for acute pancreatitis are gallbladder disease (often caused by choledocholithiasis) and chronic alcohol consumption. acute pancreatitis is defi ned as the presence of typical pancreatic abdominal pain, an increase in serum amylase/lipase more than three times of regular value, and ultrasound, ct, or mri imaging fi ndings support the diagnosis 19-21. in these patients, we found no risk factors that could explain the development of acute pancreatitis. some of the most common pancreatitis etiologies such as gallstones, alcohol, pancreatitis-causing drugs such as furosemide, thiazide, sulfa, hypertriglyceridemia, other viral infections, autoimmune, posttraumatic. no medical history of the patient seemed to cause the pancreatitis, but the use of clopidogrel might possibly trigger it. his past medical history and drugs does not support the variable causes of pancreatitis. patients with acute pancreatitis often have positive blood culture results when a systemic infection is found, especially in patients who have previously undergone intra-biliaer procedures. the results of blood culture in the most common acute pancreatitis patients were escherichia coli and klebsiella sp. those systemic infection could be fatal. in this patient we found his blood culture positive for staphylococcus sp meaning the source of infection could be anywhere that precipitate the septic state. therefore, it is consistent with several similar case reports that it is possible to injury the pancreas in a patient with covid-19 12, 19. in fact, in several observational case-control studies on pancreatic injury, there was an increase in serum amylase/lipase as a marker of pancreatic damage in 8.5-17.3% of cases. interestingly, pancreatic abnormalities have been more frequently noted in the sub-group of patients having severe covid disease 17-19, 22. several hypotheses suggest the occurrence of pancreatitis in covid-19, namely the expression of ace2 in the pancreatic ductal, acinar, and islet cells so that the virus can easily spread from the duodenal epithelium to the pancreatic gland 17, 23, 24. other studies have shown that sars-cov-2 is able to infect pancreatic-induced pluripotent stem cells (ipsc) thus they produce proinfl ammatory cytokines such as cxcl12, il-6, il-8, il-10 12. they observed that the sars-cov-2 hijacked the ribosomal machinery in the pancreatic cells and also increased the expression of some pancreatic ductal stress response genes. prominently, the genes cxcl12, nfkb1, and stat3 showed signifi cant upregulation as compared to the control. the researchers report that the transcriptional analysis of sars-cov-2 infected ipsc-derived pancreatic cultures demonstrated active viral replication and pancreas-specific covid-19 associated disease signatures. the srp-protein targeting processes were upregulated, indicating 83pradana zaky romadhon, et al.: manifestations of acute pancreatitis in severe covid-19 patients ijtid, p-issn 2085-1103, e-issn 2356-0991 open acces under cc-by-nc-sa share alike 4.0 that host cell machinery was being repurposed for viral replication 25, 26. f r o m r n a s e q u e n c i n g s t u d i e s , t h e y established that the pancreas, specifi cally the exocrine compartment (acinar and ductal cells), has a high expression of ace2. gender and age present no diff erence in the expression of the ace2 receptors. the researchers demonstrated that the ipsc-derived pancreatic cells used in this study exhibit ace2 and tmprss2 expression. both the receptors are present in the pancreas, especially in the exocrine portion 26. despite the many published cases of both the coincidence of acute pancreatitis with covid-19, acute pancreatitis caused by covid-19 is still unproven. however, if there is a case of acute pancreatitis with covid-19, the occurrence of idiopathic acute pancreatitis due to covid-19 cannot be neglected 12. conclusion cases of acute pancreatitis with covid-19 can be a coincident or idiopathic. thus far, diagnostic tool of this case is necrosis autopsy and sarscov2 pcr. through our observation, covid19 has raised suspicions of acute idiopathic pancreatitis in severe covid-19. therapy and monitoring in patients are still carried out according to the management of covid-19 and pancreatitis. we thank you to one of the private hospital in surabaya for providing proper care for the patient conflict of interest the authors declare that there is no confl ict of interest references 1. kumar m, taki k, gahlot r, sharma a, dhangar k. a chronicle of sars-cov-2: part-i -epidemiology, diagnosis, prognosis, transmission and treatment. science of 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this server. apache/2.4.41 (ubuntu) server at e-journal.unair.ac.id port 80 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