J Arthropod-Borne Dis, June 2017, 11(2): 331–337 H Ebrahimzade-Parikhani et al.: Seroprevalence of … 331 http://jad.tums.ac.ir Published Online: May 27, 2017 Original Article Seroprevalence of Visceral Leishmaniasis in Children up to 12 Years old Among Nomadic Tribes from Rural Areas of Pars Abad, Northwestern Iran: an Observational Study in 2015 Hassan Ebrahimzade-Parikhani 1, *Mehdi Mohebali 1,2, Zabiholah Zarei 1, Behnaz Akhoundi 1, Zahra Kakoei 1 1Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Center for Research of Endemic Parasites of Iran, Tehran University of Medical Sciences, Tehran, Iran (Received 3 Mar 2016; accepted 7 Jan 2017) Abstract Background: Since Pars Abad district had been known as a focus of visceral leishmaniasis (VL) in Ardabil Province but the prevalence of the disease in nomadic tribes has not been determined, thus, this study was conducted. Methods: This descriptive cross- sectional study was conducted on children up to 12yr old of nomadic tribes from Pars Abad County, Ardabil Province, Iran in 2015. For each individual, a questionnaire including age, sex, clinical manifestations, history of disease, and contact with reservoir hosts of VL were completed, separately. To determine VL seroprevalence, blood samples were collected from the children and after centrifugation, the plasma samples were tested using Direct Agglutination Test (DAT) for detection of anti-Leishmania infantum antibodies. Statistical analyses were performed using SPSS16. Results: From 776 children up to 12yr old, 2 (0.25%) showed anti-L. infantum antibodies at titers 1:1600 and only one case (0.13%) showed anti-Leishmania antibodies at titers 1:3200. The child with anti-L. infantum antibodies titers of 1:3200 showed mild fever for more than 2 months period, paleness, weakness and mild splenomegaly. After physical examination and confirmation of VL (kala-azar), the patient was treated with antileishmanial drugs. Conclusion: The findings indicated that L. infantum infection is being circulated with low prevalence in nomadic tribes of Pars Abad but it is necessary that the surveillance system is regularly monitored among physicians and pub- lic health managers in the studied areas. Keywords: Sero-prevalence, Visceral leishmaniasis, Nomadic tribes, Children, Iran Introduction Leishmaniasis has been reported from 98 countries in the world, in Iran this disease is endemic. More than 20 species of Leishmania produce diseases, and approximately 30 species of sand flies species (Phlebotomus) play a role in disease transmission (Alvar et al. 2012). The disease presents four clinical features, namely cutaneous leishmaniasis, mucocutane- ous leishmaniasis (Espondia), visceral leish- maniasis (VL or kala-azar), and post kala-azar dermal leishmaniasis (PKDL) (Arevalo et al. 2007). Visceral leishmaniasis (VL), generally produced by L. donovani complex, is a dan- gerous form of leishmaniasis and transmitted by the bite of female sandflies (Mohebali 2013). This disease is manifested by prolonged systemic symptoms such as fever, hepatomeg- aly, splenomegaly, weight loss, and pancyt- openia (Alvar et al. 2012, Mohebali 2012). Visceral leishmaniasis is projected to about 500,000 cases of morbidity and 59,000 cases of mortality every year in the world (Wer- neck 2014). This disease has high Case Fa- *Corresponding author: Dr Mehdi Mohebali, E-mail: mohebali@tums.ac.ir J Arthropod-Borne Dis, June 2017, 11(2): 331–337 H Ebrahimzade-Parikhani et al.: Seroprevalence of … 332 http://jad.tums.ac.ir Published Online: May 27, 2017 tality Rate (CFR) in untreated patients, un- derweight children and cases of HIV/ VL co- infection (Cavalcante and Vale, 2014). Vis- ceral leishmaniasis is mostly reported in six countries: India, Ethiopia, Nepal, Sudan, Bang- ladesh, and Brazil (Boelaert et al. 2000). The hazard in gaining the illness is mediated through unfortunate housing environments, nonexistence of private protective processes against the vector and migration that leads to non-immune hosts entering VL-endemic re- gions (Bern et al. 2008). Kala-azar is endem- ic in many Asian countries and the Middle East. In Iran, VL is observed in sporadic and endemic forms, so in many parts of Iran the disease is sporadic and in some parts of the provinces such as Ardabil, East Azerbaijan, Bushehr and Fars, the disease is seen as en- demic (Mohebali et al. 2011). Remarkably, from the 7204 serum sample gathered from domestic dogs in rural com- munities well-known as the endemic foci of human VL in Iran, 879 (12.2%) were sero- positive by titers of ≥1:320 (Mohebali et al. 2005, Mohebali et al. 2006, Moshfe et al. 2008, Moshfe et al. 2009). Also, in a study conducted by Moshfe et al in 2006–2007 on canine Visceral leishmaniasis in Meshkin Shahr District in northwestern Iran, 17.4% of se- rum sample were positive by DAT (1:320 and higher). Moreover, a quarter of seroposi- tive dogs displayed clinical VL symptoms, and cachexia and alopecia were the highest clinical signs in the seropositive dogs (Mohe- bali et al. 2005). Annually, approximately 100–300 new symptomatic cases of VL are registered in the Health Care System of Iran (Mohebali 2013). Out of the 31 provinces of Iran, more than 2000 cases of VL were reported up to 2012 (Mohebali 2012). In this report, 44.6% of the cases were from the northwestern re- gion of Iran (Mohebali 2013). During the last decade in Iran, an annual average inci- dence rate of disease was reported as 0.449 per 100000 at-risk populations. Here, in the northwestern part of Iran, with an incidence rate of 57 cases per 100, 000, have the maxi- mum incidence rate of the disease in Iran (Mohebali 2013). In this study, the direct agglutination test (DAT) was used as a sero diagnostic tool due to its simplicity, cost-effectiveness, appro- priate sensitivity (92–100%) and specificity (72–100%), valid test results and its suitabil- ity for use in field conditions (Elmahallawy et al. 2014). Since Ardabil Province is one of the foci of VL and the prevalence of the disease especially on nomadic tribes of this province has not been evaluated, this present study was conducted to determine the se- roprevalence of VL in the nomadic tribes of Pars Abad County to rapid case finding, and to provide suggestions for the prevention of the disease among nomadic tribes in the study area. Materials and Methods Study area This survey was carried out in the nomadic tribes of Pars Abad district from 21 March 2014 to 20 March 2015.Pars Abad district is located in the north of Ardabil Province, northwestern Iran. The study area has mild weather condition in the summer and cold in the winter with an average altitude of 32m above the sea level and a landmass of 1383 km2 (14 percent of the Ardabil Province area). Pars Abad area is found in the northern part of the Ardabil Province, located between the orbits of 39 degrees and 12min to 39 °C and 42min north latitude and 47 degrees and 10 min to 48 degrees and 21min east of the Green- wich meridian. Pars Abad area have 3 dis- tricts, 2 cities and 6 villages. According to the 2011 Iranian census, the population of this county was 88,924 (https://www.amar.org.ir). Blood sampling This study was conducted on children ≤12yr old of nomadic tribes from rural areas J Arthropod-Borne Dis, June 2017, 11(2): 331–337 H Ebrahimzade-Parikhani et al.: Seroprevalence of … 333 http://jad.tums.ac.ir Published Online: May 27, 2017 of Pars Abad, northwestern Iran. Altogether, the blood samples were prepared from 776 children. For each that entered the study, questionnaires that included individual char- acteristics such as age, sex, location, clinical signs, history of the disease, and contact with dog were provided and filled. For serologic studies, blood samples were collected, and the plasma samples were separated by centrifuga- tion and stored at -20 °C until their examina- tions. Performance of direct agglutination test (DAT) The L. infantum antigens for this study were prepared in the Leishmaniasis Laboratory of the School of Public Health, Tehran Univer- sity of Medical Sciences, Tehran, Iran. The principal phases of the procedure for prepar- ing direct agglutination test antigen were mass production of promastigotes of Iranian strain of L. infantum [MCAN/IR/07/Moheb- gh. (GenBank accession no. FJ555210)] in RPMI1640 medium (Biosera, South Amer- ica), plus 10% fetal bovine serum (Biosera, South America), followed by tripsinization of the parasites, staining with Coomassie bril- liant blue R-250 (Sigma, USA) and fixing with formaldehyde 1.2% (El Harith et al. 1988, Zijlstra et al. 1991, Mohebali et al. 2006). All the collected plasma samples were tested by DAT. The titration of Leishmania- specific antibodies was performed by fol- lowing the general procedures described by Mohebali et al. (2006). Primarily, two dilutions of 1:800 and 1:3200 were made and tested for screening. The samples that were posi- tive with the titer of 1:800 were diluted up to 1:102400 in a V-shaped microtiter plate into a dilution fluid containing 0.9% saline and 0.78% 2-mercaptoethanol. One equal volume (50μl) of antigen suspension was added to each well. The results were read after 18–24 h incubation in a wet room at room tempera- ture. The highest dilution at which agglutina- tion was still visible in comparison with pos- itive and negative controls titer was defined as the titer of the sample. Compact blue dots were scored as negative and large diffused blue mats as positive. The characteristics of seropositive cases of the disease were reported to the health cen- ters of Pars Abad District to receive suitable cure, if necessary. Antigen control well (an- tigen and diluent plasma only) and the known negative and positive controls were tested in each plate daily. Based on prior studies (El Harith et al. 1988, Mohebali et al. 2006), titers of ≥ 1:3200 were considered as seropositive, and persons with suspected (a titer of 1:1600) or lysed samples were re-sampled 2 to 3 weeks later (Schallig et al. 2001, Mohebali et al. 2006, Kakooei et al. 2014). Ethical considerations Informed written consent was obtained from the parents of the children examined. This study was approved by the Research Ethical Review Committee of Tehran Uni- versity of Medical Sciences, Tehran, Iran. Statistical analysis Chi-squared and Fisher exact tests were used for the assessment of the relation between two quantitative variables (sero-prevalence val- ues in relation to sex and age group). Statisti- cal significance was assumed if P< 0.050. All reported P values are two-sided. Statistical anal- yses were performed using SPSS software (version 16, SPSS Inc., Chicago, IL, USA). Results Altogether, 776 blood samples were collect- ed from the children up to 12 years old from nomadic tribes of north of Ardabil Province. Four hundred and three of them (51.9%) were male, and 373 (48.1%) were female. The sex ratio (male/female) of the studied population was 1.08. From the 776 children, 3 (0.3%) showed anti-Leishmania antibodies at titers ≥ J Arthropod-Borne Dis, June 2017, 11(2): 331–337 H Ebrahimzade-Parikhani et al.: Seroprevalence of … 334 http://jad.tums.ac.ir Published Online: May 27, 2017 1:1600, and from these only one case showed anti- L. infantum antibodies at titers of 1:3200 (Table 1). Hence, the prevalence of the dis- ease (Titers of 1:3200) in the studied areas was estimated as 1.28 per 1000 at-risk popu- lations. Furthermore, the seropositive case pre- senting anti-Leishmania antibodies at titers 1:3200 was a 10 year old female from AGH Ghabagh (Hosein Gheshlagh) village with a positive history of the disease with remittent fever, mild splenomegaly and cutaneous scar in the face in two or three months prior to her disease diagnosis. This patient was hospital- ized in the pediatric hospital and was success- fully treated with Glucantime® as the first line of anti-leishmanial drugs. The relation between human Leishmania infection titers and sex and age group was not statistically significant, with P-values of (P= 0.197) and P= 0.332), respectively prob- ably because of the low number of seroposi- tivities. The frequency of anti-Leishmania an- tibody titers with DAT according to the age groups is shown in Table 2. Age group and sex distribution of samples is presented in Table 3. In Table 4, the age group, sex, loca- tion and anti-Leishmania antibody titers of 3 seropositive cases of VL has been showed. Table 1. Seroprevalence of human visceral Leishmania infantum infection by gender in children up to 13 years old of nomadic tribes of Pars Abad County from Ardabil Province, Iran in 2015 Anti-Leishmania infantum antibody titers ≥ 1:32001:16001:800Negative (%)Number of examined Gender Number (%)Number (%)Number (%) 0 (0%)0 (0%)0 (0%)403 (100%)403Male 1 (0.3%)2 (0.5%)0 (0%)370 (99.2%)373Female 1 (0.1%)2 (0.3%)0 (0%)773 (99.6%)776Total Table 2. Seroprevalence of human Leishmania infantum infection by age group in nomadic tribes of Pars Abad County from Ardabil Province, Iran in 2015 Anti-Leishmania infantum antibody titers 1:32001:16001:800Negative (%)Number of examined Age group (yr) Number (%)Number (%)Number (%) 0 (0%)1 (0.5%)0 (0%)201 (99.5%)202<4 0 (0%)0 (0%)0 (0%)280 (100)2805–8 1 (0.5%)1 (0.5%)0 (0%)292 (99%)2949–12 1 (0.1%)2 (0.3%)0 (0%)773 (99.6%)776Total Table 3. Anti-Leishmania infantum antibody titers of three seropositive cases of visceral Leishmania infantum infection by direct agglutination test with respect to their age, gender and locality in nomadic tribes of Pars Abad from Ardabil Province, Iran 2015 Case No. Age (yr) Gender Location Tribe Antibody Titer 1 9 Female Pars Abad Asad Kandi 1:1600 2 ≤1 Female Pars Abad Griloo 1:1600 3 10 Female Pars Abad Hossin Gheshlagh 1:3200* *This patient had showed clinical signs and symptoms J Arthropod-Borne Dis, June 2017, 11(2): 331–337 H Ebrahimzade-Parikhani et al.: Seroprevalence of … 335 http://jad.tums.ac.ir Published Online: May 27, 2017 Fig. 1. Situation of Ardabil province in Iran and location of study areas in Ardabil Province (Pars Abad) Discussion This study was conducted to assess the epidemiological characteristics of VL in Pars Abad in the north of Ardabil Province (north- western Iran). The prevalence of visceral L. infantum infection in the Pars Abad district was 1.28 per 1000 at-risk populations. Fe- males and those belonging to the age group of 9–12 years old are at higher risk of dis- ease, in comparison with the other groups. In recent years, various and scattered stud- ies about several aspects of VL have been conducted in Ardabil Province in northwest- ern Iran. The results showed that VL is en- demic in some areas of the province, and most of cases of VL were reported from Meshkin Shahr District (Soleimanzadeh et al. 1993, Edrissian et al. 1996, Mohebali et al. 2006). In this study, we observed that from the 776 persons that entered the study, 3 (0.3%) of them showed anti- L. infantum antibodies at titers ≥ 1:1600, and from these only one case (0.1%) showed anti-Leishmania antibod- ies at titers ≥ 1:3200. In a sero-epidemiologi- cal study that was conducted by in our study, only one seropositive case of the disease with titers of ≥1:3200 was diagnosed, which in- cluded a female in the age group of 9–12yr old. In a study performed by Abbaszadeh- Afshar et al. (2015) 0.50% of 6 seropositive cases were males and 0.50% were female, and children belonging to the age group of 5–8yr old showed the highest seroprevalence rate (4.1%) of the disease, A similar result was found by Mahmoudvand et al. (2011) in which 41.66% (5 cases) of 14 seropositive cases were females and 58.34% (9 cases) were males. Conclusion Leishmania infantum infection is being cir- culated with low prevalence in the nomadic tribes of Pars Abad area, however, it is nec- essary for the surveillance system to regular- ly monitor the infection in the studied areas. Appropriate counseling should be provided to military personnel, researchers, and other groups of travelers who may be exposed to sand flies in the endemic areas. Early case de- tection and treatment can reduce the impact of severe illness and death. Further studies on domestic dogs as principal animal reser- voir host of VL in the nomadic tribes of Pars Abad are recommended. Acknowledgements This study was financially supported by Tehran University of Medical Sciences (Grant No: 93-03-160-27323). We extend our thanks to Mr Ojagh Rudy, Mr Shahram Dastori, and Mrs Sorour Charehdar for their valuable field and laboratory techniques. The authors are also grateful to the directors and staff of the Health and Medical Center in Pars Abad district for their co-operation. The authors declare that there is no conflict of interest. References Abbaszadeh-Afshar M, Mohebali M, Sharifi I, Akhoundi B, Aflatoonian M, Bahreini M, Mahmoudvand H (2015) Seroepide- miological survey of Visceral leishman- iasis among nomadic tribes of Kerman J Arthropod-Borne Dis, June 2017, 11(2): 331–337 H Ebrahimzade-Parikhani et al.: Seroprevalence of … 336 http://jad.tums.ac.ir Published Online: May 27, 2017 Province, Southeastern Iran: An obser- vational study for implication to health policy. J Biostat Epidemiol. 1: 105–111. Alvar J, Velez I, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, Den Boer M, Team W (2012) Leishmaniasis worldwide and global estimates of its incidence. PloS One. 7: e35671. Arevalo J, Ramirez L, Adaui V, Zimic M, Tulliano G, Miranda-Verástegui C, Lazo M, Loayza-Muro R, De Doncker S, Maurer A (2007) Influence of Leishma- nia (Viannia) species on the response to antimonial treatment in patients with American tegumentary leishmaniasis. J Infect Dis. 195: 1846–1851. Asgari Q, Fakhar M, Motazedian M (2006) Nomadic Kala-Azar In South of Iran. Iran J Public Health. 35: 85–86. Bern C, Maguire J, Alvar J (2008) Com- plexities of assessing the disease bur- den attributable to leishmaniasis. PLoS Negl Trop Dis. 2: e313. Boelaert M, Criel B, Leeuwenburg J, Van Damme, W, Le Ray D, Van Der Stuyft P (2000) Visceral leishmaniasis con- trol: a public health perspective. Trans R Soc Trop Med Hyg. 94: 465–471. Cavalcante M, Vale M (2014) Epidemiological aspects of visceral leishmaniasis (kala- azar) in Ceará in the period 2007 to 2011. Rev Bras Epidemiol. 17: 911–924. Edrissian G, Hajaran H, Mohebali M, So- leimanzaehg, Bokaei S (1996) Applica- tion and evaluation of direct agglutination test in sero-diagnosis of visceral leish- maniasis in man and canine reservoirs in Iran. Iran J Med Sci. 21: 119–124. Edrissian Gh, Ahanchin A, Gharachahi A, Ghorbani M, Nadim A, Ardehali S (1993) Seroepidemiological studies of visceral leishmaniasis and search for animal reservoirs in Fars Province, south- ern Iran. Iran J Med Sci. 18: 99–105. El Harith A, Kolk A, Leeuwenburg J, Muigai R, Huigen E, Jelsma T, Kager P (1988) Improvement of a direct agglutination test for field studies of visceral leish- maniasis. J Clin Microbiol. 26: 1321– 1325. Elmahallawy Ek, Martinez A, Rodriguez- Granger J, Hoyos-Mallecot Y, Agil A, Mari Jm, Fernandez J (2014) Diagnosis of leishmaniasis. J Infect Dev Ctries. 8: 961–972. Fakhar M, Mohebali M, Barani M (2004) Identification of Endemic Focus of Kala azar and Seroepidemiologcial Studyـ of Visceral Leishmania Infection in Human and Canine in Qom Province, Iran. Ar- maghane Danesh. 9: 43–52. Mahmoudvand H, Mohebali M, Sharifi I, Keshavarz H, Hajjaran H, Akhoundi B, Jahanbakhsh S, Zarean M, Javadi A (2011) Epidemiological aspects of vis- ceral leishmaniasis in Baft District, Ker- man Province, Southeast of Iran. Iran J Parasitol. 6: 1–11. Mohebali M (2012) Epidemiological status of visceral leishmaniasis in Iran: expe- riences and review of literature. J Clin Exp Pathol. S3: 003. Mohebali M (2013) Visceral leishmaniasis in Iran: review of the epidemiological and clinical features. Iran J Parasitol. 8: 348– 358. Mohebali M, Edrissian GhH, Shirzadi MR, Akhoundi B, Hajjaran H, Zarei Z, Molaei S, Sharifi I, Mamishi S, Mah- moudvand H, Torabi V, Moshfe AA, Malmasi AA, Motazedian MH, Fakhar M (2011) An observational study on the current distribution of visceral leishman- iasis in different geographical zones of Iran and implication to health policy. Travel Med Infect Dis. 9: 67–74. Mohebali, Edrissian G, Nadim A, Hajjaran H, Akhoundi B, Hooshmand B, Zarei Z, Arshi S, Mirsamadi N, Naeini K (2006) Application of direct agglutina- tion test (DAT) for the diagnosis and seroepide-miological studies of viscer- J Arthropod-Borne Dis, June 2017, 11(2): 331–337 H Ebrahimzade-Parikhani et al.: Seroprevalence of … 337 http://jad.tums.ac.ir Published Online: May 27, 2017 al leishmaniasis in Iran. Iran J Parasi- tol. 1: 15–25. Mohebali M, Hajjaran H, Hamzavi Y, Mobedi I, Arshi S, Zarei Z, Akhoundi B, Naeini KM, Avizeh R, Fakhar M (2005) Epi- demiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran. Vet Parasitol. 129: 243–251. Moshfe A, Mohebali M, Edrissian G, Zarei Z, Akhoundi B, Kazemi B, Jamshidi S, Mahmoodi M (2008) Seroepidemiolog- ical study on canine visceral leishman- iasis in Meshkin-Shahr District, Arda- bil Province, northwest of Iran during 2006-2007. Iran J Parasitol. 3: 1–10. Moshfe A, Mohebali M, Edrissian G, Zarei Z, Akhoundi B, Kazemi B, Jamshidi S, Mahmoodi M (2009) Canine visceral leishmaniasis: asymptomatic infected dogs as a source of L. infantum infec- tion. Acta Trop. 112: 101–105. Nadim A, Javadian E, Mohebali M, Momeni A (2008) Leishmania parasite and leish- maniasis. Acad Publish Cent, Tehran, Iran. Markaze Nashre Daneshgahi Publ. Schallig H, Schoone G, Kroon C, Hailu A, Chappuis F, Veeken H (2001) Devel- opment and application of'simple'diag- nostic tools for visceral leishmaniasis. Med Microbiol Immunol. 190: 69–71. Soleimanzadeh G, Edrissian G, Movahhed- Danesh A, Nadim A (1993) Epidemio- logical aspects of kala-azar in Mesh- kin-Shahr, Iran: human infection. Bull World Health Organ. 71: 759. Werneck G (2014) Visceral leishmaniasis in Brazil: rationale and concerns related to reservoir control. Rev Saude Pub- lica. 48: 851–856. Kakooei Z, Mohebali M,Akoundi B, Rahimi Foroshani A (2014) Stability of Freeze- Dried Sera Stored at Different Tem- peratures for the Detection of Anti- Leishmania infantum Antibodies Using Direct Agglutination Test. Iran J Pub- lic Health. 43(11): 1557–1562. Zijlstra E, Ali M, El-Hassan A, El-Toum I, Satti M, Ghalib H, Kager P (1991) Di- rect agglutination test for diagnosis and sero-epidemiological survey of Kala- Azar in the Sudan. Trans R Soc Trop Med Hyg. 85: 474–476.