Background: Visceral leishmaniasis (VL) or kala-azar is a parasitic disease caused by the species of Leishmania donovani complex. Mediterranean type of the disease is endemic in some parts of Iran and more than 95% of cases were reported in children up to 12 years of age. This study was performed to determine the seroprevalence of VL in the rural areas of the Dashti district from Bushehr province.Materials and Methods: In this cross-sectional study, a randomized cluster sampling method was used for the collection of blood samples from children up to 12 years old from rural areas of Dashti district. Before sampling; a questionnaire was filled out for each case. All the collected blood samples were examined after the serum separating by Direct Agglutination Test (DAT) for detection of anti-Leishmania infantum antibodies. The cutoff titers of ≥1: 3200 with specific clinical features were supposed to be considered as VL.Results: Altogether, 24 out of 1221 (1.96%) blood samples showed titers between 1:800 and 1:1600 which considered as suspicious cases. None of the suspicious cases had a history of kala-azar. None of 1221 collected blood samples showed anti Leishmania infantum (L. infantum) at titer ≥1:3200.Conclusion: This study confirms the circulation of L. infantum in Dashti district and highlights the sporadic pattern of VL in the studied areas which necessitates the surveillance system to be monitored by health authorities

Abbaszadeh Afshar, Mohammad javad

Akhoundi, Behnaz

Azargashsb, Eznollah

Gorgipoor, Mohammad

Kazemi, Bahram

Khazaei, Sasan

Khazan, Hooshang

Mohebali, Mehdi

English

Journals Portal, Shahid Beheshti University of Medical Sciences

  Novelty in Biomedicine       NBM 54   Novelty in Biomedicine 2017, 2, 54-8 Original Article   Human Visceral Leishmaniasis: a Serological Survey in Rural Areas of Dashti District of Bushehr Province, Southern Iran   Mohammad Gorgipour1, Mehdi Mohebali2,3, Behnaz Akhoundi2, Mohammad javad Abbaszadeh Afshar2, Bahram Kazemi5, Sasan Khazaei1, Eznollah Azargashsb5, Hooshang Khazan1*  1 Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2 Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3 Center for Research of Endemic Parasites of Iran (CREPI), Tehran University of Medical Sciences, Tehran, Iran 4 Department of Biotechnology, Shahid Beheshti University of Medical Sciences, Tehran, Iran 5 Department of Social Medicine, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran  Received: 09 August, 2016; Accepted:   31 January, 2017 Abstract Background: Visceral leishmaniasis (VL) or kala-azar is a parasitic disease caused by the species of Leishmania donovani complex. Mediterranean type of the disease is endemic in some parts of Iran and more than 95% of cases were reported in children up to 12 years of age. This study was performed to determine the seroprevalence of VL in the rural areas of the Dashti district from Bushehr province. Materials and Methods: In this cross-sectional study, a randomized cluster sampling method was used for the collection of blood samples from children up to 12 years old from rural areas of Dashti district. Before sampling; a questionnaire was filled out for each case. All the collected blood samples were examined after the serum separating by Direct Agglutination Test (DAT) for detection of anti-Leishmania infantum antibodies. The cutoff titers of ≥1: 3200 with specific clinical features were supposed to be considered as VL. Results: Altogether, 24 out of 1221 (1.96%) blood samples showed titers between 1:800 and 1:1600 which considered as suspicious cases. None of the suspicious cases had a history of kala-azar. None of 1221 collected blood samples showed anti Leishmania infantum (L. infantum) at titer ≥1:3200. Conclusion: This study confirms the circulation of L. infantum in Dashti district and highlights the sporadic pattern of VL in the studied areas which necessitates the surveillance system to be monitored by health authorities. Keywords: Visceral leishmaniasis, Direct Agglutination Test, human, Iran  *Corresponding Author: Hooshang Khazan, Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Email: Khazan_h36@yahoo.co.in  Please cite this article as: Gorgipour M, Mohebali M, Akhoundi B, Abbaszadeh Afshar M J, Kazemi B, Khazaei S, Azargashsb E, Khazan H. Human Visceral Leishmaniasis: a Serological Survey in Rural Areas of Dashti District of Bushehr Province, Southern Iran. Novel Biomed. 2017;5(2):54-8.  Introduction Visceral leishmaniasis (VL) so-called kala-azar caused by the species of Leishmania donovani complex is a systemic parasitic disease which is transmitted by Phlebotominae sand flies. Canis familiaris are principal VL reservoir hosts in Mediterranean type of VL1,2. This disease is responsible about 500,000 new cases each year and about 59,000 deaths annually. Approximately, 100-300 new cases of VL are reported in Iran every year2,3. VL is common (over 98%) among children Human Visceral Leishmaniasis: a Serological Survey in Rural Areas of Dashti District …                            Gorgipour et al. NBM 55                      Novelty in Biomedicine 2017, 2, 54-8 under 12 years old in different endemic foci in Iran and adult cases frequently present with subclinical and asymptomatic forms in endemic regions4,5. Kala-azar is characterized by fever, weight loss, mucosal ulcers, fatigue, anemia and substantial hyperplasia of the liver and spleen and even death in most of untreated cases1,6. The first report of VL in Iran was from the Mazandaran province in 19497. At least six endemic foci of VL have been detected in some areas of Ardabil, East Azerbaijan, Fars, Bushehr and recently from Qom and northern Khorasan provinces2,8. Other parts of Iran are considered as sporadic areas for VL. In this study, Direct Agglutination Test (DAT) was used as sero-diagnostic tool since it is a valid, cost effective, sensitive/specific, and user friendly test2,9. From 1991 to 1997, 92 cases of kala azar were diagnosed in Dashti and Dashtestan district of Bushehr province. These districts are near southern part of Fars province, areas known to be endemic foci of kala-azar in Iran10. The current investigation was performed to determine the sero-prevalence of VL in Dashti district of Bushehr province. Methods Study area: Dashti district with 77,530 populations and 50,028 square kilometers has 4 urban centers and 93 villages. This district is situated on the southern slope of the Zagros range of mountains, nearly the south of the Bushehr province (Figure1). The study area has a tropical climate with an average altitude of 65 meters above the sea level (Available at: https://en.wikipedia.org/wi-ki/Jiroft_County). Blood Sampling: This cross-sectional study was carried out as a descriptive survey during March 2014 to February 2015 and a randomized cluster sampling method was used for the sample collection. A questionnaire was completed for each individual to recording demographic characteristics such as age, gender, village, symptoms, history of VL and close contact with the dog. Sixteen villages (cluster) from 93 villages in four geographical zones of Dashti district were randomly selected. Blood samples (0.5-1 ml) were collected from 1221 children up to 12 years old in tubes containing heparin as anticoagulant and processed them 4-10 h after collection. Serum samples were separated by centrifugation at 800 ×g for 5 minutes and stored at −20°C for further serological examination. Direct Agglutination Test: All the collected serum samples were examined by DAT. In order to determine the Leishmania-specific antibodies titers a procedure described by Harith et al, was used11. Initially, for screening purposes, two dilutions of 1:800 and 1:3200 were provided and tested. The samples that were positive with titers of 1:800 were diluted up to 1:102400 in a V-shaped micro-titer plate into a dilution fluid containing 0.9% saline and 0.78% 2-mercaptoethanol. An equal volume (50 µl) of antigen suspension was added to each well. The results were read after 18-24 hours' incubation in a wet room at room temperature. The highest dilution at which agglutination was still visible in comparison with positive and negative control titers was defined as the sample titer. Compact blue dots were scored as negative and large diffuse blue mats as positive. The known negative and positive controls were tested in each plate daily. Titers of ≥1: 3200 were considered as seropositive cases8, 12. The cutoff was based on previous studies4,11,13-15. DAT Leishmania antigen was prepared in the protozoology unit of the School of Public Health, Tehran University of Medical Sciences.  Statistical analysis: Chi-squared and Fisher exact tests were used to compare Sero-prevalence values relative to gender and age groups. Analyses were performed with SPSS (version 18), with a probability p value of <0.05 considered as statistically significant. Results Totally, 1221 blood samples collected from children up to 12 years old by Dashti district from Bushehr province [634 (52%) for males and 587 (48%) from females]. After screening, 24 cases (1.96%) were shown L. infantum specific antibodies at titers 1:800. Five (0.4%) out of 24 cases were diagnosed as suspicious cases with titers of 1:1600 (Table 1). Also, none of the evaluated subjects were found to be DAT positive (at titers ≥1: 3200). There was no significant difference between male and female at 1:800 and 1: 1600 titers of anti-Leishmania specific antibodies (P>0.05). Although, the number of human cases with 1:800 and 1:1600 titers in males were higher than females. Frequency of anti-Leishmania antibody titers Gorgipour et al.                              Human Visceral Leishmaniasis: a Serological Survey in Rural Areas of Dashti District … NBM                                                                            56                                       Novelty in Biomedicine 2017, 2, 54-8 with DAT according to the age groups is indicated in Table 1. Age and gender distribution of samples are shown in Table (2). The highest percentage of seropositivity was observed in age groups of 9-12 years old (3%). Meanwhile, re-sampling of all suspicious cases and testing they did not show   Figure 1. The map showing location of Dashti district as a subpart of Bushehr province in Iran used as target population of the study.  Table 1: Seroprevalence of human visceral Leishmaniasis by Direct Agglutination Test with anti-Leishmania infantum antibodies based on gender and age groups in Dashti district, 2014. parameters No. of sera        Antibody Titer              Total          1:800         1:1600     No. of sera %   No. of sera % No.of sera % Gender          Male 634 13 2   5 0.8 18 2.8 Female 587 6 1   0 0 6 1 Age (yr)          ≤ 4 364 6 1.6   1 0.3 7 1.92 5-8 462 4 0.8   1 0.2 5 1 9-12 395 9 2.3   3 0.7 12 3 Total 1221 19 1.6   5 0.4 24 1.96  Table 2: Distribution of target population for detection of seroprevalence of human visceral leishmaniasis according to gender and age groups in Dashti district, 2014. Age (yr)             Male           Female              Total  No. of   sera % No. of   sera % No. of   sera % ≤ 4 191 52.4 173 47.6 364 30 5-8 254 55 208 45 462 38 9-12 189 48 206 52 395 32 Total 634 52 587 48 1221 100  Human Visceral Leishmaniasis: a Serological Survey in Rural Areas of Dashti District …                            Gorgipour et al. NBM 57                      Novelty in Biomedicine 2017, 2, 54-8 increased titers of L. infantum specific antibodies. None suspicious cases had clinical signs. Discussion Bushehr province is one of the endemic parts of visceral leishmaniasis in Iran12. In this province a high number of cases of kala-azar were recorded during previous decades. From 1991 to 1997, 92 cases of kala-azar were diagnosed in the Dashti and Dashtestan districts in Bushehr. These districts are near south of Fars province, areas known to be endemic foci of kala-azar in Iran10. Since no serological study had been performed in last decade in this province, the present study was carried out to evaluate the epidemiological aspects of VL in this area. Because of its highly sensitive and specific results for detection of L. infantum infection in humans, DAT analysis was applied as a main tool of this survey8.  The sensitivity and specificity of this method varies in different studies between 90–100% and 72–100% respectively9. Of the 1221 samples, 24 cases (1.96%) showed titers between 1: 800 and 1: 1600 by DAT, although none of those were found DAT positive (at titers ≥1:3200). Most cases that showed titers between 1: 800 and 1: 1600 were detected in children of 9-12 years old (3%). Based on our results, currently, VL occurs with sporadic pattern in Dashti district of Bushehr province. However, according to a previous study made by Mohebali et al, in 2001, Dashti and Dashtastan were reported as endemic areas of kala-azar where They found 3.4% of sero-positive cases in Dashti and Dashtestan districts10. These differences seem to be associated with a proper control of the disease over the years by health authorities of the strict or differences in method of sampling, number of population and the presence or absence of clinical symptoms. In 2006, Asgari et al. found 1.86% of sero-positivity in Qashqai tribes in south of Fars province. These people travel with their flocks each year from the summer highland pastures “Yailaq” in south of Fars province to winter quarters, on lower (and warmer) lands “Qishlaq” in Dashti and Dashtestan districts in Bushehr province. It seems that L.infantum infection among people of Dashti district is in association with the presence of Qashqai tribes in this district in part of year. In Qom province, Fakhar et al. found 1.7% of sero-positivity in 8 villages of Ghahan in which three over seven seropositive cases had a previous history of VL16. In our study None of the cases with 1: 800 and 1: 1600 titers of anti-Leishmania-specific antibodies presented a history of kala-azar. Conclusion This study confirms the circulation of L. infantum in Dashti district and highlights the sporadic pattern of VL in the studied areas. Further investigations regarding the sand file's fauna and wild canines as reservoir hosts of the disease are recommended. Acknowledgment The authors thank Protozoology Unit of the School of Public Health and Institute of Public Health research, Tehran University of Medical Sciences for their helps in providing laboratory facilities for preparation of DAT antigens. We thank honorable colleagues and staff of  Bushehr University of Medical Sciences and all staff at the health care centres in Bushehr Province for their cooperation in selecting study subjects and providing samples. References 1. Organization WH. International travel and health: situation as on 1 January 2010: World Health Organization; 2010. 2. Mohebali M. Visceral leishmaniasis in Iran: review of the epidemiological and clinical features. Iranian journal of parasitology. 2013;8(3):348. 3. Alvar J, Velez ID, Bern C, et al. Leishmaniasis worldwide and global estimates of its incidence. PloS one. 2012;7(5):e35671. 4. Mohebali M, Hajjaran H, Hamzavi Y. Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran. Veterinary parasitology. 2005;129(3):243-51. 5. EDRISIAN GH, Nadim A, Alborzi A, AREDEHALI S. Visceral leishmaniasis; the Iranian experience. 1998. 6. Ready PD. Epidemiology of visceral leishmaniasis. Clin Epidemiol. 2014;6(6):147-54. 7. Mahmoudvand H, Mohebali M, Sharifi I, et al. Epidemiological aspects of visceral leishmaniasis in Baft district, Kerman Province, Southeast of Iran. Iranian journal of parasitology. 2011;6(1):1. 8. Mohebali M, Edrissian G, Nadim A, et al. Application of direct agglutination test (DAT) for the diagnosis and seroepide-miological studies of visceral leishmaniasis in Iran. Iranian journal of parasitology. 2006;1(1):15-25. 9. Werneck GL. Visceral leishmaniasis in Brazil: rationale and concerns related to reservoir control. Revista de saude publica. 2014;48(5):851-6. 10. Mohebali M, Hamzavi Y, Edrissian GH, Forouzani A. Seroepidemiological study of visceral leishmaniasis among humans and animal reservoirs in Bushehr province, Islamic Republic of Iran. 2001. Gorgipour et al.                              Human Visceral Leishmaniasis: a Serological Survey in Rural Areas of Dashti District … NBM                                                                            58                                       Novelty in Biomedicine 2017, 2, 54-8 11. el Harith A, Slappendel R, Reiter I, et al. Application of a direct agglutination test for detection of specific anti-Leishmania antibodies in the canine reservoir. Journal of clinical microbiology. 1989;27(10):2252-7. 12. Mohebali M, Edrissian GH, Shirzadi MR, et al. An observational study on the current distribution of visceral leishmaniasis in different geographical zones of Iran and implication to health policy. Travel medicine and infectious disease. 2011;9(2):67-74. 13. Abbaszadeh-Afshar M, Mohebali M, Sharifi I, et al. Seroepidemiological survey of Visceral leishmaniasis among nomadic tribes of Kerman Province, Southeastern Iran: An observational study for implication to health policy. Journal of Biostatistics and Epidemiology. 2015;1(2):105-11. 14. Harith A, Kolk A, Kager P, et al. A simple and economical direct agglutination test for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1986;80(4):583-6. 15. Soleimanzadeh G, Edrissian GH, Movahhed-Danesh A, Nadim A. Epidemiological aspects of kala-azar in Meshkin-Shahr, Iran: human infection. Bulletin of the world health organization. 1993;71(6):759. 16. Fakhar M, Mohebali M, Barani M. Identification of Endemic Focus of Kala ـ azar and Seroepidemiologcial Study of Visceral Leishmania Infection in Human and Canine in Qom Province, Iran. Armaghane danesh. 2004;9(1):43-52.    

Human Visceral Leishmaniasis: a Serological Survey in Rural Areas of Dashti District of Bushehr Province, Southern Iran

Background: Visceral leishmaniasis (VL) or kala-azar is a parasitic disease caused by the species of Leishmania donovani complex. Mediterranean type of the disease is endemic in some parts of Iran and more than 95% of cases were reported in children up to 12 years of age. This study was performed to determine the seroprevalence of VL in the rural areas of the Dashti district from Bushehr province.Materials and Methods: In this cross-sectional study, a randomized cluster sampling method was used for the collection of blood samples from children up to 12 years old from rural areas of Dashti district. Before sampling; a questionnaire was filled out for each case. All the collected blood samples were examined after the serum separating by Direct Agglutination Test (DAT) for detection of anti-Leishmania infantum antibodies. The cutoff titers of ≥1: 3200 with specific clinical features were supposed to be considered as VL.Results: Altogether, 24 out of 1221 (1.96%) blood samples showed titers between 1:800 and 1:1600 which considered as suspicious cases. None of the suspicious cases had a history of kala-azar. None of 1221 collected blood samples showed anti Leishmania infantum (L. infantum) at titer ≥1:3200.Conclusion: This study confirms the circulation of L. infantum in Dashti district and highlights the sporadic pattern of VL in the studied areas which necessitates the surveillance system to be monitored by health authorities.</p

Human Visceral Leishmaniasis: a Serological Survey in Rural Areas of Dashti District of Bushehr Province, Southern Iran

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