Visceral leishmaniasis among children in an endemic area of northwestern Iran between 2016 and 2017: An epidemiological study

Objective: To clarify the epidemiological aspects of visceral leishmaniasis in Kaleybar and Khoda-Afarin districts, north-west of Iran. Methods: A total of 1 420 human (children under 12 years) samples, 101 domestic dogs samples (Canis familiaris), and 577 female sand fly samples were collected. Sera of human and dogs were tested using the direct agglutination test, and sand flies were identified at species level using the microscopic method. Furthermore, a structured questionnaire was applied to evaluate the correlation between the potential risk factors and the related clinical signs/ symptoms with the human and dogs' seropositivity. Results: Totally, 2.18% of human samples were positive at titers≥: 800; among them, 13 cases (41.94%) were above 1:3 200, and clinical symptoms were observed in all of them except for an 11-year old girl. Anti-Leishmania infantum antibodies were found at titers ≥1: 320 in 9.90% of dogs' samples, half of them had at least one sign of canine visceral leishmaniasis. Moreover, 10 Phlebotomus species were identified in the study areas, and Phlebotomus (Larroussius) major group was the predominant species. There are significant correlations between the presence of anti-Leishmania infantum antibodies and the fever (P<0.001), anemia (P=0.001) and weight loss (P=0.016) in children. On the other hand, significant correlations were revealed between the Leishmania infection and the shelter (P=0.039), cutaneous lesion (P=0.005), lymphadenopathy (P=0.001) and weight loss (P<0.001) in the infected dogs. Conclusions: Visceral Leishmania infection is prevalent in rural areas of Kaleybar and Khoda- Afar districts located in East-Azerbaijan province, therefore active detection and treatment of visceral leishmaniasis cases should not be neglected.

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

Seroprevalence survey of viscereal leishmaniasis among children up to 12 years old and domestic dogs in rural areas of Dehloran district, Ilam province of west part of Iran, 2014

Background: Visceral leishmaniasis [VL], caused by Leishmania infantum [L. infantum], is a life-threatening vector-borne parasitic disease is distributed in some parts of the world. The disease is endemic in some parts of Iran. This study was aimed to determine the seroprevalence of VL among children and domestic dogs [as a reservoir of the parasite] in Dehloran, west of Iran

Materials and Methods: This cross-sectional study was carried out in Dehloran County. The blood samples of 872 children up to 12 years old and 52 dogs were collected from 10 villages of Dehloran using randomly-clustered sampling method. Sera were separated from all peripheral blood samples and tested by direct agglutination test [DAT]. Anti-Leishmania infantum antibodies at titers of >/=1:800 and >/=1:80 were considered as Leishmania infantum infection in human and dog, respectively

Results: In general, among 872 human samples, 1.03% of samples had anti-Leishmania antibody with 1:1600 titers and 1.26% had 1:800 titers. In addition, from 52 dog samples, 21.15% of dogs had a titer of >/=1:320 and 25% had 1:80 and 1:160 titers

Conclusion: Our findings indicate that the seropositive dogs in the studied areas are considerable and L. infantum may be circulated between human and domestic dog in the studied area. Further study of isolation and molecular identification of Leishmania spp. is recommended

Visceral Leishmaniasis in Rural Areas of Alborz Province of Iran and Implication to Health Policy

Visceral leishmaniasis (VL) or kala-azar mainly affects children in endemic areas. This study was conducted to determine the seroprevalence of VL using direct agglutination test (DAT) in children living in rural districts of Alborz Province located 30 km from Tehran capital city of Iran. Multi-stage cluster random sampling was applied. Blood samples were randomly collected from 1,007 children under 10 years of age in the clusters. A total of 37 (3.7%) of the studied population showed anti-Leishmania infantum antibodies with titers of > or =1:800. There was a significant association between positive sera and various parts of the rural areas of Alborz Province (P or =1:3,200 indicated kala-azar clinical features and treated with anti-leishmaniasis drugs in pediatric hospital. The findings of this study indicated that Leishmania infection is prevalent in rural areas of Alborz Province. Therefore, it is necessary to increase the awareness and alertness among physicians and public health managers, particularly in high-risk rural areas of the province in Iran.

[Sero-prevalence study of visceral leishmaniasis using Direct Agglutination Test (DAT) in children up to 12 years old in Delphan City, Lorestan Province: introduce a new focus of VL In Iran]

Background and Objectives: Two clinical forms of leishmaniasis exist in Iran: cutaneous and visceral. According to the sporadic reports of new cases of Visceral Leishmaniasis [VL] in Lorestan province, real status of VL is not clear, so this study aimed to describe the seroprevalence of VL in Delphan city

Materials and Methods: In this descriptive analytic study, blood samples were collected from children /= 1/3200 accompanied with clinical symptoms was considered as VL diseas

Results: 800 collected serum samples, 21[2.62%] showed anti-Leishmania antibodies at titers of 1/800 and 1/1600, whereas 5[0.62%] showed anti-Leishmania antibodies at titers of ?1/3200. But just one of them showed clinical symptoms [anemia and large abdominal] which is under treatment with miltefosine

Conclusion: A new focus of VL with low endemicity is going to be formed in our region, which showed that further studies on vector and reservoirs is necessary in the region and other parts of Lorestan province

Evaluation of a novel herbal immunomodulator drug [IMOD] in treatment of experimental canine visceral leishmaniasis

Toxicity and drug resistance against pentavalent antimonials, medications of choice in treatment of leishmaniasis for more than 5 decades, have become important subjects globally. This study was a randomized, open labeled trial that was designed to determine efficacy and safety of IMOD as a novel herbal immunomodulator drug for treatment of canine visceral leishmaniasis [CVL]. Twenty healthy mongrel dogs were infected with Iranian strain of L. Infantum amastigotes and randomly divided to 5 groups with four animals for each included on: I: negative control [non-infected] II: Glucantime[registered sign] III: Glucantime[registered sign] plus IMOD [immune-chemotherapy] IV: IMOD and V: positive control [non-treated]. Physical examination, hematological, biochemical, serological, parasitological, pathological and imaging evaluations were performed pre-/post- interventions every month for 3 months. Comparing with control groups [IandV], immune-chemotherapy group [Glucantime[registered sign] plus IMOD] showed significantly higher efficacy in resolving the clinical signs and hematobiochemistry factors. Based on our results, using IMOD in combination with meglumine antimoniate [Glucantime[registered sign]] has significantly improved CVL than the latter drug alone. So, it seems this new herbal medicine is useful as adjuvant therapy for canine visceral leishmaniasis

Assessment of the effects of a novel herbal immunomodulator drug [IMOD] on cytokine profiles in experimental canine visceral leishmaniasis: a preliminary survey

Cytokines play a fundamental role in the regulation of immune responses in remission and/or relapsing of leishmaniasis. Therefore, immunotherapy for the treatment of canine visceral leishmaniasis [CVL] has represented a principle approach in control of the infection. The present research aimed to evaluating the immunotherapeutic potential of a novel herbal immunomodulator drug [IMOD] on CVL. Twelve mongrel dogs were intravenously infected with Iranian strain of L. infantum and randomly divided into three groups; 1: negative control [non-infected], 2: immunotherapy with IMOD and 3: positive control [non-treated]. Cell proliferation and Th1-/Th2-type cytokines were measured in peripheral blood mononuclear cell [PBMC] by cell proliferation kit I [MTT] and enzyme-linked immunospot [ELISpot] assays, respectively. At the 60 days follow-up assessment, no adverse effects were observed in treated interventional group. Cellular proliferation assay indicated that PBMCs of IMOD group had higher stimulation index [SI] than positive control group [p <0.05]. Enhancement of CD4+ T cells such as IL-2, IL-4 and IL-10 were detected in negative control group due to in vitro IMOD stimulation 30 days post-treatment. In accordance to decreasing trends of Th1 and Th2 cytokines in positive control group, the mean number of IFN-gamma, IL-2, IL-4 and IL-10 spot forming cells [SFCs] down regulated for IMOD group during the study. These data indicate that IMOD had immunomodulatory potential but is not sufficient for total parasitic cure due to balance of Th1/Th2 cytokines. This is a preliminary study and we propose to undertake a series of experiments to evaluate the CVL due to in vitro modulatory effects of IMOD

Stability of freeze-dried sera stored at different temperatures for the detection of anti-leishmania infantum antibodies using direct agglutination test

This study aimed to evaluate freeze-dried sera as an alternative to non-freeze dried for detection of anti- Leishmania infantum antibodies over the course of 11 months using the direct agglutination test [DAT]. Altogether, 60 serum samples [30 from humans and 30 from dogs] were collected from various geographical locations in Iran. All the collected sera were pooled and each pooled serum sample contained 10 different sera. In the beginning, the human and dog pooled sera were categorized as positive [weak and strong] and negative based on anti-L. infantum antibodies using the DAT. All the freeze-dried and non-freeze-dried sera were stored at -70[degree sign] C, -20[degree sign] C, 4[degree sign] C, 22-28[degree sign] C and 56[degree sign] C for 11 months. The positive and negative human and dog pooled sera were separately tested using the DAT each month and the results were compared to non-freeze-dried sera kept under the same conditions. We found strong agreement [100%] between the results obtained from freeze-dried human and dog in strong DAT positive sera kept at -70[degree sign] C, -20[degree sign] C, 4[degree sign] C and 22-28[degree sign] C during this study. The human and dog pooled sera stored at 56[degree sign] C were corrupted after 2 weeks. The DAT results were highly reproducible using freeze-dried human pooled sera in the beginning and month 11 of this study [CV = 0.036]. Freeze-dried human and dog strong DAT positive sera are highly stable under different temperature conditions, are easy to transport and are safe for use as positive and negative serum controls in laboratories

Preparation and evaluation of a glycerol-preserved direct agglutination antigen for long-term preservation: a comparative study of the detection of anti-Leishmania infantum antibodies in human and dog

OBJECTIVE@#To prepare and evaluate a glycerol-preserved antigen from an Iranian strain of Leishmania infantum (L. infantum) for use in glycerol-preserved direct agglutination tests (GP-DAT) as an alternative to freeze dried direct agglutination tests (FD-DAT) that use freeze-dried antigen.@*METHODS@#Glycerol-preserved DAT antigen was prepared and stored at different temperatures. We tested antigen stored at 4 °C, 22-37 °C and 50 °C over a period of 365 days. Seven hundred twenty-nine serum samples were collected from different geographical zones of Iran from 2007-2009, and 80 of these samples were pooled to produce sera. Each pooled serum contained 10 sera. All positive and negative pooled sera were separately tested for anti-L. infantum antibodies with GP-DAT, FD-DAT and formaldehyde-fixed direct agglutination test (FF-DAT) antigens; tests were performed on both human and dog sera over a period of 12 months.@*RESULTS@#There was strong agreement between the results obtained using GP-DAT and FD-DAT antigens stored at 22-37 °C for 12 months for both human (100%) and dog (100%) pooled sera. The direct agglutination test results were highly reproducible (weighted kappa: GP=0.833, FD=0.979 and FF=0.917).@*CONCLUSIONS@#Because GP-DAT antigen is highly stable over a range of temperatures and is easy to transport in the field, this type of antigen may be particularly useful in areas with endemic visceral leishmaniasis.

Comparison of leishmanin skin test and direct smear for the diagnosis of cutaneous leishmaniasis

Cutaneous leishmaniasis [CL] is an endemic disease in some parts of Iran and it has high morbidity in some areas of the country. The disease is detected by parasitological examinations including direct microscopic and culture tests. This comparative study aimed to evaluate the relationship between positivity of the leishmanin skin test [LST], microscopically examination and clinical forms of CL for the diagnosis of human cutaneous leishmaniasis. This study was performed on 66 patients suspected to cutaneous leishmaniasis. CL cases evaluated by both microscopical examination and leishmanin skin test. In this study, 1 ml of leishmanin fluid [lot no 121/1, produced in Pasteur institute of Iran] was injected intradermally in forearms of all patients and indurations were measured after 72 hours. Induration of 5 mm and higher was considered as positive results. The collected data were statistically analyzed using the SPSS version 13.5. From 66 CL patients who were evaluated in this study, 30 [45.5%] of them had positive microscopically results while 28 [42/4%] of them had showed positive leishmanin skin test [>/=5mm diameter]. From 36 [54.5%] patients who had negative microscopical examination, only 6 [16/6%] of them had positive leishmanin skin test. The agreement between two tests was 87.9% by kappa analysis [p< 0.01]. In attention to the results of this study, it seems the LST would be used as an alternative diagnosis method when there is a strong clinical doubt to cutaneous leishmaniasis even there is no parasite in direct smear