Visceral Leishmaniasis in Iran: An Update on Epidemiological Features from 2013 to 2022.

Background: Visceral leishmaniasis (VL) is one of the most important neglected tropical diseases. The zoonotic form of VL is endemic in some areas of Iran. We aimed to determine the status of VL identified in humans and canines in different parts of Iran from 2013 to 2022. Method: A national representative cross-sectional study was conducted in 10 provinces of Iran, including the national leishmaniasis reference lab. We employed the direct agglutination test (DAT) as a reliable serological method to detect anti-Leishmania infantum antibodies in humans and animal reservoir hosts. Additionally, a narrative literature review was conducted to identify relevant studies on VL seroprevalence in Iran from 2013 to 2023. Results: The results of 21281 human and 5610 canine serum samples from 2013 to 2022 are reported. Altogether, 448 (2.1%, 95%CI: 2.0-2.3) human serum samples showed anti-L. infantum antibody levels of ≥1:3200. Of these samples, 13716 (64.5%) were collected actively, which showed a seroprevalence of 0.6% (95% CI: 0.5-0.8) and 7565 (35.5%) were collected passively, which showed a seroprevalence of 4.8% (95%CI: 4.3-5.3). Overall, 1035 (20.1%, 95%CI: 19.0-21.2) of 5160 domestic dogs (Canis familiaris) samples showed anti-L. infantum antibody levels of ≥1:320. Northwest (2.8%) and northeast (0.96%) regions had the highest human VL seroprevalence, while northwest (21.5%) and south (14.4%) regions had the highest canine VL seroprevalence. Conclusion: Zoonotic VL, an endemic parasitic disease, is still present in several different distinct areas across Iran. While human VL cases have shown a declining trend over the last decade, the prevalence of canine VL remains significant.

Allelic Variations of Plasmodium vivax Apical Membrane Antigen-1 (Pv AMA-1) in Malarious Areas of Southeastern Iran Using PCR-RFLP Technique.

BACKGROUND: Although Plasmodium vivax is usually known as benign malaria, some variations of the parasite can result in acute and sever infection. In this study we tried to determine some genetic variations in PvAMA-1 antigen among the samples were collected form southeastern Iran. METHODS: About two ml blood samples were collected into EDTA pre-dosed tubes from 30 P. vivax-infected patients individually between 2011 and 2013. A Giemsa stained thick and thin blood film was prepared from each of the patients. A PCR-RFLP technique was employed using EcoR-1, Pvu-II and Hind3 restriction enzymes to determine the allelic variations of the antigen. RESULTS: A 1300bp gene corresponding to PvAMA-1 was selected for the amplification process. Among the total cases identified in this study 90% showed similar bounds when exposed to the restriction enzymes. Nine isolates (accession numbers: KF435081-KF435083 and JF682785-JF682790) were identified and registered in Gene bank. Identity among isolates was more than 96% in nucleotide level. Dendrogram clarified a close relationship among the clusters in spite of geographical distribution of the parasite. CONCLUSION: This study increased our data about prevalence and variation of PvAMA-1 alleles amongst P. vivax isolates in southeastern parts of Iran where besides native population bears considerable Afghan and Pakistani immigrants.

History of Medical Parasitology and Parasitic Infections in Iran.

Parasites and parasitic diseases have been prevalent in Iran according to Iranian ancient scholars and physicians' inscriptions dating back to 865-1496. Some protozoan diseases such as malaria and cutaneous leishmaniasis have been introduced by clinical manifestations and helminthic infections by size and morphology of the worms. Scientific studies of Parasitology started in Iran from 1833, first by foreign physicians and continued from 1909 by Iranian researchers. The pioneer medical parasitologists of Iran were Dr N. Ansari and Dr. Sh. Mofidi who established the Department of Medical Parasitology in the School of Medicine, University of Tehran, 1939. Afterward, a considerable number of researchers and professors of parasitology have been active in training and research works in the fields of medical parasitology throughout the entire nation. At present, some significant parasitic diseases such as bilharsiasis and dracunculiasis are more or less eradicated and malaria is in the elimination phase. The prevalence of most helminthic infections has considerably decreased. Most of the departments of medical Parasitology in Iran are active in training MD, MSPH and PhD students. The Iranian Society of Parasitology established in 1994 is active with many eligible members and its creditable publication, the Iranian Journal of Parasitology, published seasonally since 2006. From 1833, when the scientific studies of Parasitology have started in Iran up to 2013, many researchers have been done on various fields of medical Parasitology and parasitic diseases in Iran and 2517 papers in English and 1890 papers in Persian have been published in national and international scientific journals. In addition, more than 420 books related in the field of medical parasitology field have been published in Persian language.

Diagnosing Malaria Cases Referred to the Malaria Reference Laboratory in Tehran University of Medical Science, Iran.

BACKGROUND: The number of malaria cases is declining worldwide; however, it remains as a serious health problem. Diagnosing unusual cases is the most important issue to manage the problem. This study designed to describe the number of falciparum and vivax malaria infected patients referred to Malaria Reference Laboratory in Tehran University of Medical Science from 2000 to 2012. METHODS: A retrospective study was conducted based on the collected questionnaires from each patient referred to the laboratory. Diagnosing results and demographic information for positive cases were analyzed using SPSS software. Problematic cases were evaluated for any difficulties in diagnosis or in clinical signs. Scanning and molecular methods were performed whenever there was an atypical case referred to the laboratory. Some of the samples had various difficulties for diagnosing such as presence of fussed gametocytes and schizonts of Plasmodium falciparum in peripheral blood and CCHF like hemoragic disorders. RESULTS: Plasmodium vivax caused a large proportion of the cases (76.1%) in contrast with P. falciparum that included smaller proportion (22.8%) and the rest (1.1) belonged to mixed infection. Most of the positive cases (69.6%) were belonged to Afghani people. Men (94.6%) showed more infection than women (5.4%), moreover the most infection (44.5%) was seen at a range of 21-30 yr. CONCLUSION: In the case of existing atypical issues to diagnose, it is needed to perform more precise microscopical examination beyond the current standard conditions. Sometimes molecular method is required to verify the exact agent of the disease.

Downregulation of Calcineurin Gene Is Associated with Glucantime(®) Resiatance in Leishmania infantum.

BACKGROUND: Pentavalent antimonials are the first line drugs for the treatment of leishmaniasis. Unresponsiveness of Leishmania spp. to antimonial drugs is a serious problem in some endemic areas. Investigations on molecular mechanisms involved in drug resistance are essential for monitoring and managing of the disease. Cal-cineurin is an essential protein phosphatase for number of signal transduction pathways in eukaryotic cells and it has a mediated role in apoptosis. This study aimed to determine of biomarker(s) in Glucantime(®) resiatance strain of L. infan-tum. METHODS: We used cDNA amplified fragment length polymorphism (cDNA-AFLP) and real time-RT PCR assays to compare gene expression profiles at the mRNA levels in resistant and susceptible L. infantum field isolates. RESULTS: The cDNA-AFLP results showed downlegulation of calcineurin in resis-tant isolate in comparison with susceptible one. Significant downregulation of cal-cineurin (0.42 fold) (P<0.05) was found in resistant isolate compared to susceptible one by Real time-RT PCR. CONCLUSION: This is the first report of calcineurin implication in Glucantime(®) drug resistance of field (natural) isolate of L. infantum. Downregulation of calcineurin could protect parasites from antimonial-induced apoptosis.

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.

Rapid detection of human Leishmania infantum infection: a comparative field study using the fast agglutination screening test and the direct agglutination test.

This study aimed to evaluate the performance of a fast agglutination screening test (FAST) for serodiagnosis of human Leishmania infantum infection in Iran. FAST is based on the direct agglutination test (DAT) but combines with a higher parasite concentration and is performed with only one serum dilution. The validity of FAST for the detection of L. infantum infection in the field was compared with the direct agglutination test on 110 confirmed or patients suspected of infection with leishmaniasis, 177 healthy individuals and 41 patients with other infectious diseases who were from northwestern and southern parts of Iran. In this study, we found a 1:1600 cut-off point empirically by seeking the best correlation (90.8) between sera confirmed with visceral leishmaniasis and healthy control sera. A sensitivity of 95.4% (95% CI, 91.4-99.4) and specificity of 88.5% (95% CI, 84.2-92.8) were found with 1:1600 as a cut-off titer when DAT-confirmed cases were compared with the control groups. A good degree of agreement was found between FAST and DAT (90.8%) by Kappa analysis. FAST requires 2 h for reading the results versus the 12-18 h needed for DAT. As FAST is simple, rapid, sensitive and non-invasive and does not require a higher volume of antigens or much expertise, it can be used for screening and serodiagnosis of human L. infantum infection.

Canine visceral leishmaniasis: asymptomatic infected dogs as a source of L. infantum infection.

Clinically infected dogs have been identified as the main reservoir hosts of visceral leishmaniasis (VL) caused by Leishmania infantum in the Mediterranean region. The objective of this study was to determine the potential of asymptomatic infected dogs compared with symptomatic ones as a source of L. infantum infection to golden hamster. For this purpose, anti-Leishmania antibodies were detected with direct agglutination test (DAT) in 13 symptomatic (7 seropositive =>or=1:320) and 53 asymptomatic (9 seropositive =>or=1:320 and 44 seronegative =<1:320) ownership dogs. DNA of Leishmania sp. was extracted from skin and peripheral blood tissues of each dog and tested by PCR. Sixty-six Syrian golden hamsters (Mesocricetus auratus) were used for the determination of infectivity and pathogenicity of L. infantum, isolated from the dogs. We used the internal transcribed spacer 2 (ITS 2) rDNA sequence analysis. The results showed that 22 and 11 out of 66 inoculated golden hamsters were positive by PCR and parasitological examinations, respectively. From 22 PCR positive hamsters, 17 were related to asymptomatic dogs and 5 were from symptomatic ones. There was no significant difference between symptomatic and asymptomatic dogs in producing Leishmania infection in the susceptible animal model (P=0.66). Smears and cultures of 5 dogs from 13 symptomatic dogs (38.5%) and 6 dogs from 53 asymptomatic ones (11.3%) were found to be positive at parasitological examination. All the L. infantum isolates from symptomatic and asymptomatic dogs were similar in sequencing. In conclusion, asymptomatic infected dogs as well as symptomatic ones can harbor L. infantum in their blood and skins which are virulent and infectious for inoculated golden hamster.