Unresponsiveness to Glucantime Treatment in Iranian Cutaneous Leishmaniasis due to Drug-Resistant Leishmania tropica Parasites

BACKGROUND: Recent circumstantial evidence suggests that an increasing number of Iranian patients with cutaneous leishmaniasis are unresponsive to meglumine antimoniate (Glucantime), the first line of treatment in Iran. This study was designed to determine whether the clinical responses (healing, or non-healing) were correlated with the susceptibility of Leishmania parasites to Glucantime. METHODS AND FINDINGS: In vitro susceptibility testing was first performed on 185 isolated parasites in the intracellular mouse peritoneal macrophage model. A strong correlation between the clinical outcome and the in vitro effective concentration 50% (EC (50)) values was observed. Parasites derived from patients with non-healing lesions had EC (50) values at least 4-fold higher than parasites derived from lesions of healing patients. A selection of these strains was typed at the molecular level by pulsed-field gels and by sequencing the pteridine reductase 1 (PTR1) gene. These techniques indicated that 28 out of 31 selected strains were Leishmania tropica and that three were Leishmania major. The L. major isolates were part of a distinct pulsed-field group, and the L. tropica isolates could be classified in three related additional pulsed-field groups. For each pulsed-field karyotype, we selected sensitive and resistant parasites in which we transfected the firefly luciferase marker to assess further the in vitro susceptibility of field isolates in the monocyte cell line THP1. These determinations confirmed unequivocally that patients with non-healing lesions were infected with L. tropica parasites resistant to Glucantime. Additional characterization of the resistant isolates showed that resistance is stable and can be reversed by buthionine sulfoximine, an inhibitor of glutathione biosynthesis. CONCLUSIONS: To the authors' knowledge, this is the first report of proven resistant parasites contributing to treatment failure for cutaneous leishmaniasis and shows that primary Glucantime-resistant L. tropica field isolates are now frequent in Iran

Treatment Failure in Cutaneous Leishmaniasis Patients Referred to the School of Public Health, Tehran University of Medical Sciences During 2008-2017

Background: Cutaneous leishmaniasis (CL) is a vector borne disease predominantly found in tropical and subtropical countries, including Iran. For more than 6 decades, pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis, but over the past few years, clinical resistance to these medications has increased. In this study, we evaluated CL patients who did not show any desirable responses to the anti-leishmanial treatment within a 10-year period (2008 to 2017). Methods: All patients from different parts of Iran suspected of having cutaneous leishmaniasis, who were referred to the laboratory of leishmaniosis in Tehran University of Medical Sciences from 2008-2017 were parasitological examined. Results: During this period, a total of 1480 suspected CL patients were referred to the laboratory of leishmaniosis. Samples from 655 patients (70.8) suspected of having CL were positive microscopically. The failure rate in patients treated with anti-leishmaniasis medications for a minimum of three complete treatment periods was 1.83 (12 cases). There was no association between the number and size of skin lesions and patient characteristics. Also, the route of drug administration had no significant effect on the number and size of lesions. Conclusion: In the present study, treatment failure was found in some confirmed CL patients treated with meglumine antimoniate. Over the past few years, it seems that had been increased in resistance to these medications. So, a review of the correct implementation of the treatment protocol and/or a combination therapy may be helpful in preventing an increase in the rate of treatment failure

Disseminated Leishmaniasis Caused by Leishmania tropica in a Puppy from Karaj, Central Iran

A 5-month old puppy with muco-cutaneous lesions in the chin, around lips and eyes was examined physically and microscopically for leishmaniasis. Muco-cutaneous lesions containing a large number of amastigotes of Leishmania spp. were observed. Amastigotes were also detected in liver and spleen of the puppy. The animal was positive with Dipstick rK39 kit and high level of anti-Leishmania antibodies was detected by direct agglutination test (DAT). DNA, Using PCR-RFLP technique extracted from cultured Leishmania promastigotes and L. tropica was identified. This is the first report of concurrent mucosal and visceral involvement of L. tropica in a puppy from Iran

Epidemiological Aspects of Visceral Leishmaniasis in Baft District, Kerman Province, Southeast of Iran

Background: Visceral leishmaniasis (kala-azar) is an endemic disease in some areas of Iran. A cross- sectional study was conducted for sero-epidemiological survey of visceral leishmaniasis (VL) in Baft district from Kerman Province, southeast of Iran.Methods: Blood samples were collected from children up to 12 years old and 10% of adult population from Baft villages with a multi-stage randomized cluster sampling. In addition, blood samples were collected from 30 domestic dogs from the same areas. All the collected blood sam­ples were tested by direct agglutination test (DAT) for the detection of anti-Leishmania antibod­ies in both human and dog using the cut-off value of ≥1:3200 and ≥ 1:320, respectively. Parasitologi­cal, molecular, and pathological were performed on infected dogs. Chi-square and Fisher exact tests were used to compare sero-prevalence values.Results: From 1476 collected human serum samples, 23 (1.55%) showed anti-Leishmania antibod­ies at titers of 1:800 and 1:1600 whereas 14 (0.95%) showed anti-Leishmania infantum antibodies at titers of ≤ 1:3200. No statistically significant difference was found between male (1.18 %) and female (0.69%) sero-prevalence (P=0.330). Children of 5-8 years showed the high­est sero-prevalence rate (3.22%). Seven out of 30 domestic dogs (23%) showed anti-Leishmania antibodies at titers ≤1:320. Leishmania infantum was identified in five infected dogs by nested - PCR assay.Conclusion: It seems that visceral leishmaniasis is being endemic in southern villages of Baft district, southeast of Iran

Molecular and parasitological study of cutaneous leishmaniasis in Bushehr province, southwest of the Islamic Republic of Iran: a cross-sectional study during 2009–2012

Cutaneous leishmaniasis (CL) is one of the most important parasitic disease in Iran. CL is distributed among more than half of 31 provinces of Iran. Studies on epidemiological aspects of the disease and Leishmania species identification among infected humans are necessary for providing a comprehensive prevention and control program thus; this descriptive cross-sectional study was conducted on all CL suspected patients who referred to Health Centers of Bushehr province from 2009 to 2012. Physical examinations were carried out in suspected individuals and CL cases were confirmed by microscopical examinations. Prepared slides from suspicious cases of CL were fixed with absolute methanol and stained by Giemsa 10 %. All the Giemsa-stained slides examined under a light microscope with high magnification (1,0009) and classified them based on grading of Leishmania parasites. DNA from each slide was extracted, separately. The ribosomal internal transcribed spacer 1 was amplified with specific primers and PCR products were digested by restrict enzymes (HaeIII), run them in 3 % gel agarose for electrophoresis and visualized on a UV transilluminator after staining with ethidium bromide. SPSS version 21 was used for data analyses. A total of 726 suspected CL cases were referred to Health Centers of Bushehr province from 2009 to 2012 and samples were only prepared from 188 of the patients whereas 43 (5.9 %) of them were microscopy positive. The most frequent of CL was observed in November (14 %) and December (12 %). The most distribution of CL lesions were observed on hands (32 %), feet (26 %), and face (21 %), respectively. The highest frequency of CL was observed in 1–9 years old (30 %). Altogether, 50 % of the patients showed one skin lesion and 2–10 skin lesions were occurred in the remained CL patients. Totally, 27 out of 43 (63 %) of the Giemsa stained slides were positive by PCR–RFLP assay because all the PCR–RFLP negative slides were prepared 3–4 years ago and kept without cover slip, and also observed scarce amastigotes during microscopy observations. Leishmania species were identified in 21 desirable slides which 14 of them were L. major and 7 of the remained isolates were identified L. tropica using PCR–RFLP

Comparative proteomic profiling of Leishmania tropica: investigation of a case infected with simultaneous cutaneous and viscerotropic leishmaniasis by 2-dimentional electrophoresis and mass spectrometry

Background: Viscerotropic leishmaniasis caused by Leishmania tropica poses a significant prob­lem in the diagnosis and treatment management. Since differential gene expression is more im­portant in outcome of the infection, we employed proteomic approach to identify potential pro­teins involved in visceralization of L. tropica. Methods: The proteomes profiling of L. tropica isolated from cutaneous and visceral tissues of one host were compared by 2-DE/MS proteomics study. Moreover, the transcript level of some identified proteins was confirmed using real-time RT-PCR. Results: Of the 700 protein spots that were detected reproducibly on each gel, 135 were found to be differentially expressed (P≤ 0.05). Most of responsive proteins in visceral isolate changed in less abundant compared to cutaneous isolate. Among differentially expressed proteins, 56 proteins were confidently identified and classified according to the biological process. The larg­est groups consist of proteins involved in carbohydrate metabolism and protein synthesis. Most of the identified proteins, which implicated in energy metabolism, cell signaling and virulence were down-regulated, whereas some proteins that have a role in protein folding, antioxidant defense and proteolysis were up-regulated in visceral form. Moreover, the transcript level of some identified proteins such as co-chaperon was confirmed using real-time RT-PCR. Conclusion: L. tropica probably uses different mechanisms for survival and multiplication in viscera to establish viscerotropic leishmaniasis. The current study provides some clues into the mechanisms underlying the dissemination of L. tropica

Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran

An epidemiological study to examine the sero-prevalence of zoonotic visceral leishmaniosis (ZVL) among domestic and wild canines in endemic foci of Iran was carried out during 1999-2003 to assess the distribution of the disease and the possible association between infection in dogs, wild canines and people. Anti-leishmanial antibodies were detected by the direct agglutination test (DAT). Parasitological study was performed for all captured wild canines and were detected in some of the seropositive dogs with specific clinical signs (n = 107). Serum samples (n = 1568) were collected from domestic dogs in villages that are known endemic foci of human visceral leishmaniosis (HVL). Wild canine sera were collected from jackals (Canis aureus, n = 10), foxes (Vulpes vulpes, n = 10) and wolves (Canis lupus, n = 10). Of the 1568 serum sampled collected from domestic dogs, 222 (14.2%) were positive by DAT (1:320 and above). No statistically significant difference was found between male (15.2%) and female (11.8%) sero-prevalence (P = 0.083). Dogs of 8 years and above showed the highest sero-prevalence (40.6%). Only 23.9% of the seropositive domestic dogs had clinical signs. Parasitology and serology tests that were performed in 30 wild canines showed 10% these animals were infected by Leishmania infantum. Ten out of 11 Leishmania spp. isolated from the dogs and wild canines were identified as L infantum and one other as L. tropica by molecular and biochemical techniques. For the first time in Iran, L. infantum and L. tropica were isolated from viscera of both a wolf and a domestic dog. (c) 2005 Elsevier B.V. All rights reserved

Comparative proteomic profiling of Leishmania tropica: investigation of a case infected with simultaneous cutaneous and viscerotropic leishmaniasis by 2-dimentional electrophoresis and mass spectrometry

Background: Viscerotropic leishmaniasis caused by Leishmania tropica poses a significant prob­lem in the diagnosis and treatment management. Since differential gene expression is more im­portant in outcome of the infection, we employed proteomic approach to identify potential pro­teins involved in visceralization of L. tropica. Methods: The proteomes profiling of L. tropica isolated from cutaneous and visceral tissues of one host were compared by 2-DE/MS proteomics study. Moreover, the transcript level of some identified proteins was confirmed using real-time RT-PCR. Results: Of the 700 protein spots that were detected reproducibly on each gel, 135 were found to be differentially expressed (P≤ 0.05). Most of responsive proteins in visceral isolate changed in less abundant compared to cutaneous isolate. Among differentially expressed proteins, 56 proteins were confidently identified and classified according to the biological process. The larg­est groups consist of proteins involved in carbohydrate metabolism and protein synthesis. Most of the identified proteins, which implicated in energy metabolism, cell signaling and virulence were down-regulated, whereas some proteins that have a role in protein folding, antioxidant defense and proteolysis were up-regulated in visceral form. Moreover, the transcript level of some identified proteins such as co-chaperon was confirmed using real-time RT-PCR. Conclusion: L. tropica probably uses different mechanisms for survival and multiplication in viscera to establish viscerotropic leishmaniasis. The current study provides some clues into the mechanisms underlying the dissemination of L. tropica

Western Blot Analysis of Leishmania infantum Antigens in Se-ra of Patients with Visceral Leishmaniasis

Background: Visceral leishmaniasis (VL) is endemic in the northwest and south of Iran. Untreated cases of VL could cause death. The aim of the present study was to evaluate the diagnostic performance of western blotting to detect a specific immunodominant proteins pattern for Leishmania infantum infection using human sera infected with VL. Methods: We studied a panel of 122 cryopreserved human serum samples from the leishmaniasis Research Laboratory, Tehran University of Medical Sciences, Tehran, Iran from 2010 to 2017. Serum samples were collected from visceral (Group I, n: 43) and cutaneous leishmaniasis (CL) (Group II, n: 8) patients, healthy individuals from endemic (Group III, n: 13) and non-endemic (Group IV, n: 16) areas for VL, and patients with other infectious diseases (Group V, n: 42). Total antigens were prepared from the Iranian strain of L. infantum promastigote form. Results: In western blotting method, 34 protein bands of 14 to 163 kDa were recognized using the sera of VL pa­tients. The polypeptide fractions with the highest frequency including 29, 51, and 62 kDa fractions were detected using 81.4%, 79%, and 81.4% of the sera, respectively. These bands were not detected using the sera of the negative control. Moreover, 19-23, 27, 31-35, 143-163, and 109 kDa fractions were detected specifically using the sera of the patients with VL. Conclusion: This technique could be a primary step for further exploration of VL immunodominant antigens for cloning (or any technique) further investigations for future planning

Evaluation of rK39 rapid diagnostic tests for canine visceral leishmaniasis : longitudinal study and meta-analysis

Canine visceral leishmaniasis is a vector-borne disease caused by the intracellular parasite Leishmania infantum. It is an important veterinary disease, and dogs are also the main animal reservoir for human infection. The disease is widespread in the Mediterranean area, and parts of Asia and South and Central America, and is potentially fatal in both dogs and humans unless treated. Diagnosis of canine infections requires serological or molecular tests. Detection of infection in dogs is important prior to treatment, and in epidemiological studies and control programmes, and a sensitive and specific rapid diagnostic test would be very useful. Rapid diagnostic tests (RDTs) have been developed, but their diagnostic performance has been reported to be variable. We evaluated the sensitivity of a RDT based on serological detection of the rK39 antigen in a cohort of naturally infected Brazilian dogs. The sensitivity of the test to detect infection was relatively low, but increased with time since infection and the severity of infection. We then carried out a meta-analysis of published studies of rK39 RDTs, evaluating the sensitivity to detect disease and infection. The results suggest that rK39 RDTs may be useful in a veterinary clinical setting, but the sensitivity to detect infection is too low for operational control programmes