First report on isolation of Leishmania tropica from sandflies of a classical urban Cutaneous leishmaniasis focus in southern Iran

Shiraz district in south of Iran is a classical focus of Cutaneous leishmaniasis (CL) and previous research has consistently documented the etiologic agent to be Leishmania tropica and Leishmania major in urban and rural areas, respectively. However, none of the Phlebotomus sergenti, a known vector for L. tropica, of the region has been found infected. We report the first isolation of L. tropica from sandflies in urban community of southern part of Shiraz city. Parasite polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and gene sequencing analyses indicate CL cases in this community were caused by either L. major or L. tropica. Sandflies of P. sergenti were infrequent, however, three out of 10 (30.0%) females captured in urban area were found infected with L. tropica. But, no human cases were found to be infected with L. tropica. Phlebotomus papatasi were found the most dominant and infected species where 41 out of 207 (20%) tested individuals harboring L. major in suburb area of the city. Patients have been lived in the suburb area of the city where people keep normally domestic animals in their houses which provide appropriate environment for completion of sandfly life cycle and expansion of CL disease in the region.     

Detection, identification and molecular typing of Leishmania major in Phlebotomus papatasi from a focus of zoonotic cutaneous leishmaniasis in central of Iran

Leishmania major is the causative agent and Phlebotomus papatasi is the main vector of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran and elsewhere. Nested PCR protocols were used to amplify a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA gene) in female P. papatasi. In the current investigation, L. major was found in Natanz, Isfahan province in centre of Iran, in a focus of rural ZCL. Ten (1.8%) out of 549 female P. papatasi was found to be infected with L. major based on the PCR detection and sequencing of parasite ITS-rDNA.Nine (1.8%) out of 498 female P. papatasi infected with L. major came from animal shelters, inside houses and yards. And one (1.9%) out of 51 female P. papatasi infected with L. major came from gerbil borrows. Infection rates were higher for females containing red blood meals, large eggs (semi-mature and mature) than for those without either blood meals or eggs. From the 10 infections detected three different haplotypes of L. major were identified. Two haplotypes were found to be novel. The other haplotypes of L. major was found to be identical to that of isolates of L. major from Iran and in elsewhere using GenBank data. Comparisons of infection rates between habitats will be inaccurate when the proportions of blood-fed and gravid flies differ among sandfly samples.     

Successful treatment of lupoid cutaneous leishmaniasis with glucantime and topical trichloroacetic acid (a case report)

Lupoid leishmaniasis is a unique form of cutaneous leishmaniasis characterized by unusual clinical features and a chronic relapsing course, mostly caused by infection with Leishmania tropica. In this clinical form, 1-2 yr after healing of the acute lesion, new papules and nodules appear at the margin of the remaining scar. Herein, we describe a case of this clinical form that was resistant to 2 courses of treatments: systemic glucantime and then a combination therapy with allopurinol and systemic glucantime. However, marked improvement was seen after a combination therapy with topical trichloroacetic acid solution (50%) and systemic glucantime, and there were no signs of recurrence after 1 yr of follow-up.     

Isolation and detection of Leishmania species among naturally infected Rhombomis opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran

In Iran, three species of Leishmania have been incriminated as the causative agents of human leishmaniasis, Leishmania (L.) major, Leishmania tropica, and Leishmania infantum.Rhombomis opimus have been incriminated as a principal reservoirs of the parasitic protozoan Leishmania major, the causative agent of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran. Rodents captured and examined to find Leishmania species using conventional methods including direct impression smear and microscopic observation inoculation samples to Balb/c and culture in NNN medium. Also molecular method was employed to detect Leishmania in rodents by amplifying a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA–ITS2) using Nested PCR. Leshmania species were specified by DNA sequences. 36 (38.3%) of R. opimus were Leishmania positive using at least one conventional methods. Many more ITS-rDNA fragments were amplified from R. opimus but only 65 out of 74 PCR products contained enough DNA for direct sequencing or readable sequences. The PCR assays detected in Iranian R. opimus not only Leishmania major in 59 (79.7%) rodents but also Leishmania turanica in 6 (8.1%) rodents, another parasite of the great gerbil. These parasites were found in Turkemen Sahara, North East of Iran, in a focus of rural (ZCL). L. major and L. turanica in R. opimus firmly identified from Turkemen Sahara. Nine rodents with Leishmania infections unidentified which some were unreadable sequences, these could be mixed infections of L. major, L. turanica, Leishmania gerbillisensu lato and Leishmania close to L. gerbilli or a related species reported in sandflies previously from this location. The haplotypes of L. major and L. turanica were found to be identical to that of isolates of L. major and L. turanica from Iran and in GenBank elsewhere. R. opimus is probably the key reservoir in this ZCL focus because of its abundance and its infection rates with both L. major and L. turanica.     

Identification of the causative agent of cutaneous leishmaniasis in Chichaoua province, Morocco

Cutaneous leishmaniasis (CL) in Morocco is caused by three species, Leishmania major, L. tropica and L. infantum. CL has been known in Chichaoua province since 2000. Using DNA extracted from microscopic slides and parasite cultures, collected in the years 2006 and 2009, we identified for the first time L. tropica as the causative agent of CL in this region. Species identification was achieved by performing the ITS1-PCR-RFLP approach. By using this method it was possible to identify parasites in Giemsa stained slides containing less than five parasites per oil-immersion field even they were conserved for up to four months.     

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 ≥1:800. There was a significant association between positive sera and various parts of the rural areas of Alborz Province (P<0.002). Two children with anti-Leishmania infantum antibodies titers of ≥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.     

An Epidemiological Study of Cutaneous Leishmaniasis in Al-Jabal Al-Gharbi,Libya

Cutaneous leishmaniasis (CL) is an endemic parasitic infection in the Mediterranean region, including Libya and its Al-jabal Al-gharbi province. We aimed at studying the occupational relevance as well as other epidemiological aspects of CL. We investigated 140 CL cases who attended at Gharyan outpatient polyclinic during a period of 6 months in 2009. CL infection was clinically diagnosed and confirmed by demonstration of Leishmania parasites on smears from lesions. Our findings showed that males were more affected than females (P=0.04), and people above 10-years were more affected than younger ones (P=0.0001). A significant percent of CL cases belonged to Al-Kawasem subprovince (P=0.0001). Farm-related activities were the most frequent occupations among CL cases (P=0.04). In addition to farm workers, housewives and students are at risk groups since they are engaged at farm activities. Moreover, those who have occupations that require staying outdoors for a part of night, e.g., policemen, are also at risk. Compared to children, adult CL patients had multiple lesions (P=0.001) that were more prevalent in their upper and lower extremities than the face (P=0.0001). We conclude that CL is a major health problem in Al-jabal Al-gharbi province of Libya. The presence of rodents and sandflies makes it a suitable environment for Leishmania to spread in an endemic epidemiological pattern. Being engaged in farming activities or outdoor occupations increases the risk of infection. Various clinical patterns of CL suggest the presence of more than 1 species of Leishmania at Al-jabal Al-gharbi province. We propose that the 2 species responsible for CL in this area are L. major and L. tropica. Further investigations to identify the leishmanial species responsible for CL at Al-jabal Al-gharbi together with adoption of preventive and control programs are needed.     

Detection of Leishmania (Viannia) DNA in blood from patients with American cutaneous leishmaniasis

The aim of the present study was to investigate the presence of Leishmania (Viannia) subgenus DNA in peripheral blood from patients with cutaneous lesions due to American cutaneous leishmaniasis. The buffy coats from 68 blood samples were analyzed by polymerase chain reaction using the MP1L/MP3H primers. The parasite DNA was detected in 2 (3.4%) out of 59 patients who had amastigotes present in samples taken from lesions. The presence of Leishmania (Viannia) DNA in the blood of these patients indicates hematogeneous parasite dissemination.     

Leishmania major: activity of tamoxifen against experimental cutaneous leishmaniasis

Leishmaniasis is a family of diseases caused by protozoan parasites of the genus Leishmania. Various Leishmania species can cause human infection, producing a spectrum of clinical manifestations. The current treatments are unsatisfactory, and in absence of a vaccine, there is an urgent need for effective drugs to replace/supplement those currently in use. Recent studies have shown that the antineoplastic drug, tamoxifen, had direct leishmanicidal effect on several Leishmania species in vitro. Moreover, in vivo testing was carried out on some of the species and showed promising results. The authors have carried out the present work to complement previous published studies by investigating in vivo activity of tamoxifen in an experimental model of cutaneous leishmaniasis (CL) caused by Leishmania major. Groups of infected mice were given tamoxifen, orally, at a dose of 20 mg/kg/day for 15 days. Efficacy was assessed clinically, parasitologically, histopathologically by light and transmission electron microscope (TEM). Results showed that untreated infected mice suffered from autoamputation of the inoculated foot pad. However, those which received tamoxifen showed marked improvement of the cutaneous lesions and reduction of parasite burden. TEM of the cutaneous lesions from infected mice revealed the fine structure of normal Leishmania amastigotes, whereas those from infected mice treated with tamoxifen showed considerable changes. All male mice that received tamoxifen showed scrotal swelling with evident histopathological changes in the testes that could seriously compromise fertility of male mice. In conclusion, although tamoxifen causes significant side effects to the male reproductive system in the mouse model, it could provide an alternative to current agents. Results of this study demonstrated in vivo activity of tamoxifen against Leishmania major, thus, suggesting that tamoxifen is a suitable lead for the synthesis of more effective and less toxic antileishmanial derivatives.     

Distribution frequency of pathogenic bacteria isolated from cutaneus leishmaniasis lesions

Cutaneous leishmaniasis (CL) is a parasitic disease characterized by single or multiple ulcerations. Secondary bacterial infections are one of the complications that can increase the tissue destruction and the resulting scar. To better determine the incidence of real secondary bacterial infections in CL, we designed the current study. This was a cross-sectional study performed in Skin Diseases and Leishmaniasis Research Centre, Isfahan, Iran. A total of 1,255 patients with confirmed CL enrolled in the study. Sterile swaps were achieved for ulcer exudates and scraping was used for non-ulcerated lesions. All samples were transferred to tryptic soy broth medium. After 24 hr of incubation at 37 degrees C they were transferred to eosin methylene blue agar (EMB) and blood agar. Laboratory tests were used to determine the species of bacteria. Among 1,255 confirmed CL patients, 274 (21.8%) had positive cultures for secondary bacterial infections. The bacteria isolated from the lesions were Staphylococcus aureus in 190 cases (69.3%), coagulase negative Staphylococcus in 63 cases (23.0%), E. coli in 10 cases (3.6%), Proteus sp. in 6 cases (2.2%), and Klebsiella sp. in 5 cases (1.9%). The results show that the overall incidence of secondary bacterial infections in the lesions of CL was 21.8%, considerably high. The incidence of secondary bacterial infections was significantly higher in ulcerated lesions compared with non-ulcerated lesions.     

Leishmania (V.) braziliensis: detection by PCR in biopsies from patients with cutaneous leishmaniasis

Cutaneous leishmaniases present similar clinical appearances, but differing prognosis in the course of infection. Ulcers caused by parasites of the subgenus Viannia are more aggressive than ulcers caused by parasites of the subgenus Leishmania. Another problem is distinguishing between true Leishmania infection and other skin diseases in endemic areas, where cutaneous lesions and a single positive Montenegro intradermal test are enough to submit patients to specific treatment for cutaneous leishmaniasis. This study evaluated the efficacy of PCR in detecting in Leishmania in patients with cutaneous lesions. Leishmania (V.) braziliensis complex was determined by a primer pair from the multicopy spliced leader RNA. The results were compared to those of traditional methods. We analyzed biopsies of 109 patients with cutaneous lesions in the second most endemic region of Sao Paulo State, Brazil. Definitive diagnosis was established by clinical and “consensus laboratory criteria” (positive culture, stained tissue smears or PCR). Of 52 patients with cutaneous leishmaniasis, 96% had positive PCR, 69%, positive parasitological tests and 100%, positive Montenegro intradermal tests. Histopathological examination (only in 32 samples) were positive in 14 samples, suggestive in 14 and negative in 4 samples. All 57 patients with other etiologies had negative results in parasitological methods, PCR and histopathological examination (in 39 samples), but Montenegro intradermal tests were positive in 35%. PCR was highly sensitive and specific for L. (V.) braziliensis complex detection compared with other laboratory methods. Despite the specificity of the parasitological tests, the sensitivity was less than 70%. Montenegro intradermal reaction was highly sensitive, but with low specificity, only 65%. As suggestive results in histopathological examinations were shown in 14 samples, it was difficult to determine the true result. PCR applied to biopsies proved to be useful for differential diagnosis of cutaneous lesions of other etiologies in patients living in endemic areas. The advantages are most striking in clinical specimens with scarce amastigotes for which conventional methods have low sensitivity and should be considered for clinical and epidemiological patterns. On the other hand, both Montenegro intradermal test and parasitological methods are only modestly effective in cutaneous leishmaniasis diagnosis.     

Effect of chlorpyrifos on healing of cutaneous leishmaniasis lesions after treatment with Pentostam®

The insecticide chlorpyrifos (CPF) is widely used in the Kingdom of Saudi Arabia (KSA) to control agricultural pests. The present work is a preliminary investigation of the effect of CPF on healing of cutaneous leishmaniasis (CL) lesions, caused by Leishmania major in farmers exposed to this insecticide, after treatment with Pentostam^®. Lesion diameters were measured and CPF concentrations in the blood plasma of farmer and non-farmer CL patients in Al-Ahsa were detected by gas chromatography/mass spectrometry/mass spectrometry before and 6 weeks after treatment with Pentostam^®. CPF concentrations in the blood of farmer patients ranged between 4.570 and 7.096 ng/μl (mean = 6.19 ± 0.881 ng/μl) before and after treatment with Pentostam^®. The mean lesion diameter in these patients decreased by a factor of 2.21 after treatment with Pentostam^®; they measured 1.85–11.75 mm, (mean = 6.165 ± 3.500 mm) before treatment and 0.22–6.10 mm (mean = 2.796 ± 2.102 mm) after treatment. Lesion diameter increased exponentially with the increase of CPF concentration in the patients’ blood. CPF was not detected in the non-farmer patients before or after treatment. Their mean lesion diameter decreased by a factor of 6.86 after treatment with Pentostam^®; they measured 1.33–7.10 mm (mean = 2.882 ± 1.764 mm) before treatment and 0.11–0.92 mm (mean = 0.425 ± 0.277 mm) after treatment. The mean lesion diameter in farmer patients was much greater than that of non-farmer patients both before (2.14×) and after (6.657×) treatment with Pentostam^®. Chronic exposure to low levels of the pesticide aggravates the development and delays the healing of CL lesions due to immunotoxicity and/or peripheral neurotoxicity caused by CPF. Further detailed studies would assess CPF effect on the severity of infection with CL in agricultural workers continuously exposed to this insecticide in different areas of KSA in conformity of their finding.     

Leishmania species: Detection and identification by nested PCR assay from skin samples of rodent reservoirs

Many rodent species act as reservoir hosts of zoonotic cutaneous leishmaniasis in endemic areas. In the present study a simple and reliable assay based on nested PCR was developed for the detection and identification of Leishmania parasites from rodent skin samples. We designed Leishmania-specific primers that successfully amplified ITS regions of Leishmania major, Leishmania gerbilli and Leishmania turanica using nested PCR. Out of 95 field collected Rhombomys opimus, 21 were positive by microscopic examination and 48 by nested PCR. The percentage of gerbils infected with L. major, L. gerbilli and L. turanica was 3.2%, 1.1% and 27.4%, respectively. In 15.8% of the rodents, we found mixed natural infections by L. major and L. turanica, 1.1% by L. major and L. gerbilli, and 2.1% by the three species. We concluded that this method is simple and reliable for detecting and identifying Leishmania species circulating in rodent populations.     

Vectors of cutaneous leishmaniasis in north-central Venezuela

Abstract. An entomological survey was undertaken from January 1991 to February 1992 in El Ingenio, Miranda State, Venezuela, an endemic area of cutaneous leishmaniasis: prevalence of 10.7 cases per 100,000 inhabitants.
A total of 4863 female sandflies (Phlebotominae) of fourteen species were collected in Shannon traps, then dissected and examined for leishmanial infections.
Lutzomyia ovallesi (85.4%) and Lu. gomezl (11.2%) were the predominant anthropophilic species of sandfly. Fifty-one (1.19%) Lu. ovallesi and two(0.47%) Lu. gomezi had natural infection with Leishmania promastigotes. Identification of the parasites was done by using the polymerase chain reaction (PCR) and DNA hybridization. Two isolates from Lu. gomezi and forty-nine from Lu. ovallesi were typed as Leishmania braziliensis and three of the latter reacted with Le. mexicana also. This is the first report of Lu. gomezi with parasites typed as Le. braziliensis.
We concluded that Lu. ovallesi is the primary vector of cutaneous leishmaniasis in the north-central area of Venezuela and Lu. gomezi should be regarded as an additional vector.     

Protective effect of lectin from Synadenium carinatum on Leishmania amazonensis infection in BALB/c mice

The protective effect of the Synadenium carinatum latex lectin (ScLL), and the possibility of using it as an adjuvant in murine model of vaccination against American cutaneous leishmaniasis, were evaluated. BALB/c mice were immunized with the lectin ScLL (10, 50, 100 microgram/animal) separately or in association with the soluble Leishmania amazonensis antigen (SLA). After a challenge infection with 10(6) promastigotes, the injury progression was monitored weekly by measuring the footpad swelling for 10 weeks. ScLL appeared to be capable of conferring partial protection to the animals, being most evident when ScLL was used in concentrations of 50 and 100 microgram/animal. Also the parasite load in the interior of macrophages showed significant reduction (61.7%) when compared to the control group. With regard to the cellular response, ScLL 50 and 100 microgram/animal stimulated the delayed-type hypersensitivity (DTH) reaction significantly (P < 0.05) higher than SLA or SLA plus ScLL 10 weeks after the challenge infection. The detection of high levels of IgG2a and the expression of mRNA cytokines, such as IFN-gamma, IL-12, and TNF-alpha (Th1 profiles), corroborated the protective role of this lectin against cutaneous leishmaniasis. This is the first report of the ScLL effect on leishmaniasis and shows a promising role for ScLL to be explored in other experimental models for treatment of leishmaniasis.     

Sandflies (Diptera: Psychodidae) in rural and urban environments in an endemic area of cutaneous leishmaniasis in southern Brazil

The high proportion of cases of cutaneous leishmaniasis reported amongst residents in the city of Bandeirantes, in the state of Paraná, Brazil, led the authors to investigate the phlebotomine fauna in both urban and rural environments. The sandflies were captured with automatic light traps from 07:00 pm-07:00 am fortnightly in 11 urban peridomiciles from April 2008-March 2009 and monthly in three ecotopes within four rural localities from April 2009-March 2010. In one of these latter localities, sandfly capture was conducted with white/black Shannon traps during each of three seasons: spring, summer and fall. A total of 5,729 sandflies of 17 species were captured. Nyssomyia neivai (46.7%) and Nyssomyia whitmani (35.3%) were the predominant species. In this study, 3,865 specimens were captured with automatic light traps: 22 (0.083 sandflies/trap) in the urban areas and 3,843 (26.69 sandflies/trap) in the rural areas. Ny. neivai was predominant in urban (68.2%) and rural (42.8%) areas. A total of 1,864 specimens were captured with the white/black Shannon traps and Ny. neivai (54.5%) and Ny. whitmani (31.4%) were the predominant species captured. The small numbers of sandflies captured in the urban areas suggest that the transmission of Leishmania has occurred in the rural area due to Ny. neivai and Ny. whitmani as the probable vectors.     

Multilocus microsatellite typing shows three different genetic clusters of Leishmania major in Iran

Ten polymorphic microsatellite markers were used to analyse 25 strains of Leishmania major collected from cutaneous leishmaniasis cases in different endemic areas in Iran. Nine of the markers were polymorphic, revealing 21 different genotypes. The data displayed significant microsatellite polymorphism with rare allelic heterozygosity. Bayesian statistic and distance based analyses identified three genetic clusters among the 25 strains analysed. Cluster I represented mainly strains isolated in the west and south-west of Iran, with the exception of four strains originating from central Iran. Cluster II comprised strains from the central part of Iran, and cluster III included only strains from north Iran. The geographical distribution of L. major in Iran was supported by comparing the microsatellite profiles of the 25 Iranian strains to those of 105 strains collected in 19 Asian and African countries. The Iranian clusters I and II were separated from three previously described populations comprising strains from Africa, the Middle East and Central Asia whereas cluster III grouped together with the Central Asian population. The considerable genetic variability of L. major might be related to the existence of different populations of Phlebotomus papatasi and/or to differences in reservoir host abundance in different parts of Iran.     

Viscerotropic growth pattern of Leishmania tropica in BALB/c mice is suggestive of a murine model for human viscerotropic leishmaniasis

Leishmania (L.) tropica is a causative agent of cutaneous leishmaniasis, and occasionally of visceral or viscerotropic leishmaniasis in humans. Murine models of Leishmania infection have been proven to be useful for elucidation of mechanisms for pathogenesis and immunity in leishmaniasis. The aim of this study was to establish a murine model for human viscerotropic leishmaniasis, and the growth pattern of L. tropica was studied in different tissues of BALB/c mice in order to find out whether the parasite visceralizes in this murine model. L. major was used as a control as this species is known to cause a progressive infection in BALB/c mice. L. tropica or L. major was injected into the footpad of mice, and thickness of footpad, parasite loads in different tissues, and the weight of the spleen and lymph node were determined at different intervals. Results showed that L. tropica visceralizes to the spleen and grows there while its growth is controlled in footpad tissues. Dissemination of L. tropica to visceral organs in BALB/c mice was similar to the growth patterns of this parasite in human viscerotropic leishmaniasis. The BALB/c model of L. tropica infection may be considered as a good experimental model for human diseases.     

Effects of combined therapy with thalidomide and glucantime on leishmaniasis induced by Leishmania major in BALB/c mice

For treating Leishmania major infection in BALB/c mice, we used thalidomide in conjunction with glucantime. Groups of mice were challenged with 510(3) metacyclic promastigotes of L. major subcutaneously. A week after the challenge, drug treatment was started and continued for 12 days. Thalidomide was orally administrated 30 mg/kg/day and glucantime was administrated intraperitoneally (200 mg/kg/day). It was shown that the combined therapy is more effective than single therapies with each one of the drugs since the foot pad swelling in the group of mice received thalidomide and glucantime was significantly decreased (0.9 +/- 0.2 mm) compared to mice treated with either glucantime, thalidomide, or carrier alone (1.2 +/- 0.25, 1.4 +/- 0.3, and 1.7 +/- 0.27 mm, respectively). Cytokine study showed that the effect of thalidomide was not dependent on IL-12; however, it up-regulated IFN-gamma and down-regulated IL-10 production. Conclusively, thalidomide seems promising as a conjunctive therapy with antimony in murine model of visceral leishmaniasis.