First report on natural Leishmania infection of Phlebotomus sergenti due Leishmania tropica by high resolution melting curve method in Southeastern Iran

Objective:To identify the Leishmania species in infected sand flies by Real-time PCR coupled with HRM analysis.Methods:Real-time PCR coupled with HRM analysis targeting the first internal transcribed spacer(ITS1)of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish Lelthmania species in sand flies specimens.Results:Three out of 115females of Phlebotomus sergenti(P.sergenti)(2.6%)were positive to Leishmania tropica(L.tropica).Conclusions:This is the first report on P.sergenti as the main and proven vector of anthroponitic cutaneous leishmaniasis in Dehbakri County using Real-time PCR coupled with HUM analysis.This method is rapid,sensitive and specific for diagnosing of parasites in infected Sand flies and ideal for large scale genotyping projects.     

Human cutaneous leishmaniasis due to Leishmania infantum in the Sidi Bourouis focus (Northern Tunisia): epidemiological study and isoenzymatic characterization of the parasites

The authors report an increase of the number of case of cutaneous leishmaniasis in the Sidi Bourouis region community of Siliana Governorate (Tunisia), with 38 cases diagnosed in 6 months (1st November 2000-30th April 2001), contrary to its usual sporadic character. The isoenzymatic identification of 15 isolated strains emphasizes the role of the Leishmania infantum zymodeme MON-1 as an important factor in the genesis of the sporadic cutaneous leishmaniasis of Northern Tunisia. In fact it was isolated in 6 cases while L. infantum MON-24, the usual agent was isolated in 9 cases.     

Experimental canine leishmaniasis: evolution of infection following re-challenge with Leishmania infantum

The aim of the study was to assess the clinical, parasitological and immunological effect of a second inoculation of amastigotes in dogs previously inoculated with Leishmania infantum. Three dogs primarily inoculated with amastigotes (Group I) and four with cultured virulent stationary phase promastigotes (Group II) were afterwards re-inoculated with 2x10(9) amastigotes per kg. Three other groups of dogs were used as controls: Group III was infected only once with amastigotes, Group IV only once with promastigotes and Group V was non infected. The animals were followed up by clinical and parasitological examinations, hematological and serum protein analysis, anti-leishmanial antibody levels and proliferative assays of specific peripheral blood mononuclear cells over a period up to 50 months. Parasites were isolated from lymph node of three animals during primary amastigote infection and in five animals (Group I and II) after re-challenge. Group I dogs presented a strong increase of the humoral immune response while Group II animals displayed no significant or significantly low antileishmanial antibodies titres, after re-challenge. The detection, only after challenge, of positive specific lymphoproliferation in two animals of Group II that had the longest primary infection interval (more than 26 months), indicates the requirement of a long time interval to obtain specific lymphocyte sensitization. A previous exposure to virulent cultured L. infantum promastigotes seems to confer some degree of resistance against an amastigote 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 = ≥1:320) and 53 asymptomatic (9 seropositive = ≥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.     

Leishmania infantum MON-98: infection in a dog from Alto Douro,Portugal

We report the isolation of Leishmania infantum zymodeme MON-98 in Portugal, its first known occurrence outside the focus of El Agamy, Egypt. One dog found to be infected with Acanthocheilonema dracunculoides during a survey of canine filariosis in the Alto Douro region, north-east Portugal, was also discovered to have Leishmania in bone marrow. The isolated strain was identified by isoenzyme analysis. A search for other possible cases of L. infantum MON-98 infection in dogs, vectors and particularly humans is necessary to establish the real epidemiological importance of this zymodeme in the endemic region of Alto Douro.     

Antigenuria in visceral leishmaniasis: detection and partial characterisation of a carbohydrate antigen

The detection of antigen in the urine is increasingly being used for diagnosis of parasitic infections. A urinary antigen has recently been demonstrated in visceral leishmaniasis (VL), using a latex agglutination test. The results of our study show that the detected antigen is: heat-stable, precipitates with acetone and ethanol but not TCA, is sensitive to periodate and acid hydrolysis but not to pronase E, lipase, or neuraminidase. The antigen is a low molecular weight glycoconjugate that can be extracted by phenol-water, partitions into the aqueous phase when extracted with Triton X-114 or chloroform/methanol, and can be labelled by biotin hydrazide. Since this urinary antigen cannot be characterised by conventional SDS-PAGE and Western blotting, we used an affinity transfer blotting system in which antigens were captured onto nitro-cellulose paper previously coated with a specific antibody. Using this system a low molecular weight antigen (LMWA) spanning an area of the nitro-cellulose membrane corresponding to molecular weight of 5-20 kDa was detected in the urine of VL patients (from Nepal, Sudan, Brazil, Yemen and Spain) and of experimentally infected animals. No LMWA was detected in the urine of patients with malaria, schistosomiasis, or other nonparasitic diseases including typhoid and brucellosis. Immunoprecipitation, using antibody-coated latex, followed by immunoblotting showed that the LMWA is the target antigen in the previously described latex agglutination test ('KATEX'). The antigen is detectable in both the promastigote and amastigote stages of the parasite. Monoclonal antibodies (mAbs) against Leishmania glycoconjugates strongly react with this molecule. These results suggest that the detected antigen is highly specific and diagnostic for VL.     

Follow-up of Leishmania infantum naturally infected dogs treated with allopurinol: immunofluorescence antibody test,ELISA and Western blot

Fourteen dogs naturally infected with Leishmania infantum and treated with allopurinol were monitored clinically and serologically with immunofluorescent antibody test (IFAT, amastigotes and promastigotes), enzyme linked-immunosorbent-assay (ELISA, IgG1 and IgG2) and Western blotting (WB). In all dogs therapy lead to clinical improvement together with decreasing specific antibodies in IFAT, ELISA and WB, demonstrating the usefulness of serology for follow-up. Although IgG1 and IgG2 varied considerably between individual animals, IgG2 of all dogs was predominantly in both ELISA and WB. This suggests the value of monitoring the IgG2 response (especially against 29 and 67 kDa antigens) in the follow-up of treated dogs.     

Palestinian infantile visceral leishmaniasis caused by a genetic variant of Leishmania infantum belonging to a new zymodeme

The parasites causing a Palestinian case of infantile visceral leishmaniasis (IVL) and those from four dogs from the Jenin District were identified serologically, biochemically and molecular biologically as Leishmania infantum, showing dogs act as a reservoir. The strain from the human case was distinct because of its unique 200-bp kDNA-polymerase chain reaction (PCR) component in its restriction fragment length polymorphism (RFLP) profile after digestion with the endonuclease RsaI, and by the electrophoretic mobility of its malate dehydrogenase (MDH(140)), designating it the reference strain of a new zymodeme of L. infantum, MON-281.     

High infection frequency,low diversity of Leishmania major and first detection of Leishmania turanica in human in northern Iran

Smears of suspected patients infected with zoonotic cutaneous leishmaniasis (ZCL) were stained and examined under a light microscopic observation. DNA of parasites within human ulcers was extracted directly from their smears. Nested PCR was used to amplify a fragment containing the internal transcribed spacers of the ribosomal RNA genes (ITS-rDNA) of Lesihmania parasites in human from Turkemen Sahara located in the northeastern part of Iran. Based on RFLP method by digesting BsuRI restriction enzyme and more precisely sequencing of DNA ITS-rDNA was shown to be species-specific. The infection rates of Leishmania parasites were high with 154 (93.9%) infections out of 164 suspected patients using microscopic observations. Only from 128 suspected patients out of 164, ITS-rDNA fragments were amplified and 125 samples had enough DNA to digest BsuRI restriction enzyme and do DNA sequencing. The Nested PCR assays detected not only Leishmania major but also Leishmania turanica for the first time, another parasite of the great gerbil in human. The density of L. major was high but the diversity was low with only 2 haplotypes. The overall ratio of L. major (123 infections) to L. turanica (2 infections) was significantly higher (Chi-squared test: p < 0.05). Infections of L. turanica are not reported only and/or not known to cause human disease. Our analytical framework conveys a clear understanding of both L. major and L. turanica which can only be approved as causative agents of ZCL by more extensive sampling and followed by standardized molecular diagnosis.     

Evaluation of three recombinant Leishmania infantum antigens in human and canine visceral leishmaniasis diagnosis

Visceral leishmaniasis (VL) is a neglected disease and is fatal if untreated. Dogs serve as reservoirs for Leishmania infantum (syn. L. chagasi) due to their susceptibility to infection and high skin parasitism. Therefore, VL control in Brazil involves the elimination of seropositive dogs, among other actions. However, the most frequently used serological tests have limitations regarding sensitivity and specificity. In this study, we have selected three Leishmania antigens (C1, C8 and C9) and have produced them as recombinant proteins using pET-28a-TEV vector and Escherichia coli BL-21 as expression system. When tested in ELISA with human samples, the C9 antigen was the one showing the most promising results, with 68% sensitivity and 78% specificity. When testing canine samples, the C1, C8 and C9 antigens showed a sensitivity range from 70% to 80% and specificity range from 60% to 90%. The C1 antigen presented higher sensitivity (80%) and the C8 antigen presented higher specificity (90%). Due to it, we decided to mix and test C1 and C8 antigens together, resulting in the C18 antigen. The mix also yielded high percentages of detected symptomatic and asymptomatic dogs however it did not improve the performance of the diagnostic. Comparison of our tests with the tests recommended by the Brazilian Ministry of Health revealed that our antigens’ sensitivities and the percentage of detected asymptomatic dogs were much higher. Our results suggest that the C1, C8, C18 and C9 recombinant proteins are good antigens to diagnose canine visceral leishmaniasis and could potentially be used in screening tests. To diagnose human visceral leishmaniasis, the C9 antigen presented reasonable results, but more optimization must be performed for this antigen to provide better performance.     

The burden of Leishmania chagasi infection during an urban outbreak of visceral leishmaniasis in Brazil

First noted in the city of Teresina in 1981, the last decades have witnessed a remarkable increase in urban transmission of American visceral leishmaniasis (VL) in many Brazilian cities. Teresina, the site of this study, has faced two large outbreaks of VL. The first occurred from 1981-1985 when almost 1000 new cases were reported. The second started in the 1990s, and between 1993 and 1996 more than 1200 new cases were detected. This report describes the prevalence of infection with Leishmania chagasi in Teresina at the end of the second outbreak and gives estimates of the number of people who became infected during the epidemic. Between June 1995 and May 1996, 200 households were chosen at random from a list of addresses covering about 93% of Teresina's urban households. In each household, one person over the age of 1 year was screened for Leishmania antibodies and skin-tested. Nearly 50% of persons had a positive leishmanin reaction, but only 13.9% had detectable antibodies to L. chagasi. While prevalence estimates based on the leishmanin skin-test increased with age (P<0.001), those based on serological tests showed a lesser, and non significant, variation with age (P=0.31). Using a geometric growth equation, and assuming that the annual distribution of clinical cases may serve as an approximation to what would have been the distribution of infections by year, we estimated that over 320000 persons were infected during the epidemic. Little is known about the epidemiology of VL in urban areas, where social networks, population density, and relationships of housing with the natural environment are more varied and complex than in the rural scene. In those areas, control interventions have failed to eliminate transmission of the parasite and prevent new epidemics. Further epidemiological studies of VL in urban areas might be needed to inform control actions.     

Treatment of mucosal leishmaniasis (L. infantum) with miltefosine in a patient with Good syndrome

We report on a 76-year old patient with recurrent mucosal leishmaniasis. Multiple treatment regimens were administered. After the second relapse, immunologic workup and review of the patient's history revealed the presence of Good syndrome, characterized by immunodeficiency in patients with thymoma. The third relapse was treated with oral miltefosine with complete resolution of the lesions. Miltefosine is an option for treating Old World leishmaniasis (Leishmania infantum) and immunodeficiency should be considered in patients with recurrent leishmaniasis.     

Emergence of a new focus of anthroponotic cutaneous leishmaniasis due to Leishmania tropica in rural communities of Bam district after the earthquake, Iran

Objectives To describe a new emerging focus of anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica in rural areas of Dehbakry county, south‐eastern Iran, after the earthquake of 2003. Methods House‐to‐house survey of 3884 inhabitants for active leishmaniasis lesions or scars. The diagnosis was confirmed by smears, cultures and identification of the parasite by polymerase chain reaction (PCR). Results All age groups were affected, although patients ≤10 years of age showed the highest rate of infection (P = 0.0001). The overall prevalence rate was 5.3%; 6.3% in females and 4.3% in males. Of 204 cases, 1.8% had active sores and 3.5% had scars, with a significant difference between the sexes (P = 0.005). 47% of the lesions were on the face and 77.9% had one lesion. The incidence rose gradually 2004–2005, but grew exponentially 2006–2008. Electrophoresis of PCR products indicated that L. tropica was the causative agent. Conclusions The current emergence was unexpected in this rural locality, where no previous history of CL was recorded. According to our knowledge this is the first report of a gradually establishing new ACL focus in rural communities after the 2003 earthquake.     

SLC11A1 polymorphisms and susceptibility to visceral leishmaniasis in Moroccan patients

Human visceral leishmaniasis is endemic in the Mediterranean basin. Since most infections are sub-clinical or asymptomatic, host genetics can provide concrete evidence in determining disease outcome. SLC11A1/NRAMP1 is a candidate gene that may be related to host susceptibility versus resistance to intracellular pathogens. This study aimed to determine possible association of SLC11A1 polymorphisms with visceral leishmaniasis among Moroccan children. A total of 106 children who developed visceral leishmaniasis due to Leishmania infantum were enrolled in this study. The control group was composed of 137 unrelated children, 97 asymptomatic subjects (DTH+) and 42 healthy individuals (DTH) who had no evidence of present or past infection. Four polymorphisms were studied by PCR–RFLP and sequencing: (GT)n microsatellite in the 5′ exon 1; silent substitutions 469 + 14G/C in intron 4; amino acid substitution D543N in exon 15 and 823C/T polymorphism in exon 8. Thereafter, the frequencies of genotypes, alleles and haplotypes were estimated. Two polymorphisms were each significantly associated in the genotypes with visceral leishmaniasis: 823C/T in exon 8 and D543N in exon 15 when comparing visceral leishmaniasis and DTH+ groups. The results of haplotype frequencies suggested an evidence of association with resistance to visceral leishmaniasis for the “286GTG” and “288GCA” haplotypes, whereas, the “286GCG” haplotype appears to increase the risk to visceral leishmaniasis susceptibility.Our data provide insights into the possible role of SLC11A1 variation in visceral leishmaniasis susceptibility. These results must be regarded as preliminary but suggestive that further study with larger populations is worthwhile.     

First detection of Leishmania major in peridomestic Phlebotomus papatasi from Isfahan province, Iran: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA

Two PCR methods were compared for their sensitivity in detecting cultured Leishmania major, before being used to estimate infection rates in female sandflies (Phlebotomus papatasi) collected from peridomestic animal shelters and the nearby burrows of the gerbil reservoir hosts, Rhombomys opimus, in Isfahan province, central Iran. A semi-nested PCR was used to amplify a fragment of minicircle kinetoplast (k) DNA with a length and sequence diagnostic for L. major, and a nested PCR was developed to amplify a fragment containing the internal transcribed spacers of the ribosomal RNA genes (ITS-rDNA) with a sequence diagnostic for L. major. The semi-nested PCR was less sensitive than the nested PCR when using DNA extracted from cultured promastigotes of L. major, but it was more sensitive for detecting L. major in wild-caught sandflies. At the edges of two Isfahan villages, infection rates were significantly higher in P.papatasi collected outside gerbil burrows (14/28) compared with those from peridomestic animal shelters (2/21). This is the first record of L. major detected in P.papatasi from peridomestic sites in Isfahan province.     

The burden of the Leishmania chagasi/infantum infection in a closed rural focus of visceral leishmaniasis in Lara state, west-central Venezuela

A prospective study was conducted in El Brazilar, Curarigua, Lara State, Venezuela, a small rural focus of visceral leishmaniasis (VL) to investigate the burden and the evolution of Leishmania infection in the human and canine population. The incidence of the disease from February 1998 to February 2002 was recorded and two cross-sectional surveys using the leishmanin skin test (LST) and immunofluorescent antibody test (IFAT) were carried out. The dipstick test with recombinant r-K39 antigen was also applied in 2002. The incidence of the disease per year among the population (n=118) during the period of study was 0.004. The rate of new infections per year was 0.07 [95% confidence interval (95% CI): 0.15-1.09]. The overall prevalence of infection measured by LST was not significantly higher in 2002 (43.2%) than in 1998 (28.3%), but it was with IFAT [16.3%vs. 4.6%; odds ratio (OR): 4.01; 95% CI: 1.03-22.78; P=0.022] which would indicate an increasing transmission. The dipstick test only detected infection in children up to 10 years (19.4%). Prevalence in dogs was not significantly different in 2002 (57%) vs. 1998 (33%). The parasite was isolated from dogs and identified by a polymerase chain reaction based on telomeric sequences as Leishmania chagasi/infantum.     

Contrasting effects of Trichinella spiralis and Trichuris muris antigens on the infection by Leishmania infantum in BALB/c mice

The interaction of Trichinella spiralis and Trichuris muris derived antigens with the infection by Leishmania infantum was investigated in BALB/c mice. Infection with 10(6) promastigotes of L. infantum did not induce relevant serum antibody (IgG subclasses), nor cytokine (IFN-gamma, IL-4) responses despite that mice could partially control the infection. Immunization with T. spiralis activated a moderate IgG1 and secondarily an IgG2a anti-leishmanial response whereas immunization with T. muris elicited only a weak and late activation of IgG1 anti-leishmanial response. Immunization with T. muris caused an elevation of serum IFN-gamma levels which was drastically reinforced by the L. infantum infection, and that was accompanied by almost complete parasitological cure of infected mice. Immunization with T. spiralis induced an elevation of serum IL-4 levels but this response was greatly (about 60%) neutralized by the infection with L. infantum, and this was associated to exacerbation of the infection.