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.     

Natural Leishmania infantum infection in Migonemyia migonei (França, 1920) (Diptera:Psychodidae:Phlebotominae) the putative vector of visceral leishmaniasis in Pernambuco State, Brazil

A study of the natural infection of phlebotomine sand flies by Leishmania (Leishmania) infantum was conducted in an area of visceral leishmaniasis in São Vicente Férrer, located in the northern part of the Atlantic rain forest region in the State of Pernambuco, Brazil. In a previous study, Migonemyia migonei have been found predominantly in peridomiciles and houses in this endemic area. The analysis of M. migonei, collected by CDC light trap, by multiplex PCR assay coupled to non-isotopic hybridization showed that 2 females out of 50 were infected by L. infantum. This is the first finding of natural infection of M. migonei by L. infantum suggesting that M. migonei may be the vector of L. infantum in areas of visceral leishmaniasis where Lutzomyia longipalpis, the usual vector, is absent.     

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.     

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.     

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.     

Visceral leishmaniasis in pregnancy: a case report

Visceral leishmaniasis (VL) is endemic in the island of S?o Luis, State of Maranh?o, Brazil. Despite an increase in the number of VL cases, the frequency of the disease is low among pregnant women. We present here the case of a pregnant woman followed up by our group, who was treated with amphotericin B with excellent outcome.     

Candidate gene study of susceptibility to cutaneous leishmaniasis in Sri Lanka

Objectives To investigate the association between selected single nucleotide polymorphisms (SNPs) in TNF, LTA and SLC11A1 genes and risk of endemic cutaneous leishmaniasis (CL) in Sri Lanka through a case–control disease association study. Methods An anonymized DNA resource representative of the Sri Lankan population was genotyped initially to establish baseline parameters. This was followed up by genotyping 200 patients and 200 matched controls. Published or modified PCR/RFLP methods were employed for genotyping. Results Comparison of the different ethnic groups showed the distribution of alleles of LTA +252 A>G to differ significantly in Tamils and Moors when compared with Sinhalese. The differences seen at allele level were also reflected in the haplotypes defined by these SNPs at the TNF locus. The case–control analysis did not show an association between the SNPs or the haplotypes investigated and CL. The distribution of these variant alleles in other populations, where they are positively associated with leishmaniasis, differed significantly from the Sri Lankan study cohort. Conclusions The selected polymorphisms do not predispose to CL in the Sri Lankan population. The study of extended haplotypes at these loci using a sufficiently powered sample collection would elaborate the findings of this study. In the face of an evolving disease pattern in the country with other forms of leishmaniasis now being reported, prevalence of polymorphisms predisposing to these forms calls for heightened surveillance and preparedness.     

Interleukin 10 receptor blockade--pentavalent antimony treatment in experimental visceral leishmaniasis

Interleukin 10 (IL-10), a suppressive Th2 cell-type cytokine, promotes disease progression in experimental visceral leishmaniasis. To extend testing the therapeutic effects of applying IL-10 receptor (IL-10R) blockade with antileishmanial chemotherapy, BALB/c mice with established intracellular Leishmania donovani infection were injected once with anti-IL-10R mAb at the time low-dose, daily pentavalent antimony (Sb) therapy was initiated. In this treatment model, simultaneous administration of anti-IL-10R enhanced overall antileishmanial activity in the liver in an interferon-gamma-dependent fashion, and accelerated the kinetics of Sb-associated killing, induced a >10-fold Sb dose-sparing effect and shortened the required duration of Sb treatment. These results suggest the possibility of using mAb-induced IL-10R blockade to develop low-dose and/or short-course immunochemotherapeutic regimens in visceral leishmaniasis.     

Lutzomyia migonei as putative vector of visceral leishmaniasis in La Banda, Argentina

Four autochthonous cases of human visceral leishmaniasis (VL) were reported in La Banda, Santiago del Estero from June 2007 to May 2008. In the vicinity of these cases there were 3/47 rK39 sero-positive dogs, and another 4 dogs with VL were reported by passive surveillance. The sero-positive dogs and infected humans lived within a 3.1 km radius. Phebotomine sand fly captures were performed twice during November/December 2007 and April 2008. In 20 of the 59 sampled sites in the areas of the human and canine cases (220 night/traps) 151 phlebotomine sand flies were collected and consisted of: Lutzomyia migonei 93%, Lutzomyia cortelezzii 5.6% and Lutzomyia neivai 1.4%. We propose that there was an enzootic cycle of VL with accidental human transmission due to L. migonei and suggest that there be a surveillance of human isolated cases of VL within the L. migonei dispersion area.     

Entomological investigation following the resurgence of human visceral leishmaniasis in southern Algeria

Visceral and cutaneous leishmaniasis are the main endemic vector born diseases in Algeria. In the Hoggar region (extreme south of the country) human visceral leishmaniasis (HVL) is known to be sporadic but during the last decade the number of cases has increased significantly. In 2010, a peak of HVL cases was registered mostly among children. Therefore an entomological survey and a retrospective study on HVL cases were carried out in order to explore the transmission of the disease. Among the sand fly caught Phlebotomus bergeroti was the most frequent species (68%) followed by Sergentomyia schwetzi (22%). In this work we describe the presence of Phlebotomus (Paraphlebotomus) kazeruni for the first time in the Hoggar region.     

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.     

Relationship between Crohn＇s disease, infection with Mycobacterium avium subspecies paratuberculosis and SLC11A1 gene polymorphisms in Sardinian patients

AIM: To study the association between Crohn's disease (CD), Mycobacterium avium subspecies paratuberculosis (MAP), and genetic factors by examining the role of natural resistance-associated macrophage protein 1 (NRAMP1) gene polymorphisms (now SLC11A1) in Sardinian patients with CD and controls. METHODS: Thirty-seven CD patients and 34 controls with no inflammatory bowel disease (IBD) were recruited at the University of Sassari after giving written consent. Six SCL11A1 polymorphisms previously reported to be the most significantly associated with IBD were searched. M. paratuberculosis was identified by IS900 PCR and sequencing. Logistic regression was used to calculate odds ratios (OR) for the associations among CD, presence of MAP, and 6 loci described above. RESULTS: For the first time, a strong association was observed between polymorphisms at NRAMP1 locus 823C/T and CD. While CD was strongly associated with both NRAMP1 and MAP, NRAMP1 polymorphisms and MAP themselves were not correlated. CONCLUSION: Combined with previous work on the NOD2/CARD15 gene, it is clear that the interplay of genetic, infectious, and immunologic factors in the etiology of CD is complex.     

Experimental canine leishmaniasis: Clinical, parasitological and serological follow-up

Canine leishmaniasis (CanL) caused by Leishmania infantum is transmitted by the bite of phlebotomine sand flies and affects millions of dogs in Europe, Asia, North Africa and South America. Canis familiaris is the major host for these parasites, and the main reservoir for human visceral infection. The development of effective molecules for therapy and immunoprophylaxis, would be an important tool in the control of this zoonosis. The aim of this study was to characterize an experimental CanL model in order to determine the best challenge model and which parameters are the most reliable to evaluate the efficacy of new drugs or vaccine candidates against L. infantum infection. The intravenous challenge with purified amastigotes used in this study allowed the development of infection in all animals inoculated (as confirmed by the detection of parasite in the different tissues and organs collected 6 months after inoculation). Molecular and serologic techniques were efficient methods for the follow-up. Lymph node and bone marrow aspirates were suitable clinical samples to detect the presence of Leishmania parasites. Despite ELISA was highly sensitive in detecting specific anti-Leishmania antibodies the use of two tests can improve the sensitivity and specificity of serological diagnosis.     

Leishmania infantum DNA detection in Phlebotomus tobbi in a new northern focus of visceral leishmaniasis in Iran

Objective

To identify the vector(s), the parasite and the species composition of sand flies in the district during May-October 2012.

Methods

For reaching our objectives we used polymerase chain reaction of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism.

Results

Two species of Phlebotomus sergenti and Phlebotomus tobbi were the most prevalent among 8 species identified comprising 51.1% and 32.9% respectively. Among the 160 specimens of female sand flies tested by polymerase chain reaction of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphisms, only 1 out of 80 Phlebotomus tobbi (1.25%) were positive to Leishmania infantum parasites.

Conclusions

Our finding showed that Phlebotomus tobbi may play as a vector to circulate the parasite of Leishmania infantum among reservoir(s) and human.     

Experimental and field investigations on the role of birds as hosts of Leishmania infantum, with emphasis on the domestic chicken

In this study, 19 chickens were experimentally infected by Leishmania infantum and tissue samples, collected at different times, were cultured and subjected to conventional PCR and/or real time PCR (qPCR) to assess their susceptibility to infection. In addition, 121 serum samples from rural chickens (n = 73) and backyard birds (n = 48) were tested for anti-L. infantum antibodies by indirect immunofluorescence test. All the 19 animals showed to be molecularly positive at least at one tissue sample. In particular, 26 tissue samples from the experimentally infected chickens were positive on conventional PCR and/or qPCR but no clinical signs or seroconversion were detected and all tissue cultures were negative. Accordingly, all serum samples from rural chickens were negative whereas four (8.4%) from game birds (three Anser anser and one Phasianus colchicus) were positive. These results indicate that chickens are not suitable hosts for L. infantum under experimental condition. The occurrence of anti-L. infantum antibodies in domestic gooses (A. anser) and in a pheasant (P. colchicus) points out their possible role in the epidemiology of visceral leishmaniasis.     

Differences in transmission seasons as an epidemiological tool for characterization of anthroponotic and zoonotic cutaneous leishmaniasis in northern Afghanistan

Regional epidemiological data, when available from Afghan or international health authorities, usually include cutaneous leishmaniasis cases without further elaboration. Scientific reports from Afghanistan mainly focus on the current status of war and refugee-related anthroponotic cutaneous leishmaniasis (ACL), but little is known about zoonotic cutaneous leishmaniasis (ZCL), its regional and seasonal distribution, or other disease characteristics. Multiple field investigations revealed that both ACL and ZCL are widespread in Afghanistan and may show sharp differences in specific epidemiology and incubation periods. The previously unknown transmission dynamics and differing seasonality of ZCL, with maximum clinical cases in September and October, as opposed to the ACL peak in March and April, are here described, thus permitting for the first time prediction of the causative Leishmania species in undiscriminated CL reports. Results show that epidemiological differences may serve as a convenient tool for discriminating between ACL and ZCL, at least in northern and central Afghanistan, which can be important because specific treatment and control measures may be different.     

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.