Lenea Campino

Spread of Vector-borne Diseases and Neglect of Leishmaniasis, Europe

Exotic vector-borne diseases are gaining attention at the expense of leishmaniasis.

Methods for diagnosis of canine leishmaniasis and immune response to infection

Canine leishmaniasis (CanL) caused by Leishmania infantum (syn. L. chagasi, in Latin America), which is transmitted by the bite of phlebotomine sand flies, is endemic and affects millions of dogs in Europe, Asia, North Africa and South America. It is an emergent disease in North America. Early detection and treatment of infected animals may be critical in controlling the spread of the disease and is an essential part of human zoonotic visceral leishmaniasis control. The laboratory diagnosis of CanL still poses a challenge, despite progress made in the development of several direct and indirect methods. An effective diagnosis test, apart of being able to confirm a clinical suspicion in a single patient as well as to detect infection in asymptomatic dogs, should have high sensitivity, specificity and reproducibility; it must be simple, easy to perform, non-expensive, feasible in regional laboratories or adaptable for field conditions. Ideally, it should detect all Leishmania-infected dogs, preferentially using non-invasive collection of biological samples. In this paper we review the advantages and shortcomings of the available procedures for CanL diagnosis in the different phases, e.g. pre-patent and patent period of the infection and methods to determine the related immune response.

An experimental model for canine visceral leishmaniasis.

Summary Seven mixed‐breed dogs were challenged with either promastigotes or amastigotes of Leishmania donovani infantum strains recently isolated from naturally infected dogs. Different routes and numbers of parasites were utilized and each dog was monitored for at least 1 year post‐infection. Anti‐parasite specific antibody levels were measured by enzyme‐linked immunosorbence, immunofluorescence, crossed‐immune electrophoresis and Western blotting on crude antigen. Western blotting on two pure parasite proteins, dp72 and gp70‐2. was also done. Mitogenic and antigen‐specific stimulation of peripheral blood lymphocytes was monitored; and the haematological, clinical and parasitological parameters measured. Dogs challenged with amastigotes exhibited a more pronounced humoral response to leishmanial antigens. Only in one case was strong antigen‐specific proliferation detected. Clinical signs of disease, including hypergammaglobulinaemia, enlarged lymph nodes and the presence of parasites, were also more apparent in the dogs challenged with amastigotes. None of the seven dogs died. Serum antibodies to leishmanial antigens were apparent between 15 to 3 months following challenge and correlated with the appearance of enlarged lymph nodes, hypergammaglobulinaemia and the presence of parasites in tissue biopsies. Serum antibodies remained chronically high in these dogs throughout the period of the study. Only one dog (1/3) challenged intravenously with promastigotes and the dog challenged intradermally with amastigotes produced transient antibody responses to leishmanial antigen.

PCR as a rapid and sensitive tool in the diagnosis of human and canine leishmaniasis using Leishmania donovani s.l.-specific kinetoplastid primers

This study was performed in order to test the efficacy of a new polymerase chain reaction (PCR) assay for the diagnosis of both human and canine leishmaniasis caused by Leishmania infantum. The new primers were developed on the basis of a complete DNA sequence of the L. infantum kinetoplast minicircle. Specificity and sensitivity were evaluated by testing bone marrow spots on filter paper and skin biopsy samples, and the PCR results were compared to data from in vitro cultures. Leishmania strains from different foci, as well as other trypanosomatids and opportunistic pathogenic micro-organisms, were also included in this study. The results show that the primers are highly specific, detecting only L. donovani s.l. DNA, and sensitive for the detection of parasite DNA in biological samples from three different geographical regions of Portugal (north, centre and south) and from Brazil.

Differentiation and Gene Flow among European Populations of Leishmania infantum MON-1

Background Leishmania infantum is the causative agent of visceral and cutaneous leishmaniasis in the Mediterranean region, South America, and China. MON-1 L. infantum is the predominating zymodeme in all endemic regions, both in humans and dogs, the reservoir host. In order to answer important epidemiological questions it is essential to discriminate strains of MON-1. Methodology/Principal Findings We have used a set of 14 microsatellite markers to analyse 141 strains of L. infantum mainly from Spain, Portugal, and Greece of which 107 strains were typed by MLEE as MON-1. The highly variable microsatellites have the potential to discriminate MON-1 strains from other L. infantum zymodemes and even within MON-1 strains. Model- and distance-based analysis detected a considerable amount of structure within European L. infantum. Two major monophyletic groups—MON-1 and non-MON-1—could be distinguished, with non-MON-1 being more polymorphic. Strains of MON-98, 77, and 108 were always part of the MON-1 group. Among MON-1, three geographically determined and genetically differentiated populations could be identified: (1) Greece; (2) Spain islands–Majorca/Ibiza; (3) mainland Portugal/Spain. All four populations showed a predominantly clonal structure; however, there are indications of occasional recombination events and gene flow even between MON-1 and non-MON-1. Sand fly vectors seem to play an important role in sustaining genetic diversity. No correlation was observed between Leishmania genotypes, host specificity, and clinical manifestation. In the case of relapse/re-infection, only re-infections by a strain with a different MLMT profile can be unequivocally identified, since not all strains have individual MLMT profiles. Conclusion In the present study for the first time several key epidemiological questions could be addressed for the MON-1 zymodeme, because of the high discriminatory power of microsatellite markers, thus creating a basis for further epidemiological investigations.

QUANTIFICATION OF LEISHMANIA INFANTUM PARASITES IN TISSUE BIOPSIES BY REAL-TIME POLYMERASE CHAIN REACTION AND POLYMERASE CHAIN REACTION–ENZYME-LINKED IMMUNOSORBENT ASSAY

Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan® probes and to compare the efficacy of detection of this technique with a PCR–enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum–infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR– ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR–ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan® PCR.

Drug regimens for visceral leishmaniasis in Mediterranean countries

Until the early 1990s, pentavalent antimony was the only documented first‐line drug employed for the treatment of zoonotic visceral leishmaniasis (VL) in the Mediterranean, with reported cure rates exceeding 95% in immunocompetent patients. The emergence of antimony resistance in other endemic settings and the increase in drug options have stimulated re‐evaluation of the current therapeutic approaches and outcomes in Mediterranean countries. A scientific consortium (‘LeishMed’ network) collected updated information from collaborating clinical health centres of 11 endemic countries of Southern Europe, Northern Africa and the Middle East. In contrast with the previous situation, VL is now treated differently in the region, basically through three approaches: (1) In Northern Africa and in part of the Middle East, pentavalent antimony is still the mainstay for therapy, with no alternative drug options for treating relapses; (2) In some European countries and Israel, both pentavalent antimony and lipid‐associated amphotericin B (AmB) formulations are used as first‐line drugs, although in different patients’ categories; (3) In other countries of Europe, mainly liposomal AmB is employed. Importantly, cure rates exhibited by different drugs, including antimonials in areas where they are still in routine use, are similarly high (≥95%) in immunocompetent patients. Our findings show that antimony resistance is not an emerging problem in the Mediterranean. A country’s wealth affects the treatment choice, which represents a balance between drug efficacy, toxicity and cost, and costs associated with patient’s care.

Importance of cats in zoonotic leishmaniasis in Portugal.

Leishmaniasis, caused by Leishmania infantum, is an endemic zoonosis in the Mediterranean basin. Dogs are considered the major host for these parasites, as well as the main reservoir for human visceral infection. In recent years, asymptomatic infection or clinical disease caused by L. infantum in cats has been reported in several countries where zoonotic leishmaniasis is present. The aim of the present study was to perform a leishmaniasis survey in cats from an endemic focus. Twenty-three adult stray cats were surveyed by clinical examination, and peripheral blood samples for serological and molecular analysis were collected. In 7 of the 23 cats (30.4%) Leishmania DNA was detected in blood. A low level of fluorescent antibodies was detected in four serum samples. All the animals were asymptomatic. Taking into account the high rate of asymptomatic feline leishmaniasis in this survey, it can be suggested that cats may act as a habitual reservoir host of L. infantum infection in endemic areas. Furthermore, it will be important in the future to add this parasitosis to the differential diagnosis of feline infections from leishmaniasis foci in cats. Feline leishmaniasis diagnosis should be accessed by molecular tools.

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis.

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.

Risk factors for canine leishmaniasis in an endemic Mediterranean region.

Human visceral leishmaniasis is an emergent/re-emergent parasitic zoonotic disease in Europe caused by Leishmania infantum, with domestic dog as its main reservoir host. This study presents the results of a canine epidemiological survey in a mediterranean region where human and canine leishmaniasis (CanL) are endemic - Portugal. The main goal was to identify risk factors, which can be relevant for Leishmania infection control. The national survey was carried out in January 2009 with a screening of 3974 dogs from all 18 districts of mainland Portugal. Direct Agglutination Test was used for the detection of anti-Leishmania antibodies in canine blood. An overall CanL true prevalence of 6.31% was observed. Apparent prevalence at district level ranged from 0.88% to 16.16%, with the highest prevalence in the interior regions. Identified risk factors for positivity were: dogs of 2 years and older (adjusted odds ratio OR=5.39); spending exclusively/most of the time outdoors (OR=2.51); origin from the interior of Portugal in comparison to littoral/coast districts (OR=2.51); not having long fur (OR=2.03); and being pure exotic (OR=1.67). The results confirm the leishmaniasis endemicity in Portugal and the dynamic character of prevalence as new foci emerged and old foci lost their importance. The dogs age, fur size, district and living outdoors as opposed to indoors were more important than dog breeds and insecticide treatment in the transmission of Leishmania infection. The future of CanL prevention and control rely on an integrated approach involving veterinarians, dog owners and health authorities in order to reduce the canine infection risk and consequently, the human zoonotic visceral leishmaniasis.