Diseño y evaluación de nuevas técnicas moleculares para la detección y diagnóstico de leishmania spp. Y su aplicación en estudios epidemiológicos

Resumen

Cutaneous leishmaniasis (CL) is a disfiguring and stigmatising disease occurring in more than 70 countries across de world including Spain and Morocco. The use of molecular diagnostic methods, such as PCR, are highly sensitive and specific assays for the detection of Leishmania DNA and the differentiation of species, therefore they are considered the cornerstone in the diagnosis of leishmaniasis. However, there is no consensus concerning the most suitable protocol for molecular diagnosis taking into account the diversity of epidemiological scenarios, this lack of standardization is the main drawback of these useful diagnostic tools. The sensitivities and specificities of parasitological methods and four PCRs were compared in cutaneous samples from 77 patients from Spanish (PSH) and Moroccan hospitals (PMH). Exudates and fresh or paraffin-embedded tissue biopsies were used. None of the PCRs used in this study allows the diagnosis of all CL cases, showing also some drawbacks. Lmj4/Uni21-PCR displayed the best sensitivity with PMH but it did not provide positive results in PSH, although they were positive with other PCRs. Conversely, JW13/JW14¬-PCR and L. infantum-PCR-ELISA displayed good sensitivities with PSH that were not achieved with PMH. Nested-ITS-1-PCR showed higher sensitivity with PSH than PMH. False negative results were obtained in 19% of PSH due to unspecific hybridizations of ITS-1 primers with human chromosome 1. PCR should be routinely used in patients with cutaneous lesions compatible with CL being advisable to combine the use of two PCR techniques. The selection of these PCRs will be influenced by the epidemiological scenario: in areas where L. infantum is endemic, the use of the PCR-ELISA joint with JW13/JW14-PCR or ITS-1 seems an appropriate choice whereas in areas like Morocco, Lmj4/Uni21 and ITS-1 provide satisfactory results. None of the PCRs previously used allows the diagnosis of all CL cases in both epidemiological scenarios analysed. Although the use of a highly sensitive and specific technique, such as the PCR-ELISA, might be enough in Spain, the possibility of imported cases requires the use of another technique capable of detecting other Leishmania species (in the present work 6 imported CL cases due to L. major were detected). However, a PCR-ELISA is hardly implementable in a hospital. This led us to design a new PCR technique capable of differentiating among the three species analyzed in the present work in a real-time setting, characterized by its greater simplicity, rapidity and that would be suitable for a hospital use. This novel PCR is in process of trade mark registration and patent under the name of GRANALEISH Multiplex qPCR. Through the use of serial dilutions, the sensitivity of GRANALEISH Multiplex qPCR is similar to that of PCR-RFLP ITS-1 when L. tropica is the species involved; slightly higher in L. infantum cases and lower at detecting L. major. In clinical samples, the diagnostic efficacy showed by both techniques is similar except for the use of paraffin-embedded samples, for which PCR-RFLP ITS-1 is not satisfactory, in contrast to the good results obtained with our novel PCR. GRANALEISH Multiplex PCR allows the identification of the Leishmania species as satisfactorily as the PCR-RFLP ITS-1 and even better considering that it can differentiate clearly between L. tropica and L. infantum. This novel PCR can be used with a variety of samples, including sandflies and hair, allowing its use in epidemiological studies. CL is underestimated in Spain as in other European countries due to the polymorphism of its clinical manifestations and histopathological features discouraging doctors from suspecting leishmaniasis. Due to CL may be masked as different granulomatous diseases and be overlooked in L. infantum endemic areas, our aim is to verify this misdiagnosing and contribute to the improvement of CL diagnosis. A retrospective study involving skin biopsies with histopathological features of granulomatous lesions of unknown origin (GLUO) detected 17 patients with these characteristics. Additionally, our study included 13 patients diagnosed with CL that were used as positive controls, 9 patients with other confirmed diseases used as negative controls and 7 patients with histological features suggestive of CL or mucosal leishmaniasis (ML) without confirmation. All the samples were formalin-fixed paraffin-embedded (FFPE) tissue biopsies. Clinical and histopathological features were recorded from archived medical histories. Molecular analysis was blindly performed using two different PCR techniques. The use of PCR enabled the detection of 15 CL cases in which the diagnosis of this parasitic disease was neither clinically nor histologically suspected. In addition, leishmaniasis was confirmed in 7 patients in whom this disease was suspected but classical techniques had failed to detect the parasite. L. infantum species was identified in all cases. The mean age of GLUO patients was higher than that of patients in whom leishmaniasis was suspected (68 versus 58 years). A systematic literature review of CL cases in GLUO patients from European countries identified 45 reported cases. In L. infantum endemic areas, a high percentage of GLUO are due to Leishmania infection. The main consequences are delayed diagnosis and underestimation of the real incidence. PCR performed on paraffin-embedded tissue proved to be a reliable tool for diagnosis of CL and must be performed routinely in any granulomatous dermatitis, even when the morphological features are no stereotypical of CL. Molecular techniques have contributed to the study of the epidemiology of leishmaniasis for decades, and the present thesis has followed this trend with the aim of disentangling diverse epidemiological topics that drawn or attention, such as the possible introduction in Spain of other Leishmania species requiring a well-established population of the sandfly vector species of the parasite. No autochthonous cases of anthroponotic cutaneous leishmaniasis have been detected in southwestern Europe, and Leishmania infantum is the only causative agent of leishmaniasis in this area. Phlebotomus sergenti, the main vector of Leishmania tropica, is commonly found in the Iberian Peninsula at sufficient densities to be able to act as a vector. It is characterised by high genetic diversity and classified in four mitochondrial lineages. Our aim was to analyse the composition and distribution of P. sergenti mitochondrial lineages in southwestern Europe given the possibility of phenotypic differences of biomedical importance between them. Sandflies were captured in the Iberian Peninsula and on the Canary and Balearic Islands. Mitochondrial lineage identification of 137 P. sergenti was performed using a novel PCR-RFLP that avoids the necessity of gene sequencing. Two lineages were evidenced, the typical Iberian one (lineage I) and another, held in common with North Africa (lineage III), that show a distinctive distribution. P. sergenti lineage I shows a better correlation to the bioclimatic diversity in southwestern Europe. Conversely, P. sergenti lineage III prefers warmer temperatures and less precipitation, which are typical of the Mediterranean. Therefore, Lineage I seems to have adaptive advantages given its wider tolerance to temperature and altitude than lineage III, and it would seem more suitable to lead a potential geographical expansion towards the rest of Europe. Current knowledge on the eco-epidemiology of a number of parasitic diseases, such as leishmaniasis, required much effort during the last century in order to identify possible reservoirs involved in the life cycle of the parasite in different geographical regions where it is present. Classic parasitological methods have been traditionally used in the diagnosis of leishmaniasis, but the use of molecular techniques, such as PCR, and the availability of a variety of genetic markers have increased the sensitivity in the detection of the parasite. However, the relevance of other potential reservoirs should be taken into account because of their involvement in the control of the disease. Until recently, no study had provided clear evidence of the existence of any relevant reservoir but the dog in the life cycle of L. infantum, a fact that does not discard its existence. The sampling of animals for a parasitic infection is the first step in the identification of new reservoirs. To date Leishmania spp. has been identified and isolated in more than 70 domestic and wild animals belonging to a wide variety of families. Therefore, an epidemiological study was carried out in a well-known canine leishmaniasis (CanL) focus where we captured rabbits (Oryctolagus cuniculus) and wild rodents with the aim of investigating the natural infection by L. infantum and its role as possible reservoirs. For wild rabbits, samples of blood, bone marrow, liver, spleen, heart and skin were taken and analysed through parasitological, serological and molecular techniques in order to detect Leishmania and Trypanosoma. 20.7% of the rabbits were infected with L. infantum and 82.4% with Trypanosoma nabiasi, and 14.8% of mixed infections were detected. Both parasites were found in all the animal organs analysed, a factor which, along with the presence of serological cross-reactions, must be taken into account in epidemiological studies on leishmaniasis. O. cuniculus is an abundant and gregarious species, with a long enough average lifespan to ensure L. infantum transmission. The presence of the parasite in the skin and blood of these rabbits with no acute manifestation of disease ensures its contact with the vector, which finds in their warrens a suitable biotope to inhabit. The rabbit therefore seems to meet the most of conditions for being considered a reservoir host of L. infantum. For wild rodents, blood, liver, spleen, bone marrow, and skin from 37 specimens (24 Apodemus sylvaticus, 9 Rattus rattus, and 4 Mus musculus) were analysed by optical microscopy, culture, and two different polymerase chain reactions. L. infantum DNA was found in 27% (10 out of 37) of the trapped rodents. High prevalences of L. infantum infection were found in the three investigated rodent species. The presence of other trypanosomatids was also evidenced. These rodent species are abundant, widely distributed in Europe, and have a long enough lifespan to overcome the low sandfly activity season. They live in a suitable habitat for sandflies and serve as blood sources for these insects, which can become infected when induced to feed on Leishmania-infected animals. Whether they are reservoirs or just irrelevant incidental hosts, it is clear that the epidemiology of L. infantum is more complex than previously thought, and so is its control. The classic epidemiological cycle dog-sandfly-human is turning into a network of animal species that collaborate with the dog in the maintenance of the parasite under natural conditions and probably showing local differences. Trypanosomes are widespread haemoflagellate protozoans, commonly found in all groups of vertebrates and usually transmitted by arthropods. Non-pathogenic species are those that cause little or no apparent negative effects in the host and it is accepted that Trypanosoma nabiasi is the species that infects the domestic and wild rabbit, Oryctolagus cuniculus. Knowledge about genetic variability, in vitro cultivation and infectivity of this parasite is very scarce, so the aim was to provide an insight on them. The parasite was detected in all the type of samples of 121 wild rabbits. Epimastigotes were visualized and isolated from all the organ cultures types except from skin, and twenty-six strains were isolated and grown in mass. Epimastigote infectivity was assessed in vitro and in vivo. Amastigotes were obtained in infected macrophages from cultured epimastigotes. Furthermore, trypomastigotes were found in the peripheral bloodstream of an experimentally infected naïve domestic rabbit with cultured epimastigotes at the fourth day after infection. The rising titre of antibodies led to the disappearance of the parasite from blood. In addition, this study reports the existence of two T. nabiasi genetic lineages in southern Spain. Phylogenetic analysis places T. nabiasi in the same clade as T. lewisi and other rodent trypanosomes of the subgenus Herpetosoma.