Cutaneous leishmaniasis by Leishmania infantum: behind granulomatous lesions of unknown aetiology.

BACKGROUND: Cutaneous leishmaniasis (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. Mucosal manifestations (ML) are misdiagnosed due to the fact that they often mimic cancer. OBJECTIVES: Given that leishmaniasis may be masked as different granulomatous diseases in Leishmania infantum endemic areas, the aim of this study was to verify this misdiagnosing and contributes to the improvement of CL/ML diagnosis. METHODS: A retrospective study involving formalin-fixed paraffin-embedded tissue biopsies with histopathological features of granulomatous lesions of unknown origin (GLUO) detected in 17 patients. This study included 13 patients with CL that was used as positive controls, nine patients with other confirmed diseases used as negative controls and seven patients with histological features suggestive of CL or ML without confirmation. Molecular analysis was blindly performed using two different PCR techniques. RESULTS: The PCR detected 15 CL cases in which the diagnosis was neither clinically nor histologically suspected. Leishmaniasis was confirmed in seven suspected patients in whom the classical techniques failed to detect the parasite. L. infantum was identified in all cases. A systematic review of CL cases in GLUO patients from European countries identified 45 reported cases. CONCLUSIONS: 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/ML and must be performed routinely in any granulomatous dermatitis, even when the morphological features are no stereotypical of leishmaniasis.

Comparison of PCR-based methods for the diagnosis of cutaneous leishmaniasis in two different epidemiological scenarios: Spain and Morocco.

BACKGROUND: Cutaneous leishmaniasis (CL) is a disfiguring and stigmatising disease occurring in more than 70 countries across the world including Spain and Morocco. The use of sensitive tests that can differentiate Leishmania species is advised. OBJECTIVE: To evaluate the influence of the epidemiological scenario on the reliability of the PCR techniques and contribute to the selection of the most efficient one for CL diagnosis. METHODS: The sensitivities 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. RESULTS: None of the PCRs used in this study allowed 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 with CL confirmed by 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 did not show enough sensitivity with paraffin-embedded tissue biopsies. False-negative results were obtained in 19% of PSH due to unspecific hybridizations of ITS-1 primers with human chromosome1. CONCLUSIONS: PCR should be routinely used in patients with cutaneous lesions compatible with CL and furthermore, the combination of two PCR techniques is advisable. 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 seems an appropriate choice, whereas in areas such as Morocco, Lmj4/Uni21 and ITS-1 provide satisfactory results.

Phlebotomus langeroni Nitzulescu (Diptera, Psychodidae) a new vector for Leishmania infantum in Europe.

Burrows of the wild rabbit, Oryctolagus cuniculus, a lagomorph that has been recently suggested as a Leishmania infantum reservoir, constitute an unspoilt biotope in phlebotomine studies in Europe. We hypothesize that Phlebotomus langeroni, a proven vector of L. infantum in North Africa, is associated with rabbits and may have been overlooked in Europe. Sandfly captures were carried out with CDC light traps in an L. infantum endemic area of southern Spain with a high density of lagomorphs and a large numbers of burrows. The stable, permanent, and highly abundant presence of P. langeroni was assessed. After morphological identification, this sandfly species was characterized by comparing it with P. perniciosus and other P. langeroni populations from North Africa through molecular techniques. P. langeroni had not been found in southern Spain to date, despite being a highly investigated area, except for this particular biotope. Its activity period turned out to begin in mid-July, ending in late October, accounting for a maximum activity during this month. This study shows that P. langeroni is associated with the existence of rabbit burrows and has been overlooked in Europe. L. infantum DNA was found in almost half of the female specimens (47.6%) captured inside a biotope where wild rabbits are infected as well.

The sesquiterpene (-)-α-bisabolol is active against the causative agents of Old World cutaneous leishmaniasis through the induction of mitochondrial-dependent apoptosis.

Cutaneous leishmaniasis treatment remains challenging due to the absence of a satisfactory treatment. The screening of natural compounds is a valuable strategy in the search of new drugs against leishmaniasis. The sesquiterpene (-)-α-bisabolol is effective in vivo against visceral leishmaniasis due to Leishmania infantum, but its mechanism of action remains elusive. The aim of this study is to validate this promising compound against the causative species of Old World cutaneous leishmaniasis and to get an insight into its antileishmanial mode of action. The compound was evaluated on L. tropica promastigotes and intracellular amastigotes using bone marrow-derived macrophages and its cytotoxicity was evaluated on L929 fibroblasts. The reactive oxygen species generation was evaluated using a sensitive probe. Mitochondrial depolarization was assessed evaluating the fluorescence due to rhodamine 123 in a flow cytometer. Apoptosis was investigated by measuring the fluorescence due to annexin V and propidium iodide in a flow cytometer. The ultrastructure of treated promastigotes and intracellular amastigotes was analysed through transmission electron microscopy. (-)-α-Bisabolol was active against L. tropica intracellular amastigotes displaying an inhibitory concentration 50 % of 25.2 µM and showing low cytotoxicity. This compound induced time and dose-dependent oxidative stress, mitochondrial depolarization and phosphatidilserine externalization (a marker of apoptosis). These effects were noticed at a low concentration and short exposure time. In the ultrastructural analyses, the treated parasites showed mitochondrial disruption, presence of electron-dense structures and chromatin condensation. These results suggest that this natural compound induces oxidative stress and mitochondrial-dependent apoptosis on Leishmania without disturbing the plasma membrane.

Differential ecological traits of two Phlebotomus sergenti mitochondrial lineages in southwestern Europe and their epidemiological implications.

OBJECTIVES: The introduction of leishmaniasis in a new area requires 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Leishmania infantum in wild rodents: reservoirs or just irrelevant incidental hosts?

Wild rodents constitute a very large biomass of potential reservoirs for Leishmania spp. Therefore, an epidemiological study was carried out in a well-known focus of canine leishmaniasis from southern Spain, with the objective of detecting and characterizing Leishmania infantum infection in wild rodents. Blood, liver, spleen, bone marrow, and skin from 37 rodents (24 Apodemus sylvaticus, 9 Rattus rattus, and 4 Mus musculus) were analyzed 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, in a variety of tissues: bone marrow, spleen, or healthy skin (ear lobe). 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.

Effectiveness of the sesquiterpene (-)-α-bisabolol in dogs with naturally acquired canine leishmaniosis: an exploratory clinical trial.

The use of natural products is a promising approach for treating visceral leishmaniosis. (-)-α-Bisabolol is a sesquiterpene that have been proved active in vivo on Leishmania infantum-infected mice without showing toxicity. A single-centre, parallel-group, randomized, exploratory study was designed to assess its efficacy in a canine leishmaniosis model involving naturally infected dogs. In this clinical trial, 12 dogs were allocated into two groups and were treated with either meglumine antimoniate (100 mg/kg) through subcutaneous route or (-)-α-bisabolol (30 mg/kg) through oral route for two treatment series of 30 days, separated by a 30-day interval. A 4-month follow-up period was established as well. Parasite loads in bone marrow, lymph node and blood were estimated through quantitative PCR. Antibody titres were determined through immunofluorescence antibody test and cytokine expression values were estimated through real-time reverse transcription-PCR. Treatment safety was assessed through the evaluation of weight, gastrointestinal alterations and hematological and biochemical parameters in blood. Analyses were performed before and after treatment, and after a 4-months follow-up period. Treatment with the sesquiterpene was effective at decreasing parasite loads and increasing gamma-interferon expression level. Dogs treated with (-)-α-bisabolol did not show any toxicity sign. These results were better than those obtained using the reference drug, meglumine antimoniate. The natural compound seemed to induce a Th1 immune response that led to parasitological and clinical improvement without showing any safety issue, suggesting a high potential for the treatment of canine and human visceral leishmaniosis.

Genetic variability and infective ability of the rabbit trypanosome, Trypanosoma nabiasi Railliet 1895, in southern Spain.

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 of this study 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.

Hair parasite load as a new biomarker for monitoring treatment response in canine leishmaniasis.

Canine leishmaniasis treatment focuses on the reduction of parasite load, the clinical improvement of the animal, and the avoidance of relapses, in a scenario where the definitive parasite clearance is not achievable. Therefore, monitoring is crucial during the treatment of this disease. Quantitative PCR has been shown as an ideal tool for the treatment monitoring when quantifying parasite load in target organs such as lymph node or bone marrow, tissues that are too invasive for regular evaluation. This study aims to prove the potential of hair parasite load in the treatment monitoring of canine leishmaniasis. Six dogs were treated with meglumine antimoniate and monitored up to four months after the end of the treatment. Parasite loads in bone marrow, blood, lymph node and hair were quantified by real-time quantitative PCR. Total IgG, IgG1, and IgG2 antibody titres were analysed by immunofluorescent assay and a clinical assessment was carried out. Treatment consisted of two 28-day courses of meglumine antimoniate (100mg/kg/day) separated by an one-month interval. Analyses were performed before (day 0), during (day 60) and after treatment (day 120), and at the end of a follow-up period (day 210, four months after the end of treatment). Hair parasite load turned out to be strongly correlated with bone marrow, lymph node and blood parasite loads and with the clinical score and the IgG1 antibody titre. The evolution of this biomarker reflects the evolution of the parasitological, immunological and clinical state of the dog, highlighting its potential as a non-invasive marker for the treatment monitoring in canine leishmaniasis.

High rates of Leishmania infantum and Trypanosoma nabiasi infection in wild rabbits (Oryctolagus cuniculus) in sympatric and syntrophic conditions in an endemic canine leishmaniasis area: epidemiological consequences.

Leishmania infantum infection has been reported in various host species, both domestic and wild, in some cases with high prevalence rates. However, until the recent discovery of infected hares, no studies had provided clear evidence of any significant reservoir other than domestic dogs. Our focus was on another lagomorph, Oryctolagus cuniculus or wild rabbit. This species is native to the Iberian Peninsula and its presence and abundance gave rise to the name of Spain. In an endemic area for canine leishmaniasis in the southeast of Spain, 150 rabbits were captured over a period of three years. 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.