Molecular diagnosis of cutaneous leishmaniasis and identification of the causative Leishmania species in Morocco by using three PCR-based assays

Background: The diagnosis of cutaneous leishmaniasis (CL) might be difficult, in particular in endemic areas where different species of Leishmania can cause lesions of very similar appearance and where other skin diseases with similar clinical symptoms occur. Even today, the parasitological diagnosis of CL remains the gold standard and it is based on the direct identification of amastigotes in microscopy smears and/or culture of promastigotes from infected tissues. Although these techniques are highly specific, they are not sensitive enough. The objective of this study is to contribute to improving the diagnosis of CL and the identification of Leishmania species in Morocco by comparing three PCR-based assays applied directly on dermal samples. Methods: A total of 58 patients presenting with cutaneous lesions suggestive of CL were sampled for parasitological diagnosis by direct examination (DE), culture in NNN medium, two kinetoplast DNA (kDNA) PCRs (Lmj4/Uni21 and 13A/13B primers) and one rRNA gene internal transcribed spacer 1 (ITS1) PCR (LITSR/L5.8S primers). The techniques were statistically analyzed and compared. Results: According to our consensus positive, 44 out of 58 samples were true positives. The 13A/13B-PCR and ITS1-PCR showed the highest sensitivities (100%). Parasite microscopy and culture detected 43% and 29% of the true positives, respectively, while culture and microscopy together improved sensitivity to 52%. PCRs 13A/13B and ITS1 were associated to four and one false positives, respectively, while the other assays were 100% specific. Furthermore, the ITS1-PCR-RFLP assay clearly identified the Leishmania species for all the true positives (44/44), whereas Lmj4/Uni21-PCR identified 35/44 samples. The comparison between the Leishmania molecular characterizations and the expected species according to the national data from the Ministry of Health indicate 7 discrepant results. Conclusions: The PCR-based assays tested on our samples increased the speed and sensitivity of the diagnosis of CL compared to the conventional techniques. Furthermore, we showed that we can not base the species identification on the national data from the Ministry of Health. Finally, we suggest the use of PCR-ITS1-RFLP for diagnosis and simultaneous identification of the species in the Moroccan epidemiological context, but also in similar areas of the Mediterranean Basin.This study was supported by the University of Granada, Spain (CICODE program), the Faculty of Medicine and Pharmacy of Casablanca (Morocco), and the PHC Volubilis program

Morphology does not allow differentiating the species of the Phlebotomus perniciosus complex: Molecular characterization and investigation of their natural infection by Leishmania infantum in Morocco

Morphological and DNA-based complemented approaches were applied for characterization of sympatric populations of Phlebotomus longicuspis and Phlebotomus perniciosus in Morocco. Both sand fly species are generally recorded in sympatry in North Africa but on few occasions have been molecularly characterized. The diagnostic confusion of these species has led to errors in their geographical distribution and probably, in the assignment of their role in the transmission of L. infantum. Sand flies were caught inside households in El Borouj, central Morocco, in 2014–2015. For female sand flies, detection of L. infantum natural infection and blood meal identification were carried out. According to morphological identification, Phlebotomus longicuspis s.l. (34.7%) was the second most abundant Phlebotomus species after P. sergenti, followed by atypical Phlebotomus perniciosus (7.1%); 11.6% of the male specimens of P. longicuspis s.l. were identified as P. longicuspis LCx according to the number of coxite setae. The density of Larroussius species was very high (31 Larroussius/light trap/night) in the peripheral neighbourhood of Oulad Bouchair (p = 0.001) where the first case of cutaneous leishmaniasis due to Leishmania infantum was detected in 2017. Phylogenetic trees based on three independent genes highlighted three well-supported clusters within P. perniciosus complex that could be interpreted as corresponding to P. perniciosus, P. longicuspis s.s. and an undescribed species, all coexisting in sympatry. Some females with typical morphology of P. longicuspis were genetically homologous to P. perniciosus. The taxa cannot be differentiated by morphological methods but characterized by a distinctive genetic lineage for which the synapomorphic characters are described. Leishmania infantum was detected in females of all clusters with a low parasite load. Population genetics will help to assess the threat of the geographical spreadUniversity of Granada (Centro de Iniciativas de Cooperación al Desarrollo, CICODE, 2013)Universidad de Granada/CBU

Concomitant visceral and localized cutaneous leishmaniasis in two Moroccan infants

Background: Leishmaniases are vector-borne diseases caused by the protozoa of the Leishmania genus. The clinical spectrum of these diseases extends from benign dermal lesions to visceral forms. In the Mediterranean region, zoonotic visceral leishmaniasis (ZVL) is caused by L. infantum. If untreated within two years, the disease usually leads to death. In Morocco, ZVL is endemic in the north, with a hundred cases notified each year, mostly in children aged below five years. Here, we report on two clinical observations in infants presenting unusual concomitant VL and cutaneous leishmaniasis (CL) in Morocco. Case presentation: In this case study, we report on two infants aged nine and 12 months old. They both have a history of febrile splenomegaly, anemia, and pallor of mucous membranes. Visceral leishmaniasis was confirmed by parasitological diagnosis (positive bone marrow smear and screening of anti-L. infantum antibodies). However, the clinical examination also showed cutaneous lesions that suggested the presence of CL. This was reinforced by the patients having a history of living or traveling to endemic foci. Thus, direct examination, culture, and PCR-RFLP (ITS1- Hae 3) were carried out on the patients’ dermal exudates. In one of the infants, CL was associated with L. infantum, while in the other it was associated with L. tropica. The infants were treated as according to the recommendations of the Ministry of Health. Both patients were cured in two months; defervescence, reduction of splenomegaly, and healing of cutaneous lesions were all observed. Conclusions: These singular patients illustrate the clinical polymorphism of CL and the necessity of updating the differential diagnosis of leukemia-like syndromes, including VL, in children living in or travelling to known endemic areas. These observations suggest a change in the Mediterranean VL phenotype that may be associated with CL

Molecular diagnosis of Pseudoterranova decipiens s.s in human, France

Background: Anisakis and Pseudoterranova are the main genera involved in human infections caused by nematodes of the Anisakidae family. Species identification is complicated due to the lack of differential morphological characteristics at the larval stage, thus requiring molecular differentiation. Pseudoterranova larvae ingested through raw fish are spontaneously eliminated in most cases, but mechanical removal by means of endoscopy might be required. To date, only very few cases of Pseudoterranova infection have been reported in France. Case presentation: A 19-year-old woman from Northeastern France detected, while brushing her teeth, a larva exiting through her mouth. The patient who presented with headache, diarrhea, and abdominal cramps reported having eaten baked cod. The worm was a fourth-stage larva with a size of 22 × 0.9 mm, and molecular biology identified it as Pseudoterranova decipiens sensu stricto (s. s.). In a second P. decipiens infection case, occurring a few months later, a worm exited through the patient’s nose after she had eaten raw sea bream. Conclusion: These two cases demonstrate that Pseudoterranova infection is not uncommon among French patients. Therefore, molecular techniques should be more widely applied for a better characterization of anisakidosis epidemiology in France

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

The objectives of the present work are: To evaluate the influence of the epidemiological scenario from two endemic areas, Spain and Morocco, on the reliability of the PCR techniques and contribute to the selection of the most efficient one for cutaneous leishmaniosis and typing Leishmania species. To contribute to the improvement in the diagnosis of cutaneous and mucosal leishmaniasis through the establishment of a diagnosis protocol from histological samples. To analyse the composition and distribution of Phlebotomus sergenti mitochondrial lineages in Southwestern Europe using a novel PCR-RFLP for the Cytochrome B population analysis. To investigate Leishmania infantum infection in wild rabbits (Oryctolagus cuniculus) and discuss their potential role as reservoirs in a recognized endemic area with ample epidemiological knowledge, as is the case of Southeastern Spain. The sympatric and syntrophic presence of other kinetoplastid, Trypanosoma nabiasi, was taken into consideration to avoid confusion. To detect and characterize L. infantum infection in wild rodents in an epidemiological study in a well-known focus of canine leishmaniasis due to L. infantum in Southern Spain. To provide an insight into the genetic variability of Trypanosoma nabiasi, its in vitro cultivation and infectivity, and finally investigate the course of infection in its host (O. cuniculus).Tesis Univ. Granada. Programa Oficial de Doctorado en FarmaciaEl grupo de investigación de la Junta de Andalucía BIO-176El proyecto del CICODE, Universidad de Granada “Mejora del diagnóstico y la caracterización de la leishmaniosis cutánea en Settat y Sidi Kacem (Marruecos). IX Convocatoria de proyectos de cooperación universitaria para el desarrollo, transferencia de conocimientos en el ámbito de la acción social y sensibilización y educación para el desarrollo” de un año de duración (2010).El proyecto del Ministerio de Ciencia e Innovación “Sobre el posible establecimiento de Leishmania tropica en España y la expansión altitudinal de los vectores de Leishmania infantum en el sudeste de España” del Ministerio (CGL2010-22368-C02-02), de 3 años de duración (2011-2014).El proyecto del Ministerio de Economía y Competitividad y Fondos FEDER “Riesgo de Leishmaniosis en Andalucía Oriental y Canarias: Prevalencia, Factores de Riesgo y Desarrollo de Nuevos Fármacos”, (PI14/01024) de la convocatoria 2014 de “Proyectos de Investigación en Salud” del Instituto de Salud Carlos III, de 3 años de duración (2015-2017)

Evaluation of different Mediterranean essential oils as prophylactic agents in anisakidosis

Context: Anisakis Dujardin 1845 (Anisakidae) nematodes can cause gastrointestinal and allergic diseases when humans eat raw or undercooked seafood containing larvae. There is currently no drug available in the market against this parasitic disease, and the study of plant-derived molecules could be useful in the discovery of effective compounds. Objective: This research assesses the biocidal activity of a range of essential oils (EOs) from some Mediterranean plants against larvae found in the musculature of fresh fish. Materials and methods: EOs composition was analyzed by gas chromatography-mass spectroscopy. All the EOs were diluted at 5% v/v in olive oil to cover the fish with the solutions for 24 h. The larvae that abandoned the muscle and the larvae obtained from the artificial digestion of the fish were collected. Controls were carried out in parallel. Furthermore, Wistar rats were infected with the live larvae collected from the in vitro trials in order to find any larvae that may have penetrated the gastrointestinal wall. Results: Between 60.8% and 87.6% of parasites treated with EOs abandoned the fish muscle, and the highest in vitro mortality rate was achieved with oregano EO (53.9%). Rats previously treated with oregano, cumin and Spanish lavender EOs showed no detectable lesions in the digestive tract due to the infection with larvae. Conclusions: Oregano (Origanum vulgare L. Lamiaceae), cumin (Cuminum cyminum L. Apiaceae) and Spanish lavender (Lavender stoechas L. Lamiaceae) EOs could be used as promising ingredients in the development of products for the control of anisakiasis

Implementación y evaluación de un curso práctico piloto para la captura y recolección de parásitos en grados que contemplen la parasitología como estrategia para innovar en el proceso enseñanza-aprendizaje de esta disciplina

Afrontamos el reto de innovar en la enseñanza-aprendizaje de la Parasitología diseñando una metodología adaptada al nuevo enfoque del EESS, buscando: la participación activa del estudiante, la adquisición y evaluación de competencias y la retroalimentación. Las enseñanzas prácticas de la Parasitología son deficitarias en recogida de muestras y sin embargo estas funciones las realizan nuestros egresados. El modelo de enseñanza/aprendizaje que proponemos supone la realización de tareas fuera de las aulas, proporcionando a los alumnos experiencias de aprendizaje enriquecedoras. Acercamos a los alumnos al entorno de los parásitos y buscamos su competentencia en la toma de muestras en diferentes contexto

Additional file 1: of Concomitant visceral and localized cutaneous leishmaniasis in two Moroccan infants

Multilingual abstracts in the five official working languages of the United Nations. (PDF 346 kb