BackgroundThe 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.MethodsA 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.ResultsAccording 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.ConclusionsThe 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.

BackgroundNasopharyngeal carcinoma (NPC) is a malignant tumor which arises in surface epithelium of the posterior wall of the nasopharynx. There's is evidence that Epstein Barr virus (EBV) is associated to NPC development. However, many epidemiologic studies point to a connection between viral infections by the human papillomavirus (HPV) and NPC.MethodSeventy Moroccan patients with NPC were screened for EBV and HPV. EBV detection was performed by PCR amplification of BZLF1 gene, encoding the ZEBRA (Z Epstein-Barr Virus Replication Activator) protein, and HPV infection was screened by PCR amplification with subsequent typing by hybridization with specific oligonucleotides for HPV types 16, 18, 31, 33, 35, 45 and 59.ResultsThe age distribution of our patients revealed a bimodal pattern. Sixty two cases (88.9%) were classified as type 3 (undifferentiated carcinoma), 6 (8.6%) as type 2 (non keratinizing NPC) and only 2 (2.9%) cases were classified as type 1 (keratinizing NPC). EBV was detected in all NPC tumors, whereas HPV DNA was revealed in 34% of cases (24/70). Molecular analysis showed that 20.8% (5/24) were infected with HPV31, and the remaining were infected with other oncogenic types (i.e., HPV59, 16, 18, 33, 35 and 45). In addition, statistical analysis showed that there's no association between sex or age and HPV infection (P > 0.1).ConclusionOur data indicated that EBV is commonly associated with NPC in Moroccan patients and show for the first time that NPC tumours from Moroccan patients harbour high risk HPV genotypes.

Disease manifestation after infection with cutaneous Leishmania species is the result of a complex interplay of diverse factors, including the immune status of the host, the infecting parasite species, or the parasite load at the lesion site. Understanding how these factors impact on the pathology of cutaneous leishmaniasis (CL) may provide new targets to manage the infection and improve clinical outcome. We quantified the relative expression of 170 genes involved in a diverse range of biological processes, in the skin biopsies from patients afflicted with CL caused by infection with either L. major or L. tropica. As compared to healthy skin, CL lesions bear elevated levels of transcripts involved in the immune response, and conversely, present a significant downregulation in the expression of genes involved in epidermal integrity and arginine or fatty acid metabolism. The expression of transcripts encoding for cytotoxic mediators and chemokines in lesions was inversely correlated with the expression of genes involved in epidermal integrity, suggesting that cytotoxicity is a major mediator of CL pathology. When comparing the transcriptional profiles of lesions caused by either L. major or L. tropica, we found them to be very similar, the later presenting an aggravated inflammatory/cytotoxic profile. Finally, we identified genes positively correlated with the parasite load in lesions. Among others, these included Th2 or regulatory cytokines, such as IL4 or IL10. Remarkably, a single gene among our dataset, encoding for tryptophan-2,3-deoxygenase (TDO), presented a negative correlation with the parasite load, suggesting that its expression may restrict parasite numbers in lesions. In agreement, treatment of macrophages infected with L. major in vitro with a TDO inhibitor led to an increase in parasite transcripts. Our work provides new insights into the factors that impact CL pathology and identifies TDO as a restriction factor for cutaneous Leishmania.

BackgroundLeishmaniases 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 presentationIn 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (10.1186/s40249-018-0413-8) contains supplementary material, which is available to authorized users.

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