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

Particle and Fibre Toxicology

Myriam Riyad²  Joaquina Martin-Sanchez¹  Hassan Fellah³  Soumiya Chiheb²  Gema Merino-Espinosa¹  Manuel Morales-Yuste¹  Tarik Mouttaki² 

[1] Department of Parasitology, Faculty of Pharmacy, Granada, Spain;Research Team on Cutaneous Leishmaniasis, Faculty of Medicine and Pharmacy, 19 rue Tarik Ibn Ziad, BP. 9154 Casablanca, Morocco;Centre for Doctoral Studies in Health Sciences, Faculty of Medicine and Pharmacy, Casablanca, Morocco

关键词: Morocco;    LITSR/L5.8S primer;    Lmj4/Uni21 primer;    13A/13B primer;    PCR-RFLP;    PCR;    Culture;    Direct examination;    L. infantum;    L. major;    L. tropica;    Cutaneous leishmaniasis;   

Others  :  1150534
DOI  :  10.1186/1756-3305-7-420

 received in 2014-04-22, accepted in 2014-08-28,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Mouttaki et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

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