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

Abstract

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.

Efficiency of noninvasive sampling methods (swab) together with Polymerase Chain Reaction (PCR) for diagnosing American Tegumentary Leishmaniasis

“…33 is around 10,000 copies per parasite. However, false-positive results, due to extremely sensitive reactions, are a concern 29 .…”

Inadequacy of 12-Week Miltefosine Treatment for Indian Post-Kala-Azar Dermal Leishmaniasis

“…Among the antigen-based studies, PCR is considered as more sensitive than microscopy. 8 At active disease, all the 18 analyzable patients were positive but became negative for ITS1-PCR after 4 weeks of treatment. To validate this, we selected another region (116-bp fragment of the minicircle kDNA) of L. donovani and performed normal PCR and RT-PCR (40 cycles), based on the kDNA copy number being higher in Leishmania parasites, the latter being considered more sensitive than ITS1-PCR 8 ; here too, PCR was negative after 4 weeks of treatment.…”

Clinicopathological presentation of cutaneous leishmaniasis and its diagnostic algorithm

“…PCR technique is rapid, sensitive and specific but not easily available [32,33]. 70%-75% of estimated incidence of global cutaneous leishmaniasis are seen in Afghanistan, Algeria, Colombia, Brazil, Iran, Syria, Ethiopia, North Sudan, Costa Rica and Peru [34] (Table 3).…”