Genetic Detection of Leishmania Tropica in Clinical Samples from Patients with Cutaneus Leishmaniasis by Using Convenntial PCR and RT-Time PCR

Abstract

Background: cutaneous leishmaniais is an endemic parasitic disease in Iraq.

Objectives:

We applied two kinds of PCR technique: convinential PCR, and RT-PCR on paraffin-embedded skin biopsies to estimate which Leishmania spp. is most prevelence in iraqi population. The detection of Leishmania  by Traditional serological methods was not accurtate, a pair of primers were used to amplify a region within 5.8s ribosomal RNA gene, then the amplified products were sequenced by macrogen company. The Real-time PCR were done to detect the presence of Leishmania troppica sp. by targeting spesific gene ITS. This technique were done to compare its accuricy to the conventional pcr technique. The phylogenetic tree was studided to show the distence among the spesies in Iraq and other countries.

Methods: A total of 26 specimens collected from patients with cutaneous ulcers suggestive of leishmaniasis with age ranging from 5 to 14 years. All those patients were attending hospital and health centers in north Baghdad from July to September of 2019. After the DNA extraction it has been ita has been visuilized by DNA electrophoresis, Real-time PCR has been used to explore the presence of cutaneous Leishmania DNA in samples. Conventional PCR were also done and the amplified products were sent to sequencing.

Results: the results proved that using ITS gene as detection gene is accurate enough. The phylogenetic tree  was shown 48% share identical with isolate from Iran (MH488993) and the least identical percentage showed 20% with isolates from Spain and USA (MN604128 and FJ948452).

Conclusions: This study was proved that RT-PCR has  less invasive sampling, more sensitivity, and specificity than traditional diagnostic methods and recommend it to be used for detection of leishmaniasis in hospitals and research centers in Iraq.