Detection the effect of the treatment of pentostam on LPG, CPA, and PPG virulence factors of cutaneous Leishmania by real time-PCR

Abstract

The aim of this study was to evaluate the effectiveness of pentostam treatment through effect on some of the virulence factors of cutaneous Leishmania parasite by using the real-time PCR within five weeks, as well as the determination of parasite type by three virulence factors lipophosphoglycan, Cysteine protease and proteophosphoglycans by using nested PCR technique, since that these factors are already were diagnosed in Leishmania parasite. Fifty-five biopsies were taken from skin ulcers which clinically diagnosed as cutaneous leishmaniasis from patients attending Al-Diwaniyah education hospital in Al-Qadisiyah Province for the period between 1 \ 11 \ 2012-1 \ 5 \ 2013 ,whose age ranged from (10-80) years. The DNA was extracted, and then amplified by using primers selected on repetitive kDNA for identification of a Leishmania parasite; it is L. major species and the above three virulence factors, after that DNA electrophoresis was done for the amplified DNA. The results showed that Leishmania parasite was detected in 89.09% of samples. The L. major species was identified in the majority of Leishmania parasite positive samples 98%. RNA was extracted from positive samples collected before and after the treatment with pentostam to measure the amount of gene expression of virulence factors after treatment and to measure the effect of the drug on these factors. After amplification using four of primers, the first three of them were the target genes LPG, CPA, PPG whereas the fourth primer is called the initiator, which is GAPDH. These primers are designed using the NCBI gene bank database. Results showed that there was a high difference for each gene during the period of treatment in a level of the possibility of (P≤0.05) and by comparing the three genes it was found that there was a significant difference between cysteine protease and the other two factor lipophosphoglycan, proteophosphoglycan, whereas no significant difference between lipophosphoglycan, proteophosphoglycan. Hence, it has been clear that the treatment with pentostam has a high effect on parasite by its effect on the virulence factors (LPG, CPA, and PPG) and this explains that possibility of infection may happen again after having the treatment.