Detection of Plasmodium falciparum and P. vivax Mixed Infection Level and Identification of Pfcrt and Pfmdr1 Resistance Mutations in North-West Ethiopia

Abstract

This study was conducted to assess the level of mixed infections and prevalence of chloroquine transporter gene point mutations that are important determinants of drug resistance level in P. falciparum. A total of 7,343 patients were diagnosed for malaria and the level of mixed infections of P. falciparum and P. vivax was also determinedmicroscopically. Dried blood spots were prepared from 168 positive samples and parasite DNA extracted by commercial kit and used for real-time PCR analysis. Out of the total 7, 343 samples 1,802 (24.54%) microscopically positive for plasmodium, (67.48%) were P. falciparum, (30.68%) P. vivax and (1.8%) mixed infection of both species. Among 168 positive samples that were selected for further analysis, 7 (4.17%) were P. vivax, 158 (94.05%) were P. falciparum and 3 (1.80%) were mixed infection of both species. The real-time PCR analysis of these 168 positive samples showed that 21 (12.50%) were P. falciparum and P. vivax mixed infections, 17 (10.12%) were P. ovale, 10 (5.95%) were P. vivax and 112 (66.67%) were P. falciparum. The genotyping of real-time PCRP. falciparum positive samples for (Pfcrt, K76T), (Pfmdr1, N86Y) and copy number variation showed (54.88%) were confirmed as mutant (Pfcrt, 76T) and the rest (45.11%) were wild type (Pfcrt, K76). The analysis of (Pfmdr1, N86Y) showed (45.86%) had single copy and the rest (54.13%) had multi-copy higher than 1.5 copies per genome. The results indicated (27.38%) difference and disagreement between microscopy and real- time PCR for mixed species detection and misdiagnosis. The present study showed a high prevalence and fixation of (Pfcrt, 76T) mutations 10 years after chloroquine withdrawal, but statistically insignificant (P=0.635, P>0.05). A few samples showed xvii mutant Pfmdr1, butprevalence of Pfmdr1 copy number variants was high suggesting the presence of inducing factors other than chloroquine for emergence of P. falciparum strains with higher copy numbers. However, Prevalence of (Pfmdr1, N86Y) copy number variant was not statistically significant (P= 0.455, P>0.05). This study recommends regular clinical diagnosis quality control and molecular surveillances on drug resistance malaria. The use of chloroquine for P. vivax treatment under poor diagnosis facilities should be discontinued. Key words: Mixed infections, Plasmodium falciparum, P. vivax, microscopic diagnosis, real-time PCR.