SCREENING AND MOLECULAR IDENTIFICATION OF CHLOROQUINE RESISTANT GENES IN PLASMODIUM FALCIPARUM FROM MUZAFFARGARH, PAKISTAN

Abstract

The current research work reports on the incidences of Plasmodium infections and its chloroquine resistant genes from Muzaffargarh, Pakistan. Samples collection continued from November, 2008 to November, 2010 (25 months). The analysis focused on the inter relationship of Plasmodium vivax and P.falciparum infection with particular months, seasons, genders, age groups, socio-economic status, symptoms and the Plasmodium stages. Another core objective of the analysis was the scrutiny of mutant and wild types of pfcrt (codon 72-76) and pfmdr1 N86Y, and their association with different months, seasons, genders, age groups, socio-economic status, symptoms and the Plasmodium stages. The overall positivity rates that consisted of slide positivity rate (SPR), P.vivax positivity rate (VPR) and P.falciparum positivity rate (FPR) were 21.40%, 19.37% and 2.03% respectively. The difference between P.vivax infection (90.49%) and P.falciparum infection (9.51%) was highly significant (χ2=1456; p<0.001). Month- wise variations in incidences of Plasmodium infection were highly significant (χ2=8306.63; p<0.001) and association between P.vivax and P.falciparum infection with monthly variations was highly significant (χ2= 69.8; p<0.001). Season-wise analysis revealed that variations in incidence of Plasmodium infection were highly significant (χ2=1886.08; p<0.001). The association between the incidences of P.vivax and P.falciparum infection with seasonal variations was also found to be highly significant (χ2=44.99; p<0.001). Gender based analyses evidenced that Plasmodium infection was significantly higher (χ2=344.08; p<0.001) in males (69.68%) than females (30.32%). The association between the incidence of P.vivax and P.falciparum infection with gender was found to be non-significant (χ2=0.103; p>0.0.05). Plasmodium infection showed highly significant difference (χ2 =1216.4; p<0.001), when it was analyzed age-wise, whereas, a non-significant (χ2=1.895; p>0.05) association, between the incidence of P.vivax and P.falciparum infection with age groups was noted. People aging between 16 and 30 years were affected far more, both in the case of P.vivax infection (48.78%) and in that of P.falciparum (45.02%). Age group 0 to 5 years was the least hit: 1.00% by P.vivax infection and 0.95% by the P.falciparum. The disease frequency was significantly higher (χ 2=12.41; p<0.001) in lower income class (53.74%) and was comparatively lower (46.26%) in the mediocre. Analysis further discovered that people in the lower socio-economic class were more endangered, both in case of P.vivax infection (53.86%) and in that of P.falciparum infection (52.61%). The association between the incidence of P.vivax and P.falciparum infection with socio-economic status was found to be non-significant (χ2=0.120; p>0.05). In the course of this research work, symptom specific analyses were conducted for both types of the infection. The difference in symptoms was found to be highly significant (χ2=1149.49; p<0.001). Symptoms such as periodic fever, chill and headache were observed more frequently both in P.vivax (82.63%) and P.falciparum infection (82.46%). Symptoms of continuous fever, vomiting and weakness were observed at a lesser degree, both in cases of P.vivax infection (7.72%) and in the P.falciparum (5.21%). The association between the incidence of P.vivax and P.falciparum infections with symptoms was found to be non-significant (χ2=2.97; p>0.05). In case of malarial infection, maximum number (77.75%) of stages observed consisted of trophozoites with gametocytes, whereas the minimum number (0.86%) of stages observed were gametocytes. The difference analyzed in stages was highly significant (χ2=6081.24; p<0.001). Highly significant (χ2=20.60; p<0.001) association was found between the incidences of P.vivax and P.falciparum infection with stages of Plasmodium. Molecular analysis of the P.falciparum positive cases showed that presence of gene pfcrt (codon 72-76) contained in sequence of SagtVMNT was 100%. Sequencing results of pfmdr1 gene fragment showed that wild type pfmdr1 N86 (TAT) existed 33% and pfmdr1 Y86 (AAT) existed 84.30%. The difference in numbers of mutant and wild type was found to be highly significant (χ2=99.64; p<0.001). No significant (p>0.05) association was found between mutant (pfmdr1 Y86) and wild type (pfmdr1N86) with different months, seasons, genders, ages, socioeconomic status, disease symptoms and Plasmodium stages. However, more studies are required to find Patterns of antimalarial drug resistant mutations, especially in endemic areas.