Objectives: The objectives of this study were: to evaluate malaria diagnostic methods, therapeutic efficacy of artemether-lumefantrine (AL), the prevalence of N86Y, Y184F, S1034C and N1042D SNPs in Pfmdr1 gene and their association with AL resistance in Plasmodium falciparum malaria infections in Nigeria.
Methods: This study was carried out within 14 months. One hundred and sixty (160) malaria patients that met criteria were recruited through rapid diagnostic testing (RDT) using first response kit and blood film microscopy after which each patient received a 3-day complete dose of AL treatment. Patients were monitored and followed up on days 0, 3, 7, 14 and 28. Body temperature was taken and 2ml of blood was sampled from each patient into labeled EDTA bottle. Genomic DNA was extracted from each sample by saponin heamolysis. Samples were analyzed by nested polymerase chain reaction (PCR) and real time PCR for identification and quantification of Plasmodium falciparum. Genotyping of Pfmdr 1 gene for specific genetic variants: N86Y, Y184F, S1034C and N1042D were done using Restriction Fragment Length Polymorphism (RFLP-PCR).
The overall performances of diagnostic methods used were analyzed using sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) obtained (with PCR as the gold standard). Data were analyzed using both Graph Pad (Prism 5) and SPSS (version 16). Descriptive statistics presented as counts, percentages, means, and standard deviations, as appropriate, were used to compare the demographic characteristics of the study population and their initial clinical and biological characteristics (such as temperature and geometric mean parasitaemia). Differences in proportions of treatment outcome and frequency in occurrence of Pfmdr 1 gene mutation in pretreatment and post treatment groups were analyzed using the paired T test, Chi-square test or Fisher’s exact test. While the differences in parasitic densities of the various groups were analyzed using ANOVA and inter group comparison was done using Post Hoc test.
Results: Sensitivity, specificity, positive and negative predictive values of 75 %, 75 %, 95 %, 31 % and 92 %, 50 %, 91 %, 55 % were obtained by RDT and microscopy respectively using PCR as gold standard. For therapeutic efficacy, 106 (68.8 %) patients had body temperature of ≥ 37.5 °C with mean temperature of 38.4 °C on day 0 while on day 3, 3.3 % of the patients had temperature of 37.1-38 °C with mean temperature of 36.2 °C. There was significant decrease in temperature from day 0 to day 28 (P < 0.0001). There was significant difference (P < 0.0001) between parasite mean densities on day 0 compared to days 3,7,14 and 28, with high prevalence of delayed parasite clearance. There were 4 cases of late clinical failure for both 14 and 28 day. Adequate clinical and parasitological responses, (PCR uncorrected) were 80 % and 75 % respectively for day 14 and 28. Out of 60 samples with treatment failure that were successfully genotyped, 26 (24.3 %), 24 (23.0 %) and 9 (7.0 %) had Pfmdr 1 N86Y, Y184F and S1034C respectively. Of the sixty, 59 (98.3 %) had one form of mutation or the other. Of the 56 that had good response, 3 (5.4 %) were mutated. The prevalence of Pfmdr 1 mutation was significantly associated with treatment failure (P < 0.001). There was significant increase (P < 0.001) in the prevalence of Pfmdr 1 N86Y mutation from six (20.0 %) to twenty four (92.3 %), Pfmdr 1 Y184F mutation from eight (5.2 %) to twenty five (92.6 %) and Pfmdr 1 S1034C mutation from four (44 %) to nine (100 %) in pre-treatment compared to post-treatment group respectively.Conclusion: Microscopy is more reliable than first response (RDT) using PCR as standard. Therapeutic efficacy of AL in adults has reduced in Enugu State; South Eastern Nigeria. There is high prevalence of Pfmdr 1 N86Y and F184Y mutation with no incidence of Pfmdr 1 N1042D in Enugu, Nigeria. Presence of individual and multiple mutations in the study population is associated to AL resistance by P. falciparum. The presence of AL drug significantly induced genetic variation in the plasmodial genes