Increased prevalence of the pfdhfr/phdhps quintuple mutant and rapid emergence of pfdhps resistance mutations at codons 581 and 613 in Kisumu, Kenya

<p>Abstract</p> <p>Background</p> <p>Anti-malarial drug resistance in Kenya prompted two drug policy changes within a decade: sulphadoxine-pyrimethamine (SP) replaced chloroquine (CQ) as the first-line anti-malarial in 1998 and artemether-lumefantrine (AL) replaced SP in 2004. Two cross-sectional studies were conducted to monitor changes in the prevalence of molecular markers of drug resistance over the period in which SP was used as the first-line anti-malarial. The baseline study was carried out from 1999-2000, shortly after implementation of SP, and the follow-up study occurred from 2003-2005, during the transition to AL.</p> <p>Materials and methods</p> <p>Blood was collected from malaria smear-positive, symptomatic patients presenting to outpatient centers in Kisumu, Kenya, during the baseline and follow-up studies. Isolates were genotyped at codons associated with SP and CQ resistance. <it>In vitro </it>IC₅₀values for antifolates and quinolones were determined for isolates from the follow-up study.</p> <p>Results</p> <p>The prevalence of isolates containing the <it>pfdhfr </it>N51I/C59R/S108N/<it>pfdhps </it>A437G/K540E quintuple mutant associated with SP-resistance rose from 21% in the baseline study to 53% in the follow-up study (p < 0.001). Isolates containing the <it>pfdhfr </it>I164L mutation were absent from both studies. The <it>pfdhps </it>mutations A581G and A613S/T were absent from the baseline study but were present in 85% and 61%, respectively, of isolates from the follow-up study. At follow-up, parasites with mutations at five <it>pfdhps </it>codons, 436, 437, 540, 581, and 613, accounted for 39% of isolates. The CQ resistance-associated mutations <it>pfcrt </it>K76T and <it>pfmdr1 </it>N86Y rose from 82% to 97% (p = 0.001) and 44% to 76% (p < 0.001), respectively, from baseline to follow-up.</p> <p>Conclusions</p> <p>During the period in which SP was the first-line anti-malarial in Kenya, highly SP-resistant parasites emerged, including isolates harboring <it>pfdhps </it>mutations not previously observed there. SP continues to be widely used in Kenya; however, given the highly resistant genotypes observed in this study, its use as a first-line anti-malarial should be discouraged, particularly for populations without acquired immunity to malaria. The increase in the <it>pfcrt </it>K76T prevalence, despite efforts to reduce CQ use, suggests that either these efforts are not adequate to alleviate CQ pressure in Kisumu, or that drug pressure is derived from another source, such as the second-line anti-malarial amodiaquine.</p

The role of Pfmdr1 and Pfcrt in changing chloroquine, amodiaquine, mefloquine and lumefantrine susceptibility in western-Kenya P. falciparum samples during 2008-2011.

Single Nucleotide Polymorphisms (SNPs) in the Pfmdr1, and Pfcrt, genes of Plasmodium falciparum may confer resistance to a number of anti-malaria drugs. Pfmdr1 86Y and haplotypes at Pfcrt 72-76 have been linked to chloroquine (CQ) as well as amodiaquine (AQ) resistance. mefloquine (MQ) and lumefantrine (LU) sensitivities are linked to Pfmdr1 86Y. Additionally, Pfcrt K76 allele carrying parasites have shown tolerance to LU. We investigated the association between Pfmdr1 86/Pfcrt 72-76 and P. falciparum resistance to CQ, AQ, MQ and LU using field samples collected during 2008-2011 from malaria endemic sites in western Kenya. Genomic DNA from these samples was genotyped to examine SNPs and haplotypes in Pfmdr1 and Pfcrt respectively. Additionally, immediate ex vivo and in vitro drug sensitivity profiles were assessed using the malaria SYBR Green I fluorescence-based assay. We observed a rapid but steady percent increase in wild-type parasites with regard to both Pfmdr1 and Pfcrt between 2008 and 2011 (p<0.0001). Equally, a significant reciprocate decrease in AQ and CQ median IC50 values occurred (p<0.0001) during the same period. Thus, the data in this study point to a significantly rapid change in parasite response to AQ and CQ in the study period. This may be due to releasing of drug pressure on the parasite from reduced use of AQ in the face of increased Artemisinin (ART) Combination Therapy (ACT) administration following the intervention of the Global Fund in 2008. LU has been shown to select for 76K genotypes, thus the observed increase in 76K genotypes coupled with significant cross resistance between LU and MQ, may herald emergence of tolerance against both drugs in future

Median IC_50s for CQ, AQ, LU and MQ per Year during 2008–2011.

<p>Medians were calculated in Graphpad prism Version 5. Statistical significance was determined using Kruskal Wallis H test.</p

<i>Pfcrt</i> K76T SNP compared against drug IC₅₀ (in nM).

<p>A. Comparison of <i>Pfcrt</i> K76T SNP against CQ IC₅₀ stratified by year; B. Comparison of <i>Pfcrt</i> K76T SNP against AQ IC₅₀ stratified by year; C. Comparison of <i>Pfcrt</i> K76T SNP against MQ IC₅₀ stratified by year; D. Comparison of <i>Pfcrt</i> K76T SNP against LU IC₅₀ stratified by year. Median values are shown. *indicates data is significant.</p

Pearson product-moment correlation for CQ, AQ, MQ and LU.

<p>Correlation was determined in Graphpad prism Version 5 based on IC₅₀ values for each pair of drugs per isolate.</p

<i>Pfmdr1</i> N86Y SNP compared against drug IC₅₀s (in nM).

<p>A. Comparison of <i>Pfmdr1</i> N86Y SNP against CQ IC₅₀ stratified by year; B. Comparison of <i>Pfmdr1</i> N86Y SNP against AQ IC₅₀ stratified by year; C. Comparison of <i>Pfmdr1</i> N86Y SNP against MQ IC₅₀ stratified by year; D. Comparison of <i>Pfmdr1</i> N86Y SNP against LU IC₅₀ stratified by year. Median values are shown. *indicates data is significant.</p

Frequency of <i>Pfcrt</i> 72-76 haplotype and <i>Pfmdr1</i> N86Y SNP.

<p>A. <i>Pfcrt</i> 72-76 haplotype frequency per year; B. <i>Pfmdr1</i> N86Y SNP frequency per year. Statistical significance was determined by Chi-square.</p