The prevalence and antifolate drug resistance profiles of Plasmodium falciparum in study participants randomized to discontinue or continue cotrimoxazole prophylaxis

Abstract

Objective: Cotrimoxazole prevents opportunistic infections including falciparum malaria in HIV-infected individuals but there are concerns of cross-resistance to other antifolate drugs such as sulphadoxine-pyrimethamine (SP). In this study, we investigated the prevalence of antifolate-resistance mutations in Plasmodium falciparum that are associated with SP resistance in HIV-infected individuals on antiretroviral treatment randomized to discontinue (STOP-CTX), or continue (CTX) cotrimoxazole in Western Kenya.

Design: Samples were obtained from an unblinded, non-inferiority randomized controlled trial where participants were recruited on a rolling basis for the first six months of the study, then followed-up for 12 months with samples collected at enrollment, quarterly, and during sick visits.

Method: Plasmodium DNA was extracted from blood specimens. Initial screening to determine the presence of Plasmodium spp. was performed by quantitative reverse transcriptase real-time PCR, followed by genotyping for the presence of SP-resistance associated mutations by Sanger sequencing.

Results: The prevalence of mutant haplotypes associated with SP-resistant parasites in pfdhfr (51I/59R/108N) and pfdhps (437G/540E) genes were significantly higher (P = 0.0006 and P = 0.027, respectively) in STOP-CTX compared to CTX arm. The prevalence of quintuple haplotype (51I/59R/108N/437G/540E) was 51.8% in STOP-CTX vs. 6.3% (P = 0.0007) in CTX arm. There was a steady increase in mutant haplotypes in both genes in STOP-CTX arm overtime through the study period, reaching statistical significance (P < 0.0001).

Conclusion: The frequencies of mutations in pfdhfr and pfdhps genes were higher in STOP-CTX arm compared to CTX arm, suggesting cotrimoxazole effectively controls and selects against SP-resistant parasites.

Trial registration: ClinicalTrials.gov NCT01425073.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Prevalence of haplotype mutations in pfdhfr and pfdhps genes in subjects continuing with cotrimoxazole prophylaxis therapy (CTX) and those who stopped CTX therapy (STOP CTX).

The prevalence was based on the total number of P. falciparum positive samples in each arm. The statistical difference in parasite prevalence between the two arms was determined. A) Haplotype mutations (51I, 59R, 108N and 164L) in pfdhfr gene; B) haplotype mutations (437G, 540E and 581G) in pfdhps gene and; C) haplotype mutations present in both genes. There were statistical significant differences between the STOP CTX and CTX arms in the pfdhfr gene haplotype 51I/59R/ 108N (P = 0.0006), in pfdhps gene haplotype 437G/540E (P = 0.027) and in both genes haplotype 51I/59R/108N/437G/540E (P = 0.0007).

Fig 2. Temporal trends of haplotype mutations in the pfdhfr and pfdhps genes in the STOP-CTX arm.

The prevalence is based on the total number of samples collected at each time point. Temporal trends shown for haplotype mutations in pfdhfr gene (51I/59R/108N), pfdhps gene (437G/540E) and both genes combined (51I/59R/108N/437G/540E).