Plasmodium falciparum DHFR and DHPS Mutations Are Associated With HIV-1 Co-Infection and a Novel DHPS Mutation I504T Is Identified in Western Kenya.

Brandi K Torrevillas,Shirley Luckhart,Karina I Rivas,James T Van Leuven,Nicholas J Hathaway,Dharmeshkumar Patel,Alexander J Mckeeken,John N Waitumbi,Jeffrey A Bailey,Sarah M Garrison,Nathaniel I Dizon,Janet Oyieko,V Ann Stewart,Carolyne M Kifude

doi:10.3389/fcimb.2020.600112

Abstract

Antifolate resistance is significant in Kenya and presumed to result from extensive use and cross-resistance between antifolate antimalarials and antibiotics, including cotrimoxazole/Bactrim used for HIV-1 chemotherapy. However, little is known about antifolate-resistant malaria in the context of newly diagnosed HIV-1 co-infection prior to administration of HIV-1 chemotherapy. Blood samples from a cross-sectional study of asymptomatic adult Kenyans enrolled during voluntary HIV testing were analyzed by PCR for Plasmodium spp. More than 95% of volunteers with identifiable parasite species (132 HIV-1 co-infected) were infected with Plasmodium falciparum alone or P. falciparum with Plasmodium ovale and/or Plasmodium malariae. Deep sequencing was used to screen for mutations in P. falciparum dihydrofolate reductase (dhfr) (N51I, C59R, S108N, I164L) and dihydropteroate synthase (dhps) (S436H, A437G, K540E, A581G) from 1133 volunteers. Individual mutations in DHPS but not DHFR correlated with HIV-1 status. DHFR haplotype diversity was significantly different among volunteers by gender and HIV-1 status. DHPS haplotype diversity by HIV-1 status was significantly different between volunteers paired by age and gender, indicating that patterns of resistance were independent of these variables. Molecular simulations for a novel DHPS mutation (I504T) suggested that the mutated protein has increased affinity for the endogenous ligand DHPPP and decreased affinity for drug binding. A sub-group of monoclonal infections revealed that age and parasitemia were not correlated and enabled identification of a rare septuple-mutant haplotype (IRNL-HGEA). In our study, adult Kenyans newly diagnosed with HIV-1 infection were predominantly infected with moderately resistant P. falciparum, with patterns of infecting parasite genotypes significantly associated with HIV-1 status. Together with the discovery of DHPS I504T, these data indicate that antifolate resistance continues to evolve in Kenya. Further, they highlight the need to understand the effects of associated mutations on both fitness and resistance of P. falciparum in the context of HIV-1 co-infection to better inform treatment for asymptomatic malaria.

Highlights

Repeated episodes of malaria typically result in partial protective immunity in regions of stable and holoendemic transmission
A total of 1762 individuals were enrolled. 1133 yielded detectable signal from blood samples in a quantitative PCR assay for the Plasmodium genus-specific 18S ribosomal RNA gene and 132 were HIV-1 co-infected; among volunteers for which infection could be determined to species (n=670), more than 90% were positive for P. falciparum infection only (n=606), while an additional 6.0% were positive for infection with P. falciparum in combination with P. malariae (n= 28) or P. ovale (n=9), or infected with all three (n=3)
Interpretable sequence data for DHFR (N51I, C59R, S108N, I164L) and DHPS (S436A/H, A437G, K540E, A581G) mutations were collected from 680 volunteers yielding 749 haplotype sequences, including 332 haplotype sequences for dhfr and 417 haplotype sequences for dhps

Summary

Introduction

Repeated episodes of malaria typically result in partial protective immunity in regions of stable and holoendemic transmission. In this context, partial immunity can facilitate chronic parasite carriage and asymptomatic infection, challenging efforts to reduce transmission. Partial immunity can facilitate chronic parasite carriage and asymptomatic infection, challenging efforts to reduce transmission Such persistent, sub-patent infections can sustain parasite genetic diversity, including those genotypes that are drug-resistant, as the predominant reservoir for parasite transmission (Tadesse et al, 2018). A higher prevalence of drug-resistant parasites was detected in a 2014 study of asymptomatic parasitemic Ugandan children relative to infected children with fever (Tukwasibwe et al, 2014). Of particular relevance for our studies, Rutto et al noted higher mean parasite densities in HIV1 co-infected individuals relative to HIV-1 negative individuals in western Kenya (Rutto et al, 2015)

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Results

Conclusion