"Plasmodium falciparum" drug resistance, molecular genotyping and generation of a malaria resistance genogram by DNA microarray-based technology

Abstract

Prior to the 2001 malarial treatment policy change in Tanzania, studies were conducted to assess the efficacy of sulfadoxine-pyrimethamine (SP) and usefulness of molecular markers in monitoring SP resistance. In these studies the 1996 WHO protocol (with 14 days follow-up) was used to assess treatment responses. The findings show that SP failure rates were 6.8 – 13.5% and P. falciparum triple-Pfdhfr mutant genotype (18.6 – 21.8 %) were already prevalent prior to the change. Mkuzi site, which due to high CQ failure rate, had been using SP against pediatric malaria since 1984, had exceptionally high failure rate (23.6%) and prevalence of triple-Pfdhfr mutant genotype (80%). Therefore, the study suggested that the drug may have a short useful therapeutic life (UTL) in Tanzania. Hence SP was adopted as an interim first line antimalarial drug in 2001 while combination therapies were being evaluated for long-term use. The molecular findings also pointed to the potential of the triple-pfdhfr mutant genotype as an early warning tool for increasing SP resistance. These data formed the baseline SP efficacy and molecular markers profile in Tanzania prior to the policy change. SP efficacy monitoring studies conducted using the WHO 2002 protocol (with 28 days follow-up) after widespread use of SP showed high (~40%) SP failure rates in Tanzania. Therefore, these findings provided evidence for withdrawing SP use in Tanzania and highly justified the recommendation of Ministry of Health of switching the first line treatment to artemether-lumefantrine (AL). Concurrent with efficacy studies, community surveys were conducted in the health facilities’ catchment areas to assess the profile of all molecular markers of P. falciparum resistance to withdrawn and in-use antimalarial drugs as well as those that have never been officially deployed for use. Results show that molecular markers of SP resistance are more prevalent compared to those recorded prior to adoption of SP, with triple-Pfdhfr mutant genotype ranging from 54 – 74%. The triple- Pfdhfr mutant genotype showed some little evidence of depiction of SP failure rate observed at health facilities. This marker may be applicable as a tool in community-based surveillance of dynamics of SP resistance. However, its usefulness must be further explored by assessing its dynamics relative to SP failure rates in many sites, preferably with different failure rates while taking into account linkage disequilibria of the observed mutations. The frequencies of the main mediators of CQ resistance markers (Pfcrt 76 and Pfmdr1 86 Asn) have decreased following suspension of CQ use in Tanzania, but seems to be influenced by uncontrolled use of QN and AQ for uncomplicated malaria. The lack of the ATPase6 mutation suggest that resistance to artemisinin has not been selected in Tanzania. This observation further supports the decision to adopt AL. The capacity to detect many SNPs in many resistance conferring genes makes DNA micorarray technology a potential tool for monitoring dynamics of P falciparum resistance to both withdrawn, “in-use” and undeployed drugs. However, in order to better appreciate parasite genotypic dynamics following alteration of drug pressure, the interval between assessments should be relatively longer than the one used in this study. In the framework of WHO/TDR combination therapy (ACT) trials of uncomplicated P. falciparum malaria in Sub-Sahara African children, we assessed genotyping performance and use-effectiveness. Parasitological failures were adjusted by stepwise genotyping the P. falciparum glutamine rich protein (glurp), merozoite surface protein 1 (msp1) and 2 (msp2) in Day 0 and post-Day14 recurrent parasitaemias. Recurrences on or before Day 14 were assumed to be recrudescent and were not genotyped. Molecular genotyping refined parasitological outcomes, with differences between crude and adjusted outcomes in most sites >10%. The overall and laboratory performances, (69%) and (78%), respectively, of the stepwise genotyping system were better. However, diligence is needed in sample collection and analysis in order to reduce loss of genotyping data and hence failure to resolve recurrences. Additional genotyping of pre-Day 14 recurrences in Uganda site identified many more new infections and further reduced the PCR-adjusted parasitological failure rate by 8%. Therefore, the study recommends that all recurrent infections in malaria treatment trials/studies in high transmission areas should be genotyped. The stepwise genotyping approach, coupled with more advanced DNA extraction methods needs to be validated and considered for adoption as a standard integral part in malaria drug efficacy studies.