Evidence on sulfadoxine-pyrimethamine resistance molecular markers from India: interpret with caution.

BACKGROUND: Sulfadoxine-pyrimethamine (SP), as a partner to artesunate as ACT is the treatment of choice for uncomplicated P. falciparum infections in the majority of India and SP-resistance has a potential to lead to ACT failure. In the lack of robust surveillance of therapeutic efficacy of SP, validate molecular markers of SP-resistance offer a hint of failing SP. However, studies reporting these validated markers often suffer from certain pitfalls that warrant a careful interpretation. MAIN BODY: Critical analyses of the results and their reported interpretations from a recent study and other studies conducted on the WHO-validated molecular markers of SP-resistance in India were analysed and the main problems with studying and reporting of these markers are presented here. It was noted that almost all studies analysed flawed either on the usage, estimation and/or interpretation of the standardized classification of the studies SP mutations. These flaws not only impart spatiotemporal incomparability of the published data but also have the potential of being misunderstood and wrongly translated. CONCLUSION: Based on this universal problem in studying, reporting and interpreting the data from the studies on molecular markers of SP-resistance, it is stressed that the future studies should be conducted with utmost caution so that robust evidence may be generated and correctly translated to policy.

Impact of Sulfadoxine-Pyrimethamine Resistance on Effectiveness of Intermittent Preventive Therapy for Malaria in Pregnancy at Clearing Infections and Preventing Low Birth Weight.

BACKGROUND: Owing to increasing sulfadoxine-pyrimethamine (SP) resistance in sub-Saharan Africa, monitoring the effectiveness of intermittent preventive therapy in pregnancy (IPTp) with SP is crucial. METHODS: Between 2009 and 2013, both the efficacy of IPTp-SP at clearing existing peripheral malaria infections and the effectiveness of IPTp-SP at reducing low birth weight (LBW) were assessed among human immunodeficiency virus-uninfected participants in 8 sites in 6 countries. Sites were classified as high, medium, or low resistance after measuring parasite mutations conferring SP resistance. An individual-level prospective pooled analysis was conducted. RESULTS: Among 1222 parasitemic pregnant women, overall polymerase chain reaction-uncorrected and -corrected failure rates by day 42 were 21.3% and 10.0%, respectively (39.7% and 21.1% in high-resistance areas; 4.9% and 1.1% in low-resistance areas). Median time to recurrence decreased with increasing prevalence of Pfdhps-K540E. Among 6099 women at delivery, IPTp-SP was associated with a 22% reduction in the risk of LBW (prevalence ratio [PR], 0.78; 95% confidence interval [CI], .69-.88; P < .001). This association was not modified by insecticide-treated net use or gravidity, and remained significant in areas with high SP resistance (PR, 0.81; 95% CI, .67-.97; P = .02). CONCLUSIONS: The efficacy of SP to clear peripheral parasites and prevent new infections during pregnancy is compromised in areas with >90% prevalence of Pfdhps-K540E. Nevertheless, in these high-resistance areas, IPTp-SP use remains associated with increases in birth weight and maternal hemoglobin. The effectiveness of IPTp in eastern and southern Africa is threatened by further increases in SP resistance and reinforces the need to evaluate alternative drugs and strategies for the control of malaria in pregnancy.

Low prevalence of dihydro folate reductase (dhfr) and dihydropteroate synthase (dhps) quadruple and quintuple mutant alleles associated with SP resistance in Plasmodium vivax isolates of West Bengal, India.

BACKGROUND: Emergence of chloroquine resistant Plasmodium vivax is a serious obstacle towards malaria control in India. This study elucidates the temporal pattern of antifolate [sulfadoxine-pyrimethamine (SP)] resistance in P. vivax infection by means of genetic polymorphisms, especially analysing the single nucleotide polymorphisms of dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) gene among the field isolates of urban Kolkata Municipal Corporation and rural Purulia region of West Bengal, India. METHODS: Blood samples were collected from 99 microscopically diagnosed P. vivax patients (52 from Kolkata Municipal Corporation and 47 from Purulia). Parasitic DNA was extracted followed by polymerase chain reaction and sequencing of different codons of pvdhfr gene (15, 33, 50, 57, 58, 61, 64, 117, and 173 codons) and pvdhps gene (373, 380, 382, 383, 384, 512, 553, 585, and 601 codons) were performed to identify the mutations. RESULTS: Prevalence of double mutant dhfr A15P33N50F57 R 58 T61V64 N 117 I173 allele (53.85 %) was observed in Kolkata Municipal Corporation (KMC) whereas in Purulia, wild dhfr A15P33N50F57S58T61V64S117I173 allele was predominated (48.94 %). In pvdhps gene a significant number of isolates (17.31 %) in KMC contained the double mutant S373E380S382 G 383 P384K512 G 553 V585M601 allele. pvdhfr and pvdhps combination haplotype revealed the emergence of quadruple (13.46 %) and quintuple (3.84 %) mutant allele in KMC, which might result in poor clinical response against antifolate drugs. CONCLUSION: The study reveals that P. vivax parasites in rural Purulia may still be susceptible to SP but additional caution should be taken for treatment of vivax malaria in KMC to limit the blooming of quadruple and quintuple mutant allele in the remainder of the West Bengal, India.

Genomic characterization of Plasmodium falciparum genes associated with anti-folate drug resistance and treatment outcomes in eastern India: A molecular surveillance study from 2008 to 2017.

Introduction: After being used vigorously for the previous two decades to treat P. falciparum, chloroquine and sulfadoxine-pyrimethamine were replaced in 2009 with an artemisinin-based combination therapy (artesunate-sulfadoxine-pyrimethamine) in an effort to combat multidrug-resistant parasites. Methods: We set out to assess the genetic variants of sulfadoxine-pyrimethamine resistance and the effectiveness of its treatment in eastern India prior to, during, and 6 to 8 years following the introduction of the new pharmacological regime. In 2008-2009, 318 P. falciparum-positive patients got the recommended doses of sulfadoxine-pyrimethamine. We used 379 additional isolates from 2015 to 2017 in addition to the 106 isolates from 2010. All 803 isolates from two study sites underwent in vitro sulfadoxine-pyrimethamine sensitivity testing and genomic characterisation of sulfadoxine-pyrimethamine resistance (pfdhfr and pfdhps). Results: In Kolkata and Purulia, we observed early treatment failure in 30.7 and 14.4% of patients, respectively, whereas recrudescence was found in 8.1 and 13.4% of patients, respectively, in 2008-2009. In 2017, the proportion of in vitro pyrimethamine and sulfadoxine resistance steadily grew in Kolkata and Purulia despite a single use of sulfadoxine-pyrimethamine. Treatment failures with sulfadoxine-pyrimethamine were linked to quintuple or quadruple pfdhfr- pfdhps mutations (AICII-AGKAT, AICII-AGKAA, AICII-SGKGT, AICII-AGKAA, AICNI-AGKAA) in 2008-2009 (p < 0.001). The subsequent spread of mutant-haplotypes with higher in vitro sulfadoxine-pyrimethamine resistance (p < 0.001), such as the sextuple (dhfr-AIRNI+dhps-AGEAA, dhfr-ANRNL+dhps-AGEAA) and septuple (dhfr-AIRNI+dhps-AGEAT), mutations were observed in 2015-2017. Discussion: This successive spread of mutations with high in vitro sulfadoxine-pyrimethamine resistance confirmed the progressive increase in antifolate resistance even after an 8-year withdrawal of sulfadoxine-pyrimethamine.

Selective sweep and phylogenetic models for the emergence and spread of pyrimethamine resistance mutations in Plasmodium vivax.

Pyrimethamine resistance is a major concern for the control of human haemoprotozoa, especially Plasmodium species. Currently, there is little understanding of how pyrimethamine resistance developed in Plasmodium vivax in the natural field conditions. Here, we present for the first time evidence of positive selection pressure on a dihydrofolate reductase locus and its consequences on the emergence and the spread of pyrimethamine resistance in P. vivax in the Punjab province of Pakistan. First, we examined the dihydrofolate reductase locus in 38 P. vivax isolates to look for evidence of positive selection pressure in human patients. The S58R (AGA)/S117N (AAC) double mutation was most common, being detected in 10/38 isolates. Single mutation S117N (AAC), I173L (CTT) and S58R (AGA) SNPs were detected in 8/38, 2/38 and 1/38 isolates, respectively. The F57L/I (TTA/ATA) and T61M (ATG) SNPs were not detected in any isolates examined. Although both soft and hard selective sweeps have occurred with striking differences between isolates, there was a predominance of hard sweeps. A single resistance haplotype was present at high frequency in 9/14 isolates, providing a strong evidence for single emergence of resistance by the single mutation, characteristics of hard selective sweeps. In contrast, 5/14 isolates carried multiple resistance haplotypes at high frequencies, providing an evidence of the emergence of resistance by recurrent mutations, characteristics of soft selective sweeps. Our phylogenetic relationship analysis suggests that S58R (AGA)/S117N (AAC) and S117N (AAC) mutations arose multiple times from a single origin and spread to multiple different cities in the Punjab province through gene flow. Interestingly, the I173L (CTT) mutation was present on a single haplotype, suggesting that it arises rarely and has not spread between cities. Our work shows the need for responsible use of existing and new antimicrobial drugs and their combinations, control the movement of infected patients and mosquito vector control strategies.

High prevalence of sulphadoxine-pyrimethamine resistance-associated mutations in Plasmodium falciparum field isolates from pregnant women in Brazzaville, Republic of Congo.

Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) has not been evaluated in the Republic of Congo since its implementation in 2006 and there is no published data on molecular markers of SP resistance among Plasmodium falciparum isolates from pregnant women. This first study in this country aimed to describe the prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) point mutations and haplotypes in P. falciparum isolates collected from pregnant women with asymptomatic infection. From March 2012 to December 2013, pregnant women attending Madibou health centre (in Southern Brazzaville) for antenatal visits were enrolled in this study after obtaining their written informed consent. Blood samples were collected and P. falciparum infections were characterized using PCR. A total of 363 pregnant women were enrolled. P. falciparum infection was detected in 67 (18.4%) samples as their PCR amplification of dhfr and dhps genes yielded bands and all the PCR products were successfully digested. Out of these 67 isolates, 59 (88%), 57 (85%) and 53 (79.1%) carried 51I, 59R and 108N dhfr mutant alleles, respectively. The prevalence of dhps 436A, 437G and 540E mutations were 67.1% (45/67), 98.5% (66/67) and 55.2% (37/67), respectively. More than one-half of the isolates carried quintuple mutations, with highly resistant haplotype dhfr51I/59R/108N + dhps437G/540E detected in 33% (22/67) whereas 25% (17/67) were found to carry sextuple mutations. We observed significantly higher frequencies of triple dhps mutations 436A/437G/540E and quintuple mutations dhfr51I/59R/108N+dhps437G/540E in isolates from women who received IPTp-SP than those who did not. Overall, this study shows high prevalence rates of SP-associated resistance mutations in P. falciparum isolates collected from pregnant women. The presence of the dhps mutant allele 540E and the high prevalence of isolates carrying quintuple dhfr/dhps mutations are here reported for the first time in the Republic of Congo. The increasing prevalence of multiple mutant alleles observed in this study is alarming and may present a challenge for the future interventions including IPTp-SP in the country.

Molecular monitoring of resistant dhfr and dhps allelic haplotypes in Morogoro and Mvomero districts in south eastern Tanzania.

BACKGROUND: Resistance to the antimalarial drug sulfadoxine-pyrimethamine (SP) emerged in Plasmodium falciparum from Asia in the 1960s and subsequently spread to Africa. In Tanzania, SP use as a national policy began in 1983 as a second line to chloroquine (CQ) for the treatment of uncomplicated malaria, until August 2001 when it was approved to replace CQ as a national first line. OBJECTIVE: The present study assesses the frequency of resistant dhfr and dhps alleles in Morogoro-Mvomero district in south eastern Tanzania and contrast their rate of change during 17 years of SP second line use against five years of SP first line use. METHODOLOGY: Cross sectional surveys of asymptomatic infections were carried out at the end of rainy season during July-September of 2000, when SP was the national second line (CQ was the first line) and 2006 when SP was the national first line antimalarial treatment. Genetic analysis of SP resistance genes was carried out on 1,044 asymptomatic infections and the effect of the two policies on SP evolution compared. RESULTS: The frequency of the most resistant allele, the double dhps-triple dhfr mutant genotype, increased by only 1% during 17 years of SP second line use, but there was a dramatic increase by 45% during five years of SP first line use. CONCLUSION: We conclude that National policy change from second line to first line SP, brought about an immediate shift in treatment practice and this in turn had a highly significant impact on drug pressure. The use of SP in specific programs only such as intermittent preventive treatment of infants (IPTi) and intermittent preventive treatment of pregnant women (IPTp) will most likely reduce substantially SP selection pressure and the SP resistance alleles alike.