Sulfadoxine-pyrimethamine Resistance Research Articles

Chloroquine and Sulfadoxine-Pyrimethamine Resistance in Sub-Saharan Africa-A Review.

Malaria is a great concern for global health and accounts for a large amount of morbidity and mortality, particularly in Africa, with sub-Saharan Africa carrying the greatest burden of the disease. Malaria control tools such as insecticide-treated bed nets, indoor residual spraying, and antimalarial drugs have been relatively successful in reducing the burden of malaria; however, sub-Saharan African countries encounter great challenges, the greatest being antimalarial drug resistance. Chloroquine (CQ) was the first-line drug in the 20th century until it was replaced by sulfadoxine–pyrimethamine (SP) as a consequence of resistance. The extensive use of these antimalarials intensified the spread of resistance throughout sub-Saharan Africa, thus resulting in a loss of efficacy for the treatment of malaria. SP was replaced by artemisinin-based combination therapy (ACT) after the emergence of resistance toward SP; however, the use of ACTs is now threatened by the emergence of resistant parasites. The decreased selective pressure on CQ and SP allowed for the reintroduction of sensitivity toward those antimalarials in regions of sub-Saharan Africa where they were not the primary drug for treatment. Therefore, the emergence and spread of antimalarial drug resistance should be tracked to prevent further spread of the resistant parasites, and the re-emergence of sensitivity should be monitored to detect the possible reappearance of sensitivity in sub-Saharan Africa.

Historical trends and new surveillance of Plasmodium falciparum drug resistance markers in Angola

BackgroundPlasmodium falciparum resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) has historically posed a major threat to malaria control throughout the world. The country of Angola officially replaced CQ with artemisinin-based combination therapy (ACT) as a first-line treatment in 2006, but malaria cases and deaths have recently been rising. Many classic resistance mutations are relevant for the efficacy of currently available drugs, making it important to continue monitoring their frequency in Angola.MethodsPlasmodium falciparum DNA was sampled from the blood of 50 hospital patients in Cabinda, Angola from October-December of 2018. Each infection was genotyped for 13 alleles in the genes crt, mdr1, dhps, dhfr, and kelch13, which are collectively involved in resistance to six common anti-malarials. To compare frequency patterns over time, P. falciparum genotype data were also collated from studies published from across Angola in the last two decades.ResultsThe two most important alleles for CQ resistance, crt 76T and mdr1 86Y, were found at respective frequencies of 71.4% and 6.5%. Historical data suggest that mdr1 N86 has been steadily replacing 86Y throughout Angola in the last decade, while the frequency of crt 76T has been more variable across studies. Over a third of new samples from Cabinda were ‘quintuple mutants’ for SP resistance in dhfr/dhps, with a sixth mutation at dhps A581G present at 9.6% frequency. The markers dhfr 51I, dhfr 108N, and dhps 437G have been nearly fixed in Angola since the early 2000s, whereas dhfr 59R may have risen to high frequency more recently. Finally, no non-synonymous polymorphisms were detected in kelch13, which is involved in artemisinin resistance in Southeast Asia.ConclusionsGenetic markers of P. falciparum resistance to CQ are likely declining in frequency in Angola, consistent with the official discontinuation of CQ in 2006. The high frequency of multiple genetic markers of SP resistance is consistent with the continued public and private use of SP. In the future, more complete haplotype data from mdr1, dhfr, and dhps will be critical for understanding the changing efficacy of multiple anti-malarial drugs. These data can be used to support effective drug policy decisions in Angola.

High prevalence of genotypes associated with sulfadoxine/pyrimethamine resistance in the rural area of Fougamou, Gabon

Pregnancy-associated malaria (PAM) is a complex form of malaria. To prevent PAM, several African countries have adopted intermittent preventive treatment with sulfadoxine/pyrimethamine (IPT-SP). However, resistance to SP has been reported, associated with mutations in the genes Plasmodium falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr). The aim of this study was to investigate the prevalence of mutations in Pfdhfr and Pfdhps in P. falciparum isolates from rural areas of Gabon. A cross-sectional survey of febrile patients (n=202) who consulted Fougamou Health Center between February-May 2016 was performed. DNA was extracted from patient samples and the Pfdhfr and Pfdhps genes were genotyped using PCR-RFLP. Statistical analyses were performed. The malaria prevalence in febrile patients included in the study was 60.4% (122/202). The main parasite species was P. falciparum (96.7%; 118/122), followed by Plasmodium malariae (3.3%; 4/122). Genotypes on codons 16, 51, 59 and 108 of Pfdhfr were highly mutated (>96%). In Pfdhps, codons 436, 437, 540 and 613 also expressed high mutation rates. The prevalence of triple mutations of Pfdhfr VIRNI and AIRNI was 12.1% and 84.5%, respectively. The prevalence of mutant haplotypes of Pfdhps SGEA, SGKA and AGEA was 37.9%, 25.9% and 12.1%, respectively. The prevalence of quadruple mutants IRN-A and IRN-G was 20.0% and 93.1%, respectively, whereas quintuple mutants were found at 57.8% (IRN-GE) and 5.0% (IRN-AE). Our data show a high prevalence of genotypes associated with SP resistance. Clinical trials to investigate the efficacy of IPT-SP are much needed.

Temporal evolution of sulfadoxine-pyrimethamine resistance genotypes and genetic diversity in response to a decade of increased interventions against Plasmodium falciparum in northern Ghana

BackgroundAnti-malarial drug resistance remains a key concern for the global fight against malaria. In Ghana sulfadoxine-pyrimethamine (SP) is used for intermittent preventive treatment of malaria in pregnancy and combined with amodiaquine for Seasonal Malaria Chemoprevention (SMC) during the high malaria season. Thus, surveillance of molecular markers of SP resistance is important to guide decision-making for these interventions in Ghana.MethodsA total of 4469 samples from uncomplicated malaria patients collected from 2009 to 2018 was submitted to the Wellcome Trust Sanger Institute, UK for DNA sequencing using MiSeq. Genotypes were successfully translated into haplotypes in 2694 and 846 mono infections respectively for pfdhfr and pfdhps genes and the combined pfhdfr/pfdhps genes across all years.ResultsAt the pfdhfr locus, a consistently high (> 60%) prevalence of parasites carrying triple mutants (IRNI) were detected from 2009 to 2018. Two double mutant haplotypes (NRNI and ICNI) were found, with haplotype NRNI having a much higher prevalence (average 13.8%) than ICNI (average 3.2%) across all years. Six pfdhps haplotypes were detected. Of these, prevalence of five fluctuated in a downward trend over time from 2009 to 2018, except a pfdhps double mutant (AGKAA), which increased consistently from 2.5% in 2009 to 78.2% in 2018. Across both genes, pfdhfr/pfdhps combined triple (NRNI + AAKAA) mutants were only detected in 2009, 2014, 2015 and 2018, prevalence of which fluctuated between 3.5 and 5.5%. The combined quadruple (IRNI + AAKAA) genotype increased in prevalence from 19.3% in 2009 to 87.5% in 2011 before fluctuating downwards to 19.6% in 2018 with an average prevalence of 37.4% within the nine years. Prevalence of parasites carrying the quintuple (IRNI + AGKAA or SGEAA) mutant haplotypes, which are highly refractory to SP increased over time from 14.0% in 2009 to 89.0% in 2016 before decreasing to 78.9 and 76.6% in 2017 and 2018 respectively. Though quintuple mutants are rising in prevalence in both malaria seasons, together these combined genotypes vary significantly within season but not between seasons.ConclusionsDespite high prevalence of pfdhfr triple mutants and combined pfdhfr/pfdhps quadruple and quintuple mutants in this setting SP may still be efficacious. These findings are significant as they highlight the need to continuously monitor SP resistance, particularly using deep targeted sequencing to ascertain changing resistance patterns.

Evaluation of health system readiness and coverage of intermittent preventive treatment of malaria in infants (IPTi) in Kambia district to inform national scale-up in Sierra Leone

BackgroundIntermittent preventive treatment of malaria in infants (IPTi) with sulfadoxine-pyrimethamine (SP) is a proven strategy to protect infants against malaria. Sierra Leone is the first country to implement IPTi nationwide. IPTi implementation was evaluated in Kambia, one of two initial pilot districts, to assess quality and coverage of IPTi services.MethodsThis mixed-methods evaluation had two phases, conducted 3 (phase 1) and 15–17 months (phase 2) after IPTi implementation. Methods included: assessments of 18 health facilities (HF), including register data abstraction (phases 1 and 2); a knowledge, attitudes and practices survey with 20 health workers (HWs) in phase 1; second-generation sequencing of SP resistance markers (pre-IPTi and phase 2); and a cluster-sample household survey among caregivers of children aged 3–15 months (phase 2). IPTi and vaccination coverage from the household survey were calculated from child health cards and maternal recall and weighted for the complex sampling design. Interrupted time series analysis using a Poisson regression model was used to assess changes in malaria cases at HF before and after IPTi implementation.ResultsMost HWs (19/20) interviewed had been trained on IPTi; 16/19 reported feeling well prepared to administer it. Nearly all HFs (17/18 in phase 1; 18/18 in phase 2) had SP for IPTi in stock. The proportion of parasite alleles with dhps K540E mutations increased but remained below the 50% WHO-recommended threshold for IPTi (4.1% pre-IPTi [95%CI 2–7%]; 11% post-IPTi [95%CI 8–15%], p < 0.01). From the household survey, 299/459 (67.4%) children ≥ 10 weeks old received the first dose of IPTi (versus 80.4% for second pentavalent vaccine, given simultaneously); 274/444 (62.5%) children ≥ 14 weeks old received the second IPTi dose (versus 65.4% for third pentavalent vaccine); and 83/217 (36.4%) children ≥ 9 months old received the third IPTi dose (versus 52.2% for first measles vaccine dose). HF register data indicated no change in confirmed malaria cases among infants after IPTi implementation.ConclusionsKambia district was able to scale up IPTi swiftly and provide necessary health systems support. The gaps between IPTi and childhood vaccine coverage need to be further investigated and addressed to optimize the success of the national IPTi programme.

Low prevalence of highly sulfadoxine\u2010resistant dihydropteroate synthase alleles in Plasmodium falciparum isolates in Benin

BackgroundIn 2004, in response to high levels of treatment failure associated with sulfadoxine-pyrimethamine (SP) resistance, Benin changed its first-line malaria treatment from SP to artemisinin-based combination therapy for treatment of uncomplicated Plasmodium falciparum malaria. Resistance to SP is conferred by accumulation of single nucleotide polymorphisms (SNPs) in P. falciparum genes involved in folate metabolism, dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. Because SP is still used for intermittent preventive treatment in pregnant women (IPTp) and seasonal malaria chemoprevention (SMCP) in Benin, the prevalence of Pfdhfr and Pfdhps SNPs in P. falciparum isolates collected in 2017 were investigated.MethodsThis study was carried out in two sites where the transmission of P. falciparum malaria is hyper-endemic: Klouékanmey and Djougou. Blood samples were collected from 178 febrile children 6–59 months old with confirmed uncomplicated P. falciparum malaria and were genotyped for SNPs associated with SP resistance.ResultsThe Pfdhfr triple mutant IRN (N51I, C59R, and S108N) was the most prevalent (84.6%) haplotype and was commonly found with the Pfdhps single mutant A437G (50.5%) or with the Pfdhps double mutant S436A and A437G (33.7%). The quintuple mutant, PfdhfrIRN/PfdhpsGE (A437G and K540E), was rarely observed (0.8%). The A581G and A613S mutant alleles were found in 2.6 and 3.9% of isolates, respectively. Six isolates (3.9%) were shown to harbour a mutation at codon I431V, recently identified in West African parasites.ConclusionsThis study showed that Pfdhfr triple IRN mutants are near fixation in this population and that the highly sulfadoxine-resistant Pfdhps alleles are not widespread in Benin. These data support the continued use of SP for chemoprevention in these study sites, which should be complemented by periodic nationwide molecular surveillance to detect emergence of resistant genotypes.

Efficacy and safety of malarial prophylaxis with mefloquine during pregnancy in Kisangani, Democratic Republic of Congo: A randomized clinical trial

Kisangani is an area with intense malaria transmission and sulfadoxine-pyrimethamine resistance. Alternative antimalaria prophylaxis medication and protocols are needed, particularly with pregnant individuals. In this study, we compare the tolerance and effectiveness of mefloquine regimen as a split dose with a meal vs. sulfadoxine-pyrimethamine for the intermittent preventive treatment in pregnant individuals in Kisangani. This study was conducted from 15 May to 30 November 2019 as a single-blind, randomized clinical trial comparing 2 regimens of intermittent preventive treatment during pregnancy. The first regimen consisted of 4 doses of sulfadoxine-pyrimethamine, and the second of 2 doses of mefloquine taken as a split dose with meal. The occurrence of major or minor side-effects among patients treated with mefloquine and those treated with sulfadoxine-pyrimethamine were not statistically significant (major side effects: Fisher exact = 0.5014; minor side effects: P = .0961). Intermittent preventive treatment using mefloquine significantly reduced the risk of placental malaria (risk ratio [RR]: 0.4315, 95% confidence interval [CI]: 0.2201-0.8460), maternal peripheral parasitaemia (RR: 0.4397, 95% CI: 0.2377-0.8132) and low birth weight (RR: 0.4708, 95% CI: 0.2455-0.9029). Splitting dose and intake with a meal increased mefloquine tolerability while keeping its efficacy higher compared to sulfadoxine-pyrimethamine. Intermittent preventive treatment during pregnancy using mefloquine reduces the risk of placental malaria, maternal peripheral parasitaemia and low birth weight, compared to sulfadoxine-pyrimethamine. Thus, mefloquine is a good alternative to intermittent preventive treatment in pregnancy.

Single Nucleotide Polymorphisms of Pfdhfr and Pfdhps Genes: Implications for Malaria Prophylactic Strategies in Maiduguri, Northeast Nigeria

Background. The success of Intermittent Preventive Treatment in Pregnancy (IPTp), Intermittent Preventive Treatment in Infancy (IPTi), and Seasonal Malaria Chemoprevention (SMC) depends on sulfadoxine-pyrimethamine (SP) efficacy. Objective. The study determined Single Nucleotide Polymorphisms (SNPs) of Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) in Maiduguri, Northeast Nigeria. Materials and Methods. Giemsa-stained blood smears, capillary blood, and dried blood spot samples were collected from 63 subjects with uncomplicated malaria in Maiduguri between May and October 2018. Plasmodium species was determined and parasite density (PD) was estimated using the smears. Genomic DNA (gDNA) of P. falciparum was extracted from the dried blood spot samples using QIAamp DNA Mini Kit. The gDNA was subjected to nested PCR followed by restriction fragment length polymorphism (RFLP) to determine SNPs at Pfdhfr codons N51I, C59R, and S108N and Pfdhps codons S436A/F, A437G, and K540E. Results. The subjects’ mean age ± standard deviation was 23.6 ± 8.7 (2.0–67.0) years with a geometric mean PD of 8,948 (2,100–13,400) asexual parasites/µl blood. SNPs prevalence at any of the six Pfdhfr and Pfdhps codons was 85.7% (54/63); the prevalence was higher ( p &lt; 0.05 ) in Pfdhfr (82.5%; 52/63) than Pfdhps (58.7%; 37/63). Pfdhfr allele 108N (82.5%; 52/63) was the highest ( p &lt; 0.05 ) mutant when compared with alleles 51I (60.3%; 38/63) and 59R (66.7%; 42/63). Triple Pfdhfr mutation was observed in 60.3% (38/63) of the isolates and was higher ( p &lt; 0.05 ) among female subjects and SP recipients. Prevalence of Pfdhps allele 436A (28.6%; 18/63) was similar ( p &gt; 0.05 ) to allele 437G (34.9%; 22/63), with double mutation recorded in 4.8% (3/63). K540E mutation was not observed. Conclusion. Pfdhfr and Pfdhps mutations observed in Maiduguri are suggestive of SP resistance level, and this could constitute a setback to malaria prophylactic strategies in the region if unchecked. Thus, there is a need to investigate the clinical efficacy of SP.

Pneumococal community-acquired pneumonia in the intensive care unit: Azithromycin remains protective despite macrolide resistance

BackgroundStreptococcus pneumoniae (SP) remains the leading pathogen in community-acquired pneumonia (CAP). Despite the increasing prevalence of macrolide resistance in SP, guidelines recommend the use of macrolides as part of a combination regiment for intensive care unit (ICU) patients with CAP. We sought to describe if macrolide resistance effects outcomes in SP CAP in the ICU and if macrolides remain associated with a mortality advantage in an era of greater resistance. MethodsWe identified all patients with SP CAP admitted to the ICU between January 2012 and December 2016, and hospital mortality represented the primary endpoint. We recorded markers of acute and chronic disease severity (eg, Charlson score, need for mechanical ventilation and/or vasopressors) along with infection-related variables including the presence of macrolide resistance. We compared subjects treated with azithromycin to those not given this agent. ResultsThe cohort included 140 subjects (89.2% on mechanical ventilation, 14.3% crude mortality). Macrolide resistance occurred often (60.8%) and, in univariate analyses, was associated with higher mortality while azithromycin use appeared linked to fewer death. In multivariate analysis controlling for multiple confounders including macrolide resistance and the timeliness and appropriateness of antibiotic therapy, treatment with azithromycin resulted in fewer death (Adjusted odds ratio 0.27, 95% confidence interval: 0.09–0.85, p = 0.024). Macrolide resistance, however, was not independently related to mortality. ConclusionsMacrolide resistance appears frequently in SP ICU CAP. The addition of azithromycin to the antibiotic regimen in this scenario is significantly associated with a reduction in in-hospital mortality independent of multiple co-variates.

Evaluation of a single screen and treat strategy to detect asymptomatic malaria among pregnant women from selected health facilities in Lindi region, Tanzania

BackgroundIn areas of high transmission, malaria in pregnancy (MiP) primarily causes asymptomatic infections; these infections nonetheless increase the risk of adverse maternal and fetal outcomes. In 2014, Tanzania initiated a single screening and treatment (SST) strategy for all pregnant women at their first antenatal care (ANC) visit using malaria rapid diagnostic tests (RDT) for surveillance purposes. However, there is paucity of data on the effectiveness of SST in the prevention of MiP. The objective of this study was to estimate the number of asymptomatic infections among pregnant women detected by SST, which would have been missed in the absence of the policy.MethodsData from pregnant women attending their first ANC visits between October 2017 and June 2018, including gestational age, history of fever, and RDT results, were abstracted from ANC registers in eight health centres in two randomly selected districts, Kilwa and Lindi, in Lindi Region. The proportion of symptomatic (with history of fever in the past 48 h) and asymptomatic pregnant women with positive RDTs were calculated and stratified by trimester (first, second and third). The study areas were categorized as low transmission with prevalence < 10% or moderate/high with ≥ 10%.ResultsOver the study period, 1,845 women attended their first ANC visits; 22.1% were in the first trimester (< 12 weeks gestation age). Overall 15.0% of the women had positive RDTs, and there was a trend towards higher malaria prevalence in the first (15.9%) and second (15.2%) trimesters, compared to the third (7.1%), although the differences were not statistically significant (p = 0.07). In total, 6.9% of women reported fever within the past 48 h and, of these, 96.1% were RDT positive. For every 100 pregnant women in the moderate/high and low transmission areas, SST identified 60 and 26 pregnant women, respectively, with asymptomatic infections that would have otherwise been missed. Among the 15.9% of women detected in the first trimester, 50.7% were asymptomatic.ConclusionIn areas of moderate/high transmission, many infected women were asymptomatic, and would have been missed in the absence of SST. The benefits on maternal and fetal birth outcomes of identifying these infections depend heavily on the protection afforded by treatment, which is likely to be greatest for women presenting in the first trimester when intermittent preventive treatment (IPTp) with sulfadoxine-pyrimethamine (SP) is contraindicated, and in areas with high SP resistance, such as most parts of Tanzania. An evaluation of the impact and cost-effectiveness of SST across different transmission strata is warranted.

Molecular surveillance of anti-malarial resistance Pfdhfr and Pfdhps polymorphisms in African and Southeast Asia Plasmodium falciparum imported parasites to Wuhan, China

BackgroundAnti-malarial drug resistance is a severe challenge for eventual control and global elimination of malaria. Resistance to sulfadoxine-pyrimethamine (SP) increases as mutations accumulate in the Pfdhfr and Pfdhps genes. This study aimed to assess the polymorphisms and prevalence of mutation in these genes in the Plasmodium falciparum infecting migrant workers returning to Wuhan, China.MethodsBlood samples were collected for 9 years (2011–2019). Parasite genomic DNA was extracted from blood spots on filter paper. The mutations were evaluated by nested PCR and sequencing. The single-nucleotide polymorphisms (SNPs) and haplotypes of the Pfdhfr and Pfdhps genes were analysed.ResultsPfdhfr codon 108 showed a 94.7% mutation rate, while for Pfdhps, the rate for codon 437 was 79.0%. In total, five unique haplotypes at the Pfdhfr locus and 11 haplotypes at the Pfdhps locus were found while the Pfdhfr-Pfdhps combined loci revealed 28 unique haplotypes. A triple mutant (IRNI) of Pfdhfr was the most prevalent haplotype (84.4%). For Pfdhps, a single mutant (SGKAA) and a double mutant (SGEAA) were detected at frequencies of 37.8 and 22.3%, respectively. Among the combined haplotypes, a quadruple mutant (IRNI-SGKAA) was the most common, with a 30.0% frequency, followed by a quintuplet mutant (IRNI-SGEAA) with a frequency of 20.4%.ConclusionThe high prevalence and saturation of Pfdhfr haplotypes and the medium prevalence of Pfdhps haplotypes demonstrated in the present data will provide support for predicting the status and progression of antifolate resistance in malaria-endemic regions and imported malaria in nonendemic areas. Additional interventions to evaluate and prevent SP resistance should be continuously considered.

Carbon flow conversion induces alkali resistance and lipid accumulation under alkaline conditions based on transcriptome analysis in Chlorella sp. BLD

Alkaline environments are abundant globally and cause damage to most organisms, while some microalgae can grow well and accumulate lipids under those conditions. Here the mechanisms of alkali resistance and lipid accumulation in the alkaliphilic microalgae Chlorella sp. BLD were explored using physiological-biochemical and transcriptome analysis. When cultivated at alkaline pH, Chlorella sp. BLD exhibited good alkali-resistance ability and increased biomass (0.97 g L−1). The biochemical composition of Chlorella sp. BLD changed significantly (lipid content increased 39% and protein content decreased 19.5%) compared with pH 7.5. Through transcriptome analysis, we found that pathways related to carbon metabolism such as photosynthesis, glycolysis, and the TCA cycle were significantly regulated under alkaline conditions. Genes that encoding the key enzyme in carbon-related metabolism such as Rubisco, AMY, PK, ME, CS, ACAT, KAS, and DGAT were identified. Transcriptional regulation of these genes results in carbon flow switching from starch and protein to cell wall metabolism, organic acid synthetic and lipid accumulation in response to alkaline conditions. These results reveal the alkali resistance mechanism of Chlorella sp. BLD and provide a theoretical basis for microalgae oil production under alkaline conditions.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Plasmodium falciparum resistance to sulfadoxine-pyrimethamine in Africa: a systematic analysis of national trends

IntroductionThe rising burden of drug resistance is a major challenge to the global fight against malaria. We estimated national Plasmodium falciparum resistance to sulfadoxine-pyrimethamine (SP) across Africa, from 2000 to...

Cost-effectiveness of intermittent preventive treatment with dihydroartemisinin–piperaquine for malaria during pregnancy: an analysis using efficacy results from Uganda and Kenya, and pooled data

SummaryBackgroundPrevention of malaria infection during pregnancy in HIV-negative women currently relies on the use of long-lasting insecticidal nets together with intermittent preventive treatment in pregnancy with sulfadoxine–pyrimethamine (IPTp-SP). Increasing sulfadoxine–pyrimethamine resistance in Africa threatens current prevention of malaria during pregnancy. Thus, a replacement for IPTp-SP is urgently needed, especially for locations with high sulfadoxine–pyrimethamine resistance. Dihydroartemisinin–piperaquine is a promising candidate. We aimed to estimate the cost-effectiveness of intermittent preventive treatment in pregnancy with dihydroartemisinin–piperaquine (IPTp-DP) versus IPTp-SP to prevent clinical malaria infection (and its sequelae) during pregnancy.MethodsWe did a cost-effectiveness analysis using meta-analysis and individual trial results from three clinical trials done in Kenya and Uganda. We calculated disability-adjusted life-years (DALYs) arising from stillbirths, neonatal death, low birthweight, mild and moderate maternal anaemia, and clinical malaria infection, associated with malaria during pregnancy. Cost estimates were obtained from data collected in observational studies, health-facility costings, and from international drug procurement databases. The cost-effectiveness analyses were done from a health-care provider perspective using a decision tree model with a lifetime horizon. Deterministic and probabilistic sensitivity analyses using appropriate parameter ranges and distributions were also done. Results are presented as the incremental cost per DALY averted and the likelihood that an intervention is cost-effective for different cost-effectiveness thresholds.FindingsCompared with three doses of sulfadoxine–pyrimethamine, three doses of dihydroartemisinin–piperaquine, delivered to a hypothetical cohort of 1000 pregnant women, averted 892 DALYs (95% credibility interval 274 to 1517) at an incremental cost of US$7051 (2653 to 13 038) generating an incremental cost-effectiveness ratio (ICER) of $8 (2 to 29) per DALY averted. Compared with monthly doses of sulfadoxine–pyrimethamine, monthly doses of dihydroartemisinin–piperaquine averted 534 DALYS (−141 to 1233) at a cost of $13 427 (4994 to 22 895), resulting in an ICER of $25 (−151 to 224) per DALY averted. Both results were highly robust to most or all variations in the deterministic sensitivity analysis.InterpretationOur findings suggest that among HIV-negative pregnant women with high uptake of long-lasting insecticidal nets, IPTp-DP is cost-effective in areas with high malaria transmission and high sulfadoxine–pyrimethamine resistance. These data provide a comprehensive overview of the current evidence on the cost-effectiveness of IPTp-DP. Nevertheless, before a policy change is advocated, we recommend further research into the effectiveness and costs of different regimens of IPTp-DP in settings with different underlying sulfadoxine–pyrimethamine resistance.FundingMalaria in Pregnancy Consortium, which is funded through a grant from the Bill & Melinda Gates Foundation to the Liverpool School of Hygiene and Tropical Medicine.

Improved Herbicide Resistance of 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19 through Directed Evolution.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) has attracted extensive interest as a promising target for the genetic engineering of herbicide-resistant crops. However, naturally occurring HPPDs are generally very sensitive to HPPD inhibitors. In this study, random mutagenesis was performed to increase the HPPD inhibitors' resistance of Sphingobium sp. HPPD (SpHPPD). Two mutants, Q258M and Y333F, with improved resistance were obtained. Subsequently, a double-mutant (Q258M/Y333F) was generated through combined mutation. Q258M/Y333F exhibited the highest resistance to four HPPD inhibitors [topramezone, mesotrione, tembotrione, and diketonitrile (DKN)]. The enzyme fitness of Q258M/Y333F to topramezone, mesotrione, tembotrione, and DKN was increased by 4.0-, 4.1-, 4.2-, and 3.2-folds, respectively, in comparison with that of the wild-type. Molecular modeling and docking revealed that Q258M mutation leads to the decrease of enzyme-inhibitor-binding strength by breaking the hydrogen bond between the enzyme and the inhibitor, and Y333F mutation changes the conformational balance of the C-terminal helix H11, which hinders the binding of the inhibitor to the enzyme and thus would contribute to improved herbicide resistance. This study helps to further elucidate the structural basis for herbicide resistance and provides better genetic resources for the genetic engineering of herbicide-resistant crops.

Multi-metal resistance and potential of Alcaligenes sp. MMA for the removal of heavy metals

Pollution of water bodies is increasing because of toxic metals. In view of this, the present study examines the removal efficiency of heavy metals Cu2+, Cd2+, Cr6+, Ni2+, and Zn2+ by a bacterial strain isolated from River Yamuna. Initially, three strains were isolated, and further examined for the maximum tolerance index. Among the three isolates, strain MMA showed the highest tolerance to the heavy metals and was selected. The biochemical characterization and phylogenetic analysis based on 16S rRNA gene sequencing showed that the strain belonged to the genus Alcaligenes. Metal removal by the strain was observed at 20 mg/L in both individual and multi-metal experimental setup. The highest metal removal within 72 h was observed for Cu2+(88.45%) followed by Ni2+(82.45%), Zn2+ (69.99%), Cd2+ (63.04%), and least for Cr6+ (48.93%). Bacterial biosorption studies were done using SEM–EDX and FT-IR. Along with the metal removal, the heavy metal assessment of the River was also done for the year September 2017–August 2018, representing the post-monsoon, monsoon, and pre-monsoon. Cu2+, Cd2+, Cr6+, and Zn2+ were detected in the water sample due to the uncontrolled discharge of effluents into the river.

Changes in the frequencies of Plasmodium falciparum dhps and dhfr drug-resistant mutations in children from Western Kenya from 2005 to 2018: the rise of Pfdhps S436H

BackgroundSulfadoxine-pyrimethamine (SP) is the only anti-malarial drug formulation approved for intermittent preventive treatment in pregnancy (IPTp). However, mutations in the Plasmodium falciparum dhfr (Pfdhfr) and dhps (Pfdhps) genes confer resistance to pyrimethamine and sulfadoxine, respectively. Here, the frequencies of SP resistance-associated mutations from 2005 to 2018 were compared in samples from Kenyan children with malaria residing in a holoendemic transmission region.MethodsPartial sequences of the Pfdhfr and Pfdhps genes were amplified and sequenced from samples collected in 2005 (n = 81), 2010 (n = 95), 2017 (n = 43), and 2018 (n = 55). The frequency of known mutations conferring resistance to pyrimethamine and sulfadoxine were estimated and compared. Since artemisinin-based combination therapy (ACT) is the current first-line treatment for malaria, the presence of mutations in the propeller domain of P. falciparum kelch13 gene (Pfk13) linked to ACT-delayed parasite clearance was studied in the 2017/18 samples.ResultsAmong other changes, the point mutation of Pfdhps S436H increased in frequency from undetectable in 2005 to 28% in 2017/18. Triple Pfdhfr mutant allele (CIRNI) increased in frequency from 84% in 2005 to 95% in 2017/18, while the frequency of Pfdhfr double mutant alleles declined (allele CICNI from 29% in 2005 to 6% in 2017/18, and CNRNI from 9% in 2005 to undetectable in 2010 and 2017/18). Thus, a multilocus Pfdhfr/Pfdhps genotype with six mutations (HGEAA/CIRNI), including Pfdhps S436H, increased in frequency from 2010 to 2017/18. Although none of the mutations associated with ACT-delayed parasite clearance was observed, the Pfk13 mutation A578S, the most widespread Pfk13 SNP found in Africa, was detected in low frequency (2.04%).ConclusionsThere were changes in SP resistance mutant allele frequencies, including an increase in the Pfdhps S436H. Although these patterns seem consistent with directional selection due to drug pressure, there is a lack of information to determine the actual cause of such changes. These results suggest incorporating molecular surveillance of Pfdhfr/Pfdhps mutations in the context of SP efficacy studies for intermittent preventive treatment in pregnancy (IPTp).

Surveillance of molecular markers for antimalarial resistance in Zambia: Polymorphism of Pfkelch 13, Pfmdr1 and Pfdhfr/Pfdhps genes

Antimalarial resistance is an inevitable feature of control efforts and a key threat to achieving malaria elimination. Plasmodium falciparum, the deadliest of several species causing human malaria, has developed resistance to essentially all antimalarials. This study sought to investigate the prevalence of molecular markers associated with resistance to sulfadoxine-pyrimethamine (SP) and artemether-lumefantrine (AL) in Southern and Western provinces in Zambia. SP is used primarily for intermittent preventive treatment during pregnancy, while AL is the first-line antimalarial for uncomplicated malaria in Zambia. Blood samples were collected from household members of all ages in a cross-sectional survey conducted during peak malaria transmission, April to May of 2017, and amplified by polymerase chain reaction (PCR). Amplicons were then analysed by high-resolution melt following PCR to identify mutations associated with SP resistance in the P. falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps) genes and lumefantrine resistance in the P. falciparum multi-drug resistance 1 (Pfmdr1) gene. Finally, artemether resistance was assessed in the P. falciparum Kelch 13 (PfK13) gene using nested PCR followed by amplicon sequencing. The results showed a high frequency of genotypic-resistant Pfdhps A437G (93.2%) and Pfdhfr C59R (86.7%), N51I (80.9%), and S108N (80.8%) of which a high proportion (82.4%) were quadruple mutants (Pfdhfr N51I, C59R, S108N +Pfdhps A437G). Pfmrd1 N86Y, Y186F, and D1246Y - NFD mutant haplotypes were observed in 41.9% of isolates. The high prevalence of quadruple dhps/dhfr mutants indicates strong antifolate drug pressure from SP or other drugs (e.g., co-trimoxazole). Three samples contained PfK13 mutations, two synonymous (T478 and V666) and one non-synonymous (A578S), none of which have been associated with delayed clearance. This suggests that artemisinin remains efficacious in Zambia, however, the moderately high prevalence of approximately 40% Pfmdr1 NFD mutations calls for close monitoring of AL.

Transcriptomic analyses reveal the pathways associated with the volatilization and resistance of mercury(II) in the fungus Lecythophora sp. DC-F1

The biotic enzymatic reduction of mercury II [Hg(II)] to elemental Hg [Hg(0)] is an important pathway for Hg detoxification in natural ecosystems. However, the mechanisms of Hg(II) volatilization and resistance in fungi have not been understood completely. In the present study, we investigated the mechanisms of Hg(II) volatilization and resistance in the fungus Lecythophora sp. DC-F1. Hg(II) volatilization occurred during the investigation via the reduction of Hg(II) to Hg(0) in DC-F1. Comparative transcriptome analyses of DC-F1 revealed 3439 differentially expressed genes under 10 mg/L Hg(II) stress, among which 2770 were up-regulated and 669 were down-regulated. Functional enrichment analyses of genes and pathways further suggested that the Hg(II) resistance of DC-F1 is a multisystem collaborative process with three important transcriptional responses to Hg(II) stress: a mer-mediated Hg detoxification system, a thiol compound metabolism, and a cell reactive oxygen species stress response system. The phylogenetic analysis of merA protein homologs suggests that the Hg(II) reduction by merA is widely distributed in fungi. Overall, this study provides evidence for the reduction of Hg(II) to Hg(0) in fungi via the mer-mediated Hg detoxification system and offers a comprehensive explanation for its role within Hg biogeochemical cycling. These findings offer a strong theoretical basis for the application of fungi in the bioremediation of Hg-contaminated envionments.