Increase in the prevalence of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum isolates collected from early to late pregnancy in Nanoro, Burkina Faso.
ABSTRACT: Pregnant women are a high-risk group for Plasmodium falciparum infections, which may result in maternal anaemia and low birth weight newborns, among other adverse birth outcomes. Intermittent preventive treatment with sulfadoxine-pyrimethamine during pregnancy (IPTp-SP) is widely implemented to prevent these negative effects of malaria. However, resistance against SP by P. falciparum may decrease efficacy of IPTp-SP. Combinations of point mutations in the dhps (codons A437, K540) and dhfr genes (codons N51, C59, S108) of P. falciparum are associated with SP resistance. In this study the prevalence of SP resistance mutations was determined among P. falciparum found in pregnant women and the general population (GP) from Nanoro, Burkina Faso and the association of IPTp-SP dosing and other variables with mutations was studied.Blood spots on filter papers were collected from pregnant women at their first antenatal care visit (ANC booking) and at delivery, from an ongoing trial and from the GP in a cross-sectional survey. The dhps and dhfr genes were amplified by nested PCR and products were sequenced to identify mutations conferring resistance (ANC booking, n = 400; delivery, n = 223; GP, n = 400). Prevalence was estimated with generalized estimating equations and for multivariate analyses mixed effects logistic regression was used.The prevalence of the triple dhfr mutation was high, and significantly higher in the GP and at delivery than at ANC booking, but it did not affect birth weight. Furthermore, quintuple mutations (triple dhfr and double dhps mutations) were found for the first time in Burkina Faso. IPTp-SP did not significantly affect the occurrence of any of the mutations, but high transmission season was associated with increased mutation prevalence in delivery samples. It is unclear why the prevalence of mutations was higher in the GP than in pregnant women at ANC booking.The high number of mutants and the presence of quintuple mutants in Burkina Faso confirm concerns about the efficacy of IPTp-SP in the near future. Other drug combinations to tackle malaria in pregnancy should, therefore, be explored. An increase in mutation prevalence due to IPTp-SP dosing could not be confirmed.
Project description:BACKGROUND: Resistance to sulphadoxine-pyrimethamine (SP) in Plasmodium falciparum parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes and has spread worldwide. SP remains the recommended drug for intermittent preventive treatment for malaria in pregnancy (IPTp) and information on population prevalence of the SP resistance molecular markers in pregnant women is limited. METHODS: Temporal trends of SP resistance molecular markers were investigated in 489 parasite samples collected from pregnant women at delivery from three different observational studies between 1996 and 2009 in Kenya, where SP was adopted for both IPTp and case treatment policies in 1998. Using real-time polymerase chain reaction, pyrosequencing and direct sequencing, 10 single-nucleotide polymorphisms (SNPs) of SP resistance molecular markers were assayed. RESULTS: The prevalence of quintuple mutant (dhfr N51I/C59R/S108N and dhps A437G/K540E combined genotype) increased from 7% in the first study (1996-2000) to 88% in the third study (2008-2009). When further stratified by sample collection year and adoption of IPTp policy, the prevalence of the quintuple mutant increased from 2.4% in 1998 to 44.4% three years after IPTp policy adoption, seemingly in parallel with the increase in percentage of SP use in pregnancy. However, in the 1996-2000 study, more mutations in the combined dhfr/dhps genotype were associated with SP use during pregnancy only in univariable analysis and no associations were detected in the 2002-2008 and 2008-2009 studies. In addition, in the 2008-2009 study, 5.3% of the parasite samples carried the dhps triple mutant (A437G/K540E/A581G). There were no differences in the prevalence of SP mutant genotypes between the parasite samples from HIV + and HIV- women over time and between paired peripheral and placental samples. CONCLUSIONS: There was a significant increase in dhfr/dhps quintuple mutant and the emergence of new genotype containing dhps 581 in the parasites from pregnant women in western Kenya over 13 years. IPTp adoption and SP use in pregnancy only played a minor role in the increased drug-resistant parasites in the pregnant women over time. Most likely, other major factors, such as the high prevalence of resistant parasites selected by the use of SP for case management in large non-pregnant population, might have contributed to the temporally increased prevalence of SP resistant parasites in pregnant women. Further investigations are needed to determine the linkage between SP drug resistance markers and efficacy of IPTp-SP.
Project description:The effectiveness of sulphadoxine-pyrimethamine (SP) intermittent preventive treatment of malaria in pregnancy (IPTp) might be compromised by high prevalence of resistance-associated Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations. As a proxy for IPTp-SP effectiveness, the in vivo efficacy of SP to clear parasitaemia and prevent reinfection in asymptomatic parasitaemic pregnant women in an area with high SP resistance prevalence was assessed.Pregnant women 16-26 weeks' gestation with asymptomatic parasitaemia presenting for antenatal care were given IPTp-SP and followed for 42 days. The primary outcome was polymerase chain reaction (PCR) uncorrected 42-day survival rate; the per cent of patients without recrudescence or reinfection by day 42. PCR was used to distinguish recrudescence from reinfection. DNA was sequenced to detect resistance-associated dhfr and dhps mutations.Of 245 pregnant women included in the intention-to-treat analysis, 93.9% cleared their parasitaemia by day 7. The day 42 PCR-uncorrected survival rate was 58.1% (95% confidence interval (CI) 51.5-65.7) and day 42 PCR-corrected survival was 68.7% (CI 61.4-76.0). Recrudescence was more common among primi- than among multigravid women; recrudescence rate 33.3% (CI 25.1-42.4%) versus 21.4% (CI 15.0-29.0%) (log rank test p-value 0.006). The quintuple mutant was present in nearly all samples (95%), while 2% were sextuple mutants with an additional mutation at dhps A581G.SP efficacy for acute malaria treatment has been compromised by resistance, but SP retains partial activity among pregnant women with asymptomatic parasitaemia, and thus might be useful for IPTp. Nonetheless, research on non-SP IPTp regimens should continue.ClinicalTrials.gov NCT01120145 .
Project description:In 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3-59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (<?5 years old) with uncomplicated malaria. The dhfr triple mutant I<sub>51</sub>R<sub>59</sub>N<sub>108</sub>, dhps double mutant G<sub>437</sub>G<sub>581</sub> and quadruple dhfr I<sub>51</sub>R<sub>59</sub>N<sub>108</sub>?+?dhps G<sub>437</sub> mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I<sub>51</sub>R<sub>59</sub>N<sub>108</sub>?+?dhps G<sub>437</sub>E<sub>540</sub> and sextuple dhfr I<sub>51</sub>R<sub>59</sub>N<sub>108</sub>?+?dhps G<sub>437</sub>E<sub>540</sub>G<sub>581</sub> mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.
Project description:<h4>Background</h4>Sulfadoxine-pyrimethamine (SP) is a cornerstone of malaria chemoprophylaxis and is considered for programmes in the Democratic Republic of Congo (DRC). However, SP efficacy is threatened by drug resistance, that is conferred by mutations in the dhfr and dhps genes. The World Health Organization has specified that intermittent preventive treatment for infants (IPTi) with SP should be implemented only if the prevalence of the dhps K540E mutation is under 50%. There are limited current data on the prevalence of resistance-conferring mutations available from Eastern DRC. The current study aimed to address this knowledge gap.<h4>Methods</h4>Dried blood-spot samples were collected from clinically suspected malaria patients [outpatient department (OPD)] and pregnant women attending antenatal care (ANC) in four sites in North and South Kivu, DRC. Quantitative PCR (qPCR) was performed on samples from individuals with positive and with negative rapid diagnostic test (RDT) results. Dhps K450E and A581G and dhfr I164L were assessed by nested PCR followed by allele-specific primer extension and detection by multiplex bead-based assays.<h4>Results</h4>Across populations, Plasmodium falciparum parasite prevalence was 47.9% (1160/2421) by RDT and 71.7 (1763/2421) by qPCR. Median parasite density measured by qPCR in RDT-negative qPCR-positive samples was very low with a median of 2.3 parasites/µL (IQR 0.5-25.2). Resistance genotyping was successfully performed in RDT-positive samples and RDT-negative/qPCR-positive samples with success rates of 86.2% (937/1086) and 55.5% (361/651), respectively. The presence of dhps K540E was high across sites (50.3-87.9%), with strong evidence for differences between sites (p?<?0.001). Dhps A581G mutants were less prevalent (12.7-47.2%). The dhfr I164L mutation was found in one sample.<h4>Conclusions</h4>The prevalence of the SP resistance marker dhps K540E exceeds 50% in all four study sites in North and South Kivu, DRC. K540E mutations regularly co-occurred with mutations in dhps A581G but not with the dhfr I164L mutation. The current results do not support implementation of IPTi with SP in the study area.
Project description:Background:We investigated whether adding community scheduled malaria screening and treatment (CSST) with artemether-lumefantrine by community health workers (CHWs) to standard intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) would improve maternal and infant health. Methods:In this 2-arm cluster-randomized, controlled trial, villages in Burkina Faso, The Gambia, and Benin were randomized to receive CSST plus IPTp-SP or IPTp-SP alone. CHWs in the intervention arm performed monthly CSST during pregnancy. At each contact, filter paper and blood slides were collected, and at delivery, a placental biopsy was collected. Primary and secondary endpoints were the prevalence of placental malaria, maternal anemia, maternal peripheral infection, low birth weight, antenatal clinic (ANC) attendance, and IPTp-SP coverage. Results:Malaria infection was detected at least once for 3.8% women in The Gambia, 16.9% in Benin, and 31.6% in Burkina Faso. There was no difference between study arms in terms of placenta malaria after adjusting for birth season, parity, and IPTp-SP doses (adjusted odds ratio, 1.06 [95% confidence interval, .78-1.44]; P = .72). No difference between the study arms was found for peripheral maternal infection, anemia, and adverse pregnancy outcomes. ANC attendance was significantly higher in the intervention arm in Burkina Faso but not in The Gambia and Benin. Increasing number of IPTp-SP doses was associated with a significantly lower risk of placenta malaria, anemia at delivery, and low birth weight. Conclusions:Adding CSST to existing IPTp-SP strategies did not reduce malaria in pregnancy. Increasing the number of IPTp-SP doses given during pregnancy is a priority. Clinical Trials Registration:NCT01941264; ISRCTN37259296.
Project description:There are few published reports of mutations in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes in P. falciparum populations in Nigeria, but one previous study has recorded a novel dhps mutation at codon 431 among infections imported to the United Kingdom from Nigeria. To assess how widespread this mutation is among parasites in different parts of the country and consequently fill the gap in sulfadoxine-pyrimethamine (SP) resistance data in Nigeria, we retrospectively analysed 1000 filter paper blood spots collected in surveys of pregnant women and children with uncomplicated falciparum malaria between 2003 and 2015 from four sites in the south and north. Genomic DNA was extracted from filter paper blood spots and placental impressions. Point mutations at codons 16, 50, 51, 59, 108, 140 and 164 of the dhfr gene and codons 431, 436, 437, 540, 581 and 613 of the dhps gene were evaluated by nested PCR amplification followed by direct sequencing. The distribution of the dhps-431V mutation was widespread throughout Nigeria with the highest prevalence in Enugu (46%). In Ibadan where we had sequential sampling, its prevalence increased from 0% to 6.5% between 2003 and 2008. Although there were various combinations of dhps mutations with 431V, the combination 431V + 436A + 437G+581G+613S was the most common. All these observations support the view that dhps-431V is on the increase. In addition, P. falciparum DHPS crystal structure modelling shows that the change from Isoleucine to Valine (dhps-431V) could alter the effects of both S436A/F and A437G, which closely follow the 2nd ?-strand. Consequently, it is now a research priority to assess the implications of dhps-VAGKGS mutant haplotype on continuing use of SP in seasonal malaria chemoprevention (SMC) and intermittent preventive treatment in pregnancy (IPTp). Our data also provides surveillance data for SP resistance markers in Nigeria between 2003 and 2015.
Project description:BACKGROUND:Tanzania adopted the revised World Health Organization policy in 2013 recommending a minimum of ?3 doses of Intermittent Preventive Treatment during pregnancy with Sulfadoxine-Pyrimethamine (IPTp-SP) to protect against malaria. A study in Tanzania in 2014 reported low (9%) uptake. We investigated health workers knowledge about IPTp-SP and factors that influenced uptake of >?3 doses of IPTp-SP among pregnant women. METHODS:We conducted a cross-sectional study in 2017 among post-delivery women and health care workers from nine randomly-selected public health facilities in three Districts of Arusha Region. Probability proportional to size methodology was used to determine number of participants per facility. We used a structured questionnaire to collect socio-demographic and obstetric data, information on doses of SP received, and knowledge of SP for IPTp. Health care workers were interviewed about their knowledge for IPTp- SP and challenges encountered in its uptake and use. RESULTS:We interviewed 556 persons (median age 26?years, range 16-42?years) with the response rate of 99.3%. Of these, 484 (87.1%) had >?3 Antenatal Care (ANC) visits. A total of 402 (72.3%) were multigravida with 362 (65.1%) having given birth at least once. Of the 556 participants, 219 (39.4%) made their first ANC booking at <?17?weeks of pregnancy and 269 (48.4%) had received >?3 doses of SP-IPTp. Factors associated with uptake of >?3 doses of IPTp-SP included having secondary or higher education [Adjusted Odds Ratio (AOR) =1.6, 95%CI 1.1-2.4], attending ?4 ANC visits [AOR?=?3.1, 95%CI 2.1-4.6], having first antenatal booking at <?17?weeks [AOR?=?1.8, 95%CI 1.4-2.3], and adequate knowledge on IPTp-SP [AOR?=?2.7, 95%CI 1.9-3.9]. Among 36 health care workers interviewed, 29(80.6%) had adequate knowledge about IPTp-SP. SP was available in seven (87.5%) of the visited health facilities and was administered under Direct Observed Therapy (DOT) in six (75%) facilities. Health care workers reported that stock outs of SP was a challenge. CONCLUSIONS:Fewer than half of the women interviewed reported uptake of >?3 doses of IPTp-SP. That is below the Tanzania national target of 80%. Making >?4 ANC visits, having secondary or higher education, making an early first ANC visit and having adequate knowledge on IPTp-SP promoted uptake of >?3 doses. Further qualitative studies are needed to explore factors that might contribute to low uptake of SP.
Project description:BACKGROUND:Plasmodium falciparum-resistance to sulphadoxine-pyrimethamine (SP) has been largely reported among pregnant women. However, the profile of resistance markers to SP dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) in the general population are varied and not frequently monitored. Currently, SP is used as partner drug for artemisinin combination therapy (SP-artesunate) in some sub-Saharan African countries or as a prophylactic drug in intermittent preventive treatment of malaria during pregnancy and infants and in seasonal malaria chemoprevention (SMC). Profiling of P. falciparum-resistant genotypes to SP is dynamic and critical in providing data that would be useful for malaria control programmes. This study assessed the profile of dhfr and dhps genes genotypes among individuals with malaria in Lagos, Nigeria. METHODS:Molecular markers of SP resistance were identified by nested PCR and sequenced among malaria positive dried blood spots (DBS) that were collected from individuals attending health facilities from January 2013 to February 2014 and during community surveys from October 2010 to September 2011 across different Local Government Areas of Lagos State, Nigeria. RESULTS:A total of 242 and 167 samples were sequenced for dhfr and dhps, respectively. Sequence analysis of dhfr showed that 95.5% (231/242), 96.3% (233/242) and 96.7% (234/242) of the samples had N51I, C59R and S108N mutant alleles, respectively. The prevalence of dhps mutation at codons A437G, A613S, S436A, A581G, I431V and K540E were 95.8% (160/167), 41.9% (70/167), 41.3% (69/167), 31.1% (52/167), 25.1% (42/167), and 1.2% (2/167) respectively. The prevalence of triple mutations (CIRNI) in dhfr was 93.8% and 44.3% for the single dhps haplotype mutation (SGKAA). Partial SP-resistance due to quadruple dhfr-dhps haplotype mutations (CIRNI-SGKAA) and octuple haplotype mutations (CIRNI-VAGKGS) with rate of 42.6% and 22.0%, respectively has been reported. CONCLUSIONS:There was increased prevalence in dhfr triple haplotype mutations when compared with previous reports in the same environment but aligned with high prevalence in other locations in Nigeria and other countries in Africa. Also, high prevalence of dhfr and dhps mutant alleles occurred in the study areas in Lagos, Nigeria five to eight years after the introduction of artemisinin combination therapy underscores the need for continuous monitoring.
Project description:Accumulation of mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) is strongly associated with sulfadoxine-pyrimethamine (SP) treatment failure. Routine surveillance for these resistance markers was conducted annually at 26 sentinel sites in Maputo Province, Mozambique, before and after the phased deployment of artesunate plus SP (AS-SP), with 15,758 children sampled between 2004 and 2008. Mean asexual parasite prevalence, polymerase chain reaction (PCR) corrected, decreased from 44.2% in 2004 to 3.8% in 2008 (P < 0.0001). Among the 2,012 PCR-confirmed falciparum samples, the dhfr triple mutation remained close to fixation, whereas both dhps double and dhfr/dhps "quintuple" mutations increased from 11.0% in 2004, to 75.0% by 2008 (P < 0.0001). Adding artesunate to SP did not retard the spread of SP-resistant parasites. The high "quintuple" mutation prevalence suggests a limited useful therapeutic lifespan of AS-SP for treating uncomplicated malaria, and may curb efficacy of SP-monotherapy for intermittent preventive treatment in Mozambique.
Project description:BACKGROUND: Mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes of Plasmodium falciparum are associated with resistance to anti-folate drugs, most notably sulphadoxine-pyrimethamine (SP). Molecular studies document the prevalence of these mutations in parasite populations across the African continent. However, there is no systematic review examining the collective epidemiological significance of these studies. This meta-analysis attempts to: 1) summarize genotype frequency data that are critical for molecular surveillance of anti-folate resistance and 2) identify the specific challenges facing the development of future molecular databases. METHODS: This review consists of 220 studies published prior to 2009 that report the frequency of select dhfr and dhps mutations in 31 African countries. Maps were created to summarize the location and prevalence of the highly resistant dhfr triple mutant (N51I, C59R, S108N) genotype and dhps double mutant (A437G and K540E) genotype in Africa. A hierarchical mixed effects logistic regression was used to examine the influence of various factors on reported mutant genotype frequency. These factors include: year and location of study, age and clinical status of sampled population, and reporting conventions for mixed genotype data. RESULTS: A database consisting of dhfr and dhps mutant genotype frequencies from all African studies that met selection criteria was created for this analysis. The map illustrates particularly high prevalence of both the dhfr triple and dhps double mutant genotypes along the Kenya-Tanzania border and Malawi. The regression model shows a statistically significant increase in the prevalence of both the dhfr triple and dhps double mutant genotypes in Africa. CONCLUSION: Increasing prevalence of the dhfr triple mutant and dhps double mutant genotypes in Africa are consistent with the loss of efficacy of SP for treatment of clinical malaria in most parts of this continent. Continued assessment of the effectiveness of SP for the treatment of clinical malaria and intermittent preventive treatment in pregnancy is needed. The creation of a centralized resistance data network, such as the one proposed by the WorldWide Antimalarial Resistance Network (WWARN), will become a valuable resource for planning timely actions to combat drug resistant malaria.