Protocol > Research Protocol
de Wit MBK, Rao B, Lassovski M, Ouabo A, Badjo C, et al.
1 July 2018
Primary Objective: To measure the prevalence of molecular markers of SP resistant malaria in North and South Kivu, DRC.
Sulfadoxine/pyrimethamine (SP) forms the backbone of most malaria chemoprevention programmes in high endemicity settings, including intermittent preventative therapy in pregnancy and infants (IPTp and IPTi respectively) as well as seasonal malaria chemoprevention (SMC). P. falciparum parasite resistance to SP threatens recent triumphs preventing malaria infection in the most vulnerable risk groups. WHO guidance is that chemoprevention using SP may not be implemented when prevalence of the dhps K540E gene denoting SP resistance are greater than 50%. Simple, robust polymerase chain reaction (PCR) - based methods for molecular surveillance of resistance to SP have the potential to indicate whether SP-based chemoprevention programmes would be effective in areas where surveillance was conducted, but also to identify early stages of emerging resistance in order to advocate for alternative chemoprevention strategies.
A minimum of 750 samples will be collected per province. Three sites per province will provide 250 samples assuming an estimated prevalence of 50% prevalence of dhps K540E gene with 95% confidence and 5% precision. This is also sufficient for robust estimation of the prevalence of dhps 581, an alternative critical marker. This sample size is calculated to estimate regional prevalence, i.e. for both South Kivu and North Kivu, and hence this study requires samples from multiple MSF sites (including from different MSF Operating Centre missions) e.g. Baraka, Kimbi and Lulingu amongst others in South Kivu and Mweso, Rutsuru and Walikale in North Kivu with a minimum total of 750 per province. If estimating specific prevalence in only one limited site, a large sample size would be required.
Sulfadoxine/pyrimethamine (SP) forms the backbone of most malaria chemoprevention programmes in high endemicity settings, including intermittent preventative therapy in pregnancy and infants (IPTp and IPTi respectively) as well as seasonal malaria chemoprevention (SMC). P. falciparum parasite resistance to SP threatens recent triumphs preventing malaria infection in the most vulnerable risk groups. WHO guidance is that chemoprevention using SP may not be implemented when prevalence of the dhps K540E gene denoting SP resistance are greater than 50%. Simple, robust polymerase chain reaction (PCR) - based methods for molecular surveillance of resistance to SP have the potential to indicate whether SP-based chemoprevention programmes would be effective in areas where surveillance was conducted, but also to identify early stages of emerging resistance in order to advocate for alternative chemoprevention strategies.
A minimum of 750 samples will be collected per province. Three sites per province will provide 250 samples assuming an estimated prevalence of 50% prevalence of dhps K540E gene with 95% confidence and 5% precision. This is also sufficient for robust estimation of the prevalence of dhps 581, an alternative critical marker. This sample size is calculated to estimate regional prevalence, i.e. for both South Kivu and North Kivu, and hence this study requires samples from multiple MSF sites (including from different MSF Operating Centre missions) e.g. Baraka, Kimbi and Lulingu amongst others in South Kivu and Mweso, Rutsuru and Walikale in North Kivu with a minimum total of 750 per province. If estimating specific prevalence in only one limited site, a large sample size would be required.
Journal Article > ResearchFull Text
Trop Med Int Health. 1 October 2006; Volume 11 (Issue 10); DOI:10.1111/j.1365-3156.2006.01710.x
Swarthout TD, van den Broek IVF, Kayembe G, Montgomery JM, Pota H, et al.
Trop Med Int Health. 1 October 2006; Volume 11 (Issue 10); DOI:10.1111/j.1365-3156.2006.01710.x
We undertook a trial of artesunate + amodiaquine (AS + AQ) and artesunate + sulphadoxine-pyrimethamine (AS + SP) in 180 children of age 6-59 months with uncomplicated malaria in Democratic Republic of Congo. Children were randomly allocated to receive 3 days observed treatment of AS + AQ (n = 90) or 3 days of AS + SP (n = 90). Primary efficacy outcomes were 28-day parasite recurrence rates, and recrudescence rates were adjusted by genotyping to distinguish new infection and recrudescence. In addition, we determined the prevalence of molecular markers of resistance to sulphadoxine and pyrimethamine. Day 28 parasite recurrence rates were 16.9% (14/83; 95% CI: 9.5-26.7) in the AS + AQ group and 34.6% (28/81; 95% CI: 24.3-46.0) in the AS + SP group (P = 0.009). After PCR correction, recrudescence rates were 6.7% (5/74; 95% CI: 2.2-15.1) for AS + AQ and 19.7% (13/66; 95% CI: 10.9-31.3) for AS + SP (P = 0.02). There was no significant difference between the two arms in time to parasite clearance, fever clearance and gametocyte clearance. Parasite genotyping showed high frequencies of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) molecular SP-resistance markers, with 57% of the samples showing more than three mutations linked to SP resistance, and 27% with triple-dhfr/double-dhps haplotype, confirming that SP treatment failure rates are likely to be high. AS + AQ had significantly higher efficacy than AS + SP. These results contributed to the subsequent change to AS + AQ as first-line regimen in the country. Efforts to properly implement the new protocol and maintain adherence at acceptable levels should include health staff and patient sensitization. The 6.8% recrudescence rate indicates that AS + AQ should be monitored closely until a more effective artemisinin combination therapy regimen is needed and can be introduced.
Journal Article > ResearchFull Text
Malar J. 18 December 2019; Volume 18 (Issue 1); 430.; DOI:10.1186/s12936-019-3057-7
van Lenthe M, van der Meulen R, Lassovsky M, Ouabo A, Bakula E, et al.
Malar J. 18 December 2019; Volume 18 (Issue 1); 430.; DOI:10.1186/s12936-019-3057-7
BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
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.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
Journal Article > ResearchFull Text
Emerg Infect Dis. 24 July 2014; Volume 20 (Issue 8); DOI:10.3201/eid2008.131897
Alifrangis M, Nag S, Schousboe ML, Ishengoma D, Lusingu J, et al.
Emerg Infect Dis. 24 July 2014; Volume 20 (Issue 8); DOI:10.3201/eid2008.131897
Super-resistant Plasmodium falciparum threatens the effectiveness of sulfadoxine-pyrimethamine in intermittent preventive treatment for malaria during pregnancy. It is characterized by the A581G Pfdhps mutation on a background of the double-mutant Pfdhps and the triple-mutant Pfdhfr. Using samples collected during 2004-2008, we investigated the evolutionary origin of the A581G mutation by characterizing microsatellite diversity flanking Pfdhps triple-mutant (437G+540E+581G) alleles from 3 locations in eastern Africa and comparing it with double-mutant (437G+540E) alleles from the same area. In Ethiopia, both alleles derived from 1 lineage that was distinct from those in Uganda and Tanzania. Uganda and Tanzania triple mutants derived from the previously characterized southeastern Africa double-mutant lineage. The A581G mutation has occurred multiple times on local Pfdhps double-mutant backgrounds; however, a novel microsatellite allele incorporated into the Tanzania lineage since 2004 illustrates the local expansion of emergent triple-mutant lineages.
Journal Article > ResearchFull Text
PLOS Med. 14 April 2009; Volume 6 (Issue 4); DOI:10.1371/journal.pmed.1000055
Pearce RJ, Pota H, Evehe M-SB, Ba EH, Mombo-Ngoma G, et al.
PLOS Med. 14 April 2009; Volume 6 (Issue 4); DOI:10.1371/journal.pmed.1000055
BACKGROUND: Although the molecular basis of resistance to a number of common antimalarial drugs is well known, a geographic description of the emergence and dispersal of resistance mutations across Africa has not been attempted. To that end we have characterised the evolutionary origins of antifolate resistance mutations in the dihydropteroate synthase (dhps) gene and mapped their contemporary distribution. METHODS AND FINDINGS: We used microsatellite polymorphism flanking the dhps gene to determine which resistance alleles shared common ancestry and found five major lineages each of which had a unique geographical distribution. The extent to which allelic lineages were shared among 20 African Plasmodium falciparum populations revealed five major geographical groupings. Resistance lineages were common to all sites within these regions. The most marked differentiation was between east and west African P. falciparum, in which resistance alleles were not only of different ancestry but also carried different resistance mutations. CONCLUSIONS: Resistant dhps has emerged independently in multiple sites in Africa during the past 10-20 years. Our data show the molecular basis of resistance differs between east and west Africa, which is likely to translate into differing antifolate sensitivity. We have also demonstrated that the dispersal patterns of resistance lineages give unique insights into recent parasite migration patterns.