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Ten countries have reported pfhrp2/pfhrp3 gene deletions since the first observation of pfhrp2 -deleted parasites in 2012. In a previous study (Watson et al., 2017), we characterised the drivers selecting for pfhrp2/3 deletions and mapped the regions in Africa with the greatest selection pressure. In February 2018, the World Health Organization issued guidance on investigating suspected false-negative rapid diagnostic tests (RDTs) due to pfhrp2/3 deletions. However, no guidance is provided regarding the timing of investigations. Failure to consider seasonal variation could cause premature decisions to switch to alternative RDTs. In response, we have extended our methods and predict that the prevalence of false-negative RDTs due to pfhrp2/3 deletions is highest when sampling from younger individuals during the beginning of the rainy season. We conclude by producing a map of the regions impacted by seasonal fluctuations in pfhrp2/3 deletions and a database identifying optimum sampling intervals to support malaria control programmes., Competing Interests: OW, RV, AG, TG, JC, AT, MM, SM, JP, HS No competing interests declared, (© 2019, Watson et al.)

Background: Resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine threatens the antimalarial effectiveness of intermittent preventive treatment during pregnancy (IPTp) in sub-Saharan Africa. We aimed to assess the associations between markers of sulfadoxine-pyrimethamine resistance in P falciparum and the effectiveness of sulfadoxine-pyrimethamine IPTp for malaria-associated outcomes., Methods: For this systematic review and meta-analysis, we searched databases (from Jan 1, 1990 to March 1, 2018) for clinical studies (aggregated data) or surveys (individual participant data) that reported data on low birthweight (primary outcome) and malaria by sulfadoxine-pyrimethamine IPTp dose, and for studies that reported on molecular markers of sulfadoxine-pyrimethamine resistance. Studies that involved only HIV-infected women or combined interventions were excluded. We did a random-effects meta-analysis (clinical studies) or multivariate log-binomial regression (surveys) to obtain summarised dose-response data (relative risk reduction [RRR]) and multivariate meta-regression to explore the modifying effects of sulfadoxine-pyrimethamine resistance (as indicated by Ala437Gly, Lys540Glu, and Ala581Gly substitutions in the dhps gene). This study is registered with PROSPERO, number 42016035540., Findings: Of 1097 records screened, 57 studies were included in the aggregated-data meta-analysis (including 59 457 births). The RRR for low birthweight declined with increasing prevalence of dhps Lys540Glu (p trend =0·0060) but not Ala437Gly (p trend =0·35). The RRR was 7% (95% CI 0 to 13) in areas of high resistance to sulfadoxine-pyrimethamine (Lys540Glu ≥90% in east and southern Africa; n=11), 21% (14 to 29) in moderate-resistance areas (Ala437Gly ≥90% [central and west Africa], or Lys540Glu ≥30% to <90% [east and southern Africa]; n=16), and 27% (21 to 33) in low-resistance areas (Ala437Gly <90% [central and west Africa], or Lys540Glu <30% [east and southern Africa]; n=30; p trend =0·0054 [univariate], I 2 =69·5%). The overall RRR in all resistance strata was 21% (17 to 25). In the analysis of individual participant data from 13 surveys (42 394 births), sulfadoxine-pyrimethamine IPTp was associated with reduced prevalence of low birthweight in areas with a Lys540Glu prevalence of more than 90% and Ala581Gly prevalence of less than 10% (RRR 10% [7 to 12]), but not in those with an Ala581Gly prevalence of 10% or higher (pooled Ala581Gly prevalence 37% [range 29 to 46]; RRR 0·5% [-16 to 14]; 2326 births)., Interpretation: The effectiveness of sulfadoxine-pyrimethamine IPTp is reduced in areas with high resistance to sulfadoxine-pyrimethamine among P falciparum parasites, but remains associated with reductions in low birthweight even in areas where dhps Lys540Glu prevalence exceeds 90% but where the sextuple-mutant parasite (harbouring the additional dhps Ala581Gly mutation) is uncommon. Therapeutic alternatives to sulfadoxine-pyrimethamine IPTp are needed in areas where the prevalence of the sextuple-mutant parasite exceeds 37%., Funding: US Centers for Disease Control and Prevention, the Malaria in Pregnancy Consortium (funded through a grant from the Bill & Melinda Gates Foundation to the Liverpool School of Tropical Medicine), Worldwide Antimalarial Resistance Network, European and Developing Countries Clinical Trials Partnership., (Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: New diagnostic tools for malaria are required owing to the changing epidemiology of malaria, particularly among pregnant women in sub-Saharan Africa. Real-time PCR assays targeting Plasmodium falciparum lactate dehydrogenase (pfldh) gene may facilitate the identification of a high proportion of pregnant women with a P. falciparum parasitaemia below the threshold of microscopy. These molecular methods will enable further studies on the effects of these submicroscopic infections on maternal health and birth outcomes., Methods: The pfldh real-time PCR assay and conventional microscopy were compared for the detection of P. falciparum from dried blood spots and blood smears collected from the peripheral blood of 475 Malawian women at delivery. A cycle threshold (Ct) of the real-time PCR was determined optimizing the sensitivity and specificity of the pfldh PCR assay compared to microscopy. A real-time PCR species-specific assay was applied to identify the contribution to malaria infections of three Plasmodium species (P. falciparum P. ovale and P. malariae) in 44 discordant smear and pfldh PCR assay results., Results: Of the 475 women, P. falciparum was detected in 11 (2.3%) by microscopy and in 51 (10.7%) by real-time PCR; compared to microscopy, the sensitivity of real-time PCR was 90.9% and the specificity 91.2%. If a Ct value of 38 was used as a cut-off, specificity improved to 94.6% with no change in sensitivity. The real-time PCR species-specific assay detected P. falciparum alone in all but four samples: two samples were mixed infections with P. falciparum and P. malariae, one was a pure P. malariae infection and one was a pfldh PCR assay-positive/species-specific assay-negative sample. Of three P. malariae infections detected by microscopy, only one was confirmed by the species-specific assay., Conclusions: Although microscopy remains the most appropriate method for clinical malaria diagnosis in field settings, molecular diagnostics such as real-time PCR offer a more reliable means to detect malaria parasites, particularly at low levels. Determination of the possible contribution of these submicroscopic infections to poor birth outcomes and maternal health is critical. For future studies to investigate these effects, this pfldh real-time PCR assay offers a reliable detection method.

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.

The resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) is an emerging public health threat. Resistance to these drugs is associated with point mutations in the genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). We describe here an assay using real-time PCR and sequence-specific probes that detects these mutations. Using DNA from plasmids, cultured strains, and clinical samples, real-time PCR could distinguish four DHPS polymorphisms (codons 437, 540, 581, and 613) and three DHFR polymorphisms (codons 51, 59, and 108). This assay is rapid and sensitive, with a detection limit of 10 copies in most cases. This assay is amenable to large-scale studies of drug resistance.

Since the first reports of chloroquine-resistant falciparum malaria in southeast Asia and South America almost half a century ago, drug-resistant malaria has posed a major problem in malaria control. By the late 1980s, resistance to sulfadoxine-pyrimethamine and to mefloquine was also prevalent on the Thai-Cambodian and Thai-Myanmar (Thai-Burmese) borders, rendering them established multidrug-resistant (MDR) areas. Chloroquine resistance spread across Africa during the 1980s, and severe resistance is especially found in east Africa. As a result, more than ten African countries have switched their first-line drug to sulfadoxine-pyrimethamine. Of great concern is the fact that the efficacy of this drug in Africa is progressively deteriorating, especially in foci in east Africa, which are classified as emerging MDR areas. Urgent efforts are needed to lengthen the lifespan of sulfadoxine-pyrimethamine and to identify effective, affordable, alternative antimalarial regimens. Molecular markers for antimalarial resistance have been identified, including pfcrt polymorphisms associated with chloroquine resistance and dhfr and dhps polymorphisms associated with sulfadoxine-pyrimethamine resistance. Polymorphisms in pfmdr1 may also be associated with resistance to chloroquine, mefloquine, quinine, and artemisinin. Use of such genetic information for the early detection of resistance foci and future monitoring of drug-resistant malaria is a potentially useful epidemiological tool, in conjunction with the conventional in-vivo and in-vitro drug-sensitivity assessments. This review describes the various features of drug resistance in Plasmodium falciparum, including its determinants, current status in diverse geographical areas, molecular markers, and their implications.