Different origin and dispersal of sulfadoxine-resistant Plasmodium falciparum haplotypes between Eastern Africa and Democratic Republic of Congo

https://doi.org/10.1016/j.ijantimicag.2016.12.007Get rights and content

Highlights

  • The origin and genetic structure of Pfdhps haplotypes are defined.

  • Double- and triple-mutant Pfdhps haplotypes are predominant in East Africa, whilst single mutants are common in DR Congo.

  • Limited dissemination of double- and triple-mutant Pfdhps haplotypes between East Africa and DR Congo is highlighted.

  • Parasite genetic structure and clustering of Pfdhps resistance haplotypes differ between East Africa and DR Congo.

  • Implications for SP implementation in malaria chemoprevention strategies in the region are highlighted.

Abstract

Sulfadoxine/pyrimethamine (SP) is still used for malaria control in sub-Saharan Africa; however, widespread resistance is a major concern. This study aimed to determine the dispersal and origin of sulfadoxine resistance lineages in the Democratic Republic of the Congo compared with East African Plasmodium falciparum dihydropteroate synthetase (Pfdhps) haplotypes. The analysis involved 264 isolates collected from patients with uncomplicated malaria from Tanzania, Uganda and DR Congo. Isolates were genotyped for Pfdhps mutations at codons 436, 437, 540, 581 and 613. Three microsatellite loci (0.8, 4.3 and 7.7 kb) flanking the Pfdhps gene were assayed. Evolutionary analysis revealed a shared origin of Pfdhps haplotypes in East Africa, with a distinct population clustering in DR Congo. Furthermore, in Tanzania there was an independent distinct origin of Pfdhps SGEGA resistant haplotype. In Uganda and Tanzania, gene flow patterns contribute to the dispersal and shared origin of parasites carrying double- and triple-mutant Pfdhps haplotypes associated with poor outcomes of intermittent preventive treatment during pregnancy using SP (IPTp-SP). However, the origins of the Pfdhps haplotypes in DR Congo and Eastern Africa sites are different. The genetic structure demonstrated a divergent and distinct population cluster predominated by single-mutant Pfdhps haplotypes at the DR Congo site. This reflects the limited dispersal of double- and triple-mutant Pfdhps haplotypes in DR Congo. This study highlights the current genetic structure and dispersal of high-grade Pfdhps resistant haplotypes, which is important to guide implementation of SP in malaria chemoprevention strategies in the region.

Introduction

The widespread emergence of Plasmodium falciparum resistance to antimalarial drugs is a major public health concern threatening malaria control efforts. Sulfadoxine/pyrimethamine (SP) and amodiaquine were adopted to replace chloroquine as the first-line treatment against uncomplicated malaria following widespread resistance to chloroquine [1]. However, a few years after the adoption of SP, escalating treatment failure rates indicated that SP resistance was spreading. This prompted policy changes to adopt artemisinin-based combination therapies (ACTs) as the first-line treatment in malaria-endemic countries in Africa.

SP is still recommended by the World Health Organization (WHO) in sub-Saharan Africa for intermittent preventive treatment during pregnancy (IPTp-SP) to protect women and to improve foetal outcomes against the consequences of malaria infection. It is also used in seasonal malaria chemoprevention (SMC) strategies in combination with amodiaquine in areas prone to high seasonal malaria transmission across the Sahel subregion as well as in intermittent preventive treatment in infants (IPTi-SP) in areas of moderate to high transmission [2], [3]. In some countries (including the Democratic Republic of the Congo), SP is used in combination with artemisinin for the treatment of uncomplicated malaria [4]. However, due to widespread SP resistance, concerns have been raised about whether this strategy is still effective [5], [6].

Pyrimethamine resistance is conferred by point mutations in the P. falciparum dihydrofolate reductase (Pfdhfr) gene leading to substitutions at codons N51I, C59R and S108N [7]. Sulfadoxine resistance is mediated by substitutions in the P. falciparum dihydropteroate synthetase (Pfdhps) at codons S/A436F, A437G, K540E, A581G and A613S/T [8], [9]. Evidence suggests that SP resistance tends to increase as a result of stepwise accumulation of single nucleotide polymorphisms (SNPs) in the PfdhpsPfdhfr genes [10]. Interestingly, parasite isolates from East Africa were shown to harbour Pfdhps haplotypes with double mutations (at codons A437G and K540E) leading to the SGEAA haplotype, whereas in Western African countries the mutations are mainly limited to a single A437G mutation [11], [12]. Recently, in East Africa, expansion of Pfdhps mutation at codons A581G and K540E has worsened SP resistance leading to the emergence of the triple-mutant haplotype (SGEGA) [13]. Pfdhfr–Pfdhps haplotypes confer high-grade resistance associated with poor outcomes of IPTp-SP [6], [14]. Recently, a Pfdhps sextuple haplotype, defined as a combination of the triple Pfdhfr and triple Pfdhps mutations, was associated with reduced birth weight [6]. In addition, a recent meta-analysis estimated that if the prevalence of Pfdhps 581G is >10.1%, then IPTp-SP is no longer protective against low birth weight and alternative strategies should be considered [15]. In the East African region where the levels of SGEAA and SGEGA haplotypes are expanding, the effectiveness of IPTp-SP in controlling parasite growth and improving pregnancy outcomes is deteriorating [6], [14], [15]. In contrast, in West Africa, the majority of haplotypes are either Pfdhps wild-type or a single A437G (SGKAA) or S436A (AAKAA) mutation, with limited occurrence of the K540E and A581G mutants, and thus IPTp-SP retains its effectiveness [11]. These observations underscore the need for monitoring the distribution of parasite populations in order to mitigate the dispersal of resistant haplotypes in the region.

Studies from Southeast Asia revealed multiple and limited origins of Pfdhps mutant alleles [16], [17], and analysis of the Pfdhps mutant alleles in malaria parasites from East and West Africa demonstrated that resistance emerged independently in multiple sites in Africa [17]. After segregating the Pfdhps triple mutant SGEG haplotype lineages by country, the Pfdhps haplotypes seemed of local origin [18]. In DR Congo, Taylor et al [19] demonstrated genetically distinct resistant Pfdhps haplotypes between Eastern and Western provinces. However, owing to the escalating levels of Pfdhps A581G and K540E mutations associated with poor IPTp outcomes in Eastern Africa, there is a need to define the genetic structure and dispersal of Pfdhps haplotypes in the Central African corridor and their relatedness to the Eastern African lineages. Therefore, this study determined the origin and dispersal of sulfadoxine-resistant lineages in Central Africa compared with Eastern Africa Pfdhps haplotypes.

Section snippets

Sample collection

Samples for this study were collected between 2012 and 2014 in Uganda (Kihurura) and DR Congo (Lisungi-Kinshasa) as part of the QuinACT clinical trial [20]. Samples from Tanzania were collected in 2014 in Muheza District, Tanga Region, as part of a study to assess the efficacy and safety of artemether/lumefantrine versus dihydroartemisinin/piperaquine for the treatment of uncomplicated malaria (Fig. 1). Children aged 6 months to 10 years with uncomplicated falciparum malaria were enrolled.

Frequency and pattern of Pfdhps single nucleotide polymorphism haplotypes

A total of 264 samples were analysed for Pfdhps SNPs at codons 436, 437, 540, 581 and 613. Of these, 140 samples could be constructed into haplotypes, as the rest were mixed infections at one or more of the Pfdhps SNPs; DR Congo, n = 37; Tanzania, n = 41; and Uganda, n = 62. In total, six Pfdhps SNP haplotypes were detected: SAKAA (wild-type); SGKAA, AGKAA and SAEAA (single mutants); and SGEAA and SAEGA (double mutants); and SGEGA (triple mutant) (Fig. 2). The pattern varied, with single-mutant

Discussion

Molecular analysis of Pfdhps revealed a high presence of double (SGEAA) and triple (SGEGA) mutant haplotypes predominantly in the East African region (Tanzania and Uganda), whilst in DR Congo the single Pfdhps mutants were predominant. In contrast, the frequency of the Pfdhps SGEGA observed was somewhat low compared with recent studies in the same area of Northern Eastern Tanzania [32]. Nonetheless, the level of the Pfdhps triple mutation correlated with the increasing level of Pfdhps A581G

Conclusion

In Uganda and Tanzania, gene flow patterns contribute the dispersal and shared the origin of parasites carrying double- and triple-mutant Pfdhps haplotypes associated with poor IPTp-SP outcomes. However, the origins of the Pfdhps haplotypes in DR Congo and Eastern Africa sites are different. The genetic structure demonstrated a divergent and distinct population cluster predominated by single-mutant Pfdhps haplotypes in the DR Congo site. This reflects the limited dispersal of double- and

Acknowledgements

The study participants are thanked for agreeing to take part in this study. NIMR Tanga (Tanzania) staff involved in the WB project are thanked for their role in field sample collection. This work would not have been possible without the medical and study team involved in the QuinACT trial at Lisungi Health Centre (DR Congo) and Kihuru Kazo Health Centre (Uganda). Ulla Abildtrup at the Centre for Medical Parasitology (Copenhagen, Denmark) is thanked for the technical support during the

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      It would therefore be speculative to imagine the presence or absence of other mutations that were not analyzed (e.g., dhps I431V and A581G). However, though the prevalence of dhps A581G is expected to be lower in Kinshasa than in East Africa, we acknowledge that this specific mutation is worth being investigated in future given its clinical relevance for IPTp-SP failures and the growing concern raised by its emergence in other regions of Africa (Baraka et al., 2017; Mandoko et al., 2018; Minja et al., 2013; van Eijk et al., 2019). Secondly, the survey could not avoid the potential information and memory biases inherent in any self-reporting of information, even if the use of medical files and ANC booklets could have reduced these biases.

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      Interestingly, about 12% of the haplotypes (of the Pfdhps gene), although in a low frequency, are restricted to one of the other population and are thus considered to be particular to that population. In line with this study is the detection of some of the Pfdhps haplotypes (IAGKAA, ISGKAA, IAAKAA, ISAKAA, ISGKGA, IAGKAS, IAGKGA, IAGKGS, VAGKAS, VAGKGS and VAGKGA) from previous studies conducted in Democratic Republic of Congo, Tanzania as well as in India (Baraka et al., 2017; Oguike et al., 2016; Minja et al., 2013). Only four haplotypes (two wild and two mutant, two in Pfcrt and one each in Pfdhfr and Pfdhps) were found to be highly prevalent in the southern part of Nigeria with a minimum frequency of 25% (Pfdhps - ISGKAA in Edo) to >45% for Pfdhfr (AIRNI in Lagos).

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      The four mutations S436A, A437G, K540E and A581G have been found among samples from asymptomatic pregnant women in the south of the Republic of the Congo [36]. These same mutations were found in the neighbouring Democratic Republic of the Congo [53,54], whereas the K540E mutation is rare and A581G is completely absent in Gabon, its other neighbouring country [17,55]. The A613S mutation is rare in Africa and was not observed [13,56,57].

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