Prevalence of Plasmodium falciparum antimalarial drug resistance genes in Southeastern Gabon from 2011 to 2014

Introduction

Malaria is the most important parasitic disease in the world and affects children and pregnant women in particular. Africa has the most substantial burden, especially sub-Saharan African countries, with ~216 million cases of illness and 445,000 deaths in 2016.¹ The emergence of drug resistance remains a major obstacle in the fight against malaria. Given the spread of resistance to the major antimalarial drugs, the World Health Organization (WHO) has recommended the use of artemisinin-based combination therapies (ACTs) to treat uncomplicated malaria and sulfadoxine–pyrimethamine (SP) as an intermittent preventive treatment during pregnancy (IPTp). Consequently, since 2010, the mortality rates due to malaria have fallen by 32%.¹ However, the emergence of artemisinin resistance, manifested by delayed parasite clearance after monotherapy with artesunate or ACTs, was described in Southeast Asia.^2,3 The withdrawal of previous antimalarial drugs and the switch to ACTs were followed in some countries by a reemergence of some wild-type alleles that were associated with susceptibility to the previous drug treatment. This was reported for the Pfcrt chloroquine marker (Plasmodium falciparum chloroquine resistance transporter) in Tanzania. The K76 wild-type haplotype increased after abandoning chloroquine use and implementing artemether–lumefantrine treatment.⁴

Two genes, Pfdhfr (P. falciparum dihydrofolate reductase) and Pfdhps (P. falciparum dihydropteroate synthase), have been associated with pyrimethamine and sulfadoxine resistance, respectively, around the world.⁵ The triple Pfdhfr mutation N51I, C59R, and S108N in combination with the double A437G and K540E Pfdhps mutant formed a quintuple mutant haplotype, which confers a high risk of treatment failure with SP.⁶ Recent studies have shown an increase in Pfdhps mutations at A581G, thus further escalating the risk for even higher levels of resistance and significant decreases in the effectiveness of SP as IPTp.^7,8

The P. falciparum kelch 13 (PfK13) propeller gene has been associated with artemisinin resistance in Southeast Asia since 2013.⁹ Currently, 17 mutations have been highlighted and associated with artemisinin in Southeast Asia. Among these mutations, the C580Y, Y493H, R539T, and I543T mutations are strongly associated with slow-clearing parasites.^2,9–11 Many studies have been conducted in Africa but none of the specific Asian PfK13 mutations associated with artemisinin resistance were found.^10,12–17 The A578S mutation was often reported in Africa ^{10,12,17–20} but all the patients carrying these mutant parasites were cured by artemether–lumefantrine or artesunate-amodiaquine (ASAQ) before day 3.^{10,12,17–23}

In Gabon, a hyperendemic country, the use of ACT as the first line of treatment for uncomplicated malaria has been effective since 2005; whereas ASAQ and artemether–lumefantrine have been the first-line treatment and dihydroarte-misinin-piperaquine (DHA-PPQ) has been a second-line treatment for uncomplicated malaria. Severe malaria is treated with injectable quinine or artesunate. Consequently, a significant decrease in the malaria burden has been observed in Libreville and Franceville.^24,25 Moreover, SP is used as an IPTp for pregnant women.²⁴ This measure contributed to a significant drop in malaria in pregnant women.²⁶ Chloroquine was the drug used before the introduction of ACT, and its resistance was well described in Gabon. A high level of K76T mutation in Pfcrt was reported.^25,27,28 Before the implementation of IPTp with SP, studies reported treatment failures with SP.^24,29 During the period of implementation of IPTp with SP in Libreville and Lambarene, a preliminary study reported a high prevalence of multiple mutations in Pfdhfr (nearly 98.0%).²⁴ In North Gabon, 91.9% of Pfdhfr triple mutants and 64.8% of quadruple mutants (Pfdhps A437G) were observed 3 years after this implementation.³⁰ The frequencies of Pfdhfr triple and quintuple mutants (Pfdhps S436A and A437G) increased from 92.9% to 100% and from 17.9% to 75.6%, respectively, between 2005 and 2011 in pregnant women treated after IPTp with SP.³¹ A recent exploration of PfK13 at Libreville did not detect any mutation.¹⁸ There are no available data on the prevalence of PfK13 mutations in South Gabon.

The aim of this retrospective study was to determine the evolution of the prevalence of wild-type/mutant haplotypes for different genes linked to resistance, such as Pfcrt (codons 72 to 76), Pfdhfr (51, 59, 108, 164), Pfdhps (436, 437, 540, 581, 613), and PfK13 between 2011 and 2014 in Lastourville, Koulamoutou, and Franceville, three cities located in Southeast Gabon.

Methods

Results

Discussion

To investigate the evolution of antimalarial drug resistance, children younger than 6 and 168 months, with a mean age between 36 and 79 months, were identified at the three sites. We retrospectively analyzed samples from 2011 to 2014, but the data were only from 2013 to 2014 for Lastourville and Koulamoutou.

There was a significant difference in the mean age between 2013 and 2014 at the three sites. The children enrolled in Lastourville and Koulamoutou (rural and semi-urban areas, respectively) were younger than those from the urban site of Franceville. This was consistent with previous studies, which reported the association of an increase of the mean age of P. falciparum-infected children with the drop of malaria prevalence in urban localities, such as Franceville, and in other endemic areas, such as Gambia.^36–39 This increase is due to the delay of immunity acquisition. Indeed, the decrease in the malaria burden has led to a decrease in the number of infective bites, which consequently limits immune stimulation. It was previously shown that the malaria prevalence was higher in Lastourville than in Franceville and Koulamoutou, leading to a higher antimalarial immunity in Lastourville than in Franceville and Koulamoutou.³⁷ Indeed, parasitemia was higher in the rural site in 2013 and 2014. A probable hypothesis is the better availability of antimalarial treatment and preventive measures in urban areas. These findings can also be associated with the prevalence of other parasitic or infectious diseases in rural areas that increased the anti-infectious disease immunity, as previously reported.^40–43

P. falciparum drug resistance remains a challenge for controlling the malarial burden. The analysis of Pfcrt (chloroquine resistance marker) revealed a significant increase in the chloroquine-susceptible wild-type haplotype CVMNK between 2012 and 2014 in Franceville (the urban area), despite a high level of the resistant haplotype CVIET. However, although the results were significant, these data were determined in a small number of samples (1/17 in 2011, 1/25 in 2012, 2/19 in 2013, and 5/17 in 2014), suggesting an absence of a noticeable reemergence of strains susceptible to chloroquine. The prevalence of resistant strains to chloroquine was still 70.6%. In the semi-urban and rural sites, no significant difference was observed, and the prevalence of the resistant haplotype CVIET remained high since 2000.²⁷ Despite the implementation of ACT and the withdrawal of chloroquine, the frequency of the resistant allele 76T remained high in Franceville (93.8% in 2004, 96% in 2009, and from 70.6% to 96.3% between 2011 and 2014) and in all of Gabon (100% from 1995 until 2002, 97% from 2005 until 2007, and above 70% since 2008).^25,27,28,38 Recent studies conducted in Gabon from 1995 to 2008 showed an increased but non-significant amount of the wild-type allele K76, as shown in other studies in Sub-Saharan Africa.^{27,28,44–46} In South Gabon, the frequency of the resistant allele 76T was higher at 2009.⁴⁷ There can be a long delay in the selection of the T76 Pfcrt genotype, as shown in previous data from Zanzibar, where this selection was not observed in 2005 but clearly appeared in 2009.^4,48 Indeed, recent data from Franceville showed a significant decrease of T76 prevalence around 10 years after chloroquine withdrawal.³⁷ These findings suggest that the selection of T76 Pfcrt takes more time after changes in the antimalarial policy. So, some explanations of the non-significant variation of T76 prevalence between 2011 and 2014 could be the short time and also the smallness of the study effectives here. Furthermore, the T76 prevalence found at 2014 was similar to the previous findings.³⁷ The high frequency of the resistant allele 76T in the south of Gabon may also be due to cross resistance with amodiaquine used in combination with artesunate despite the withdrawal of chloroquine.^49–51. On other hand, some reports highlighted that an increase in chloroquine resistance has been observed in recent years following a decrease due to the withdrawal of chloroquine and the introduction of ACT in 2002 in Senegal.

Because of the implementation of IPTp with SP, the Pfdhfr and Pfdhps haplotypes associated with SP resistance were investigated. According to published data from Gabon, the most frequent Pfdhfr haplotype found was the triple-mutant IRNI, which is present in the three localities. A previous study showed that IRNI was the most prevalent Pfdhfr haplotype found both in Lambarene (>92%) in 2007 in Northwest Gabon and in Libreville in 2011 in pregnant women after IPTp with SP (100%).^24,29,30,52 These high prevalences of IRNI (>90%) were also observed in Central Africa.^53–55 The withdrawal of IPT with SP induced the decline of the IRNI haplotype in North-western Ethiopia, suggesting that the high prevalence of IRNI is maintained due to the use of pyrimethamine.⁵⁶

Among the successfully analyzed Pfdhps sequence, only mutations at codons 436 and/or 437 have been observed in the three localities. The SGKAA haplotype bearing the 437 single mutation is predominant (50%–100%). These data were consistent with the selection of the 437G mutation reported recently between 2005 and 2008 in North Gabon.³⁰ A non-significant increase in the haplotype with the double mutations 436 and 437 AGKAA has been observed since 2013 in Franceville and since 2014 in Koulamoutou and Lastourville. No haplotype with the 540 and 581 mutations has been observed, contrary to other African countries, such as the Congo, which is a neighbor of Gabon.⁵⁷ Indeed, the K540E mutation was found in Libreville between 2005 and 2006 and in Lambarene.^24,29 However, previous data from the Haut Ogooué province did not report mutations in codons 540 and 581 in 2000.⁵⁸ The K540E and A581G mutations contributed strongly to reducing the effectiveness of SP during pregnancy.^5,7,59 The A613S mutation, which is rarely found in Africa, was present in only one isolate. No A613T mutations described in Asia and East Africa were found in our study.^24,60,61

The high prevalence of the IRNI haplotype of Pfdhfr and the SGKAA of Pfdhps concomitantly in urban and rural areas suggested a similar level of adhesion of IPT with SP in urban and rural areas. The absence of high-grade resistance mutant haplotypes (Pfdhfr [51+59+108] + Pfdhps [437+540+581]) associated with IPTp-SP failures during pregnancy in the three localities suggests the efficiency of SP as IPTp.⁶² This efficiency was associated with a decrease in the prevalence of microscopic P. falciparum infection during pregnancy following IPTp-SP implementation in the urban cities of Gabon.⁶³ However, the highest level of the triple Pfdhfr mutant plus Pfdhps 437 mutant, considered as marker for SP failure, call for the need of IPTp-SP efficacy monitoring.

Monitoring the emergence and spread of artemisinin resistance is a great challenge. However, the emergence of artemisinin resistance, as manifested by delayed parasite clearance after monotherapy with artesunate or ACT, was described in Southeast Asia.^2,3 Several mutations in the PfK13 propeller gene and, more specifically, the C580Y, Y493H, R539T, and I543T mutations were associated with artemisinin resistance in Southeast Asia.^2,9–11 None of the mutations described in Southeast Asia that were linked to artemisinin resistance were detected in our samples. The use of bitherapies in the treatment of malaria can delay the apparition of drug resistance. Also, the very short half-life of artemisinin is not favorable for the selection of resistant parasite. These two factors could explain the fact that, less than 10 years after ACT implementation, no molecular markers for artemisinin resistance was observed. Those results were consistent with previous studies in Africa.^{10,12–17,64} The A578S mutation was detected in one isolate (0.4%). This mutation was often reported in Africa but it had a low prevalence (<2%).^{10,12,17–20,22,23} This mutation was also identified in Bangladesh.⁶⁵ However, A578S mutation was not associated with artemisinin treatment failure and all the patients carrying the mutant parasites were cured by artemether–lumefantrine or ASAQ before day 3.^10,21 Furthermore, the absence of a role for the A578S mutation in artemisinin resistance was confirmed in an in vitro study using site-directed mutagenesis.¹⁰ Two others mutations, V494A (0.4%) and A504V (0.9%), were identified for the first time in two isolates. A non-synonymous mutation V494I was observed in Angola and in Mozambique but was not associated with artemisinin resistance.⁶⁶ This study described new PfK13 polymorphisms, but the lack of an in vitro assay did not allow us to determine the absence of resistance to artemisinin in the southeast of Gabon. However, the role of PfK13 polymorphisms in artemisinin derivatives in Africa is still debated. Recent publications reported that parasites collected from African patients who were still parasitemic at day 3 and beyond did not carry mutations in PfK13 after ACT treatment.^67–70

Conclusion

This study showed a high prevalence of a Pfcrt-resistant haplotype in Gabon between 2011 and 2014 (>70%), even though chloroquine was no longer used. Despite a great diversity and variable distribution of the alleles linked to SP susceptibility, no resistant quintuple mutation haplotypes associated with IPTp-SP failure have been found in this period. Since ACT implementation, there is not yet artemisinin resistance in Southeast Gabon. However, the spread of antimalarial drug resistance from Southeast Asia to Africa, as described for chloroquine and SP, requires an intensive epidemiological survey.^71,72