Therapeutic efficacy of artesunate–amodiaquine and artemether–lumefantrine for the treatment of uncomplicated falciparum malaria in Chad: clinical and genetic surveillance

Abstract

Background Artesunate–amodiaquine (AS–AQ) and artemether–lumefantrine (AL) are the currently recommended first-and second-line therapies for uncomplicated Plasmodium falciparum infections in Chad. This study assessed the efficacy of these artemisinin-based combinations, proportion of day 3 positive patients, proportions of molecular markers associated with P. falciparum resistance to anti-malarial drugs and variable performance of HRP2-based malaria rapid diagnostic tests (RDTs). Methods A single-arm prospective study assessing the efficacy of AS–AQ and AL at three sites (Doba, Kelo and Koyom) was conducted between November 2020 to January 2021. Febrile children aged 6 to 59 months with confirmed uncomplicated P. falciparum infection were enrolled sequentially first to AS–AQ and then AL at each site and followed up for 28 days. The primary endpoint was PCR-adjusted adequate clinical and parasitological response (ACPR). Samples collected on day 0 were analysed for mutations in pfkelch13, pfcrt, pfmdr-1, pfdhfr, pfdhps genes and deletions in pfhrp2/pfhrp3 genes. Results By the end of 28-day follow-up, per-protocol PCR corrected ACPR of 97.8% (CI 95% 88.2–100) in Kelo and 100% in Doba and Kayoma were observed among AL treated patients. For ASAQ, 100% ACPR was found in all sites. All, but one patient, did not have parasites detected on day 3. Out of the 215 day 0 samples, 96.7% showed pfkelch13 wild type allele. Seven isolates carried nonsynonymous mutations not known to be associated artemisinin partial resistance (ART-R). Most of samples had a pfcrt wild type allele (79% to 89%). The most prevalent pfmdr-1 allele detected was the single mutant 184F (51.2%). For pfdhfr and pfdhps mutations, the quintuple mutant allele N51I/C59R/S108N + G437A/540E responsible for SP treatment failures in adults and children was not detected. Single deletion in the pfhrp2 and pfhrp3 gene were detected in 10/215 (4.7%) and 2/215 (0.9%), respectively. Dual pfhrp2/pfhrp3 deletions, potentially threatening the efficacy of HRP2-based RDTs, were observed in 5/215 (2.3%) isolates. Conclusion The results of this study confirm that AS–AQ and AL treatments are highly efficacious in study areas in Chad. The absence of known pfkelch13 mutations in the study sites and the high parasite clearance rate at day 3 suggest the absence of ART-R. The absence of pfdhfr/pfdhps quintuple or sextuple (quintuple + 581G) mutant supports the continued use of SP for IPTp during pregnancy. The presence of parasites with dual pfhrp2/pfhrp3 deletions, potentially threatening the efficacy of HRP2-based RDTs, warrants the continued surveillance. Trial registration ACTRN12622001476729

Impact of pfhrp2 and pfhrp3 deletions in diagnosis of asymptomatic malaria among school children in Osun State, Nigeria

“…However, the frequency of deletions detected (41.53% pfhrp2 deletion, 52.72% pfhrp3 deletion) in this study is higher than the reported in previous studies in Nigeria [9,10,17] and in its border countries, such as Chad (12.3% pfhrp2 deletion) [23], Cameroon (1.5% pfhrp2 deletion) [24] and Benin (0% pfhrp2 deletion) [10]. That could be explained by different factors.…”

Efficacy of artemether-lumefantrine and dihydroartemisinin-piperaquine and prevalence of molecular markers of anti-malarial drug resistance in children in Togo in 2021

“…For the Pfmdr-1 gene, the single 184F mutation, which has no clear impact on ACT drug efficacy, was highly prevalent in Togo (75.9%). This is usually reported in many African settings such as Ghana [ 65 ], Uganda [ 66 ], Madagascar [ 67 ], Tanzania [ 68 ], and Equatorial Guinea [ 69 ], Chad [ 44 ],…”

“…As previously described [ 44 ], multiplex nested PCR assays with indexed primers with unique sample-specific tags (barcodes of individual 8-base indices) specific to the sample and adapter sequences (14 or 15 bases), which enabled the final PCR product to bind to the sequencing flow cell, were used to generated amplicons of targeted sequences were produced. To increase the sample volume and reduce the amount of sample for subsequent protocol steps, 4 µL of PCR reactions for each sample was pooled (96 samples).…”

Assessing the histidine-rich protein 2/3 gene deletion in Plasmodium falciparum isolates from Burkina Faso

“…The eluted DNA was then quantified by fluorometric quantification (Qubit, Thermo Fisher), adjusted to 20 ng/μL and stored at − 20 °C until further use. Amplicons from the target regions (exon 2 of the hrp2 and hrp3 genes) were generated using multiplexed nested PCR assays with indexed primers containing specific tags (barcodes)[ 29 – 31 ]. A total of 4 μL of PCR reactions from each sample were pooled (96 samples) to increase the sample volume and to minimize the use of samples for the downstream steps of the protocol.…”