Emergence of artemisinin resistance in East Africa has greatly necessitated surveillance of artemisinin resistance in other African countries with great malaria burden. Specific mutations within the β-propeller and BTB/Poz domains of Pfkelch13 gene are validated markers of artemisinin resistance. Sparse information exists on mutations outside these domains that may also contribute to artemisinin resistance. This study evaluated the occurrence and frequency of mutations in all domains of Pfkelch13 gene, and their impact on treatment outcome in Ibadan pre- and post-adoption of Artemisinin-based Combination Therapies (ACTs) in Nigeria. Dry blood spots prepared from blood samples obtained from P. falciparum-infected patients during retrospective (2000–2005) and prospective (2021) studies were analysed. Treatment outcomes with dihydroartemisinin-piperaquine were evaluated in a cohort of patients from the prospective study during a 42-day follow-up. Nested amplifications of Pfkelch13 gene fragments were done and subjected to Sanger dideoxy sequencing. Mutations were identified by sequence alignment and correlated with treatment outcome parameters including parasite clearance time (PCT) and day 2 parasite reduction ratio (PRRD₂) among others. Mean PCT was 2.1 ± 0.6 days (95%CI: 1.97–2.24) while PRRD₂ was 4815 with 100% adequate clinical and parasitological response. Altogether, 64 (11 retrospective/53 prospective) samples were successfully sequenced. None of the β-propeller domain mutations validated as artemisinin resistance markers were found within the analysed samples. However, four distinct mutations, K189T (64.2%), K189N (1.9%), R255K (3.8%), and N217H (1.9%) were identified within the N-terminal domain of the prospective samples while the K189T mutation was identified in a retrospective sample obtained in 2003. The K189T and R255K mutations correlated significantly with longer parasite clearance time in treated patients (P < 0.002). There was no evidence of validated molecular marker of artemisinin resistance within the ß-propeller domain of Pfkelch13. However, frequency of K189T mutation and its significant correlation with longer PCT may suggest possibilities of geographical variations in genetic drivers of artemisinin resistance.

Objectives: The invasive nature of the current malaria diagnostic techniques impairs compliance to diagnosis, especially for on-field detection. Adapting non-invasive methods of biological sample collection for rapid diagnosis of malaria infections may provide a more efficient approach to case management and epidemiological studies of malaria. This study was designed to evaluate the detection of Plasmodium falciparum Histidine-rich Protein II (PfHRP-2) in urine samples and optimization as diagnostic markers for P. falciparum infection. Methods: One hundred (100) microscopically confirmed patients with Plasmodium falciparum infection and 25 P. falciparum negative controls were recruited for the study. Blood samples of all participants were tested for the presence of PfHRP-2 using Rapid Diagnostic Test (RDT) kits. In addition, urine samples of the confirmed malaria-infected patients were analyzed for PfHRP-2 using the CareStartTM and Global Devices (USA) Malaria kits. The diagnostic performances of the RDT kits were evaluated. Results: Overall, the two brands of malaria rapid diagnostics demonstrated 71% sensitivity (95%CI=62.1-79.9%) and 96% specificity (95%CI=88.3-103.7%) for PfHRP-2 detection in urine. The sensitivities of the tests in urine at asexual parasitemia ≤ 2000 μL-1 and asexual parasitemia > 2000 μL-1 were 69.6% (95%CI=56.3-82.9%) and 72.2% (95%CI=60.3-84.2%) respectively. Global Devices and CareStartTM kits had individual sensitivities of 80% (95%CI= 65.7-94.3%) and 67.1% (95%CI= 56.1-78.1%) respectively for PfHRP-2 detection in urine (P= 0.072). Conclusion: Findings revealed that urine-based RDTs have limited capacities for malaria diagnosis due to their low sensitivity and require more optimizations to meet required diagnostic standards. J Microbiol Infect Dis 2022; 12(3):97-107.

The replacement of chloroquine with artemisinin-based combination therapies (ACTs) for over a decade has had varying impacts on the ability of malaria parasite to sustain its chloroquine resistance prowess in different malaria-endemic regions. We evaluated the frequency of Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations in an endemic area of southwest Nigeria 17 years after replacement of chloroquine with ACTs for malaria treatment. Genomic DNA was isolated from dried blood spot samples obtained from 129 patients (aged 1-35 years) with microscopically confirmed P. falciparum infection. PfCRT fragments covering codons 72-76, CVMNK (wildtype) and A220 were amplified and sequenced. Two mutant PfCRT haplotypes on residues 72-76 (CVIET and CVINT) were identified with a prevalence of 18.6% and 2.3%, respectively. Interestingly, the CVINT haplotype was identified for the first time in this region. A220S changes were found in 16.3% of samples occurring concurrently with the CVIET haplotype, while a Q271E mutation occurred in a wildtype isolate. The reduced prevalence of the PfCRT mutant alleles in this study may suggest a gradual disappearance of chloroquine-resistant malaria parasites following reduced drug pressure. It may also be an indicator of the ability of malaria parasites to develop resistance gradually against the current first-line regimen.