Osoti, V. | Search bioRxiv

bioRxiv2025

Convenient screening for drug resistance mutations from historical febrile malaria samples across Kenya

BackgroundHistorically, chloroquine resistance emerged that was driven by mutations in the chloroquine resistance transporter (Pfcrt) gene. This led to the global withdrawal of chloroquine in 1998 and its subsequent replacement with sulfadoxine-pyrimethamine, whose efficacy was compromised by a high prevalence of mutations in the dihydrofolate reductase and the dihydropteroate synthase genes by 2004. Consequently, artemisinin-based combination therapies (ACTs) were introduced in 2006. Since then, thirteen mutations in the kelch 13 (Pfk13) propeller domain have emerged and validated by the World Health Organization (WHO) as markers of partial artemisinin resistance. This study aimed to characterize temporal trends in both established, Pfcrt and Pfk13 and less well-described potential markers, cysteine desulfurase (Pfnfs) and Pfcoronin, using febrile malaria samples collected across diverse regions of Kenya between 2013 and 2022. MethodsThe temporal trend of these markers of resistance were assessed by screening archived P. falciparum positive dried blood spots (DBS). A total of 1,750 DBS samples collected from Therapeutic Efficacy Studies (TES) conducted in: Kwale (2013, n=350), Kisumu (2015, n=314), Busia (2016, n=334), Kisii (2017, n=314), Kwale (2018, n=150), and a hrp2 study conducted Kisii (2022, n=288). Parasite genomic DNA was extracted using the Chelex-saponin method and confirmed by a Pf 18S RT-PCR. Pfk13, Pfcrt, Pfnfs and Pfcoronin PCR amplicons were sequenced using capillary electrophoresis, Illumina Miseq or the Oxford Nanopore (GridION) platform. ResultsThe prevalence of Pfcrt mutations declined over time and no WHO validated Pfk13 mutations associated with artemisinin resistance were detected. However, synonymous substitutions at WHO-validated codons C469C and P553P were identified. In the PfCoronin gene, non-synonymous mutations distinct from those reported in West Africa were observed at high frequencies (>75%). Notably, the Pfnfs-K65Q mutation, previously associated with reduced lumefantrine sensitivity in West Africa, was detected in over 80% of samples. Our findings reveal differences in some antimalarial resistance genetic markers between observations made in The Gambia and Senegal (West Africa) and Kilifi (East Africa). Based on the convenient sample set, there were no WHO validated k13 mutations up until 2022, suggesting continued ACT efficacy in Kenya. This study underscores the importance of continued molecular surveillance and suggests that resistance may evolve through different pathways in East compared to West Africa and Southeast Asia.

genomics↗

bioRxiv2022

Validation of saline, PBS and a locally produced VTM at varying storage conditions to detect the SARS-CoV-2 virus by qRT-PCR

Coronavirus Disease-2019 tests require a Nasopharyngeal (NP) and/or Oropharyngeal (OP) specimen from the upper airway, from which virus RNA is extracted and detected through quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR). The viability of the virus is maintained after collection by storing the NP/OP swabs in Viral Transport Media (VTM). We evaluated the performance of four transport media: locally manufactured ("REVITAL") Viral Transport Media (RVTM), Standard Universal Transport Media (SUTM), PBS and 0.9% (w/v) NaCl (normal saline). We used laboratory cultured virus to evaluate: i) viral recovery and maintaining integrity at different time periods and temperatures; ii) stability in yielding detectable RNA consistently for all time points and conditions; and iii) their overall accuracy. Four vials of SARS-CoV-2 cultured virus (2 high and 2 low concentration samples) and 1 negative control sample were prepared for each media type (SUTM, RVTM, PBS and normal saline) and stored at the following temperatures, -80{degrees}C, 4{degrees}C, room temperature (25{degrees}C) and 37{degrees}C for 7 days. Viral Ribonucleic acid (RNA) extractions and qRT-PCR were done on the following days after inoculation with the cultured virus, days 1, 2, 3, 4 and 7 to assess virus stability and viral recovery. CT values fell over time at room temperature, but normal saline, PBS, RVTM and SUTM all showed comparable performance in maintaining virus integrity and stability allowing for the detection of SARS-CoV-2 viral RNA. Overall, this study demonstrated that normal saline, PBS and the locally manufactured VTM can be used for COVID-19 sample collection and testing, thus expanding the range of SARS-CoV-2 viral collection media.

molecular biology↗