Novel Plasmodium falciparum K13 Gene Polymorphisms from Kisii County, Kenya during an era of Artemisinin-Based Combination Therapies (ACTs) deployment

Currently, Chemotherapy stands out as the major malaria intervention strategy, however, antimalarial resistance may hamper global elimination programs. Artemisinin Combined Therapies (ACTs) stands as the drug of choice for treatment of Plasmodium falciparum malaria. P. falciparum Kelch13 gene mutations are associated with artemisinin resistance. Thus, this study was aimed at evaluating the circulation of P. falciparum K13 Gene Polymorphisms from Kisii County, Kenya during an era of ACTs deployment. was extracted A nested reaction (PCR) was and the were sequenced Sequenced were analyzed using DNAsp software and then on to the NCBI for K13 propeller gene sequence identity using the Basic Local Alignment Search Tool (BLAST). To assess the selection pressure in P. falciparum parasite population, Tajima’ D statistic and Fu & Li’s D test in DnaSP software 5.10.01 was used. excluded from the study. Before collecting blood specimen, a complete medical examination and demographic information were obtained. All patients who had been suspected of having malaria infection by having fever ( ≥ 38°C) or having a history of fever in the past 24 h, were con�rmed for the presence of P. falciparum using microscopy (as a con�rmatory test). Brie�y, thick and thin blood smears were stained with 2% Giemsa for 30 min. A smear was considered negative if no parasites were observed after examination under 100 high-powered �elds. For Giemsa stained thin smears, they were �xed in methanol solution before the examination. Blood samples were collected by obtaining 1 ml of venous blood for the participants older than 2 years. 100 µL �nger-pricked blood samples were collected in the case of children below 2 years of age. This procedure was repeated during the consequent follow-up visits. The blood spots were made on chromatography �lter paper (ET31CHR; Whatman Limited, Kent, UK) and labeled well with the participant identi�cation number. Malaria-positive participants were followed up for a period of 28 days by evaluating clinical and parasitological parameters on days 1, 3, 7, 14, and 28, respectively, after ACTs (A-L) treatment initiation. Finger pricks for follow-up were taken on days 1, 3, 7, 14 and 28 to check for the presence of P. falciparum [17]. To distinguish between recrudescence and re-infection, 4 drops of blood from malaria-positive patients were collected on �lter paper on day zero before treatment, and on any day of recurrent P. falciparum malaria. Molecular analysis was conducted following the previously described method [18], with slight modi�cations. Brie�y, blood spotted �lter papers were soaked for 24 h in 1 mL of 0.5% saponin-1 phosphate buffered saline. The mixture was washed twice in 1-mL PBS and boiled for 8 min in 100 mL PCR-grade water to release DNA from the cells. To elute the extracted DNA, 150 µL Buffer AE was added to each well using a multichannel pipette and incubated for 1 min at room temperature. This setup was then centrifuged at 2608 RCF for 8 min. DNA was recovered and stored at -80°C. Nested PCR was performed on the extracted DNA for subsequent genotyping of P. falciparum polymorphic gene loci encoding Merozoite surface protein 2 (MSP-2) using the method described by [19]. A master mix was prepared according to manufacturer instructions (New England Bio Labs, Massachusetts, USA). 24 µL of the Master Mix was added to the PCR 96 well plate and 25 µL of the master mix was also added to the negative PCR control. The plates were sealed using a thermo seal plate sealer and placed in the PCR thermo-cycler. Ampli�cation was then performed under the following conditions; denaturation (94°C), annealing (55°C), and extension (72°C). Ampli�cation was con�rmed by running the nested PCR product together with a DNA ladder on the QIAxcel capillary electrophoresis. The result was classi�ed as recrudescence if at least one identical MSP2 allele was detected in both ACTs pre-treatment and ACTs post-treatment blood samples. Blood samples where MSP2 alleles did not match ACTs pre- and ACTs post-treatment were classi�ed as new infections. Any sample, which failed to amplify was classi�ed as undetermined. Blood samples, which showed 0.5 mL forward primer (10 mM), 0.5 mL reverse primer (10 mM), and sterile ultrapure water to a �nal volume of 20 mL. For the second round of PCR, 0.5 mL primary PCR products were ampli�ed with a 40 mL reaction system, including 20 mL PCR Mix, 1.0 mL forward primer (10 mM), 1.0 mL reverse primer (10 mM), and H2O. The ampli�cation conditions were maintained at 95°C for 3 min; followed by 30 cycles (95°C for 30 s, 55°C for 30 s, 72°C for 30 s); 72°C. For 5 min; then stored at 12°C. Sequencing was done using Sanger’s method described by (Ocan et al., 2016), with slight modi�cations. The second round PCR products were puri�ed by using a jet quick PCR product puri�cation kit. 5 µL of the puri�ed second round PCR products were then run on 1.0% (w/v) agarose gel stained with 0.05 µg/mL of Ethidium Bromide (Sigma Aldrich, USA) to counter-check for the presence and concentration of PCR products. This was followed by Bi-directional cycle sequencing of K13 using the second round K133 Forward and K132 Reverse PCR primers (Euro�ns Genomics, Germany), using the Big Dye terminator v3.1 cycle sequencing kit (Applied Bio systems, USA). Cycle sequencing PCR was performed in a total reaction volume of 20 µL. 6.0 µL of the Big Dye terminator v3.1 5X sequencing buffer (Applied Bio systems, This was accomplished by mixing the above total reaction volume with; 2.0 µL Big Dye terminator v3.1 (Applied Bio systems, USA), 1.0uL of 10 ng/µL (K133 forward or K132 Reverse) primers and 6.0uL of nuclease-free water. Finally, 5.0 uL of 5.0 ng/µL of the puri�ed second round PCR products were then added to make up the total volume.