Novel Plasmodium falciparum k13 gene polymorphisms from Kisii County, Kenya during an era of artemisinin-based combination therapy deployment

This study was conducted in Kisii County, Kenya in the year 2021, during the months of February to June. The county has nine sub-counties. The county is located approximately 306 kms from the capital city, Nairobi. It lies at latitude: (0.41°) South, longitude: (34.46°) East. According to the 2019 Kenya population and housing census, the county population size is 1,266,660 persons [16]. The main economic activity is agriculture. The county is characterized by hilly topography interspersed with ridges and valleys. The county is characterized by seasonal and permanent rivers which flow into Lake Victoria. The county exhibits a highland equatorial climate with an average rainfall of 1500 mm/year. The average temperature range is between 21 and 30 °C. Most of the population lives in rural areas, residing in local houses. The county health system consists of government and private-based health facilities. The government sector has one teaching and referral hospital (KTRH), which serves as a regional reference hospital and a teaching hospital for Kisii University Medical School. This county contains 14 sub-county hospitals. The county also contains 84 dispensaries, 28 health centres and 32 community health units, which serve as centres for minor health cases [17]. The county records three rain seasons namely; April–May, August–September and November–December. The main killer disease is malaria. The main malaria intervention approaches used to combat malaria in this region include proper case management with anti-malarial drugs, such as artemisinin-based combinations, intermittent prophylaxis during pregnancy (IPTp) and the use of mosquito nets. The current drug of choice for treating uncomplicated malaria is artemether-lumefantrine. Diagnosis and treatment services of malaria are available in all government health facilities and a few private facilities. The current study was conducted in hospitals selected from 4 sub-counties (Kenyenya, Marani, Bonchari,Nyamache) of Kisii County (Fig. 1). A molecular study was conducted at the Molecular Biology and Immunology Laboratory, School of Health Sciences, Makerere University, Kampala, Uganda.

This was a cross-sectional health point prospective study. The study enrolled participants who before the start of the study were presented with malaria clinical characteristics and had resided in Kisii County for at least the last six months. However, the study excluded those who were reluctant to give consent to the study. In addition, participants with febrile clinical illnesses initiated by pathogens other than malaria were 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 a fever (≥ 38 °C) or having a history of fever in the past 24 h, were confirmed for the presence of P. falciparum using microscopy (as a confirmatory test). Briefly, 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 fields. Thin smears were fixed in methanol before Giemsa-staining. Blood samples were collected by obtaining 1 ml of venous blood for the participants older than 2 years. 100 μL finger-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 filter paper (ET31CHR; Whatman Limited, Kent, UK) and labelled with the participant identification 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 AL 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 [18].

To distinguish between recrudescence and re-infection, 4 drops of blood from malaria-positive patients were collected on filter paper on day zero before treatment, and on any day of recurrent P. falciparum malaria. Molecular analysis was conducted following the previously described method [19], with slight modifications. Briefly, blood spotted filter 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 [20]. 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. Amplification was then performed under the following conditions; denaturation (94 °C), annealing (55 °C), and extension (72 °C). Amplification was confirmed by running the nested PCR product together with a DNA ladder on the QIAxcel capillary electrophoresis. The result was classified as recrudescence if at least one identical MSP2 allele was detected in both ACT pre-treatment and ACT post-treatment blood samples. Blood samples where MSP2 alleles did not match ACT pre- and ACT post-treatment were classified as new infections. Any sample, which failed to amplify was classified as undetermined. Blood samples, which showed recrudescence of parasites during any follow up day were further genotyped for P. falciparum k13 resistance markers. The primers used in this protocol are shown in Table 1.

DNA was extracted from blood spotted filter papers by using the chelex suspension method described by [20], with slight modifications. Blood spotted filter papers were soaked for 24 h in 1 mL of 0.5% saponin-1 phosphate buffered saline. The mixture was washed 2 times in 1-mL PBS and boiled for 8 min in 100 mL PCR-grade water. 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 centrifuged at 2608 RCF for 8 min to recover the DNA and stored at -30 °C. K13-propeller genes were amplified by the nested PCR protocol described previously [21] by using the primers listed in Table 1. For the first round of PCR, 0.5-mL DNA was amplified with 10 mL PCR Mix (1.25 U/mL, Taq DNA Polymerase, 0.4 mMdNTP Mixture, PCR buffer, and 4 mM Mg2þ), 0.5 mL forward primer (10 mM), 0.5 mL reverse primer (10 mM), and sterile ultrapure water to a final volume of 20 mL. For the second round of PCR, 0.5 mL primary PCR products were amplified 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 amplification 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 the Sanger method described by [22], with slight modifications. The second round PCR products were purified by using a jet quick PCR product purification kit. 5 μL of the purified 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 (Eurofins Genomics, Germany), using the Big Dye terminator v3.1 cycle sequencing kit (Applied Biosystems, 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 Biosystems, USA). This was accomplished by mixing the above total reaction volume with; 2.0 μL Big Dye terminator v3.1 (Applied Biosystems, USA), 1.0uL of 10 ng/μL (K133 forward or K132 Reverse) primers and 6.0 μL of nuclease-free water. Finally, 5.0 μL of 5.0 ng/μL of the purified second round PCR products were then added to make up the total volume. The following sequencing conditions were used. One cycle of polymerase activation at 96 °C for 60 s followed by 35 cycles of; denaturation at 96 °C for 10 s, annealing at 53 °C for 30 s and extension at 60 °C for 300 s (Gene Amp 9700 PCR system, USA). The amplified products were then stored at 4 °C until the next step of extension. The extension PCR products were then followed by purification using Dye Ex 2.0 spin Kit (QIAGEN, Maryland, USA). Subsequently, 5.0 μL of the purified cycle sequencing PCR products were then mixed with 5.0 μL of De-ionized formamide (Sigma Aldrich, USA) and then loaded in the 3130 genetic analyzer (Applied Bio systems, USA). Finally, the products the products were bi-sequenced with POP-7™ (Applied Bio systems, USA) as a sequencing Polymer. The primers used in this protocol are shown in Table 2.

The DNA sequences were analysed using the sequence analysis software 5 and then blasted on to the NCBI sequence database to confirm the k13 propeller gene sequence identity by using the Basic Local Alignment Search Tool (BLAST) at http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi. The sequences were exported to bio edit sequence alignment editor 7.2.5 for manual editing and then in to MEGA 5 software version 5.10 for detection of polymorphism using the PF3D7_1343700 and K13-propeller gene sequences present in the NCBI database were used as the reference sequence. Additional single-nucleotide polymorphism (SNPs) analysis within the K13 propeller gene was performed using the DnaSP software version 5.10.01. To assess the selection pressure in P. falciparum parasite population in Kisii County, Tajima’ D statistic and Fu & Li’s D test in DnaSP software 5.10.01 were used. In this analysis, the study evaluated whether the P. falciparum k13 propeller domain sequence data show evidence of deviation from the neutrality theory of molecular evolution. The analysis was done using commands in the DnaSP software. In the DnaSP software, the probability of Tajima’s D and Fu & Li’s D are estimated by simulation. The test uses information on the frequency of mutations (allelic variation) [23]. Tajima’s D and Fu & Li’s D test is based on the fact that under the neutral model, estimates of the number of polymorphic sites and the average number of nucleotide differences are correlated. The critical values (Tajima’s D and Fu & Li’s D) obtained were used in interpreting the findings under the neutrality assumption [24].

where, π, Mean pairwise differences; S, Number of segregating sites; p, total number of mutations.

where, ∩ e, Expected number of derived mutations that are present only once in the sample; S, Number of segregating sites

Ethical approval was sought from the University of East Africa, Baraton Institutional Review Board (UEAB/REC/4/2/2021, research permit was issued by Kenya National Commission for Science, Technology and Innovation (NACOSTI) License No: NACOSTI/P/21/8974 and Kisii County government (DTR/4/27). The guidelines outlined in the Declaration of Helsinki were followed as follows; written informed consent was obtained from the adult participants. The consent of those below 18 years of age was provided by the parents or the care-givers. Permission for conducting this study was granted by different sub-counties before starting the study. Moreover, participation was voluntary. The participants were coded instead of reflecting their names to maintain confidentiality. Participants who were malaria positive were given antimalarial treatment according to the WHO regulations and they were reimbursed for the travel cost, lost earnings and food expenses. Participants were respected in relation to their right of their cultural beliefs and rights. Participants were allowed to withdraw from the research without any condition. Approval from local leaders was obtained before beginning the study. Since this study was conducted during the COVID-19 pandemic, standard operating procedures were followed as stipulated by the WHO.

A total of 275 participants were recruited in 2021 during the months of February to June. More female (60.0%) participants were enrolled compared to males (40%). The mean age of the recruited participants was 27.60 ± 0.92 years. 69% (189.75 ± 0.25) of the participants were adults, with a nearly equal proportion between males and females. 84% (231.0 ± 0.84) of the participants completed the efficacy profiling on AL from day 0 to day 28. The temperature recorded at enrollment (day 0) varied across the sites, with Kenyenya recording 37.6 °C ± 1.1, Marani recording 37.5 °C ± 1.1, Bonchari recording 37.8 °C ± 1.1 and Nyamache recording 37.3 °C ± 1.1 respectively. However, the temperatures recorded during 28 days of study did not vary across the sites. The geometric mean parasite density (asexual parasites/µL) was significantly higher at Bonchari, having recorded 13,531 (9,242–15,603), (p = 0.047) compared to the other sites; patients enrolled at Nyamache had the lowest parasitaemia. The mean weight and median age range varied across all sub-counties (Table 3).

After a complete follow-up of the participants, 13/231 (5.6%) had P. falciparum on day 28. For those samples with the occurrence of parasites on day 28 after treatment had the following PCR; 77% of the samples had bands on both day 0 and day 28, hence were classified as recrudescence. About 23% of the samples had no bands on both days 0 and the respective days of parasite recurrence, hence classified as new infections (Fig. 2). The 77% of samples showing recrudescence in this study were stored for further P. falciparum kelch13 sequencing to evaluate the presence of any mutational polymorphisms conferring ACT resistance.

A total of 13 blood samples confirmed for day 28 recrudescence using microscopy correction were processed for Pfkelch13 PCR amplifications. Parasites DNA of 5 (38%) samples were successfully amplified as shown in Fig. 3 and Table 4 by using nested PCR reaction for the k13-propeller gene mutations and hence were sequenced for mutations.

K13 propeller single-nucleotide polymorphisms were compared with the 3D7 reference strain (PF3D7-1343700). K13-propeller non-synonymous polymorphisms recorded in the current study include R539T, N458Y, R561H, N431S, and A671V. All mutations with an exception of R561H were reported in 1 sample, while R561H mutation was reported in 2 samples, respectively. However, mutation on K13 propeller gene was not detected at positions 580, 578, 574, 568, 553, 543, 539, 537, 533, 527, 508, 493, 481, 476, and 474, respectively. A detailed analysis of the samples is shown in Table 5. In total, there were six (6) polymorphic sites identified in the 13 samples analysed.

The mutations in the samples analysed were unselectively neutral, as shown by the negative Tajima’s D statistic (− 1.72305) and Fu & Li’s D test (− 1.74248). There was no significant haplotype/gene diversity (P = 0.305) with a variance in the diversity of 0.00363 and a standard deviation of 0.078 (Table 6).