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

BACKGROUND: Currently, chemotherapy stands out as the major malaria intervention strategy, however, anti-malarial resistance may hamper global elimination programs. Artemisinin-based combination therapy (ACT) stands as the drug of choice for the treatment of Plasmodium falciparum malaria. Plasmodium 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 ACT deployment. METHODS: Participants suspected to have malaria were recruited. Plasmodium falciparum was confirmed using the microscopy method. Malaria-positive patients were treated with artemether-lumefantrine (AL). Blood from participants who tested positive for parasites after day 3 was kept on filter papers. DNA was extracted using chelex-suspension method. A nested polymerase chain reaction (PCR) was conducted and the second-round products were sequenced using the Sanger method. Sequenced products were analysed using DNAsp 5.10.01 software and then blasted on 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. RESULTS: Out of 275 enrolled participants, 231 completed the follow-up schedule. 13 (5.6%) had parasites on day 28 hence characterized for recrudescence. Out of the 13 samples suspected of recrudescence, 5 (38%) samples were positively amplified as P. falciparum, with polymorphisms in the k13-propeller gene detected. Polymorphisms detected in this study includes R539T, N458T, R561H, N431S and A671V, respectively. The sequences have been deposited in NCBI with bio-project number PRJNA885380 and accession numbers SAMN31087434, SAMN31087433, SAMN31087432, SAMN31087431 and SAMN31087430 respectively. CONCLUSIONS: WHO validated polymorphisms in the k13-propeller gene previously reported to be associated with ACT resistance were not detected in the P. falciparum isolates from Kisii County, Kenya. However, some previously reported un-validated k13 resistant single nucleotide polymorphisms were reported in this study but with limited occurrences. The study has also reported new SNPs. More studies need to be carried out in the entire country to understand the association of reported mutations if any, with ACT resistance.

Trend of Malaria Burden Among Residents of Kisii County, Kenya After More Than a Decade Usage of Artemisinin Combined Therapies, 11-Year Laboratory Based Retrospective Study.

Background: Malaria remains a major vector borne disease globally, with the majority of the casualties reported in Africa. Despite this fact, there is drastic reduction in malaria infection using Artemisinin combined therapies (ACTs). Malaria is characterized by significant inconsistency in different geographical locations due to different confounding factors. There is need to identify zone-specific malaria trends and interventions to completely eliminate the disease. Thus the study was aimed at assessing the 11-year trend of microscopically confirmed malaria cases in Kisii County, Kenya, so as to devise area-specific evidence-based interventions, informed decisions, and to track the effectiveness of malaria control programs. Methods: This was a retrospective study carried out to determine 11-year malaria trend rates centered on the admission and laboratory records from health facilities located at four Sub-Counties in Kisii County, Kenya. Parasitological positivity rates of malaria were determined by comparing with the register records in health facilities which recorded confirmed malaria cases with the total number of monthly admissions over the entire year. Data was analyzed by using descriptive tools and chi-square test. Results: There were 36,946 suspect cases, with 8449 (22.8%) confirmed malaria cases reported in this study. The overall malaria slide positivity rate over the last 11 years in the study area was 22.6%. The months of April and August showed the largest number of malaria cases (63%). The age group of ≥18 years contained the most positive confirmed cases, having a prevalence rate of 2953 (35.45%). Out of the confirmed malaria cases, 2379 (28.1%) were males and 6070 (71.9%) were females The highest malaria prevalence rate was recorded in 2014, with Marani Sub-County recording the highest positivity rate of 37.94%. Conclusion: From the observed trends, malaria prevalence and transmission still remains stable in the study area. Thus more interventions need to be scaled up.

Limited Polymorphism in Plasmodium falciparum Artemisinin Resistance Kelch13-Propeller Gene Among Clinical Isolates from Bushenyi District, Uganda.

INTRODUCTION: Drug resistance remains a major challenge in malaria treatment, especially after the emergence of resistance to artemisinin-based combined therapies. Plasmodium falciparum Kelch13 gene mutations are implicated in conferring artemisinin resistance. Thus, this study was aimed at determining the occurrence of Kelch13 (K13) propeller resistance gene polymorphism mutations in Bushenyi district, Uganda. METHODS: Participants suspected to have malaria were recruited. P. falciparum was confirmed using antigen histidine-rich protein 2 (HRP2) (Pf) (Access Bio, Inc, USA) and microscopy. Malaria-positive patients were treated with artemeter-lumefantrine (AL). Blood was withdrawn from participants who tested positive for parasites after day 3 and kept in blood filter papers (ET31CHR; Whatman Limited, Kent, UK). DNA was extracted using chelex-suspension method. Nested polymerase chain reaction (PCR) was conducted and the second-round products sequenced using Sanger's method. Sequenced products were analyzed using DNAsp 5.10.01 software and then blasted on to the NCBI for K13-propeller gene sequence identity using the Basic Local Alignment Search Tool (BLAST). RESULTS: Out of 283 enrolled participants, 194 completed the follow-up schedule. A total of 134 (69%) had no parasites on day 3, while 60 (31%) had parasites on that day. Out of the 60 samples, 40 (62%) were positively amplified as P. falciparum, with polymorphisms in the K13-propeller gene detected in 3 (7.5%) out of the 40 amplicons. Polymorphisms at codon 1929, 1788 and 1801 were detected separately in one sample each. Sequences have been deposited in NCBI with accession numbers PRJNA720348 and PRJNA720800. CONCLUSION: Polymorphisms in the K13-propeller gene previously reported to be associated with artemisinin resistance were not detected in the P. falciparum isolates from Bushenyi district, Uganda. More studies need to be conducted on the new mutations detected so as to understand their association, if any, with ACT resistance.

Fluoroquinolone resistant bacterial isolates from the urinary tract among patients attending hospitals in Bushenyi District, Uganda.

INTRODUCTION: bacterial resistance to fluoroquinolones is on the rise globally, bacteria causing urinary tract infections (UTIs) are no exception to this fact. Judicious use of the current antibiotics by clinicians is therefore deemed necessary to combat development of resistance. This study determined fluoroquinolone resistant profiles, multiple antibiotic resistance indices (MARI), factors associated with fluoroquinolone resistance and their strength among patients attending hospitals in Bushenyi District, Uganda. METHODS: this was a cross-sectional study in which a total of 86 bacterial uropathogens isolated previously by standard microbiological methods were subjected to antibiotic susceptibility testing using Kirby Bauer disk diffusion method. Data for factors suspected to be associated with fluoroquinolone resistant UTI were obtained by use of questionnaires. RESULTS: the most resisted fluoroquinolone was ofloxacin with 29/83 (34.9%), followed by moxifloxacin 27/83 (32.5%), levofloxacin 24/86 (27.9%) and ciprofloxacin 23/86 (26.7%). The bacterial uropathogens that exhibited the highest frequency of fluoroquinolone resistant strains were P. mirabilis with 2/3 (66.7%) and E. faecalis with 2/3 (66.7%), followed by E. coli 19/36 (52.8%), S. aureus 13/27 (48.1%), K. oxytoca 2/6 (33.3%), K. pneumoniae 2/10 (20.0%) and P. vulgaris 0/1 (0.0%). All the bacterial uropathogens tested showed MARI of ≥ 0.2. Hospitalization, history of fluoroquinolones use in the last 12 months and wrong prescription of antibiotics were found to bear statistically significant relationships (p < 0.05) with fluoroquinolone resistant UTI. CONCLUSION: antibiotic susceptibility testing of the first generation quinolones such as nalidixic acid in hospitalized patients, patients with history of fluoroquinolones' use in the last 12 months and wrong prescription of antibiotics should be adopted to avoid fluoroquinolone abuse. For empiric treatment of UTIs in Bushenyi District, ciprofloxacin still remains the first line of choice among the fluoroquinolone class of antibiotics.