Strain structure analysis of Mycobacterium tuberculosis circulating among HIV negative, positive and drug resistant TB patients attending chest clinics in Western Kenya.

BACKGROUND: Despite global tuberculosis (TB) interventions, the disease remains one of the major public health concerns. Kenya is ranked 15th among 22 high burden TB countries globally. METHODS: A cross-sectional study was conducted in Western Kenya, which comprises 10 counties. A multistage sampling method was used where a single sub-county was randomly selected followed by sampling two high volume health facility from each sub-county. Identification of spoligotype profiles and their family distribution and lineage level were achieved by comparison with SITVIT database. RESULTS: Lineage distribution pattern revealed that the most predominant lineage was CAS 220 (39.8%) followed by Beijing 128 (23.1%). The other lineages identified were T, LAM, H, X, S and MANU which were quantified as 87 (15.7%), 67 (12.1%), 16 (2.8%), 10 (1.8%), 8 (1.4%) and 5 (0.9%) respectively. CAS and Beijing strains were the most predominant lineage in both HIV negative and positive TB patients. The Beijing lineage was also the most predominant in resistant M. tuberculosis strains as compared to wild type. A total of 12 (2.0%) were orphaned M. tuberculosis strains which were spread across all the 10 counties of the study site. In multivariate logistic regression adjusting for potential cofounders three potential risk factors were significant. HIV status (OR = 1.52, CI = 0.29-3.68 and P value of 0.001), Alcohol use (OR = 0.59, CI = 0.43-3.12 and P-value =0.001) and cross border travel (OR = 0.61, CI = 0.49-3.87 and P value = 0.026). Most M. tuberculosis clinical isolates showed genetic clustering with multivariate logistic regression indicating three potential risk factors to clustering. HIV status (OR = 1.52, CI = 0.29-3.68 and P value of 0.001), Alcohol use (OR = 0.59, CI = 0.43-3.12 and P-value =0.001) and cross border travel (OR = 0.61, CI = 0.49-3.87 and P value = 0.026). CONCLUSION: There exist diverse strains of M. tuberculosis across the 10 counties of Western Kenya. Predominant distribution of clustered genotype points to the fact that most TB cases in this region are as a result of resent transmission other than activation of latent TB.

Distribution patterns of drug resistance Mycobacterium tuberculosis among HIV negative and positive tuberculosis patients in Western Kenya.

INTRODUCTION: Globally anti-tuberculosis drug resistance is one of the major challenges affecting control and prevention of tuberculosis. Kenya is ranked among 30 high burden TB countries globally. However, there is scanty information on second line antituberculosis drug resistance among tuberculosis patients. Therefore, this study aimed at determining Mycobacterium tuberculosis drug resistant strain distribution pattern in 10 counties of Western Kenya among HIV positive and negative patients. METHOD: A cross-sectional study was conducted in Western Kenya, which comprises 10 counties. A multistage sampling method was used where a single sub-county was randomly selected followed by sampling one high volume health facility from each sub-county. Consenting study subjects with at least two smear positive sputum at the time of enrolment were randomly selected. The collected sputum was decontaminated with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) and then stained with Ziehl Neelsen Stain before visualizing the presence of bacilli under microscope at ×100 magnification with oil immersion. Further, the identified bacilli were cultured and susceptibility test carried out using known first and second line antimycobacterial tuberculosis. HIV testing was carried out using Determine® HIV-1/2 rapid test (Abbot Diagnostics, Maidenhead, United Kingdom). Those who had smear converted were dropped from the study. Finally, drug susceptibility pattern across the 10 counties of Western Kenya was evaluated. RESULTS: Our study showed that Mycobacterium tuberculosis drug resistance among HIV negative and positive cases in Western Kenya was prevalent in all the 10 counties surveyed. Based on the drug susceptibility tests, 53.2% and 42.7% of the study samples were resistant to at least one antituberculosis drug among HIV negative and HIV positive patients respectively. The data analysis revealed that among the HIV-positive and HIV-negative patients, resistance to INH was predominant (28.5%, and 23.6%, respectively), followed by RIF (16.4% and 14.6% respectively). Second-line drug resistant strains identified among HIV negative patients included Ethionamide (0.3%), Gatifloxacin (0.3%), Amikacin (0.3%) and Capreomycin (0.3%). There was no second line drug monoresistance among HIV positive TB patients. Multi/poly drug resistance were noted among HIV-negative patients in, INH + AMK (0.7%), INH + PZA (1%), INH + GFX (0.7%, INH + ETO (0.7%, STY + ETO (1%), ETH + ETO (1.0%), INH + KAN (0.7%) and INH + CAP (0.7%) strains/cases at 95% confidence interval. Among HIV positive patients INH + GFX (1.1%), INH + ETO (0.4%) and INH + KAN (0.4%) strains of M. tuberculosis were identified with a confidence interval of 95%. Geographical distribution patterns analysis of M. tuberculosis drug polyresistant strains across the 10 counties were recorded. Among HIV TB patients, resistant strains were identified in Nyamira (INH + GFX, INH + KAN), Bungoma ((ETO + STY), Busia (ETH + ETO and STY + ETO) Homabay (RIF + AMK. ETO + ETH and ETO + STY), Kisumu (ETH + ETO and PZA + ETO) and in Kakamega, Kisii and Vihiga (INH + KAN and RIF + AMK). There was no M. tuberculosis polyresistant strain identified in Migori and Siaya counties. Among HIV positive TB patients, M. tuberculosis resistant strains were identified in three counties, Nyamira (INH + KAN) Homabay (INH + GFX and INH + AMK) and Kakamega (INH + GFX). There was no polyresistant M. tuberculosis strain identified in Migori, Bungoma, Kisii, Vihiga, Busia, Siaya and Kisumu Counties. DISCUSSION: The distribution patterns of M. tuberculosis drug resistance among HIV negative and positive TB patients could be as a result of reported high prevalence of HIV in Western Kenya counties especially the area under study. Tuberculosis is one of the opportunistic diseases that have been shown to be the major cause of AIDS among HIV infected patients. Resent reports by National AIDS Control Council shows that Kisumu, Siaya, Homabay, Migori, Busia have the overall leading in HIV prevalence in Kenya. The low prevalence of drug resistant strains among HIV tuberculosis patients could be as a result of drug adherence attitude adopted by HIV patients, availability of continuous counselling and close follow up and notification by healthcare workers and community health volunteers. CONCLUSION: Drug resistant M. tuberculosis strains prevalence is still high among HIV negative and positive patients in Western Kenya with the most affected being HIV negative TB patients. It is therefore probable that the existing control measures are not adequate to control transmission of drug resistant strains. Further, miss diagnosis or delayed diagnosis of TB patients could be contributing to the emergence of M. tuberculosis drug polyresistant strains. RECOMMENDATION: Based on the result of this study, regular TB drug resistance surveillance should be conducted to ensure targeted interventions aimed at controlling increased transmission of the tuberculosis drug resistant strains among HIV/AIDS and HIV negative patients. There is also need for improved drug resistant infection control measures, timely and rapid diagnosis and enhanced and active screening strategies of tuberculosis among suspected TB patients need to be put in place. Further, studies using a larger patient cohort and from counties across the country would shed much needed insights on the true national prevalence of different variants of M. tuberculosis drug resistance.

Profiling of RNA Viruses in Biting Midges (Ceratopogonidae) and Related Diptera from Kenya Using Metagenomics and Metabarcoding Analysis.

Vector-borne diseases (VBDs) cause enormous health burden worldwide, as they account for more than 17% of all infectious diseases and over 700,000 deaths each year. A significant number of these VBDs are caused by RNA virus pathogens. Here, we used metagenomics and metabarcoding analysis to characterize RNA viruses and their insect hosts among biting midges from Kenya. We identified a total of 15 phylogenetically distinct insect-specific viruses. These viruses fall into six families, with one virus falling in the recently proposed negevirus taxon. The six virus families include Partitiviridae, Iflaviridae, Tombusviridae, Solemoviridae, Totiviridae, and Chuviridae. In addition, we identified many insect species that were possibly associated with the identified viruses. Ceratopogonidae was the most common family of midges identified. Others included Chironomidae and Cecidomyiidae. Our findings reveal a diverse RNA virome among Kenyan midges that includes previously unknown viruses. Further, metabarcoding analysis based on COI (cytochrome c oxidase subunit 1 mitochondrial gene) barcodes reveal a diverse array of midge species among the insects used in the study. Successful application of metagenomics and metabarcoding methods to characterize RNA viruses and their insect hosts in this study highlights a possible simultaneous application of these two methods as cost-effective approaches to virus surveillance and host characterization. IMPORTANCE The majority of the viruses that currently cause diseases in humans and animals are RNA viruses, and more specifically arthropod-transmitted viruses. They cause diseases such as dengue, West Nile infection, bluetongue disease, Schmallenberg disease, and yellow fever, among others. Several sequencing investigations have shown us that a diverse array of RNA viruses among insect vectors remain unknown. Some of these could be ancient lineages that could aid in comprehensive studies on RNA virus evolution. Such studies may provide us with insights into the evolution of the currently pathogenic viruses. Here, we applied metagenomics to field-collected midges and we managed to characterize several RNA viruses, where we recovered complete and nearly complete genomes of these viruses. We also characterized the insect host species that are associated with these viruses. These results add to the currently known diversity of RNA viruses among biting midges as well as their associated insect hosts.

Evaluation of Analgesic Activities of Extracts of Two Marine Molluscs: Tympanotonus fuscatus var radula (Linnaeus) and Pachymelania aurita (Müller).

PURPOSE AND METHODS: In this study, the analgesic activity of the crude alcohol (acetone-methanol) and aqueous (in PBS, pH 7.2) extracts of the marine molluscs, Pachymelania aurita and Tympanotonus fuscatus, has been evaluated using the formalin test (for chronic antinociceptive) and the tail-flick (acute antinociceptive) pain models in male swiss albino mice. RESULTS: The results show that the extracts of P. aurita and T. fuscatus demonstrated high safety margins as single doses of up to 2000 mg/kg bwt proved to be well tolerated and non-lethal, although the alcohol extract of P. aurita caused necrosis in the liver and kidney when administered at a dose level of 2000 mg/kg bwt. In the formalin test, treatment with the aqueous extracts of P. aurita and T. fuscatus as well as the alcohol extract of T. fuscatus 30 min before the subcutaneous injection of 5% formalin to the paw of the mice resulted in a significant time- and dose-dependent reduction in total and phase 2a pain-related behavior and thus nociception. The extracts had no analgesic effect in tail-flick test up to the highest dose tested. CONCLUSION: Hence, the results from both models indicate that the site of their analgesic action is probably peripheral.

In vitro antiproliferative studies of extracts of the marine molluscs: Tympanatonus fuscatus Var radula (linnaeus) and Pachymelania aurita (muller).

This study aimed to investigate the antimitotic and antiproliferation activities of crude acetone-methanol and aqueous extracts of two marine molluscs commonly found in the Niger Delta region of Nigeria; T.fuscatus and P.aurita, against human cancerous cell lines (DU145, Hep-2, and HCC1395) cell lines in vitro. The antimitotic activity of the extracts was evaluated using Allium cepa root meristematic cells. Antiproliferative activity of the plant extracts against the cancerous cell lines was compared with normal cell line (VeroE6). Doxorubicin was used as a positive control. Gene expression studies using qPCR for the proapoptotic genes, CASP3, CASP8 and P53 were also carried out. The alcohol extract of T.fuscatus (TFAC) exhibited the most promising activity against all the cancer cell lines tested (DU145 IC50 = 96.48 ± 1.36 µg/ml, HCC 1395 IC50 = 61.44 ± 2.45 µg/ml, Hep2 IC50 = 0.52 ± 0.36 µg/ml) and also had the highest selectivity index of 4.94, 7.78 and 921.97 for DU145, HCC 1395 and Hep-2 cells respectively. Furthermore, TFAC was the only extract that significantly upregulated the expression of caspase 3, caspase 8 and P53. Thus, these findings suggest potential exploitation of TFAC as an anticancer agent.

Biodegradability of polyethylene by bacteria and fungi from Dandora dumpsite Nairobi-Kenya.

This study aimed at isolating and identifying bacteria and fungi with the capacity to degrade low density polyethylene (LDPE). The level of biodegradation of LDPE sheets with bacterial and fungal inoculums from different sampling points of Dandora dumpsite was evaluated under laboratory conditions. Incubation of the LDPE sheets was done for sixteen weeks at 37°C and 28°C for bacteria and fungi respectively in a shaker incubator. Isolation of effective candidates for biodegradation was done based on the recorded biodegradation outcomes. The extent of biodegradation on the polyethylene sheets was assessed by various techniques including weight loss analysis, Fourier Transform Infrared Spectroscopy (FTIR) and GC-MS. Fourier Transform Infra-Red spectroscopy (FTIR) analysis revealed the appearance of new functional groups attributed to hydrocarbon degradation after incubation with the bacteria and fungi. Analysis of the 16S rDNA and 18S rDNA sequences for bacteria and fungi respectively showed that bacteria belonging to genera Pseudomonas, Bacillus, Brevibacillus, Cellulosimicrobium, Lysinibacillus and fungi of genus Aspergillus were implicated as polyethylene degraders. An overall analysis confirmed that fungi are generally better degraders of polyethylene than bacteria. The highest fungal degradation activity was a mean weight reduction of 36.4±5.53% attributed to Aspergillus oryzae strain A5, 1 (MG779508). The highest degradation activity for bacteria was a mean of 35.72± 4.01% and 20.28± 2.30% attributed to Bacillus cereus strain A5,a (MG645264) and Brevibacillus borstelensis strain B2,2 (MG645267) respectively. Genus Aspergillus, Bacillus and Brevibacillus were confirmed to be good candidates for Low Density Poly Ethene bio-degradation. This was further confirmed by the appearance of the aldehyde, ether and carboxyl functional groups after FTIR analysis of the polythene sheets and the appearance of a ketone which is also an intermediary product in the culture media. To improve this degrading capacity through assessment of optimum conditions for microbial activity and enzyme production will enable these findings to be applied commercially and on a larger scale.

Molecular Characterization of Low-Density Polyethene (LDPE) Degrading Bacteria and Fungi from Dandora Dumpsite, Nairobi, Kenya.

This study aimed at molecular and biochemical characterization of low-density polyethene (LDPE) degrading fungi and bacteria from Dandora dumpsite, Nairobi. Twenty bacterial and 10 fungal isolates were identified using 16S rDNA and 18S rDNA sequences for bacteria and fungi, respectively. The highest fungal degradation was attributed to Aspergillus oryzae strain A5,1 while the highest bacterial degradation was attributed to Bacillus cereus strain A5,a and Brevibacillus borstelensis strain B2,2, respectively. Isolates were screened for their ability to produce extracellular laccase and esterase; Aspergillus fumigatus strain B2,2 exhibited the highest presence of laccase (15.67 mm) while Aspergillus oryzae strain A5,1 exhibited the highest presence of esterase (14.33 mm). Alkane hydroxylase-encoding genes were screened for using primer AlkB 1 which amplified the fragment of size 870 bp. Four bacterial samples were positive for the gene. Optimum growth temperature of the fungal isolates was 30°C. The possession of laccase, esterase, and alkane hydroxylase activities is suggested as key molecular basis for LDPE degrading capacity. Knowledge of optimum growth conditions will serve to better utilize microbes in the bioremediation of LDPE. The application of Aspergillus oryzae strain A5,1 and Bacillus cereus strain A5,a in polyethene degradation is a promising option in this kind of bioremediation as they exhibited significantly high levels of biodegradation. Further investigation of more alkane degrading genes in biodegrading microbes will inform the choice of the right microbial consortia for bioaugmentation strategies.

Genetically Determined Response to Artemisinin Treatment in Western Kenyan Plasmodium falciparum Parasites.

Genetically determined artemisinin resistance in Plasmodium falciparum has been described in Southeast Asia. The relevance of recently described Kelch 13-propeller mutations for artemisinin resistance in Sub-Saharan Africa parasites is still unknown. Southeast Asia parasites have low genetic diversity compared to Sub-Saharan Africa, where parasites are highly genetically diverse. This study attempted to elucidate whether genetics provides a basis for discovering molecular markers in response to artemisinin drug treatment in P. falciparum in Kenya. The genetic diversity of parasites collected pre- and post- introduction of artemisinin combination therapy (ACT) in western Kenya was determined. A panel of 12 microsatellites and 91 single nucleotide polymorphisms (SNPs) distributed across the P. falciparum genome were genotyped. Parasite clearance rates were obtained for the post-ACT parasites. The 12 microsatellites were highly polymorphic with post-ACT parasites being significantly more diverse compared to pre-ACT (p < 0.0001). The median clearance half-life was 2.55 hours for the post-ACT parasites. Based on SNP analysis, 15 of 90 post-ACT parasites were single-clone infections. Analysis revealed 3 SNPs that might have some causal association with parasite clearance rates. Further, genetic analysis using Bayesian tree revealed parasites with similar clearance phenotypes were more closely genetically related. With further studies, SNPs described here and genetically determined response to artemisinin treatment might be useful in tracking artemisinin resistance in Kenya.

Molecular characterization of the cytochrome b gene and in vitro atovaquone susceptibility of Plasmodium falciparum isolates from Kenya.

The prevalence of a genetic polymorphism(s) at codon 268 in the cytochrome b gene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227 Plasmodium falciparum parasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC50) and parasite genetic polymorphism; mutant alleles had higher IC50s than the wild type.