Human brucellosis in Baringo County, Kenya: Evaluating the diagnostic kits used and identifying infecting Brucella species.

Human brucellosis diagnosis has been a challenge in Brucella-endemic areas. In Kenya, diagnosis is usually carried out using Febrile Brucella Antigen agglutination test (FBAT) whose performance is not well documented. This paper reports on the sensitivity and specificity of the FBAT used for brucellosis diagnosis on blood samples/serum collected in three healthcare facilities in Baringo County, Kenya, and on Brucella species present in the study area. The FBAT test results at the hospitals were used to guide patient management. Patients who visited the hospital's laboratory with a clinician's request for brucellosis testing also filled a questionnaire to assess knowledge and attitudes associated with transmission of the disease in the study area. The remaining serum samples were tested again using FBAT and Rose Bengal Plate Test (RBPT) within a month of blood collection at the University Nairobi Laboratory. The two rapid tests were then compared, with respect to brucellosis diagnostic sensitivity and specificity. To identify infecting Brucella species, a proportion 43% (71/166) of the blood clots were analyzed by multiplex polymerase chain reaction (PCR) using specific primers for B. abortus, B. melitensis, B. ovis and B. suis. Out of 166 serum samples tested, 26.5% (44/166) were positive using FBAT and 10.2% (17/166) positive using RBPT. The sensitivity and specificity of FBAT compared to RBPT was 76.47% and 71.19%, respectively while the positive and negative predictive values were 29.55% and 96.72%, respectively. The FBAT showed higher positivity then RBPT. The difference in sensitivity and specificity of FBAT and RBPTs was relatively low. The high FBAT positivity rate would be indication of misdiagnosis; this would lead to incorrect treatment. Brucella abortus was detected from 9.9% (7/71) of the blood clots tested; no other Brucella species were detected. Thus human brucellosis, in Baringo was mainly caused by B. abortus.

Implementation of a customised antimicrobial resistance laboratory scorecard in Cameroon, Ethiopia and Kenya.

Background: In low-resource settings, antimicrobial resistance (AMR) is detected by traditional culture-based methods and ensuring the quality of such services is a challenge. The AMR Scorecard provides laboratories with a technical assessment tool for strengthening the quality of bacterial culture, identification, and antimicrobial testing procedures. Objective: To evaluate the performance of the AMR Scorecard in 11 pilot laboratory evaluations in three countries also assessed with the Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) checklist. Methods: Pilot laboratory evaluations were conducted in Cameroon, Ethiopia and Kenya between February 2019 and March 2019. Assessors with previous SLIPTA and microbiology experience were trained. Assessors performed the laboratory assessments using the SLIPTA and AMR Scorecard tools. Results: Weaknesses in technical procedures and the quality management systems were identified in all areas and all laboratories. Safety had the highest mean performance score (SLIPTA: 68%; AMR Scorecard: 73%) while management review had the lowest (SLIPTA: 32%; AMR Scorecard: 8%) across all laboratories. The AMR Scorecard scores were generally consistent with SLIPTA scores. The AMR Scorecard identified technical weaknesses in AMR testing, and SLIPTA identified weaknesses in the quality management systems in the laboratories. Conclusion: Since the AMR Scorecard identified important gaps in AMR testing not detected by SLIPTA, it is recommended that microbiology laboratories use SLIPTA and the AMR Scorecard in parallel when preparing for accreditation. Expanding the use of the AMR Scorecard is a priority to address the need for quality clinical microbiology laboratory services in support of optimal patient care and AMR surveillance.

Antinutrient to mineral molar ratios of raw common beans and their rapid prediction using near-infrared spectroscopy.

The presence of antinutrients in common beans negatively affects mineral bioavailability. Therefore, this study aimed to predict the antinutrient to mineral molar ratios (proxy-indicators of in vitro mineral bioavailability) of a wide range of raw bean types, using near-infrared (NIR) spectroscopy. Iron, zinc, phytate and tannin concentrations and, antinutrient to mineral molar ratios were determined. Next, model calibration using NIR spectra from milled beans was performed. This entailed wavelength selection, pre-processing and partial least squares regression. Bean type had a significant effect on tannin content. The average values of phytate to iron (Phy:Fe), phytate to zinc (Phy:Zn), tannins to iron (Tan:Fe) and phytate and tannins to iron (Phy + Tan:Fe) MRs were 27.6, 61.7, 16.0 and 43.6, respectively. With determination coefficients for test set prediction above 75%, the PLS-R models for Phy:Zn, Tan:Fe and Phy + Tan:Fe molar ratios are useful for screening purposes.

Evaluation of the detection of staA, viaB and sopE genes in Salmonella spp. using the polymerase chain reaction (PCR).

Typhoid fever is caused by the bacteria Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) and remains a significant health problem in many developing countries. Lack of adequate diagnostic capabilities has contributed greatly in making typhoid fever endemic in these regions. Reliable and inexpensive diagnostic tests are needed to improve the management of this disease burden. We evaluated the ability of staA, viaB and sopE genes to detect and differentiate between the three most prevalent Salmonella spp. in Kenya (S. Typhi, S. Typhimurium and S. Enteritidis) using conventional polymerase chain reaction (PCR). The staA primers and viaB primers were found to be specific only for the different strains of S. Typhi, producing PCR products of 585 bp and 540 bp, respectively. The sopE primers was demonstrated to be specific for all Salmonella spp. producing a 465 bp PCR product with no amplification with E. coli and S. boydii bacterial strains.

Effect of Lb. plantarum BFE 5092 Fermentation on Antinutrient and Oligosaccharide Composition of Whole Red Haricot Bean (Phaseolus vulgaris L).

Common beans are a leguminous plant of the genus Phaseolus. They are rich in protein, energy, and minerals. They confer a wide range of health benefits when consumed. Utilization of common bean has however been poor due to high antinutrient content that results in reduced nutrient digestibility and mineral bioavailability. Flatulence after consumption is also a huge deterrent to common bean consumption. Lactic acid fermentation is the most common form of food fermentation with the Lactobacilli spp dominating most spontaneous fermentations. The objective of this study was to determine the effect of lactic acid bacteria (LAB) on the antinutrient and flatulence causing oligosaccharide composition of red haricot bean. A factorial research design was used in the study. Red haricot beans were sorted and soaked for 15 h. The soaked beans were fermented in 2% salt-sugar solutions for 120 h. Experimental batch was inoculated with Lb. plantarum BFE 5092 (IF), and the control batch was spontaneously fermented (SF). Microbial growth and pH were monitored every 24 h during fermentation. After fermentation, the beans were dried and milled, and the flours were subjected to biochemical analysis. ANOVA was done using SPSS statistics 23. The pH decreased significantly (P < 0.05) from 6.06 to 3.9 in both batches at the end of fermentation. The LAB counts significantly increased (P < 0.05) in both batches, whereas coliform counts decreased significantly (P < 0.05). Fungi were not detected in both batches. Soaking lowered tannins and phytates and raffinose concentrations significantly but had no significant effect on stachyose concentration. At the end of 120 h of fermentation, the tannin content was 109.50 and 54.04 mg/100 g in IF and SF, respectively. Phytates were at 242.52 and 163.43 mg/100 g in IF and SF, respectively. Raffinose content was 32.85 and 32.58 mg/100 g in IF and SF, respectively, while stachyose content was 593.33 and 467.49 mg/100 g in IF and SF, respectively. This research showed that LAB is able to ferment soaked whole red haricot and lower the tannin, phytate, raffinose, and stachyose content significantly. Spontaneous fermentation lowered these antinutrients and oligosaccharides better than inoculation with Lb. plantarum BFE 5092.

Molecular characterization and antibiotic resistance profiles of Salmonella isolated from fecal matter of domestic animals and animal products in Nairobi.

BACKGROUND: Salmonella has significant public health implications causing food borne and zoonotic diseases in humans. Treatment of infections due to Salmonella is becoming difficult due to emergence of drug resistant strains. There is therefore need to characterize the circulating non-typhoidal Salmonella (NTS) serovars in domestic animals and animal products in Kenya as well as determine their antibiotic resistance profiles. METHODS: A total of 740 fecal samples were collected from cows (n = 150), pigs (n = 182), chicken (n = 191) and chicken eggs (n = 217) from various markets and abattoirs in Nairobi. The prevalence of NTS serovars using culture techniques and biochemical tests, antimicrobial sensitivity testing using disc diffusion method of the commonly prescribed antibiotics and phylogenetic relationships using 16S rRNA were determined. RESULTS: The results showed that the overall prevalence of Salmonella was 3.8, 3.6, 5.9 and 2.6% for pigs, chicken, eggs and cows respectively. Two serovars were isolated S. Typhimurium (85%) and S. Enteritidis (15%) and these two serovars formed distinct clades on the phylogenetic tree. Forty percent of the isolates were resistant to one or more antibiotics. CONCLUSION: The isolation of Salmonella Typhimurium and Salmonella Enteritidis that are resistant to commonly used antibiotics from seemingly healthy animals and animal products poses a significant public health threat. This points to the need for regular surveillance to be carried out and the chain of transmission should be viewed to ascertain sources of contamination.

Analyses of mitochondrial genes reveal two sympatric but genetically divergent lineages of Rhipicephalus appendiculatus in Kenya.

BACKGROUND: The ixodid tick Rhipicephalus appendiculatus transmits the apicomplexan protozoan parasite Theileria parva, which causes East coast fever (ECF), the most economically important cattle disease in eastern and southern Africa. Recent analysis of micro- and minisatellite markers showed an absence of geographical and host-associated genetic sub-structuring amongst field populations of R. appendiculatus in Kenya. To assess further the phylogenetic relationships between field and laboratory R. appendiculatus tick isolates, this study examined sequence variations at two mitochondrial genes, cytochrome c oxidase subunit I (COI) and 12S ribosomal RNA (rRNA), and the nuclear encoded ribosomal internal transcribed spacer 2 (ITS2) of the rRNA gene, respectively. RESULTS: The analysis of 332 COI sequences revealed 30 polymorphic sites, which defined 28 haplotypes that were separated into two distinct haplogroups (A and B). Inclusion of previously published haplotypes in our analysis revealed a high degree of phylogenetic complexity never reported before in haplogroup A. Neither haplogroup however, showed any clustering pattern related to either the geographical sampling location, the type of tick sampled (laboratory stocks vs field populations) or the mammalian host species. This finding was supported by the results obtained from the analysis of 12S rDNA sequences. Analysis of molecular variance (AMOVA) indicated that 90.8 % of the total genetic variation was explained by the two haplogroups, providing further support for their genetic divergence. These results were, however, not replicated by the nuclear transcribed ITS2 sequences likely because of recombination between the nuclear genomes maintaining a high level of genetic sequence conservation. CONCLUSIONS: COI and 12S rDNA are better markers than ITS2 for studying intraspecific diversity. Based on these genes, two major genetic groups of R. appendiculatus that have gone through a demographic expansion exist in Kenya. The two groups show no phylogeographic structure or correlation with the type of host species from which the ticks were collected, nor to the evolutionary and breeding history of the species. The two lineages may have a wide geographic distribution range in eastern and southern Africa. The findings of this study may have implications for the spread and control of R. appendiculatus, and indirectly, on the transmission dynamics of ECF.

Detailed analysis of seed coat and cotyledon reveals molecular understanding of the hard-to-cook defect of common beans (Phaseolus vulgaris L.).

The hard-to-cook (HTC) defect in legumes is characterized by the inability of cotyledons to soften during the cooking process. Changes in the non-starch polysaccharides of common bean seed coat and cotyledon were studied before and after development of the HTC defect induced by storage at 35°C and 75% humidity for 8months. Distinct differences in the yields of alcohol insoluble residues, degree of methoxylation (DM), sugar composition, and molar mass distribution of non-starch polysaccharides were found between the seeds coat and cotyledons. The non-starch polysaccharide profiles, both for seed coats and cotyledons, significantly differed when comparing HTC and easy-to-cook (ETC) beans. In conclusion, differences in the structure, composition and extractability of non-starch polysaccharides between the ETC and HTC beans confirmed the significant role of pectin polysaccharides in interaction with divalent ions in the HTC development, which consequently affect their cooking behaviors.

Multi-locus genotyping reveals absence of genetic structure in field populations of the brown ear tick (Rhipicephalus appendiculatus) in Kenya.

Rhipicephalus appendiculatus is an important tick vector of several pathogens and parasitizes domestic and wild animals across eastern and southern Africa. However, its inherent genetic variation and population structure is poorly understood. To investigate whether mammalian host species, geographic separation and resulting reproductive isolation, or a combination of these, define the genetic structure of R. appendiculatus, we analyzed multi-locus genotype data from 392 individuals from 10 geographic locations in Kenya generated in an earlier study. These ticks were associated with three types of mammalian host situations; (1) cattle grazing systems, (2) cattle and wildlife co-grazing systems (3) wildlife grazing systems without livestock. We also analyzed data from 460 individuals from 10 populations maintained as closed laboratory stocks and 117 individuals from five other species in the genus Rhipicephalus. The pattern of genotypes observed indicated low levels of genetic differentiation between the ten field populations (FST=0.014±0.002) and a lack of genetic divergence corresponding to the degree of separation of the geographic sampling locations. There was also no clear association of particular tick genotypes with specific host species. This is consistent with tick dispersal over large geographic ranges and lack of host specificity. In contrast, the 10 laboratory populations (FST=0.248±0.015) and the five other species of Rhipicephalus (FST=0.368±0.032) were strongly differentiated into distinct genetic groups. Some laboratory bred populations diverged markedly from their field counterparts in spite of originally being sampled from the same geographic locations. Our results demonstrate a lack of defined population genetic differentiation in field populations of the generalist R. appendiculatus in Kenya, which may be a result of the frequent anthropogenic movement of livestock and mobility of its several wildlife hosts between different locations.

Hydration properties and texture fingerprints of easy- and hard-to-cook bean varieties.

The objective of this study was to understand the factors that affect the hydration and cooking profiles of different bean varieties. During this study, nine bean varieties were classified as either easy-to-cook (ETC) or hard-to-cook (HTC) based on a subjective finger pressing test and an objective cutting test. Rose coco, Red haricot, and Zebra beans were classified as ETC, while Canadian wonder, Soya fupi, Pinto, non-nodulating, Mwezi moja, Gwaku, and New mwezi moja were HTC. The effect of different soaking (pre)-treatments on the cooking behavior and/or water absorption of whole or dehulled beans was investigated. Dehulling, soaking in high pH and monovalent salt solutions reduced the cooking time of beans, while soaking in low pH and CaCl2 solutions increased the cooking time. Moisture uptake was faster in ETC and dehulled beans. Soaking at high temperatures also increased the hydration rate. The results point to pectin-related aspects and the rate of water uptake as possible factors that influence the cooking rate of beans.

Extraction and characterization of pectic polysaccharides from easy- and hard-to-cook common beans (Phaseolus vulgaris).

The occurrence of the hard-to-cook (HTC) defect in legumes is characterized by the inability of cotyledons to soften during the cooking process. This phenomenon may be influenced by pectin properties. The objective of this study was to characterize the pectic polysaccharides comprised in the alcohol insoluble residue (AIR) extracted from easy-to-cook (Rose coco) and hard-to-cook (Pinto) common beans. This would provide an insight in the relationship between the pectin properties and HTC defect. The AIR was extracted from raw, half-cooked hard, half-cooked soft and fully-cooked bean samples. Subsequently, it was fractionated into water-, chelator- and Na2CO3-soluble pectin fractions and a hemicellulose fraction. For the AIR and the pectin fractions, determination of the galacturonic acid content, neutral sugars, degree of methylesterfication (DM), degree of acetylation (DAc) and molar mass (MM) distribution was performed. Results on the pectin fractions, MM distribution and pectin content profile, revealed that Rose coco pectin generally showed higher pectin solubility than Pinto. Neutral sugar profiles indicated that Pinto contained higher amounts of branched pectin (i.e. arabinans) than Rose coco. There was no difference between the DM of Pinto and Rose coco, however, the DAc was higher in Rose coco. In conclusion, the differences in pectin structure and solubility properties between easy- and hard-to-cook common beans might contribute to the differences in their cooking behavior.

Predicted HIV-1 coreceptor usage among Kenya patients shows a high tendency for subtype d to be cxcr4 tropic.

BACKGROUND: CCR5 antagonists have clinically been approved for prevention or treatment of HIV/AIDS. Countries in Sub-Saharan Africa with the highest burden of HIV/AIDS are due to adopt these regimens. However, HIV-1 can also use CXCR4 as a co-receptor. There is hence an urgent need to map out cellular tropism of a country's circulating HIV strains to guide the impending use of CCR5 antagonists. OBJECTIVES: To determine HIV-1 coreceptor usage among patients attending a comprehensive care centre in Nairobi, Kenya. METHODS: Blood samples were obtained from HIV infected patients attending the comprehensive care centre, Kenyatta National Hospital in years 2008 and 2009. The samples were separated into plasma and peripheral blood mononuclear cells (PBMCs). Proviral DNA was extracted from PBMCs and Polymerase Chain reaction (PCR) done to amplify the HIV env fragment spanning the C2-V3 region. The resultant fragment was directly sequenced on an automated sequencer (ABI, 3100). Co-receptor prediction of the env sequences was done using Geno2pheno [co-receptor], and phylogenetic relationships determined using CLUSTALW and Neighbor Joining method. RESULTS: A total of 67 samples (46 treatment experienced and 21 treatment naive) were successfully amplified and sequenced. Forty nine (73%) sequences showed a prediction for R5 tropism while 18(27%) were X4 tropic. Phylogenetic analysis showed that 46(69%) were subtype A, 11(16%) subtype C, and 10(15%) subtype D. No statistical significant associations were observed between cell tropism and CD4+ status, patient gender, age, or treatment option. There was a tendency for more X4 tropic strains being in the treatment experienced group than the naive group: Of 46 treatment experiencing participants, 14(30%) harboured X4, compared with 4(19%) of 21 of the treatment-naïve participants, the association is however not statistically significant (p = 0.31). However, a strong association was observed between subtype D and CXCR4 co- receptor usage (p = 0.015) with 6(60%) of the 10 subtype D being X4 tropic and 4(40%) R5 tropic. CONCLUSION: HIV-1 R5 tropic strains were the most prevalent in the study population and HIV infected patients in Kenya may benefit from CCR5 antagonists. However, there is need for caution where subtype D infection is suspected or where antiretroviral salvage therapy is indicated.

Micro- and minisatellite-expressed sequence tag (EST) markers discriminate between populations of Rhipicephalus appendiculatus.

Biological differences, including vector competence for the protozoan parasite Theileria parva have been reported among populations of Rhipicephalus appendiculatus (Acari: Ixodidae) from different geographic regions. However, the genetic diversity and population structure of this important tick vector remain unknown due to the absence of appropriate genetic markers. Here, we describe the development and evaluation of a panel of EST micro- and minisatellite markers to characterize the genetic diversity within and between populations of R. appendiculatus and other rhipicephaline species. Sixty-six micro- and minisatellite markers were identified through analysis of the R. appendiculatus Gene Index (RaGI) EST database and selected bacterial artificial chromosome (BAC) sequences. These were used to genotype 979 individual ticks from 10 field populations, 10 laboratory-bred stocks, and 5 additional Rhipicephalus species. Twenty-nine markers were polymorphic and therefore informative for genetic studies while 6 were monomorphic. Primers designed from the remaining 31 loci did not reliably generate amplicons. The 29 polymorphic markers discriminated populations of R. appendiculatus and also 4 other Rhipicephalus species, but not R. zambeziensis. The percentage Principal Component Analysis (PCA) implemented using Multiple Co-inertia Analysis (MCoA) clustered populations of R. appendiculatus into 2 groups. Individual markers however differed in their ability to generate the reference typology using the MCoA approach. This indicates that different panels of markers may be required for different applications. The 29 informative polymorphic micro- and minisatellite markers are the first available tools for the analysis of the phylogeography and population genetics of R. appendiculatus.

Emergence of new types of Theileria orientalis in Australian cattle and possible cause of theileriosis outbreaks.

Theileria parasites cause a benign infection of cattle in parts of Australia where they are endemic, but have, in recent years, been suspected of being responsible for a number of outbreaks of disease in cattle near the coast of New South Wales. The objective of this study was to identify and characterize the species of Theileria in cattle on six farms in New South Wales where disease outbreaks have occurred, and compare with Theileria from three disease-free farms in Queensland that is endemic for Theileria. Special reference was made to sub-typing of T. orientalis by type-specific PCR and sequencing of the small subunit (SSU) rRNA gene, and sequence analysis of the gene encoding a polymorphic merozoite/piroplasm surface protein (MPSP) that may be under immune selection. Nucleotide sequencing of SSU rRNA and MPSP genes revealed the presence of four Theileria genotypes: T. orientalis (buffeli), T. orientalis (ikeda), T. orientalis (chitose) and T. orientalis type 4 (MPSP) or type C (SSU rRNA). The majority of animals showed mixed infections while a few showed single infection. When MPSP nucleotide sequences were translated into amino acids, base transition did not change amino acid composition of the protein product, suggesting possible silent polymorphism. The occurrence of ikeda and type 4 (type C) previously not reported to occur and silent mutation is thought to have enhanced parasite evasion of the host immune response causing the outbreak.

Rapid discrimination and quantification of Theileria orientalis types using ribosomal DNA internal transcribed spacers.

We report the population structure analysis of Theileria orientalis types (Ikeda, Buffeli and Chitose), the causative agent of theileriosis in cattle and its cohorts, using ITS1 and ITS2 spacers by fragment genotyping. We utilized primers flanking the two ribosomal RNA internal transcribed spacers (ITS1 and ITS2). Due to varying degrees of sequence polymorphism in the ITS regions found within and between species, we exploited the insertions and or deletions in these regions which resulted in different fragment sizes. On the basis of fragment size polymorphism, we could discriminate the three commonly found types of T. orientalis. ITS1 was capable of discriminating all three types (Ikeda-251 bp, Chitose-274 bp and Buffeli-269 bp) in one single reaction by fragment genotyping. In contrast, using ITS2, Ikeda (133-bp) a more pathogenic type was distinguishable from Buffeli/Chitose (139-bp). When compared with previous PCR detection method using, ITS1 and ITS2 genotyping was found to be more sensitive method with high specificity in population analysis and can be deployed in molecular epidemiology studies.