Bacterial etiology of urinary tract infections in patients treated at Kenyan health facilities and their resistance towards commonly used antibiotics.

BACKGROUND: Evidence-based empirical antibiotic prescribing requires knowledge of local antimicrobial resistance patterns. The spectrum of pathogens and their susceptibility strongly influences guidelines for empirical therapies for urinary tract infections (UTI) management. OBJECTIVE: This study aimed to determine the prevalence of UTI causative bacteria and their corresponding antibiotic resistance profiles in three counties of Kenya. Such data could be used to determine the optimal empirical therapy. METHODS: In this cross-sectional study, urine samples were collected from patients who presented with symptoms suggestive of UTI in the following healthcare facilities; Kenyatta National Hospital, Kiambu Hospital, Mbagathi, Makueni, Nanyuki, Centre for Microbiology Research, and Mukuru Health Centres. Urine cultures were done on Cystine Lactose Electrolyte Deficient (CLED) to isolate UTI bacterial etiologies, while antibiotic sensitivity testing was done using the Kirby-Bauer disk diffusion using CLSI guidelines and interpretive criteria. RESULTS: A total of 1,027(54%) uropathogens were isolated from the urine samples of 1898 participants. Staphylococcus spp. and Escherichia coli were the main uropathogens at 37.6% and 30.9%, respectively. The percentage resistance to commonly used drugs for the treatment of UTI were as follows: trimethoprim (64%), sulfamethoxazole (57%), nalidixic acid(57%), ciprofloxacin (27%), amoxicillin-clavulanic acid (5%), and nitrofurantoin (9%) and cefixime (9%). Resistance rates to broad-spectrum antimicrobials, such as ceftazidime, gentamicin, and ceftriaxone, were 15%, 14%, and 11%, respectively. Additionally, the proportion of Multidrug-resistant (MDR) bacteria was 66%. CONCLUSION: High resistance rates toward fluoroquinolones, sulfamethoxazole, and trimethoprim were reported. These antibiotics are commonly used drugs as they are inexpensive and readily available. Based on these findings, more robust standardised surveillance is needed to confirm the patterns observed while recognising the potential impact of sampling biases on observed resistance rates.

Biofilm formation, antimicrobial susceptibility and virulence genes of Uropathogenic Escherichia coli isolated from clinical isolates in Uganda.

INTRODUCTION: Uropathogenic E. coli is the leading cause of Urinary tract infections (UTIs), contributing to 80-90% of all community-acquired and 30-50% of all hospital-acquired UTIs. Biofilm forming Uropathogenic E. coli are associated with persistent and chronic inflammation leading to complicated and or recurrent UTIs. Biofilms provide an environment for poor antibiotic penetration and horizontal transfer of virulence genes which favors the development of Multidrug-resistant organisms (MDRO). Understanding biofilm formation and antimicrobial resistance determinants of Uropathogenic E. coli strains will provide insight into the development of treatment options for biofilm-associated UTIs. The aim of this study was to determine the biofilm forming capability, presence of virulence genes and antimicrobial susceptibility pattern of Uropathogenic E. coli isolates in Uganda. METHODS: This was a cross-sectional study carried in the Clinical Microbiology and Molecular biology laboratories at the Department of Medical Microbiology, Makerere University College of Health Sciences. We randomly selected 200 Uropathogenic E. coli clinical isolates among the stored isolates collected between January 2018 and December 2018 that had significant bacteriuria (> 105 CFU). All isolates were subjected to biofilm detection using the Congo Red Agar method and Antimicrobial susceptibility testing was performed using the Kirby disk diffusion method. The isolates were later subjected PCR for the detection of Urovirulence genes namely; Pap, Fim, Sfa, Afa, Hly and Cnf, using commercially designed primers. RESULTS: In this study, 62.5% (125/200) were positive biofilm formers and 78% (156/200) of these were multi-drug resistant (MDR). The isolates were most resistant to Trimethoprim sulphamethoxazole and Amoxicillin (93%) followed by gentamycin (87%) and the least was imipenem (0.5%). Fim was the most prevalent Urovirulence gene (53.5%) followed by Pap (21%), Sfa (13%), Afa (8%), Cnf (5.5%) and Hyl (0%). CONCLUSIONS: We demonstrate a high prevalence of biofilm-forming Uropathogenic E. coli strains that are highly associated with the MDR phenotype. We recommend routine surveillance of antimicrobial resistance and biofilm formation to understand the antibiotics suitable in the management of biofilm-associated UTIs.

First confirmed case of bovine besnoitiosis in Rwanda.

An-eighteen months (one and half years) old heifer was presented with a progressive loss of weight leading to cachexia and was attended to by a team of doctors from the ambulatory clinic of the School of Animal Science and Veterinary Medicine, University of Rwanda. Clinical examination revealed scleroderma of both sides of the neck and pinpoint cysts in the sclera and conjunctiva as well as corneal opacity of the left eye. Histological examination of skin biopsy showed a mononuclear dermatitis with several cysts of 100-300 µm filled up with bradyzoites in the subcutis and dermis. These lesions are consistent with bovine besnoitiosis, a disease caused by Besnoitia besnoiti in cattle. To the best of our knowledge, this is the first confirmed case of bovine besnoitiosis in Rwanda.

Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays.

BACKGROUND: Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read-thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. METHODS AND RESULTS: Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × 10-4-1 × 10-5 (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × 105) patient sample. CONCLUSIONS: Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h.

Prevalence and Characterization of Carbapenem-Resistant Enterobacteriaceae Isolated from Mulago National Referral Hospital, Uganda.

INTRODUCTION: Carbapenemases have increasingly been reported in enterobacteriaceae worldwide. Most carbapenemases are plasmid encoded hence resistance can easily spread. Carbapenem-resistant enterobacteriaceae are reported to cause mortality in up to 50% of patients who acquire bloodstream infections. We set out to determine the burden of carbapenem resistance as well as establish genes encoding for carbapenemases in enterobacteriaceae clinical isolates obtained from Mulago National Referral Hospital, Uganda. METHODS: This was a cross-sectional study with a total of 196 clinical isolates previously collected from pus swabs, urine, blood, sputum, tracheal aspirates, cervical swabs, endomentrial aspirates, rectal swabs, Vaginal swabs, ear swabs, products of conception, wound biopsy and amniotic fluid. All isolates were subjected to phenotypic carbapenemase screening using Boronic acid-based inhibition, Modified Hodge and EDTA double combined disk test. In addition, all the isolates were subjected to PCR assay to confirm presence of carbapenemase encoding genes. RESULTS: The study found carbapenemase prevalence of 22.4% (44/196) in the isolates using phenotypic tests, with the genotypic prevalence slightly higher at 28.6% (56/196). Over all, the most prevalent gene was blaVIM (21,10.7%), followed by blaOXA-48 (19, 9.7%), blaIMP (12, 6.1%), blaKPC (10, 5.1%) and blaNDM-1 (5, 2.6%). Among 56 isolates positive for 67 carbapenemase encoding genes, Klebsiella pneumonia was the species with the highest number (52.2%). Most 32/67(47.7%) of these resistance genes were in bacteria isolated from pus swabs. CONCLUSION: There is a high prevalence of carbapenemases and carbapenem-resistance encoding genes among third generation cephalosporins resistant Enterobacteriaceae in Uganda, indicating a danger of limited treatment options in this setting in the near future.

Long-term dominance of Mycobacterium tuberculosis Uganda family in peri-urban Kampala-Uganda is not associated with cavitary disease.

BACKGROUND: Previous studies have shown that Mycobacterium tuberculosis (MTB) Uganda family, a sub-lineage of the MTB Lineage 4, is the main cause of tuberculosis (TB) in Uganda. Using a well characterized patient population, this study sought to determine whether there are clinical and patient characteristics associated with the success of the MTB Uganda family in Kampala. METHODS: A total of 1,746 MTB clinical isolates collected from 1992-2009 in a household contact study were genotyped. Genotyping was performed using Single Nucleotide Polymorphic (SNP) markers specific for the MTB Uganda family, other Lineage 4 strains, and Lineage 3, respectively. Out of 1,746 isolates, 1,213 were from patients with detailed clinical data. These data were used to seek associations between MTB lineage/sub-lineage and patient phenotypes. RESULTS: Three MTB lineages were found to dominate the MTB population in Kampala during the last two decades. Overall, MTB Uganda accounted for 63% (1,092/1,746) of all cases, followed by other Lineage 4 strains accounting for 22% (394/1,746), and Lineage 3 for 11% (187/1,746) of cases, respectively. Seventy-three (4 %) strains remained unclassified. Our longitudinal data showed that MTB Uganda family occurred at the highest frequency during the whole study period, followed by other Lineage 4 strains and Lineage 3. To explore whether the long-term success of MTB Uganda family was due to increased virulence, we used cavitary disease as a proxy, as this form of TB is the most transmissible. Multivariate analysis revealed that even though cavitary disease was associated with known risk factors such as smoking (adjusted odds ratio (aOR) 4.8, 95% confidence interval (CI) 3.33-6.84) and low income (aOR 2.1, 95% CI 1.47-3.01), no association was found between MTB lineage and cavitary TB. CONCLUSION: The MTB Uganda family has been dominating in Kampala for the last 18 years, but this long-term success is not due to increased virulence as defined by cavitary disease.

Species and genotypic diversity of non-tuberculous mycobacteria isolated from children investigated for pulmonary tuberculosis in rural Uganda.

BACKGROUND: Smear microscopy, a mainstay of tuberculosis (TB) diagnosis in developing countries, cannot differentiate M. tuberculosis complex from NTM infection, while pulmonary TB shares clinical signs with NTM disease, causing clinical and diagnostic dilemmas. This study used molecular assays to identify species and assess genotypic diversity of non-tuberculous mycobacteria (NTM) isolates from children investigated for pulmonary tuberculosis at a demographic surveillance site in rural eastern Uganda. METHODS: Children were investigated for pulmonary tuberculosis as part of a TB vaccine surveillance program (2009-2011). Two cohorts of 2500 BCG vaccinated infants and 7000 adolescents (12-18 years) were recruited and followed up for one to two years to determine incidence of tuberculosis. Induced sputum and gastric aspirates were processed by the standard N-acetyl L-cysteine (NALC)-NaOH method. Sediments were cultured in the automated MGIT (Becton Dickson) liquid culture system and incubated at 37°C for at least six weeks. Capilia TB assay was used to classify mycobacteria into MTC and NTM. The GenoType CM/AS assays were performed to identify species while Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR genotyping was used to assess genetic diversity of the strains within each species. RESULTS: Among 2859 infants and 2988 adolescents screened, the numbers of TB suspects were 710 and 1490 infants and adolescents respectively. The prevalence of NTM in infant suspects was 3.7% (26/710) (95% CI 2.5-5.2) while that in adolescent suspects was 4.6% (69/1490) (95% CI 3.6-5.8). On culture, 127 isolates were obtained, 103 of which were confirmed as mycobacteria comprising of 95 NTM and eight M. tuberculosis complex. The Genotype CM/AS assay identified 63 of the 95 NTM isolates while 32 remained un-identified. The identified NTM species were M. fortuitum (40 isolates, 63.5%), M. szulgai (9 isolates, 14.3%), M. gordonae (6 isolates, 9.5%), M. intracellulare (3 isolates, 4.7%), M. scrofulaceum (2 isolates, 3.2%), M. lentiflavum (2 isolates, 3.2%), and M. peregrinum (1 isolate, 1.6%). Genotyping did not reveal any clustering in M. intracellulare, M. gordonae and M. szulgai species. M. fortuitum, on the other hand, had two clusters, one with three isolates of M. fortuitum 1 and the other with two isolates of M. fortuitum 2 subspecies. The remaining 35 of the 40 isolates of M. fortuitum had unique fingerprint patterns. CONCLUSION: M. fortuitum is the most common cause of infection by NTM among Infants and adolescents in rural Uganda. There is a varied number of species and genotypes, with minimal clustering within species, suggesting ubiquitous sources of infection to individuals in this community.

Performance of frontloading for smear microscopy in the diagnosis of pulmonary tuberculosis: a cross-sectional study at a referral hospital in Uganda.

OBJECTIVE: To compare the performance of frontloading and the standard WHO method for diagnosis of pulmonary TB at Mulago Hospital in order to validate the technique in this setting. METHODS: This was a cross-sectional study in which 229 adult (≥18 years) TB suspects were consecutively enrolled. Suspects submitted three sputum samples as follows: at initial presentation, one hour after the first sample, and the next morning. The first and next morning samples formed the standard WHO method, while the first and the one hour later samples formed the frontloading method. Sample processing was by the standard N-acetyl L-cystein (NALC)-NaOH method, and fluorescent microscopy was done for both methods, while cultures of the first sample on Lowenstein-Jensen slants acted as a gold standard. The sensitivity, specificity and predictive values for the WHO standard and frontloading methods were compared. RESULTS: The sensitivity of both the frontloading and standard schemes was 91.1% while their specificities were 86.2% and 91.7% respectively. There was excellent agreement between the diagnostic capacity of the two methods (kappa statistic = 0.87, P<0.0001). The positive predictive value for the frontloading scheme was 87.2% and that for the standard approach was 91.9%, while the negative predictive values were 90.4% and 90.9%, respectively. Among the HIV positive patients, frontloading identified 59/79 (74.7%) culture positive samples while the standard approach identified 55/79 (69.6%). In the HIV sero-negative category, on the other hand, front-loading identified 48/110 (43.6%) culture positive samples compared to 45/110 (40.9%) by the standard approach. CONCLUSION: Frontloading based on smear examination of two same-day sputum samples has a similar performance to the current standard method and would not be associated with any significant missed diagnosis. It may therefore be advocated for use in our setting so as to reduce time to completion of diagnosis and patient loss to follow-up.