Unravelling patient pathways in the context of antibacterial resistance in East Africa.

BACKGROUND: A key factor driving the development and maintenance of antibacterial resistance (ABR) is individuals' use of antibiotics (ABs) to treat illness. To better understand motivations and context for antibiotic use we use the concept of a patient treatment-seeking pathway: a treatment journey encompassing where patients go when they are unwell, what motivates their choices, and how they obtain antibiotics. This paper investigates patterns and determinants of patient treatment-seeking pathways, and how they intersect with AB use in East Africa, a region where ABR-attributable deaths are exceptionally high. METHODS: The Holistic Approach to Unravelling Antibacterial Resistance (HATUA) Consortium collected quantitative data from 6,827 adult outpatients presenting with urinary tract infection (UTI) symptoms in Kenya, Tanzania, and Uganda between February 2019- September 2020, and conducted qualitative in-depth patient interviews with a subset (n = 116). We described patterns of treatment-seeking visually using Sankey plots and explored explanations and motivations using mixed-methods. Using Bayesian hierarchical regression modelling, we investigated the associations between socio-demographic, economic, healthcare, and attitudinal factors and three factors related to ABR: self-treatment as a first step, having a multi-step treatment pathway, and consuming ABs. RESULTS: Although most patients (86%) sought help from medical facilities in the first instance, many (56%) described multi-step, repetitive treatment-seeking pathways, which further increased the likelihood of consuming ABs. Higher socio-economic status patients were more likely to consume ABs and have multi-step pathways. Reasons for choosing providers (e.g., cost, location, time) were conditioned by wider structural factors such as hybrid healthcare systems and AB availability. CONCLUSION: There is likely to be a reinforcing cycle between complex, repetitive treatment pathways, AB consumption and ABR. A focus on individual antibiotic use as the key intervention point in this cycle ignores the contextual challenges patients face when treatment seeking, which include inadequate access to diagnostics, perceived inefficient public healthcare and ease of purchasing antibiotics without prescription. Pluralistic healthcare landscapes may promote more complex treatment seeking and therefore inappropriate AB use. We recommend further attention to healthcare system factors, focussing on medical facilities (e.g., accessible diagnostics, patient-doctor interactions, information flows), and community AB access points (e.g., drug sellers).

Nasopharyngeal carriage, spa types and antibiotic susceptibility profiles of Staphylococcus aureus from healthy children less than 5 years in Eastern Uganda.

BACKGROUND: Staphylococcus aureus carriage is a known risk factor for staphylococcal disease. However, the carriage rates vary by country, demographic group and profession. This study aimed to determine the S. aureus carriage rate in children in Eastern Uganda, and identify S. aureus lineages that cause infection in Uganda. METHODS: Nasopharyngeal samples from 742 healthy children less than 5 years residing in the Iganga/Mayuge Health and Demographic Surveillance Site in Eastern Uganda were processed for isolation of S. aureus. Antibiotic susceptibility testing based on minimum inhibitory concentrations (MICs) was determined by the BD Phoenix™ system. Genotyping was performed by spa and SCCmec typing. RESULTS: The processed samples yielded 144 S. aureus isolates (one per child) therefore, the S. aureus carriage rate in children was 19.4% (144/742). Thirty one percent (45/144) of the isolates were methicillin resistant (MRSA) yielding a carriage rate of 6.1% (45/742). All isolates were susceptible to rifampicin, vancomycin and linezolid. Moreover, all MRSA were susceptible to vancomycin, linezolid and clindamycin. Compared to methicillin susceptible S. aureus (MSSA) isolates (68.8%, 99/144), MRSA isolates were more resistant to non-beta-lactam antimicrobials -trimethoprim/sulfamethoxazole 73.3% (33/45) vs. 27.3% (27/99) [p < 0.0001]; erythromycin 75.6% (34/45) vs. 24.2% (24/99) [p < 0.0001]; chloramphenicol 60% (27/45) vs. 19.2% (19/99) [p < 0.0001]; gentamicin 55.6% (25/45) vs. 25.3% (25/99) [p = 0.0004]; and ciprofloxacin 35.6% (16/45) vs. 2% (2/99) [p < 0.0001]. Furthermore, 42 MRSA (93.3%) were multidrug resistant (MDR) and one exhibited high-level resistance to mupirocin. Overall, 61 MSSA (61.6%) were MDR, including three mupirocin and clindamycin resistant isolates. Seven spa types were detected among MRSA, of which t037 and t064 were predominant and associated with SCCmec types I and IV, respectively. Fourteen spa types were detected in MSSA which consisted mainly of t645 and t4353. CONCLUSIONS: S. aureus carriage rate in healthy children in Eastern Uganda is high and comparable to rates for hospitalized patients in Kampala. The detection of mupirocin resistance is worrying as it could rapidly increase if mupirocin is administered in a low-income setting. S. aureus strains of spa types t064, t037 (MRSA) and t645, t4353 (MSSA) are prevalent and could be responsible for majority of staphylococcal infections in Uganda.

Epidemiology and antibiotic susceptibility of Vibrio cholerae associated with the 2017 outbreak in Kasese district, Uganda.

BACKGROUND: Uganda is among the 51 countries where cholera outbreaks are common with epidemics occurring predominantly along the western border with Democratic Republic of Congo (DRC), Kampala city slums, Busia district which is a border town with Western Kenya, Mbale district and the Karamoja Sub-region. This report summarizes findings from the epidemiologic investigation, which aimed at identifying the mode of transmission and antibiotic susceptibility patterns of the Vibrio cholerae isolated in Kasese district, Uganda. METHODS: A descriptive cross-sectional study was carried out between 2017 and 2018 to describe the epidemiology of the cholera epidemic in Kasese district, Uganda. Rectal swabs were collected from 69 suspected case-persons and cultured on Thiosulphate-Citrate-Bile-Salts Sucrose (TCBS™; SEIKEN Japan) agar and incubated at 37 °C for 18-24 h. The isolates were serotyped with polyvalent 01 antiserum and monovalent serotype Inaba and Ogawa antisera (Denka Seiken, Tokyo, Japan) to determine which serotype was responsible for the outbreak. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar. A list of discharged patients was obtained from the isolation units of Bwera hospital and Kagando hospital and the individuals were followed to the community where they live. Questionnaires were administered to a total of 75 participants who were either the cases or relatives to the case. GPS points of the homes of the cases and pictures of potential source infection were also taken and cases were mapped. RESULTS: A total of 222 cases were recorded in the Kasese District outbreak between the month of September 2017 and January 2018 with the case fatality rate (CFR) of 1.4%. Children below the age of 14 years contributed the biggest proportion of the cases (70%) and out of these, 33% were aged below 5 years. Culture isolated 69 V. cholerae 01 serotype Inaba from the total of 71 samples. Salmonella typhi was Isolated from the other two samples which were negative for V. cholerae. Antibiotic susceptibility using Kirby-Bauer disc diffusion method was done on isolates from 69 participants and showed 100% resistance to Ampicillin and over 50% were resistant to trimethoprim/Sulfamethoxazole whereas gentamicin showed 100% susceptibility. Environmental assessment revealed rampant cases of open defecation. CONCLUSION: Though we did not culture water to confirm contamination with Vibrio cholerae, we hypothesize that the cholera epidemic in Kasese 2017 was sparked off by consumption of contaminated water following the heavy floods that washed away latrines into water sources in Bwera, Isango and Nakiyumbu sub-counties. V. cholerae was also highly resistant to the commonly used antibiotics.

Prevalence and molecular characteristics of Staphylococcus aureus, including methicillin resistant strains, isolated from bulk can milk and raw milk products in pastoral communities of South-West Uganda.

BACKGROUND: Staphylococcus aureus strains are now regarded as zoonotic agents. In pastoral settings where human-animal interaction is intimate, multi-drug resistant microorganisms have become an emerging zoonotic issue of public health concern. The study of S. aureus prevalence, antimicrobial resistance and clonal lineages in humans, animals and food in African settings has great relevance, taking into consideration the high diversity of ethnicities, cultures and food habits that determine the lifestyle of the people. Little is known about milk carriage of methicillin resistant S. aureus strains (MRSA) and their virulence factors in Uganda. Here, we present the prevalence of MRSA in bulk can milk and raw milk products in pastoral communities of south-west Uganda. We also present PFGE profiles, spa-types, as well as frequency of enterotoxins genes. METHODS: S. aureus was identified by the coagulase test, susceptibility testing by the Kirby-Bauer disc diffusion and E-test methods and MRSA by detection of the mecA gene and SCCmec types. The presence of Panton - Valentine Leucocidin (PVL) genes and staphylococcal enterotoxins was determined by PCR, while genotyping was by PFGE and spa typing. RESULTS: S. aureus were isolated from 30/148 (20.3%) milk and 11/91(12%) sour milk samples. mecA gene carriage, hence MRSA, was detected in 23/41 (56.1%) of the isolates, with 21 of the 23 (91.3%) being SCCmec type V; while up to 30/41 (73.2%) of the isolates were resistant to tetracycline. Only five isolates carried the PVL virulence gene, while PFGE typing revealed ten clusters (ranging from two seven isolates each) that comprised 83% of the sample, and only eight isolates with unique pulsotypes. The largest PFGE profile (E) consisted of seven isolates while t7753, t1398, and t2112 were the most common spa-types. Thirty seven of the 41 strains (90.2%) showed at least one of the eight enterotoxin genes tested, with sem 29 (70.7%), sei 25 (61%) and seg 21 (51.2%) being the most frequently observed genes. CONCLUSION: This is the first study to demonstrate MRSA and enterotoxin genes in raw milk and its products in Uganda. The fact that over 90% of the isolates carried at least one gene encoding enterotoxins shows a high risk of spread of foodborne diseases through milk in this setting.

Molecular epidemiology of Panton-Valentine Leukocidin-positive community-acquired methicillin resistant Staphylococcus aureus isolates in pastoral communities of rural south western Uganda.

BACKGROUND: The emergence of multidrug resistant Staphylococcus aureus strains, including methicillin resistant (MRSA), is a global concern. Treatment of bacterial infections in Uganda's health care settings is largely empirical, rarely accompanied by laboratory confirmation. Here we show the burden, characteristics of MRSA and epidemiology of Panton-Valentine Leukocidin (PVL) positive strains in asymptomatic carriers in pastoral households of south-west Uganda. METHODS: Nasal swabs from 253 participants were cultured following standard methodology. MRSA strains were identified by detection of the mecA gene and SCCmec typing, and PVL genes detected by PCR. Pulsed Field Gel Electrophoresis (PFGE) was done to evaluate possible transmission patterns. Spa typing of PVL positive isolates was done to study the epidemiology of virulent strains in this setting. RESULTS: S. aureus was isolated in 29% (n = 73) of the participants, of which 48 were MRSA by mecA typing. PVL-encoding genes were found in 49.3% (n = 36) of the 73 isolates, of which 25 were also mecA positive. Among the PVL negative strains (n = 37), 62.2% (n = 23) carried the mecA gene. The most common SCCmec type was V, detected in 39 (18 PVL positive and 21 PVL negative) isolates. PFGE clustered 21/36 (58.3%) PVL positive isolates divided in four pulsotypes and 18/37 (48.6%) PVL negative isolates divided in eight pulsotypes. The most prevalent Spa types were t318 (26.5%, n = 9) and t645 (20.6%, n = 7); while other common Spa types were t11656 (n = 3), t127 (n = 3) and t355 (n = 3). CONCLUSION: The study shows a high prevalence of community acquired (CA)-MRSA, and PVL-positive isolates with two predominant spa types in rural Uganda, further complicating infection control strategies in these underprivileged communities.

The T2 Mycobacterium tuberculosis genotype, predominant in Kampala, Uganda, shows negative correlation with antituberculosis drug resistance.

Surveillance of the circulating Mycobacterium tuberculosis complex (MTC) strains in a given locality is important for understanding tuberculosis (TB) epidemiology. We performed molecular epidemiological studies on sputum smear-positive isolates that were collected for anti-TB drug resistance surveillance to establish the variability of MTC lineages with anti-TB drug resistance and HIV infection. Spoligotyping was performed to determine MTC phylogenetic lineages. We compared patients' MTC lineages with drug susceptibility testing (DST) patterns and HIV serostatus. Out of the 533 isolates, 497 (93.2%) had complete DST, PCR, and spoligotyping results while 484 (90.1%) participants had results for HIV testing. Overall, the frequency of any resistance was 75/497 (15.1%), highest among the LAM (34.4%; 95% confidence interval [CI], 18.5 to 53.2) and lowest among the T2 (11.5%; 95% CI, 7.6 to 16.3) family members. By multivariate analysis, LAM (adjusted odds ratio [OR(adj)], 5.0; 95% CI, 2.0 to 11.9; P < 0.001) and CAS (OR(adj), 2.9; 95% CI, 1.4.0 to 6.3; P = 0.006) families were more likely to show any resistance than was T2. All other MTC lineages combined were more likely to be resistant to any of the anti-TB drugs than were the T2 strains (OR(adj), 1.7; 95% CI, 1.0 to 2.9; P = 0.040). There were no significant associations between multidrug resistance and MTC lineages, but numbers of multidrug-resistant TB strains were small. No association was established between MTC lineages and HIV status. In conclusion, the T2 MTC lineage negatively correlates with anti-TB drug resistance, which might partly explain the reported low levels of anti-TB drug resistance in Kampala, Uganda. Patients' HIV status plays no role with respect to the MTC lineage distribution.

Knowledge and perceptions of brucellosis in the pastoral communities adjacent to Lake Mburo National Park, Uganda.

BACKGROUND: Brucellosis is one of the most common zoonotic infections globally. Lack of knowledge about brucellosis may affect the health-seeking behavior of patients, thus leading to sustained transmission in these communities. Our study assessed knowledge and perceptions of brucellosis among pastoral communities adjacent to Lake Mburo National Park (LMNP), Kiruhura District, Uganda. METHODS: A community cross-sectional questionnaire survey involving 371 randomly selected household heads from three sub-counties neighboring LMNP were interviewed between June and August 2012. Data collected included communities' knowledge on causes, symptoms, transmission, treatment, prevention and risk factors of brucellosis. Multivariable logistic regression analysis was performed to explore strength of association between overall knowledge of brucellosis and various individual factors using odds ratios and 95% confidence intervals. RESULTS: Only 70 (19%) knew the symptoms of brucellosis in animals, and three quarters (279, 75.5%) mentioned joint and muscle pain as a common symptom in humans. Almost all participants (370, 99.3%) had ever heard about brucellosis, majority (311, 84.7%) believed it affects all sexes and two thirds (67.7%) of the respondents believed close proximity to wildlife contributes to the presence of the disease. Almost all (352, 95.4%) knew that brucellosis in humans could be treatable using modern drugs. The main routes of infection in humans such as consumption of unpasteurized dairy products were known by 97% (360/371); eating of half-cooked meat by 91.4% and eating contaminated pasture in animals by 97.4%. There was moderate overall knowledge of brucellosis 197 (53.1%). Factors associated with higher overall knowledge were being agro-pastoralists (aOR: 2.08, CI: 1.17-3.71) compared to pure pastoralists while those who reported that the disease was a health problem (aOR: 0.18, CI: 0.06-0.56) compared to those who said it was not were less likely to be knowledgeable. CONCLUSIONS: There was moderate overall knowledge of human and animal brucellosis among the participants. Majority of the participants believed that close proximity to wildlife contributes to the presence of the disease in the area. There is a need for collaboration between the public health, veterinary and wildlife sectors to provide health education on brucellosis for better management of the disease in the communities.

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.

Klebsiella pneumoniae carbapenamases in Escherichia coli isolated from humans and livestock in rural south-western Uganda.

BACKGROUND: The accumulation of resistance genes in Escherichia coli (E. coli) strains imposes limitations in the therapeutic options available for the treatment of infections caused by E.coli. Production of Klebsiella pneumoniae carbapenemase (KPC) by E. coli renders it resistant to broad-spectrum ß-lactam antibiotics. Globally there is existing evidence of spread of carbapenem-resistant E. coli in both humans and livestock driven by acquisition of the several other carbapenemase genes. Overall, there is little information regarding the extent of KPC gene distribution in E. coli. We set out to determine the prevalence, and evaluate the phenotypic and genotypic patterns of KPC in E. coli isolated from humans and their livestock in rural south western Uganda. METHODS: A laboratory-based, descriptive cross-sectional study was conducted involving 96 human and 96 livestock isolates collected from agro-pastoralist communities in Mbarara district in south western Uganda. Phenotypic and molecular methods (PCR) were used for presence and identification of KPC genes in the E. coli isolates. A chi-square test of independence was used to evaluate the differences in resistant patterns between carbapenems and isolates. RESULTS: The overall prevalence of carbapenem resistance by disk diffusion susceptibility testing (DST) for both humans and livestock isolates were 41.7% (80/192). DST-based resistance was identical in both human and livestock isolates (41.7%). The prevalence of carbapenem resistance based on Modified Hodge Test (MHT) was 5% (2/40) and 10% (4/40) for humans and livestock isolates respectively. Both human and livestock isolates, 48.7% (95/192) had the KPC gene, higher than phenotypic expression; 41.7% (80/192). blaKPC gene prevalence was overall similar in human isolates (51%; 49/96) vs livestock isolates (47.9%; 46/96). Approximately, 19% (15/80) of the isolates were phenotypically resistant to carbapenems and over 70% (79/112) of the phenotypically sensitive strains harbored the blaKPC gene. CONCLUSION: Our results suggest that both human and livestock isolates of E. coli in our setting carry the blaKPC gene with a high percentage of strains not actively expressing the blaKPC gene. The finding of fewer isolates carrying the KPC gene than those phenotypically resistant to carbapenems suggests that other mechanisms are playing a role in this phenomenon, calling for further researcher into this phenomenon.

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.

A first insight into the genotypic diversity of Mycobacterium tuberculosis from Rwanda.

BACKGROUND: Mycobacterium tuberculosis complex (MTC) is the causative agent of tuberculosis (TB). Globally, increasing evidence shows that in M. tuberculosis, transmission varies from strain to strain and that different strains exhibit a range of geographical and host specificities, pathogenicity, and drug susceptibility. Therefore rapid and accurate differentiation of the members of MTC is critical in guiding treatment and public health decisions. We carried out a study at different health units and the National Reference Laboratory in Rwanda identify Mycobacterium tuberculosis complex species prevalent in TB patients in Rwanda. We further characterized the isolates using spoligotyping in order to gain an insight into the strain diversity of drug resistant and susceptible isolates of M. tuberculosis in this setting. METHODS: A total of 151 isolates from culture positive sputum samples were harvested, heat killed at 80°C for two hours, and then shipped to Makerere University College of Health Sciences, Uganda, for speciation and typing. Species identification was achieved by regions of difference (RD) analysis, while Spoligotyping was done to identify strain types. RESULTS: Region of difference analysis identified all the 151 isolates as M. tuberculosis. Spoligotyping revealed predominance of the T2 family (58.3%, 88/151), with SIT 52 being the most prevalent strain (31.8%, 48/151). Among the 151 isolates, 64 (42.4%) were multidrug resistant (MDR) with 3 cases on mono-resistance. Of 94 retreatment cases, 48 (51.1%) were MDR and of 46 newly presenting cases 14 (30.4%) were MDR. There was a significant difference (p=0.01) in anti-TB drug resistance between new and retreatment cases in the sample. However, there was no significant relationship between HIV serostatus and the two major strain types SIT 52 (p =0.15and SIT 152 (p = 0.41). CONCLUSION: Mycobacterium tuberculosis is the most prevalent species of Mycobacterium tuberculosis complex in Rwanda, and SIT 52 (T2) the predominant strain. There is significantly more MDR in the retreatment cases but no significant difference was observed by HIV status in relation to any spoligotypes.

Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in South-Western Uganda.

BACKGROUND: Determination of the prevalence and drug susceptibility of the M. tuberculosis strains is important in tuberculosis control. We determined the genetic diversity and susceptibility profiles of mycobacteria isolated from tuberculosis patients in Mbarara, South Western Uganda. METHODS: We enrolled, consecutively; all newly diagnosed and previously treated smear-positive TB patients aged≥18 years. The isolates were characterized using regions of difference (RD) analysis and spoligotyping. Drug resistance against rifampicin and isoniazid were tested using the Genotype® MDRTBplus assay and the indirect proportion method on Lowenstein-Jensen media. HIV-1 testing was performed using two rapid HIV tests. RESULTS: A total of 125 isolates from 167 TB suspects (60% males) with a mean age 33.7 years and HIV prevalence of 67.9% (55/81) were analyzed. Majority (92.8%) were new cases while only 7.2% were retreatment cases. All the 125 isolates were identified as M. tuberculosis strict sense with the majority (92.8%) of the isolates being modern strains while seven (7.2%) isolates were ancestral strains. Spoligotyping revealed 79 spoligotype patterns, with an overall diversity of 63.2%. Sixty two (49.6%) of the isolates formed 16 clusters consisting of 2-15 isolates each. A majority (59.2%) of the isolates belong to the Uganda genotype group of strains. The major shared spoligotypes in our sample were SIT 135 (T2-Uganda) with 15 isolates and SIT 128 (T2) with 3 isolates. Sixty nine (87%) of the 79 patterns had not yet been defined in the SpolDB4.0.database. Resistance mutations to either RIF or INH were detected in 6.4% of the isolates. Multidrug resistance, INH and RIF resistance was 1.6%, 3.2% and 4.8%, respectively. The rpoß gene mutations seen in the sample were D516V, S531L, H526Y H526D and D516V, while one strain had a Δ1 mutation in the wild type probes. There were three strains with katG (codon 315) gene mutations only while one strain showed the inhA promoter gene mutation. CONCLUSION: The present study shows that the TB epidemic in Mbarara is caused by modern M. tuberculosis strains mainly belonging to the Uganda genotype and anti-TB drug resistance rate in the region is low.

The simple direct slide method is comparable to indirect Lowenstein Jensen proportion culture for detecting rifampicin resistant tuberculosis.

Introduction. Drug resistant tuberculosis remains a worldwide problem that requires prompt diagnosis.Hypothesis/Gap statement. The WHO recommended direct, rapid Xpert MTB/RIF is prohibitively costly, therefore, there is a need to validate a rapid, affordable DST for use in low- and middle-income settings.Aim. The technical performance and time to results of a simple, direct microscopy-based slide DST (SDST) assay for diagnosis of rifampicin-resistant TB was evaluated in Uganda.Methodology. Sputum samples from 122 smear-positive re-treatment TB patients presenting to the TB treatment centre at Uganda's National Referral Hospital, Mulago, Kampala, Uganda were examined. The sputum samples were tested by the direct SDST which was compared to the indirect Lowenstein Jensen Proportion Method (LJDST) method as the gold standard. The time to results was defined as the time from DST setting to results interpretation. The results were further analysed for sensitivity and specificity as well as agreement between LJDST and SDST for rifampicin resistance determination.Results. A total of 117 smear positive sputum samples with valid results for both tests were compared. The median time to results for SDST was 14 days with an interquartile range (IQR) of 10-14 days compared to 60 days with IQR of 60-75 days for LJDST. The number for rifampicin resistance by the gold standard LJDST was 26. The SDST had a sensitivity of 96 % (95 %; CI 81-99 %) and a specificity of 97.8 % (95 %; CI 93-100 %). The Positive Predictive and Negative Predictive values for SDST were 92.3 % (95 %; CI 76.8-99 %) and 98.9 % (95 %; CI 94-100 %), respectively. The kappa agreement between SDST and LJDST was 92.3 %.Conclusion. The SDST was found to be a rapid and accurate direct test for the detection of rifampicin resistance among retreatment TB cases in low-income settings.

Protocol for an interdisciplinary cross-sectional study investigating the social, biological and community-level drivers of antimicrobial resistance (AMR): Holistic Approach to Unravel Antibacterial Resistance in East Africa (HATUA).

INTRODUCTION: Antimicrobial resistance (AMR) is a global health threat that requires urgent research using a multidisciplinary approach. The biological drivers of AMR are well understood, but factors related to treatment seeking and the social contexts of antibiotic (AB) use behaviours are less understood. Here we describe the Holistic Approach to Unravel Antibacterial Resistance in East Africa, a multicentre consortium that investigates the diverse drivers of drug resistance in urinary tract infections (UTIs) in East Africa. METHODS AND ANALYSIS: This study will take place in Uganda, Kenya and Tanzania. We will conduct geospatial mapping of AB sellers, and conduct mystery client studies and in-depth interviews (IDIs) with drug sellers to investigate AB provision practices. In parallel, we will conduct IDIs with doctors, alongside community focus groups. Clinically diagnosed patients with UTI will be recruited from healthcare centres, provide urine samples and complete a questionnaire capturing retrospective treatment pathways, sociodemographic characteristics, attitudes and knowledge. Bacterial isolates from urine and stool samples will be subject to culture and antibiotic sensitivity testing. Genomic DNA from bacterial isolates will be extracted with a subset being sequenced. A follow-up household interview will be conducted with 1800 UTI-positive patients, where further environmental samples will be collected. A subsample of patients will be interviewed using qualitative tools. Questionnaire data, microbiological analysis and qualitative data will be linked at the individual level. Quantitative data will be analysed using statistical modelling, including Bayesian network analysis, and all forms of qualitative data analysed through iterative thematic content analysis. ETHICS AND DISSEMINATION: Approvals have been obtained from all national and local ethical review bodies in East Africa and the UK. Results will be disseminated in communities, with local and global policy stakeholders, and in academic circles. They will have great potential to inform policy, improve clinical practice and build regional pathogen surveillance capacity.

Detection of multiple strains of Mycobacterium tuberculosis using MIRU-VNTR in patients with pulmonary tuberculosis in Kampala, Uganda.

BACKGROUND: Many studies using DNA fingerprinting to differentiate Mycobacterium tuberculosis (MTB) strains reveal single strains in cultures, suggesting that most disease is caused by infection with a single strain. However, recent studies using molecular epidemiological tools that amplify multiple targets have demonstrated simultaneous infection with multiple strains of MTB. We aimed to determine the prevalence of MTB multiple strain infections in Kampala, and the impact of these infections on clinical presentation of tuberculosis (TB) and response to treatment. METHODS: A total of 113 consecutive smear and culture positive patients who previously enrolled in a house-hold contact study were included in this study. To determine whether infection with multiple MTB strains has a clinical impact on the initial presentation of patients, retrospective patient data (baseline clinical, radiological and drug susceptibility profiles) was obtained. To determine presence of infections with multiple MTB strains, MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeats) -PCR was performed on genomic DNA extracted from MTB cultures of smear positive sputum samples at baseline, second and fifth months. RESULTS: Of 113 patients, eight (7.1%) had infection with multiple MTB strains, coupled with a high rate of HIV infection (37.5% versus 12.6%, p = 0.049). The remaining patients (105) were infected with single MTB strains. The proportions of patients with MTB smear positive cultures after two and five months of treatment were similar. There was no difference between the two groups for other variables. CONCLUSION: Infection with multiple MTB strains occurs among patients with first episode of pulmonary tuberculosis in Kampala, in a setting with high TB incidence. Infection with multiple MTB strains had little impact on the clinical course for individual patients. This is the first MIRU-VNTR-based study from in an East African country.

Use of the GenoType(R) MTBDRplus assay to assess drug resistance of Mycobacterium tuberculosis isolates from patients in rural Uganda.

BACKGROUND: Drug resistance levels and patterns among Mycobacterium tuberculosis isolates from newly diagnosed and previously treated tuberculosis patients in Mbarara Uganda were investigated. METHODS: We enrolled, consecutively, all newly diagnosed and previously treated smear-positive TB patients aged >/= 18 years. Isolates were tested for drug resistance against rifampicin (RIF) and isoniazid (INH) using the Genotype(R) MDRTBplus assay and results were compared with those obtained by the indirect proportion method on Lowenstein-Jensen media. HIV testing was performed using two rapid HIV tests. RESULTS: A total of 125 isolates from 167 TB suspects with a mean age 33.7 years and HIV prevalence of 67.9% (55/81) were analysed. A majority (92.8%) of the participants were newly presenting while only 7.2% were retreatment cases. Resistance mutations to either RIF or INH were detected in 6.4% of the total isolates. Multidrug resistance, INH and RIF resistance was 1.6%, 3.2% and 4.8%, respectively. The rpobeta gene mutations seen in the sample were D516V, S531L, H526Y H526 D and D516V, while one strain had a Delta1 mutation in the wild type probes. There were three strains with katG (codon 315) gene mutations while only one strain showed the inhA promoter region gene mutation. CONCLUSION: The TB resistance rate in Mbarara is relatively low. The GenoType(R) MTBDRplus assay can be used for rapid screening of MDR-TB in this setting.

Antimicrobial resistance at the human-animal interface in the Pastoralist Communities of Kasese District, South Western Uganda.

Intensive usage of antimicrobials in the management of animal diseases leads to selection for resistance among microorganisms. This study aimed to assess antimicrobial use and to describe factors associated with the transmission of antimicrobial resistance between humans and animals in pastoralist communities of Kasese district. A mixed-methods approach was employed in this study. Rectal swabs were collected from the participants and cattle and transported in Carry-Blaire transport medium to the laboratory within 24 h of collection for culture and sensitivity to confirm carriage of multi-drug resistant bacteria. In-depth interviews were conducted among veterinary officers, veterinary drug vendors, human health facility in-charges in both public and private health facilities, and operators of human pharmacies and drug shops. Carriage of multi-drug resistant bacteria among humans was 88 (93%) and 76(80%) among cattle. Consumption of lakeshore water and carriage of multi-drug resistant bacteria in cattle were associated with carriage of multi-drug resistant bacteria in the human population. The prevalence of multi-drug resistance among organisms Isolated from both humans and animals was high. There is a high likelihood of transmission of multi-drug resistance between humans and animals.

Whole genome sequences of multi-drug resistant Escherichia coli isolated in a Pastoralist Community of Western Uganda: Phylogenomic changes, virulence and resistant genes.

BACKGROUND: The crisis of antimicrobial resistance is already here with us, affecting both humans and animals alike and very soon, small cuts and surgeries will become life threatening. This study aimed at determine the whole genome sequences of multi-drug resistant Escherichia coli isolated in a Pastoralist Community of Western Uganda: phylogenomic changes, virulence and resistant genes. METHODS: This was a laboratory based cross sectional study. Bacterial isolates analyzed in this study were 42 multidrug resistant E. coli isolated from stool samples from both humans (n = 30) and cattle (n = 12) in pastoralist communities collected between January 2018-March 2019. Most of the isolates (41/42) were resistant to three or more antibiotics (multi-drug resistant) and 21/42 isolates were ESBL producers; 13/30 from human and 8/12 from cattle. Whole Genome Sequencing (WGS) was carried out at the facilities of Kenya Medical Research Institute-Wellcome trust, Kilifi, to determine the phylogenomic changes, virulence and resistant genes. RESULTS: At household level, the genomes from both human and animals clustered away from one another except for one instance where two human isolates from the same household clustered together. However, 67% of the E. coli isolated from cattle were closely related to those found in humans. The E. coli isolates were assigned to eight different phylogroups: A, B1, B2, Cladel, D, E, F and G, with a majority being assigned to phylogroup A; while most of the animal isolates were assigned to phylogroup B1. The carriage of multiple AMR genes was higher from the E. coli population from humans than those from cattle. Among these were Beta-lactamase; blaOXA-1: Class D beta-lactamases; blaTEM-1, blaTEM-235: Beta-lactamase; catA1: chloramphenicol acetyl transferase; cmlA1: chloramphenicol efflux transporter; dfrA1, dfrA12, dfrA14, dfrA15, dfrA17, dfrA5, dfrA7, dfrA8: macrolide phosphotransferase; oqxB11: RND efflux pump conferring resistance to fluoroquinolone; qacL, qacEdelta1: quinolone efflux pump; qnrS1: quinolone resistance gene; sul1, sul2, sul3: sulfonamide resistant; tet(A), tet(B): tetracycline efflux pump. A high variation of virulence genes was registered among the E. coli genomes from humans than those of cattle origin. CONCLUSION: From the analysis of the core genome and phenotypic resistance, this study has demonstrated that the E. coli of human origin and those of cattle origin may have a common ancestry. Limited sharing of virulence genes presents a challenge to the notion that AMR in humans is as a result of antibiotic use in the farm and distorts the picture of the directionality of transmission of AMR at a human-animal interface and presents a task of exploring alternative routes of transmission of AMR.

Species and drug susceptibility profiles of staphylococci isolated from healthy children in Eastern Uganda.

Staphylococci are a key component of the human microbiota, and they mainly colonize the skin and anterior nares. However, they can cause infection in hospitalized patients and healthy individuals in the community. Although majority of the Staphylococcus aureus strains are coagulase-positive, some do not produce coagulase, and the isolation of coagulase-positive non-S. aureus isolates in humans is increasingly being reported. Therefore, sound knowledge of the species and characteristics of staphylococci in a given setting is important, especially isolates from children and immunocompromised individuals. The spectrum of Staphylococcus species colonizing children in Uganda is poorly understood; here, we aimed to determine the species and characteristics of staphylococci isolated from children in Eastern Uganda. Seven hundred and sixty four healthy children less than 5 years residing in Iganga and Mayuge districts in Eastern Uganda were enrolled. A total of 513 staphylococci belonging to 13 species were isolated from 485 children (63.5%, 485/764), with S. aureus being the dominant species (37.6%, 193/513) followed by S. epidermidis (25.5%, 131/513), S. haemolyticus (2.3%, 12/513), S. hominis (0.8%, 4/513) and S. haemolyticus/lugdunensis (0.58%, 3/513). Twenty four (4.95%, 24/485) children were co-colonized by two or more Staphylococcus species. With the exception of penicillin, antimicrobial resistance (AMR) rates were low; all isolates were susceptible to vancomycin, teicoplanin, linezolid and daptomycin. The prevalence of methicillin resistance was 23.8% (122/513) and it was highest in S. haemolyticus (66.7%, 8/12) followed by S. aureus (28.5%, 55/193) and S. epidermidis (23.7%, 31/131). The prevalence of multidrug resistance was 20.3% (104/513), and 59% (72/122) of methicillin resistant staphylococci were multidrug resistant. Four methicillin susceptible S. aureus isolates and a methicillin resistant S. scuiri isolate were mupirocin resistant (high-level). The most frequent AMR genes were mecA, vanA, ant(4')-Ia, and aac(6')-Ie- aph(2'')-Ia, pointing to presence of AMR drivers in the community.