Treatment seeking behaviours, antibiotic use and relationships to multi-drug resistance: A study of urinary tract infection patients in Kenya, Tanzania and Uganda.

Antibacterial resistance (ABR) is a major public health threat. An important accelerating factor is treatment-seeking behaviour, including inappropriate antibiotic (AB) use. In many low- and middle-income countries (LMICs) this includes taking ABs with and without prescription sourced from various providers, including health facilities and community drug sellers. However, investigations of complex treatment-seeking, AB use and drug resistance in LMICs are scarce. The Holistic Approach to Unravel Antibacterial Resistance in East Africa (HATUA) Consortium collected questionnaire and microbiological data from adult outpatients with urinary tract infection (UTI)-like symptoms presenting at healthcare facilities in Kenya, Tanzania and Uganda. Using data from 6,388 patients, we analysed patterns of self-reported treatment seeking behaviours ('patient pathways') using process mining and single-channel sequence analysis. Among those with microbiologically confirmed UTI (n = 1,946), we used logistic regression to assess the relationship between treatment seeking behaviour, AB use, and the likelihood of having a multi-drug resistant (MDR) UTI. The most common treatment pathway for UTI-like symptoms in this sample involved attending health facilities, rather than other providers like drug sellers. Patients from sites in Tanzania and Uganda, where over 50% of patients had an MDR UTI, were more likely to report treatment failures, and have repeat visits to providers than those from Kenyan sites, where MDR UTI proportions were lower (33%). There was no strong or consistent relationship between individual AB use and likelihood of MDR UTI, after accounting for country context. The results highlight the hurdles East African patients face in accessing effective UTI care. These challenges are exacerbated by high rates of MDR UTI, suggesting a vicious cycle of failed treatment attempts and sustained selection for drug resistance. Whilst individual AB use may contribute to the risk of MDR UTI, our data show that factors related to context are stronger drivers of variations in ABR.

Predominance of multidrug-resistant bacteria causing urinary tract infections among symptomatic patients in East Africa: a call for action.

Background: In low- and middle-income countries, antibiotics are often prescribed for patients with symptoms of urinary tract infections (UTIs) without microbiological confirmation. Inappropriate antibiotic use can contribute to antimicrobial resistance (AMR) and the selection of MDR bacteria. Data on antibiotic susceptibility of cultured bacteria are important in drafting empirical treatment guidelines and monitoring resistance trends, which can prevent the spread of AMR. In East Africa, antibiotic susceptibility data are sparse. To fill the gap, this study reports common microorganisms and their susceptibility patterns isolated from patients with UTI-like symptoms in Kenya, Tanzania and Uganda. Within each country, patients were recruited from three sites that were sociodemographically distinct and representative of different populations. Methods: UTI was defined by the presence of >104 cfu/mL of one or two uropathogens in mid-stream urine samples. Identification of microorganisms was done using biochemical methods. Antimicrobial susceptibility testing was performed by the Kirby-Bauer disc diffusion assay. MDR bacteria were defined as isolates resistant to at least one agent in three or more classes of antimicrobial agents. Results: Microbiologically confirmed UTI was observed in 2653 (35.0%) of the 7583 patients studied. The predominant bacteria were Escherichia coli (37.0%), Staphylococcus spp. (26.3%), Klebsiella spp. (5.8%) and Enterococcus spp. (5.5%). E. coli contributed 982 of the isolates, with an MDR proportion of 52.2%. Staphylococcus spp. contributed 697 of the isolates, with an MDR rate of 60.3%. The overall proportion of MDR bacteria (n = 1153) was 50.9%. Conclusions: MDR bacteria are common causes of UTI in patients attending healthcare centres in East African countries, which emphasizes the need for investment in laboratory culture capacity and diagnostic algorithms to improve accuracy of diagnosis that will lead to appropriate antibiotic use to prevent and control AMR.

Evaluation of three protocols for direct susceptibility testing for gram negative-Enterobacteriaceae from patient samples in Uganda with SMS reporting.

In Uganda, the challenge of generating and timely reporting essential antimicrobial resistance (AMR) data has led to overreliance on empirical antibiotic therapy, exacerbating the AMR crisis. To address this issue, this study aimed to adapt a one-step AMR testing protocol alongside an SMS (Short Message Service) result relay system (SRRS), with the potential to reduce the turnaround time for AMR testing and result communication from 4 days or more to 1 day in Ugandan clinical microbiology laboratories. Out of the 377 samples examined, 54 isolates were obtained. Notably, E. coli (61%) and K. pneumoniae (33%) were the most frequently identified, majority testing positive for ESBL. Evaluation of three AMR testing protocols revealed varying sensitivity and specificity, with Protocol A (ChromID ESBL-based) demonstrating high sensitivity (100%) but no calculable specificity, Protocol B (ceftazidime-based) showing high sensitivity (100%) and relatively low specificity (7.1%), and Protocol C (cefotaxime-based) exhibiting high sensitivity (97.8%) but no calculable specificity. ESBL positivity strongly correlated with resistance to specific antibiotics, including cefotaxime, ampicillin, and aztreonam (100%), cefuroxime (96%), ceftriaxone (93%), and trimethoprim sulfamethoxazole (87%). The potential of integrating an SRRS underscored the crucial role this could have in enabling efficient healthcare communication in AMR management. This study underscores the substantial potential of the tested protocols for accurately detecting ESBL production in clinical samples, potentially, providing a critical foundation for predicting and reporting AMR patterns. Although considerations related to specificity warrant careful assessment before widespread clinical adoption.

Treatment seeking and antibiotic use for urinary tract infection symptoms in the time of COVID-19 in Tanzania and Uganda.

Background: There is still little empirical evidence on how the outbreak of coronavirus disease 2019 (COVID-19) and associated regulations may have disrupted care-seeking for non-COVID-19 conditions or affected antibiotic behaviours in low- and middle-income countries (LMICs). We aimed to investigate the differences in treatment-seeking behaviours and antibiotic use for urinary tract infection (UTI)-like symptoms before and during the pandemic at recruitment sites in two East African countries with different COVID-19 control policies: Mbarara, Uganda and Mwanza, Tanzania. Methods: In this repeated cross-sectional study, we used data from outpatients (pregnant adolescents aged >14 and adults aged >18) with UTI-like symptoms who visited health facilities in Mwanza, Tanzania and Mbarara, Uganda. We assessed the prevalence of self-reported behaviours (delays in care-seeking, providers visited, antibiotics taken) at three different time points, labelled as 'pre-COVID-19 phase' (February 2019 to February 2020), 'COVID-19 phase 1' (March 2020 to April 2020), and 'COVID-19 phase 2' (July 2021 to February 2022). Results: In both study sites, delays in care-seeking were less common during the pandemic than they were in the pre-COVID phase. Patients in Mwanza, Tanzania had shorter care-seeking pathways during the pandemic compared to before it, but this difference was not observed in Mbarara, Uganda. Health centres were the dominant sources of antibiotics in both settings. Over time, reported antibiotic use for UTI-like symptoms became more common in both settings. During the COVID-19 phases, there was a significant increase in self-reported use of antibiotics like metronidazole (<30% in the pre-COVID-19 phase to 40% in COVID phase 2) and doxycycline (30% in the pre-COVID-19 phase to 55% in COVID phase 2) that were not recommended for treating UTI-like symptoms in the National Treatment Guidelines in Mbarara, Uganda. Conclusions: There was no clear evidence that patients with UTI-like symptoms attending health care facilities had longer or more complex treatment pathways despite strict government-led interventions related to COVID-19. However, antibiotic use increased over time, including some antibiotics not recommended for treating UTI, which has implications for future antimicrobial resistance.

Evaluation of the diagnostic performance of the urine dipstick test for the detection of urinary tract infections in patients treated in Kenyan hospitals.

Introduction: Culture is the gold-standard diagnosis for urinary tract infections (UTIs). However, most hospitals in low-resource countries lack adequately equipped laboratories and relevant expertise to perform culture and, therefore, rely heavily on dipstick tests for UTI diagnosis. Research gap: In many Kenyan hospitals, routine evaluations are rarely done to assess the accuracy of popular screening tests such as the dipstick test. As such, there is a substantial risk of misdiagnosis emanating from inaccuracy in proxy screening tests. This may result in misuse, under-use or over-use of antimicrobials. Aim: The present study aimed to assess the accuracy of the urine dipstick test as a proxy for the diagnosis of UTIs in selected Kenyan hospitals. Methods: A hospital-based cross-sectional method was used. The utility of dipstick in the diagnosis of UTIs was assessed using midstream urine against culture as the gold standard. Results: The dipstick test predicted 1416 positive UTIs, but only 1027 were confirmed positive by culture, translating to a prevalence of 54.1 %. The sensitivity of the dipstick test was better when leucocytes and nitrite tests were combined (63.1 %) than when the two tests were separate (62.6 and 50.7 %, respectively). Similarly, the two tests combined had a better positive predictive value (87.0 %) than either test alone. The nitrite test had the best specificity (89.8 %) and negative predictive value (97.4 %) than leucocytes esterase (L.E) or both tests combined. In addition, sensitivity in samples from inpatients (69.2 %) was higher than from outpatients (62.7 %). Furthermore, the dipstick test had a better sensitivity and positive predictive value among female (66.0 and 88.6 %) than male patients (44.3 and 73.9 %). Among the various patient age groups, the dipstick test's sensitivity and positive predictive value were exceptionally high in patients ≥75 years old (87.5 and 93.3 %). Conclusion: Discrepancies in prevalence from the urine dipstick test and culture, the gold standard, indicate dipstick test inadequacy for accurate UTI diagnosis. The finding also demonstrates the need for urine culture for accurate UTI diagnosis. However, considering it is not always possible to perform a culture, especially in low-resource settings, future studies are needed to combine specific UTI symptoms and dipstick results to assess possible increases in the test's sensitivity. There is also a need to develop readily available and affordable algorithms that can detect UTIs where culture is not available.

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.

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).

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.

Treatment seeking behaviours, antibiotic use and relationships to multi-drug resistance: A study of urinary tract infection patients in Kenya, Tanzania and Uganda.

Antibacterial resistance (ABR) is a major public health threat. An important accelerating factor is treatment-seeking behaviours, including inappropriate antibiotic (AB) use. In many low- and middle-income countries (LMICs) this includes taking ABs with and without prescription sourced from various providers, including health facilities and community drug sellers. However, investigations of complex treatment-seeking, AB use and drug resistance in LMICs are scarce. The Holistic Approach to Unravel Antibacterial Resistance in East Africa (HATUA) Consortium collected questionnaire and microbiological data from 6,827 adult outpatients with urinary tract infection (UTI)-like symptoms presenting at healthcare facilities in Kenya, Tanzania and Uganda. Among 6,388 patients we analysed patterns of self-reported treatment seeking behaviours ('patient pathways') using process mining and single-channel sequence analysis. Of those with microbiologically confirmed UTI (n=1,946), we used logistic regression to assessed the relationship between treatment seeking behaviour, AB use, and likelihood of having a multi-drug resistant (MDR) UTI. The most common treatment pathways for UTI-like symptoms included attending health facilities, rather than other providers (e.g. drug sellers). Patients from the sites sampled in Tanzania and Uganda, where prevalence of MDR UTI was over 50%, were more likely to report treatment failures, and have repeated visits to clinics/other providers, than those from Kenyan sites, where MDR UTI rates were lower (33%). There was no strong or consistent relationship between individual AB use and risk of MDR UTI, after accounting for country context. The results highlight challenges East African patients face in accessing effective UTI treatment. These challenges increase where rates of MDR UTI are higher, suggesting a reinforcing circle of failed treatment attempts and sustained selection for drug resistance. Whilst individual behaviours may contribute to the risk of MDR UTI, our data show that factors related to context are stronger drivers of ABR.

Antibiotic dispensing practices during COVID-19 and implications for antimicrobial resistance (AMR): parallel mystery client studies in Uganda and Tanzania.

BACKGROUND: Over-the-counter antibiotic access is common in low-and-middle-income countries and this may accelerate antimicrobial resistance. Our study explores critical aspects of the drug seller-client interaction and antibiotic dispensing patterns for simulated COVID-19 symptoms during the pandemic in two study sites in Tanzania and Uganda, countries with different government responses to the pandemic. METHODS: Research assistants posing as clients approached different types of drug sellers such as pharmacies (Pharms), drug shops (DSs), and accredited drug dispensing outlets (ADDOs) in Mwanza, Tanzania (nPharms = 415, nADDOs = 116) and Mbarara, Uganda (nPharms = 440, nDSs = 67), from June 10 to July 30, 2021. The mystery clients held no prescription and sought advice for simulated COVID-19 symptoms from the drug sellers. They documented the quality of their interaction with sellers and the type of drugs dispensed. RESULTS: Adherence to COVID-19 preventive measures and vigilance to COVID-19 symptoms was low in both sites but significantly higher in Uganda than in Tanzania. A higher percentage of drug sellers in Mbarara (Pharms = 36%, DSs = 35%, P-value = 0.947) compared to Mwanza (Pharms = 9%, ADDOs = 4%, P-value = 0.112) identified the client's symptoms as possibly COVID-19. More than three-quarters of drug sellers that sold prescription-only medicines in both Mbarara (Pharms = 86%, DSs = 89%) and Mwanza (Pharms = 93%, ADDOs = 97%) did not ask the MCs for a prescription. A relatively high percentage of drug sellers that sold prescription-only medicines in Mwanza (Pharms = 51%, ADDOs = 67%) compared to Mbarara (Pharms = 31%, DSs = 42%) sold a partial course without any hesitation. Of those who sold antibiotics, a higher proportion of drug sellers in Mbarara (Pharms = 73%, DSs = 78%, P-value = 0.580) compared to Mwanza (Pharms = 40% ADDOs = 46%, P-value = 0.537) sold antibiotics relevant for treating secondary bacterial infections in COVID-19 patients. CONCLUSION: Our study highlights low vigilance towards COVID-19 symptoms, widespread propensity to dispense prescription-only antibiotics without a prescription, and to dispense partial doses of antibiotics. This implies that drug dispensing related to COVID-19 may further drive AMR. Our study also highlights the need for more efforts to improve antibiotic stewardship among drug sellers in response to COVID-19 and to prepare them for future health emergencies.

The role of multidimensional poverty in antibiotic misuse: a mixed-methods study of self-medication and non-adherence in Kenya, Tanzania, and Uganda.

BACKGROUND: Poverty is a proposed driver of antimicrobial resistance, influencing inappropriate antibiotic use in low-income and middle-income countries (LMICs). However, at subnational levels, studies investigating multidimensional poverty and antibiotic misuse are sparse, and the results are inconsistent. We aimed to investigate the relationship between multidimensional poverty and antibiotic use in patient populations in Kenya, Tanzania, and Uganda. METHODS: In this mixed-methods study, the Holistic Approach to Unravelling Antimicrobial Resistance (HATUA) Consortium collected data from 6827 outpatients (aged 18 years and older, or aged 14-18 years and pregnant) with urinary tract infection (UTI) symptoms in health-care facilities in Kenya, Tanzania, and Uganda. We used Bayesian hierarchical modelling to investigate the association between multidimensional poverty and self-reported antibiotic self-medication and non-adherence (ie, skipping a dose and not completing the course). We analysed linked qualitative in-depth patient interviews and unlinked focus-group discussions with community members. FINDINGS: Between Feb 10, 2019, and Sept 10, 2020, we collected data on 6827 outpatients, of whom 6345 patients had complete data; most individuals were female (5034 [79·2%]), younger than 35 years (3840 [60·5%]), worked in informal employment (2621 [41·3%]), and had primary-level education (2488 [39·2%]). Antibiotic misuse was more common among those least deprived, and lowest among those living in severe multidimensional poverty. Regardless of poverty status, difficulties in affording health care, and more familiarity with antibiotics, were related to more antibiotic misuse. Qualitative data from linked qualitative in-depth patient interviews (n=82) and unlinked focus-group discussions with community members (n=44 groups) suggested that self-medication and treatment non-adherence were driven by perceived inconvenience of the health-care system, financial barriers, and ease of unregulated antibiotic access. INTERPRETATION: We should not assume that higher deprivation drives antibiotic misuse. Structural barriers such as inefficiencies in public health care, combined with time and financial constraints, fuel alternative antibiotic access points and treatment non-adherence across all levels of deprivation. In designing interventions to reduce antibiotic misuse and address antimicrobial resistance, greater attention is required to these structural barriers that discourage optimal antibiotic use at all levels of the socioeconomic hierarchy in LMICs. FUNDING: UK National Institute for Health Research, UK Medical Research Council, and the Department of Health and Social Care.

Pan-Resistome Characterization of Uropathogenic Escherichia coli and Klebsiella pneumoniae Strains Circulating in Uganda and Kenya, Isolated from 2017-2018.

Urinary tract infection (UTI) develops after a pathogen adheres to the inner lining of the urinary tract. Cases of UTIs are predominantly caused by several Gram-negative bacteria and account for high morbidity in the clinical and community settings. Of greater concern are the strains carrying antimicrobial resistance (AMR)-conferring genes. The gravity of a UTI is also determined by a spectrum of other virulence factors. This study represents a pilot project to investigate the burden of AMR among uropathogens in East Africa. We examined bacterial samples isolated in 2017-2018 from in- and out-patients in Kenya (KY) and Uganda (UG) that presented with clinical symptoms of UTI. We reconstructed the evolutionary history of the strains, investigated their population structure, and performed comparative analysis their pangenome contents. We found 55 Escherichia coli and 19 Klebsiella pneumoniae strains confirmed uropathogenic following screening for the prevalence of UTI virulence genes including fimH, iutA, feoA/B/C, mrkD, and foc. We identified 18 different sequence types in E. coli population while all K. pneumoniae strains belong to ST11. The most prevalent E. coli sequence types were ST131 (26%), ST335/1193 (10%), and ST10 (6%). Diverse plasmid types were observed in both collections such as Incompatibility (IncF/IncH/IncQ1/IncX4) and Col groups. Pangenome analysis of each set revealed a total of 2862 and 3464 genes comprised the core genome of E. coli and K. pneumoniae population, respectively. Among these are acquired AMR determinants including fluoroquinolone resistance-conferring genes aac(3)-Ib-cr and other significant genes: aad, tet, sul1, sul2, and cat, which are associated with aminoglycoside, tetracycline, sulfonamide, and chloramphenicol resistance, respectively. Accessory genomes of both species collections were detected several ß-lactamase genes, blaCTX-M, blaTEM and blaOXA, or blaNDM. Overall, 93% are multi-drug resistant in the E. coli collection while 100% of the K. pneumoniae strains contained genes that are associated with resistance to three or more antibiotic classes. Our findings illustrate the abundant acquired resistome and virulome repertoire in uropathogenic E. coli and K. pneumoniae, which are mainly disseminated via clonal and horizontal transfer, circulating in the East African region. We further demonstrate here that routine genomic surveillance is necessary for high-resolution bacterial epidemiology of these important AMR pathogens.

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.

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.

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.

Environmental determinants influencing anthrax distribution in Queen Elizabeth Protected Area, Western Uganda.

Bacillus anthracis, the bacteria that causes anthrax, a disease that primarily affects herbivorous animals, is a soil borne endospore-forming microbe. Environmental distribution of viable spores determines risky landscapes for herbivore exposure and subsequent anthrax outbreaks. Spore survival and longevity depends on suitable conditions in its environment. Anthrax is endemic in Queen Elizabeth Protected Area in western Uganda. Periodic historical outbreaks with significant wildlife losses date to 1950s, but B. anthracis ecological niche in the ecosystem is poorly understood. This study used the Maximum Entropy modeling algorithm method to predict suitable niche and environmental conditions that may support anthrax distribution and spore survival. Model inputs comprised 471 presence-only anthrax occurrence data from park management records of 1956-2010, and 11 predictor variables derived from the World Climatic and Africa Soil Grids online resources, selected considering the ecology of anthrax. The findings revealed predicted suitable niche favoring survival and distribution of anthrax spores as a narrow-restricted corridor within the study area, defined by hot-dry climatic conditions with alkaline soils rich in potassium and calcium. A mean test AUC of 0.94 and predicted probability of 0.93 for anthrax presence were registered. The five most important predictor variables that accounted for 93.8% of model variability were annual precipitation (70.1%), exchangeable potassium (12.6%), annual mean temperature (4.3%), soil pH (3.7%) and calcium (3.1%). The predicted suitable soil properties likely originate from existing sedimentary calcareous gypsum rocks. This has implications for long-term presence of B. anthracis spores and might explain the long history of anthrax experienced in the area. However, occurrence of suitable niche as a restricted hot zone offers opportunities for targeted anthrax surveillance, response and establishment of monitoring strategies in QEPA.

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.

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.