Tuberculosis Molecular Bacterial Load Assay Reveals Early Delayed Bacterial Killing in Patients With Relapse.

Bacterial killing in patients with tuberculosis (TB) relapse was compared to that in patients achieving cure, measured by TB molecular bacterial load assay (TB-MBLA) or mycobacteria growth indicator tube (MGIT) time to positivity (TTP). TB-MBLA in 4 relapsed patients was significantly different compared to 132 cured patients after 2 weeks of treatment; MGIT TTP showed a significant difference from week 8.

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).

Mycobactericidal Effects of Different Regimens Measured by Molecular Bacterial Load Assay among People Treated for Multidrug-Resistant Tuberculosis in Tanzania.

Rifampin or multidrug-resistant tuberculosis (RR/MDR-TB) treatment has largely transitioned to regimens free of the injectable aminoglycoside component, despite the drug class' purported bactericidal activity early in treatment. We tested whether Mycobacterium tuberculosis killing rates measured by tuberculosis molecular bacterial load assay (TB-MBLA) in sputa correlate with composition of the RR/MDR-TB regimen. Serial sputa were collected from patients with RR/MDR- and drug-sensitive TB at days 0, 3, 7, and 14, and then monthly for 4 months of anti-TB treatment. TB-MBLA was used to quantify viable M. tuberculosis 16S rRNA in sputum for estimation of colony forming units per ml (eCFU/ml). M. tuberculosis killing rates were compared among regimens using nonlinear-mixed-effects modeling of repeated measures. Thirty-seven patients produced 296 serial sputa and received treatment as follows: 13 patients received an injectable bedaquiline-free reference regimen, 9 received an injectable bedaquiline-containing regimen, 8 received an all-oral bedaquiline-based regimen, and 7 patients were treated for drug-sensitive TB with conventional rifampin/isoniazid/pyrazinamide/ethambutol (RHZE). Compared to the adjusted M. tuberculosis killing of -0.17 (95% confidence interval [CI] -0.23 to -0.12) for the injectable bedaquiline-free reference regimen, the killing rates were -0.62 (95% CI -1.05 to -0.20) log10 eCFU/ml for the injectable bedaquiline-containing regimen (P = 0.019), -0.35 (95% CI -0.65 to -0.13) log10 eCFU/ml for the all-oral bedaquiline-based regimen (P = 0.054), and -0.29 (95% CI -0.78 to +0.22) log10 eCFU/ml for the RHZE regimen (P = 0.332). Thus, M. tuberculosis killing rates from sputa were higher among patients who received bedaquiline but were further improved with the addition of an injectable aminoglycoside.

The global prevalence and trend of human intestinal carriage of ESBL-producing Escherichia coli in the community.

OBJECTIVES: Intestinal colonization by ESBL Escherichia coli and its association with community-acquired MDR infections is of great concern. This review determined the worldwide prevalence of human faecal ESBL E. coli carriage and its trend in the community over the past two decades. METHODS: A systematic literature search was conducted using PubMed, EMBASE and Google Scholar to retrieve articles published between 1 January 2000 and 13 February 2020 that contained data on the prevalence of faecal carriage of ESBL E. coli among healthy individuals. A cumulative (for the whole period) meta-analysis was used to estimate the global and regional pooled prevalence rates. Articles were grouped into study periods of 3 years, and subgroup meta-analyses were undertaken to examine the global pooled prevalence over time. RESULTS: Sixty-two articles covering 29 872 healthy persons were included in this meta-analysis. The cumulative (2003-18) global pooled prevalence of ESBL E. coli intestinal carriage in the community was 16.5% (95% CI 14.3%-18.7%; P  <  0.001). The pooled prevalence showed an upward trend, increasing from 2.6% (95% CI 1.6%-4.0%) in 2003-05 to 21.1% (95% CI 15.8%-27.0%) in 2015-18. Over the whole period, the highest carriage rate was observed in South-East Asia (27%; 95% CI 2.9%-51.3%), while the lowest occurred in Europe (6.0%; 95% CI 4.6%-7.5%). CONCLUSIONS: Globally, an 8-fold increase in the intestinal carriage rate of ESBL E. coli in the community has occurred over the past two decades. Prevention of its spread may require new therapeutic and public health strategies.

Tuberculosis bacillary load, an early marker of disease severity: the utility of tuberculosis Molecular Bacterial Load Assay.

In this comparative biomarker study, we analysed 1768 serial sputum samples from 178 patients at 4 sites in Southeast Africa. We show that tuberculosis Molecular Bacterial Load Assay (TB-MBLA) reduces time-to-TB-bacillary-load-result from days/weeks by culture to hours and detects early patient treatment response. By day 14 of treatment, 5% of patients had cleared bacillary load to zero, rising to 58% by 12th week of treatment. Fall in bacillary load correlated with mycobacterial growth indicator tube culture time-to-positivity (Spearmans r=-0.51, 95% CI (-0.56 to -0.46), p<0.0001). Patients with high pretreatment bacillary burdens (above the cohort bacillary load average of 5.5log10eCFU/ml) were less likely to convert-to-negative by 8th week of treatment than those with a low burden (below cohort bacillary load average), p=0.0005, HR 3.1, 95% CI (1.6 to 5.6) irrespective of treatment regimen. TB-MBLA distinguished the bactericidal effect of regimens revealing the moxifloxacin-20 mg rifampicin regimen produced a shorter time to bacillary clearance compared with standard-of-care regimen, p=0.008, HR 2.9, 95% CI (1.3 to 6.7). Our data show that the TB-MBLA could inform clinical decision making in real-time and expedite drug TB clinical trials.

Model-Based Relationship between the Molecular Bacterial Load Assay and Time to Positivity in Liquid Culture.

The molecular bacterial load (MBL) assay is a new tuberculosis biomarker which provides results in ∼4 hours. The relationship between MBL and time-to-positivity (TTP) has not been thoroughly studied, and predictive models do not exist. We aimed to develop a model for MBL and identify the MBL-TTP relationship in patients. The model was developed on data from 105 tuberculosis patients from Malawi, Mozambique, and Tanzania with joint MBL and TTP observations quantified from patient sputum collected for 12 weeks. MBL was quantified using PCR of mycobacterial RNA and TTP using the mycobacterial growth indicator tube (MGIT) 960 system. Treatment consisted of isoniazid, pyrazinamide, and ethambutol in standard doses together with rifampin 10 or 35 mg/kg of body weight. The developed MBL-TTP model included several linked submodels, a component describing decline of bacterial load in sputum, another component describing growth in liquid culture, and a hazard model translating bacterial growth into a TTP signal. Additional components for contaminated and negative TTP samples were included. Visual predictive checks performed using the developed model gave good description of the observed data. The model predicted greater total sample loss for TTP than MBL due to contamination and negative samples. The model detected an increase in bacterial killing for 35 versus 10 mg/kg rifampin (P = 0.002). In conclusion, a combined model for MBL and TTP was developed that described the MBL-TTP relationship. The full MBL-TTP model or each submodel was used separately. Second, the model can be used to predict biomarker response for MBL given TTP data or vice versa in historical or future trials.

Heat Inactivation Renders Sputum Safe and Preserves Mycobacterium tuberculosis RNA for Downstream Molecular Tests.

The World Health Organization End Tuberculosis (TB) strategy has called for the development of-and increased access to-effective tools for diagnosis and treatment of TB disease. Mycobacterium tuberculosis , the causative agent of TB, is categorized as a highly infectious agent. Consequently, diagnostic tests that involve comprehensive manipulation of specimens from presumed tuberculosis cases must be performed in a category 3 laboratory. We have evaluated the use of heat inactivation to render TB samples safe to work with while preserving RNA for downstream molecular tests. Using Mycobacterium bovis bacillus Calmette-Guérin (BCG) cultures and TB-positive sputum samples, we show that boiling for 20 min at 80, 85, and 95°C inactivates all M. tuberculosis bacilli. The efficiency of inactivation was verified by culturing heat-treated and untreated (live) fractions of BCG and TB sputum samples for 42 days. No growth was observed in the cultures of heat-treated samples. In contrast, the optical density of untreated BCG in Middlebrook 7H9 broth rose from 0.04 to 0.85, and the untreated sputum samples flagged positive at 3 days of incubation in mycobacterial growth indicator tubes. Quantification of reference genes 16S rRNA, transfer-messenger RNA (tmRNA), pre-16S rRNA, and rpoB by reverse transcriptase quantitative PCR (RT-qPCR) showed minimal loss in estimated bacterial load. The loss was RNA species dependent, <1 log10, 1.1 log10, 1.3 log10, and 2.4 log10 estimated CFU/ml for 16S rRNA, tmRNA, pre-16S rRNA, and rpoB, respectively. The RNA loss was independent of inactivation temperature. These findings show that heat inactivation could obviate the need for category 3 laboratories to perform RNA-based testing of TB samples.

Molecular Bacterial Load Assay Concurs with Culture on NaOH-Induced Loss of Mycobacterium tuberculosis Viability.

Effective methods to detect viable Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB), are urgently needed. To date, cultivation of M. tuberculosis is the gold standard, which depends on initial sample processing with N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH), chemicals that compromise M. tuberculosis viability and, consequently, the performance of downstream tests. We applied culture and the novel molecular bacterial load assay (MBLA) to measure the loss of M. tuberculosis viability following NALC-NaOH treatment of M. tuberculosis H37Rv pure culture and clinical sputum samples from pulmonary TB patients. Compared to the bacterial loads of untreated controls, NALC-NaOH treatment of M. tuberculosis reduced the MBLA-detectable bacillary load (estimated number of CFU [eCFU] per milliliter) by 0.66 ± 0.21 log10 at 23°C (P = 0.018) and 0.72 ± 0.08 log10 at 30°C (P = 0.013). Likewise, NALC-NaOH treatment reduced the viable count on solid culture by 0.84 ± 0.02 log10 CFU/ml at 23°C (P < 0.001) and 0.85 ± 0.01 log10 CFU/ml at 30°C (P < 0.001), respectively. The reduction in the viable count was reflected by a corresponding increase in the time to positivity of the mycobacterial growth indicator tube (MGIT) liquid culture: 1.2 days at 23°C (P < 0.001) and 1.1 days at 30°C (P < 0.001). This NaOH-induced M. tuberculosis viability loss was replicated in clinical sputum samples, with the bacterial load dropping by 0.65 ± 0.17 log10 from 5.36 ± 0.24 log10 eCFU/ml to 4.71 ± 0.16 log10 eCFU/ml for untreated and treated sputa, respectively. Applying the model of Bowness et al. (R. Bowness, M. J. Boeree, R. Aarnoutse, R. Dawson, et al., J Antimicrob Chemother 70:448-455, 2015, https://doi.org/10.1093/jac/dku415) revealed that the treated MGIT time to culture positivity of 142 ± 7.02 h was equivalent to 4.86 ± 0.28 log10 CFU, consistent with the MBLA-measured bacterial load. Our study confirms the contribution of NALC-NaOH treatment to the loss of viable bacterial counts. Tests that obviate the need for decontamination may offer an alternative option for the accurate detection of viable M. tuberculosis and treatment response monitoring.

Quantifying Viable M. tuberculosis Safely Obviating the Need for High Containment Facilities.

Mycobacterium tuberculosis is an infectious pathogen that requires biosafety level-3 laboratory for handling. The risk of transmission is high to laboratory staff, and to manage the organism safely, it is necessary to construct high containment laboratory facilities at great expense. This limits the application of tuberculosis diagnostics to areas where there is insufficient capital to invest in laboratory infrastructure. In this method, we describe a process of inactivating sputum samples by either heat or guanidine thiocyanate (GTC) that renders them safe without affecting the quantification of viable bacteria. This method eliminates the need for level 3 containment laboratory for the tuberculosis molecular bacterial load assay (TB-MBLA) and is applicable in low- and middle-income countries.

Improved Diagnosis and Treatment Monitoring of Tuberculosis Using Stool and the Tuberculosis Bacterial Load Assay (TB-MBLA).

The diagnosis and monitoring of tuberculosis treatment is difficult as many patients are unable to produce sputum. This means that many patients are treated on the basis of clinical findings and consequently some will be exposed to anti-tuberculosis drugs unnecessarily. Moreover, for those appropriately on treatment and unable to produce a sputum sample, it will be impossible to monitor the response to treatment. We have shown that stool is a potential alternative sample type for diagnosis of tuberculosis. Currently, available protocols like the Xpert MTB/RIF use DNA as a target to detect Mycobacterium tuberculosis in stool but DNA survives long after the organism is dead so it is not certain whether a positive test is from an old or a partially treated infection. The TB MBLA only detects live organisms and thus, can be used to follow the response to treatment. In this chapter, we describe a protocol for TB-MBLA, an RNA-based assay, and apply it to quantify TB bacteria in stool.

Suppression of host gene expression is associated with latent TB infection: a possible diagnostic biomarker.

The World Health Organization End TB strategy aims for a 90% reduction of tuberculosis (TB) incidence by 2035. Systematic testing and treatment of latent TB infection (LTBI) among contacts of active TB patients is recommended as one of the ways to curtail TB incidence. However, there is a shortage of tools to accurately diagnose LTBI. We assessed the appropriateness of whole blood host transcriptomic markers (TM) to diagnose LTBI among household contacts of bacteriologically confirmed index cases compared to HIV negative healthy controls (HC). QuantiFERON-TB Gold Plus Interferon gamma release assay (IGRA) and reverse-transcriptase quantitative PCR were used to determine LTBI and quantify TM expression respectively. Association between TM expression and LTBI was evaluated by logistic regression modelling. A total of 100 participants, 49 TB exposed (TBEx) household contacts and 51 HC, were enrolled. Twenty-five (51%) TBEx individuals tested positive by IGRA, and were denoted as LTBI individuals, and 37 (72.5%) HC were IGRA-negative. Expression of 11 evaluated TM was significantly suppressed among LTBI compared to HC. Out of the 11 TM, ZNF296 and KLF2 expression were strongly associated with LTBI and successfully differentiated LTBI from HC. Paradoxically, 21 (49%) TBEx participants who tested IGRA negative exhibited the same pattern of suppressed TM expression as IGRA positive (LTBI-confirmed individuals). Results suggest that suppression of gene expression underlies LTBI and may be a more sensitive diagnostic biomarker than standard-of-care IGRA.

Developing biomarker assays to accelerate tuberculosis drug development: defining target product profiles.

Drug development for tuberculosis is hindered by the methodological limitations in the definitions of patient outcomes, particularly the slow organism growth and difficulty in obtaining suitable and representative samples throughout the treatment. We developed target product profiles for biomarker assays suitable for early-phase and late-phase clinical drug trials by consulting subject-matter experts on the desirable performance and operational characteristics of such assays for monitoring of tuberculosis treatment in drug trials. Minimal and optimal criteria were defined for scope, intended use, pricing, performance, and operational characteristics of the biomarkers. Early-stage trial assays should accurately quantify the number of viable bacilli, whereas late-stage trial assays should match the number, predict relapse-free cure, and replace culture conversion endpoints. The operational criteria reflect the infrastructure and resources available for drug trials. The effective tools should define the sterilising activity of the drug and lower the probability of treatment failure or relapse in people with tuberculosis. The target product profiles outlined in this Review should guide and de-risk the development of biomarker-based assays suitable for phase 2 and 3 clinical drug trials.

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.

Accuracy of the tuberculosis molecular bacterial load assay to diagnose and monitor response to anti-tuberculosis therapy: a longitudinal comparative study with standard-of-care smear microscopy, Xpert MTB/RIF Ultra, and culture in Uganda.

BACKGROUND: In 2018, the tuberculosis molecular bacterial load assay (TB-MBLA), a ribosomal RNA-based test, was acknowledged by WHO as a molecular assay that could replace smear microscopy and culture for monitoring tuberculosis treatment response. In this study, we evaluated the accuracy of TB-MBLA for diagnosis and monitoring of treatment response in comparison with standard-of-care tests. METHODS: For this longitudinal prospective study, patients aged 18 years or older with presumptive tuberculosis (coughing for at least 2 weeks, night sweats, and weight loss) were enrolled at China-Uganda Friendship Hospital Naguru (Kampala, Uganda). Participants were evaluated for tuberculosis by TB-MBLA in comparison with Xpert MTB/RIF Ultra (Xpert-Ultra) and smear microscopy, with Mycobacteria Growth Indicator Tube (MGIT) culture as a reference test. Participants who were positive on Xpert-Ultra were enrolled on a standard 6-month anti-tuberculosis regimen, and monitored for treatment response at weeks 2, 8, 17, and 26 after initiation of treatment and then 3 months after treatment. FINDINGS: Between Nov 15, 2019, and June 15, 2022, 210 participants (median age 35 years [IQR 27-44]) were enrolled. 135 (64%) participants were male and 72 (34%) were HIV positive. The pretreatment diagnostic sensitivities of TB-MBLA and Xpert-Ultra were similar (both 99% [95% CI 95-100]) but the specificity was higher for TB-MBLA (90% [83-96]) than for Xpert-Ultra (78% [68-86]). Ten participants were Xpert-Ultra trace positive, eight (80%) of whom were negative by TB-MBLA and MGIT culture. Smear microscopy had lower diagnostic sensitivity (75% [65-83]) but higher specificity (98% [93-100]) than TB-MBLA and Xpert-Ultra. Among participants who were smear microscopy negative, the sensitivity of TB-MBLA was 96% (95 CI 80-100) and was 100% (95% CI 86-100) in those who were HIV positive. 129 (61%) participants were identified as tuberculosis positive by Xpert-Ultra and these individuals were enrolled in the treatment group and monitored for treatment response. According to TB-MBLA, 19 of these patients cleared bacillary load to zero by week 2 of treatment and remained negative throughout the 6-month treatment follow-up. Positivity for tuberculosis decreased with treatment as measured by all tests, but the rate was slower with Xpert-Ultra. Consequently, 31 (33%) of 95 participants were still Xpert-Ultra positive at the end of treatment but were clinically well and negative on TB-MBLA and culture at 6 months of treatment. Two patients were still Xpert-Ultra positive with a further 3 months of post-treatment follow-up. The rate of conversion to negative of the DNA-based Xpert-Ultra was 3·3-times slower than that of the rRNA-based TB-MBLA. Consequently for the same patient, it would take 13 weeks and 52 weeks to reach complete tuberculosis negativity by TB-MBLA and Xpert-Ultra, respectively. Participants who were positive on smear microscopy at 8 weeks, who received an extra month of intensive treatment, had a similar TB-MBLA-measured bacillary load at 8 weeks to those who were smear microscopy negative. INTERPRETATION: TB-MBLA has a similar performance to Xpert-Ultra for pretreatment diagnosis of tuberculosis, but is more accurate at detecting and characterising the response to treatment than Xpert-Ultra and standard-of-care smear microscopy. FUNDING: European and Developing Countries Clinical Trials Partnership, Makerere University Research and Innovation Fund, US National Institutes of Health.

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.

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.

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.

Localization and phenotyping of tuberculosis bacteria using a combination of deep learning and SVMs.

Successful treatment of pulmonary tuberculosis (TB) depends on early diagnosis and careful monitoring of treatment response. Identification of acid-fast bacilli by fluorescence microscopy of sputum smears is a common tool for both tasks. Microscopy-based analysis of the intracellular lipid content and dimensions of individual Mycobacterium tuberculosis (Mtb) cells also describe phenotypic changes which may improve our biological understanding of antibiotic therapy for TB. However, fluorescence microscopy is a challenging, time-consuming and subjective procedure. In this work, we automate examination of fields of view (FOVs) from microscopy images to determine the lipid content and dimensions (length and width) of Mtb cells. We introduce an adapted variation of the UNet model to efficiently localising bacteria within FOVs stained by two fluorescence dyes; auramine O to identify Mtb and LipidTox Red to identify intracellular lipids. Thereafter, we propose a feature extractor in conjunction with feature descriptors to extract a representation into a support vector multi-regressor and estimate the length and width of each bacterium. Using a real-world data corpus from Tanzania, the proposed method i) outperformed previous methods for bacterial detection with a 8% improvement (Dice coefficient) and ii) estimated the cell length and width with a root mean square error of less than 0.01%. Our network can be used to examine phenotypic characteristics of Mtb cells visualised by fluorescence microscopy, improving consistency and time efficiency of this procedure compared to manual methods.

A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory.

BACKGROUND: Mycobacterium tuberculosis is a category B infectious pathogen requiring level-3-containment laboratories for handling. We assessed the efficacy of heat and Guanidine thiocyanate (GTC) to inactivate M. tuberculosis prior to performance of tuberculosis Molecular Bacterial Load Assay (TB-MBLA). METHOD: We performed in vitro experiments using M.tb, H37Rv reference strain and replicated in sputum specimens. A 0.5 MacFarland standard of M. tuberculosis was serially diluted to 1x101 CFU/mL and pooled sputum was homogenised prior to serial dilutions and Xpert MTB/RIF Ultra. Three replicates for each containing 1 mL for M. tuberculosis and sputum were inactivated at 80 °C for 20 min and with GTC for 15 min. Inactivated samples were processed for culture and TB-MBLA. RESULTS: No M. tuberculosis growth was observed in MGIT for GTC or heat treated H37Rv cultures. All untreated H37Rv dilutions were MGIT positive except the most diluted specimens. Heat and GTC treatment of H37Rv reduced TB-MBLA load by 2.1log10 (P = 0.7) and 1.8log10 (P = 0.7) respectively, compared to controls. In contrast, heat treated sputum had TB-MBLA bacterial load of 3.47 ± 3.53 log10 compared to 5.4 ± 3.1 log10 eCFU/mL for GTC (p = 0.57). All heat and GTC treated sputum were culture negative. CONCLUSION: Heat or GTC renders M. tuberculosis non-viable and eliminates the need for BSL3 laboratory for performing TB-MBLA in routine healthcare settings.

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.