Short reads-based characterization of pathotype diversity and drug resistance among Escherichia coli isolated from patients attending regional referral hospitals in Tanzania.

BACKGROUND: Escherichia coli is known to cause about 2 million deaths annually of which diarrhea infection is leading and typically occurs in children under 5 years old. Although Africa is the most affected region there is little information on their pathotypes diversity and their antimicrobial resistance. OBJECTIVE: To determine the pathotype diversity and antimicrobial resistance among E. coli from patients attending regional referral hospitals in Tanzania. MATERIALS AND METHODS: A retrospective cross-section laboratory-based study where a total of 138 archived E. coli isolates collected from 2020 to 2021 from selected regional referral hospitals in Tanzania were sequenced using the Illumina Nextseq550 sequencer platform. Analysis of the sequences was done in the CGE tool for the identification of resistance genes and virulence genes. SPSS version 20 was used to summarize data using frequency and proportion. RESULTS: Among all 138 sequenced E. coli isolates, the most prevalent observed pathotype virulence genes were of extraintestinal E. coli UPEC fyuA gene 82.6% (114/138) and NMEC irp gene 81.9% (113/138). Most of the E. coli pathotypes observed exist as a hybrid due to gene overlapping, the most prevalent pathotypes observed were NMEC/UPEC hybrid 29.7% (41/138), NMEC/UPEC/EAEC hybrid 26.1% (36/138), NMEC/UPEC/DAEC hybrid 18.1% (25/138) and EAEC 15.2% (21/138). Overall most E. coli carried resistance gene to ampicillin 90.6% (125/138), trimethoprim 85.5% (118/138), tetracycline 79.9% (110/138), ciprofloxacin 76.1% (105/138) and 72.5% (100/138) Nalidixic acid. Hybrid pathotypes were more resistant than non-hybrid pathotypes. CONCLUSION: Whole genome sequencing reveals the presence of hybrid pathotypes with increased drug resistance among E. coli isolated from regional referral hospitals in Tanzania.

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.

Nanopore sequencing technology for clinical diagnosis of infectious diseases where laboratory capacity is meager: A case report.

In resource-limited settings, patients are often first presented to clinical settings when seriously ill and access to proper clinical microbial diagnostics is often very limited or non-existing. On February 16th, 2022 we were on a field trip to test a completely field-deployable metagenomics sequencing set-up, that includes DNA purification, sequencing, and bioinformatics analyses using bioinformatics tools installed on a laptop for water samples, just outside Moshi, Tanzania. On our way to the test site, we were contacted by the nearby Machame hospital regarding a child seriously ill with diarrhea and not responding to treatment. Within the same day, we conducted an onsite metagenomics examination of a fecal sample from the child, and Campylobacter jejuni was identified as the causative agent. The treatment was subsequently changed, with almost immediate improvement, and the child was discharged on February 21st.

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.

Unlocking the health system barriers to maximise the uptake and utilisation of molecular diagnostics in low-income and middle-income country setting.

BACKGROUND: Early access to diagnosis is crucial for effective management of any disease including tuberculosis (TB). We investigated the barriers and opportunities to maximise uptake and utilisation of molecular diagnostics in routine healthcare settings. METHODS: Using the implementation of WHO approved TB diagnostics, Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) and Line Probe Assay (LPA) as a benchmark, we evaluated the barriers and how they could be unlocked to maximise uptake and utilisation of molecular diagnostics. RESULTS: Health officers representing 190 districts/counties participated in the survey across Kenya, Tanzania and Uganda. The survey findings were corroborated by 145 healthcare facility (HCF) audits and 11 policy-maker engagement workshops. Xpert MTB/RIF coverage was 66%, falling behind microscopy and clinical diagnosis by 33% and 1%, respectively. Stratified by HCF type, Xpert MTB/RIF implementation was 56%, 96% and 95% at district, regional and national referral hospital levels. LPA coverage was 4%, 3% below culture across the three countries. Out of 111 HCFs with Xpert MTB/RIF, 37 (33%) used it to full capacity, performing ≥8 tests per day of which 51% of these were level five (zonal consultant and national referral) HCFs. Likewise, 75% of LPA was available at level five HCFs. Underutilisation of Xpert MTB/RIF and LPA was mainly attributed to inadequate-utilities, 26% and human resource, 22%. Underfinancing was the main reason underlying failure to acquire molecular diagnostics. Second to underfinancing was lack of awareness with 33% healthcare administrators and 49% practitioners were unaware of LPA as TB diagnostic. Creation of a national health tax and decentralising its management was proposed by policy-makers as a booster of domestic financing needed to increase access to diagnostics. CONCLUSION: Our findings suggest higher uptake and utilisation of molecular diagnostics at tertiary level HCFs contrary to the WHO recommendation. Country-led solutions are crucial for unlocking barriers to increase access to diagnostics.

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.

Boosting effect of IL-7 in interferon gamma release assays to diagnose Mycobacterium tuberculosis infection.

BACKGROUND: A quarter of the world's population is estimated to be infected with Myobacterium tuberculosis (Mtb). Infection is detected by immune response to M. tuberculosis antigens using either tuberculin skin test (TST) and interferon gamma release (IGRA's), tests which have low sensitivity in immunocompromised. IL-7 is an important cytokine for T-cell function with potential to augment cytokine release in in-vitro assays. This study aimed to determine whether the addition of IL-7 in interferon-gamma release assays (IGRAs) improves its diagnostic performance of Mtb infection. METHODS: 44 cases with confirmed TB and 45 household contacts without TB were recruited and 1ml of blood was stimulated in two separate IGRA's tube set: one set of standard Quantiferon TB gold tubes mitogen, TB antigen and TB Nil; one set of customized Quantiferon TB gold tubes with added IL-7. Following IFN-γ and IP-10 release was determined using ELISA. RESULTS: We found that the addition of IL-7 led to significantly higher release of IFN-γ in individuals with active TB from 4.2IU/ml (IQR 1.4-6.9IU/ml) to 5.1IU/ml (IQR 1.5-8.1IU/ml, p = 0.0057), and we found an indication of a lower release of both IFN-γ and IP-10 in participants with negative tests. CONCLUSIONS: In TB cases addition of IL-7 in IGRA tubes augments IFN-γ but not IP-10 release, and seems to lower the response in controls. Whether IL-7 boosted IGRA holds potential over standard IGRA needs to be confirmed in larger studies in high and low TB incidence countries.

Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6.

There is a need for an improved vaccine for tuberculosis. ESAT-6 is a cardinal vaccine antigen with unique properties and is included in several vaccine candidates in development. ESAT-6 is also the core antigen in the IFN-γ release assays (IGRA) used to diagnose latent infection, rendering IGRA tests unspecific after vaccination. This challenge has prompted the development of a companion diagnostic for ESAT-6 based vaccines, an ESAT-6 free IGRA. We screened a panel of seven potential new diagnostic antigens not recognized in BCG vaccinated individuals. Three highly recognized antigens EspC, EspF and Rv2348c were identified and combined with CFP10 in an ESAT-6 free antigen cocktail. The cocktail was prepared in a field-friendly format, lyophilized with heparin in ready-to-use vacutainer tubes. The diagnostic performance of the ESAT-6 free IGRA was determined in a cross-validation study. Compared IGRA, the ESAT-6 free IGRA induced a comparable magnitude of IFN-γ release, and the diagnostic performance was on par with Quantiferon (sensitivity 84% vs 79%; specificity 99% vs 97%). The comparable performance of the ESAT-6 free IGRA to IGRA suggests potential as companion diagnostic for ESAT-6 containing vaccines and as adjunct test for latent infection.