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.

Genomic characterization of methicillin-resistant Staphylococcus aureus isolated from patients attending regional referral hospitals in Tanzania.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) colonization increases the risk of subsequent infection by MRSA strain complex interlinking between hospital and community-acquired MRSA which increases the chance of drug resistance and severity of the disease. OBJECTIVE: Genomic characterization of Staphylococcus aures strains isolated from patients attending regional referral hospitals in Tanzania. METHODOLOGY: A laboratory-based cross-sectional study using short read-based sequencing technology, (Nextseq550,Illumina, Inc. San diego, California, USA). The samples used were collected from patients attending selected regional referral hospitals in Tanzania under the SeqAfrica project. Sequences were analyzed using tools available in the center for genomic and epidemiology server, and visualization of the phylogenetic tree was performed in ITOL 6.0. SPSS 28.0 was used for statistical analysis. RESULTS: Among 103 sequences of S. aureus, 48.5% (50/103) carry the mecA gene for MRSA. High proportions of MRSA were observed among participants aged between 18 and 34 years (52.4%), in females (54.3%), and among outpatients (60.5%). The majority of observed MRSA carried plasmids rep5a (92.0%), rep16 (90.0%), rep7c (90.0%), rep15 (82.0%), rep19 (80.0%) and rep10 (72.0%). Among all plasmids observed rep5a, rep16, rep20, and repUS70 carried the blaZ gene, rep10 carried the erm(C) gene and rep7a carried the tet(K) gene. MLST and phylogeny analysis reveal high diversity among MRSA. Six different clones were observed circulating at selected regional hospitals and MRSA with ST8 was dominant. CONCLUSION: The study reveals a significant presence of MRSA in Staphylococcus aureus strains from Tanzanian regional hospitals, with nearly half carrying the mecA gene. MRSA is notably prevalent among young adults, females, and outpatients, showing high genetic diversity and dominance of ST8. Various plasmids carrying resistance genes indicate a complex resistance profile, highlighting the need for targeted interventions to manage MRSA infections in Tanzania.

Whole genome-based antimicrobial resistance and virulence profiling of Staphylococcus aureus isolates from chronic leg ulcer patients in Kilimanjaro, Tanzania.

BACKGROUND: Chronic leg ulcers are hard to treat and can be a burden, particularly in resource-limited settings where diagnosis is a challenge. Staphylococcus aureus is among the common bacteria isolated from chronic wounds with a great impact on wound healing, particularly in patients with co-morbidities. Antimicrobial resistance genes and virulence factors in Staphylococcus aureus isolates were assessed to support healthcare professionals to make better therapeutic choices, and importantly to curb the development and spread of antibiotic resistance. METHODS: A cross-sectional study involved both inpatients and outpatients with chronic leg ulcers was conducted from August 2022 to April 2023 in 2 health facilities in Kilimanjaro region in Tanzania. Antimicrobial susceptibility testing was done using the disk diffusion method. Further, whole genome sequencing was performed to study the genotypic characteristics of the isolates. RESULTS: A total of 92 participants were recruited in which 9 participants were only positive for 10 Staphylococcus aureus isolates upon culture. Five STs among 9 isolates were identified. Most of them belonged to ST8 (44%), with 1 isolate does not belong to any ST. Additionally, 50% of the isolates were methicillin-resistant Staphylococcus aureus (MRSA). All S. aureus isolates had almost similar virulence factors such as hemolysin, proteases and evasions that promote toxin production, protease production and host immune evasion respectively. Moreover, all mecA positive S. aureus isolates were phenotypically susceptible to cefoxitin. CONCLUSION: Presence of mecA positive S. aureus isolates which are also phenotypically susceptible to cefoxitin implies the possibility of classifying MRSA as MSSA. This may result in the possible emergence of highly cefoxitin - resistant strains in health care and community settings when subsequently exposed to beta-lactam agents. Therefore, combination of whole genome sequencing and conventional methods is important in assessing bacterial resistance and virulence to improve management of patients.

Status and future prospects for mobile phone-enabled diagnostics in Tanzania.

INTRODUCTION: Diagnosis is a key step towards the provision of medical intervention and saving lives. However, in low- and middle-income countries, diagnostic services are mainly centralized in large cities and are costly. Point of care (POC) diagnostic technologies have been developed to fill the diagnostic gap for remote areas. The linkage of POC testing onto smartphones has leveraged the ever-expanding coverage of mobile phones to enhance health services in low- and middle-income countries. Tanzania, like most other middle-income countries, is poised to adopt and deploy the use of mobile phone-enabled diagnostic devices. However, there is limited information on the situation on the ground with regard to readiness and capabilities of the veterinary and medical professionals to make use of this technology. METHODS: In this study we survey awareness, digital literacy and prevalent health condition to focus on in Tanzania to guide development and future implementation of mobile phoned-enable diagnostic tools by veterinary and medical professionals. Data was collected using semi-structured questionnaire with closed and open-ended questions, guided in-depth interviews and focus group discussion administered to the participants after informed consent was obtained. RESULTS: A total of 305 participants from six regions of Tanzania were recruited in the study. The distribution of participants across the six regions was as follows: Kilimanjaro (37), Arusha (31), Tabora (68), Dodoma (61), Mwanza (58), and Iringa (50). Our analysis reveals that only 48.2% (126/255) of participants demonstrated significant awareness of mobile phone-enabled diagnostics. This awareness varies significantly across age groups, professions and geographical locations. Interestingly, while 97.4% of participants own and can operate a smartphone, 62% have never utilized their smartphones for health services, including disease diagnosis. Regarding prevalent health condition to focus on when developing mobile phone -enabled diagnostics tools for Tanzania; there was disparity between medical and veterinary professionals. For medical professionals the top 4 priority diseases were Malaria, Urinary Tract Infections, HIV and Diabetes, while for veterinary professionals they were Brucellosis, Anthrax, Newcastle disease and Rabies. DISCUSSION: Despite the widespread ownership of smartphones among healthcare providers (both human and animal), only a small proportion have utilized these devices for healthcare practices, with none reported for diagnostic purposes. This limited utilization may be attributed to factors such as a lack of awareness, absence of policy guidelines, limited promotion, challenges related to mobile data connectivity, and adherence to cultural practices. CONCLUSION: The majority of medical and veterinary professionals in Tanzania possess the necessary digital literacy to utilize mobile phone-enabled diagnostics and demonstrate readiness to adopt digital technologies and innovations to enhance diagnosis. However, effective implementation will require targeted training and interventions to empower them to effectively apply such innovations for disease diagnosis and other healthcare applications.

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.