Antimicrobial resistance and heterogeneity of Neisseria gonorrhoeae isolated from patients attending sexually transmitted infection clinics in Lusaka, Zambia.

BACKGROUND: Antimicrobial resistance (AMR) of Neisseria gonorrhoeae is a threat to public health as strains have developed resistance to antimicrobials available for the treatment of gonorrhea. Whole genome sequencing (WGS) can detect and predict antimicrobial resistance to enhance the control and prevention of gonorrhea. Data on the molecular epidemiology of N. gonorrhoeae is sparse in Zambia. This study aimed to determine the genetic diversity of N. gonorrhoeae isolated from patients attending sexually transmitted infection (STI) clinics in Lusaka, Zambia. METHODS: A cross-sectional study that sequenced 38 N. gonorrhoeae isolated from 122 patients with gonorrhea from 2019 to 2020 was conducted. The AMR profiles were determined by the E-test, and the DNA was extracted using the NucliSens easyMaG magnetic device. Whole genome sequencing was performed on the Illumina NextSeq550 platform. The Bacterial analysis pipeline (BAP) that is readily available at: https://cge.cbs.dtu.dk/services/CGEpipeline-1.1 was used for the identification of the species, assembling the genome, multi-locus sequence typing (MLST), detection of plasmids and AMR genes. Phylogeny by single nucleotide polymorphisms (SNPs) was determined with the CCphylo dataset. RESULTS: The most frequent STs with 18.4% of isolates each were ST7363, ST1921 and ST1582, followed by ST1583 (13%), novel ST17026 (7.9%), ST1588 (7.9%), ST1596 (5.3%), ST11181 (5.3%), ST11750 (2.6/%) and ST11241 (2.6%) among the 38 genotyped isolates. The blaTeM-1B and tetM (55%) was the most prevalent combination of AMR genes, followed by blaTeM-1B (18.4%), tetM (15.8%), and the combination of blaTeM-1B, ermT, and tetL was 2.6% of the isolates. The AMR phenotypes were predicted in ciprofloxacin, penicillin, tetracycline, azithromycin, and cefixime. The combination of mutations 23.7% was gryA (S91F), parC (E91G), ponA (L421) and rpsJ (V57M), followed by 18.4% in gyrA (S91F), ponA (L421P), rpsJ (V57M), and 18.4% in gyrA (D95G, S91F), ponA (L421P), and rpsJ (V57M). The combinations in gyrA (D95G, S91F) and rpsJ (V57M), and gyrA (D95G, S91F), parC (E91F), ponA (L421P) and rpsJ (V57M) were 13.2% each of the isolates. Plasmid TEM-1 (84.2%), tetM (15.8%), and gonococcal genetic island (GGI) was detected in all isolates. CONCLUSION: This study revealed remarkable heterogeneity of N. gonorrhoeae with blaTEM-1, tetM, ponA, gyrA, and parC genes associated with high resistance to penicillin, tetracycline, and ciprofloxacin demanding revision of the standard treatment guidelines and improved antimicrobial stewardship in Zambia.

Using WGS to identify antibiotic resistance genes and predict antimicrobial resistance phenotypes in MDR Acinetobacter baumannii in Tanzania.

BACKGROUND: Reliable phenotypic antimicrobial susceptibility testing can be a challenge in clinical settings in low- and middle-income countries. WGS is a promising approach to enhance current capabilities. AIM: To study diversity and resistance determinants and to predict and compare resistance patterns from WGS data of Acinetobacter baumannii with phenotypic results from classical microbiological testing at a tertiary care hospital in Tanzania. METHODS AND RESULTS: MLST using Pasteur/Oxford schemes yielded eight different STs from each scheme. Of the eight, two STs were identified to be global clones 1 (n = 4) and 2 (n = 1) as per the Pasteur scheme. Resistance testing using classical microbiology determined between 50% and 92.9% resistance across all drugs. Percentage agreement between phenotypic and genotypic prediction of resistance ranged between 57.1% and 100%, with coefficient of agreement (κ) between 0.05 and 1. Seven isolates harboured mutations at significant loci (S81L in gyrA and S84L in parC). A number of novel plasmids were detected, including pKCRI-309C-1 (219000 bp) carrying 10 resistance genes, pKCRI-43-1 (34935 bp) carrying two resistance genes and pKCRI-49-1 (11681 bp) and pKCRI-28-1 (29606 bp), each carrying three resistance genes. New ampC alleles detected included ampC-69, ampC-70 and ampC-71. Global clone 1 and 2 isolates were found to harbour ISAba1 directly upstream of the ampC gene. Finally, SNP-based phylogenetic analysis of the A. baumannii isolates revealed closely related isolates in three clusters. CONCLUSIONS: The validity of the use of WGS in the prediction of phenotypic resistance can be appreciated, but at this stage is not sufficient for it to replace conventional antimicrobial susceptibility testing in our setting.

Exploring the Role of IL-32 in HIV-Related Kaposi Sarcoma.

The intracellular proinflammatory mediator IL-32 is associated with tumor progression; however, the mechanisms remain unknown. We studied IL-32 mRNA expression as well as expression of other proinflammatory cytokines and mediators, including IL-1α, IL-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, the proangiogenic and antiapoptotic enzyme cyclooxygenase-2, the IL-8 receptor C-X-C chemokine receptor (CXCR) 1, and the intracellular kinase focal adhesion kinase-1. The interaction of IL-32 expression with expression of IL-6, TNF-α, IL-8, and cyclooxygenase-2 was also investigated. Biopsy specimens of 11 HIV-related, 7 non-HIV-related Kaposi sarcoma (KS), and 7 normal skin tissues (NSTs) of Dutch origin were analyzed. RNA was isolated from the paraffin material, and gene expression levels of IL-32 α, ß, and γ isoforms, IL1a, IL1b, IL6, IL8, TNFA, PTGS2, CXCR1, and PTK2 were determined using real-time quantitative PCR. Significantly higher expression of IL-32ß and IL-32γ isoforms was observed in HIV-related KS biopsy specimens compared with non-HIV-related KS and NST. The splicing ratio of the IL-32 isoforms showed IL-32γ as the highest expressed isoform, followed by IL-32ß, in HIV-related KS cases compared with non-HIV-related KS and NST. Our data suggest a possible survival mechanism by the splicing of IL-32γ to IL-32ß and also IL-6, IL-8, and CXCR1 signaling pathways to reverse the proapoptotic effect of the IL-32γ isoform, leading to tumor cell survival and thus favoring tumor progression.

Prevalence and associated risk factors for Kaposi's sarcoma among HIV-positive patients in a referral hospital in Northern Tanzania: a retrospective hospital-based study.

BACKGROUND: Kaposi's sarcoma (KS) is a multifocal angioproliferative tumor involving blood and lymphatic vessels, caused by Human Herpes Virus-8 (HHV-8). KS is an important AIDS-defining tumor with high prevalence in Sub-Saharan Africa, including Tanzania which has high HIV and HHV-8 sero-prevalence. It is critically important to monitor the prevalence of AIDS-defining tumors, such as KS, in the age of HIV/AIDS. We studied the prevalence of KS and associated risk factors among HIV-positive patients at Kilimanjaro Christian Medical Centre (KCMC), a referral hospital in northern Tanzania, over the period from January 2012 to December 2015. METHODS: This was a retrospective hospital-based cross-sectional study to determine the prevalence of KS among HIV/AIDS patients between 2012 and 2015. The study included 1100 HIV patients' data which were collected at the Infectious Disease Clinic (IDC) from patients' files. Stata version 13 (StataCorp LP, Texas 77,845 USA) was used for all statistical analyses. The prevalence of KS was calculated across levels of a number of categorical variables. Logistic regression was performed to determine relative risk of KS for all characteristics. We included all variables with p-values ≤10% in the multivariate analysis, including ART use, as this is considered to have an influence on KS. In the multivariate analysis, statistical significance was established based on a two-tailed p-value ≤5%. All patients' notes were kept confidential as per the Helsinki declaration. RESULTS: Our results revealed a 4.6% prevalence of KS at KCMC hospital, between January 2012 and December 2015, 51(4.6%) patients were diagnosed with KS out of 1100 HIV-positive patients. The study further revealed that KS in HIV patients was most associated with low CD4 cell count (less than or equal to 200 cells/µl). Moreover, women were more likely than men to diagnosed with KS, with higher odds significantly associated with KS (OR 0.42, p < 0.009). Increased age, above 35 years, among the HIV seropositive patients was significantly associated with KS (OR 25.67, p < 0.007). HIV patients who were none smokers were more likely to suffer from KS compared to HIV smokers (OR 0.41, p < 0.010). CONCLUSION: KS remains a common malignant vascular tumor commonly associated with HIV/AIDS in Tanzania. Our study highlights the need for continued efforts to combat HIV, as well as associated diseases such as KS. Continued availability of ART (Anti-Retroviral Therapy) to HIV/AIDS patients, and test reagents for CD4 cell count and viral load determination are important measures to alleviate the suffering of these patients. Furthermore, studies to gather more evidence on ART resistance are highly needed to guide treatment choices.

Molecular epidemiology of virulence and antimicrobial resistance determinants in Klebsiella pneumoniae from hospitalised patients in Kilimanjaro, Tanzania.

This study aimed to use whole-genome sequencing to determine virulence and antimicrobial resistance genes in K. pneumoniae isolated from patients in a tertiary care hospital in Kilimanjaro. K. pneumoniae isolates from patients attending Kilimanjaro Christian Medical Centre between August 2013 and August 2015 were fully genome-sequenced and analysed locally. Sequence analysis was done for identification of virulence and AMR genes. Plasmid and multi-locus sequence typing and capsular or capsular (K) typing were performed and phylogeny was done to ascertain K. pneumoniae relatedness. Stata 13 (College Station, TX, 77845, USA) was used to determine Cohen's kappa coefficient of agreement between the phenotypically tested and sequence-predicted resistance. A total of 16 (47.1%) sequence types (STs) and 10 (29.4%) K types were identified in 30 (88.2%) and 17 (50.0%) of all analysed isolates, respectively. K. pneumoniae ST17 were 6 (17.6%). The commonest determinants were blaCTX-M-15 in 16 (47.1%) isolates, blaSHV in 30 (88.2%), blaOXA-1 in 8 (23.5%) and blaTEM-1 in 18 (52.9%) isolates. Resistance genes for aminoglycosides were detected in 21 (61.8%) isolates, fluoroquinolones in 13 (38.2%) and quinolones 34 (100%). Ceftazidime and ceftriaxone showed the strongest agreement between phenotype- and sequence-based resistance results: 93.8%, kappa = 0.87 and p = 0.0002. Yersiniabactin determinant was detected in 12 (35.3%) of K. pneumoniae. The proportion of AMR and virulence determinants detected in K. pneumoniae is alarming. WGS-based diagnostic approach has showed promising potentials in clinical microbiology, hospital outbreak source tracing virulence and AMR detection at KCMC.

Prevalence and risk factors for CTX-M gram-negative bacteria in hospitalized patients at a tertiary care hospital in Kilimanjaro, Tanzania.

Emergence and spread of extended spectrum beta-lactamase (ESBL)-producing gram-negative bacteria, mainly due to CTX-M, is a major global public health problem. Patients infected with ESBL-producing gram-negative bacteria have an increased risk of treatment failure and death. We investigated the prevalence and risk factors for CTX-M gram-negative bacteria isolated from clinical specimens of patients hospitalized at a tertiary care hospital in Kilimanjaro, Tanzania. Isolated gram-negative bacteria from inpatients admitted at Kilimanjaro Christian Medical Centre (KCMC) between August 2013 and August 2015 were fully genome sequenced. The prevalence of ESBL-producing gram-negative bacteria was determined based on the presence of blaCTX-M. The odds ratio (OR) and risk factors for ESBL-producing gram-negative bacteria due to CTX-M were assessed using logistic regression models. The overall CTX-M prevalence (95% CI) was 13.6% (10.1-18.1). Adjusted for other factors, the OR of CTX-M gram-negative bacteria for patients previously hospitalized was 0.26 (0.08-0.88), p = 0.031; the OR for patients currently on antibiotics was 4.02 (1.29-12.58), p = 0.017; the OR for patients currently on ceftriaxone was 0.14 (0.04-0.46), p = 0.001; and the OR for patients with wound infections was 0.24 (0.09-0.61), p = 0.003. The prevalence of ESBL-producing gram-negative bacteria due to CTX-M in this setting is relatively low compared to other previous reports in similar settings. However, to properly stop further spread in the hospital, we recommend setting up a hospital surveillance system that takes full advantage of the available next-generation sequencing facility to routinely screen for all types of bacterial resistance genes.

Patterns of infections, aetiological agents and antimicrobial resistance at a tertiary care hospital in northern Tanzania.

OBJECTIVE: To determine the causative agents of infections and their antimicrobial susceptibility at a tertiary care hospital in Moshi, Tanzania, to guide optimal treatment. METHODS: A total of 590 specimens (stool (56), sputum (122), blood (126) and wound swabs (286)) were collected from 575 patients admitted in the medical and surgical departments. The bacterial species were determined by conventional methods, and disc diffusion was used to determine the antimicrobial susceptibility pattern of the bacterial isolates. RESULTS: A total of 249 (42.2%) specimens were culture-positive yielding a total of 377 isolates. A wide range of bacteria was isolated, the most predominant being Gram-negative bacteria: Proteus spp. (n = 48, 12.7%), Escherichia coli (n = 44, 11.7%), Pseudomonas spp. (n = 40, 10.6%) and Klebsiella spp (n = 38, 10.1%). Wound infections were characterised by multiple isolates (n = 293, 77.7%), with the most frequent being Proteus spp. (n = 44, 15%), Pseudomonas (n = 37, 12.6%), Staphylococcus (n = 29, 9.9%) and Klebsiella spp. (n = 28, 9.6%). All Staphylococcus aureus tested were resistant to penicillin (n = 22, 100%) and susceptible to vancomycin. Significant resistance to cephalosporins such as cefazolin (n = 62, 72.9%), ceftriaxone (n = 44, 51.8%) and ceftazidime (n = 40, 37.4%) was observed in Gram-negative bacteria, as well as resistance to cefoxitin (n = 6, 27.3%) in S. aureus. CONCLUSION: The study has revealed a wide range of causative agents, with an alarming rate of resistance to the commonly used antimicrobial agents. Furthermore, the bacterial spectrum differs from those often observed in high-income countries. This highlights the imperative of regular generation of data on aetiological agents and their antimicrobial susceptibility patterns especially in infectious disease endemic settings. The key steps would be to ensure the diagnostic capacity at a sufficient number of sites and implement structures to routinely exchange, compare, analyse and report data. Sentinel sites (hospitals) across the country (and region) should report on a representative subset of bacterial species and their susceptibility to drugs at least annually. A central organising body should collate the data and report to relevant national and international stakeholders.

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.

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.

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.

Complete genome sequence of Brucella abortus isolated from a human blood culture sample in Tanzania.

Brucella abortus causes infections in humans and livestock. Bacterial isolates are challenging to obtain, and very little is known about the genomic epidemiology of this species in Africa. Here, we report the complete genome sequence of a Brucella abortus isolate cultured from a febrile human in northern Tanzania.

Molecular Characterization of Staphylococcus aureus Isolated from Raw Milk and Humans in Eastern Tanzania: Genetic Diversity and Inter-Host Transmission.

Staphylococcus aureus is a common cause of infection in humans and animals, including bovine mastitis, globally. The objective of this study was to genetically characterize a collection of S. aureus isolates recovered from milk and nasal swabs from humans with and without animal contact (bovine = 43, human = 12). Using whole genome sequencing (NextSeq550), isolates were sequence typed, screened for antimicrobial resistance and virulence genes and examined for possible inter-species host transmission. Multi locus sequence typing (MLST) and single nucleotide polymorphism (SNP)-based phylogeny revealed 14 different sequence types, including the following six novel sequence types: ST7840, 7841, 7845, 7846, 7847, and 7848. The SNP tree confirmed that MLST clustering occurred most commonly within CC97, CC5477, and CC152. ResFinder analysis revealed five common antibiotic resistance genes, namely tet(K), blaZ, dfrG, erm©, and str, encoding for different antibiotics. mecA was discovered in one human isolate only. Multidrug resistance was observed in 25% of the isolates, predominantly in CC152 (7/8) and CC121 (3/4). Known bovine S. aureus (CC97) were collected in humans and known human S. aureus lineages (CC152) were collected in cattle; additionally, when these were compared to bovine-isolated CC97 and human-isolated CC152, respectively, no genetic distinction could be observed. This is suggestive of inter-host transmission and supports the need for surveillance of the human-animal interface.

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.

Circumstances for treatment and control of invasive Enterobacterales infections in eight hospitals across sub-Saharan Africa: a cross-sectional study.

Background: Bloodstream infections caused by Enterobacterales show high frequency of antimicrobial resistance (AMR) in many Low- and Middle-Income Countries. We aimed to describe the variation in circumstances for management of such resistant infections in a group of African public-sector hospitals participating in a major research study. Methods: We gathered data from eight hospitals across sub-Saharan Africa to describe hospital services, infection prevention and antibiotic stewardship activities, using two WHO-generated tools. We collected monthly cross-sectional data on availability of antibiotics in the hospital pharmacies for bloodstream infections caused by Enterobacterales. We compared the availability of these antibiotics to actual patient-level use of antibiotics in confirmed Enterobacterales bloodstream infections (BSI). Results: Hospital circumstances for institutional management of resistant BSI varied markedly. This included self-evaluated infection prevention level (WHO-IPCAF score: median 428, range 155 to 687.5) and antibiotic stewardship activities (WHO stewardship toolkit questions: median 14.5, range 2 to 23). These results did not correlate with national income levels. Across all sites, ceftriaxone and ciprofloxacin were the most consistently available antibiotic agents, followed by amoxicillin, co-amoxiclav, gentamicin and co-trimoxazole. There was substantial variation in the availability of some antibiotics, especially carbapenems, amikacin and piperacillin-tazobactam with degree of access linked to national income level. Investigators described out-of-pocket payments for access to additional antibiotics at 7/8 sites. The in-pharmacy availability of antibiotics correlated well with actual use of antibiotics for treating BSI patients. Conclusions: There was wide variation between these African hospitals for a range of important circumstances relating to treatment and control of severe bacterial infections, though these did not all correspond to national income level. For most antibiotics, patient-level use reflected in-hospital drug availability, suggesting external antibiotics supply was infrequent. Antimicrobial resistant bacterial infections could plausibly show different clinical impacts across sub-Saharan Africa due to this contextual variation.

Mortality associated with third-generation cephalosporin resistance in Enterobacterales bloodstream infections at eight sub-Saharan African hospitals (MBIRA): a prospective cohort study.

BACKGROUND: Bacteria of the order Enterobacterales are common pathogens causing bloodstream infections in sub-Saharan Africa and are frequently resistant to third-generation cephalosporin antibiotics. Although third-generation cephalosporin resistance is believed to lead to adverse outcomes, this relationship is difficult to quantify and has rarely been studied in this region. We aimed to measure the effects associated with resistance to third-generation cephalosporins in hospitalised patients with Enterobacterales bloodstream infection in Africa. METHODS: We conducted a prospective, matched, parallel cohort study at eight hospitals across sub-Saharan Africa. We recruited consecutive patients of all age groups with laboratory-confirmed Enterobacterales bloodstream infection and matched them to at least one patient without bloodstream infection on the basis of age group, hospital ward, and admission date. Date of infection onset (and enrolment) was defined as the day of blood sample collection for culturing. Patients infected with bacteria with a cefotaxime minimum inhibitory concentration of 1 mg/L or lower were included in the third-generation cephalosporin-susceptible (3GC-S) cohort, and the remainder were included in the third-generation cephalosporin-resistant (3GC-R) cohort. The primary outcomes were in-hospital death and death within 30 days of enrolment. We used adjusted multivariable regression models to first compare patients with bloodstream infection against matched patients within the 3GC-S and 3GC-R cohorts, then compared estimates between cohorts. FINDINGS: Between Nov 1, 2020, and Jan 31, 2022, we recruited 878 patients with Enterobacterales bloodstream infection (221 [25·2%] to the 3GC-S cohort and 657 [74·8%] to the 3GC-R cohort) and 1634 matched patients (420 [25·7%] and 1214 [74·3%], respectively). 502 (57·2%) bloodstream infections occurred in neonates and infants (age 0-364 days). Klebsiella pneumoniae (393 [44·8%] infections) and Escherichia coli (224 [25·5%] infections) were the most common Enterobacterales species identified. The proportion of patients who died in hospital was higher in patients with bloodstream infection than in matched controls in the 3GC-S cohort (62 [28·1%] of 221 vs 22 [5·2%] of 420; cause-specific hazard ratio 6·79 [95% CI 4·06-11·37] from Cox model) and the 3GC-R cohort (244 [37·1%] of 657 vs 115 [9·5%] of 1214; 5·01 [3·96-6·32]). The ratio of these cause-specific hazard ratios showed no significant difference in risk of in-hospital death in the 3GC-R cohort versus the 3GC-S cohort (0·74 [0·42-1·30]). The ratio of relative risk of death within 30 days (0·82 [95% CI 0·53-1·27]) also indicated no difference between the cohorts. INTERPRETATION: Patients with bloodstream infections with Enterobacterales bacteria either resistant or susceptible to third-generation cephalosporins had increased mortality compared with uninfected matched patients, with no differential effect related to third-generation cephalosporin-resistance status. However, this finding does not account for time to appropriate antibiotic treatment, which remains clinically important to optimise. Measures to prevent transmission of Enterobacterales could reduce bloodstream infection-associated mortality from both drug-resistant and drug-susceptible bacterial strains in Africa. FUNDING: Bill & Melinda Gates Foundation.

Plasmid characterization in bacterial isolates of public health relevance in a tertiary healthcare facility in Kilimanjaro region, Tanzania.

OBJECTIVES: Plasmids are infectious double stranded DNA molecules that are found within bacteria. Horizontal gene transfer promotes successful spread of different types of plasmids within or among bacteria species, making their detection an important task for guiding clinical treatment. We used whole genome sequenced data to determine the prevalence of plasmid replicon types in clinical bacterial isolates, the presence of resistance and virulence genes in plasmid replicon types, and the relationship between resistance and virulence genes within each plasmid replicon. METHODS: All bacterial sequences were de novo assembled using Unicycler before extraction of plasmids. Assembly graphs were submitted to Gplas+plasflow for plasmid contigs prediction. The predicted plasmid contigs were validated using PlasmidFinder. RESULTS: A total of 159 (56.2%) out of 283 bacterial isolates were found to carry plasmid replicons, with Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus being the most prevalent plasmid carriers. A total of 26 (86.7%) multiple-replicon types were found to carry both resistance and virulence genes compared to 4 (13.3%) single plasmid replicons. No statistically significant correlation was found between the number of antibiotic resistance and virulence genes in multiple-replicon types (r = - 0.14, P > 0.05). CONCLUSION: Our findings show a relatively high proportion of plasmid replicon-carrying isolates suggesting selection pressure due to antibiotic use in the hospital. Co-occurrence of antibiotic resistance and virulence genes in clinical isolates is a public health problem warranting attention.

Whole-genome sequencing of SARS-CoV-2 isolates from symptomatic and asymptomatic individuals in Tanzania.

Background: Coronavirus Disease-2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accounts for considerable morbidity and mortality globally. Paucity of SARS-CoV-2 genetic data from Tanzania challenges in-country tracking of the pandemic. We sequenced SARS-CoV-2 isolated in the country to determine circulating strains, mutations and phylogenies and finally enrich international genetic databases especially with sequences from Africa. Methods: This cross-sectional study utilized nasopharyngeal swabs of symptomatic and asymptomatic adults with positive polymerase chain reaction tests for COVID-19 from January to May 2021. Viral genomic libraries were prepared using ARTIC nCoV-2019 sequencing protocol version three. Whole-genome sequencing (WGS) was performed using Oxford Nanopore Technologies MinION device. In silico genomic data analysis was done on ARTIC pipeline version 1.2.1 using ARTIC nCoV-2019 bioinformatics protocol version 1.1.0. Results: Twenty-nine (42%) out of 69 samples qualified for sequencing based on gel electrophoretic band intensity of multiplex PCR amplicons. Out of 29 isolates, 26 were variants of concern [Beta (n = 22); and Delta (n = 4)]. Other variants included Eta (n = 2) and B.1.530 (n = 1). We found combination of mutations (S: D80A, S: D215G, S: K417N, ORF3a: Q57H, E: P71L) in all Beta variants and absent in other lineages. The B.1.530 lineage carried mutations with very low cumulative global prevalence, these were nsp13:M233I, nsp14:S434G, ORF3a:A99S, S: T22I and S: N164H. The B.1.530 lineage clustered phylogenetically with isolates first reported in south-east Kenya, suggesting regional evolution of SARS-CoV-2. Conclusion: We provide evidence of existence of Beta, Delta, Eta variants and a locally evolving lineage (B.1.530) from samples collected in early 2021 in Tanzania. This work provides a model for ongoing WGS surveillance that will be required to inform on emerging and circulating SARS-CoV-2 diversity in Tanzania and East Africa.

Effect of Digital Adherence Tools on Adherence to Antiretroviral Treatment Among Adults Living With HIV in Kilimanjaro, Tanzania: A Randomized Controlled Trial.

BACKGROUND: Lifelong adherence to antiretroviral treatment remains challenging for people living with HIV (PLHIV). The aim of this study was to investigate whether any of 2 digital adherence tools could improve adherence among PLHIV in Kilimanjaro, Tanzania. METHODS: We performed a parallel 3-arm, nonblinded, randomized controlled trial with 1:1:1 allocation. We included adults aged between 18 and 65 years, living in Kilimanjaro region, and who were on antiretroviral treatment for at least 6 months. Their adherence, as judged by the study nurses, had to be suboptimal. In one arm, participants received reminder short message service (SMS) texts, followed by a question SMS. In the second arm, participants received a real-time medication monitoring (RTMM) device (Wisepill) with SMS reminders. In the third arm, participants received standard care only. The primary outcome of mean adherence over 48 weeks was compared between arms using between-group t tests in a modified intention-to-treat analysis. RESULTS: In each arm, we randomized 83 participants: data of 82 participants in the RTMM arm, 80 in the SMS arm, and 81 in the standard care arm were analyzed. The average (over 48 weeks) adherence in the SMS, RTMM, and control arms was 89.6%, 90.6%, and 87.9% for pharmacy refill; 95.9%, 95.0%, and 95.2% for self-report in the past week; and 97.5%, 96.6%, and 96.9% for self-report in the past month, respectively (P values not statistically significant). CONCLUSIONS: Receiving reminder SMS or RTMM combined with feedback about adherence levels and discussion of strategies to overcome barriers to adherence did not improve adherence to treatment and treatment outcome in PLHIV. CLINICAL TRIAL NUMBER: PACTR201712002844286.

Patterns of acquired HIV-1 drug resistance mutations and predictors of virological failure in Moshi, Northern Tanzania.

Emergence of HIV drug resistance poses a serious risk of inactivity to all currently approved antiretroviral drugs. Profiles of HIV drug resistance mutations (HIVDRM) and virological failure (VF) are not extensively studied in Tanzania. This study aimed to determine HIVDRM and predictors of VF in HIV-infected individuals failing first-line HIV drugs in Moshi, Northern Tanzania. A case-control study was conducted at Kilimanjaro Christian Medical Centre, Mawenzi, Pasua and Majengo health facilities with HIV-care and treatment clinics from October, 2017 to August, 2018. Cases and controls were HIV-infected individuals with VF and viral suppression (VS) respectively. HIV-1 reverse transcriptase and protease genes were amplified and sequenced. Stanford University's HIV drug resistance database and REGA subtyping tool 3.0 determined HIVDRM and HIV-1 subtypes respectively. Odds ratios (OR) with 95% confidence interval (95% CI) investigated predictors of VF. P-value < 5% was considered statistically significant. A total of 124 participants were recruited, of whom 63 (50.8%) had VF, 61 (49.2%) had VS and 82 (66.1%) were females. Median [IQR] age and duration on ART were 45 [35-52] years and 72 [48-104] months respectively. Twenty-five out of 26 selected samples from cases were successfully sequenced. Twenty-four samples (96%) had at least one major mutation conferring resistance to HIV drugs, with non-nucleoside analogue reverse transcriptase inhibitor (NNRTI)-resistance associated mutations as the majority (92%). Frequent NNRTI-resistance associated mutations were K103N (n = 11), V106M (n = 5) and G190A (n = 5). Prevalent nucleoside analogue reverse transcriptase inhibitors-resistance associated mutations were M184V (n = 17), K70R (n = 7) and D67N (n = 6). Dual-class resistance was observed in 16 (64%) samples. Thirteen samples (52%) had at least one thymidine analogue-resistance associated mutation (TAM). Three samples (12%) had T69D mutation with at least 1 TAM. Two samples (8%) had at least one mutation associated with protease inhibitor resistance. Age [aOR = 0.94, 95% CI (0.90-0.97), p < 0.001] and occupation [aOR = 0.35, 95% CI (0.12-1.04), p = 0.059] associated with VF. In conclusion, HIV drug resistance is common among people failing antiretroviral therapy. Resistance testing will help to guide switching of HIV drugs.

Ceftriaxone use in a tertiary care hospital in Kilimanjaro, Tanzania: A need for a hospital antibiotic stewardship programme.

Excessive use of antibiotics, especially watch group antibiotics such as ceftriaxone leads to emergence and spread of antimicrobial resistance (AMR). In low and middle-income countries (LMICs), antibiotics are overused but data on consumption is scarcely available. We aimed at determining the extent and predictors of ceftriaxone use in a tertiary care university teaching hospital in Kilimanjaro, Tanzania. A hospital-based cross-sectional study was conducted from August 2013 through August 2015. Patients admitted in the medical, surgical wards and their respective intensive care units, receiving antimicrobials and other medications for various ailments were enrolled. Socio-demographic and clinical data were recorded in a structured questionnaire from patients' files and logistic regression was performed to determine the predictors for ceftriaxone use. Out of the 630 patients included in this study, 322 (51.1%) patients were on ceftriaxone during their time of hospitalization. Twenty-two patients out of 320 (6.9%) had been on ceftriaxone treatment without evidence of infection. Ceftriaxone use for surgical prophylaxis was 44 (40.7%), of which 32 (72.7%) and 9 (20.5%) received ceftriaxone prophylaxis before and after surgery, respectively. Three (6.8%) received ceftriaxone prophylaxis during surgery. Predicting factors for that the health facility administered ceftriaxone were identified as history of any medication use before referral to hospital [OR = 3.4, 95% CI (1.0-11.4), p = 0.047], bacterial infection [OR = 18.0, 95% CI (1.4-225.7, p = 0.025)], surgical ward [OR = 2.9, 95% CI (0.9-9.4), p = 0.078] and medical wards [OR = 5.0, 95% CI (0.9-28.3), p = 0.070]. Overall, a high ceftriaxone use at KCMC hospital was observed. Antimicrobial stewardship programs are highly needed to monitor and regulate hospital antimicrobial consumption, which in turn could help in halting the rising crisis of antimicrobial resistance.