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.

Leveraging Community Health Workers and a Responsive Digital Health System to Improve Vaccination Coverage and Timeliness in Resource-Limited Settings: Protocol for a Cluster Randomized Type 1 Effectiveness-Implementation Hybrid Study.

BACKGROUND: Tanzania is 1 of 20 countries where the majority of unvaccinated and undervaccinated children reside. Prior research identified substantial rural-urban disparities in the coverage and timeliness of childhood vaccinations in Tanzania, with children in rural settings being more likely to receive delayed or no vaccinations. Further research is necessary to identify effective and scalable interventions that can bridge rural-urban gaps in childhood vaccination while accounting for multifaceted barriers to vaccination. OBJECTIVE: This protocol describes a type 1 effectiveness-implementation hybrid study to evaluate Chanjo Kwa Wakati (timely vaccination in Kiswahili), a community-based digital health intervention to improve vaccination timeliness. The intervention combines human resources (community health workers), low-cost digital strategies (electronic communication, digital case management, and task automation), a vaccination knowledge intervention, and insights from behavioral economics (reminders and incentives) to promote timely childhood vaccinations. METHODS: The study will be conducted in 2 predominantly rural regions in Tanzania with large numbers of unvaccinated or undervaccinated children: Shinyanga and Mwanza. Forty rural health facilities and their catchment areas (clusters) will be randomized to an early or delayed onset study arm. From each cluster, 3 cohorts of mother-child dyads (1 retrospective cohort and 2 prospective cohorts) will be enrolled in the study. The timeliness and coverage of all vaccinations recommended during the first year of life will be observed for 1200 children (n=600, 50% intervention group children and n=600, 50% nonintervention group children). The primary effectiveness outcome will be the timeliness of the third dose of the pentavalent vaccine (Penta3). Quantitative surveys, vaccination records, study logs, fidelity checklists, and qualitative interviews with mothers and key informants will inform the 5 constructs of the reach, effectiveness, adoption, implementation, and maintenance (RE-AIM) framework. The results will be used to develop an implementation blueprint to guide future adaptations and scale-up of Chanjo Kwa Wakati. RESULTS: The study was funded in August 2022. Data collection is expected to last from February 2024 to July 2027. CONCLUSIONS: This study will address the lack of rigorous evidence on the effectiveness of community-based digital health interventions for promoting vaccination coverage and timeliness among children from sub-Saharan Africa and identify potential implementation strategies to facilitate the deployment of vaccination promotion interventions in low- and middle-income countries. TRIAL REGISTRATION: ClinicalTrials.gov NCT06024317; https://www.clinicaltrials.gov/study/NCT06024317. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/52523.

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.

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.

Using discrete choice experiments to design interventions for heterogeneous preferences: protocol for a pragmatic randomised controlled trial of a preference-informed, heterogeneity-focused, HIV testing offer for high-risk populations.

INTRODUCTION: Approximately one million undiagnosed persons living with HIV in Southern and Eastern Africa need to test for HIV. Novel approaches are necessary to identify HIV testing options that match the heterogeneous testing preferences of high-risk populations. This pragmatic randomised controlled trial (PRCT) will evaluate the efficacy of a preference-informed, heterogeneity-focused HIV counselling and testing (HCT) offer, for improving rates of HIV testing in two high-risk populations. METHODS AND ANALYSIS: The study will be conducted in Moshi, Tanzania. The PRCT will randomise 600 female barworkers and 600 male Kilimanjaro mountain porters across three study arms. All participants will receive an HIV testing offer comprised of four preference-informed testing options, including one 'common' option-comprising features that are commonly available in the area and, on average, most preferred among study participants-and three options that are specific to the study arm. Options will be identified using mixed logit and latent class analyses of data from a discrete choice experiment (DCE). Participants in Arm 1 will be offered the common option and three 'targeted' options that are predicted to be more preferred than the common option and combine features widely available in the study area. Participants in Arm 2 will be offered the common option and three 'enhanced' options, which also include HCT features that are not yet widely available in the study area. Participants in Arm 3, an active control arm, will be offered the common option and three predicted 'less preferred' options. The primary outcome will be uptake of HIV testing. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Duke University Health System IRB, the University of South Carolina IRB, the Ethics Review Committee at Kilimanjaro Christian Medical University College, Tanzania's National Institute for Medical Research, and the Tanzania Food & Drugs Authority (now Tanzania Medicines & Medical Devices Authority). Findings will be published in peer-reviewed journals. The use of rigorous DCE methods for the preference-based design and tailoring of interventions could lead to novel policy options and implementation science approaches. TRIAL REGISTRATION NUMBER: NCT02714140.

Identifying genetic variants and pathways associated with extreme levels of fetal hemoglobin in sickle cell disease in Tanzania.

BACKGROUND: Sickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common genetic variants which differ across populations and hence do not fully account for HbF variation. METHODS: We investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A, MYB, HOXA9, HBB, HBG1, HBG2, CHD4, KLF1, MBD3, ZBTB7A and PGLYRP1. RESULTS: Results revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified frameshift deletions in individuals with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis. CONCLUSIONS: This study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD interrogating multiple genomic variants associated with HbF in SCD.

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.

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.

Prescription and non-prescription antibiotic dispensing practices in part I and part II pharmacies in Moshi Municipality, Kilimanjaro Region in Tanzania: A simulated clients approach.

Antibiotic dispensing without a prescription poses a threat to public health as it leads to excessive antibiotic consumption. Inappropriate antibiotic availability to the community has been documented to be amongst drivers of antimicrobial resistance emergence. Community pharmacies are a source of antibiotics in low and middle-income countries (LMICs). We aimed at assessing antibiotic dispensing practices by community pharmacy retailers in Moshi urban, Kilimanjaro, Tanzania and recommend interventions to improve practice. Using a Simulated Client (SC) Method, an observational cross-sectional survey of antibiotic dispensing practices was conducted from 10th June to 10th July 2017. Data analysis was done using Stata 13 (StataCorp, College Station, TX, USA). A total of 82 pharmacies were visited. Part I pharmacies were 26 (31.71%) and 56 (68.29%) were part II. Overall 92.3% (95% CI 77.8-97.6) of retailers dispensed antibiotics without prescriptions. The antibiotics most commonly dispensed without a prescription were ampiclox for cough (3 encounters) and azithromycin for painful urination (3 encounters). An oral third generation cephalosporin (cefixime) was dispensed once for painful urination without prescription by a part I pharmacy retailer. Out of 21, 15(71.43%) prescriptions with incomplete doses were accepted and had antibiotics dispensed. Out of 68, 4(5.9%) retailers gave instructions for medicine use voluntarily. None of the retailers voluntarily explained drug side-effects. In Moshi pharmacies, a high proportion of antibiotics are sold and dispensed without prescriptions. Instructions for medicine use are rarely given and none of the retailers explain side effects. These findings support the need for a legislative enforcement of prescription-only antibiotic dispensing rules and regulations. Initiation of clinician and community antibiotic stewardship and educational programs on proper antibiotic use to both pharmacists and public by the regulatory bodies are highly needed.

Whole genome sequencing reveals high clonal diversity of Escherichia coli isolated from patients in a tertiary care hospital in Moshi, Tanzania.

Background: Limited information regarding the clonality of circulating E. coli strains in tertiary care hospitals in low and middle-income countries is available. The purpose of this study was to determine the serotypes, antimicrobial resistance and virulence genes. Further, we carried out a phylogenetic tree reconstruction to determine relatedness of E. coli isolated from patients in a tertiary care hospital in Tanzania. Methods: E. coli isolates from inpatients admitted at Kilimanjaro Christian Medical Centre between August 2013 and August 2015 were fully genome-sequenced at KCMC hospital. Sequence analysis was done for identification of resistance genes, Multi-Locus Sequence Typing, serotyping, and virulence genes. Phylogeny reconstruction using CSI Phylogeny was done to ascertain E. coli relatedness. Stata 13 (College Station, Texas 77,845 USA) was used to determine Cohen's kappa coefficient of agreement between the phenotypically tested and whole genome sequence predicted antimicrobial resistance. Results: Out of 38 E. coli isolates, 21 different sequence types (ST) were observed. Eight (21.1%) isolates belonged to ST131; of which 7 (87.5.%) were serotype O25:H4. Ten (18.4%) isolates belonged to ST10 clonal complex; of these, four (40.0%) were ST617 with serotype O89:H10. Twenty-eight (73.7%) isolates carried genes encoding beta-lactam resistance enzymes. On average, agreement across all drugs tested was 83.9%. Trimethoprim/sulphamethoxazole (co-trimoxazole) showed moderate agreement: 45.8%, kappa =15% and p = 0.08. Amoxicillin-clavulanate showed strongest agreement: 87.5%, kappa = 74% and p = 0.0001. Twenty-two (57.9%) isolates carried virulence factors for host cells adherence and 25 (65.7%) for factors that promote E. coli immune evasion by increasing survival in serum. The phylogeny analysis showed that ST131 clustering close together whereas ST10 clonal complex had a very clear segregation of the ST617 and a mix of the rest STs. Conclusion: There is a high diversity of E. coli isolated from patients admitted to a tertiary care hospital in Tanzania. This underscores the necessity to routinely screen all bacterial isolates of clinical importance in tertiary health care facilities. WGS use for laboratory-based surveillance can be an effective early warning system for emerging pathogens and resistance mechanisms in LMICs.

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.

Hospital Epidemiology of Methicillin-Resistant Staphylococcus aureus in a Tertiary Care Hospital in Moshi, Tanzania, as Determined by Whole Genome Sequencing.

OBJECTIVE: To determine molecular epidemiology of methicillin-resistant S. aureus in Tanzania using whole genome sequencing. METHODS: DNA from 33 Staphylococcus species was recovered from subcultured archived Staphylococcus isolates. Whole genome sequencing was performed on Illumina Miseq using paired-end 2 × 250 bp protocol. Raw sequence data were analyzed using online tools. RESULTS: Full susceptibility to vancomycin and chloramphenicol was observed. Thirteen isolates (43.3%) resisted cefoxitin and other antimicrobials tested. Multilocus sequence typing revealed 13 different sequence types among the 30 S. aureus isolates, with ST-8 (n = seven, 23%) being the most common. Gene detection in S. aureus stains were as follows: mecA, 10 (33.3%); pvl, 5 (16.7%); tst, 2 (6.7%). The SNP difference among the six Tanzanian ST-8 MRSA isolates ranged from 24 to 196 SNPs and from 16 to 446 SNPs when using the USA300_FPR3757 or the USA500_2395 as a reference, respectively. The mutation rate was 1.38 × 10-11 SNPs/site/year or 1.4 × 10-6 SNPs/site/year as estimated by USA300_FPR3757 or the USA500_2395, respectively. CONCLUSION: S. aureus isolates causing infections in hospitalized patients in Moshi are highly diverse and epidemiologically unrelated. Temporal phylogenetic analysis provided better resolution on transmission and introduction of MRSA and it may be important to include this in future routines.

Aeromonas caviae mimicking Vibrio cholerae infectious enteropathy in a cholera-endemic region with possible public health consequences: two case reports.

BACKGROUND: Aeromonas species have been documented to yield false positive results in microbiological tests for Vibrio cholerae. They share many biochemical properties with Vibrio species, with which they were jointly classified in the family Vibrionaceae until genotypic information provided new insights. Aeromonas species are increasingly associated with gastrointestinal infections, albeit with great apparent variation in pathogenicity and virulence both between and within species of the genus. We report two cases with clinically mild cholera-like symptoms, at a time when a cholera outbreak was unfolding in other regions of the country (Tanzania). These are the first cases to be reported with Aeromonas mimicking cholera in our area. CASE PRESENTATION: Two patients were admitted at the isolation unit designated by the Kilimanjaro Christian Medical Centre for emerging infectious diseases and provided informed consent about regular stool analysis and culture under the provisional diagnosis of gastroenteritis. The first patient was a 23-year-old black African woman with a 2-day history of watery diarrhea and vomiting associated with a temperature of 39.7 °C. The second patient was a 47-year-old black African woman with a 2-day history of diarrhea and vomiting with a temperature of 37.7 °C, and she was hemodynamically stable. Both patients were isolated in a specific area for infection control and treated with fluids and orally administered rehydration solution, ciprofloxacin, metronidazole, and paracetamol. Stool culture was done. The isolated colonies were reported as V. cholerae and transferred to the research laboratory of Kilimanjaro Clinical Research Institute for confirmation using whole genome sequencing. Microbiological testing determined colonies isolated from stool to be V. cholerae, and warranted the conclusion "presumptive cholera." Whole genome sequencing, however, established the presence of Aeromonas caviae rather than V. cholerae. CONCLUSIONS: The co-existence of Aeromonas species with V. cholerae in cholera-endemic regions suggests the possibility that a proportion of suspected cholera cases may be Aeromonas infections. However, with close to no epidemiological data available on Aeromonas infection in cases of diarrhea and dysentery in Sub-Saharan Africa, it is not currently possible to establish the extent of misdiagnosis to any degree of certainty. Whole genome sequencing was shown to readily exclude V. cholerae as the etiological agent and establish the presence of Aeromonas species.

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.

Meta-analysis of proportion estimates of Extended-Spectrum-Beta-Lactamase-producing Enterobacteriaceae in East Africa hospitals.

BACKGROUND: A high proportion of Extended-Spectrum-Beta-Lactamase (ESBL) producing Enterobacteriaceae is causing common infections in all regions of the world. The burden of antibiotic resistance due to ESBL in East Africa is large but information is scarce and thus it is unclear how big the problem really is. To gain insight into the magnitude and molecular epidemiology of ESBL-producing Enterobacteriaceae in East Africa a literature search was performed in PubMed on 31 July 2015 to retrieve articles with relevant information on ESBL. METHODS AND RESULTS: Meta-analysis was performed to determine overall proportion estimate of ESBL-producing Enterobacteriaceae. A total of 4076 bacterial isolates were included in the analysis. The overall pooled proportion of ESBL-producing Enterobacteriaceae among included surveys done in East African hospitals was found to be 0.42 (95 % CI: 0.34-0.50). Heterogeneity (I(2)) between countries' proportions in ESBL was significantly high (96.95 % and p < 0.001). The frequently detected genes encoding ESBL were CTX-M, TEM, SHV and OXA while the most infrequent reported genes were KPC and NDM. CONCLUSION: The available studies show a very wide variation in resistance due to ESBL between countries. This highlights a need for active surveillance systems which can help understand the actual epidemiology of ESBL, aid in formulating national or regional guidelines for proper screening of ESBL, and support developing standardized approaches for managing patients colonized with ESBL.

Surveillance of artemether-lumefantrine associated Plasmodium falciparum multidrug resistance protein-1 gene polymorphisms in Tanzania.

BACKGROUND: Resistance to anti-malarials is a major public health problem worldwide. After deployment of artemisinin-based combination therapy (ACT) there have been reports of reduced sensitivity to ACT by malaria parasites in South-East Asia. In Tanzania, artemether-lumefantrine (ALu) is the recommended first-line drug in treatment of uncomplicated malaria. This study surveyed the distribution of the Plasmodium falciparum multidrug resistance protein-1 single nucleotide polymorphisms (SNPs) associated with increased parasite tolerance to ALu, in Tanzania. METHODS: A total of 687 Plasmodium falciparum positive dried blood spots on filter paper and rapid diagnostic test strips collected by finger pricks from patients attending health facilities in six regions of Tanzania mainland between June 2010 and August 2011 were used. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to detect Pfmdr1 SNPs N86Y, Y184F and D1246Y. RESULTS: There were variations in the distribution of Pfmdr1 polymorphisms among regions. Tanga region had exceptionally high prevalence of mutant alleles, while Mbeya had the highest prevalence of wild type alleles. The haplotype YFY was exclusively most prevalent in Tanga (29.6%) whereas the NYD haplotype was the most prevalent in all other regions. Excluding Tanga and Mbeya, four, most common Pfmdr1 haplotypes did not vary between the remaining four regions (χ² = 2.3, p = 0.512). The NFD haplotype was the second most prevalent haplotype in all regions, ranging from 17% - 26%. CONCLUSION: This is the first country-wide survey on Pfmdr1 mutations associated with ACT resistance. Distribution of individual Pfmdr1 mutations at codons 86, 184 and 1246 varies throughout Tanzanian regions. There is a general homogeneity in distribution of common Pfmdr1 haplotypes reflecting strict implementation of ALu policy in Tanzania with overall prevalence of NFD haplotype ranging from 17 to 26% among other haplotypes. With continuation of ALu as first-line drug this haplotype is expected to keep rising, thus there is need for continued pharmacovigilance studies to monitor any delayed parasite clearance by the drug.

High levels of sulphadoxine-pyrimethamine resistance Pfdhfr-Pfdhps quintuple mutations: a cross sectional survey of six regions in Tanzania.

BACKGROUND: In 2006, the first-line anti-malarial drug treatment in Tanzania was changed from sulphadoxine-pyrimethamine (SP) to artemether-lumefantrine (ALu), an artemisinin-based combination (ACT), since when the use of SP has been restricted for intermittent preventive treatment in pregnancy (IPTp). A number of Plasmodium falciparum mutations are known to be associated with resistance to SP, but it is not known if the prevalence of these mutations is increasing or decreasing under the conditions of reduced levels of SP use. This study reports on the current SP resistant quintuple Pfdhfr-Pfdhps mutations in six regions of Tanzania. METHODS: Finger-prick blood on filter paper and rapid diagnostic test strips from P. falciparum-positive individuals of all age groups attending health facilities in six regions of Tanzania between June 2010 and August 2011 were obtained. Using chelex-100 extracted DNA, genotyping was done for mutations on codons 51, 59 and 108 of Pfdhfr and 437 and 540 of Pfdhps genes using PCR-RFLP technique. RESULTS: A total of 802 malaria-positive samples were screened and genotyped. The prevalence of Pfdhfr 51I, Pfdhps 437G and 540E varied between the regions (p < 0.001) whereas Pfdhfr 59R (FE 10.79, p = 0.225) and 108 N (FE 10.61, p = 0.239) did not vary between the regions. The Pfdhfr triple mutant was above 84% and close to fixation levels in all regions, whereas the Pfdhps double mutation ranged from 43.8 to 97% between the regions. The quintuple mutant (IRNGE) was the most prevalent in all regions and it varied significantly from 37.5 to 90.2% (χ(2) = 1.11, p <0.001). CONCLUSIONS: There is evidence of persistent high levels of SP resistance markers in Tanzania with evidence of quintuple mutations that are likely to become fixed in the population. This threatens the future of SP not only in IPTp programmes, but as a combination drug for ACT. Continuous monitoring of SP-IPTp efficacy should be encouraged subsequent to searching for alternative drugs for IPTp in East Africa.