Clinical application of whole-genome sequencing in the management of extensively drug-resistant tuberculosis: a case report.

BACKGROUND: Whole-genome sequencing (WGS)-based prediction of drug resistance in Mycobacterium tuberculosis has the potential to guide clinical decisions in the design of optimal treatment regimens. METHODS: We utilized WGS to investigate drug resistance mutations in a 32-year-old Tanzanian male admitted to Kibong'oto Infectious Diseases Hospital with a history of interrupted multidrug-resistant tuberculosis treatment for more than three years. Before admission, he received various all-oral bedaquiline-based multidrug-resistant tuberculosis treatment regimens with unfavourable outcomes. RESULTS: Drug susceptibility testing of serial M. tuberculosis isolates using Mycobacterium Growth Incubator Tubes culture and WGS revealed resistance to first-line anti-TB drugs, bedaquiline, and fluoroquinolones but susceptibility to linezolid, clofazimine, and delamanid. WGS of serial cultured isolates revealed that the Beijing (Lineage 2.2.2) strain was resistant to bedaquiline, with mutations in the mmpR5 gene (Rv0678. This study also revealed the emergence of two distinct subpopulations of bedaquiline-resistant tuberculosis strains with Asp47f and Glu49fs frameshift mutations in the mmpR5 gene, which might be the underlying cause of prolonged resistance. An individualized regimen comprising bedaquiline, delamanid, pyrazinamide, ethionamide, and para-aminosalicylic acid was designed. The patient was discharged home at month 8 and is currently in the ninth month of treatment. He reported no cough, chest pain, fever, or chest tightness but still experienced numbness in his lower limbs. CONCLUSION: We propose the incorporation of WGS in the diagnostic framework for the optimal management of patients with drug-resistant and extensively drug-resistant tuberculosis.

Whole genome sequencing of Mycobacterium tuberculosis isolates and clinical outcomes of patients treated for multidrug-resistant tuberculosis in Tanzania.

BACKGROUND: Tuberculosis (TB), particularly multi- and or extensive drug resistant TB, is still a global medical emergency. Whole genome sequencing (WGS) is a current alternative to the WHO-approved probe-based methods for TB diagnosis and detection of drug resistance, genetic diversity and transmission dynamics of Mycobacterium tuberculosis complex (MTBC). This study compared WGS and clinical data in participants with TB. RESULTS: This cohort study performed WGS on 87 from MTBC DNA isolates, 57 (66%) and 30 (34%) patients with drug resistant and susceptible TB, respectively. Drug resistance was determined by Xpert® MTB/RIF assay and phenotypic culture-based drug-susceptibility-testing (DST). WGS and bioinformatics data that predict phenotypic resistance to anti-TB drugs were compared with participant's clinical outcomes. They were 47 female participants (54%) and the median age was 35 years (IQR): 29-44). Twenty (23%) and 26 (30%) of participants had TB/HIV co-infection BMI < 18 kg/m2 respectively. MDR-TB participants had MTBC with multiple mutant genes, compared to those with mono or polyresistant TB, and the majority belonged to lineage 3 Central Asian Strain (CAS). Also, MDR-TB was associated with delayed culture-conversion (median: IQR (83: 60-180 vs. 51:30-66) days). WGS had high concordance with both culture-based DST and Xpert® MTB/RIF assay in detecting drug resistance (kappa = 1.00). CONCLUSION: This study offers comparison of mutations detected by Xpert and WGS with phenotypic DST of M. tuberculosis isolates in Tanzania. The high concordance between the different methods and further insights provided by WGS such as PZA-DST, which is not routinely performed in most resource-limited-settings, provides an avenue for inclusion of WGS into diagnostic matrix of TB including drug-resistant TB.

High prevalence of human immunodeficiency virus, hepatitis B and C viral infections among people who inject drugs: a potential stumbling block in the control of HIV and viral hepatitis in Tanzania.

BACKGROUND: Tanzania has witnessed a significant decrease in the prevalence of human immunodeficiency virus (HIV) and viral hepatitis in the general population attributed to several interventional measures. It is uncertain whether this decline has also occurred among people who inject drugs (PWID). This study aimed to determine the seroprevalence of HIV, Hepatitis B and C viruses infection among PWID recruited from their hotspot sites in Dar es Salaam, Tanzania. METHODS: A cross-sectional study conducted between June and September 2017 recruited PWID from pre-identified hotspot sites using a snowball referral sampling technique. A structured questionnaire was used to obtain information regarding socio-demographic characteristics, behaviour and drug use. Blood was tested for the presence of IgG antibodies against HIV and Hepatitis C virus (HCV) and hepatitis B surface antigen (HBsAg). Data were entered in the computer using excel software and analysed using Statistical Package for Social Sciences version 20. RESULTS: A total of 219 PWID were recruited, the majority of whom were males (74.9%), unmarried (60.7%), had low education (62.6%) and low income (57.1%). The median age was 39 years, with an inter-quartile range of 35-43. Approximately 32.0% had a history of drug injection for more than 3 years, 79.9% were injecting drugs more than 3 times per day and 47.5% were sharing needles. The overall prevalence of HIV, HBsAg, and HCV was 33.8, 7.8, and 50.2%, respectively. There was serologic evidence of at least one infection for 68.9%, while 22.4% had two or more infections. HIV infection was independently associated with being married, while HCV was associated with injecting drugs for more than 3 years and unprotected sex. CONCLUSION: Over two-third of PWID had serologic evidence of infection with at least one virus while 22.4% having at least two infections. The high prevalence of HIV and viral hepatitis infections among PWID may hamper initiatives of ending HIV and viral hepatitis epidemics in Tanzania.

Effects of isoniazid resistance on TB treatment outcomes under programmatic conditions in a high-TB and -HIV setting: a prospective multicentre study.

Objectives: The scale and impact of background isoniazid resistance in TB- and HIV-endemic countries requires definition to improve treatment success and guide the scale-up of isoniazid preventive therapy (IPT). We describe the effects of isoniazid resistance on TB treatment outcomes among patients with or without HIV infection in Dar es Salaam, Tanzania. Methods: A multicentre, prospective observational study was conducted among TB patients commencing WHO-recommended first-line TB treatment. In multivariate analysis we ascertained the relationship between isoniazid resistance at presentation with a composite of poor treatment outcomes (death, failure or default from TB therapy). Results: Of 861 patients, 250 (29.0%) were HIV infected and 23 (2.7%) had isoniazid resistance. Seven hundred and ninety-seven (92.6%) of the patients were successfully treated and 25 (2.9%) died. Isoniazid resistance [relative risk (RR) = 6.0; 95% CI = 1.9-18.7; P < 0.01] and HIV infection with (RR = 2.3; 95% CI = 1.0-5.2; P = 0.05) or without (RR = 3.1; 95% CI = 1.5-6.2; P < 0.01) ART were independent predictors of poor treatment outcomes. Conclusions: Background isoniazid resistance and HIV infection adversely affected TB treatment outcomes. Early laboratory detection of isoniazid resistance is important for successful TB therapy. Studies on the impact of background isoniazid resistance on the efficacy of isoniazid prophylaxis are recommended.

Improved performance of Xpert MTB/RIF assay on sputum sediment samples obtained from presumptive pulmonary tuberculosis cases at Kibong'oto infectious diseases hospital in Tanzania.

BACKGROUND: The introduction of Xpert MTB/RIF assay (Xpert) has significantly improved diagnosis of Tuberculosis (TB) in resource limited human immunodeficiency virus (HIV) endemic settings. We aimed to modify the Xpert protocol to improve the detection of Mycobacterium tuberculosis (MTB). METHODS: This cross sectional study was conducted among presumptive pulmonary tuberculosis (PTB) patients at Kibong'oto Infectious Diseases Hospital between August and November 2015. Each patient consented to provide 2 samples of raw sputa. One-sputum sample was sedimented using the Petroff's method and divided into two portions. One portion of sediment was inoculated on Lowenstein-Jensen culture media and observed for any growth for up to 8 weeks. Both, raw sputum and the portions of sediments were tested separately using Xpert with a sample reagent ratio of 1:2. Mean age of patients, prevalence of MTB, Xpert sensitivity, specificity, positive and negative predictive value were calculated. An incremental sensitivity was determined. Pearson chi-square and either an independent T or Mann-Whitney U-test were used to compared categorical and continuous variables respectively. A p- value of ≤0.05 was considered significant. RESULTS: Of the 270 presumptive PTB cases, 262 were eligible for analysis. Eight (3%) were excluded due to contaminated culture. Patients' mean age was 42.9 (±SD 15.1) years of which 173 (66%) were female. The overall prevalence of PTB was 112 (43%), of which the Xpert detected 105 (40%) in sediments and 98 (37%) in raw sputa as compared to culture which detected 85 (32%) cases of PTB. Sensitivity, specificity, positive and negative predictive values of Xpert on sputum sediments were 92%, 85%, 74% and 96% respectively. Overall, the incremental sensitivity of Xpert on sediment over raw sputum was 6%. In HIV infected Presumptive PTB, the incremental sensitivity was 12%. CONCLUSION: Lowering the sample reagent to sediment dilution ratio increases sensitivity of Xpert on MTB detection among presumptive PTB cases, especially in HIV infected individuals.

Identification and antimicrobial resistance patterns of bacterial enteropathogens from children aged 0-59 months at the University Teaching Hospital, Lusaka, Zambia: a prospective cross sectional study.

BACKGROUND: Bacterial diarrhoeal disease is among the most common causes of mortality and morbidity in children 0-59 months at the University Teaching Hospital in Lusaka, Zambia. However, most cases are treated empirically without the knowledge of aetiological agents or antimicrobial susceptibility patterns. The aim of this study was, therefore, to identify bacterial causes of diarrhoea and determine their antimicrobial susceptibility patterns in stool specimens obtained from the children at the hospital. METHODS: This hospital-based cross-sectional study involved children aged 0-59 months presenting with diarrhoea at paediatrics wards at the University Teaching Hospital in Lusaka, Zambia, from January to May 2016. Stool samples were cultured on standard media for enteropathogenic bacteria, and identified further by biochemical tests. Multiplex polymerase chain reaction was used for characterization of diarrhoeagenic Escherichia coli strains. Antimicrobial susceptibility testing was performed on antibiotics that are commonly prescribed at the hospital using the Kirby-Bauer disc diffusion method, which was performed using the Clinical Laboratory Standards International guidelines. RESULTS: Of the 271 stool samples analysed Vibrio cholerae 01 subtype and Ogawa serotype was the most commonly detected pathogen (40.8%), followed by Salmonella species (25.5%), diarrhoeagenic Escherichia coli (18%), Shigella species (14.4%) and Campylobacter species (3.5%). The majority of the bacterial pathogens were resistant to two or more drugs tested, with ampicillin and co-trimoxazole being the most ineffective drugs. All diarrhoeagenic Escherichia coli isolates were extended spectrum ß-lactamase producers. CONCLUSION: Five different groups of bacterial pathogens were isolated from the stool specimens, and the majority of these organisms were multidrug resistant. These data calls for urgent revision of the current empiric treatment of diarrhoea in children using ampicillin and co-trimoxazole, and emphasizes the need for continuous antimicrobial surveillance as well as the implementation of prevention programmes for childhood diarrhoea.

Prevalence of HIV and syphilis infections among pregnant women attending antenatal clinics in Tanzania, 2011.

BACKGROUND: The occurrence of HIV-1 and syphilis infections during pregnancy poses major health risks to the foetus due to mother-to-child transmission. We conducted surveillance of HIV and syphilis infections among pregnant women attending antenatal clinics (ANCs) in Mainland Tanzania in 2011. METHODS: This surveillance was carried out in 133 ANCs selected from 21 regions in Tanzania. In each region, six ANC sites were selected, with urban, semi-urban, and rural areas contributing two each. All pregnant women who were attending selected sentinel ANC sites for the first time at any pregnancy between September and December 2011 were enrolled. Serial ELISA assays were performed to detect HIV infection in an unlinked anonymous manner using dried blood spot (DBS) after routine syphilis testing. Data analysis was conducted using Stata v.12 software. RESULTS: A total of 39,698 pregnant women representing 2.4 % of all pregnant women (1.68 million) attending ANCs in the Mainland Tanzania were enrolled. The overall HIV prevalence was found to be 5.6 % (95 % CI: 5.4-5.8 %). The risk for HIV infection was significantly higher among women aged 25-34 (cOR = 1.97, 95 % CI: 1.79-2.16; p < 0.05), older than 35 years (cOR = 1.88, 95 % CI: 1.62-2.17; p < 0.05) and those having 1-2 and 3-4 previous pregnancies. HIV infection was less prevalent among women attending rural ANC clinics (cOR = 0.46, 95 % CI 0.4-0.52; p < 0.05). The overall syphilis prevalence was 2.5 % (95 % CI: 2.3, 3.6). The risk for syphilis infection was significantly higher among women attending semi-urban and rural clinics and those having 3-4, and 5 previous pregnancies (p < 0.05). Marital status and level of education were not statistically significant with either of the two infections. HIV and syphilis co-infections occurred in 109 of 38,928 (0.3 %). CONCLUSION: The overall prevalence of HIV infection (5.6 %) and syphilis (2.5 %) found among pregnant women attending ANC clinics in Tanzania calls for further strengthening of current intervention measures, which include scaling up the integration of prevention of mother to child transmission (PMTCT) services in Reproductive and Child Health (RCH) clinics.

Species diversity of non-tuberculous mycobacteria isolated from humans, livestock and wildlife in the Serengeti ecosystem, Tanzania.

BACKGROUND: Non-tuberculous mycobacteria (NTM), which are ubiquitous micro-organisms occurring in humans, animals and the environment, sometimes receive public health and veterinary attention as opportunistic disease-causing agents. In Tanzania, there is limited information regarding the diversity of NTM species, particularly at the human-livestock-wildlife interface such as the Serengeti ecosystem, where potential for cross species infection or transmission may exist. METHODS: Mycobacterial DNA was extracted from cultured isolates obtained from sputum samples of 472 suspect TB patients and 606 tissues from wildlife species and indigenous cattle. Multiplex PCR was used to differentiate NTM from Mycobacterium tuberculosis complex (MTBC) members. NTM were further identified to species level by nucleotide sequencing of the 16S rRNA gene. RESULTS: A total of fifty five (55) NTM isolates representing 16 mycobacterial species and 5 isolates belonging to the MTBC were detected. Overall, Mycobacterium intracellulare which was isolated from human, cattle and wildlife, was the most frequently isolated species (20 isolates, 36.4%) followed by M. lentiflavum (11 isolates, 20%), M. fortuitum (4 isolates, 7.3%) and M. chelonae-abscessus group (3 isolates, 5.5%). In terms of hosts, 36 isolates were from cattle and 12 from humans, the balance being found in various wildlife species. CONCLUSION: This study reveals a diversity of NTM species in the Serengeti ecosystem, some of which have potential for causing disease in animals and humans. The isolation of NTM from tuberculosis-like lesions in the absence of MTBC calls for further research to elucidate their actual role in causing disease. We are also suggesting a one health approach in identifying risk factors for and possible transmission mechanisms of the NTM in the agro-pastoral communities in the Serengeti ecosystem.

Tetracycline and sulphonamide residues in farmed fish in Dar es Salaam, Tanzania and human health risk implications.

In Africa, antibiotic residue investigations in animal food have primarily been focused on meat, neglecting farmed fish. This cross-sectional study conducted in Dar es Salaam, Tanzania, aimed to assess sulphonamide and tetracycline residues in farmed fish, comparing levels with Codex Alimentarius Commission's acceptable daily intake (ADI) and maximum residue limits (MRLs). A total of 84 farmed fish were sampled and analysed in the presence of tetracycline and sulphonamide residues. All samples were positive for sulphonamide residues (100%; n = 84), and 2.4% (n = 2) were positive for tetracycline and consequently also positive for both compounds. Tetracycline levels were below ADI and MRL, 28.5% (n = 24) surpassed the ADI, and 6% (n = 5) of the samples exceeded the MRL for sulphonamide. Regular monitoring of antibiotic residues in aquaculture products is crucial to mitigate health risks and expanding assessments to include other commonly used compounds is warranted.

Barriers and Enablers to Retention in HIV Care and Adherence to Antiretroviral Therapy: Evidence from Dar es Salaam, Tanzania.

Purpose: To explore the enabling factors, barriers, and strategies to improve retention in HIV care and adherence to antiretroviral therapy (ART) among adults (18 years and above) living with HIV in Dar es Salaam, Tanzania. Methods: We conducted a descriptive qualitative study to better understand and explore enablers, barriers, and strategies to improve retention in HIV care and adherence to antiretroviral therapy (ART) among PLHIV in Dar es Salaam, Tanzania. Focus group discussions (FGD) were conducted with a semi-structured discussion guide between December 2021 and June 2022. A non-random purposive sampling technique was used to select PLHIV and people involved in provision of healthcare and socioeconomic support to PLHIV. Thematic analysis was used to identify and interpret the themes. Results: Three major themes with 10 sub-themes emerged. Participants indicated that family and partner support, peer-support group/adherence clubs, and healthcare provider counselling on medication adherence facilitated retention and adherence to ART. In contrast, stigma and discrimination, financial constraints, disease outbreaks such as the COVID-19 pandemic, myths and misconceptions about HIV, and side effects of antiretrovirals were mentioned as barriers. Strengthening community and patient education about HIV and ART through peer support groups and financial support for poor PLHIV were the proposed mitigation. Conclusion: Addressing the challenges to ART adherence may require a more holistic approach. We recommend the implementation of peer support groups and financial support through small microfinance groups as interventions to increase retention in HIV care and adherence to ART in the study area.

Prevalence and risk factors for infection of bovine tuberculosis in indigenous cattle in the Serengeti ecosystem, Tanzania.

BACKGROUND: Bovine tuberculosis (bTB) is a chronic debilitating disease and is a cause of morbidity and mortality in livestock, wildlife and humans. This study estimated the prevalence and risk factors associated with bovine tuberculosis transmission in indigenous cattle at the human-animal interface in the Serengeti ecosystem of Tanzania. RESULTS: A total of 1,103 indigenous cattle from 32 herds were investigated for the presence of bTB using the Single Intradermal Comparative Tuberculin Test. Epidemiological data on herd structure, management and grazing system were also collected.The apparent individual animal prevalence of tuberculin reactors was 2.4% (95% confidence interval (CI), 1.7 - 3.5%), whereas the true prevalence was 0.6% CI, 0.6 - 0.7% as indicated by a reaction to avian tuberculin purified protein derivatives (PPD) which is more than 4 mm greater than the reaction to avian tuberculin PPD. The results showed that 10.6% (117/1,103) showed non-specific reactions (atypical mycobacterium). The herd prevalence of 50% (16/32) was found. Tuberculin skin test results were found to be significantly associated with age, location, size of the household and animal tested. Of 108 respondents, 70 (64.8%) individuals had not heard about bovine tuberculosis at all. Thirty five percent (38/108) of respondents at least were aware of bTB. About 60% (23/38) of respondents who were aware of bTB had some knowledge on how bTB is spread. Eighty one percent (87/108) of respondents were not aware of the presence of bTB in wildlife. There is regular contact between cattle and wild animals due to sharing of grazing land and water sources, with 99% (107/108) of households grazing cattle in communal pastures. CONCLUSION: The study has demonstrated a high reported interaction of livestock with wildlife and poor knowledge of most cattle owners concerning bTB and its transmission pathways among people, livestock and wildlife. Although the overall proportion of animals with bTB is relatively low, herd prevalence is 50% and prevalence within herds varied considerably. Thus there is a possibility of cross transmission of bTB at wildlife-livestock interface areas that necessitates use of genetic strain typing methods to characterize them accurately.

Neonatal sepsis at Muhimbili National Hospital, Dar es Salaam, Tanzania; aetiology, antimicrobial sensitivity pattern and clinical outcome.

BACKGROUND: Neonatal sepsis contributes significantly to morbidity and mortality among young infants. The aetiological agents as well as their susceptibility to antimicrobial agents are dynamic. This study determined aetiology, antimicrobial susceptibility and clinical outcome of neonatal sepsis at Muhimbili National Hospital. METHODS: Three hundred and thirty neonates admitted at the Muhimbili National Hospital neonatal ward between October, 2009 and January, 2010 were recruited. Standardized questionnaires were used to obtain demographic and clinical information. Blood and pus samples were cultured on MacConkey, blood and chocolate agars and bacteria were identified based on characteristic morphology, gram stain appearance and standard commercially prepared biochemical tests. Antimicrobial sensitivity testing was performed for ampicillin, cloxacillin, gentamicin, amikacin, cefuroxime and ceftriaxone on Mueller Hinton agar using the Kirby Bauer diffusion method. RESULTS: Culture proven sepsis was noted in 24% (74/330) of the study participants. Isolated bacterial pathogens were predominantly Staphylococcus aureus, Klebsiella spp and Escherichia coli. Klebsiella spp 32.7% (17/52) was the predominant blood culture isolate in neonates aged below seven days while Staphylococcus aureus 54.5% (12/22) was commonest among those aged above seven days. Staphylococcus aureus was the predominant pus swabs isolate for both neonates aged 0-6 days 42.2% (98/232) and 7-28 days 52.3% (34/65). Resistance of blood culture isolates was high to ampicillin 81.1% (60/74) and cloxacillin 78.4% (58/74), moderate to ceftriaxone 14.9% (11/74) and cefuroxime 18.9% (14/74), and low to amikacin 1.3% (1/74). Isolates from swabs had high resistance to ampicillin 89.9% (267/297) and cloxacillin 85.2 (253/297), moderate resistance to ceftriaxone 38.0% (113/297) and cefuroxime 36.0% (107/297), and low resistance to amikacin 4.7% (14/297). Sepsis was higher in neonates with fever and hypothermia (p=0.02), skin pustules (p<0.001), umbilical pus discharge and abdominal wall hyperemia (p=0.04). Presence of skin pustules was an independent predictor of sepsis OR 0.26, 95% CI (0.10-0.66) p=0.004. The overall death rate was 13.9% (46/330), being higher in neonates with sepsis 24.3% (18/74) than those without 10.9% (28/256), p=0.003. CONCLUSIONS: Staphylococcus aureus was predominant isolate followed by Klebsiella and Escherichia coli. There was high resistance to ampicillin and cloxacillin. Mortality rate due to neonatal sepsis was high in our setting. Routine antimicrobial surveillance should guide the choice of antibiotics for empirical treatment of neonatal sepsis.

Determination of Sulphonamides and Tetracycline Residues in Liver Tissues of Broiler Chicken Sold in Kinondoni and Ilala Municipalities, Dar es Salaam, Tanzania.

In Tanzania, the increased demand for animal-derived foods, particularly eggs, meat, and milk, has resulted in the intensification of farming systems with the use of antimicrobials, particularly sulphonamides and tetracyclines. According to the FAO/WHO Codex Alimentarius commission, concentrations of antimicrobial residues in food exceeding the acceptable daily intake (ADI) and maximum residual limit (MRL) pose a health risk to consumers. This cross-sectional study determined the concentrations of sulphonamide and tetracycline residues in the liver tissues of commercial broiler chicken sold in Dar es Salaam, Tanzania, to find out whether the amounts of residues were within the legally permitted and acceptable limits in food. We conveniently sampled eighty-four liver tissue samples from broiler chicken sold in two out of six large markets in Dar es Salaam. The amounts of tetracycline and sulphonamide residues were determined using an ELISA kit (Shenzhen Lvshiyuan Biotechnology Company, Shenzhen, China). The results showed that all 100% (n = 84) samples contained tetracycline residues and 21.4% (n = 18) samples contained sulphonamide residues, while 21.4% (n = 18) contained both sulphonamide and tetracycline residues. The concentrations of sulphonamide residues were within the maximum residual limit (MRL). However, 90.5% (n = 76) of the samples had tetracycline levels that exceeded the acceptable daily intake (ADI) range 0-3 µg/kg and 13.1% (n = 11) of the samples had tetracycline levels that exceeded the maximum residue limit of 300 µg/kg. The observed presence of antibiotic residues in the poultry tissues poses a health risk to consumers, and may lead to antimicrobial resistance micro-organisms, which may spread to humans and animals via the environment. Vigorous surveillance and observation of the withdrawal periods should be advocated to ensure that the food from animals is safe with regard to the residues of veterinary medicines.

Antimicrobial Resistance Pattern of Escherichia coli Isolates from Small Scale Dairy Cattle in Dar es Salaam, Tanzania.

In Tanzania, information on antimicrobial resistance in small-scale dairy cattle is scarce. This cross-sectional study was conducted to determine the different levels and pattern of antimicrobial resistance (AMR), in 121 Escherichia coli isolated from rectal swab of 201 apparently healthy small-scale dairy cattle in Dar es Salaam, Tanzania. Isolation and identification of E. coli were carried out using enrichment media, selective media, and biochemical tests. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar (Merck), according to the recommendations of Clinical and Laboratory Standards Institute (CLSI). Resistance was tested against ampicillin, gentamicin, chloramphenicol, trimethoprim-sulfamethoxazole, tetracycline, nalidixic acid, ciprofloxacin and cefotaxime. Resistance to almost all antimicrobial agents was observed. The agents to which resistance was demonstrated most frequently were ampicillin (96.7%), cefotaxime (95.0%), tetracycline (50.4%), trimethoprim-sulfamethoxazole (42.1%) and nalidixic acid (33.1%). In this case, 20 extended-spectrum beta-lactamases (ESBLs) producing E. coli were identified. 74.4% (90/121) of the isolates were Multidrug resistant (MDR), ranging from a combination of three to 8 different classes. The most frequently observed phenotypes were AMP-SXT-CTX with a prevalence of 12.4%, followed by the combination AMP-CTX with 10.7% and TE-AMP-CTX and NA + TE + AMP + CTX with 8.3% each. The high prevalence and wide range of AMR calls for prudent antimicrobial use.

Comparative Performance of Line Probe Assay and GeneXpert in the Detection of Rifampicin Monoresistance in a TB-Endemic African Country.

Rapid, accurate and reliable assays are required for timely detection of drug-resistant tuberculosis and early initiation of second-line TB treatment as well as to minimize transmission of resistant strains. This study assessed diagnostic performance characteristics of two rapid molecular assays, line probe assay (LPA) and GeneXpert (MTB/RIF), in the detection rifampicin monoresistance using the phenotypic proportion method on Lowenstein−Jensen media as the gold standard. This study involved a total of 357 isolates, 74 rifampicin-resistant and 283 rifampicin-susceptible, collected at the Central Tuberculosis Reference Laboratory (CTRL) in Dar es Salaam, Tanzania, between 2016 and 2019. Sensitivity, specificity and positive and negative predictive values were used to assess the performance characteristics of the two assays while kappa coefficient was used to determine agreement of test results. The receiver operating curve (ROC) was used to determine the discriminatory ability of the test in distinguishing resistant and susceptible TB isolates. Our results showed that GeneXpert had sensitivity, specificity and positive and negative predictive values of 93.2, 82.7, 58.5 and 97.9%, respectively; the corresponding performance for LPA was 86.5, 97.5, 90.1 and 96.5%, respectively. Compared with conventional phenotypic DST results, GeneXpert had a moderate agreement (kappa 0.621, p < 0.001), while LPA had high agreement (0.853, p < 0.001). LPA showed an accuracy of 95.2% compared to GeneXpert's 84.9%. ROC curve depicted the ability of the tests to distinguish rifampicin-sensitive and rifampicin-resistant strains to be 87.9% for GeneXpert and 92.0% for LPA. Our results indicate the superiority of LPA over GeneXpert regarding detection of rifampicin monoresistance. However, logistic challenges such as longer turnaround time and need for skilled laboratory personnel may limit its use.

Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in South-Western Uganda.

BACKGROUND: Determination of the prevalence and drug susceptibility of the M. tuberculosis strains is important in tuberculosis control. We determined the genetic diversity and susceptibility profiles of mycobacteria isolated from tuberculosis patients in Mbarara, South Western Uganda. METHODS: We enrolled, consecutively; all newly diagnosed and previously treated smear-positive TB patients aged≥18 years. The isolates were characterized using regions of difference (RD) analysis and spoligotyping. Drug resistance against rifampicin and isoniazid were tested using the Genotype® MDRTBplus assay and the indirect proportion method on Lowenstein-Jensen media. HIV-1 testing was performed using two rapid HIV tests. RESULTS: A total of 125 isolates from 167 TB suspects (60% males) with a mean age 33.7 years and HIV prevalence of 67.9% (55/81) were analyzed. Majority (92.8%) were new cases while only 7.2% were retreatment cases. All the 125 isolates were identified as M. tuberculosis strict sense with the majority (92.8%) of the isolates being modern strains while seven (7.2%) isolates were ancestral strains. Spoligotyping revealed 79 spoligotype patterns, with an overall diversity of 63.2%. Sixty two (49.6%) of the isolates formed 16 clusters consisting of 2-15 isolates each. A majority (59.2%) of the isolates belong to the Uganda genotype group of strains. The major shared spoligotypes in our sample were SIT 135 (T2-Uganda) with 15 isolates and SIT 128 (T2) with 3 isolates. Sixty nine (87%) of the 79 patterns had not yet been defined in the SpolDB4.0.database. Resistance mutations to either RIF or INH were detected in 6.4% of the isolates. Multidrug resistance, INH and RIF resistance was 1.6%, 3.2% and 4.8%, respectively. The rpoß gene mutations seen in the sample were D516V, S531L, H526Y H526D and D516V, while one strain had a Δ1 mutation in the wild type probes. There were three strains with katG (codon 315) gene mutations only while one strain showed the inhA promoter gene mutation. CONCLUSION: The present study shows that the TB epidemic in Mbarara is caused by modern M. tuberculosis strains mainly belonging to the Uganda genotype and anti-TB drug resistance rate in the region is low.

Age specific aetiological agents of diarrhoea in hospitalized children aged less than five years in Dar es Salaam, Tanzania.

BACKGROUND: This study aimed to determine the age-specific aetiologic agents of diarrhoea in children aged less than five years. The study also assessed the efficacy of the empiric treatment of childhood diarrhoea using Integrated Management of Childhood Illness (IMCI) guidelines. METHODS: This study included 280 children aged less than 5 years, admitted with diarrhoea to any of the four major hospitals in Dar es Salaam. Bacterial pathogens were identified using conventional methods. Enzyme Linked Immunosorbent Assay (ELISA) and agglutination assay were used to detect viruses and intestinal protozoa, respectively. Antimicrobial susceptibility was determined using Kirby-Bauer disk diffusion method. RESULTS: At least one of the searched pathogens was detected in 67.1% of the cases, and mixed infections were detected in 20.7% of cases. Overall, bacteria and viruses contributed equally accounting for 33.2% and 32.2% of all the cases, respectively, while parasites were detected in 19.2% patients. Diarrhoeagenic Escherichia coli (DEC) was the most common enteric pathogen, isolated in 22.9% of patients, followed by Cryptosporidium parvum (18.9%), rotavirus (18.1%) and norovirus (13.7%). The main cause of diarrhoea in children aged 0 to 6 months were bacteria, predominantly DEC, while viruses predominated in the 7-12 months age group. Vibrio cholerae was isolated mostly in children above two years. Shigella spp, V. cholerae and DEC showed moderate to high rates of resistance to erythromycin, ampicillin, chloramphenicol and tetracycline (56.2-100%). V. cholerae showed full susceptibility to co-trimoxazole (100%), while DEC and Shigella showed high rate of resistance to co-trimoxazole; 90.6% and 93.3% respectively. None of the bacterial pathogens isolated showed resistance to ciprofloxacin which is not recommended for use in children. Cefotaxime resistance was found only in 4.7% of the DEC. CONCLUSION: During the dry season, acute watery diarrhoea is the most common type of diarrhoea in children under five years in Dar es Salaam and is predominantly due to DEC, C. parvum, rotaviruses and noroviruses. Constant antibiotic surveillance is warranted as bacteria were highly resistant to various antimicrobial agents including co-trimoxazole and erythromycin which are currently recommended for empiric treatment of diarrhoea.

Occurrence of Multidrug Resistant Escherichia coli in Raw Meat and Cloaca Swabs in Poultry Processed in Slaughter Slabs in Dar es Salaam, Tanzania.

This cross-sectional study was conducted between January and June 2020, in five large poultry slaughter slabs in Dar es Salaam, Tanzania. Purposive sampling was used to select broilers and spent layers, from which meat and cloaca swabs were collected to determine the occurrence of multidrug resistant (MDR) Escherichia coli. Identification of isolates was done using API 20E, and antimicrobial susceptibility testing was performed as per CLSI (2018) guidelines. EBSL (CTX-M, TEM, SHV) and plasmid mediated quinolone (qnrA, qnrB, qnrS and aac(6')-Ib-cr) were screened using PCR. Out of 384 samples, 212 (55.2%) were positive for E. coli, of which 147 (69.3%) were resistant to multiple drugs (MDR). Highest resistance was detected to tetracycline (91.9%), followed by sulfamethoxazole-trimethoprim (80.5%), ampicillin (70.9%), ciprofloxacin (40.2%) and 25% cefotaxime, gentamycin (10.8%) and imipenem (8.6%) (95% CI, p < 0.01). Out of the E. coli-positive samples, ten (10/212) (4.7%) were ESBL producing E. coli, of which CTX-M was detected in two isolates and quinolones resistant gene (qnrS) in eight, while TEM, SHV, qnrA, qnrB and aac(6')-lb-cr were not detected. The high level of resistance and multidrug resistance imply these antibiotics are ineffective, add unnecessary cost to poultry farmers and certainly facilitate emergence and spread of resistance.

Antimicrobial Use and Resistance in Agriculture and Food Production Systems in Africa: A Systematic Review.

In Africa, there is dearth of information on antimicrobial use (AMU) in agriculture and food production systems and its consequential resistance in pathogens that affect animal, human and environmental health. Data published between 1980 and 2021 on the magnitude of AMU and AMR in agriculture and food productions systems in Africa were reviewed. Data from 13-27 countries in Africa indicate that 3558-4279 tonnes of antimicrobials were used in animals from 2015 to 2019. Tetracyclines and polypeptides contributed the largest proportion of antimicrobials used. Cattle and poultry production account for the largest consumption of antimicrobials in Africa. Although limited studies have reported AMR in crops, fish and beekeeping, AMR from a variety of farm animals has been substantially documented in Africa. Some countries in Africa have developed policies/plans to address AMU and AMR in agriculture and food production systems; however, their enforcement is challenged by weak regulations. In conclusion, although there is limited information on the quantities of antimicrobials used in agriculture and food production system, the levels of AMR are high. There is a need to strengthen regulatory authorities with a capacity to monitor AMU in agriculture and food production systems in Africa.