|Call||EDCTP-AREF Preparatory Fellowships (PF)|
TMA2017 PF Abubakar Shaaban
To determine the utility of whole-genome sequencing in predicting for drug resistance genotypes, identifying and characterizing adaptive mutations in the evolved genotypes, and approaches to study the genetic constraints on the evolution of resistance amongst M. Tuberculosis, and S. aureus isolates in Tanzania. Specifically the study aims: i. To isolate M. Tuberculosis and S. aureus isolates from TB presumptive patients attending peripheral TB diagnostic centres in Dar es Salaam, Tanzania. ii. To analyse M. Tuberculosis complex genotypes and to characterize adaptive mutations and predict their drug resistance profiles using Next Generation Sequencing. iii. To analyse S. aureus isolates genotypes and to characterize their adaptive mutations and predict their resistance profiles by using Next Generation Sequencing. iv. To investigate on the utility of Next Generation Sequencing as a routine diagnostic tool for prediction of drug resistant isolates in TB control programmes in resource limited settings of Tanzania.
A prospective population-based surveillance will be conducted in three urban hospitals in Dar es Salaam, Tanzania namely, Mwanayamala Hospital in Kinondoni district, Amana Hospital in Ilala district and Temeke Hospital in Temeke district.
The emergence of methicillin-resistant Staphylococcus aureus (MRSA) creates a serious public
health concern due to its ability to colonize and infect humans and animals. This cross-
sectional study investigated the prevalence, antibiotic susceptibility pattern of S. aureus and
MRSA isolated from bovine raw milk in the Njombe region, Tanzania. A total of 470 samples,
including 389 raw milk samples collected at farm level, 57 raw milk samples from bulk milk-can
at collection centers, and 24 swab samples from bulk milk cans. The samples were cultured on
mannitol salt agar, presumptive colonies were sub-cultured onto blood agar for the isolation
of S. aureus which was subsequently preliminarily conﬁrmed using microbiological and bio-
chemical tests. Further, conﬁrmation of isolates was done using conventional PCR targeting
the gltB gene for S. aureus and mecA gene for MRSA which was later sequenced. Isolates
were tested for antibiotic susceptibility by using the disc agar diﬀusion method. The overall
prevalence of S. aureus in the study was 22.6% (106/470), with 2.9% (14/470) being MRSA.
Both S. aureus and MRSA showed high resistance to penicillin (74%, 8.5%) and ampicillin
(78%, 11.3%), respectively. A total of 81 (77%) isolates were resistant to at least one antibiotic
and 14 isolates (13.2%) showed multidrug-resistant (MDR); with frequent antibiotic resistance
patterns being to cefoxitin, penicillin, ciproﬂoxacin, tetracycline, and erythromycin. In con-
clusion, the prevalence and the MDR patterns exhibited by S. aureus and MRSA observed in
this study provide baseline data for planning mitigation measures to safeguard public health
The proportions and similarities of extended-spectrum -lactamase (ESBL) producing K. pneumoniae (ESBL-KP) and E. coli (ESBL-EC) carrying multiple ESBL genes is poorly known at our setting. This study investigated the existence of multiple ESBL genes (blaCTX-M, blaTEM, and blaSHV) among ESBL-KP and ESBL-EC concurrently isolated from clinical, colonization, and contamination samples from neonatology units in Mwanza-Tanzania. Twenty and 55 presumptive ESBL-EC and ESBL-KP, respectively, from a previous study archived at 80 C were successfully recovered for this study. Isolates were screened and confirmed for production of ESBLs by phenotypic meth ds followed by multiplex PCR assay to determine ESBL genes. All (100%) and 97.3% of presumptive ESBL isolates were phenotypically confirmed by Clinical and Laboratory Standards Institute (CLSI) and modified double-disc synergy methods, respectively. About 93.3% (70/75) of phenotypically confirmed ESBL isolates had at least one ESBL gene, whereby for 62.9% (44/70), all ESBL genes (blaCTX-M, blaTEM, and blaSHV) were detected. Eight pairs of ESBL bacteria show similar patterns of antibiotics susceptibility and ESBL genes. ESBL-KP and ESBL- EC, concurrently isolated from clinical, colonization and contamination samples, harbored multiple ESBL genes. Further, eight pairs of ESBL isolates had similar patterns of antibiotics susceptibility and ESBL genes, suggesting transmission of and/or sharing of mobile genetic elements (MGEs) among ESBL-KP and ESBL-EC.
Objective: To determine the prevalence and risk factors associated with drug resistance
tuberculosis (TB) at facility-base level in Tanga, Tanzania.
Methods: A total of 79 Mycobacterium tuberculosis (MTB) isolates included in the
study were collected from among 372 (312 new and 60 previously treated) TB suspects
self-referred to four TB clinics during a prospective study conducted from November
2012 to January 2013. Culture and drug susceptibility test of the isolates was performed
at the institute of medical microbiology and epidemiology of infectious diseases, University
hospital, Leipzig, Germany. Data on the patient's characteristics were obtained
from structured questionnaire administered to the patients who gave informed verbal
consent. Unadjusted bivariate logistic regression analysis was performed to assess the risk
factors for drug resistant-TB. The significance level was determined at P < 0.05.
Results: The overall proportions of any drug resistance and MDR-TB were 12.7% and
6.3% respectively. The prevalence of any drug resistance and MDR-TB among new cases
were 11.4% and 4.3% respectively, whereas among previously treated cases was 22.2%
respectively. Previously treated patients were more likely to develop anti-TB drug
resistance. There was no association between anti-TB drug resistances (including MDRTB)
with the risk factors analysed.
Conclusions: High proportions of anti-TB drug resistance among new and previously
treated cases observed in this study suggest that, additional efforts still need to be done in
identifying individual cases at facility-base level for improved TB control programmes
and drug resistance survey should continuously be monitored in the country.
Increased resistance of Staphylococcus aureus isolates to existing antimicrobials constitutes a major concern in human and veterinary medicine. This study aimed at determining the prevalence, antimicrobial susceptibility proles, and molecular characteristics of S. aureus from raw bovine milk in dairy and pastoral farms in Morogoro urban and Mvomero districts, Tanzania. In a cross-sectional study, 397 raw bovine milk samples were collected and carried to the laboratory. Conventional Gram staining, colony morphology on blood agar, and mannitol salt agar, along with biochemical tests, were used for S. aureus identication. Antimicrobial susceptibility testing (AST) was performed using the disk diusion method, while multiplex polymerase chain reaction (PCR) was used to detect virulence and antimicrobial resistance genes. Data were analyzed using Epi Info (Version 7). Out of the 397 samples, S. aureus was conrmed in 124 (31.2%). Contamination of raw bovine milk by S. aureus in the study area was associated with poor milking hygienic measures. The AST revealed that all S. aureus isolates were susceptible to chloramphenicol and cefoxitin, while the highest resistance 116/124 (93.5%) was noticed for penicillin. Resistance to other antimicrobials varied between 1.6-28.2%. Of the 124 S. aureus isolates, 80 (64.5%) possessed spa gene, with 76/80(95.0%) harboring more than seven tandem repeats. One of the S. aureus isolates, 1/124 (0.8%), harbored a mecA resistance gene. The presence of antimicrobial-resistant S. aureus isolates in raw bovine milk at the farm level is alarming and requires herd health improvement interventions to protect society.
Background: Multidrug resistance (MDR) is a major clinical problem in tertiary hospitals in Tanzania and jeopardizes the life of neonates in critical care units (CCUs). To better understand methods for prevention of MDR infections, this study aimed to determine, among other factors, the role of MDR-Gram-negative bacteria (GNB) contaminating neonatal cots and hands of mothers as possible role in transmission of bacteremia at Bugando Medical Centre (BMC), Mwanza, Tanzania.
Methods: This cross-sectional, hospital-based study was conducted among neonates and their mothers in a neonatal intensive care unit and a neonatology unit at BMC from December 2018 to April 2019. Blood specimens (n=200) were subcultured on 5% sheep blood agar (SBA) and MacConkey agar (MCA) plates. Other specimens (200 neonatal rectal swabs, 200 maternal hand swabs and 200 neonatal cot swabs) were directly inoculated on MCA plates supplemented with 2 μg/ml cefotaxime (MCA-C) for screening of GNB resistant to third generation cephalosporins, r-3GCs. Conventional biochemical tests, Kirby-Bauer technique and resistance to cefoxitin 30 μg were used for identification of bacteria, antibiotic susceptibility testing and detection of MDR-GNB and screening of potential Amp-C beta lactamase producing GNB, respectively.
Results: The prevalence of culture confirmed bacteremia was 34.5% of which 85.5% were GNB. Fifty-five (93.2%) of GNB isolated from neonatal blood specimens were r-3GCs. On the other hand; 43% of neonates were colonized with GNB r-
3GCs, 32% of cots were contaminated with GNB r-3GCs and 18.5% of hands of neonates’ mothers were contaminated with GNB r-3GCs. The prevalences of MDR-GNB isolated from blood culture and GNB r-3GCs isolated from neonatal colonization, cots and mothers’ hands were 96.6, 100, 100 and 94.6%, respectively. Significantly, cyanosis (OR[95%CI]: 3.13[1.51–6.51], p = 0.002), jaundice (OR[95%CI]: 2.10[1.07–4.14], p = 0.031), number of invasive devices (OR[95%CI]: 2.52[1.08–5.85], p = 0.031) and contaminated cot (OR[95%CI]: 2.39[1.26–4.55], p = 0.008) were associated with bacteremia due to GNB. Use of tap water only (OR[95%CI]: 2.12[0.88–5.09], p = 0.040) was protective for bacteremia due to GNB.
Conclusion: High prevalence of MDR-GNB bacteremia and intestinal colonization, and MDR-GNB contaminating cots and mothers’ hands was observed. Improved cots decontamination strategies is crucial to limit the spread of MDRGNB. Further, clinical presentations and water use should be considered in administration of empirical therapy whilst awaiting culture results.
Molecular typing of Mycobacterium tuberculosis(MTB) has greatly enhanced the understanding of the
population structure of MTB isolates and epidemiology of tuberculosis (TB). To characterize prevalent
genotypes of MTB, microarrays‑based spoligotyping and mycobacterial interspersed repetitive unit‑variable
number of tandem repeats (MIRU‑VNTR) were applied on 80 isolates collected from primary
health care facilities in Tanga, North‑eastern Tanzania. A total of 18 distinct spoligotypes were identified.
The lineages by order of their predominance were EAI and CAS families (26.25%, 21 isolates each), LAM
family and T super‑family (10%, 8 isolates each), MANU family (3.75%, 3 isolates), Beijing family (2.5%, 2
isolates) and S family (1.25%, 1 isolate). Overall, sixteen (20%) strains could not be allocated to any lineage
according to the SITVIT_WEB database. The allelic diversity (h) for specific MIRU‑VNTR loci showed a
considerable variation ranging from 0.826 of VNTR locus 3192 to 0.141 of VNTR locus 2059. The allelic
diversity for 11 loci (VNTR 3192, 2996, 2165, 960, 4052, 424, 4156, 2531, 1644, 802 and 3690) exceeded
0.6, indicating highly discriminatory power. Seven loci (VNTR 2163b, 2401, 1955, 577, 4348, 2687 and
580) showed moderate discrimination (0.3 h 0.6), and three loci (VNTR3007, 154 and 2059) were less
polymorphic. The present study suggests that the TB cases in Tanga might be caused by a diverse array of
MTB strain families that may be indicative of a cosmopolitan population with frequent migration and
travel. Microarray‑based spoligotyping and MIRU‑VNTR could be reliable tools in detecting different MTB
genotypes in high burden settings.
Background: The role of nontuberculous mycobacteria (NTM) in tuberculosis (TB) diagnosis is well documented in many developing settings. However, this has not been the case in many resource poor settings like Tanzania. This study aimed at understanding the role of NTM in the diagnosis and management of TB in resource poor settings of Tanzania.
Methods: A cross-sectional study was conducted in Tanga, Tanzania. Patients with symptoms suggestive of TB self-referred to health care facilities were recruited. Two sputum samples were collected for standard direct smear microscopy. Culture was performed using BacT/Alert 3D system, Löwenstein-Jensen and Gottsacker slopes. Identification of Mycobacterium tuberculosis and NTM was done by using GenoType®MTBC and GenoType®CM/AS, respectively.
Results: A total of 372 patients were involved in the study. Eighty-one (21.8%) patients were diagnosed as having M. tuberculosis by the isolation of the organism from cultures of sputum. Further analysis of culture showed that 8.1% (30/372) were NTM with 7/372 (1.9%) cases of NTM classified as pulmonary tuberculosis (PTB) patients. Ziehl Neelsen stain had a sensitivity of 68.8% and produced 10 false negative results. On the other hand, Fluorescence stain had a sensitivity of 85.7% and gave seven false negative samples when compared with culture results. Weight loss (p = 0.0001), fatigue (p = 0.003), fever (p = 0.038) and night sweats (p = 0.004), young population (18-40 years) (p = 0.0352), males (p = 0.0025) were important risk factors for TB. Four out of 30 NTM diagnosed by culture received first line anti-TB treatment suggesting that a good proportion of patients (4/65, 6.2%) were mistreated as TB patients.
Conclusion: Inefficient screening of TB patients in resource poor settings and prevalent increase of NTM may contribute to over diagnosis of TB cases. The need to integrate NTM diagnosis in the routine management of TB is urgently needed for designing effective tuberculosis prevention and control strategies in the country.
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(60)-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 sulfamethoxazoletrimethoprim (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(60)-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.
SETTING: A national tuberculosis (TB) drug resistance survey in Tanzania.
OBJECTIVE: To compare the performance of the Genotype® MTBDRplus line-probe assay (LPA) on smear-positive sputum specimens with conventional culture and isoniazid (INH) plus rifampicin (RMP) drug susceptibility testing (DST).
DESIGN: Mycobacterium tuberculosis isolates tested at the Tanzanian Central TB Reference Laboratory (CTRL) were submitted for quality assurance of phenotypic DST to its supranational reference laboratory (SRL), together with ethanol-preserved sputum specimens for LPA DST.
RESULTS: Only 321 samples could be tested using LPA; of these, three were identified as being non-tuberculous mycobacteria using CTRL DST. Both tests had 269 sets with interpretable results. CTRL DST yielded almost the same number of interpretable results as LPA, with 90% concordance (κ = 0.612, P < 0.001). Five (1.9%) multidrug-resistant (MDR) strains, 46 (17.1%) resistant to INH only and 0 RMP only, were found by CTRL DST. For the LPA, these results were respectively 5 (1.9%), 26 (9.7%) and 2 (0.7%). With SRL DST as the gold standard, LPA was more accurate than CTRL DST for RMP, but missed almost half the INH-resistant samples.
CONCLUSION: LPA applied directly on ethanol-preserved sputum specimens was similar to phenotypic DST in terms of yield of interpretable results. Although probably more accurate for RMP and MDR-TB, it appears to seriously underestimate INH resistance. Considering speed, easy and safe specimen transportation and low infrastructure requirements, LPA DST from sputum can be recommended for surveys in resource-poor settings.
Background: Pulmonary nocardiosis mimic pulmonary tuberculosis in most clinical and radiological manifestations.
In Tanzania, where tuberculosis is one of the major public health threat clinical impact of nocardiosis as the cause
of the human disease remains unknown. The objective of the present study was to isolate and identify Nocardia
isolates recovered from TB suspects in Northeastern, Tanzania by using biochemical and molecular methods.
Methods: The study involved 744 sputum samples collected from 372 TB suspects from four periphery diagnostic
centers in Northeastern, Tanzania. Twenty patients were diagnosed as having presumptively Nocardia infections
based on microscopic, cultural characteristics and biomèrieux ID 32C Yeast Identification system and confirmed
using 16S rRNA and hsp65 gene specific primers for Nocardia species and sequencing.
Results: Biochemically, the majority of the isolates were N. asteroides (n = 8/20, 40%), N. brasiliensis (n = 4/20, 20%),
N. farcinica (n = 3/20, 15%), N. nova (n = 1/20, 5%). Other aerobic actinomycetales included Streptomyces cyanescens
(n = 2/20, 10%), Streptomyces griseus, Actinomadura madurae each (n = 1/20, 5%). Results of 16S rRNA and hsp65
sequencing were concordant in 15/17 (88. 2%) isolates and discordant in 2/17 (11.8%) isolates. Majority of the
isolates belonged to N. cyriacigeorgica and N. farcinica, four (23.5%) each.
Conclusions: Our findings suggest that Nocardia species may be an important cause of pulmonary nocardiosis that
is underdiagnosed or ignored. This underscores needs to consider pulmonary nocardiosis as a differential diagnosis
when there is a failure of anti-TB therapy and as a possible cause of human infections.
Background: Non-tuberculous mycobacteria (NTM) are increasingly reported worldwide associated with human disease.
Defining the significance of NTM in settings with endemic tuberculosis (TB) requires the discrimination of NTM
from TB in suspect patients. Correct and timely identification of NTM will impact both therapy and epidemiology of TB
and TB-like diseases. The present study aimed at determining the frequency and diversity of NTM among TB suspects
in northeastern Tanzania.
Methods: A cross-sectional study was conducted between November 2012 through January 2013. Seven hundred
and forty-four sputum samples were collected from 372 TB suspects. Detection was done by using phenotypic, Geno-
Type® Mycobacterium CM/AS kits, 16S rRNA and hsp65 gene sequencing for identification of isolates not identified by
Hain kits. Binary regression model was used to analyse the predictors of NTM detection.
Results: The prevalence of NTM was 9.7 % of the mycobacterial isolates. Out of 36 patients with confirmed NTM
infection, 12 were HIV infected with HIV being a significant predictor of NTM detection (P < 0.001). Co-infection with
Mycobacterium tuberculosis (M. tb) was found in five patients. Twenty-eight NTM isolates were identified using Geno-
Type® Mycobacterium CM/AS and eight isolates could not be identified. Identified species included M. gordonae and
M. interjectum 6 (16.7 %), M. intracelullare 4 (11.1 %), M. avium spp. and M. fortuitum 2 (5.5 %), M. kansasii, M. lentiflavum,
M. simiae, M. celatum, M. marinum 1 (2.8 %) each. Of isolates not identified to subspecies level, we identified M. kumamotonense
(2), M. intracellulare/kansasii, M. intermedium/triplex, M. acapulcensis/flavescens, M. stomatepiae, M. colombiense
and M. terrae complex (1) each using 16S rRNA sequencing. Additionally, hsp65 gene sequencing identified M.
kumamotonense, M. scrofulaceum/M. avium, M. avium, M. flavescens/novocastrense, M. kumamotonense/hiberniae, M.
lentiflavum, M. colombiense/M. avium and M. kumamotonense/terrae/hiberniae (1) each. Results of the 16S rRNA and
hsp65 gene sequencing were concordant in three and discordant in five isolates not identified by GenoType® Mycobacterium
Conclusion: NTM infections may play a vital role in causing lung disease and impact management of TB in endemic
settings. GenoType® Mycobacterium CM/AS represents a useful tool to identify clinical NTM infections. However, 16S
rRNA gene sequencing should be thought for confirmatory diagnosis of the clinical isolates. Due to the complexity
and inconsistence of NTM identification, we recommend diagnosis of NTM infections be centralized by strengthening
and setting up quality national and regional infrastructure.
In Zambia, poultry is a rapidly increasing sector contributing 4.8% of the Agricultural Gross
Domestic Product (GDP), thus providing a signicant income-generating activity. Worldwide,
poultry is a major reservoir of Salmonella with an increasing incidence of extended-spectrum
beta-lactamase (ESBL) producing strains. ESBLs are enzymes produced by bacteria and are
capable of inactivating a wide range of beta-lactam antibiotics. Salmonella enterica serovars
Enteritidis and Typhimurium are the most common food-borne serotypes in many countries,
infecting both humans and animals and are transmitted to humans through the food supply
chain. CTX-M ESBLs have been described in Salmonella Typhimurium isolates with resistant
genes located on transferable plasmids. This study aimed to detect S. Typhimurium, their an-
timicrobial resistance, and CTX-M-type ESBL producing strains in commercial poultry farms
in Copperbelt province, Zambia. Five districts were considered for this study, where one poul-
try farm per district was randomly selected for sampling. An overall number of 384 fecal
samples were analyzed using microbiological and molecular methods. S. Typhimurium was
detected at 17.7% (CI: 14.2%-21.8%) in commercial poultry farms in Copperbelt province,
of which 12.8% (CI: 9.8%-16.5%) were found harboring the CTX-M-type ESBL genes. S.
Typhimurium isolates showed 88.2% resistance to at least one antimicrobial compound. All
the isolates showed 100% resistance to tetracycline, followed by ampicillin and amoxicillin at
91.2%. These isolates also showed 58.8% resistance to cefotaxime and 54.4% to ceftazidime.
Detection of CTX-M ESBL producing S. Typhimurium suggests the contamination of chicken
food chain at farm level and calls for public health protection measures.
|Augustino Chengula||Tanzania, United Republic of||Sokoine University of Agriculture|
|Abubakar Hoza||Tanzania, United Republic of||Sokoine University of Agriculture|
|George Sanga||Tanzania, United Republic of||Sokoine University of Agriculture|