Isolation, biochemical and molecular identification of Nocardia species among TB suspects in northeastern, Tanzania; a forgotten or neglected threat?

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.

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.

Existence of Multiple ESBL Genes among Phenotypically Confirmed ESBL Producing Klebsiella pneumoniae and Escherichia coli Concurrently Isolated from Clinical, Colonization and Contamination Samples from Neonatal Units at Bugando Medical Center, Mwanza, Tanzania.

The proportions and similarities of extended-spectrum ß-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 methods 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.

Bacteremia in critical care units at Bugando Medical Centre, Mwanza, Tanzania: the role of colonization and contaminated cots and mothers' hands in cross-transmission of multidrug resistant Gram-negative bacteria.

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 sub-cultured 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 MDR-GNB. Further, clinical presentations and water use should be considered in administration of empirical therapy whilst awaiting culture results.

Molecular characterization of Mycobacterium tuberculosis isolates from Tanga, Tanzania: First insight of MIRU-VNTR and microarray-based spoligotyping in a high burden country.

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, microarrays­based spoligotyping and mycobacterial interspersed repetitive unit­variable number of tandem repeats (MIRU­VNTR) were applied on 80 isolates collected from primary health care facilities in Tanga, North­eastern 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 super­family (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 MIRU­VNTR 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. Microarray­based spoligotyping and MIRU­VNTR could be reliable tools in detecting different MTB genotypes in high burden settings.

Increased isolation of nontuberculous mycobacteria among TB suspects in Northeastern, Tanzania: public health and diagnostic implications for control programmes.

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, GenoType(®) 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 GenoType(®) 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 CM/AS. 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.

Anti-TB drug resistance in Tanga, Tanzania: A cross sectional facility-base prevalence among pulmonary TB patients.

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 MDR-TB) 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.