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      Genetic diversity and drug susceptibility profiles of Mycobacterium tuberculosis obtained from Saint Peter’s TB specialized Hospital, Ethiopia

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          Abstract

          Background

          Tuberculosis (TB) is one of the major public health problems in Ethiopia. Data on genetic diversity and resistance profile of circulating TB strains is critical for informing the national TB control program.

          Methods

          A cross-sectional study was conducted on 213 smear positive pulmonary TB patients between 2015 and 2016. Sputum samples were cultured on LJ media following the Petroff’s method. Region of difference-9 (RD9)-deletion typing and spoligo-typing were performed for molecular analysis of M. tuberculosis at species and strain levels, respectively. Drug sensitivity and mutation patterns of the isolates were assessed by the conventional indirect proportion method and molecular line probe assays (LPAs), respectively. Data were analyzed using statistical package for social sciences (SPSS) software version 20.

          Results

          Spoligo-typing of 150 M. tuberculosis isolates led to 57 different patterns of which 25 were new strains. The majority (71.6%) of the isolates were grouped in to 17 clusters consisting 2 to 24 isolates. The majority of the strains belonged to Euro-American lineage and the predominant spoligotypes were SIT 37 and SIT 149. MDR-TB was detected in 5.2% and 20.3% of new and retreatment cases, respectively. Two MDR-TB isolates exhibited additional resistance to one of the second line anti-TB drugs. Common gene mutations including S531L, S315T1 and M306V were detected in RIF, INH and EMB resistant strains, respectively.

          Conclusions

          The identification of several new strains, higher proportion of MDR-TB and higher clustering rate in this study, warrants the need for re-enforcement of the national TB control program. The detection of common gene mutations in the majority drug resistant strains might suggest the feasibility of LPAs for rapid screening of drug resistant M. tuberculosis strains in Ethiopia.

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          Most cited references25

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          Risk factors for multidrug resistant tuberculosis in Europe: a systematic review.

          The resurgence of tuberculosis (TB) in western countries has been attributed to the HIV epidemic, immigration, and drug resistance. Multidrug resistant tuberculosis (MDR-TB) is caused by the transmission of multidrug resistant Mycobacterium tuberculosis strains in new cases, or by the selection of single drug resistant strains induced by previous treatment. The aim of this report is to determine risk factors for MDR-TB in Europe. A systematic review was conducted of published reports of risk factors associated with MDR-TB in Europe. Meta-analysis, meta-regression, and sub-grouping were used to pool risk estimates of MDR-TB and to analyse associations with age, sex, immigrant status, HIV status, occurrence year, study design, and area of Europe. Twenty nine papers were eligible for the review from 123 identified in the search. The pooled risk of MDR-TB was 10.23 times higher in previously treated than in never treated cases, with wide heterogeneity between studies. Study design and geographical area were associated with MDR-TB risk estimates in previously treated patients; the risk estimates were higher in cohort studies carried out in western Europe (RR 12.63; 95% CI 8.20 to 19.45) than in eastern Europe (RR 8.53; 95% CI 6.57 to 11.06). National estimates were possible for six countries. MDR-TB cases were more likely to be foreign born (odds ratio (OR) 2.46; 95% CI 1.86 to 3.24), younger than 65 years (OR 2.53; 95% CI 1.74 to 4.83), male (OR 1.38; 95% CI 1.16 to 1.65), and HIV positive (OR 3.52; 95% CI 2.48 to 5.01). Previous treatment was the strongest determinant of MDR-TB in Europe. Detailed study of the reasons for inadequate treatment could improve control strategies. The risk of MDR-TB in foreign born people needs to be re-evaluated, taking into account any previous treatment.
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            Mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results.

            The objectives of this study were to investigate the origin of highly discordant rifampin (rifampicin) (RMP) drug susceptibility test results obtained for Mycobacterium tuberculosis strains during proficiency testing. Nine Supra-National Tuberculosis Reference Laboratories tested the RMP susceptibilities of 19 selected M. tuberculosis strains, using standard culture-based methods. The strains were classified as definitely resistant (R) (n = 6) or susceptible (S) (n = 2) or probably resistant (PR) (n = 8) or susceptible (PS) (n = 3) based on rpoB mutations and treatment outcome. All methods yielded a susceptible result for the two S and three PS strains lacking an rpoB mutation and a resistant result for one R strain with a Ser531Leu mutation and one PR strain with a double mutation. Although the remaining 12 R and PR strains had rpoB mutations (four Asp516Tyr, three Leu511Pro, two Leu533Pro, one each His526Leu/Ser, and one Ile572Phe), they were all susceptible by the radiometric Bactec 460TB or Bactec 960 MGIT methods. In contrast, only one was susceptible by the proportion method on Löwenstein-Jensen medium and two on Middlebrook 7H10 agar. Low-level but probably clinically relevant RMP resistance linked to specific rpoB mutations is easily missed by standard growth-based methods, particularly the automated broth-based systems. Further studies are required to confirm these findings, to determine the frequency of these low-level-resistant isolates, and to identify technical improvements that may identify such strains.
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              PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions.

              The classical Mycobacterium tuberculosis complex (MtbC) subspecies include Mycobacterium tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M. bovis bacillus Calmette-Guérin [BCG]), and Mycobacterium microti; increasingly recognized MtbC groupings include Mycobacterium bovis subsp. caprae and "Mycobacterium tuberculosis subsp. canettii." Previous investigations have documented each MtbC subspecies as a source of animal and/or human tuberculosis. However, study of these organisms is hindered by the lack of a single protocol that quickly and easily differentiates all of the MtbC groupings. Towards this end we have developed a rapid, simple, and reliable PCR-based MtbC typing method that makes use of MtbC chromosomal region-of-difference deletion loci. Here, seven primer pairs (which amplify within the loci 16S rRNA, Rv0577, IS1561', Rv1510, Rv1970, Rv3877/8, and Rv3120) were run in separate but simultaneous reactions. Each primer pair either specifically amplified a DNA fragment of a unique size or failed, depending upon the source mycobacterial DNA. The pattern of amplification products from all of the reactions, visualized by agarose gel electrophoresis, allowed immediate identification either as MtbC composed of M. tuberculosis (or M. africanum subtype II), M. africanum subtype I, M. bovis, M. bovis BCG, M. caprae, M. microti, or "M. canettii" or as a Mycobacterium other than MtbC (MOTT). This MtbC PCR typing panel provides an advanced approach to determine the subspecies of MtbC isolates and to differentiate them from clinically important MOTT species. It has proven beneficial in the management of Mycobacterium collections and may be applied for practical clinical and epidemiological use.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: MethodologyRole: ValidationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: MethodologyRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: MethodologyRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: ValidationRole: Visualization
                Role: Data curationRole: MethodologyRole: ValidationRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: MethodologyRole: SupervisionRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: SupervisionRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draft
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                24 June 2019
                2019
                : 14
                : 6
                : e0218545
                Affiliations
                [1 ] Department of Microbiology and Parasitology, School of Medicine, College of Health Science Addis Ababa University, Addis Ababa, Ethiopia
                [2 ] Aklilu Lemma Institute of Pathobiology, College of Health Sciences, Addis Ababa, Ethiopia
                [3 ] Division of Human Genetics, Department of Pathology, Institute of Infectious Disease and Molecular Medicine, University Cape Town, Cape Town, South Africa
                [4 ] Saint Peter’s TB specialized Hospital, Addis Ababa, Ethiopia
                [5 ] Armauer Hansen Research Institute, Addis Ababa, Ethiopia
                Jamia Hamdard, INDIA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-4594-713X
                Article
                PONE-D-19-01179
                10.1371/journal.pone.0218545
                6590806
                31233535
                da78b324-fc06-4742-b953-597d92960de2
                © 2019 Damena et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 14 January 2019
                : 4 June 2019
                Page count
                Figures: 0, Tables: 4, Pages: 12
                Funding
                We would like to thank the DELTAS Africa Initiative, Addis Ababa University and St. Peter’s TB specialized Hospital for their financial and technical supports. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Bacteria
                Actinobacteria
                Mycobacterium Tuberculosis
                Medicine and Health Sciences
                Infectious Diseases
                Bacterial Diseases
                Tuberculosis
                Medicine and Health Sciences
                Tropical Diseases
                Tuberculosis
                Medicine and Health Sciences
                Infectious Diseases
                Bacterial Diseases
                Tuberculosis
                Extensively Drug-Resistant Tuberculosis
                Medicine and Health Sciences
                Tropical Diseases
                Tuberculosis
                Extensively Drug-Resistant Tuberculosis
                People and Places
                Geographical Locations
                Africa
                Ethiopia
                Medicine and Health Sciences
                Infectious Diseases
                Bacterial Diseases
                Tuberculosis
                Multi-Drug-Resistant Tuberculosis
                Medicine and Health Sciences
                Tropical Diseases
                Tuberculosis
                Multi-Drug-Resistant Tuberculosis
                Medicine and Health Sciences
                Pharmacology
                Drug Screening
                Medicine and Health Sciences
                Pharmacology
                Drug Information
                Biology and Life Sciences
                Genetics
                Gene Identification and Analysis
                Mutation Detection
                Custom metadata
                All relevant data are within the manuscript and its Supporting Information files.

                Uncategorized
                Uncategorized

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