Browsing by Author "Stray-Pedersen, Babill"
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- ItemDiagnosis of Multi-drug Resistant Tuberculosis Mutations Using Hain Line Probe Assay and GeneXpert: A Study Done in Zimbabwe(SCIENCEDOMAIN international, 2015) Dube-Mandishora, Rachea, S.; Dhlamini, Zephaniah; Mutetwa, Reggie; Duri, Kerina; Mason, Peter; Stray-Pedersen, BabillBackground and Aims: Tuberculosis (TB) is a global public health problem and one of the leading causes of death. Worldwide, 31% of all estimated new TB cases are from Africa. Zimbabwe is one of the 22 high TB burden countries. Multi-drug resistant TB (MDR-TB) poses challenges in TB control, hence the need for rapid laboratory diagnosis of MDR-TB for optimal treatment and reducing spread. The study aim was to investigate genetic mutations associated with MDR-TB isolates from various Harare clinics using the GeneXpert MTB/RIF® by Cepheid and Genotype MTBDRplus, to improve the diagnosis and management of MDR-TB. Methods: Samples from adults aged 16 years and older, recruited from several polyclinics in the southern suburbs of Harare were used for our study. All laboratory tests prior to this study had been carried out at Biomedical research and training institute’s level three bio-safety TB laboratory from January 2008-August 2012. Ethical approval was sought from BRTI Institutional review board. A total of 69 (37 MDR-TB and 32 non MDR-TB) archived isolates processed on Genotype MTBDRplus (Hains) and corresponding 39 sputum were processed on the GeneXpert. Mutations on rpoB, katG and inhA genes were observed. The gold standard was culture. Diagnostic accuracy of both methods and their level of agreement were calculated. Results: Of the 37/69 isolates screened by culture for MDR-TB, 88.4% were confirmed by MTBDR® plus line probe assay (Hains). Within the 39 isolates tested using the Xpert MTB/RIF (GeneXpert) assay 12 were true MDR-TB. Over 8 single nucleotide polymorphisms were observed on the three genes conferring Rifampicin and Isoniazide drug resistance. The Hains and GeneXpert had an almost perfect agreement with a kappa value of 0.82. Conclusion: Genetic markers can be used in the diagnosis of MDR-TB, to complement phenotypic methods such as culture. Using the commercial methods, Hains and GeneXpert, 88.4- 94.2% of drug resistance maybe detected. Furthermore, we recommend sequencing so as to identify novel mutations and to design a kit that is custom made for the population.
- ItemMutations in rpoB and katG genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods(African Field Epidemiology Network, 2017-06-28) Takawira, Faustinos Tatenda; Mandishora, Racheal S. D.; Dhlamini, Zephaniah; Munemo, Ellen; Stray-Pedersen, BabillIntroduction: Tuberculosis remains the leading causes of death worldwide with frequencies of mutations in rifampicin and isoniazid resistant Mycobacterium tuberculosis isolates varying according to geographical location. There is limited information in Zimbabwe on specific antibiotic resistance gene mutation patterns in MTB and hence, increased rate of discordant results and mortality due to inappropriate antibiotic prescriptions. The rpoB and katG genes molecular markers are used for detecting rifampicin and isoniazid resistance respectively. Some mutations within these gene sequences are associated with drug resistance as they directly alter gene function. The objectives of this research was to determine the drug resistance profiles in M. tuberculosis isolates that are phenotypically resistant but not detected by the GeneXpert and MTBDRplus kit and also to detect mutations in the rpoB and katG genes which are not detected by the Hain Genotype MTBDRplus kit and GeneXpert diagnosis. Methods: PCR was used for the amplification of the rpoB and katG genes from MTB isolates collected from human clinical samples between 2008 and 2015. The genes were sequenced and compared to the wild type MTB H37Rv rpoB (accession number L27989) and kat G genes (KP46920), respectively. Sequence analysis results were compared to genotyping results obtained from molecular assays and culture results of all isolates. Results: The most frequent mutation responsible for rifampicin resistance was (25/92) S531L that was detected by using all molecular assays. Some inconsistencies were observed between phenotypic and genotypic assay results for both katG and rpoB genes in 30 strains. For these, eight codons; G507S, T508A, L511V, del513-526, P520P, L524L, R528H, R529Q and S531F were novel mutations. In addition, the I572P/F, E562Q, P564S, and Q490Y mutations were identified as novel mutations outside the rifampicin resistance determining region. In katG gene, amino acid changes to threonine, asparagine and isoleucine exhibited high degrees of polymorphism such as V473N, D311N, and L427I. The R463L (20/92) amino acid substitution was most common but was not associated with isoniazid resistance. Conclusion: These finding indicate that molecular assay kit diagnosis that is based on the rpoB and katG genes should be improved to cater for the genetic variations associated with the geographic specificity of the target genes and be able to detect most prevalent mutations in different areas.