Title: Genotypic Characterization of Drug Resistant Mycobacterium tuberculosis and Non-Tuberculous Mycobacteria in Clinical Isolates from Subjects Resident in Rivers State, Nigeria
Authors: Mac-Fiberesima Gborieneomie, Azuonwu Obioma, C. K. Wachukwu, S. D. Abbey
DOI: https://dx.doi.org/10.18535/jmscr/v6i12.41
Abstract
The emergence and increasing prevalence of Mycobacterium tuberculosis strains resistant to first and second line anti-tuberculous medications are exacerbating the global TB epidemic. The aim of this study was to genotypically characterize and identify resistant genes of Mycobacterium tuberculosis and non-mycobacterium tuberculosis isolates recovered from clinical specimens of patients infected with Drug Resistant Tuberculosis strains and test the isolates against first and second line anti tuberculosis drugs. Three hundred and ninety (390) sputum samples were collected from the study participants in University of Port Harcourt Teaching Hospital, Braithwaite Memorial Hospital, Bori, Ahoada, Degema and Chest Clinic Rivers State Port Harcourt. The samples were received and analyzed by decontaminating with NaOH-Citrate NaCl method before conventionally inoculating them unto Lowenstein Jensen slants and incubated at 37ᵒC for 8weeks. GeneXpert was analysed by decontaminating with isopropanol under a biosafety cabinet which were later processed in the GeneXpert machine. DNA was extracted with the chemical method using genolyse following manufacturer’s instructions. First line drug susceptibility testing was done genotypically with Genotype MTBDRplus. Second line Drug susceptibility testing was done genotypically with Genotype MTBDRsl kit and sequencing was done with 16S rNA sequence analysis. Total number of participants was higher in females (61.53%) than in males (38.46%) p<0.05 and there was no significant statistical difference in prevalence of TB with respect to site. Prevalence of MTB-NTM co-infection was 14.10%. Of the 390 samples tested, 45 (11.53%) were positive for MTB out of which 10(2.56%) were resistant for rifampicin. 52(13.33%) detected MTB out of which 14(3.58%) were resistant for rifampicin respectively. LPA and culture was also compared, of the 390 samples, 45(11.3%) were positive for MTB out of which 10(2.56%) were rifampicin resistant for LPA and 39(10.00%) were culture positive out of which 9(2.30) were rifampicin resistant. Resistance due to isoniazid was 6(1.53%) for LPA and 4(1.02%) for culture. Multiple resistance for rifampicin and isoniazide was 16(4.10) for LPA and 13(3.33) for culture. The same was done for GeneXpert and Culture. Xpert detected 52(13.33%) MTB out of which 14(3.58%) were rifampicin resistant and Culture isolated 39(10.00%) MTB out of which 9(2.30%) were rifampicin resistant. There was no statistical significance observed with respect to type of assay. 57(14.61%) were tested against second line LPA out of which 4(7.01%) were resistant to the fluoroquinolones and none was resistant to the aminoglycosides which was predominant among age 21-40 years. Seven out of the sixteen isolates were sequenced. Samples 1, 3, 4, 6 and 8 detected amplification for rpoB gene responsible for rifampicin resistance (57.14%) and samples 3, 4, and 8 (42.85%) detected amplification for katG gene. Samples 8, 9, 10, 11, and 12 were NTMs which were detected also for rpoB (100%) and sample 10 (20%) alone detected amplification for katG gene. No resistant gene was detected for inhA. 5(31.25%) NTM from the 16NMTBC isolated from the study were sequenced. Mycobacterium abscessus (40%), Mycobacterium setense (20%), Mycobacterium peregrinum (20%) and Mycobacterium conceptionense (20%). Pragmatic efforts are needed to halt drug resistance from progressing beyond what has been identified. It is recommended that people develop the habit of accessing health care early enough to stop further progress in TB.
Keywords: Genotype, drug resistance, Mycobacteriun tuberculosis, Port Harcourt, Rivers State.
References
- Global Tuberculosis Report 2013. Geneva, Switzerland: World Health Organization.
- Al-Mutairi, N.M., Ahmad, S., Mokaddas, E. (2011). First report of molecular detection of fluoroquinolone resistance associated gyrA mutations in multidrug-resistant clinical Mycobacterium tuberculosis isolates in Kuwait. Biomed Central Research Notes, 4,123-124
- Ando, H., Mitarai, S., Kondo, Y., Suetake, T., Kato, S., Mori, T. (2011). Evaluation of a line probe assay for the rapid detection of gyrA mutations associated with fluoroquinolone resistance in multidrug-resistant Mycobacterium tuberculosis. Journal of Medical Microbiology, 60(2), 184–188.
- Somoskovi, A, Jillian D, Jeremy R, Maria P, Melinda M, &Max S (2008).Direct Comparison of the GenoType MTBC and Genomic Deletion Assays in Terms of Ability to Distinguish between Members of the Mycobacterium tuberculosisComplex in Clinical Isolates and in Clinical Specimens, Journal of Clinical Microbiology, 10 (1128), 105-107
- Siddiqi, Kamran (2003). Clinical diagnosis of smear-negative pulmonary tuberculosis in low-income countries: the current evidence, The Lancet Infectious Diseases, 3,(88): 1-3.
- Kirwan, Daniela E (2012). Same-day diagnosis and treatment of tuberculosis, The Lancet Infectious Diseases, 3:1-3.
- Ameeta, S.K. & Majid, S.,Y. (2014) in Nigeria International Journal of Mycobacteriology
- Araoye, M.O. (2003). Sample Size determination. In: Research methodology with Statistics for Health and Social Sciences. Ilorin:
- Cepheid GeneXpert MTB/RIF-10.Package insert. September, 2010
- Saitou, N. & Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406-425.
- Jukes, T.H. & Cantor, C.R. (1969). Evolution of protein molecules. In Munro HN, editor, Mammalian Protein Metabolism, pp. 21-132, Academic Press, New York.
- Nwofor, A.C., Nyamngee, A., Nwabuisi, C., Iwakun, M., Gidado, M., Mensah, C., Dakum, P., Agbede, O.O., Ndembi, N., Blattner, W.A. & Abimiku, A.G. (2015). Performance of Genotype MTBDRplus in the Detection of Resistance to Rifampicin and Isoniazid among Clinical Mycobacteria Isolates in Ilorin, Nigeria.Curriculum for HIV Resistance,13(4), 308-314.
- Lawson, L., Zhang, J., Gomgnimbou, M.K., Abdurrahman, S.T., Le Moullec, S. & Mohamed, F. (2012). A Molecular Epidemiological and Genetic Diversity Study of Tuberculosis in Ibadan, Nnewi and Abuja, Nigeria. PLoS ONE, 7(6), 1-2.
- Gambo, A., Samer, S., El-Kamary, A., Abimiku, N., Ezati, I., Mosunmola, L., Hungerford, C., Brown, K. J., Tracy, J. O. & William, B. (2013) Mycobacterial Etiology of Pulmonary Tuberculosis and Association with HIV Infection and Multidrug Resistance in Northern Nigeria. Tuberc-ulosis Research and Treatment, 8(5), 3-5.
- Adjemian, J., Olivier, KN, & Prevots, D.R. (2018).Epidemiology of Pulmonary Nontuberculous Mycobacterial Sputum Positivity in Patients with Cystic Fibrosis in the United States, 2010-2014. Annals of American Thoracic Society, 15(7), 817-826.
- Chang, T. C., Hsieh, C. Y., Chang, C. D., Shen, Y. L., Huang, K. C., Tu, C., … Tsai, S. S. (2006). Pathological and molecular studies on mycobacteriosis of milkfish Chanos chanosin Taiwan. Diseases of Aquatic Organisms, 72, 147–151.
- Guz, L., Grądzki, Z., Krajewska, M., Lipiec, M., Zabost, A., Augustynowicz‐Kopeć, E. & Szulowski, K. (2013). Occurrence and antimicrobial susceptibility of Mycobacterium peregrinumin ornamental fish. Bulletin of the Veterinary Institute in Pulawy, 57, 489–492.
- Beran, V., Matlova, L., Dvorska, L., Svastova, P., & Pavlik, I. (2006). Distribution of mycobacteria in clinically healthy ornamental fish and their aquarium environment. Journal of Fish Diseases, 29, 383–393.
- Rachel, T., Carla, T., Robyn, C., Chris, C., Flavia, H. & Megan, H. (2013). Isolation of Nontuberculous Mycobacteria (NTM) from Household Water and Shower Aerosols in Patients with Pulmonary Disease Caused by NTM. Journal of Clinical Microbiology, 51(9), 3006–3011.