Back
 JTR  Vol.4 No.2 , June 2016
The Use of the PCR-Based Dot-Blot Hybridization Assay to Detect Resistance Markers to Rifampicin and Streptomycin in Mycobacterium tuberculosis Isolates from the SW Region of Cameroon
Abstract: Drug sensitivity testing to establish resistance to TB drugs takes many months to arrive at. Public health physicians have difficulties with such an approach due to long wait periods and cannot use it to establish community wide prevalence as a way to understand where resistance may be emerging faster and to limit its spread. The objective of this study was to use the dot-blot hybridization technique in the detection of resistance to rifamycin (RIF) and streptomycin (SM) in South- Western Cameroon and to compare the technique with the routine culture and drug susceptibility testing for detecting resistance in a resource poor country, Cameroon. A hospital-based study was conducted at the Regional hospitals of Buea and Limbe and Tiko Central Clinic. Tuberculosis (TB) patients aged 15 to 50 (mean age: 30.50 ± 8.33 standard deviation) were recruited for the study between December 2006 and April 2007. Cultures from 59 patients were tested for rifampicin and streptomycin sensitivity by the modified proportion method and mutational analysis for rpoB codon 516 and rrs codon 513 was performed by the dot-blot hybridization technique. Of the 59 sputum samples collected (36 were males and 23 were females) came from Buea 19 (32.2%), Limbe 20 (33.9%) and Tiko 20 (33.9%) towns respectively. Amplification for the gene showed that there was (59) 100% amplification with primers used for rpoB genes and 43 (72.9%) amplification with primers used for the rrs gene. Mutational analysis demonstrated that resistance to RIF was common in females (52.1%) than males (41.7%) while 6% of the samples were indeterminate. 12 (20.3%) samples showed phenotypic and genotypic resistance to RIF compared to 34 samples (58.1%) for SM. Phenotypic resistance and genotypic susceptibility were found in 5 (8.5%) RIF and 3 (4.7%) SM compared to phenotypic susceptibility and genotypic resistance that were found in 2 (3.5%) RIF and 3(4.7%) SM. Double mutation on rpoB and rrs genes occurred in 8 (13.6%) DNA samples. Resistance to RIF and SM due to mutations on the rpoB and rrs genes respectively in the SW region was found to be high and comparable to the drug susceptibility testing by 92%, (95% CI: 75.7 - 99.1). The Dot-blot technique will be useful in rapidly assessing the effectiveness of national TB control programs in limiting the spread of resistance strains in Cameroon.
Cite this paper: Ane-Anyangwe, I. , Mbacham, W. , Meriki, H. , Pride, T. , Nkuo-Akenji, T. , Penlap, V. , Tietcheu, L. , Anong, D. , Nji, A. and Titanji, V. (2016) The Use of the PCR-Based Dot-Blot Hybridization Assay to Detect Resistance Markers to Rifampicin and Streptomycin in Mycobacterium tuberculosis Isolates from the SW Region of Cameroon. Journal of Tuberculosis Research, 4, 72-79. doi: 10.4236/jtr.2016.42009.
References

[1]   Bloom, B.R. and Murray, C.J. (1992) Tuberculosis: Commentary on a Re-Emergent Killer. Science, 257, 1055-1064. http://dx.doi.org/10.1126/science.257.5073.1055

[2]   Dye, C., Scheele, S., Dolin, P., Pathania, V. and Raviglione, M.C. (1999) Consensus Statement. Global Burden of Tuberculosis: Estimated Incidence, Prevalence and Mortality by Country. WHO Global Surveillance and Monitoring Project. JAMA, 282, 677-668. http://dx.doi.org/10.1001/jama.282.7.677

[3]   Cohn, D.L., Bustreo, F. and Raviglione, M.C. (1997) Drug Resistant Tuberculosis: Review of the World Wide Situation and the WHO/IUATLD Global Surveillance Project. Clinical Infectious Disease, 24, 5121-5130.
http://dx.doi.org/10.1093/clinids/24.Supplement_1.S121

[4]   Ritacco, V., Di Lonardo, M. and Renierio, A. (1997) Spread of Human Immunodeficiency Virus-Related Multidrug Resistant Tuberculosis in Buenos Aires. Journal of Infectious Disease, 176, 637-642.
http://dx.doi.org/10.1086/514084

[5]   Raviglione, M.C., Snider Jr., D.E. and Kochi, A. (1995) Global Epidemiology of Tuberculosis. Morbidity and Mortality of the World Wide Epidemic. JAMA, 273, 220-226.
http://dx.doi.org/10.1001/jama.1995.03520270054031

[6]   Gillespie, S.H. (2002) Evolution of Drug Resistance in Mycobacterium tuberculosis: Clinical and Molecular Perspective. Antimicrobial Agents and Chemotherapy, 46, 267-274.
http://dx.doi.org/10.1128/AAC.46.2.267-274.2002

[7]   Ramaswamy, S.V. and Musser, J.M. (1998). Molecular Genetic Basis of Anti-Microbial Agent Resistance in Mycobacterium tuberculosis Update. Tubercle and Lung Disease, 97, 3-29.
http://dx.doi.org/10.1054/tuld.1998.0002

[8]   Van der Zanden, A.G., Hoentjen, A.H., Heilmann, F.G., Weltevreden, E.F., Schouls, L.M. and van Embden, J.D. (1998) Simultaneous Detection and Strain Differentiation of Mycobacterium tuberculosis Complex in Paraffin Wax Embedded Tissues and in Stained Microscopic Preparations. Molecular Pathology, 51, 209-214. http://dx.doi.org/10.1136/mp.51.4.209

[9]   Githui, W.A., Jordaan, A.M., Juma, E.S., Kinyanyui, M.P., .Karimi, F.G., Kimwomi, J., Meme, H., Mumbi, M.P., Steicher, E.M., Warren, R., Van Helden, P.D. and Victor, T.V. (2004) Identification of MDR-TB Beijing/w and Other Mycobacterium tuberculosis Genotypes in Nairobi Kenya. International Journal of Tuberculosis and Lung Diseases, 8, 352-360.

[10]   de Kantor, N., Kim, I., Frieden, S.J., Laszlo, T., Fluelmo, A., Norval, P.Y., Rieder, H., Valenzuela, P. and Weyer, K. (1998) Laboratory Services in Tuberculosis Control Part 11: Microscopy. WHO/TB/89.258, Jatto Associatess, Biella.

[11]   Victor, T.C., Warren, R. and Butt, J.L. (1997) Genomic and MIC Stability in Mycobacterium tuberculosis and Indications for Continuation of Use of Isoniazid in Multidrug-Resistant Tuberculosis. Journal of Medical Microbiology, 46, 847-957. http://dx.doi.org/10.1099/00222615-46-10-847

[12]   Meriki, H.D., Tufon, K.A., Atanga, P.N., Ane-Anyangwe, I.N., Anong, D.N., Cho-Ngwa, F. and Nkuo-Akenji, T. (2013) Drug Resistance Profiles of Mycobacterium tuberculosis Complex and Factors Associated with Drug Resistance in the Northwest and Southwest Regions of Cameroon. PLoS ONE, 8, e77410.
http://dx.doi.org/10.1371/journal.pone.0077410

[13]   Ratnam, S., Stead, F.A. and Howes, M. (1982) Simplified Acetylcysteine-Alkali Digestion-Decontamination Procedure for Isolation of Mycobacteria Growth Indicator Tube (MGIT) with Radiometric and Solid Culture for Recovery of Acid Fast Bacilli.

[14]   Niemann, S., Richter, E. and Rüsch-Gerdes, S. (2000) Differentiation among Members of the Mycobacterium tuberculosis Complex by Molecular and Biochemical Features: Evidence for Two Pyrazinamide-Susceptible Subtypes of M. bovis. Journal of Clinical Microbiology, 38, 152-157.

[15]   Canetti, G., Fox, W. and Khomenko, A. (1969) Advances in Techniques of Testing Mycobacterial Drug Sensitivity, and the Use of Sensitivity Tests in Tuberculosis Control Programmes. Bull World Health Organ, 41, 21-43.

[16]   Nolt, F.S., Metchock, B., McGowan, J.E., Edwads Jr., A., Okwumabua, O., Thurmond, C., Mitchell, S., Plikaytis, B. and Shinnick, T. (1993) Direct Detection of Mycobacterium tuberculosis in Sputum by Polymerase Chain Reaction and DNA Hybridization. Journal of Clinical Microbiology, 31, 1777-1782.

[17]   Victor, T., du Troit, R., Jordan, A.M. and Bester Van Helden, P.D. (1990) No Evidence for Point Mutation on Codon 12-13, and 61 of the ras Gene in a High-Incidence Area for Osophageal and Gastric Cancer. Cancer Research, 50, 4911.

[18]   World Health Organization (1991) Tuberculosis Surveillance and Monitoring. Report of a WHO Workshop. World Health Organization, Geneva.

[19]   Mitchison, D.A. (1950) Development of Streptomycine Resistant Strains of Tubercle Bacilli in Pulmonary Tuberculosis: Results of Simultaneous Sensitivity Test in Liquid and Solid Media. Thorax, 5, 144-161.

[20]   Nunes, E.A., De Capitani, E.M., Coelho, E., Joaquim, A.O., Figueiredo, I.R.O., Cossa, A.M., Panunto, M. and Carvalho-Ramos. Patterns of Anti-Tuberculosis Drug Resistance among HIV-Infected Patients in Moputo, Mozambique, 2002-2003. International Journal of Tuberculosis and Lung Disease, 9, 494-500.

[21]   Kuaban, C., Bersion, R., Jifon, G., Cunin, P. and Blackett, N.T. (2000) Acquired Anti-Tuberculosis Drug Resistance in Yaoundé, Cameroon. International Journal of Tuberculosis and Lung Diseases, 4, 427-432.

[22]   Annie, L. and Vestal, B.S. (1975) Procedures for the Isolation and Identification of Mycobacteria. CDC Publication, 81, 97.

[23]   Vashakidze, L., Salakaia, A., Shubladze, N., Cynamon, M., Barbakadze, K., Kikvidze, M., Papitashvili, L., Nonikashvili, M., Solomonia, N., Bejanishvili, N. and Khurtsilava, I. (2009) Prevalence and Risk Factors for Drug Resistance among Hospitalized TB Patients in Georgia. International Journal of Tubercle and Lung Disease, 13, 1148-1153.

[24]   Kim, S.J. (2005) Drug-Susceptibility Testing in Tuberculosis: Methods and Reliability of Results. European Respiratory Journal, 25, 564-569.

 
 
Top