ANAS Presentation

1. MOLECULAR DETECTION OF ISONIAZID AND RIFAMPICIN RESISTANCE GENES IN MYCOBACTERIUM
TUBERCULOSIS IN CLINICAL ISOLATES IN DUTSIN-MA METROPOLIS
AN M.Sc. RESEARCH PROPOSAL
BY
ANAS UMAR
MSC/19/MCB/0364
MARCH, 2021

2. Background of the Study
• Tuberculosis (TB), an ancient infectious disease caused by Mycobacterium tuberculosis, is the leading cause
of death due to an infectious agent globally. M. tuberculosis is a pathogenic bacterium initially found in 1882
by Robert Koch has a place within the Mycobacteriaceae family. It has a place with an intricate that has in
any event 9 individuals collectively known as Mycobacterium tuberculosis complex (MTBC) [1].
• When considering the mechanisms of drug resistance in M. tuberculosis, it is imperative to understand the
interplay between the molecular mechanisms, adaptive features and the innate attribute which play crucial
role in resistant-MTB strain. Pasca et al in 2010, accentuated that drug resistance in Mycobacterium
tuberculosis is due to some intrinsic factors.
• In a WHO 2016 publication, Nigeria was shown to rank fourth out of the 22 high tuberculosis burden
countries worldwide and has the highest burden of tuberculosis in Africa. According to the World Health
Organization, Nigeria is one of the countries with a high burden of tuberculosis (TB) worldwide. It is also one
of 10 countries that account for 77% of the difference between WHO estimation and actual notifications due
to underreporting and underdiagnosis[2].
• The fundamental characteristics of passive resistance to antibiotics in M. tuberculosis are due to its
impermeable cell wall and slow growth[3, 4]. M. tuberculosis has a cell wall that is composed of two main
components, these are: mycolic acids and Wax-D

3. Significance and Rationale of the Study
• Nigeria comes third behind only India and China in terms of
tuberculosis cases. Every year, around 245,000 Nigerians die from
tuberculosis (TB) and about 590,000 new cases occur (of these,
around 140,000 are also HIV-positive).
• This study will be carried out in order explore the molecular genetic
basis of isoniazid and rifampicin resistance in in the clinical isolates of
Mycobacterium tuberculosis in the study area. The result of the study
will contribute largely to data on tuberculosis and will also give
baseline information for guidelines development, treatments and
interventions towards curbing the problem of drug resistant
tuberculosis.

4. Aim and Objectives
Aim: The study is aimed at determining the molecular genetic basis of
isoniazid and rifampicin resistance in Mycobacterium tuberculosis in
clinical isolates.
Objectives of the study
• The study is aimed at determining the molecular genetic basis of
isoniazid and rifampicin resistance in Mycobacterium tuberculosis in
clinical isolates.
• To determine resistance in genes of Mycobacterium tuberculosis in
sputum associated with first and second line drug resistance by DNA
sequencing.

5. Research Questions
• What is the level of occurrence of tuberculosis in clinical samples?
• What are the resistance genes in Mycobacterium tuberculosis?

6. Materials and Method
• Study Area
• The study will be conducted in Dutsin-Ma town, Dutsin-Ma Local
Government Area of Katsina State. Dutsin-Ma Local Government lies
on the latitude 12o26N and longitude 07o29E.
• Dutsin-Ma Local Government has a size of about 552, 323 Km2 with a
total population of 169, 829 people as at 2006 National Census
(National Population and Housing Census, 2006).
Sample Size
The size of the sample to be used is determined using Fisher’s Formula
(7) as follows: N = 113

7. Ethical Clearance
• Ethical clearance will be obtained from Katsina State Ministry of
Health so as to allow collection of sputum samples from selected
facilities in Dutsin-Ma.
• Study Participants
• Patients diagnosed with tuberculosis and with sputum samples are
needed for this study, and this can be obtained from patients
attending different health facilities in Dutsin-Ma.

8. Sample Collection, Processing and Analysis
• About 113 sputum samples from patients harbouring resistant
tuberculosis bacilli will be collected in clean, wide-mouthed and leak
proof specimen containers and processed following standard
procedures for staining and culture [5, 6].

9. Laboratory Procedures
• Ziehl-Neelsen Staining [7]
• Microscopy [8, 9)]
• Culture [10]
• Drug Susceptibility Testing [11]
• Sample DNA Extraction [12]
• PCR [13]
• Electrophoresis [14]

10. Data Analysis
• Data obtained from the study will be presented using tables and
charts. Data will be analyzed using SPSS version 25.0 (P= 0.05). Chi-
square test will be used to test associations between variables that
include age, socioeconomic status and sexes of the study participants
with Mycobacterium bacilli while odds ratio will be used to determine
the strength of association amongst mentioned variables [15].

11. COSTING AND BUDGETING
S/No. Item Unit Price (₦) Quantity Total (₦) Rationale
1. LJ Medium 68, 680 1 (500g) 68, 580 Because the medium is selective
for Mycobacteria species
2. Antibiotic
Sensitivity
Disc
1000 3 3, 000 They are used for ABR assays
3. Glasswares
(Conical
flasks,
100/250/500ml
, cylinder;
100/200ml
20
20
500 10, 000
6, 000
They are used in preparing media.
They are used in transferring
required volume of water.

12. Selected References
• Campbell P.J., Morlock G.P., Sikes R.D., Dalton T.L., Metchock B., et al. (2011). Molecular detection of mutations associated with first- and second-line
drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis. Antimicrob Agents Chemother 55(5): 2032-
2041.
• Caws M, Duy PM, Tho DQ, Lan NT, Hoa DV, et al. (2006) Mutations prevalent among rifampin and isoniazid-resistant Mycobacterium tuberculosis
isolates from a hospital in Vietnam. J Clin Microbiol 44(7): 2333-2337.
• Christopher A Kerantzas, William R Jacobs (2017) Origins of Combination Therapy for Tuberculosis: Lessons for Future Antimicrobial Development and
Application. mBio 8(2): e01586-e01616.
• Chen, J.M.; Uplekar, S.; Gordon, S.V.; Cole, S.T. (2012). A point mutation in cycA partially contributes to the D-cycloserine resistance trait of
Mycobacterium bovis BCG vaccine strains. PLoS One, 7, e43467.
• Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction,
Analytical Biochemistry, vol. 162, no. 1, pp. 156–159.
• Daniel T.M. (2006). The history of tuberculosis. Respiratory Medicine.100:1862–70.
• Watt B, Edwards JR, Rayner A, Grindey AJ, Harris G (1992). In vitro activity of meropenem and imipenem against mycobacteria: development of a daily
antibiotic dosing schedule. Tuber Lung Dis. 73:134–136. [PubMed: 1421344]
• World Health Organization, Communicable Diseases Cluster (2012). Stop TB Department, World Health Organization. Tuberculosis
laboratory biosafety manual. .http://www.ncbi.nlm.nih.gov/books/NBK179135/. Accessed 1 Oct 2018.
• World Health Organization. Global tuberculosis report (2017). Geneva, Switzerland: WHO/HTM/TB/2017.23.
• World Health Organization. Global tuberculosis report (2012). Geneva, Switzerland: WHO/HTM/TB/2012.6.
http://apps.who.int/iris/handle/10665/75938. Accessed 16 Nov 2018.
• doi:10.1016/j.rmed.2006.08.006.