EVALUATION OF NITRATE REDUCTASE ASSAY FOR DETECTION OF MULTI-DRUG RESISTANT MYCOBACTERIUM TUBERCULOSIS AMONG PATIENTS AT NATIONAL TUBERCULOSIS REFERENCE LABORATORY ZARIA NGERIA

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ABSTRACT

 

 

Multi-drug resistant tuberculosis (MDR-TB) remains a major challenge for the control of tuberculosis worldwide. Susceptibility testing of Mycobacterium tuberculosis isolates to first line drugs is therefore necessary to ascertain the diagnosis of MDR-TB. A total of 437 re-treatment patients’ samples were screened for Acid Fast Bacilli (AFB), 72 were smear positive giving a prevalence of 16.47%. Out of 72 smears positive, 62 were culture positive, 6 were culture negative and 4 were contaminant using Lowenstein Jensen medium. Out of 62 cultures positive, 57 were found to be Mycobacterium tuberculosis complex (MTBC) using immunochromatographic test while 5 were negative which were considered to be NTM (Non-Tuberculosis Mycobacteria). In this study the susceptibility of 57 MTBC isolates to isoniazid (INH), rifampicin (RIF), streptomycin (STR) and Ethambutol (EMB) was determined by Lowenstein Jensen proportion method (LJPM) and Nitrate Reductase Assay (NRA). LJ PM detected 41(71.93%) MDR-TB while NRA detected 44(77.19%) MDR-TB isolates. The occurrence of poly resistance to anti-TB drugs using NRA and LJ PM was 12% and 14% while pan susceptible were of 11% and 14% by LJ PM and NRA respectively. The sensitivities and specificities of NRA compared to those of LJPM were observed to be 98% and 98%, 64% and 68%, 89% and 92%, 80% and 77% for RIF, INH, STR, and EMB respectively. Positive predictive values were 91%, 93%, 87% and 83% for RIF, INH, STR and EMB respectively. Negative values were 80%, 92%, 67% and 90% for RIF, INH, STR and EMB respectively. Good agreement was found in all the tests with κ values of 0.63, 0.61, 0.59, and 0.61for RIF, INH, STR, and EMB respectively. The HIV/TB co-infections and HIV/MDR-TB co-infections were reported to be 9.7% and 7% respectively. Highest MDR-TB among age groups of 21-30 and 31-40 years were detected by NRA and LJPM and higher MDR-TB and TB were observed in male. The NRA has the potential to be a useful tool for rapid detection of MDR-TB in resource-limited settings because of its higher sensitivity and specificity.

 

 

 

 

 

 

 

 

 

 

 

 

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TABLE OF CONTENTS

 

COVER PAGE................................................................................................................................ i

 

FLY LEAF..................................................................................................................................... ii

 

TITLE PAGE........................................................................................................... Error! Bookmark not defined.

 

CERTIFICATION........................................................................................................................ iv

 

ACKNOWLEDGEMENTS........................................................................................................... v

 

TABLE OF CONTENTS............................................................................................................. vii

 

CHAPTER ONE............................................................................................................................. 1

 

1.0 INTRODUCTION.................................................................................................................... 1

 

1.1 Background of the Study.......................................................................................................... 1

 

1.2 Statements of Problems............................................................................................................. 5

 

1.3 Justification............................................................................................................................... 6

 

1.4 Aim and Objectives................................................................................................................... 7

 

1.4.1 Aim......................................................................................................................................... 7

 

1.4.2      Objectives.......................................................................................................................... 7

 

CHAPTER TWO............................................................................................................................ 8

 

2.0 LITERATURE REVIEW...................................................................................................... 8

 

2.1 Mycobacteria............................................................................................................................ 8

 

2.1.1 Classification of the Genus Mycobacterium........................................................................ 8

 

2.1.3 Growth and Metabolic Characteristics of Mycobacteria...................................................... 10

 

2.1.4 Mycobacterial Antigens........................................................................................................ 11

 

2.1.5 The M. tuberculosis Genome................................................................................................ 11

 

2.1.6 The Origins and evolution of Mycobacterium tuberculosis.................................................. 12

 

2.1.7 Epidemiology of tuberculosis............................................................................................... 13

 

2.1.8. Pathogenesis and Virulence of Tuberculosis....................................................................... 15

 

2.1.9 Primary infection.................................................................................................................. 15

 

2.1.11 Pulmonary tuberculosis....................................................................................................... 17

 

2.1.13 Diagnosis of tuberculosis.................................................................................................... 18

 

2.1.13.1 Mantoux test..................................................................................................................... 18

 

2.1.13.2 Chest X-ray...................................................................................................................... 19

 

2.2 TB Drug Susceptibility Testing............................................................................................... 22

 

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2.2.1

General considerations .........................................................................................................

22

2.2.2

Conventional susceptibility tests..........................................................................................

23

2.2.3

New rapid susceptibility tests ..............................................................................................

25

2.3

Genotypic (Molecular) Methods of Detecting MDR-TB .......................................................

30

2.3.1

Genotyping methods used in TB Epidemiology ..................................................................

31

2.3.2

Molecular Assays ...............................................................................................................

32

2.4

Molecular Basis and Mechanisms of Resistance to First-line Drugs .....................................

33

2.4.1 Isoniazid ...............................................................................................................................

34

2.4.2

Rifampicin............................................................................................................................

35

2.4.3

Streptomycin ........................................................................................................................

37

2.4.4

Ethambutol ...........................................................................................................................

37

3.0 MATERIAL AND METHODS ..............................................................................................

40

3.1

Laboratory Settings for the study ............................................................................................

40

3.2

Study population .....................................................................................................................

40

3.4

Consent of Patients and Ethical Approval ..............................................................................

41

3.5

Inclusion criteria .....................................................................................................................

41

3.6

Exclusion criteria ....................................................................................................................

41

3.6

Sample collection ....................................................................................................................

42

3.7

Microscopy .............................................................................................................................

42

3.8

Media preparation ...................................................................................................................

42

3.9

Specimen digestion and decontamination ...............................................................................

43

3.10 Isolation.................................................................................................................................

43

3.11 Identification of Mycobacterial Isolates ...............................................................................

44

3.13 Preparation of Lowenstein- Jensen Medium with KNO3 and Drugs ....................................

44

3.14 Drug Susceptibility Testing by Proportion method ..............................................................

45

3.15 Drug Susceptibility Testing by Nitrate Reductase Assay .....................................................

46

CHAPTER FOUR .........................................................................................................................

48

4.0 RESULTS ...............................................................................................................................

48

4.1

Prevalence of TB by Microscopy ...........................................................................................

48

4.2

Culture.....................................................................................................................................

48

4.2

Drug susceptibility by LJ PM and NRA .................................................................................

48

 

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4.3 Drug susceptibility of Mycobacterium tuberculosis complex by Nitrate Reductase Assay.... 48

 

4.4 Drug Susceptibility of Mycobacterium tuberculosis Complex by LJ Proportion Method...... 53

 

4.5 Evaluation of drug susceptibility of Mycobacterium tuberculosis using Nitrate Reductase

 

Assay and LJ proportion Method.................................................................................................. 53

 

4.6 Prevalence of MDR-TB in Relation to Age Group................................................................. 56

 

4.7 Prevalence of MDR-TB in Relation to Gender....................................................................... 56

 

4.8 MDR-TB/HIV co-infection based on Age Group.................................................................. 56

 

4.9 MDR-TB/HIV Co-infections based on Gender...................................................................... 56

 

4.10 TB/HIV Co-infections patients based on age group............................................................. 61

 

4.11 TB/HIV Co-infections patients based on gender.................................................................. 61

 

CHAPTER FIVE.......................................................................................................................... 64

 

5.0 DISCUSSION........................................................................................................................ 64

 

CHAPTER SIX............................................................................................................................. 69

 

6.0 CONCLUSION AND RECOMMENDATIONS.................................................................. 69

 

6.1CONCLUSION....................................................................................................................... 69

 

6.3 RECOMMENDATIONS....................................................................................................... 70

 

REFERENCES............................................................................................................................. 71

 

APPENDICES.............................................................................................................................. 92

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ix


LIST OF FIGURES

 

Figure 4. 1: Prevalence of TB by Microscopy............................................................................... 49

 

Figure 4. 2: Percentage distribution of culture.............................................................................. 50

 

Figure 4. 3: Comparison of MDR-TB detection by Lowenstein Jensen Proportion Method and

 

Nitrate Reductase Assay based on Age group.............................................................................. 57

 

Figure 4. 4: Comparison of MDR-TB detection by Lowenstein Jensen Proportion Method and

 

Nitrate Reductase Assay based on Gender................................................................................... 58

 

Figure 4. 5: Prevalence of HIV among MDR-TB Patients based on Age Group......................... 59

 

Figure 4. 6: Prevalence of HIV among MDR-TB Patients based on Gender............................... 60

 

Figure 4. 7: Prevalence of HIV among TB Patients based on Age group..................................... 62

 

Figure 4. 8: Prevalence of HIV among TB Patients based on Gender.......................................... 63

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

x


LIST OF TABLES

 

Table 4. 1: Drug Susceptibility by LJPM and NRA...................................................................... 51

 

Table 4. 2: Drug Susceptibility of Mycobacterium tuberculosis Complex by Nitrate Reductase

 

Assay............................................................................................................................................. 52

 

Table 4. 3: Drug susceptibility of Mycobacterium tuberculosis complex by LJ Proportion method

 

54

 

Table 4. 4: Evaluation of drug susceptibility testing of Mycobacterium tuberculosis using Nitrate

 

Reductase Assay and LJ proportion Method................................................................................ 55

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

xi


LIST OF APPENDECIS

 

Appendix I : Result of Nitrate Reductase Assay showing susceptible isolates............................ 92

 

Appendix II: Nitrate Reductase test result showing Resistant isolates........................................ 93

 

Appendix III: LJPM result for resistant isolates........................................................................... 94

 

Appendix IV: LJPM result for susceptible isolates....................................................................... 95

 

Appendix V: Preparation of Working Solution of Antibiotics..................................................... 96

 

Appendix  VI:  Questionnaire    ………………………………………………………………….97

Appendix VII: Inform consent form…………………………………………………………….98

Appendix VIII: Ethnical approval …………………………………………………………….103

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

xii


 

 

LIST OF ABBREVIATIONS


 

ACP

 

AFB

 

AIDS

 

ATCC

 

BCG

 

Bp

 

BSL

 

C

 

CC

 

CFU

 

CR

 

DMSO

 

DNA

 

DR

 

DST

 

EMB

 

G

 

HIV

 

ICT

 

IJTLD

 

INH

 

InhA


Acyl carrier protein

 

Acid-fast bacilli

 

Acquired Immunodeficiency Syndrome

 

American Type Culture Collection

 

Baccile Calmette Guerin

 

Base pair

 

Biosafety level

 

Cytosine

 

Critical Concentration

 

Colony Forming Unit

 

Compliment Receptors

 

Dimethyl sulphoxide

 

Deoxyribonucleic acid

 

Drug resistant

 

Drug Susceptibility Testing

 

Ethambutol

 

guanine

 

Human immunodeficiency virus

 

Immunochromatographic Test

 

International Journal of Tuberculosis and Lung Disease

 

Isoniazid

 

Enoyl-acyl carrier protein reductase


 

 

 

xiii


IPM

 

IUATLD

 

KasA

 

KatG

 

KNO3

 

LJ

 

LJPM

 

LPA

 

MDR-TB

 

MGIT

 

MIC

 

MODS

 

MTB

 

MTBC

 

MTT

 

NADH

 

NALC

 

NAOH

 

NRA

 

NTBLCP

 

NTM

 

NTRL

 

OADC


Indirect Proportion Method

 

International Union against Tuberculosis and Lung Disease

 

β-ketoacyl-ACP synthase

 

Catalase-peroxidase enzyme

 

Potassium nitrate

 

Lowenstein-Jensen

 

Lowenstein-Jensen proportion method

 

Line probe assay

 

Multidrug resistant tuberculosis

 

Mycobacterium Growth Indicator Tube

 

Minimum inhibitory concentration

 

Microscopic observation drug susceptibility

 

Mycobacterium tuberculosis

 

Mycobacterium tuberculosis complex

 

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

 

Reduced nicotinamide adenine dinucleotide

 

N-acetyl L-cystene

 

Sodium Hydroxide

 

Nitrate Reductase assay

 

National tuberculosis and leprosy control programme

 

Non tuberculosis Mycobacteria

 

National Tuberculosis Reference Laboratory

 

Oleic Acid-Albumin-Dextrose-Catalase


 

 

 

xiv


PANTA

 

PAS

 

PBS

 

PCR

 

PG

 

PNB

 

PPG

 

RFLP

 

RIF

 

RLSs

 

RNA

 

RRDR

 

sROC

 

STR

 

TAT

 

TB

 

VNTRs

 

WHO

 

XDR-TB


Polymyxin B, Amphotericin B, Nalidixic acid, Trimethoprim and Azlocillin

 

p-aminosalicylic acid

 

Phosphate Buffer Saline

 

Polymerase chain reaction

 

Pro-Glu

 

Para-Nitrobenzoic Acid

 

Pro-Pro Glu

 

Restriction fragment length polymorphism

 

Rifampicin

 

Resource limited settings

 

Ribonucleic Acid

 

Rifampicin Resistance Determine Region

 

Summary Receiver Operator Characteristic curve

 

Streptomycin

 

Turnaround time

 

Tuberculosis

 

Variable number of tandem repeats

 

World Health Organization

 

Extensively drug resistant tuberculosis


 

 

ZN


 

Ziehl-Neelsen


 

 

 

 

 

 

 

 

 

 

xv


 

CHAPTER ONE

 

 

1.0 INTRODUCTION

 

 

1.1 Background of the Study

 

 

Tuberculosis (TB) continues to cause more deaths worldwide than any other infectious disease. The World Health Organization (WHO) estimated that in 2011, there were 8.7 million cases of TB, with nearly 1 million deaths among HIV-negative cases and 0.43 million deaths associated with HIV infection (WHO, 2012). More than 99% of deaths and 95% of new TB cases occur in middle- and low-income countries (Palomino, 2012). In the 2012 Global Tuberculosis Report estimates, expressed in rates per 100,000 populations, were 161 (25-420) for prevalence and 108 (50-186) for incidence. Case detection of all forms stood at 51% (29%-110%). The mortality rate for all forms of TB remains 27 (7-60) per 100,000 populations (46,000 deaths per year) (WHO, 2012). The majority of TB cases worldwide were in the South-East Asia (29%), African (27%) and Western Pacific (19%) regions. India and China alone accounted for 26% and 12% of total cases, respectively (WHO, 2013). Nigeria has one of the highest burdens of TB in the world and remains a major target in the global control of the disease (WHO, 2010). In 2011 an estimated 280,000 cases of TB (68%incident cases) were reported from Nigeria which corresponds to a prevalence rate of 280 per 100,000 populations according the WHO global tuberculosis report of 2012 (Gambo et al., 2013). Based on 2012 survey Nigeria ranked 4th among the highest tuberculosis burden countries in the world. The point estimates of TB prevalence rates are 318 and 524 per 100,000 population (15 years and above) respectively (WHO, 2014).

 

 

 

 

 

 

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Multidrug-resistant TB (MDR-TB) is a form of TB that is difficult and expensive to treat because it fails to respond to two important first-line drugs, specifically, rifampicin (RIF) and isoniazid (INH) (Bwanga et al., 2010). Data from more than 100 countries collected during the last decade show that 5% of all TB cases have MDR-TB. There were an estimated 500,000 new MDR-TB cases in 2007. Twenty-seven countries accounted for 85% of all MDR-TB cases. The top five countries with the largest number of MDR-TB cases are India, China, the Russian Federation, South Africa and Bangladesh, while extensively drug resistant TB (XDR-TB) has been found only in 58 countries to date (Stop TB partnership, 2009; WHO, 2010) .The number of prevalent cases of MDR-TB in many parts of the world is estimated to be much higher than the number of incident case arising annually. Globally, the median prevalence of MDR-TB is reported to be 3% among the new and 15% among the re-treatment cases (WHO, 2008).Countries and territories in Eastern Europe such as Tajikistan, Uzbekistan and parts of China have the highest MDR-TB prevalence of up to 15% among the new and 60% among the previously treated cases (Wright et al., 2009). In sub Saharan Africa, inadequacy of laboratory services makes it difficult to estimate the actual burden of MDR-TB. However, surveillance data of 2005-7 from Cote d’Ivoire, Ethiopia, Madagascar, Rwanda, and Senegal, reported a prevalence of 1-4% among the new and 4-17% among the previously treated TB cases (WHO, 2008). In WHO TB Report of 2011, the proportion of MDR-TB among TB respondent in Africa is 3.9-5% in new cases and 16.7% in retreatment cases. In the 2008 WHO report, the 27 high MDR-TB burden countries refer to those member states estimated to have at least 4000 MDR-TB cases arising annually and or at least 10% of newly registered TB cases with MDR-TB. According to the Global TB control, WHO Report 2011, the estimated prevalence of MDR-TB

 

 

 

 

2


 

in Nigeria amongst new cases is 2.2% and retreated cases is 9.4% the national survey showed a prevalence of 2.9% among new cases 14.3% for retreatment cases (WHO, 2012).

 

In low-income settings, TB diagnosis relies on sputum microscopy for acid-fast bacilli (AFB). The few facilities for culture generally use Löwenstein-Jensen (LJ) media, which can take months to give a result; wider use of LJ culture thus has limited potential to accelerate TB diagnosis (Angeby et al., 2003) Liquid media systems with early growth indicators, such as the Mycobacterial Growth Indicator Tube (MGIT), are more rapid and detect more mycobacterial isolates than LJ; (Goloubeva et al., 2001; Bemer et al., 2002; Tortoli et al., 2002). However, morphological examination of colonies alone can no longer be used for species identification, making other rapid methods more important. The Capilia TB (anti-MPB64 monoclonal) assay (TAUNS, Numazu, Japan) uses monoclonal antibodies to detect a secreted mycobacterial protein, MPB64, which can differentiate M. tuberculosis complex from non-tuberculous mycobacteria (NTM) (Caviedes, 2000; Woods 2000; Angeby et al., 2002) and shows promise as an easy and rapid tool for identifying M. tuberculosis complex in liquid cultures (Hillemann et al., 2005; Bwanga et al., 2009; WHO, 2010)

 

Determination of resistance to a given drug is performed as an in-vitro assay in the Laboratory, a process called drug susceptibility testing (DST). Where resources are limited, the WHO recommends a hierarchy of DST that should include at least RIF and INH the two most efficacious drugs that define MDR-TB (Canetti et al., 1963). For more than 40 years, DST in the developing countries has relied on conventional indirect susceptibility methods on Lowenstein-Jensen (LJ) solid medium (Canetti et al., 1963). Indirect testing involves primary isolation of pure colonies of Mycobacterium tuberculosis, which are then used as inoculums for DST. In

 

 

3


 

contrast, direct DST involves inoculation of processed smear positive samples rather than pure MTB colonies. Results of direct testing are much more rapid and help to triage MDR from non MDR-TB patients promptly.

 

The traditional method for detection of MDR TB with indirect susceptibility testing, involving isolation of the bacterium followed by drug susceptibility testing (DST), has a long turnaround time (TAT) of 10 to 12 weeks(Bwanga et al.,2010). Moreover, if the indirect DST is performed on solid medium, the TAT is longer. This long time required by the indirect methods may be a potential threat to patients, health workers, and the community (Golyshevskaia,1996) While the use of liquid systems the Mycobacteria Growth Indicator Tube (MGIT) 960 (Becton Dickinson, Cockeysville, Md.), has improved TAT to about 25–45 days (Bouyer et al., 2000; Goloubeva, et al.,2001; Bemer et al., 2002; Tortoli et al., 2002 Angeby et al., 2003) liquid culture systems require expensive substrates and equipment and are therefore not feasible in resource-poor settings (Bwanga et al.,2009). Several rapid methods for MTB diagnosis, such as DNA probes that require sophisticated equipment, have been developed (Bemer et al., 2002). The polymerase chain reaction (PCR) is an alternative method that can amplify a small fragment of DNA with high specificity for the diagnosis of infectious diseases (Goloubeva et al., 2001). PCR has also been used to detect MTB in respiratory samples (Woods, 2000;Caviedes et al.,2000 Bouyer, et al.,2000; Angeby, 2002; Tortoli, et al., 2002; Hillemann, et al.,2005 Bwanga, et al., 2009) Nitrate reductase assay (NRA) is described as a rapid, easily performed and inexpensive method for DST of Mycobacterium tuberculosis (MTB), which is based on the capability of MTB to reduce nitrate to nitrite (Bwanga et al., 2009) However, since the test is performed indirectly (by using culture isolates of MTB), it takes three to four weeks more for the isolation of the same bacterium.

 

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1.2 Statements of Problems

 

Global efforts for TB control, especially in resource limited settings, are being challenged by the lack of rapid, reliable and inexpensive techniques for the detection of M. tuberculosis (Moore et al., 2006; Shiferaw et al., 2007; Brady et al., 2008; Lazarus et al., 2012).Results from the conventional culture detection methods come too late to influence a timely decision on patient management. Early identification is the key in both patient management and controlling transmission of M. tuberculosis (Caviedes et al., 2000; Shiferaw et al., 2007). Therefore, faster, inexpensive and reliable tests are urgently needed (Moore et al., 2006; Minion et al., 2010; Leung et al., 2012).

 

There are different methods for detection of TB drug resistance. The proportion method (PM) and other conventional tests, based on the measurement of growth in culture media containing antibiotics, require several weeks to give results. The BACTEC radiometric system has the advantage of being more rapid (5–10 days), but requires the use of radioisotopes and can be costly to be performed routinely. Commercial tests (MGIT, E Test) and molecular tools (LPA) have been proposed, but are expensive and also impractical for routine use (Lemus et al., 2004; Palomino, 2005). For developing countries, it would be useful to have a simple and inexpensive test that could rapidly detect drug-resistant M. tuberculosis strains. Several methods have been reported, including colorimetric methods that use redox indicators (MTT and resazurin) and phage amplification technology (Abate et al., 1998; Martin et al., 2003; Simboli et al., 2005).

 

An alternative method is the nitrate reductase assay (NRA), previously reported as a useful tool for rapid and accurate detection of resistance to first-line antituberculosis drugs (Angeby et al., 2002; Panaiotov and Kantardjiev, 2002; Coban et al., 2004; Montoro et al., 2005).

 

 

 

 

5


 

1.3 Justification

 

Indirect susceptibility testing on Lowenstein-Jensen (LJ) medium is the most common method for detection of TB drug resistance in Africa. There is an urgent demand for early and proper detection of MDR and XDR-TB cases for the effective management and control of TB. Conventional method (proportion method) (PM) is the gold standard for DST of MTB but has some limitations, such as cumbersomeness and long-turnaround time (TAT). In recent years, a multitude of techniques for rapid DST has been designed and evaluated, such as colorimetric redox method (Franzblau et al., 1998) with this method, results take 2–3 months and during these period patients are given inappropriate drug regimens with poor responses and they continue to spread MDR strains, which might be causing MDR-TB outbreaks (Well et al., 2007). Mycobacterium Growth Indicator Tube (MGIT 960: Becton Dickinson, Sparks, Maryland) and line probe assays (HainLifescience. GenoType MTBDRplus 2009) allow more rapid detection of resistance, and have been recommended by the WHO (Innogenetics, 2009; Albert et al., 2010). However, the investment and recurrent costs is an obstacle for the broad implementation of these htechniques in the resource-limited settings (RLSs) of Africa. Therefore, the need for a rapid, affordable, accurate and easy to use test for MDR-TB in RLSs remains a priority (Bwanga et al., 2009).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6


 

1.4 Aim and Objectives

 

 

1.4.1 Aim

 

The aim was to detect multi-drug resistant Mycobacterium tuberculosis using nitrate reductase

 

assay  and  proportion   method,  among   patients   attending  National   Tuberculosis   Reference

 

Laboratory Zaria.

 

 

 

1.4.2    Objectives

 

The objectives of the study were:

 

1        To isolates and characterize Mycobacterium tuberculosis from smear positive sputum of re-treatment patients in Zaria.

 

2            To determine the prevalence of multidrug- resistant Mycobacterium tuberculosis using

 

nitrates Reductase assay in comparison with Lowenstein Jensen   proportion method.

 

3        To determine the demographic and risk factors associated with Mycobacterium tuberculosis among the study population.

 

 

 

 

 

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