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Product Code: 00007193

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The advances in diagnostic research to detect pulmonary tuberculosis have resulted in the development of a real time Polymerase Chain Reaction (PCR) assay on the gene Xpert platform that simultaneously detects rifampicin resistance, integrates sample processing and greatly simplifies testing. This is done by amplifying M. tuberculosis complex-specific region of the rpob gene which is probed with molecular beacons to detect the presence of rifampicin resistance-determining mutations. In this study, 100 (21.3%) MTB/RIF positive samples out of 470 samples from patients with presumptive TB were considered to have tuberculosis. Out of the 470 samples, 74 samples were randomly picked and cultured. Cultures of 22 patients yielded Mycobactrial growth, 17(77.3%) of the isolates were identified as M. tuberculosis complex and 5(22.7%) were Nontuberculous mycobacteria (NTM). Comparing Smear microscopy with MTB/RIF assay, it was determined that the performance of MTB/RIF assay for rapid diagnosis of pulmonary tuberculosis was higher than that of smear microscopy. MTB/RIF assay showed a detection rate of 21.3% while smear microscopy showed a detection rate of 18.7%. Again, MTB/RIF assay showed a sensitivity and specificity of 100% and 98%, respectively when compared with sputum culture, whereas smear microscopy, when compared with culture showed sensitivity and specificity of 44% and 98%, respectively. In TB/HIV co- infection, Xpert MTB/RIF assay showed a detection rate of 9.3% while Smear microscopy showed a lower rate of 4.9% out of the 126 HIV positive cases. This could be attributed to the fact that people with compromised immune system often release fewer organisms into their sputum at concentration below threshold for visual detection under the microscope. Therefore, it is concluded that Xpert MTB/RIF test has the potential to improve the diagnosis of Tuberculosis.


Title Page                                                                                                                                            i

Declaration                                                                                                                                         ii

Certification                                                                                                                            iii

Dedication                                                                                                                                           iv

Acknowledgments                                                                                                                            v

Table of Contents                                                        vi                                                                                               

List of Tables                                                                                                                           ix

List of Figures                                                                                                                                                                                      x

Abstract                                                                                                                                               xi


CHAPTER 1: INTRODUCTION                                                                                                                         1                                                   

1.1                   Background of the Study                                                                                                                                             1

1.2                   Problem Statement                                                                                                                                                     3

1.3                   Justification                                                                                                                                                                             4

1.4                   Aims and Objectives                                                                                                                                                   5

1.4.1    Specific objectives                                                                                                                                                      5


CHAPTER 2: LITERATURE REVIEW                                                                                                                 7

2.1                   Brief History of Tuberculosis                                                                                                                          7

2.2       Description and Characteristics of Mycobacterium tuberculosis                              8

2.3                   Biology of Mycobacteria                                                                                                                                            8 

2.4                   Transmission of Mycobacterium tuberculosis                                                           10

2.5                   Tuberculosis as an Infectious Disease                                                                                                            10

2.6       Pathogenesis of Tuberculosis                                                                                                                  11

2.7       Incidence of Tuberculosis in Nigeria                                                                                                  13

2.8                   Global Burden of Tuberculosis                                                                                                                       13

2.9                   TB/HIV Coinfection                                                                                                                                                     15

2.10     Diagnostic Methods for Tuberculosis                                                                                                 16

2.10.1  Sputum smear microscopy                                                                                                                             17

2.10.2  Sputum culture                                                                                                                                                                       18

2.10.3  Chest X-ray                                                                                                                                                                              19

2.10.4 Tuberculin skin test                                                                                                                                                     19

2.10.5  Nucleic acid amplification test                                                                                                                                   20

2.11     Treatment and Control of Tuberculosis                                                                                                        22


CHAPTER 3: MATERIALS AND METHODS                                                                                         25

3.1                   Study Participants                                                                                                                                                       25

3.2                   Specimen Collection                                                                                                                                                   25

3.3                   Microbiological Methods                                                                                                                                           26

3.3.1    Preparation of sputum smears                                                                                                                                   26

3.3.2    Ziehl-Neelsen staining and microscopic examination of smears                              26

3.3.3    Decontamination and concentration of sputum samples                                                      27

3.3.4    Xpert MTB/RIF test                                                                                                                                                     28

3.3.5    Preparation of Lӧwenstein-Jensen’s medium                                                                        28

3.3.6    Inoculation of Lӧwenstein-Jensen’s medium                                                                          28

3.3.7    Identification of mycobacterial isolates                                                                                                         28

3.4                   Screening for HIV                                                                                                                                                        29

3.5                   Test Performance Assessment                                                                                                           29

CHAPTER 4: RESULTS AND DISCUSSION                                                                                          31

4.1                   Results                                                                                                                                                                                     31

4.1.1    Demographical characteristics of study participants                                                            31

4.1.2     Sputum smear microscopy                                                                                                                             33

4.1.3     Sputum culture                                                                                                                                                                       36

4.1.4    Case detection rates of diagnostic tests                                                                                             38

4.1.5    Sensitivities, specificities, positive and negative predictive values

       of diagnostic tests                                                                                                                                                            40

4.1.6    Performance of diagnostic tests in TB/ HIV patients                                                             44

4.2                   Discussion                                                                                                                                                                               46


CHAPTER 5: CONCLUSION AND RECOMMENDATIONS                                          49

5.1                   Conclusion                                                                                                                                                                               49

5.2                   Recommendations                                                                                                                                                      50

References                                                                                                                                                                     51














4.1                   Demographical characteristics of study participants                                                            32


4.2                   Distribution of smear positive and negative TB suspects by

       age group and sex                                                                                                                                                        34                       

4.3                   Sputum microscopy stratified by AFB grades                                                                         35

4.4                   Categories of PTB by sputum culture                                                                                                 37

4.5                   Detection of PTB in the Study population by sputum smear

microscopy, MTB/RIF assay and culture                                                                                          39


4.6         Sensitivity and Specificity of sputum smear microscopy in

comparison with sputum culture                                                                                                           41


4.7         Sensitivity and Specificity of MTB/RIF in comparison with

sputum culture                                                                                                                                                               42


4.8          Performance of MTB/RIF assay and smear microscopy in

TB/HIV co-infection                                                                                                                                           45







4.1         Comparison of the sensitivities and specificities of sputum smear

   microscopy and MTB/RIF                                                                                                                                            43










Tuberculosis (TB) continues to be one of the greatest killers in the world among other infectious diseases, claiming over 1.8 million deaths in 2015 and 1.7 million in 2016 including 0.4million people living with HIV out of the 10.4 million people affected across the world (WHO,  2017a). In spite of substantial success in achieving standardized care and improving rates of cure in recent years, the global menace of tuberculosis (TB) remains enormous.

In recent years, the prevention, diagnosis and treatment of tuberculosis (TB) have become more complicated because of two factors changing the epidemic: HIV-associated TB and Multidrug-resistant (MDR) TB. The general consensus among international TB experts is that the existing diagnostic methods for TB are simply too slow and too cumbersome or inaccurate to meet the diagnostic expectations of the current TB control strategy. Therefore, the current global TB epidemic requires new diagnostic tools that are simple and accurate (Lalvani, 2007; Raviglione, 2007). The goal of developing new diagnostic methods that can overcome the limitations of the existing ones has been the purpose of research efforts for the past several years (Young et al, 2008). In developed countries, rapid lipid culture systems based on radiometric and non-radiometric detection of growth of M. tuberculosis and a method of nucleic acid amplification have been introduced. However, implementation of these new TB diagnostic technologies has not been feasible in most developing countries because of lack of technical expertise needed to implement them and the equipment is relatively expensive (Dorman, 2010). The diagnostic test with the greatest potential impact should be simple, accurate, inexpensive and ideally useful at point-of-care (Steingart et al., 2007).

Recently, a real-time PCR assay for Mycobacterium tuberculosis that simultaneously detects rifampicin resistance was developed on the GeneXpert platform, which integrates sample processing and greatly simplifies testing (Helb et al, 2010). In 2010, Xpert MTB/RIF was endorsed for use in TB-burdened countries by World Health Organization (WHO, 2010) and was announced as a major breakthrough in TB diagnosis. This followed several months of rigorous assessment of its field effectiveness in TB, MDR-TB and TB/HIV co-infection (Small and Pai, 2010). This test has the possibility of transforming the diagnosis of TB (Van Rie et al, 2010).


Xpert MTB/RIF is a cartridge-based, automated, user-friendly real-time Polymerase Chain Reaction (PCR) assay designed for the rapid and simultaneous identification of Mycobacterium tuberculosis and resistance to rifampicin, through nucleic acid amplification (Helb et al, 2010). The assay amplifies a M. tuberculosis complex-specific region of the rpoB gene, which is probed with molecular beacons to recognise the presence of rifampicin resistance-determining mutations (El Hajj, 2001).  The World Health Organization (WHO) recommended the use of Xpert MTB/RIF as a first test in HIV-TB co-infected patients and patients with presumptive multidrug resistant TB (WHO, 2010).


An unprecedented increase in incidence of TB in Sub-saharan Africa has been brought about by Human Immunodeficiency Virus (HIV) infection (Corbett, et al., 2003). This HIV related increase in the prevalence of active TB is compounding the problem of TB diagnosis (Mendelson, 2007; Perkin and Cunningham, 2007). Following the use of sputum microscopy, the sensitivity may be reduced further in HIV-infected individuals because HIV-positive TB patients have reduced ability to develop cavitary diseases because of immunosuppression and consequently have lower numbers of tubercle bacilli in the airway (Crampin et al., 2006; Cattamanchi et al., 2009; Davis et al., 2010). It has been suggested that as the incidence of HIV-driven TB epidemic increases, the proportion of pulmonary infection with nontuberculous mycobacteria (NTM) would increase. This is expected to affect sputum smear microscopy in terms of specificity which has been traditionally believed to be high for AFB in high TB-burdened countries (Perkins, 2000; Steingart et al., 2006). The influence of infections due to NTM on the specificity of AFB has not been investigated fully although there are reports that considerable proportion of cases of Pulmonary TB is due to these organisms (Primm et al., 2006; Mawak et al., 2006; Addo et al., 2007). In most studies in Africa and under-developed countries, the focus has been on the  M. tuberculosis complex and the role of NTM has been under-appreciated (Niobe-Eyangoh et al., 2003; Rosales et al., 2010). The impact of HIV on the performance characteristics of diagnostic tests that are commonly used for TB needs to be re-assessed (Cattamanchi et al., 2009; Dawson et al., 2010).



Despite the immense global burden of tuberculosis, case detection rates are low, posing serious hurdles for global TB control. Nigeria currently ranks 7th globally and 2nd in Africa among the 30 high burden countries designated by World Health Organization (WHO, 2015).  In recent times, the prevention, diagnosis, and treatment of tuberculosis have become more complicated because of two elements changing the epidemic: HIV-associated TB and multidrug-resistant (MDR) TB. Many people die from TB because their diagnosis is slow, and the epidemic continues to ride on due to the failure to significantly curtail transmission with current diagnosis (WHO, 2010).


 The diagnosis of TB has traditionally relied on sputum smear microscopy, sputum culture, tuberculin skin test and chest radiography. These tests have various limitations that make them inadequate for the diagnostic expectation of the current TB control strategy (Keeler et al., 2006; Perkins et al.,2006).


Sputum microscopy is laborious, it is of low sensitivity especially in situations where there is high prevalence of TB/HIV co-infection and it becomes a challenge in children less than 5 years of age who are not able to produce sputum (Kehinde et al., 2005). In addition to the problem of low sensitivity, it requires significant labour and training. These factors result in missed cases, reduced access to diagnostic services and heavy workloads to already overburdened health system. Although smear microscopy is widely used, it does not detect drug resistance and low sensitivity in HIV co-infected individuals (Getahun et al., 2007). Microscopy alone, although inexpensive, misses many patients and detects only those with relatively advanced disease (Steingart et al., 2007). Smear microscopy is highly accessible and inexpensive and together with chest X-rays has been in use for a long time by TB control agencies worldwide.


Culture technique is a more sensitive method than sputum smear microscopy and is regarded as the gold standard for definitive diagnosis of active TB (Azziz, et al., 2007; Dorman, 2010). However, culture of M. tuberculosis is not routinely done in developing countries because it requires high technical expertise and biosafety facilities that are not usually available in developing countries (WHO, 2008; Dowdy et al., 2008). However, M. tuberculosis grows slowly and results of mycobacterial culture often only become available after 2 to 8 weeks. This creates a diagnostic delay that hampers disease control, enhances transmission and increases healthcare cost (WHO, 2009).

Chest radiography method (chest x-ray) is used to complement sputum smear microscopy, especially in sputum smear negative cases. Apart from technical and cost concerns, X-ray is not specific for TB and the use of the test alone can lead to misdiagnosis or over-diagnosis with attendant problems of inappropriate or unnecessary treatment.


Tuberculin skin test (TST) is a test widely used for screening for exposure to M. tuberculosis and for detection of latent TB infection. The test is not suitable for diagnosis of active TB disease because though it can detect exposure, it cannot distinguish exposure from active disease (Farhat et al 2006). The purified protein derivative (PPD) antigen used in the test cross reacts with antigens from the M. bovis strain, Bacille Calmette Guerin (BCG) and other environmental mycobacteria. For this reason, the specificity of TST is poor when used for diagnosis of active TB (Dodd et al., 2010).



The aim of this research project is to compare the diagnostic performance of sputum microscopy and Xpert MTB/RIF for case detection of pulmonary TB.


1.4.1    Specific objectives

1.      To determine the case detection rate of pulmonary tuberculosis by Xpert MTB/RIF in Abia State.

2.       To assess the sensitivity and specificity of Xpert MTB/RIF among pulmonary TB patients.

3.      To compare the diagnostic performance of Xpert MTB/RIF with sputum-smear microscopy in routine laboratory case detection of pulmonary tuberculosis in Abia State.

4.      To compare sensitivity, specificity, positive and negative predictive values of Xpert MTB/RIF with sputum microscopy.

5.      To compare the performance of the Xpert MTB/RIF with Sputum Smear Microscopy in HIV/TB co-infection.




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