ABSTRACT
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.
TABLE OF CONTENTS
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
LIST OF TABLES
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
LIST OF FIGURE
4.1 Comparison
of the sensitivities and specificities of sputum smear
microscopy and MTB/RIF 43
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF
THE STUDY
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).
1.2 PROBLEM STATEMENT
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).
1.3 JUSTIFICATION
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).
1.4 AIMS
AND OBJECTIVES
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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