UNIVERSITY OF GHANA 

COLLEGE OF HEALTH SCIENCES  

UNIVERSITY OF GHANA MEDICAL SCHOOL 

 

 

 

THE BURDEN OF EXTENSIVELY DRUG RESISTANT AND PRE-EXTENSIVELY 

DRUG RESISTANT TUBERCULOSIS AMONG MULTIDRUG-RESISTANT 

MYCOBACTERIUM TUBERCULOSIS PATIENTS IN GHANA. 

 

 

BY 

 

 

 

 

 STEPHEN OFORI YIRENKYI  

(10877694) 

 

 

 

 

 

THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN 

PARTIAL FUFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL 

MEDICAL MICROBIOLOGY DEGREE 

 

DEPARTMENT OF MEDICAL MICROBIOLOGY 

NOVEMBER 2022 

 

 

 

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ABSTRACT 

Background 

The majority of people around the world are at risk because of the rise of extensively drug-

resistant (XDR) and pre-extensively drug-resistant (Pre-XDR) tuberculosis (TB), especially in 

developing countries like Ghana. Extensively Drug Resistant Tuberculosis, a considerably 

more difficult-to-treat type of MDR tuberculosis, had been reported in at least 46 countries as 

of February 2008. By 2017, 77 countries had reported 11 000 infections of XDR-TB with a 

treatment success rate of only 34%. In Ghana, the first case of XDR-TB was published in 2018. 

However, there is insufficient information on the prevalence of XDR-TB, pre-XDR-TB, and 

their associated resistant mutations in Ghana. The study sought to provide a baseline data on 

the burden of pre-XDR-TB and XDR-TB in Ghana. It also determined the mutations 

responsible for pre-XDR/ XDR-TB, for clinical and programmatic management of pre-XDR/ 

XDR-TB in Ghana. 

Aim of Study: The main aim of the study was to determine the proportion of extensively and 

pre-extensively Drug Resistant Mycobacterium tuberculosis among multidrug-resistant M. 

tuberculosis complex patients in Ghana  

Methods: One hundred and seventy-one (171) archived clinical isolates of MDR-TB collected 

from patients across Ghana between, January 2016 to December, 2020 were retrieved. The 

isolates were retested to confirm their phenotypic and genotypic drug susceptibility to the first- 

and second-line anti-TB drugs using the BACTEC MGIT system and MTBDRplus 96, 

MTBDRsl 96-line probe assays respectively 

Results: Of the 171 archival isolates collected, most of the isolates came from 7 regions, 

Eastern region having the highest (39.5%) followed by Greater Accra (19.8%). Majority of the 

samples were from males (78.9%). Of the 171 archival isolates collected, 81 (47.4%) were 

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confirmed to be MDR, 6 (7.4%) were Pre-XDR-TB and no XDR-TB was detected. The katG 

S315T1 (73.3%) and rpoB S531L (42.5%) were the common mutations observed among 

isoniazid and rifampicin resistant isolates respectively. Majority of the mutations and amino 

acid changes that caused pre-XDR-TB were gyrAWT3+gyrAMUT3A and gyrAMUT3A (D94A) 

(50.0%) for fluoroquinolone and (rrsWT1) C1402T (50.0%) for aminoglycosides. The other 

detected mutations with their amino acid changes were gyrA MUT1 (A90V), gyrAWT3+gyrA 

MUT3C (D94G) and gyrA MUT2 (S91P) (16.7%) each for fluoroquinolones and rrWT2 

(position 1484) (33.3%) and rrs MUT2 (G1484T) (16.7%) for the aminoglycosides. 

Conclusion: Pre-XDR-TB prevalence was marginally higher amongst MDR-TB patients in 

Ghana and no XDR-TB was detected. Nonetheless, a sustained surveillance of pre-XDR-TB 

and XDR-TB is advocated. The most common flouroquinolone mutation associated with pre-

XDR-TB was D94A 

 

 

 

 

 

 

 

 

 

 

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DEDICATION 

This work is dedicated to all patients with MDR-TB who died of Pre-XDR –TB and XDR-TB 

before the introduction of Pre XDR-TB and XDR-TB diagnosis in Ghana. It is also dedicated 

to Mr. Jonas Ofori Yirenkyi, my late father who always urged me to pursue this degree, my 

supportive wife, Lydia Merley Laryea, my mother, Victoria Agyemfrah and my children, Seth 

Ofori Yirenkyi, Stephanie O. Ofori Yirenkyi and Steve Laryea Ofori Yirenkyi. 

 

 

 

 

 

 

 

 

 

 

 

 

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ACKNOWLEDGEMENT 

My profound gratitude goes to the Almighty God in Heaven the Creator, for His abundant grace 

and making this work a possibility. 

I also express my profound gratitude and acknowledgement for the immense contributions of 

my supervisors, Prof. Japheth A. Opintan, Prof. Mercy Newman and Dr. Gloria Ivy Mensah, 

for their diligent guidance to see this study through. The Lord Jesus Christ bless you to continue 

to impact into generations. 

My heartfelt appreciation to Mr. Felix Sorvor of the National TB control Programme for 

providing me with reagents, materials, and data towards this work. 

I am also grateful to Head of laboratory of the Eastern Regional Hospital, Koforidua and Mr. 

Roger Laryea of the Eastern Regional Hospital, and Mr. Amo Omar (Head of Chest Clinic 

Lab.), Korle Bu Teaching Hospital and Mr. Honesty Ganu for their immense support especially 

for granting me access to the isolates for this work.  I wish to thank Mr. Roger Laryea, Mr. 

Felix Sorvor, Mr. Osmaun Taufik and Mr. Honesty Ganu for their various contributions in 

laboratory work, data analysis and proof reading of the write up of this work. 

I say thank you all and the Lord Jesus Christ bless you abundantly.

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Table of Contents 

ABSTRACT  ............................................................................................................................  III 

CHAPTER ONE INTRODUCTION ................................................................................................ 13 

1.1 Problem Statement .............................................................................................................. 15 

1.2 Justification ......................................................................................................................... 16 

1.3 Aim of Study ........................................................................................................................ 17 

1.4 Specific Objectives .............................................................................................................. 17 

CHAPTER TWO LITERATURE REVIEW  ................................................................................ 18 

2.1.  History of Tuberculosis ......................................................................................................... 18 

2.2.1  Tuberculosis in Ghana ........................................................................................................... 20 

2.3  Epidemiology of Tuberculosis and Drug Resistant Tuberculosis ...................................... 20 

2.5  Clinical Manifestation of Tuberculosis ................................................................................ 29 

2.6  Transmission of Tuberculosis ............................................................................................... 30 

2.7   Laboratory Diagnosis of Tuberculosis and Drug Resistant Tuberculosis ........................ 30 

2.8  Treatment/ Prevention of Drug Resistant Tuberculosis ..................................................... 38 

2.9  Genome of M. tuberculosis: ................................................................................................... 41 

2.10 Mechanism of Resistance of FLDs ......................................................................................... 42 

2.10.1  Rifampicin molecular resistant mechanism and the rpoB gene ......................................... 42 

2.10.2  Isoniazid molecular resistant mechanism and the katG and inhA gene ............................ 43 

2.11 Mechanism of Resistance of SLDs ......................................................................................... 44 

2.11.1  Mechanism of Molecular Resistance of Fluoroquinolones and gyraA and gyrB genes .... 44 

2.12   Pre-XDR-TB and XDR-TB ................................................................................................... 48 

CHAPTER THREE MATERIALS AND METHODS .................................................................... 50 

3.1 Study Design: ....................................................................................................................... 50 

3.2  Chemical and Reagents ...................................................................................................... 50 

3.3  Study Site and Sample Collection ..................................................................................... 50 

3.3.1 Patient Data Collection ....................................................................................................... 51 

3.4 Ethical Approval ................................................................................................................. 51 

3.5  Sample Size Determination ............................................................................................... 52 

3.6 Laboratory Procedures....................................................................................................... 53 

3.6.1        Isolate Recovery .................................................................................................................. 53 

3.6.1.1     BACTEC MGIT 960 Inoculation and Incubation. .......................................................... 53 

3.6.1.2     Confirmation of Mycobacterial Growth ........................................................................... 54 

3.6.1.3    Mycobacterial Growth Purity Check ................................................................................. 55 

3.6.1.4    Mycobacterium Tuberculosis Complex Identification ....................................................... 56 

3.6.1.5     Phenotypic Antimicrobial Susceptibility Testing ............................................................. 57 

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3.6.2 Molecular Method ............................................................................................................... 58 

3.6.2.1  DNA Extraction .................................................................................................................. 58 

3.6.2.2     PCR Amplification .............................................................................................................. 58 

3.6.2.3  Line Probe Assay ................................................................................................................. 60 

3.7 Quality Assurance ............................................................................................................... 61 

3.8 Biosafety ............................................................................................................................... 62 

3.9 Data Management and Statistical Analysis ...................................................................... 62 

CHAPTER FOUR  RESULTS........................................................................................................... 63 

4.1  Sample Selection ................................................................................................................. 63 

4.2  Patient Demographics ........................................................................................................ 64 

4.3  Phenotypic Drug Susceptibility Testing ............................................................................ 65 

4.4  Genotypic Drug Resistance Testing ................................................................................... 65 

4.5  Mutational Patterns Observed ........................................................................................... 68 

CHAPTER FIVE DISCUSSION AND CONCLUSION ............................................................... 72 

5.1  DISCUSSION .......................................................................................................................... 72 

5.1.1  Prevalence of MDR Among Different Gender, Age Group and Region of Residence ...... 72 

5.1.2  Resistance to first line anti-TB drugs .................................................................................... 73 

5.1.3  Resistance to second line anti-TB drugs ............................................................................... 75 

5.2  CONCLUSION ....................................................................................................................... 77 

5.3  RECOMMENDATIONS ....................................................................................................... 78 

5.4  LIMITATIONS ....................................................................................................................... 78 

6.0  REFERENCES ........................................................................................................................ 79 

APPENDICES  ............................................................................................................................. 86 

7.1  Appendix I: MATERIALS ..................................................................................................... 86 

7.2  Appendix II: REAGENT COMPOSITION AND PREPARATION .................................. 88 

7.3  Appendix III:  PROCEDURES ............................................................................................. 92 

7.4  Appendix IV: Sample results of Genotype MTBDRplus Line Probe Assay ...................... 97 

7.5  Appendix V: Sample result of Genotype MTBDRsl Line Probe Assay ............................. 98 

7.6  Appendix VI: Ethical Clearance Approval........................................................................... 88 

7.7  Appendix VII: Facility Approval Letter ............................................................................... 89 

7.8  Appendix VIII: Table of Results of Isolates ......................................................................... 89 

 

 

 

 

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LIST OF FIGURES 

Fig. 1 Global estimated tuberculosis incidence rate, 2019 

Fig. 2 Global Distribution of new TB cases with MDR/ RR-TB 

Fig. 3 Global Distribution of previously treated TB cases with MDR/RR-TB 

Fig. 4  Global distribution of MDR-TB and XDR-TB in 2019 

Fig. 5 AFBs (arrowed) in Ziehl-Neelsen stained smear (A) and Auramine O- stained Smear (B) 

Fig. 6 Mycobacterium tuberculosis growth on Lowenstein Jensen media (A) and MGIT broth (B) 

Fig. 7 Pattern of the strips for the Genotypye MTBDRplus V2 (a) and Genotype MTBDRsl V2 

(b) 

Fig. 8 Standard MTBDRplus Assay for the detection of MTBC and RIF and INH related mutations 

Fig. 9 Standard MTBDRsl Assay for the detection of MTBC and FQ and SLI related mutations 

Fig. 10 Chemical Structure of Fluoroquinolones . 

Fig. 11 Chemical Structure of Streptomycin (Aminoglycoside) 

Fig. 12 Serpentine cord of M. tuberculosis of culture-positive specimen smear stained by Ziehl-

Neelsen staining technique 

Fig. 13 Immunochromatographic cassettes for M. tuberculosis identification 

Fig. 14 Trend of Confirmed MDR-TB, 2016-2020)      

Fig. 15 Distribution of MDR-TB isolates by age 

Fig. 16 Regional Distribution of the MDR-TB Isolates 

Fig. 17 Sample result of Genotype MTBDRplus  Line Probe Assay 

Fig. 18 Sample result of Genotype MTBDRsl Line Probe Assay 

  

 

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LIST OF TABLES 

 

Table 1 TB, HIV-TB positive, MDR/RR TB Notifications, Worldwide and for WHO Regions, 

2019. 

Table 2 The adult regimen for Pre and XDR-TB 

Table 3 Pre-XDRTB and XDRTB regimen in children 

Table 4  Phenotypic drug resistance patterns 

Table 5 Genotypic drug resistance of first and second-line anti-TB drugs sand prevalence of MDR 

and pre-XDR among the patients 

Table 6 First line drug mutation patterns observed among MDR-TB by Genotype MTBDR plus 

Table 7 Second line drug mutation patterns observed among MDR-TB by Genotype MTBDR plus 

Table 8 Observed MTBDRplus assay band patterns for first line anti-TB drugs 

Table 9 Observed MTBDR assay band patterns for second line anti-TB drugs 

 

 

 

 

 

 

 

 

 

 

 

 

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LIST OF ABBREVIATIONS 

 

AFB Acid Fast Bacilli 

ADR Adverse Drug Reactions 

AM Amikacin 

BDQ Bedaquiline 

CAP Capreomycin 

Cfz Clofazimine 

Cs Cycloserine 

Dlm Delamanid 

DOT Directly Observed Therapy 

DR Drug Resistance 

DR-TB Drug Resistance Tuberculosis 

DST Drug Susceptibility Testing 

EQA External Quality Control 

E Ethambutol 

Eto Ethionamide 

FLD First-Line Anti TB medicines 

GLI Global Laboratory Initiatives 

HIV Human Immunodeficiency Virus 

INH Isoniazid 

hH High-dose Isoniazid 

Imp-Cln Imipenem-cilastatin 

LPA Line Probe Assay 

Lfx Levofloxacin 

Lzd Linezolid 

MDR-TB Multi Drug Resistance Tuberculosis 

MGIT Mycobacterium Growth Indicator Tube 

MTB Mycobacterium tuberculosis 

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MTBC Mycobacterium tuberculosis Complex 

Mfx Moxifloxacin 

Mpm Meropenem 

NTP National TB Programme 

NTM Non-Tuberculous Mycobacterium 

Pre-XDR-TB Pre- Extensively Drug Resistance Tuberculosis 

PANTA Polymycin B, Amphotericin B, Nalixidic Acid, Trimetoprim and Azloxillin 

Pto Prothionamide 

PAS p-amoxicillin acid 

PCR Polymerase Chain Reaction 

QRDR Quinolone Resistance Determining Region 

RIF Rifampicin 

RR Rifampicin Resistance 

RRDR Rifampicin Resistance Determining Region 

SLD Second-line anti TB Drugs 

SLI Second-Line Injectables 

S Streptomycin 

SIRE Streptomycin, Isoniazid, Rifampicin and Ethambutol 

TB Tuberculosis 

Trd Terizidone 

WHO World Health Organization 

XDR Extensively Drug Resistance 

ZN Ziehl-Neelsen 

Z Pyrazinamide 

 

 

 

 

 

 

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CHAPTER ONE 

1.0 INTRODUCTION 

 

Despite intensive efforts to manage it, tuberculosis (TB) continues to be a serious medical issue 

with a significant public health impact, particularly in low- and middle-income nations (Cao et 

al., 2013). Tuberculosis is still one of the most prevalent communicable diseases in the world, 

much like in Ghana where DOTS (Directly Observed Treatment Short Course) is still utilized 

to administer drugs to treat the illness  (Acheampong et al., 2018).  

Globally, national TB control programmes (NTP) are finding it increasingly difficult to control 

and manage TB cases. Significant causes include the increase in drug-resistant strains of the 

TB pathogen, Mycobacterium tuberculosis, as well as inferior disease treatment outcomes. 

Poor medical management of patients, lack of directly observed treatment, limited, or 

interrupted drug supplies, poor drug quality, widespread availability of anti-tuberculosis drugs 

without prescription, and lack of uniformity between the public and private health sectors 

regarding drug resistance may all contribute to the development of drug-resistant TB and 

subsequent transmission of drug-resistant strains in the community (Lambregts-van et al.,  

1998).  

The spread of resistant strains, particularly multidrug-resistant (MDR) TB strains, which 

challenge national control efforts, increases the burden of this contagious deadly infection 

(Vaziri et al., 2019). Evidence demonstrates that multidrug-resistant tuberculosis (MDR-TB), 

which poses a severe danger to global TB prevention and burdens developing nations with 

costly and toxic therapies, worsens the tuberculosis epidemic (Liu et al., 2021). The 

tuberculosis epidemic has gotten worse over the past ten years due to the progression of drug 

resistance in failing MDR-TB treatment regimens towards pre-extensively drug resistance (pre-

XDR) and extensively drug resistant tuberculosis (XDR-TB) (Oudghiri et al., 2021). According 

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to the World Health Organization, MDR-TB is defined as mycobacterium tuberculosis strain 

which is resistant to Rifampicin and Isoniazid, pre-XDR-TB is defined as MDR-TB strain and 

also rifampicin-resistant with resistance to fluoroquinolone (FQ), on the other hand, 

extensively drug-resistant (XDR)-TB denotes MDR-TB strain with further resistance to any 

FQs [ofloxacin levofloxacin or moxifloxacin] and other group A drugs (Bedaquilline, and 

Linezolid) ( Sinha et al., 2017; Chakaya et al., 2022). 

Extensively drug-resistant (XDR)-TB, a considerably more difficult-to-treat type of MDR-TB, 

was initially recorded in 46 countries in 2008, however, 77 countries  reported  XDR-TB cases 

with an efficacy rate of only 34% as reported by WHO in 2017  (WHO, 2018).  

With the detection of a possible link between mutations influencing expression and functioning 

of chromosome-encoded targets and medication resistance to TB, superior techniques such as 

molecular technologies have replaced traditional methods for sensitive, and more reliable 

diagnosis of TB and assessment of the mycobacterial resistance status (Oudghiri et al., 2018). 

Rifampicin (RIF), Isoniazid (INH), Fluoroquinolone (FQ), and second-line injectable anti-TB 

medicine resistance have all been extensively demonstrated over the years in a variety of 

studies. (Angelina et al., 2021; Oladimeji et al., 2022; Ssengooba et al., 2016). Molecular 

research also suggested that point mutations in the rpoB gene are mostly responsible for RIF 

resistance, while mutations in the katG, ahpC, and inhA genes are responsible for INH 

resistance (Ali et al., 2011; Oudghiri et al., 2018; Rana et al., 2022). Furthermore, it is 

hypothesized that point mutations in the genes encoding the two DNA subunits, gyrA and gyrB, 

are the main source of FQs resistance. (Province et al., 2019). Other earlier publications 

indicated that the majority of the mutations causing FQ resistance are concentrated in a brief 

region of the gyrA gene known as the Quinolone Resistance Determining Region (QRDR) 

(Mujuni et al., 2022; Province et al., 2019). In general, it is understood that mutations in the 

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rrs genes are frequently known to confer resistance to injectable medicines, however mutations 

in the tlyA gene and eis gene have also been mentioned (Ali et al., 2011; Diriba et al., 2022).  

1.1 Problem Statement 

The advent of M. tuberculosis strains which are resistant to powerful anti-tuberculosis 

medications has endangered global attempts to speed up the END TB Strategy by 2030 adopted 

by UN in the Sustainable Development Goals. (Goyal et al., 2017; WHO, 2020). The 

proportion of MDR-TB patients who have XDR-TB or pre-XDR-TB has seen considerable 

increase over the past few decades in most countries especially high burden TB countries and 

developing countries like Ghana and continues to rise steadily (WHO, 2019). In 2018, it was 

projected that 6.2% of MDR-TB infections had extensively drug resistance (Id et al., 2020). 

Hence, estimating the burden of XDR-TB/ pre-XDR-TB among MDR-TB holds immense 

epidemiological and preventive importance especially in endemic areas like Ghana, 

Finding drug resistance-causing mutations in M. tuberculosis in a specific geographic area is 

crucial for epidemiological purposes (Diriba et al., 2022). In light of this, several studies have 

been carried out worldwide mostly in high TB burden countries to determine burden and 

mutations within the M. tuberculosis genome especially within the hot spot regions of genes 

associated with XDR and pre-XDR TB (Ali et al., 2011; Mujuni et al., 2022; Salvato et al., 

2019). However, in Ghana, there is inadequate data on the burden of XDR and pre-XDR-TB 

with the associated gene mutations and the patterns of the mutations responsible for this deadly 

form of already devastating disease condition. If this is not properly studied and utilized could 

undermine the END TB strategy being undertaken by the National Tuberculosis Control 

Programme (NTP). It is against this background that the present study is designed to investigate 

the burden of extensively drug resistance and pre-extensively drug resistance tuberculosis 

among multidrug-resistant mycobacterium tuberculosis patients in Ghana. 

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1.2 Justification  

According to a recent global T.B report, 3.3% of new TB diagnosis and 18% of TB diagnoses 

that had previously been treated globally developed MDR/ RR-TB out which 17.9% had pre-

XDR/ XDR-TB  (Diriba et al., 2022; WHO, 2022). In Africa, in 2017 it was reported that for 

both newly diagnosed cases and patients that had already been treated, the rates of RR/MDR-

TB were 2.7%and 14%, respectively with 727 XDR-TB cases and in 2021, 20000 cases of 

MDR-TB were reported in Africa with 5.5% pre-XDR/ XDR-TB (WHO, 2018; WHO, 2022). 

Osei-Wusu and colleagues reported Ghana's first case of XDR-TB in 2018 (Osei-Wusu, et al, 

2018).  

To execute the necessary measures to reduce risk episodes and related consequences in MDR-

TB patients, it is crucial to provide a baseline description of the burden of extensively and pre-

extensively drug resistant tuberculosis. The study will provide a baseline data on the burden of 

pre-XDR-TB and XDR-TB in Ghana and will further provide information on the mutations 

that are associated with pre-XDR/ XDR-TB and the mutational patterns of these strains in the 

country. The data would set the platform for policy reforms in clinical and programmatic 

management of MDR-TB, and XDR-TB/ pre-XDR-TB in Ghana to the benefit of drug resistant 

TB patients and the general populace. 

 

 

 

 

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1.3 Aim of Study  

The main aim of the study is to determine the proportion of Pre-XDR/ XDR-TB among MDR-

TB patients in Ghana 

1.4 Specific Objectives  

The specific objectives are to: 

i. To determine phenotypic drug resistance of MDR-TB isolates to first line anti-TB drugs 

ii. To identify genotypic drug resistance of first- and second-line anti-TB drugs and the 

mutations associated to pre-XDR/ XDR M. tuberculosis. 

 

 

 

 

 

 

 

 

 

 

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CHAPTER TWO 

LITERATURE REVIEW 

2.1.  History of Tuberculosis  

One of the first infectious diseases that man is aware of is tuberculosis, which first appeared 

around 3400 BC. It was the root of the "White Plague" that afflicted Europe in the seventeenth 

and eighteenth centuries. During this time, over 100% of the European population was sick, 

and TB was responsible for a quarter of all adult deaths (Todar K, 2009;Schiffman, 2009). 

Tuberculosis (TB) is an ancient infection that has been recorded for thousands of years, from 

studies of ancient human bones. Its origin remained unknown until March 24, 1882, when Dr. 

Robert Koch announced his discovery of the causative bacillus, later known as Mycobacterium 

tuberculosis (Nakaoka et al., 2006;Baddeley, 2020). At the time its etiologic agent was 

discovered in 1882, about one-seventh of all fatalities in Europe were caused by TB. Today, it 

persists as one of the commonest infections and the leading cause of mortality caused by a 

single infectious agent (Nakaoka et al., 2006;WHO, 2020). Drug Resistant TB (DR-TB) is 

giving rise to lots of challenges comparable to those faced prior to the development of 

chemotherapy, such as the inability to cure TB, high mortality and morbidity, unabated 

transmission posing a major public health threat and unsustainable treatment cost (Demile et 

al., 2018). 

2.2  Global Burden of Tuberculosis 

According to WHO ( WHO, 2020) in its 2020 report, approximately more than a quarter of the 

world population are living with  M. tuberculosis and despite all the efforts by National T.B 

control programmes with the END TB strategy adopted by WHO, there was an estimated 10.6 

million TB incidences in 2021 with 1.4 million deaths of HIV-negative people and 187, 000 

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deaths of HIV-positives people. The number of new cases of drug-resistant TB diagnoses in 

2021 was about 450 000.(WHO, 2022).  

Worldwide, more than 10 million persons with an incidence of both new and relapsed TB 

infections were diagnosed in 2021 and reported to WHO through various national programs, 

and other relevant organisations. A rise from 5.8 million in 2020 (WHO, 2022) 

A great portion of the global TB cases (Fig. 1) are concentrated in developing and low-

income nations.

 

Figure 1. Global estimated tuberculosis incidence rate, 2019. (Source: Baddeley, 2020; Shana, 

2021). 

The highest rates of TB cases in 2019 were seen in the WHO regions of South-East Asia (44 

percent), Africa (25 percent), and the Western Pacific (18 percent). The lowest rates were found 

in the Americas (2.9%), Europe (2.5%), and the Eastern Mediterranean (8.2%). Eight countries, 

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including Bangladesh (3.6%), India (26%), Indonesia (8.5%), China (8.4%), the Philippines 

(6.0%), Pakistan (5.7%), Nigeria (4.4%), and South Africa (3.6%), accounted for two thirds of 

the global total (Baddeley, 2020; Chakaya et al., 2021). 

2.2.1 Tuberculosis in Ghana 

A serious concern to public health in Ghana and around the world is tuberculosis (Bonsu et al., 

2020) Based on current data, tuberculosis (TB) is a highly prevalent disease in Ghana, 

contributing significantly to both major disability and death rates in the nation (Addo et al., 

2018). According to a recent national TB prevalence survey, 111 (95% CI: 76–145) adults had 

smear-positive TB per 100,000 people. 356 (95% CI: 288–425) cases of bacteriologically 

proven tuberculosis were reported for per 100,000 people (Angelina et al., 2021: Chakaya et 

al., 2022). MDR-TB has been documented to have emerged in Ghana, according to several 

research. The first cases of extensively drug-resistant (XDR) tuberculosis were reported in 

2018 (Angelina et al., 2021). According to the WHO global TB 2022 report (WHO, 2022), the 

burden of TB in Ghana was 136 per 100 000 population, total incidence of MDR/RR-TB was 

3.6 per 100 000 population whiles there was only one (1) reported case of XDR-TB 

2.3  Epidemiology of Tuberculosis and Drug Resistant Tuberculosis 

TB continuous to be among the top ten causes of mortality globally and the leading cause of 

mortality from a single microbial agent since 2007 and ranks higher than HIV/AIDS. In 2021, 

approximately 10.6 million persons were diagnosed with tuberculosis (TB) worldwide and 

approximately 1.6 million people died of the infection (Helwig et al., 2022). Of the estimated 

10.6 million persons diagnosed with TB, 5.5 million (56%) were men, 3.2 million (32%) were 

women, 1.2 million (12%) were children (Smith, 2021). Two-thirds (2/3) of the global burden 

were in eight countries; Indonesia, India, China, Nigeria the Philippines, Democratic Republic 

of Congo, Pakistan, and Bangladesh (WHO, 2022; Smith, 2021). Tuberculosis is found all over 

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the world, however it is more prevalent in underdeveloped and emerging countries (Baddeley, 

2020). In 2019, the World Health Organization (WHO) areas of South-East Asia (44%), Africa 

(25%), and the Western Pacific (18%) had the most TB cases, with lesser numbers in the 

Eastern Mediterranean (8.2%), the Americas (2.9%), and Europe (2.5%) (Baddeley, 2020). In 

Africa, Southeast Asia, the Middle East, Latin America, and Eastern Europe, tuberculosis is 

widespread. The prevalence of tuberculosis varies greatly depending on the nation, age, race, 

gender, and socioeconomic position of the individual. Infection with the human 

immunodeficiency virus (HIV) is a substantial risk factor for active tuberculosis. HIV-positive 

people are 400 times more likely than HIV-negative people to get tuberculosis (Baddeley, 

2020). People living with HIV accounted for 8.2% of all TB cases (Chakaya et al., 2021). The 

most current global tuberculosis report states that in 2018 3.4% of new TB infections and 18% 

of cases of TB were diagnosed as Rifampicin Resistant (RR)/MDR-TB. In 2021, around 

450000 people globally acquired RR/MDR-TB (WHO, 2022). 

A total of 450 000 MDR/RR-TB cases were diagnosed and reported globally in 2021, an 

increase of 10% from 186 883 cases in 2018 (WHO, 2020) 

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 Figure 2.  Global Distribution of new TB cases with MDR/ RR-TB (Source, WHO, 2020; 

Smith, 2021). 

 

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Figure 3. Global Distribution of previously treated TB cases with MDR/RR-TB (Source, 

WHO, 2020; Smith, 2021). 

In 2021, it was estimated that 17.9% of MDR-TB cases worldwide had XDR-TB (Id et al., 

2020: WHO, 2022). In Africa it was reported that the rates of RR/MDR-TB for fresh cases and 

those who had already had treatment were 2.7%and 14%, respectively with 727 XDR-TB cases 

and in 2021 there were 20 000 cases of MDR-TB with 5.5% pre-XDR/ XDR-TB (WHO, 2018; 

WHO, 2022).  The first XDR-TB was isolated in Ghana in 2018 (Osei-wusu et al., 2018). 

TB, HIV-TB positive, MDR/RR-TB, and XDR-TB cases have been reported in 2019 

worldwide and for each WHO area. The notifications are listed in the table below. 

 

 

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Table 1.0: TB, HIV-TB positive, MDR/RR TB, and XDR TB Notifications, Worldwide and 

for WHO Regions, 2019.  

 

(Source: WHO, 2020) 

 

Figure 4.  Global distribution of MDR-TB and XDR-TB in 2019 (Source,  CDC, 2020) 

 

 

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2.4 Pathophysiology and Pathogenesis of Tuberculosis 

The bacillus Mycobacterium tuberculosis causes tuberculosis, which most commonly affects 

the lungs (pulmonary tuberculosis), but can also affect other parts of the body (extrapulmonary 

TB) (Baddeley, 2020). Alveolar macrophages in the lungs take up M. tuberculosis, which are 

unable to digest it. By inhibiting the bridge molecule early endosomal auto-antigen 1, the 

bacterium cell wall stops the phagosome from fusing with a lysosome (EEA1). The fusion of 

vesicles containing nutrients is not prevented by this blockage. As a result, the bacteria grow 

uncontrollably inside the macrophage and elude macrophage destruction by neutralizing 

reactive nitrogen intermediates. The bacteria also have the UreC gene, which stops the 

phagosome from becoming acidic ((SS Munsiff et al, 2008; Bell, 2005). Tuberculosis may 

affect almost all the body's organs, and the disease's onset and course might differ from one 

location to the other. The most frequent and contagious kind of tuberculosis is pulmonary TB. 

It damages lung tissues, from which M. tuberculosis can spread to other organs by blood 

circulation, causing milliary and extrapulmonary tuberculosis. (Todar, 2009) 

Disease development is determined by the M. tuberculosis complex strain, previous exposure, 

immunization, infectious dosage, and the infected host's immunological condition (Todar K, 

2009). 

There are five phases involved in the development and course of the disease. 

Phase 1: Uninfected people inhale small droplet nuclei that can stay airborne for several hours 

after they are ejected by a person with active TB infection. The nuclei of the inhaled droplets 

may go to the lungs' air sacs, or alveoli, where infection starts. Alveolar macrophages take up 

the germs in a general way. However, because they are not active, the macrophages cannot 

eliminate the intracellular pathogens (Todar K, 2009; Baron et al., 1999). 

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i. Phase 2 starts between 7 and 21 days after the initial infection. In inactive macrophages, 

Mycobacterium tuberculosis grows almost unchecked until the macrophages explode. 

To phagocytose the bacteria, more macrophages start to extravasate from peripheral 

blood into the alveoli, but these are similarly inactivated and cannot kill the germs 

(Todar K, 2009; Baron et al., 1999; V. Balasubramanian et al, 2004; American Thoracic 

Society, 2000). 

ii. Phase 3: Lymphocytes start to infiltrate alveoli. The M. tuberculosis antigen is 

recognised by lymphocytes, in particular T cells, when it is digested and presented in 

the presence of MHC molecules. T-cells become activated as a result, and cytokines 

such gamma interferon (IFN-) are released (Ganu, 2016; Todar K, 2009; Baron et al., 

1999). The release of IFN- γ results in the activation of macrophages, making them able 

to eradicate M. tuberculosis. At this point, the person tests positive for tuberculin (Ganu, 

2016; Todar K, 2009; Baron et al., 1999). The host's robust cell-mediated immune 

response is what causes this positive tuberculin reaction. To control an infection, a cell-

mediated immune response must be mounted. An antibody-mediated immune response 

will not assist in the prevention of MTB disease since this organism is intracellular and, 

if extracellular, is resistant to complement killing because of the high lipid 

concentration in its cell envelop. (Ganu, 2016; Todar K, 2009). Even though a cell-

mediated immune response is essential for preventing TB infection, it is also largely to 

blame due to the pathology connected to TB. The release of lytic enzymes and reactive 

intermediates by activated macrophages may promote the emergence of immunological 

disease tuberculin (Ganu, 2016; Todar K, 2009; Baron et al., 1999). 

TNF, gamma IFN, and Interleukin 1 (IL-1) are cytokines that are secreted by activated 

macrophages and T cells and may contribute to the emergence of immunological 

disease (Ganu, 2016; Todar K, 2009). The creation of tubercles starts at this point. The 

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"caseous necrosis" that gives the tubercle its semi-solid or "cheesy" consistency is what 

gives it its tubercular centre. The anoxic environment and low pH of these tubercles 

prevent Mycobacterium tuberculosis from growing there. However, the bacteria can 

survive for a long time inside these tubercles (Ganu, 2016; Todar K, 2009; Baron et al., 

1999). 

iii. Phase 4: Although there are a lot of activated macrophages around the tubercles. Many 

other macrophages are still inactive or only marginally activated. These macrophages 

are used by Mycobacterium tuberculosis to reproduce, which causes the tubercle to 

develop (Ganu, 2016; Todar K, 2009; Baron et al., 1999). A bronchus may be invaded 

by the developing tubercle. If this takes place, M. tuberculosis infection may spread to 

different lung regions. The tubercle may also encroach on an artery or other blood 

vessels in a similar manner. Extra pulmonary tuberculosis may develop through the 

haematogenous spread of M. tuberculosis, systemic or milliary tuberculosis (Ganu, 

2016; Todar K, 2009). Although secondary TB lesions can develop practically 

everywhere in the body, they typically affect the lymph nodes, genitourinary system, 

joint, peritoneum and bones. Exudative and granulomatous lesions are the two 

categories. The build-up of polymorphonuclear neutrophils (PMNs) around MTB 

causes exudative lesions (Ganu, 2016; Todar K, 2009; Baron et al., 1999). A "soft 

tubercle" develops as a result of the bacteria reproducing here with almost no resistance. 

When the host develops tuberculoprotein hypersensitivity, granulomatous lesions take 

place. A "hard tubercle" develops as a result of this circumstance (Ganu, 2016; Todar 

K, 2009; Baron et al., 1999). 

iv. Phase 5: The tubercles' caseous centres liquefy for an unidentified reason. Because of 

how well-suited this fluid is essential for the growth of M. tuberculosis, the bacterium 

quickly multiplies extracellularly (Ganu, 2016; Todar K, 2009; Baron et al., 1999). 

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After a while, the high antigen load causes the adjacent bronchial walls to burst and 

necrotize. Cavity creation follows from this. Additionally, M. tuberculosis can travel 

quickly to different lung regions and contaminate adjacent airways, as only 10% of M. 

tuberculosis infections result in illness, and an even lower percentage proceed to an 

advanced state (Ganu, 2016; Todar K, 2009; Baron et al., 1999). 

Usually, the infection will eventually be brought under control by the host. The initial 

lesion becomes fibrous and calcified as it recovers. When this happens, the lesion is 

referred to as the Ghon complex. The Ghon complex might never go away, depending 

on how big and severe it is. On a chest X-ray, the Ghon complex is typically easily 

noticeable (Ganu, 2016; Todar K, 2009). 

 

Most patients only have one main lesion. Bacilli spread through the pulmonary lymphatics as 

the initial lesion grows, eventually reaching the lymph nodes, which can expand. During the 

intracellular bacilli's growth, the lymph nodes enlarge. allowing bacilli to escape from the 

leaky, larger lymph node (Graham R. Stewart, 2003; Ganu, 2016). It's common to refer to TB 

as progressive primary TB if it arises straight from the primary disease's parenchymal or lymph 

node component (V. Balasubramanian et al, 2004; Ganu, 2016). The apical parts of the lung 

are where post-primary illness most frequently develops, even though original lesions can 

occur anywhere in the lung (Graham R. Stewart, 2003; Ganu, 2016). 

Small metastatic foci that have few M. tuberculosis cells may also calcify. These foci will 

typically contain live creatures, nevertheless. These points are known as Simon foci. Chest X-

rays can also show the Simon foci, which are frequently where the disease reactivates (Ganu, 

2016; Todar K, 2009; Baron et al., 1999). 

 

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2.5 Clinical Manifestation of Tuberculosis 

The organ involved, the bacterium's features, the surroundings, the host, and also interactions 

between both host and the organism, all have an enormous effect on the TB symptoms. 

(American Thoracic Society, 2000; Ganu, 2016). Sometimes symptoms do not appear until the 

disease has progressed significantly far. Initial symptoms could be attributed to other illnesses 

that have the same signs as TB. The most symptomatic form of TB, pulmonary, is marked by 

a chronic cough with progressive phlegm production, chest pains, exhaustion, dyspnea, weight 

loss, and hemoptysis. Other signs include fever, appetite loss, nocturnal sweats, chills, and 

paleness (WHO, 2016: Ganu, 2016). Most patients with pulmonary TB also have an abnormal 

chest X-ray, anaemia, and an elevated erythrocyte sedimentation rate (ESR) (Bonsu et al., 

2016); Ganu, 2016).  

Extrapulmonary TB has fewer symptoms and is more challenging to identify. Affected bones 

may enlarge and hurt from tuberculosis of the bones and spine, but the vertebrae may collapse 

and cause paralysis. If the joints are affected, you can have symptoms similar to arthritis. While 

intestinal TB causes abdominal swelling and soreness as well as pain that is similar to an 

appendicitis, bladder and renal TB can induce painful micturition and haematuria. Typically, 

pain is caused by organ damage at the affected areas (Ganu, 2016; Todar K, 2009). 

Isocitrate lyase (ICL), an enzyme necessary for the metabolism of fatty acids, was found to 

increase M. tuberculosis persistence in host tissues in a mouse research by (McKenny et al., 

2000; Ganu, 2016). In immune-competent mice, ICL gene disruption decreased bacterial 

persistence and virulence without influencing bacterial growth during the acute phase of 

infection. The regained pathogenicity of delta ICL bacteria in interferon-gamma (IFN) 

knockout mice demonstrated a relationship between the necessity for ICL and the 

immunological state of the host. At the level of the infected macrophages, this connection was 

obvious. ICL expression was increased when infected macrophages were activated, and the 

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delta ICL mutant was significantly reduced for survival in active macrophages but not in resting 

macrophages (McKenny et al., 2000; Ganu, 2016). This suggests that the host's reaction to 

infection has a significant impact on M. tuberculosis metabolism in vivo, which has substantial 

implications for the management of chronic TB (Ganu, 2016). 

The M. tuberculosis complex strain, prior exposure, immunisation, infectious dose, and the 

immunological condition of the infected host all have a role in disease progression (Ganu, 

2016; Todar K, 2009). 

 

2.6  Transmission of Tuberculosis 

Through the intake of air contaminated with droplet nuclei bearing M. tuberculosis bacilli with 

a diameter of 1 to 5µm, tuberculosis is spread from one person to another. The fluid evaporating 

from these tiny droplet nuclei causes the live tubercle bacillus to float for a considerable amount 

of time until being breathed (ATS, 2000; Sonal et al., 2008). Drug-resistant tuberculosis can 

spread by primary direct transmission or secondary transmission as a result of inadequate TB 

treatment over a protracted period of time. (Id et al., 2020; Palmero D. et al, 2015). 

 

2.7  Laboratory Diagnosis of Tuberculosis and Drug Resistant Tuberculosis 

The clinical sample used to diagnose pulmonary tuberculosis is sputum. In the study of extra-

pulmonary tuberculosis, stools, urine, cerebrospinal fluid (CSF), and aspirates from bone and 

joints may be utilized to identify M. tuberculosis. Microscopy, culture procedures, nucleic acid 

amplification methods, and immunological tests such as the tuberculin skin test can all be used 

in the laboratory to diagnose tuberculosis. M. tuberculosis may be detected as acid-fast bacilli 

(AFB) using the Ziehl-Neelsen and fluorescence microscopy procedures for the presumptive 

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diagnosis of tuberculosis (Frieden et al., 2003; Sonal et al., 2008). Only half of all pulmonary 

tuberculosis patients have Acid Fast Bacilli (AFB) in their sputum (Madigan et al., 2005). 

Microscopy requires an excess of 10,000 bacilli per ml of sputum to identify M. tuberculosis 

(Todar, 2009). The slightly curved long acid – fast bacilli are stained bright red in the regularly 

used Ziehl-Neelsen staining procedure, which contrasts sharply against a blue background. If 

accessible, fluorescent microscopy with Auramine staining is a somewhat more sensitive 

approach than Ziehl-Neelsen and could be used if available (Flowers, 1995; Ganu, 2016).  

                 

Figure 5: AFBs (arrowed) in Ziehl-Neelsen stained smear (A) and Auramine O stained Smear 

(B) (Source; Ganu, 2016) 

2.7.1 BACTEC MGIT 960 Liquid Culture System/ Phenotypic DST 

Isolation of M. tuberculosis on egg-based selective culture medium such as Lowenstein-Jensen 

and Ogawa media, which form cream-colored cauliflower-like colonies, can be used to provide 

a definitive diagnosis. However, because the bacterium takes 6 to 8 weeks to develop, this 

procedure is rather slow. Broth media, such as Middlebrook medium, and the BACTEC MGIT 

960 automated culture system now provide a speedier outcome for both isolation and 

phenotypic drug susceptibility testing (Chien, 2004). The sensitivity of M. tuberculosis culture 

A B 

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techniques is 10 bacilli/ml of sputum sample. M. tuberculosis develops cream coloured 

colonies that are dry with rough surfaces resembling cauliflower on solid medium. They create 

flakes or floccules in the liquid media of broth cultures (Sonal et al, 2008; Ganu, 2016; Bonsu 

et al, 2012).  

 

                      

Figure 6: Mycobacterium tuberculosis growth on Lowenstein Jensen media (A) and MGIT 

broth (B) (Source: Bonsu, 2013; (Ganu, 2016) 

 

The MGIT 960 System is a liquid, non-radiometric approach for isolating M. tuberculosis and 

determining its treatment susceptibility. It is an incubation and automated growth detection 

method that isolates mycobacteria using Middlebrooks 7H9 broth with additional enrichments 

(Ganu, 2016; WHO, 2020).  

Based on how much oxygen is used inside the tube, growth is detected. At the bottom of the 

tube, silicon gel contains an oxygen-quenched flourochrome, tris-4, 7-diphenyl-1, 10-

phenonthroline ruthenium chloride pentahydrate. Free oxygen is used by the bacteria as they 

develop and is exchanged for carbon dioxide. Since the flourochrome is no longer inhibited by 

the lack of free oxygen, there is fluorescence under ultraviolet (UV) light, at which at a 

A B 

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determined threshold it is interpreted as positive growth (MGIT Manual). The MGIT system 

uses qualitative percentage testing to evaluate if isolates of M. tuberculosis are susceptible to 

anti-TB medicines at specific critical concentrations. Before inoculating the growing medium 

with a suspension of the bacteria isolate, drug solutions are added. Drugs that are effective 

against M. tuberculosis isolates prevent their growth, which reduces the amount of fluorescence 

in those tubes. In contrast, drug-free tubes (growth control) allow isolates to proliferate 

unhindered (Bonsu, 2013; Ganu, 2016) 

In the isolation of M. tuberculosis from clinical specimens followed by DST, several 

researchers have compared the BACTEC Mycobacterial growth indicator tube (MGIT) 960 

method with other liquid radiometric methods and solid media such as Lowenstein-Jensen 

medium and 2% Ogawa egg medium and found the MGIT 960 method to be a better method 

(Chien et al., 2000; Goloubeva et al., 2001; P. Idigoras et al, 2000; Jayakumar et al, 1998; Lee 

et al., 2005; Ganu, 2016).  When comparing the nonradiometric techniques available, a study 

that was included in the Journal of Clinical Microbiology's January 2006 issue found that the 

MGIT 960 was the most effective method for MTB isolation and drug susceptibility testing 

(Piersimoni et al, 2006; Ganu, 2016). 

 

2.7.2 Molecular Diagnosis of Drug Resistant Tuberculosis 

Many molecular technologies currently exist that allow for the quick and simultaneous 

identification and typing of M. tuberculosis in clinical specimens, as well as the detection of 

treatment resistance genes, shortening the period between suspicion and confirmation of the 

disease from months to hours (Kamerbeek et al.,1997). The common ones widely used and 

recommended by WHO are GeneXpert, Line Prop Assay and Whole Genome Sequencing 

(WHO, 2020) 

 

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2.7.3 The MTB/RIF Ultra and Xpert MTB/RIF  

A completely automated real-time PCR assay for semiquantitative diagnosis of M. tuberculosis 

and detection of rifampicin resistance is available from Cepheid in Sunnyvale, California, 

USA. It is called the Xpert MTB/RIF assay. The sensitivity of Xpert MTB/RIF Ultra has 

increased for the detection of M. tuberculosis complex in mixed infections and smear-negative 

cases. This cartridge-based technology detects frequent mutations in both sputum smear-

positive and negative samples in the M. tuberculosis codon positions 428 and 452 of the rpoB 

gene's Rifampicin Resistance Determining Region (RRDR)..(WHO, 2020) 

2.7.4 Line Probe Assay 

Traditionally, M. tuberculosis strains have been grown in liquid or solid media to test their 

antibiotic susceptibility. Although it is capable of identifying RIF or INH resistance, second-

line anti-TB medicine resistance testing is less precise and complex. (Length, 2021). The World 

Health Organization currently recommends using line probe assays (LPA) besides the standard 

culture and antimicrobial susceptibility testing technique, there are several ways to quickly 

identify first- and second-line drug resistance (Ali et al., 2011; WHO, 2008; Ling D. et al., 

2008). DNA extraction, preparing the master mix, performing the PCR, and reverse 

hybridization are all steps in the LPA process for both first and second line anti-TB drugs using 

Genotype MDRTBplus and Genotype MDRTBsl, respectively (Addo et al., 2017). The 

MTBDRplus detects mutations for first line drugs in the rpoB and KatG, inhA hot spot regions 

for Rifampicin and Isoniazid respectively. The MTBDRsl line probe assay detects second-line 

drug resistance quickly and detects mutations in the gyrA, gyrB, and  rrs and eis hotspot regions 

for flouroquinolones and second line injectables (that is the aminoglycosides) respectively 

(Length, 2021). This is shown in Figure 7. 

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Figure 7: Pattern of the strips for the Genotypye MTBDRplus V2 (a) and Genotype MTBDRsl 

V2 (b) (Source: Line Probe Assays for Drug- Resistant Tuberculosis Detection, 

www.stoptb.org/wg/gli) 

The rpoB gene, which includes four rpoB mutant probes [rpoB MUT1 (D516V), rpoB MUT2B 

(H526D),  rpoB MUT2A (H526Y), and rpoB MUT3 (S531L) mutations] and eight rpoB wild-

type probes, was found to have the most significant alterations, which allowed for the 

identification of RIF resistance (Ganu, 2016; Bang et al., 2006).  

To validate the testing processes, the GenoType® MTBDRplus V2 strip includes 22 probes in 

addition to amplification and hybridization controls as well as control probes for gene loci for 

rpoB, katG, and inhA. The 20 probe Genotype® MTBDRsl V2 strip also includes control 

probes for the gryA, gyrB, rrs, and eis gene loci, as well as controls for pcr and hybridization 

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to confirm the test processes. The TUB probe identifies strains of the MTB complex. 8 rpoB 

wild-type probes (probes WT1 to WT8) cover the area of the rpoB gene expressing amino acids 

505 to 533 to determine RIF resistance. The most prevalent mutations that confer RIF 

resistance are particularly targeted by four probes (rpoB MUT1 D516V, rpoB MUT2A H526Y, 

rpoB MUT2B H526D, and rpoB MUT3 S531L). AGC-to-ACC (S315T1) and AGC-to-ACA 

(S315T2) mutations are assessed by two additional probes (katG MUT1 and MUT2), while the 

wild-type S315 region of katG is covered by one probe for the detection of INH resistance. 

Additionally, the inhA gene's promoter region includes areas between locations -15 and -16 for 

the inhA WT1 probe and positions -8 for the inhA WT2 probe. With the inhA MUT1, MUT2, 

MUT3A, and MUT3B probes, four mutations (-15C/T, -16A/G, -8T/C, and -8T/A) can be 

targeted. Once more, evidence of a resistant strain was the presence/staining of one or more 

mutant probes, or the absence of one or more wild-type probe(s) (Figure 8), (Ganu 2016). 

 

Figure 8: Standard MTBDRplus Assay for the detection of MTBC and RIF and INH related 

mutations (Source: Ganu 2016) 

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The Genotype® MTBDRsl Version 2 contains the quinolone-resistance determining region 

(QRDR) of the genes gyrA (from codon 85 to 96) and gyrB (from codon 536 to 541) (16) for 

detecting fluoroquinolone resistance, as well as the rrs (nucleic acid positions 1401, 1402, and 

1484) and the eis promoter region (from -37 to -2 nucleotides upstream) for resistance to SLI 

medicines. Eight probes (gyrA MUT1 A90V, gyrA MUT2 S91P, gyrA MUT3A D94A, 

gyrA MUT3B D94N, D94Y gyrA MUT3C D94G, gyrA MUT3D D94H, gyrB MUT1 N538D 

(N499D) and gyrB MUT2 E540V (E201V)) target the most prevalent mutations that confer 

fluoroquinolone resistance. In order to identify aminoglycoside resistance, rrs MUT1 A1401G 

and rrs MUT2 G1484T are the common mutations responsible, one mutation for the eis 

promoter region eis MUT1 C-14T is responsible for low-level kanamycin resistance. And 

again, evidence of a resistant strain was either the absence of one or more wild-type probe(s) 

or the appearance of one or more mutant probes (Figure 9). 

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Figure 9: Standard MTBDRsl Assay for the detection of MTBC and FQ and SLI related 

mutations (Source: gli LPA interpretation)  

 

2.8 Treatment/ Prevention of Drug Resistant Tuberculosis 

Treatment plans for MDR/RR-TB patients now available are by no means sufficient. Compared 

to treatments for drug-susceptible TB strains, these regimens need a longer course of treatment, 

a larger tablet dosage , and the use of drugs with a higher toxicities. Additionally, patients may 

have severe adverse events and have less favorable treatment outcomes. About 15% of 

MDR/RR-TB patients still die from the condition, and 26% of those deaths are inflicted on by 

XDR-TB patients, despite increased treatment outcomes internationally. (WHO, 2020). 

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Group A drugs (clofazimine, linezolid, high-dose INH, bedaquiline, and delamanid) are 

frequently used to treat pre-XDR and XDR-TB patients since they are frequently resistant to 

the majority of potent therapies and have subpar clinical outcomes (Id et al., 2020; Palmero D. 

et al, 2015).  

When a patient's drug resistance profile is not available, a typical treatment plan—which may 

differ from nation to nation depending on the drug resistance survey (DRS) data for the 

region—may be chosen. When the DST findings are known, this may be revised to an empirical 

or a unique regimen (Ganu, 2016). 

The following tactical principles are used to choose the medications that will be part of a 

treatment plan (WHO, 2020; Guideline for MDR-TB, 2019). 

i. The patient’s history of anti-TB drug use, the country profile of drug resistance and/or 

the DST profile of the patient. 

ii. A minimum of four new core drugs different from ones previously used that are known 

or expected to be effective 

iii. The Injectable drug forms the backbone of the 4 core drugs and should be used in the 

intensive phase 

iv. An effective fluoroquinolone should be selected. 

v. Include a first-line medication that the strain is responsive to. 

vi. Cross-resistance may occur between drugs of the same group and this is taken into 

consideration. 

vii. Drugs are administered daily under strict directly observed treatment (DOT) course 

throughout the injectable and continuation phases (Guideline for MDR-TB, 2019; 

Ganu, 2016). 

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Two phases make up the Pre-XDR/XDR-TB treatment regimen: the intensive phase and the 

continuation phase. The drugs used are chosen based on the type of resistant strain being 

treated. The duration of the treatment is usually twenty (20) months. The course of treatment 

of Pre-XDR/XDR-TB is summarised in the tables below for adult and children respectively 

(Guideline for MDR-TB, 2019). 

Table 2.  The adult regimen for Pre and XDR-TB 

 

(Source; Guideline for MDR-TB, 2019). 

Imipenem/clavulanic acid may be substituted for Linezolid for a period of six months in XDR-

TB patients who have Adverse Drug Reactions (ADRs) from Linezolid (Lzd). (Guideline for 

MDR-TB, 2019; WHO, 2020) 

 

 

 

 

 

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Table 3.  Pre-XDRTB and XDRTB regimen in children 

 

(Source: Guideline for MDR-TB, 2019). 

Infection prevention and control, as well as immunization of children with the bacille Calmette-

Guérin (BCG) vaccine, are the main methods of preventing M. tuberculosis transmission 

(WHO, 2020).  

 

2.9 Genome of M. tuberculosis:  

Circular chromosomes of M. tuberculosis have a base pair count of 4,411,532 and a Guanidine 

+ Cytosine composition of roughly 65% (Quellet et al, 2010; The Sanger Institute, 2014; Ganu, 

2016). Only 41% of the over 4000 genes in the bacterial genome have been described, and 44% 

of these genes have speculated functions (The Sanger Institute, 2014; Ganu, 2016). The lipid 

metabolism of the bacteria, which is crucial for their survival, occupies about 8% of their 

genome (Mohn et al., 2008; The Sanger Institute, 2014; Ganu, 2016). 

Recent research suggests that some drug resistance genes and intergenic areas may be 

implicated in resistance to many drugs, as well as new linkages and drug resistance genes that 

were not previously related (Zhang et al., 2013; Ganu, 2016). The RNA synthesis, catalase-

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peroxidase activity, and cell wall synthesis genes, respectively, have been linked to the rpoB 

gene, the katG gene, and the inhA gene for Rifampicin and Isoniazid respectively (Zhang et al., 

2009). By altering ribosome structures at the 16S rRNA, the second-line injectable anti-TB 

medicines Kanamycin (KM) and amikacin (AM), a KM derivative, inhibit protein synthesis. 

(Zhang et al., 2009). High levels of resistance to KM and AM are associated with mutations at 

16S rRNA (rrs) position 1400. Variable cross-resistance among KM, AMK, Capreomycin 

(CPM) or viomycin (VM) may be seen. The rrs gene may have either a C1402T or a G1484T 

mutation in individuals who are resistant to CPM, KM, and VM. (Zhang et al., 2009). 

Fluoroquinolones (FQs) cause the death of microorganisms by inhibiting DNA gyrase 

(topoisomerase II) and topoisomerase IV. GyrA and GyrB, which encode the A and B subunits, 

are present only in M. tuberculosis. The conserved genes gyrA (320 bp) and gyrB (375 bp), 

which have the quinolone-resistance-determining region (QRDR), have been shown to have a 

significant part in M. tuberculosis developing FQ resistance (Zhang et al., 2009; Laurenzo et 

al, 2011). 

2.10 Mechanism of Resistance of FLDs 

2.10.1 Rifampicin molecular resistant mechanism and the rpoB gene 

First-line anti mycobacterial treatment involves the semi-synthetic rifampin (RIF), a rifamycin 

derivative. Because of its highly efficient bactericidal action, the medication is a central part of 

anti-TB therapy. Mycobacterial genes are translated and expressed by the enzyme ribonucleic 

acid (RNA) polymerase (rpoB), which is bound by RIF. This inhibition of bacterial 

transcription activity results from rpoB's binding to RIF (Ahmad et al., 2014). Mutations have 

been discovered in the M. tuberculosis resistant strains of the rpoB gene, which codes for the 

b-subunit of RNA polymerase. Rifampicin resistance testing is a helpful surrogate sign for 

MDR-TB because resistance to rifampicin alone is quite uncommon (Ahmad et al., 2014; 

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Zhang et al., 2009). RIF inhibits the synthesis of mRNA in M. tuberculosis by physically 

limiting the formation of the phosphodiester bond in the RNA backbone and impeding 

extension of RNA. It does this by attaching to the b-subunit of the RNA polymerase (Zhang et 

al., 2009; Laurenzo David, 2011; Ahmad et al., 2014). 

The rifampicin resistance determining region, or RRDR, of the rpoB gene (codons 507–533), 

which codes for 27 amino acids, has 35 different point mutations or small insertions/deletions, 

according to data from several studies. Nearly 95% of epidemiologically unrelated rifampicin-

resistant clinical M. tuberculosis isolates carry these mutations (Ahmad et al., 2014; Zhang et 

al., 2009)  

 

2.10.2 Isoniazid molecular resistant mechanism and the katG and inhA gene 

A first-line synthetic drug isoniazid (INH), which is primarily used to treat infections caused 

by M. tuberculosis complex members (M. tuberculosis, M. bovis, M. africanum, and M. 

microti) because all other mycobacteria and other prokaryotes are resistant to it (Ahmad et al., 

2014; Laurenzo et al., 2011).  

Four different M. tuberculosis genes—katG, which encodes catalase peroxidase, inhA, which 

encodes the enoyl acyl carrier protein (ACP) reductase, kasA, which encodes b-ketoacyl ACP 

synthase, and ahpC, which encodes alkyl hydroperoxide reductase—are the source of the more 

complex molecular basis for Isoniazid resistance (Ahmad et al., 2014; Zhang et al., 2009; 

Laurenzo et al., 2011). According to genetic and biochemical evidence, changes in the ahpC 

gene that result from catalase/peroxidase activity decrease are compensatory and do not 

directly contribute to isoniazid resistance (Ahmad et al., 2014).  

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The two primary molecular causes of INH resistance are katG and inhA gene mutations, or its 

promoter region. KatG and inhA gene mutations account for more than 95% of all INH 

resistances (Maurya et al., 2013; Vijdea et al., 2008; Al-Mutairi et al., 2019; Guo et al., 2006). 

Isoniazid resistance is produced by mutations in inhA that occur in the upstream regulatory 

region, which leads to increased inhA protein production and elevated drug target levels via 

titration mechanisms resulting to low level resistance. There have also been reports of low – 

level isoniazid-resistant M. tuberculosis isolates with mis-sense mutations in the inhA structural 

gene, which lower the enzyme's affinity for decreased nicotinamide adenine dinucleotide 

(NADH). Mycolic acid biosynthesis is stopped as a result (Ahmad et al., 2014; Laurenzo et al., 

2011). Small deletions, insertions, or mis-sense or nonsense mutations within the katG gene 

are mostly responsible for clinically significant high level resistance to isoniazid. The codons 

315 and 463 of the katG gene are the locations of the most frequent genetic mutations in strains 

that are resistant to isoniazid (Ahmad et al., 2014; Palomino et al., 2014). 

 

2.11 Mechanism of Resistance of SLDs 

2.11.1  Mechanism of Molecular Resistance of Fluoroquinolones and gyraA and gyrB 

genes 

As second-line TB medications, the fluoroquinolones (moxifloxacin, gatifloxacin, 

sparfloxacin, levofloxacin, ofloxacin, and ciprofloxacin) are bactericidal antibiotics with high 

activity against M. tuberculosis (Kolyva et al., 2012). Treatment of MDR-TB and XDR-TB 

involves the use of 3rd generation fluoroquinolones such a Levofloxacin, moxifloxacin and 

gatifloxacin. When compared to other tuberculosis medications, fluoroquinolones have a 

relatively low side-effect profile, and their potent bactericidal effects have caused their use to 

increase recently. The resistance to fluoroquinolones has, regrettably, increased concurrently 

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(Laurenzo et al, 2011). The action of mycobacterial DNA gyrase, which is encoded by gyrA 

and gyrB, is inhibited by all fluoroquinolones in a manner that is similar. The quinolone 

resistance determining region (QRDR) in gyrA has been identified as the major location of 

mutations that lead to quinolone resistance. 60–70% of MTB strains that are resistant to 

quinolones have alterations in the gyrA QRDR region. (Laurenzo et al, 2011; Zhang et al., 

2009).        

    

                 Moxifloxacin            Ciprofloxacin 

Figure 10: Chemical Structure of Fluoroquinolones (Source: Kolyva et al., 2012). 

The specific amino acid substitution in the QRDR and the quantity of resistance mutations 

determine the level of fluoroquinolone resistance. Therefore, whereas a single gyrA mutation 

may provide low-level resistance, numerous gyrA mutations or concurrent gyrA and gyrB 

mutations are typically needed to offer high-level resistance to fluoroquinolones. (Kolyva et 

al., 2012; Zhang et al., 2009).   

A study conducted by (Laurenzo et al, 2011), the majority of mutations occur at codon 94 

(around 60%), then at A90V, S91P, or G88C substitution substitutions (24% , 11% and 3% 

clinical isolates, respectively). In a different research by (Ahmad et al, 2014), the results 

demostrated that, of the 97 isolates, 73 (73/97, or 75.3%) had mutations in the gyrA gene, 

namely in codons 89, 90, 91, and 94. The codon 94 mutation, which was responsible for 49.5% 

(48/97) of the isolates, had four distinct variants of the amino acid, including D94G (23/97, 

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23.7%), D94A (6/97, 6.2%), D94Y (8/97, 8.2%), and D94N (11/97, 11.3%).The second most 

common mutation was the A90V, which was present in 22.7 percent (22/97) of isolates. 

 

2.11.2  Mechanism of Molecular Resistance of Aminoglycosides; rrs and eis genes 

The first significant advancement in TB chemotherapy came with the discovery of 

streptomycin (an aminoglycoside) in the early 1940s. Kanamycin and amikacin are two other 

aminoglycosides that exhibit strong antimycobacterial properties. Aminoglycosides are being 

utilised as second-line medications primarily to treat MDR-TB/ XDR-TB (Kolyva et al., 2012; 

Laurenzo et al.,  2011).  

 

Figure 11. Chemical Structure of Streptomycin (Aminoglycoside) (Source: Kolyva et al., 

2012) 

Similar to other bacteria, mycobacterial species are susceptible to the mode of action of 

aminoglycosides through their binding to the 30S ribosomal subunit, which affects polypeptide 

synthesis and, in turn, inhibits translation (Kolyva et al., 2012;  Zhang et al., 2009). 

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Amikacin and Kanamycin resistance arises from changes in rrs similarly to streptomycin 

resistance. The main contributors to resistance to these drugs have been determined to be two 

mutations, A1400G and A1401G (Laurenzo et al, 2011; Alangaden et al., 1998). In a survey 

by (Alangaden et al., 1998), 13 clinical isolates (10 distinct strains) having MICs at 

concentrations of 0.256 mg/mL,  they observed that all 13 isolates carried the A1400G 

mutation. 

Amikacin, kanamycin, and capreomycin resistance are all caused by alterations in the 16S 

rRNA gene (rrs), particularly at locations 1484, 1402, and 1401, which result in CAP, KAN, 

and AMK resistance, respectively (Ali et al., 2011;Cui Z. et al, 2011; Alangaden G. J. et al, 

1998). High levels of resistance to KM and AM are associated with mutations at 16S rRNA 

(rrs) position 1400. Variable cross-resistance between KM, AMK, Capreomycin, or viomycin 

may be seen (VM). The rrs gene may have either a C1402T or a G1484T mutation in 

individuals who are resistant to CPM, KM, and VM. (Zhang et al., 2009). 

In a different research by (Province et al., 2019), the most prevalent mutation was found in 

33.3 percent (21/63) of the 63 SLI Drug resistant isolates, changing the rrs gene's location 1401 

from A to G. Three other isolates had mutations in the rrs gene at positions T1491C, G1454A, 

and A1499G. G10A (3/63, 4.8%) with C14T (2/63, 3.2%) were two amino acid changes found 

in the eis promoter region. However, only CAP resistant isolates had mutations in tlyA that 

resulted in the amino acid changes A119E (1/63, 1.6%), K69E (1/63, 1.6%), and K189N (1/63, 

1.6%). Each of the isolates A1128G, A1138G, C1209T, and C1483T is one., out of 193 SLID 

isolates, were found in rrs. Additionally, only one isolate had a shift from G to T at position 37 

of the eis promoter region. 

 

 

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2.12 Pre-XDR-TB and XDR-TB 

The emergence of pre-extensively (Pre-XDR) and XDR-TB is threatening management of 

MDR-TB patients for most TB control programmes worldwide, and especially developing 

countries like Ghana.  

Fluoroquinolone (FQ) is the most efficient second-line anti-tuberculosis medicine, and it’s 

mostly used for the treatment of MDR-TB patients (Id et al., 2020; Malik S. et al., 2012). 

Treatment for MDR-TB patients is compounded further by fluoroquinolone resistance (Pre-

XDR-TB), which leads to prolonged treatment times, fewer treatment alternatives, and a poor 

outcome. Early diagnosis of Pre-XDR/ XDR-TB would go a long way to help clinicians adapt 

their MDR-TB treatment regimens to include effective medications and avoid treatment failure 

(Kerléguer et al., 2021; S.E. Smith et al., 2015). 

FQ resistance genetic changes in the 320- and 375-bp hypervariable areas of the gyrA and gyrB 

genes, which encode DNA gyrase, are responsible for 50 to 90% of phenotypic FQ-resistant 

isolates. 

 Genetic changes in the 16S rRNA gene (rrs), notably at locations 1401, 1402, and 1484, are 

likely to be linked to resistance to the antibiotics capreomycin and amikacin and kanamycin, 

resulting in AMK, KAN, and CAP resistance, respectively (Ali et al., 2011;Cui Z. et al, 2011; 

Alangaden G. J. et al, 1998). 

M. tuberculosis strains are typically grown in liquid or solid media to test their susceptibility 

to various drugs, though this is capable of detecting RIF or INH resistance, it is less accurate 

and complex when it comes to second-line anti-TB medications (Length, 2021). In order to 

quickly identify first-line medicine resistance, the World Health Organization currently advises 

employing line probe assays (INNO-LiPA Rif and MTBDRplus) in addition to the standard 

culture susceptibility testing approach (Ali et al., 2011; WHO, 2008; Ling D. et al., 2008). The 

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MTBDRsl line probe assay detects second-line drug resistance quickly. These tests all look for 

genetic changes in the rpoB, katG, inhA, gyrA, gyrB, rrs and eis hotspot regions (Length, 2021) 

Several studies have been carried out worldwide especially in high burden TB countries to 

determine burden and mutations within the M. tuberculosis genome especially within the hot 

spot regions of genes associated with XDR-TB and pre-XDR TB. In Pakistan, 4.5% of XDR-

TB was reported among MDR in 2009, where 49 of 50 isolates had mutations affecting rpoB's 

4 amino acid codons, 531 (68%), 516 (24%), 526 (4%), and 513 (2%) (Id et al., 2020). In 

Myanmar, 13.5% XDR-TB and 27% pre-XDR-TB with two mutations in gyrB (T500P and 

E5014A) together with mutations in gyrA as well as mutations in rrs (A1401G) in 

aminoglycosides were reported (Ei et al., 2018).   

In a work done by Gehre et al., 2016, to determine the rise of pre-extensively drug-resistant 

TB in West Africa, there were no XDR-TB found and 2 pre-XDR-TB strains and 11 pre-XDR-

TB were found among new MDR-TB patients and retreatment MDR-TB patients respectively.  

 

 

 

 

 

 

 

 

 

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CHAPTER THREE 

 MATERIALS AND METHODS 

  

3.1 Study Design: 

The study was a retrospective and cross-sectional experimental research design. Archived 

clinical samples of MDR-TB strains of M. tuberculosis complex collected between January 

2016 to December, 2020 were used for the study. 

3.2  Chemical and Reagents 

All materials used are listed in Appendix I and the details of all reagent preparation procedures 

and composition of commercially acquired kits are in Appendix II. 

3.3  Study Site and Sample Collection 

One hundred and seventy-one (171) archival clinical isolates of M. tuberculosis complex 

collected from MDR-TB patients visiting the two major TB diagnostic laboratories involved 

in MDR-TB diagnosis in Ghana (ie, Chest Clinic TB Laboratory, Korle Bu Teaching Hospital, 

Accra and TB Laboratory, Eastern Regional Hospital, Koforidua) between 2016 and 2020 and 

kept in Tryptophan soy glycerol broth at -20°C at these laboratories were obtained.  

 

Chest Clinic T.B Laboratory of the Korle Bu Teaching Hospital, Accra, and the TB Laboratory 

of the Eastern Regional Hospital, Koforidua were the two main TB laboratory networks in the 

diagnosis of Drug Resistance TB in Ghana Health Service. Samples from these regions (Bono, 

Ahafo, Bono East, Northen, North East, Savannah, Upper East, Upper West, Volta, Oti and 

Greater Accra) were sent to Chest Clinic TB Lab, Korle Bu Teaching Hospital, whiles samples 

from Western, Western North, Ashanti and Eastern Regions were sent to Eastern Regional 

Hospital T.B Lab. However, on some occasions samples from other regions were also sent to 

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the Eastern Regional Hospital T.B lab depending on the functionality of the Korle Bu Teaching 

Hospital, Chest Clinic T.B lab.  

3.3.1 Patient Data Collection 

Patient treatment category and demographic data were extracted from the laboratory patient 

registration book at both sites. This included age, sex, Region of referring health facility, 

mycobacterial culture and DST results. 

3.3.2 Inclusion and Exclusion Criteria  

Only isolates with confirmed phenotypic and genotypic resistance to at least rifampicin/ 

rifampicin and isoniazid were selected for the study. Selected isolates that failed to grow on 

subculture or were not truly MTB complex were excluded from the study. Only isolates that 

were confirmed to be MDR strains of M. tuberculosis complex after phenotypic and genotypic 

DST retesting were included in the studies.  

 

3.4 Ethical Approval  

Ethical clearance for this study was sought from and duly approved by the Ethical and Protocol 

Review Committee (EPRC) of the College of Health Sciences, University of Ghana. 

 

 

 

 

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3.5  Sample Size Determination 

The minimum sample size for this study was determined using the Cochran (1963:75) 

formula,  

n= Z2pq/e2  

Using a 95% confidence interval, 0.14 is the proportion of the prevalence rate, and a ±5% 

precision; where  

n = Minimum sample size  

Z = Z Score  

p = An estimated proportion of an attribute that is present in the population  

e = Level of precision  

q = 1-p  

n = Z2pq/e2 = (1.96)2(0.14) (0.5)/(0.05)2 = 108.0 

The minimum sample size (n) for this study was 108.0 archived MDR-TB isolates.  

171 archived MDR-TB isolates were collected for this study from a total of 230 samples, using 

simple random sampling. Simple random sampling was done by assigning numbers to all of 

the isolates identified. The numbers were ranked and isolates selected from a table of random 

numbers, giving equal chance for an isolate to be selected and minimize bias. 

 

 

 

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3.6 Laboratory Procedures  

The laboratory analysis was carried out at the Eastern Regional Hospital, Koforidua TB 

Laboratory. All biosafety recommendations regarding the handling of M. tuberculosis were 

followed. Isolates from the Korle Teaching Hospital Chest Clinic TB lab. were transported to 

Eastern Regional Hospital TB lab in a cold chain.  

3.6.1 Isolate Recovery 

MDR-TB Isolates were sub-cultured to revive and recover pure viable isolates with the use of 

the BACTEC™ MGIT™ liquid culture system from Becton, Dickinson (BD) Company for 

phenotypic antimicrobial resistance retesting. The manufacturer’s instructions were strictly 

followed. Positive cultures were examined for mycobacterial growth by the appearance of 

white flakes and by microscopic examination using Ziehl-Neelsen stained smears from the 

broth culture. Purity check was performed on confirmed mycobacterial growths by sub 

culturing on blood agar plate streaked with the positive broth culture and incubated at 37°C for 

up to 48 hours. Pure mycobacterial colonies were tested for M. tuberculosis complex using 

BACTEC™ MGIT™ TBc Identification kit from BD to exclude Non-Tuberculous 

Mycobacteria (NTM). Using a sterile Pasteur pipette, a drop of the positive broth was put in 

the well of the TBc Identification kit after which the kit was incubated for 15mins. After 15 

minutes the TBc identification kit was read; a red line at the test zone and control zone 

indicating the presence of the MPT64 antigen of the M. tuberculosis, hence positive for MTB. 

The kit manufacture’s protocol was strictly followed. Those confirmed to be MTBCs were 

prepared for phenotypic DST and aliquots used for DNA extraction for the molecular 

procedures. 

3.6.1.1 BACTEC MGIT 960 Inoculation and Incubation.  

The MGIT PANTA vial containing five lyophilized antibiotics (Polymyxin B, Amphotericin 

B, Nalidixic Acid, Trimethoprim and Azlocillin) was reconstituted by adding 15ml of 

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supplemented medium called OADC (Oleic acid, Albumin, Dextrose, and Catalase) (see 

Appendix II). Eight hundred microliters (800 µl) of the resultant solution was added to 

Mycobacteria Growth Indicator Tube (MGIT) containing 7ml improved Middlebrook 7H9 

broth base to complete the growth medium. The PANTA suppress growth of contaminating 

bacteria and makes the medium selective for mycobacterial growth. The growth supplement is 

a source of nutritional growth requirement for M. tuberculosis and other Mycobacteria species.  

 

The MDR- TB isolates were removed from storage, thawed and allowed to reach room 

temperature. Five hundred microliters (500µl) of the bacteria suspensions were transferred to 

reconstituted MGIT media and the tubes were cupped tightly, labelled, disinfected and loaded 

into an automated BACTEC MGIT 960 instrument by scanning the tube barcodes for 

incubation at 37°C. The instrument monitored the tubes for growth at 15-minute intervals and 

flagged tubes with growth as positive (+) indicated on a computer monitor, instrument drawer 

and tube stations. The BACTEC MGIT 960 equipment uses fluorescence detection to identify 

growth. After forty-two (42) days of incubation, tubes that had not grown were marked 

negatively (-). For verification of the instrument result, all negative tubes were visually 

inspected. 

3.6.1.2 Confirmation of Mycobacterial Growth 

Instrument positive MGIT tubes were unloaded by scanning them out of the BACTEC 

®MGIT® 960 machine and examined for mycobacterial growth. All positive tubes were 

visually inspected for mycobacterial growth. M. tuberculosis growth and other mycobacterial 

growths in MGIT appears as flakes or floccules in a clear broth with non-homogenous turbidity. 

Contaminating bacteria produce a hazy or cloudy turbidity whiles fungal contaminations are 

seen as cotton ball- like growths in the broth.  

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Drops of material from the positive tubes were transferred onto clean grease-free slides, air 

dried and stained by the Zeihl-Neelsen (ZN) staining method (see Appendix III). The ZN-

stained slides were examined microscopically for AFBs and the presence of serpentine cord 

formation if AFBs were present and results documented. The presence of AFBs confirmed 

mycobacterial growth and serpentine cord formation (Figure 9), provided a presumptive 

identification of MTBC. 

 

Figure 12:  Serpentine cord of M. tuberculosis of culture –positive specimen smear stained by 

Ziehl- Neelsen staining technique (Source: Ganu, 2016). 

3.6.1.3 Mycobacterial Growth Purity Check 

All the MGIT positive cultures were sub-cultured onto Blood Agar plates and incubated at 

37˚C ±1˚C for up to 48 hours and examined for the growth of contaminating bacteria. 

Contaminated cultures were decontaminated using the NaOH – NALC procedure (Appendix 

IV) and inoculated into fresh MGIT growth media for re-incubation.  

 

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3.6.1.4 Mycobacterium Tuberculosis Complex Identification 

To distinguish M. tuberculosis complex from non-tuberculous mycobacteria, the MPT64 

antigen of M. tuberculosis complex was identified using the BD MGIT MTBc Identification 

kit, a lateral flow immunochromatography assay. Sterile transfer pipettes were used to transfer 

100ul (one drop) of broth from the instrument positive tube (after 15 seconds vortexing) into 

the sample wells of the test cassettes. The cassettes were incubated at room temperature for 15 

to 20 minutes and the results were read. Only tubes with pure uncontaminated mycobacterial 

growths were tested by this method for MTBC. M. tuberculosis complex produces MPT-64 

antigens captured as a reddish-pink band on test region (T) of the cassette with an internal 

control band (C) which validates the test (Figure 10). Test Positive External Quality Control 

strains were included as internal quality controls. Five hundred microlitres (500 µl) of bacteria 

suspension from tubes confirmed to be MTBC were transferred into 1000 µl Eppendorf vails 

for DNA extraction and the remaining bacteria suspension used for phenotypic drug 

susceptibility testing. 

 

 

 

 

 

 

 

Figure 13: Immunochromatographic cassettes for M. tuberculosis identification (Source: 

Ganu, 2016) 

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3.6.1.5 Phenotypic Antimicrobial Susceptibility Testing 

Pure colonies of M. tuberculosis complex were tested for their antimicrobial susceptibility to 

the first-line anti-TB drugs (Streptomycin, Isoniazid, Rifampicin and Ethambutol) at critical 

concentrations by the broth dilution method using BACTEC™ MGIT™ SIRE® kit on the 

BACTEC™ MGIT™ system from BD. The instrument and kit manufacturer instruction was 

observed. Drug-free growth control tubes were also included. The BACTEC™ MGIT™ 960 

SIRE Growth Supplement was added (800µl) to the MGIT media. Four milliliters of sterile, 

distilled water were used to reconstitute each lyophilized drug and 100µl of each transferred 

to the reconstituted media labelled for each drug. The test bottles' ultimate medication 

concentrations were as follows: 1.00µg/ml for Streptomycin, 0.10µg/ml for INH, 1.00µg/ml 

for Rifampicin and 5.00µg/ml for Ethambutol. 500µl of the bacteria suspension each was 

transferred to each of the drug tubes. Five hundred microlitres (500µl) of a 100-fold dilution 

of the bacteria inoculum were added to the growth control tube (GC).  The tubes were arranged 

in 5-set DST carrier in the order Growth Control, Streptomycin, INH, RIF and Ethambutol.  

The BACTEC MGIT 960 instrument was loaded with the DST sets by scanning the carrier set 

barcode for incubation and automated reading of DST results for 13 – 14 days. Results were 

interpreted as follows; the Growth Unit (GU) of the drug-free growth control tube was observe; 

a ≥400 GU of the growth control tube was compared with the drug containing tubes. The drug-

containing tube with a GU ≥100 was resistant, where GU of a drug-containing tube ≤100 was 

susceptible. Tested Positive External Quality Control (EQA) panel strains were included as 

quality controls. 

Isolates with confirmed resistance to RIF only, and RIF and INH was tested for their 

susceptibility to second-line anti-TB medications (Fluoroquinolones and aminoglycosides) 

using the Line Probe Assay from Hain Lifescience (Nehren, Germany) 

 

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3.6.2 Molecular Method 

In order to identify mutations linked to rifampicin, isoniazid, and fluoroquinolone as well as 

mutations linked to aminoglycosides and fluoroquinolones in MDR TB isolates using DNA-

STRIP technology, this study used the Hain's Test, a line probe test. Pure isolates of the bacteria 

were used to extract the DNA of M. tuberculosis, which was then amplified by multiplex PCR 

using biotinylated primers for specific target regions of the rpoB, katG, and inhA genes as well 

as the gyrA, gryB, rrs, and eis genes. Then, using reverse hybridization and an enzymatic colour 

reaction, the PCR products were located on a membrane strip. 

3.6.2.1 DNA Extraction  

The GenoLyse extraction kit Version 2.0 (Hain Life Science, Germany) was used to extract 

DNA from the M. tuberculosis isolates using the heat-alkaline procedure. 1 ml aliquots of the 

bacteria suspension were concentrated by centrifugation (at 10000rpm) for 15mins. after which 

the supernatant was decanted to leave the pellet of the bacteria cells. The bacteria pellet was 

suspended in a 100µl alkaline Lyse solution (A-LYS) and incubated in a heat block at 95℃ for 

5 min to rupture the bacteria cell and consequently release the bacteria nucleic acid material 

into solution. The reaction was neutralized with a 100µl neutralizing buffer (A-NB), the 

mixture was centrifuged at higher speed for five minutes, and the DNA extract from the 

bacterium lysate in the supernatant was used for the PCR process and stored at -20°C. 

3.6.2.2 PCR Amplification  

The GenoType® MTBDRplus Version 2 LPA kit was used to target four (4) genes of interest; 

TUB, rpoB, katG and inhA genes and the Genotype® MTBDRsl Version 2 LPA kit was used 

to target five genes of interest; TUB, gryA, gryB, rrs and eis.  The TUB identified MTBC 

whereas the rpoB, katG and inhA gene mutations related with Rif, high and low level INH 

resistance respectively and gyrA, gryB, rrs, and eis gene changes related to fluoroquinolone, 

aminoglycoside and low level Kananycin resistance respectively. 

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The Genotype MTBDRplus VER 2.0® and Genotype MTBDRsl VER 2.0® LPA is a multiplex 

PCR method that uses 22 different primer sets in the primer nucleotide mix. (PNM) were used 

for the DNA amplification. These includes primers to the TUB gene, eight (8) rpoB wild-type 

(WT 1 to WT8) gene locus and four (4) common rpoB mutant (rpoB MUT1, MUT2A, MUT2B 

and MUT3) gene regions. Three primers were included to target katG mutations for katG WT1 

locus, katG MUT1 and katG MUT2 mutant genes. Six (6) inhA gene mutation targets were also 

included; inhA WT1, inhA WT2, inhA MUT1, MUT2, MUT3A and MUT3B for the first line 

anti- TB drugs (Rifampicin and Isoniazid). The primers for the second line anti-TB drugs 

(fluoroquinolone and aminoglycosides) also included TUB gene and three (3) gryA wild-type 

(WT1 to WT3) gene locus and six (6) common gryA mutant (gyrA MUT1, MUT2, MUT3A, 

MUT3B, MUT3C and MUT3D) gene regions, one (1) gryB wild-type and two (2) gyrB mutant 

(gyrB MUT1, and MUT2). Primers for rrs locus two (2) rrs wild-type (WT1 to WT2) and two 

rrs mutant (rrs MUT1 and MUT2) were included. Primers for eis locus, three (3) eis wild-type 

(WTI to WT3) and one (1) mutant (eis MUT1) were also included.  

  

The total PCR volume was 50μL; 10µl amplification mix A/AM-A (amplification buffer 

containing 2.5mM MgCl2 and Hot Start Taq DNA polymerase) was pipetted into a pcr reaction 

tube, after which 35µl amplification mix B /AM-B (PNM containing 200 nmol/L of each 

primer, dNTPs [dATP, dCTP, dGTP and dTTP]) was added to in a DNA hood to obtain a 

master mix. 5μl of the bacterial lysate containing the target DNA was then pipetted into the 

master mix after which the pcr solution was loaded into the themocycler.  

 

The PCR process was hot started. Prior to the start of the PCR cycle, primmer antibodies that 

limit the activity of Hot Start DNA polymerase were inactivated by an initial lengthy 95°C 

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incubation period lasting 15 minutes. This was done in order to rule out non-specific primer 

binding to the DNA template and the creation of primer dimers prior to PCR. 

 

The GTQ Cycler® 96, a thermal cycler, was used to perform the PCR. The amplification 

protocol consisted of 15 minutes of denaturation at 95°C, followed by 10 cycles comprising 

30s at 95°C and 120s at 58°C; an additional 20 repeated cycles comprising 25 seconds 

denaturation of dsDNA at 95°C, 40 seconds annealing of forward and reverse primers at 53°C 

and 40 seconds, DNA Taq polymerase mediated elongation or extension by the incorporation 

of dNTPs (nucleotides) at 70°C (see Appendix III). The Taq DNA polymerase transcription of 

the strands by the incorporation of dNTPs was aided by the PCR buffer and 2.5mM MgCl2 as 

a source of magnesium (Mg2+), a co-factor for the polymerase. The amplicons were further 

held at the 70°C extension temperature after thermal cycling for 8 minutes and stored at 4°C 

until post PCR processing. The kit manufacture’s product instruction was strictly adhered to. 

3.6.2.3 Line Probe Assay 

After PCR, the GT Blot 48® machine was used for the line probe assay; a reverse hybridization 

process. The amplification products of biotin-labelled dsDNA amplicons of the genes of 

interest were denatured by pipetting 20µl of the amplification product into a trough and adding 

a 20µl NaOH denaturation solution (DEN) to break the hydrogen bonds between the paired 

nucleotides after which Deoxyribonucleic acid (DNA) strip (labelled with sample ID) with 

probes (reaction zones) of unlabelled complementary sequences that were bands immobilized 

on a nitrocellulose membrane strips that are positively charged, and they were suspended in 

the amplification product and DEN mixture.  1000µl hybridization solution (HYB) pre-warmed 

at 45°C was added and incubated at 45°C for 30 minutes for hybridization to occur. The 

hybridization solution was replaced with 1000µl alkaline stringent solution (STRN) and 

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incubated at 45°C for 15 minutes after which stringent washing was done for the removal of 

unbound or non-specifically bound DNA and further rinsed at room temperature. 

Following hybridization of the biotin-labelled amplicons to the reaction zones, the strips were 

treated with 1000µl streptavidin-alkaline phosphatase enzyme conjugate for 20mins. Band sites 

on the strip where hybridization had happened are where binding takes place because the 

ligands biotin and streptavidin have a high affinity for one another. A 1000µl hydrogen 

peroxide substrate solution was added after three washing stages, and it was left to incubate for 

20 minutes at room temperature. In order to detect the bound biotin-streptavidin combination 

colorimetrically, the streptavidin-phosphatase enzyme conjugate reacts with the hydrogen 

peroxide substrate. Two washings were done and all the strips air-dried after which the strips 

were attached to the assessment sheets provided with the kits. Visual inspection of the strips 

that all contained internal controls for amplification and conjugation procedures, including loci 

controls for the rpoB, katG, and inhA genes and the gyrA, gyrB, rrs, and eis genes, were used 

to find the precise gene sections (wild-type or mutant) that are present in the target ssDNA's 

heterogeneous mixture. The hybridization procedure was repeated for strips with 

uninterpretable and/or very faint bands. Only samples with readable results were analysed. 

 

3.7 Quality Assurance  

Quality check of MGIT reagents and all other reagents was done before use. Mycobacterium 

tuberculosis External quality control strains, obtained from Korle Bu Teaching Hospital Chest 

clinic, Laboratory, was included in the MGIT liquid culture procedures and the molecular 

procedures. 

Incubator temperature was monitored daily to ensure that the optimum incubation temperature 

was maintained. The BD MGIT MTBc Identification kit and LPA DNA strips had internal 

positive control bands. Growth media, NaOH-NALC solution and buffers used were sterilized. 

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All procedures except Z-N staining procedure were performed aseptically and in DNA-free 

environment where applicable to exclude contaminations. Stains were filtered before use to 

avoid the deposition of artefacts. Acid fast bacilli positive and negative control slides were 

included in each batch of staining procedure as controls. 

3.8 Biosafety 

All biosafety procedures recommended by the Centre for Disease Control (CDC) Atlanta, 

USA, for the handling of infectious materials and Mycobacteria were duly observed. Protective 

clothing and gadgets such as laboratory coats, examination gloves (powder-free where 

necessary) and N95 respirator were worn where necessary. Procedures involving the opening, 

closing and mixing of specimen were done carefully to reduce the creation of aerosols. Tubes 

were tightly capped before centrifugation in air-tight buckets. Opening of specimen tubes, 

smear preparation, and all inoculation procedures were done in a Class II biological safety 

cabinet. All contaminated materials were autoclaved before leaving the lab for incineration. 

3.9 Data Management and Statistical Analysis 

Data was analysed using Statistical Package for Social Sciences (SPSS version 14) SPSS Inc., 

Chicago, IL and Prism V%.0 (GraphPad Software) and tables and graphs were used to display 

the results for both first line and second line anti-TB medications, the frequency with the 

percentages of detected mutations in the MDR-TB, XDR-TB, and pre-XDR-TB isolates were 

calculated 

 

 

 

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CHAPTER FOUR 

RESULTS 

4.1 Sample Selection 

A total of 171 archival isolates of MDR strain of M. tuberculosis were collected for the study. 

Twenty-six (15.2%) of the isolates were obtained from the Chest Clinic TB laboratory, Korle 

Bu Teaching Hospital, and one hundred and forty-five (84.8%) were obtained from the 

Koforidua regional Hospital TB laboratory. 

Out of the 171 archival isolates sub cultured, 90 (52.6%) were recovered to be M. tuberculosis 

complex out of which 81 of the MTBCs were truly MDRs (resistant to at least Rif and INH) 

after phenotypic drug susceptibility testing to confirm MDR. Nine isolates were resistant to 

Isoniazid only and thus not MDR and were excluded from the analysis. A total of eighty-one 

(81) isolates were confirmed to be MDR-TB and hence suitable for analysis and were thus 

analysed in this study. Figure 14 shows the trend of the confirmed MDR-TB isolates for the 

study period. 

 

 

Fig. 14 Trend of Confirmed MDR-TB, 2016 – 2020. 

2.6%

14.3% 15.7% 16.9%

31.6%

0%

5%

10%

15%

20%

25%

30%

35%

2016 2017 2018 2019 2020

C
as

e
s

Year

TREND OF CONFIRMED MDR-TB, 2016-2020 

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4.2 Patient Demographics  

The 81 isolates used for the analysis consisted of 70 (86.07%) males and 11 (14.0%) females. 

Their ages ranged from 5 years to 84 years with a mean age of 42.7±15.2 years. Age groups of 

41-50 years and 31-40 years had a frequency of 26 (32.1%) and 24 (24.7%) respectively, whilst 

21-30 years and 51 – 60 years were 12 (14.8%) and 9 (11.1%) respectively. (Fig. 15) 

 

Fig. 15 Distribution of MDR-TB isolates by age 

Eastern region had the highest number of patients (39.5%) followed by Greater Accra region 

(19.8%) whilst the Volta region had the lowest number of patients (1.2%). (Fig. 16) 

 

Fig. 16 Regional Distribution of the MDR-TB Isolates 

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

1-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 Blank

P
er

ce
n

ta
g

e

Age Group

Distribution of MDR-TB Isolates by Age

21.6

6.4
13.5

50.3

0.6
6.4

1.2
0.0

10.0

20.0

30.0

40.0

50.0

60.0

Ashanti Central G. Accra Eastern Volta Western Upper

West

P
er

ce
n

ta
g
e

Region

Regional Distribution of the MDR-TB Isolates

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4.3 Phenotypic Drug Susceptibility Testing 

In this study, a total of 171 stored MDR-TB isolates were cultured. Out of the 171 isolates, 90 

isolates recovered but 81 of them were MDR-TB representing a growth rate of 52.6%. Among 

the MDR-TB isolates RIF+INH+ Eth and RIF+INH+ Strept. resistance patterns were seen in 

18(22.22%) and 21(25.93%) respectively, (Table 4). 

Ta