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1
Evaluation of newer drugs (Linezolid & Meropenem) and classical second line
drugs (Amikacin & Levofloxacin) in clinical isolates of MDR-TB utilizing
broth based Bactec MGIT 960 technique
By
Dr. IRFAN ALI MIRZA
MBBS, Diploma Clinical Pathology, MCPS (Clinical Pathology),
FCPS (Microbiology), FCPP (Pak)
PhD Thesis
(MICROBIOLOGY)
Presented to the Board of Advanced Studies & Research (BASR)
Of Baqai Medical University Karachi - Pakistan
in partial fulfillment
of the requirements for the degree of
doctor of philosophy (Microbiology)
2015
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Supervisor
Dr Khursheed Ali Khan
MSc, Ph.D. (Pakistan)
Professor of Microbiology
Baqai Medical University Karachi
Co Supervisor
Professor Maj.Gen(R) Farooq Ahmad Khan Hilal-e-Imtiaz (M),
MBBS, MCPS (PAK), Dip Endocrinology (London), FCPS (Pak),
FCPP (Pak), PhD (London), FRCP (Ireland), FRC Path.(UK)
EX. Commandant / Advisor in Pathology
Armed Forces Institute of Pathology (AFIP), Rawalpindi-Pakistan,
Professor of Pathology, Consultant Chemical Pathologist & Endocrinologist
Principal, Rai Medical College, Sargodha - Pakistan
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Certificate
I certify that research work included in this thesis has been carried out under my supervision. I
have read this thesis and this thesis in my opinion is comprehensive in technical scope and
adequacy as a dissertation for the degree of PhD (Microbiology).
--------------------------------------
Supervisor
Dr Khursheed Ali Khan
Msc, Ph.D. (Pakistan)
Professor of Microbiology
Baqai Medical University
----------------------------------------
Co Supervisor
Professor Maj.Gen(R) Farooq Ahmad Khan Hilal-e-Imtiaz (M),
MBBS, MCPS (PAK), Dip Endocrinology (London), FCPS (Pak),
FCPP (Pak), PhD (London), FRCP (Ireland), FRC Path.(UK) EX. Commandant / Advisor
in Pathology Armed Forces Institute of Pathology (AFIP),Rawalpindi-Pakistan,
Professor of Pathology, Consultant Chemical Pathologist & Endocrinologist
Principal, Rai Medical College, Sargodha
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CERTIFICATE
The comments and checklist for evaluation of thesis titled “Evaluation of newer drugs (Linezolid
& Meropenem) and classical second line drugs (Amikacin & Levofloxacin) in clinical isolates of
MDR-TB utilizing broth based Bactec MGIT 960 technique” by both external reviewers has been
positive. All the areas pertaining to thesis as mentioned in check list sent by Examination
department, Baqai Medical University has been answered in affirmative by both reviewers.
There were three major and two minor comments made by one reviewer which has been
addressed and explained by researcher as follows.
Sr. No Comment Explanation/Correction
1. Why Linezolid and
Meropenem are
called newer drugs?
Although both these antimicrobials have been used to treat the
infections caused by Gram positive and Gram negative
microorganisms, but in the context of treatment of
tuberculosis they are considered newer antimicrobials. WHO
has placed these drugs in latest group 5 with the caption of
agents with unclear efficacy as mentioned in Chapter 2
(literature review) Table 1, pages 36 & 37. In a report issued
by WHO in 2011 as mentioned in Chapter 5(Discussion) at
page 74, paragraph 2 that routine use of such antimicrobials
be not used until local susceptibilities have been determined
and use justified by infectious disease specialists. In the
backdrop of this fact these newer antituberculosis drugs were
tested against local MDR-TB isolates.
2. MIC’s of only
Meropenem was
determined.
Clavulanic acid can
also be added.
The observation of reviewer is valid and researcher has
mentioned in page numbers 75 &76 about the reasons for use
of Meropenem without clavulanic acid. Meropenem which is
one of the active members of carbapenem class has low
affinity substrate for the enzyme MTB-BLaC with hydrolysis
five times lower than ampicillin. The objective 1 of the study
as mentioned in chapter 1, page number 5 was thus evaluating
the in vitro efficacy of multiple breakpoint concentrations of
Meropenem. The results indicated that by serially increasing
the concentration of Meropenem in vitro more number of
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MDR TB isolates became susceptible. The researcher has
concluded in chapter 6 (Conclusion and future prospects) page
77, paragraph 1 that Meropenem can be combined with
clavulanic acid to utilize its full potential.
3. Was Linezolid and
Meropenem used in
these patients?
As mentioned in chapter 1 ( Introduction , purpose statement
& research questions) Objective 1, page number 5 that this
was laboratory based study in which in vitro efficacy of the
Linezolid and Meropenem was evaluated against clinical
isolates of MDR-TB recovered from clinical specimens in our
tertiary care diagnostic centre. As this was first study of its
kind to test these group V antituberculosis agents against
MDR TB isolates in Pakistan so the publishing of these
results can give impetus for clinicians to use such drugs for
resistant tuberculosis. Our objective was not to find the
treatment outcome. However, as suggested by reviewer,
researcher has mentioned in chapter 5 (Discussion) page
numbers 74 & 75 about the different nature of in vitro and in
vivo studies done in some developed countries.
4. Typographical &
spelling mistakes
Typographical and spelling mistakes have been corrected at
all places as pointed out in text.
5. Abbreviations &
reference formatting
All inconsistencies in abbreviations and reference formatting
have been corrected as pointed out in text.
Supervisor
Dr Khursheed Ali Khan
MSc, Ph.D. (Pakistan)
Professor of Microbiology
Baqai Medical University Karachi
Co Supervisor
Professor Maj.Gen(R) Farooq Ahmad Khan Hilal-e-Imtiaz (M),
MBBS, MCPS (PAK), Dip Endocrinology (London), FCPS (Pak),
FCPP (Pak), PhD (London), FRCP (Ireland), FRC Path.(UK)
Principal Rai Medical College Sargodha
EX. Commandant / Advisor in Pathology
Armed Forces Institute of Pathology (AFIP), Rawalpindi-
Pakistan,
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Declaration
I, Dr. Irfan Ali Mirza, hereby declare that I have produced the work presented in this thesis,
during the scheduled period of study. I also declare that I have not taken any material from any
source except referred to wherever due. I also declare that amount of plagiarism is within
acceptable range. If a violation of HEC rules on research has occurred in this thesis, I shall be
liable to punishable action under the plagiarism rules of the HEC.
Date: / / 2015 ...............................................
Dr. Irfan Ali Mirza
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Turnitin Originality Report
Evaluation of newer drugs (Linezolid & Meropenem) and classical second line drugs (Amikacin & Levofloxacin) in
clinical isolates of MDR-TB utilizing broth based Bactec MGIT 960 technique by Dr Irfan Ali Mirza (phd Thesis -
Microbiology)
From Quick Submit (Quick Submit)
Processed on 29-Oct-2014 16:26 PKT
ID: 470723326
Word Count: 18986
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1. 2% match (Internet from 15-Mar-2011)
http://diss.kib.ki.se/2010/978-91-7457-034-2/thesis.pdf
2. 2% match (publications)
Daniel, T.M.. "The history of tuberculosis", Respiratory Medicine, 200611
3. 2% match (Internet from 16-Mar-2012)
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5. 1% match (Internet from 18-Sep-2014)
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6. 1% match (publications)
H. Takiff. "Current Prospects for the Fluoroquinolones as First-Line Tuberculosis Therapy", Antimicrobial
Agents and Chemotherapy, 12/01/2011
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7. 1% match (publications)
Takushi Kaneko. "Challenges and opportunities in developing novel drugs for TB", Future Medicinal Chemistry,
09/2011
Dedication
I dedicate my work to my parents who have taught me honesty and value for hardwork. I owe a
lot for their unconditional love, prayers and visionary guidance. To my wife and daughters for
giving me strength to accomplish the task and making my life meaningful and full of happiness.
To my teachers, Professor Khursheed Ali Khan and Professor Maj Gen(R) Farooq Ahmad Khan
for their invaluable help motivation, continuous guidance and mentorship.
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Acknowledgements
1. This task would not have been possible without the support, guidance and affection of my
supervisors and mentors. Professor Khursheed Ali Khan, my supervisor has been
continuous source of inspiration and encouragement during last few years. Professor Maj
Gen ® Farroq Ahmad Khan, my co supervisor provided invaluable support and guidance
in accomplishment of this task. It has been indeed a matter of pride for me to be their
PhD student. The lessons learnt from them in various aspects of research work were
indeed a source of inspiration for me. They also emphasized and taught me the
importance of being good human being and true professional. I cannot thank enough for
their contribution of time and invaluable guidance. I experienced productive and
stimulating research work under their guidance.
2. I would like to acknowledge my colleagues and laboratory staff of Microbiology
department who have contributed immensely to my research work at Armed Forces
Institute of Pathology, Rawalpindi-Pakistan. This study would have not been possible
without the technical assistance provided by senior laboratory technologist Mr Zakir.
3. I would like to acknowledge the moral support rendered to me by my wife Dr Sabahat
and prayers of my daughters Sania, Fatima and Shazra in journey to the accomplishment
of this task.
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CONTENTS
Page
LIST OF TABLES xiv
LIST OF FIGURES xv
LIST OF ABBREVIATIONS xvi
ABSTRACT xviii
CHAPTER 1. INTRODUCTION, PURPOSE STATEMENT &
RESEARCH QUESTION
1.1 Introduction 2
1.2 Purpose statement 4
1.3 Research question 5
1.4 Objectives 5
1.5 Operational definitions 6
CHAPTER 2. LITERATURE REVIEW
2.1 History 7
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2.2 Mycobacterium: General characteristics 11
2.3 Epidemiology of tuberculosis 14
2.4 Pathogenesis of tuberculosis 16
2.5 Antituberculosis drugs 19
2.5.1 Streptomycin 19
2.5.2 Isoniazid 22
2.5.3 Rifampin 24
2.5.4 Ethambutol 26
2.5.5 Pyrazinamide 27
2.5.6 Rifabutin 29
2.5.7 Quinolones 30
2.5.8 Oxazolidinones (Linezolid) 33
2.5.9 Carbapenems & Clavulanic acid 34
2.6 Grouping of Anti tuberculosis agents 35
2.7 Development of drug resistant tuberculosis 37
2.7.1 Molecular mechanism of resistance against INH 38
2.7.2 Molecular mechanism of resistance against Rifampicin 39
2.8 Drug susceptibilities against Mycobacterium tuberculosis 39
2.8.1 Over view 39
2.8.2 Conventional methods of susceptibility testing 40
2.8.2.1 Proportion method 40
2.8.2.2 Absolute concentration method 41
2.8.2.3 Resistance ratio method 41
2.8.3 Latest phenotypic methods 41
2.8.3.1 Mycobacterial growth indicator tube (MGIT) 41
2.8.3.2 Microscopic observation drug susceptibility assay 42
2.8.3.3 Calorimetric methods 42
2.8.3.4 E test susceptibility 43
2.8.3.5 Phage based susceptibility assay 43
2.8.4 Molecular based tests 44
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2.8.4.1 Genotype MTB DR plus 44
2.8.4.2 GeneXpert 44
2.9 Building treatment regimens for MDR TB 45
CHAPTER 3. MATERIAL AND METHODS
3.1 Setting 47
3.2 Study duration 47
3.3 Sample size 47
3.4 Study design 47
3.5 Sampling technique 47
3.6 Inclusion criteria 47
3.7 Exclusion criteria 48
3.8 Sequence of study 48
3.9 Determination of MDR TB 48
3.9.1 Patient profile 48
3.9.2 Pulmonary specimen collection 48
3.9.3 Extra pulmonary specimen collection 49
3.9.3.1 Pus 49
3.9.3.2 Tissue 49
3.9.3.3 Other body fluids 49
3.9.4 Specimen processing 50
3.9.4.1 Digestion and decontamination 50
3.9.4.2 Ziehl Neelsen staining 50
3.9.5 Drug susceptibility to first line Anti TB drugs 51
3.10 Drug susceptibility testing to second line Anti TB drugs 52
3.10.1 Amikacin 52
3.10.2 Levofloxacin 53
3.10.3 Quality control 54
3.11 Drug susceptibility to newer drugs (Linezolid & Meropenem) 54
3.11.1 Linezolid 54
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3.11.2 Meropenem 54
3.12 Interpretation of results 55
CHAPTER 4. RESULTS 61
CHAPTER 5. DISCUSSION 67
CHAPTER 6. SUMMARY, CONCLUSION AND 77
FUTURE PROSPECTS
CHAPTER 7. REFERENCES 78
ANNEXURES 101
LIST OF TABLES
Description Page
Table I Groups of Antituberculosis drugs 36
Table II Stepwise building treatment regimen for MDR TB 46
Table III Percentage of XDR TB isolates 63
Table IV Susceptibility of MDR TB isolates to Levofloxacin 63
Table V Association between Susceptibilities of MDR TB isolates to
Levofloxacin and Amikacin
64
Table VI Susceptibility of MDR TB isolates to different concentrations of
Linezolid
65
Table VII Association of susceptibilities of MDR TB isolates to different
concentrations of Linezolid
65
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Table VIII Susceptibility of MDR TB isolates to different concentrations of
Meropenem
66
Table IX Association of susceptibilities of MDR TB isolates to different
concentrations of Meropenem
66
LIST OF FIGURES
Figure/
Image
Description Page
Figure I Decontamination of specimen before inoculation 57
Figure II Mycobacterial growth indicator tube ( MGIT) front view 57
Figure III MGIT inner chamber with loaded DST tubes 58
Figure IV Panel for second line drugs 58
Figure V MDR susceptible to second line drugs 59
Figure VI Resistant isolate to both Linezolid and Meropenem 60
Figure VII Gender distribution of studied population 62
Figure VIII Age distribution of studied population 62
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Figure IX XDR and Pre XDR isolates in studied population 64
LIST OF ABBREVIATIONS
TERMS DESCRIPTIONS
AFB Acid fast bacillus
AFIP Armed Forces Institute of Pathology
AK Amikacin
AUC Area under curve
ATCC American type culture collection
BCG Bacilli Calmette Guerin
BAL Bronchoalveolar lavage
CAPREO Capreomycin
CDC Centre for Disease Control & Prevention
CIP Ciprofloxacin
CSF Cerebrospinal fluid
CFU Colony forming unit
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DST Drug susceptibility testing
EPTB Extra pulmonary tuberculosis
XDR TB Extremely drug resistant tuberculosis
EMB Ethambutol
FQ Fluoroquinolones
FDA Food and drug administration
GAT Gatifloxacin
GU Growth unit
HIV Human immunodeficiency virus
INH Isoniazid
KAN Kanamycin
LEVO Levofloxacin
LJ Lowenstein Jensen
LZD Linezolid
MDR TB Multi drug resistant tuberculosis
MER Meropenem
MODS Microscopic observation drug susceptibility assay
MIC Minimum inhibitory concentration
MOX Moxifloxacin
MGIT Mycobacterial growth indicator tube
NAD Nicotinamide adenine dinuceotide
NRA Nitrate reductase assay
OADC Oleic acid, albumen, dextrose, catalase
OFX Ofloxacin
PANTA Polymyxin B, Amphotericin, Nalidixic acid, Trimethoprim & Azlocillin
PAS Para amino salicylic acid
PNB Para nitro benzoic acid
PZA Pyrazinamide
QC Quality control
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PRP,s Protein recognition receptors
RIF Rifabutin
RMP Rifampicin
SM Streptomycin
TLR,s Toll like receptors
TB Tuberculosis
WHO World Health Organization
ZN Ziehl Neelsen
ABSTRACT
Background:
With the increase in MDR-TB strains around the globe, there has been an urgent need to
carry out drug susceptibility to first and second line antituberculosis drugs. It is imperative that
treatment of patients suffering from drug resistant TB should be carried out based on quick,
reliable and quantitative measure of susceptibility testing. This endeavor is a cornerstone for
prevention of resistance in treatment of tuberculosis and a way forward for optimal exploitation
of the available antituberculosis drugs (Mukherjee et al., 2004). The increase in MDR-TB rates
has lead to pressing demands for appropriate treatment with second line antituberculosis drugs
and need to find newer compounds with potential in vitro activity against MDR-TB. A number
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of antimicrobial compounds i.e. Linezolid, Levofloxacin, Moxifloxacin, Carbepenems and
Amoxicillin/ clavulananic acid have been considered as potentially active agents against MDR-
TB (Schectoret al., 2009; Wong et al., 1988; Bozeman et al., 2005).
The reliable drug susceptibility testing method provides us with detailed knowledge on
quantitative drug resistance pattern which ultimately paves the way for empirical treatment of
drug resistant tuberculosis. During the last decade or so MGIT 960 system has been extensively
studied and validated for susceptibility testing of first line antituberculosis drugs (Bemer et al.,
2002). The multicentre laboratory validation of the BACTEC MGIT 960 technique for testing
susceptibilities of M. tuberculosis to classical second line drugs and newer antimicrobials
(Rusch-Gerdes et al., 2006) has provided us with a guideline for resource poor countries like
Pakistan to endeavor testing such compounds against our local isolates.
According to WHO global report 2013, tuberculosis culture facility in Pakistan is
possible in only seven laboratories accounting to 0.2 laboratory per 5 million population while in
whole country only four laboratories can perform drug susceptibilities accounting to only 0.1
laboratory per 5 million population. To add fuel to the fire, Pakistan also could not achieve the
target of having at least one centre for carrying out smear microscopy under the WHO global
plan to stop TB 2011-2015 (WHO, 2013 ). In the backdrop of such sorry state of affairs our
laboratory was one of the very few in Pakistan with capacity to carry out DST to first and second
line antituberculosis drugs (Ghafoor et al., 2014)). With the aim of finding the susceptibility
pattern to classical second line and newer investigational drugs, it was challenge to embark upon
journey on the guidelines provided by validation studies.
Aims & Objectives
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The study has been undertaken keeping in view two objectives.
1. To evaluate the in vitro effectiveness of multiple concentrations of newer compounds
(Linezolid and Meropenem) against MDR-TB isolates in Pakistani population.
2. To find out the susceptibilities of MDR-TB isolates against Amikacin and Levofloxacin to
find out extent of XDR & pre XDR TB cases in our set up.
Study Design
It is a quantitative cross sectional research carried out at Armed Forces Institute of Pathology,
Rawalpindi Pakistan from Sept, 2011 to Aug, 2013.
Material and Methods
The methodology of study comprised of two parts. The first part comprised of all the procedures
leading to the determination of MDR-TB, while the second part consisted of evaluation of newer
compounds (Linezolid & Meropenem) and two of the classical second line antituberculosis drugs
(Amikacin & Levofloxacin). All MDR-TB isolates were subjected to susceptibility testing
against two classical second line drugs Amikacin (AK) and Levofloxacin (LEVO). These drugs
were obtained from chemically pure form from (Sigma, Taufbirchen, Germany). Amikacin
disulfate salt 710 μg/mg cat.N. A1774 with storage at 2-8 ͦ C manufactured by Sigma and
Levofloxacin > 98% HPLC cat.N. 28266 with storage at 2-8 ͦ C manufactured by Sigma were
used. These drugs were dissolved in deionized water. The stock solutions of AMK (84μg/l and
LEVO (84μg/ml) were prepared. Before subjecting the MDR-TB isolates to the test drugs full
susceptible and quality control strain (ATCC 27294) was subjected to the critical concentration
of drug used. The drugs panel consisted of three MGIT tubes, one for growth control and two for
second line drugs. Each 7ml MGIT tube was checked for any contamination or turbidity and
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labelled properly. After mixing the growth supplement (OADC), 0.1 ml of each antibiotic stock
solution was added in respective MGIT tubes. 0.5ml of culture proven MDR-TB sample was
added to two MGIT tubes while 0.5ml of 1:100 diluted sample was added to control tube. After
bar code scanning all the inoculated tubes were entered in the instrument and incubated at a
temperature of 37oC. An un-inoculated MGIT tube was used as a negative control. All MDR-TB
isolates were separately subjected to susceptibility testing against two newer investigational
drugs Linezolid (LNZ) and Meropenem (MER). Linezolid was provided by Continental
pharmaceuticals Karachi while Meropenem was provided by Adam & Musa Jee Karachi.
Linezolid as pure substance ca.100% Cat.N. 165800-03-3 with storage recommendation at room
temperature and manufactured by Pfizer was used. These drugs were dissolved in deionized
water. The stock solutions of LNZ and MER (84 μg/ml) were prepared in sterile water as per
instructions provided in leaflets of respective drugs. The three concentrations were used for both
drugs for BACTEC MGIT 960 system. For Linezolid 0.5, 1.0 & 2.0 μg/ml and for Meropenem
4.0, 8.0 & 16μg/ml respectively (Rusch-Gerdes et al., 2006). Before subjecting the MDR-TB
isolates to the test drugs full susceptible and quality control strain (ATCC 27294) was subjected
to the three concentrations of drugs used.
The drugs panel consisted of three MGIT tubes, one for growth control and two for each of the
three concentrations of investigational drugs. Each 7ml MGIT tube was checked for any
contamination or turbidity and labelled properly. Mixing of the growth supplement (OADC),
and further processing with antibiotic stock solutions and addition of cculture proven MDR-TB
sample was similar to Second line drugs.
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The MGIT 960 system flags the completion of a DST when the growth unit (GU) of the growth
control reaches 400 and reports S for susceptible or R for resistant, as well as a GU value for
each drug-containing MGIT tube on the printout. An isolate was interpreted to be susceptible
when the GU of a drug-containing MGIT tube was equal to or less than 100 or as resistant when
the GU was greater than 100. If an isolate was interpreted to be resistant, a smear was made and
stained to prove the presence of acid-fast bacilli (AFB) with morphology compatible with that of
MTBC and the absence of contaminants.
So based on the multi centre validation studies to find out cut off concentrations of
second line and newer drugs for testing with MGIT 960 method (Rodrigues et al., 2008; Sanders
et al., 2004). Following concentrations were used for the interpretations of MDR-TB isolates
being sensitive or resistant.
1. Amikacin 1.0 μg/ml
2. Levofloxacin 2.0 μg/ml
3. Linezolid 1.0 μg/ml
4. Meropenem 4.0 μg/ml
Data management and analysis
All data was analyzed and processed using statistical software (Statistical Package for Social
Sciences; SPSS 19. Results were expressed as frequencies or as mean ± SD unless indicated
otherwise. The Pearson chi-square test was used to determine the association between classical
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second line drugs as well as susceptibilities of MDR TB isolates to different concentrations of
Linezolid and Meropenem. We considered p value less than 0.05 to be significant.
RESULTS
Out of hundred MDR-TB isolates included in the study, 64 were from men and 36 from women.
The median age was 35years (ranging 15 to 71 years). All the 100 MDR-TB isolates were tested
using AK, LEVO, LNZ & MER. Out of these, 3 (3%) turned out to be XDR-TB based upon
resistance to injectable second line antituberculosis drug and one of the Fluoroquinolones. Using
2.0 μg/ml as critical concentration of LVX, 76/100 (76%) of MDR-TB isolates were found to be
susceptible and the frequency of Pre XDR-TB cases was thus 24%. The association between
susceptibilities of MDR TB isolates to classical second line drugs was significant (p- value of
0.002).
Three concentrations of Linezolid (LZD) 0.5, 1.0 & 2.0 μg/ml were tested. Based on breakpoint
concentration of (0.5 μg/ml) used, 80/100 (80%) of the MDR-TB isolates were sensitive while
for concentrations of 1.0 μg/ml & 2.0 μg/ml 96/100 (96%) of MDR-TB isolates were susceptible.
The association of susceptibilities of MDR TB isolates to two concentrations i.e. 0.5 and 1.0
μg/ml were statistically highly significant (p-value .000).For Meropenem (MER) using
breakpoint concentrations of 4.0 μg/ml no MDR-TB isolate was susceptible, while at 8.0 μg/ml
3/100 (3%) and at 16.0 μg/ml 11/100 (11%) of MDR-TB isolates were susceptible. The
association of susceptibility of MDR TB isolates to different concentrations of Meropenem was
significant (p value .002).
Conclusions and future prospects
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In this study we evaluated one hundred MDR TB isolates against two classical second line anti
tuberculosis drugs i.e. Amikacin and Levofloxacin in recommended cut off concentrations. The
frequency of XDR TB in our set up was 3% where as 24% of our isolates were in Pre XDR
category. By evaluating three concentrations of newer compounds, LNZ & MEM against MDR
TB we found that Linezolid revealed excellent in vitro susceptibility as 96% of our MDR TB
isolates were susceptible at concentrations of 1.0 μg/ml or more. In case of Meropenem it was
found that by serially increasing the break point concentrations the more number of isolates
became susceptible. This finding thus strongly potentiates the fact that Meropenem has potential
to become effective antituberculosis drug if it is combined with Beta lactamse inhibitor agent
like Clavulanic acid to utilize its full potential.
These results have given us sufficient information about in vitro effectiveness of Linezolid and
potential of Meropenem against MDR TB isolates from Pakistan. It is hoped that relevant
quarters in National tuberculosis control programme in Pakistan would benefit from this study in
formulating the future guidelines as regards the treatment of drug resistant tuberculosis in
Pakistan are concerned. With 3% XDR TB and 24% Pre XDR TB isolates found in this study, it
should ring alarm bells because injudicious use of Quinolones without performing the drug
susceptibility testing can be very detrimental in future. It is hoped that this study would serve as
stepping stone for more research in this area specially combining the Meropenem with
Clavulanic acid and determining the break point concentrations then to compare it with the
results obtained from this study.
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Key words: Multi drug resistant tuberculosis, Extremely drug resistant tuberculosis, second
line anti tuberculosis drugs, Linezolid, Meropenem, drug susceptibility testing tuberculosis,
MGIT 960.
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CHAPTER-1
INTRODUCTION, PURPOSE STATEMENT &
RESEARCH QUESTIONS
1. 1 INTRODUCTION
One third of world population is infected with M. tuberculosis and hence has increased risk for
development of active tuberculosis. Approximately 8.8 million people are diagnosed with active
tuberculosis that causes 1.3-1.7 million deaths per year (WHO, 2010 & 2013). Tuberculosis (TB)
is a deadly infectious disease of the human beings, caused by M. tuberculosis, which is aerobic
and acid fast bacillus (AFB) due to the presence of increased amount of mycolic acid in its cell
wall. TB is also a major health problem in many parts of the world. This disease is spread mainly
by air, when patient with tuberculosis expels bacteria in air by coughing out sputum. Pulmonary
TB is defined as the disease that involves lung, while extra-pulmonary tuberculosis (EPTB) is
defined as the disease that is not associated with lung involvement except miliary tuberculosis
(WHO 2006; Butt et al., 2003). TB can affect any organ of the body, whereas about one third of
all the tuberculosis cases can be affected by extra-pulmonary tuberculosis (Ahmed and Aziz,
1998).
TB can be treated with first line primary drugs when drug resistance is not defined.
Nevertheless global increase in the incidence of extremely drug resistant tuberculosis (XDR-TB)
has posed serious challenges to the ongoing efforts to combat tuberculosis (Banerjeeet al., 2008).
Multi drug resistant tuberculosis (MDR-TB) is defined as disease caused by strains of
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M. tuberculosis that are at least resistant to treatment with isoniazid and rifampicin while XDR-
TB refers to disease caused by MDR strains that are also resistant to treatment with
fluoroquinolones and one of the injectable second line antituberculosis drugs (Amikacin,
Capreomycin and Kanamycin) (Raviglione and Smith, 2007; Nathason et al., 2010). Second line
drugs used in the treatment of MDR-TB are not only costly but have many side effects and
require long period of treatment that can limit their usage (Jacobson et al., 2010).
Pakistan is currently ranked fifth on the list of 22 high burden tuberculosis countries of
the world according to World Health Organization’s (WHO) global tuberculosis control 2013.
Each year around 460,000 new TB cases are added to the existing patient population of around
1.8 million. According to WHO global report on tuberculosis 2013, the prevalence of TB in
Pakistan is 376 in 100, 000 population and incidence of 231 cases per 100,000 population. The
incidence rate of TB in Pakistan was ranked fifth in world. The estimated percentage of new
cases suffering from MDR variety of tuberculosis is 3.5% (WHO, 2013).
A variety of drug susceptibility testing (DST) methods that employ solid media, which
includes the agar proportion method , absolute concentration method and resistance ratio method
have the inherent disadvantage of extended turnaround times. BACTEC 460 TB system which
was introduced by Becton Dickinson Diagnostic Systems, Sparks, MD was broth based
radiometric system was well established and considered “gold standard” for carrying out
susceptibility testing for both primary and secondary antituberculosis drugs
(Tenevor et al., 1993). Nevertheless due to rising tendency about use and then disposal of
radiometric material, there has been growing trend in use of commercially available non
radiometric broth based culture and susceptibility testing methods. BACTEC MGIT 960 system
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is a non radiometric method which is considered comparable to the older radiometric method in
performance. Isolation of mycobacteria from clinical specimens and susceptibility testing for the
first line antituberculosis antimicrobials has been extensively evaluated by the MGIT 960
systems (Rusch-Gerdes et al., 1999; Scarparoet al., 2004). During the last decade however,
multicenter laboratory validation of the BACTEC MGIT 960 technique for testing
susceptibilities of M. tuberculosis to classical second line drugs have also been validated
(Rusch-Gerdes et al., 2006). During recent years, The WHO and the U.S. Centers for Disease
Control and prevention (CDC) have strongly suggested to use liquid culture systems for the
identification of M. tuberculosis and carry out susceptibility testing to improve turnaround time
(Tenevor et al., 1993; WHO 2007).
The increase in MDR-TB rates has lead to pressing demands for appropriate treatment
with second line antituberculosis drugs and need to find newer compounds with potential in vitro
activity against MDR-TB. A number of antimicrobial compounds i.e. Linezolid, Levofloxacin,
Moxifloxacin, Carbepenems and Amoxicillin/ clavulananic acid have been considered as
potentially active agents against MDR-TB (Schector et al., 2009; Wong et al., 1988; Bozeman et
al., 2005).
1.2 Purpose Statement.
The purpose of this quantitative study was to evaluate the in vitro effectiveness of Linezolid and
Meropenem (Two newer compounds considered for treatment of tuberculosis) against MDR-TB
strains isolated at Armed Forces Institute of Pathology Rawalpindi (AFIP) utilizing broth based
MGIT 960 technique. In addition, MDR-TB isolates were also subjected to two classical second
line antituberculosis drugs (Amikacin & Levofloxacin) to find out percentage of extremely drug
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resistant tuberculosis (XDR) among the studied group. All the MDR-TB isolates which were
recovered from different clinical samples of patients as routine drug susceptibility testing were
used for the said purposes. The results so obtained were to inform the clinical practitioners about
the in vitro effectiveness of Linezolid and Meropenem for treating drug resistant tuberculosis and
apprise them about the extent of XDR-TB in the studied group.
1.3 Research Questions
1. What is the in vitro efficacy of Linezolid and Meropenem against MDR-TB isolates
recovered from different clinical specimens in a Pakistani population?
2. What is the percentage of XDR-TB in studied population based upon the susceptibility to
Amikacin and Levofloxacin?
1.4 Objectives
The study has been undertaken keeping in view two objectives.
1. To evaluate the in vitro effectiveness of multiple breakpoint concentrations of newer
compounds (Linezolid and Meropenem) against MDR-TB isolates in Pakistani
population.
2. To determine the susceptibilities of MDR-TB isolates against Amikacin and Levofloxacin
to find extent of XDR and pre XDR-TB in our set up.
These results can subsequently be utilized by policy makers especially in National Tuberculosis
control programme to address the global issue and finding alternative antituberculosis drugs for
drug resistant tuberculosis.
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1.5 Operational definitions
Multidrug resistant tuberculosis (MDR-TB): A type of TB which is due to bacteria that are
resistant to isoniazid (INH) and rifampicin (RMP).
Extremely drug resistant tuberculosis (XDR-TB): A variety of TB which is due to bacteria
that are resistant to INH and RMP as well as any fluoroquinolone and any of the second line anti-
TB injectable drugs (Amikacin, kanamycin or capreomycin).
Second line anti-TB drugs: These drugs include Amikacin (AMK), Ofloxacin (OFL),
Levofloxacin (LEV), Ethionamide (ETH) and Capreomycin (CAP).
MGIT 960 TB System: Mycobacterial Growth Indicator Tube (MGIT) System is a liquid based
automated system which automatically directs the placement of each tube within the instrument
and indicates positives with both a visual and an audible signal as they occur. It is non
radiometric. It uses MGIT media and patented sensors, making efficient use of advanced
fluorometric technology, which permits highly accurate detection of O2 consumption without
sharps. Automated quality control is performed continuously to ensure precise and reliable
operation. Results are provided as positive/negative and numerical growth units.
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CHAPTER-2
REVIEW OF LITERATURE
2.1 HISTORY OF TUBERCULOSIS.
TB is an ancient disease. If we trace back the history, we find that books of history are
full of details about the disease. This disease has in fact inundated humanity throughout the
historical and prehistoric period. There have been periods when the disease increased in
epidemics and then decreased as in case of other infectious diseases. It is likely that
M. tuberculosis may have taken the lives of more persons than any other infectious agent
(Daniel, 2006). It can be hypothesized that the genus Mycobacterium may have originated more
than 150 million years ago (Hayman, 1984). With advancements in recent technologies of
molecular and the genome sequencing studies, several strains of M. tuberculosis can be
accurately estimated back in the history. It has been proven through studies that biological
ancestral forms of M. tuberculosis were present in East Africa about 3 million years ago
(Gutierrez et al., 2005).
By utilizing restricted structural gene polymorphism, it is thought however that current
strains of M. tuberculosis have likely originated from a common precursor about thousands of years
ago (Sreevastan et al., 1997). Distinctive skeletal manifestations of disease including typical Pott’s
variety have been found in Egyptian mummies and have been portrayed in Egyptian art and
documented proof of this effect is traceable more than 500 years ago (Cave, 1939. Like Egypt the
historical proof of early tuberculosis is also found in America. The manifestation of disease in bones
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has also been demonstrated in Peruvian mummies and M. tuberculosis nuclear material has been
isolated from the mummified remains (Daniel, 2000).
Tuberculosis was called as phthisis in classical Greece and its manifestations were very well known.
Hippocrates is known to have evidently acknowledged the disease and explained its clinical
manifestation. He wrote in his aphorisms “phthisis makes its attacks chiefly between the age of
eighteen and thirty five”, clearly recognizing the fact that the disease has predilection for young
adults (Coar, 1982). As Europe entered into medieval period, the written and documented record of
tuberculosis became less and less. This in no way implies that the disease was absent in that era. In
fact there is archeological proof from many sites across Europe for the presence of disease during the
era after the fall of Roman Empire in fifth century (Roberts and Buikstra, 2003).
The discovery of stethoscope by Rene Theophyle Hyacinthe Laennec, a French physician in
1816 clearly explained the events leading to disease production and incorporated the thoughts of
pulmonary and extra pulmonary tuberculosis. His book initially published in French in 1819 was
translated into English in 1821. In this book, he not only explained the events of disease but also
elucidated the common features of lung disease and used the terminology which is still in use today
(Laennec, 1962). Laennec’s work in explaining the pathogenesis of disease was possible largely
because of his vast practice of carrying out post mortem of individuals who had died of the disease.
By that time TB had spread across Europe like epidemic. Death rates in major cities of Europe
approached almost 100,000/year at that time (Krause, 1928).
As the prevalence of disease rose to awesome proportions, civilization started to view the
disease in romantic fashion. Emily Bronte an English Novelist and poet described the tuberculous
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heroine in Wuthering Heights as “rather thin, but young and fresh complexioned and her eyes
sparked as bright as diamonds (Bronte, 1946).Similarly, Charles Dickens in his Novel Nicholas
Nickelby described the death of Smike as “[As] the mortal part wastes and withers away, so the spirit
grows light and sanguine” (Dickens, 1988).
In trying to encounter the patients suffering from TB, scientists and medical practitioners
were trying hard to comprehend its exact cause. In the North of Europe it was felt to be inherited
disease while in other part of Europe it was considered to be communicable in form. It was in 1865
when Frenchman, Jean Antoine Villemin for the first time established the communicable nature of
disease when he injected a rabbit with a little quantity of pus like fluid obtained from a tuberculous
cavity removed at autopsy from a patient who had died of disease. Even though the animal remained
apparently healthy, it was found to have wide spread demonstration of disease when autopsied after
few months (Major, 1945).
The history of disease witnessed an extraordinary turn when on March 24, 1882 Hermann
Heinrich Robert Koch delivered his famous speech , Die Aetiologie der Tuberculose to the forum of
Berlin Physiological Society. In that talk, Koch not only gave the detailed explanations of the
organism but also outlined his famous postulates which to date are considered standard guiding
principles for the exhibition of infectious etiology (Koch, 1932; Daniel, 2005).
As information of disease became more superior after the work by Koch and others, there
was significant decrease in the cases both in West and Northern part of America. So the death rates
began to decrease in early and middle part of Nineteenth century. Exact reasons for this decline were
not clear but improvement in living and social setting, herd immunity which resulted from natural
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selection of more resistant population and improved nutritional status were some of the probable
reasons for this decline (Davies et al., 1999). As the society witnessed decline in TB, ill patients
turned towards spas and sanitaria to seek comfort and relief. The first sanatorium was opened by
German Physician Herman Brehmer in 1859 in the mountain village of Germany where he stressed
schedule of rest, a high caloric diet and supervised exercise (Davis, 1996).
The care and comfort of Sanatorium brought joy and peace of mind to many people suffering
from TB. The editorial written in The British Journal of Tuberculosis in 1907 states “Fads and
fancies have gathered about so called open air treatment and impossible claims have been made by
inexperienced enthusiasts as to the almost miraculous efficacy accruing from sanatorium residence.
In spite of all exaggerations and failures, there can be no doubt but that the maintenance of a strictly
hygienic course of line offers the best means known to modern medical science for dealing
effectually with disease (Editorial, 1907).
Challenges faced by community due to this disease were aptly addressed by Albert Calmette
and his colleague Camile Guerin. He embarked upon the journey to develop vaccine against TB
(Sakula, 1983). At the Pasteur institute of Lille, where Calmette was the founding director they took
the daunting task to attenuate M. bovis for use as vaccine. The first recipient of BCG, (Bacille
Calmette Guerin) was baby born to a mother dying of TB in 1921. The child luckily survived and did
not develop diseases. Over the next seven years more than 100,000 children were immunized. The
vaccine was willingly acknowledged in large part of Europe except Britain. He published a paper in
British Journal of Tuberculosis in 1928 strongly advocating the use of vaccine (Calmette, 1928).
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In 1948, an extraordinary movement to combat the disease with sponsorships by
international agencies and Danish Red Cross was launched. This plan was the first disease control
program undertaken by an agency of WHO which was based on Tuberculin testing followed by BCG
vaccination of non reactors. During three years, almost 30 million people were tuberculin tested and
nearly 14 million people were vaccinated with BCG (Comstock, 1994). In 1974, the policy guidelines
for the next two decades were issued by WHO Expert Committee on Tuberculosis. According to that
document it did not encourage the radiographic screening and skin testing and advocated the use of
sputum microscopy of individuals having symptoms and also those who are at risk of acquiring the
disease (WHO, 1974). It also strongly encouraged BCG vaccination for all persons less than 15-20
years of age and setting a coverage target of 70-90% in this age group. Today WHO continues to
support and mount programs for tuberculosis control but no longer recommends BCG vaccination
except for newborns.
2.2 MYCOBACTERIUM: GENERAL CHARECTERISTICS.
The genus Mycobacterium is the only genus in the family Mycobacteriaceae and is closely related to
other mycolic acid containing bacteria. The high G+C content of DNA of Mycobacterium species
(61 to 71 mol%, except that of M. leprae are within the range of those of members of other mycolic
acid containing genera like Gordonia (63-69 mol %), Tsukmurella (68-73 mol %), Nocardia (64-72
mol %) and Rhodococcus (63-73 mol %) (Coville et al., 2005). The waxy nature of cell wall in
M. tuberculosis confers the characteristic acid fast nature, capability to remain viable even after
drying, resistance to many antimicrobials and unique capability to stimulate immune system
(Daffe and Draper, 1998).
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Mycobacteria are aerobic (though some species are able to grow under a reduced O2
atmosphere), non spore forming, non motile, slightly curved or straight rods, 0.2 to 0.6µm by 1.0 to
10 µm. There is variation among different species as regards colony morphology. This may range
from smooth to rough and from nonpigmented (non photochromogens) to pigmented ones. There are
some species which require light to form pigments (photochromogens), while there are some species
which form pigments either in light or in dark (scotochromogens). The formation of aerial filaments
is rare and is not visible without magnification. At times there may be filamentous or mycelium like
growth which on slight disturbance easily fragments into rods or coccoid elements
(Pfyffer and Palikove, 2011).
The peptidiglycolipid content of the cell wall contains meso-diaminopimelic acid, alaninine,
glutamic acid, glucosamine, muramic acid, arabinose and galactose. The high content of mycolic
acid and free lipids account for hydrophobic permeability barrier. Other important fatty acids
include waxes, phospholipids, and mycoserosic and ptheinoic acids. Various patterns of cellular
fatty acids including tuberculostearic acid, a unique cell component for a number of members of the
Actinomycetales is also unique to this organism (Kremer and Besra, 2005).
The complex nature of lipids of cell wall prevents the access of common aniline dyes.
Although mycobacteria are not readily stained by Gram’s method these are usually considered Gram
positive. Mycobacteria are also not easily decolorized even with acid alcohol i.e. they are acid fast.
This property of acid fastness can be partially or completely lost at some stages of growth by some
proportion of cells of some species, particularly the rapidly growing ones. There exists a natural
division between slow and rapidly growing species of mycobacteria. Slow growing species require
more than seven days to show colonies on solid media, while rapid growers require less than 7 days
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when sub cultured on Lowenstein-Jensen (LJ) medium. Most species of mycobacterium use
relatively simple substrates like ammonia or amino acids as nitrogen sources and glycerol as carbon
sources in the presence of mineral salts. Carbon dioxide stimulates the growth of mycobacteria,
similarly fatty acids which can be provided in the form of egg yolk or oleic acid also aids in the
growth of mycobacteria (Pfyffer and Palikove, 2011).
Optimum temperature required for growth may vary widely (˂ 30 to ˃ 45) among different
species. Growth of most mycobacterial species is slow as compared to most other species with
generation time of around 20 h on commonly used media. Depending on different species the visible
growth of colonies is seen after few days to 6 weeks of incubation under optimum conditions. The
reason of slow growth of mycobacteria is not well known; nevertheless the likely mechanisms
include difficulty in uptake of nutrients through the impermeable cell wall and slow rates of RNA
synthesis (Pfyffer and Palikove, 2011; Harshley and Ramakrishnan, 1977).
Species of mycobacteria can survive for weeks to months on inanimate objects if protected
from sunlight. Members of M. tuberculosis complex for instance have the ability to survive for
many months on surfaces, or in soil or cow dung from which other animals may be infected.
Mycobacteria can be easily killed by heat at a temperature of ˃650C for at least 30 min and by UV
(sun) light but not by freezing or desiccation. They are also more resistant to acids, alkalis and some
chemical agents. Sterilants and disinfectants like ethylene oxide, formaldehyde vapors, chlorine
compounds, 70% ethanol, 2% alkaline glutaraldehyde, peracetic acid and stabilized hydrogen
peroxide are effective in killing M. tuberculosis. Agents like alcohols which are inactivated in the
presence of organic matter cannot be relied upon to disinfect sputum and other protein containing
materials (Pfyffer and Palikove, 2011; Best et al., 1990).
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The genus Mycobacterium includes obligate pathogens, opportunistic pathogens and
saprophytes. The major habitat for M. tuberculosis complex and M. leprae is tissues of humans and
warm blooded animals. On the other hand non tuberculous mycobacteria are free living and are
usually found in association with watery habitats such as lakes, rivers and wet soil. The
M. tuberculosis complex includes, M. tuberculosis, M. bovis, (M.bovis subsp. bovis, M. bovis subsp.
Caprae, M. bovis BCG), M. africanum, M. microti, M. canetii and M. pinnipedii. Recent
comparative genomics using the complete DNA sequence of M. tuberculosis have revealed
information on regions of genome that is deleted from other members of complex (Pfyffer and
Palikove, 2011).
2.3 EPIDEMIOLOGY OF TUBERCULOSIS
According to WHO report 2013, the worldwide load of disease has remained colossal in 2012. It was
estimated that more than 8 million new cases of TB have surfaced with more than 1.3 million deaths
from the disease. Out of these, there were an estimated 0.17 million deaths occurring due to MDR-
TB, a comparatively high figure when compared with 4,50000 incident cases of MDR-TB. The
approximate percentage of cases of disease with HIV has been found to be around 13% worldwide in
2012. Even though majority of deaths occurring due to disease have been in males, the burden of
disease is high in women. During 2012 an estimated 410,000 women died from TB (250,000 among
HIV-negative women and 160,000 among HIV-positive).
More than half of total cases of disease have occurred in the countries of South-East Asia and
Western pacific. In the countries of Africa, maximum numbers of deaths have occurred relative to the
population. Populous countries like India and China had the maximum number of cases (26% and
12% of the overall total respectively). The five countries with highest number of incident cases in
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2012 were India, China, South Africa, Indonesia and Pakistan. In Pakistan the total number of
incident cases have been 0.3-0.5 million (WHO, 2013)
There have been around 12 million cases of TB prevalent in 2012 which is equivalent to 169
cases per 100,000 populations. The encouraging aspect of these statistics was the fact that this rate
had fallen 37% internationally since 1990. Regionally the prevalence rates are decreasing in all six
WHO regions. In America, by 2004 the prevalence rate of disease has been reduced to half of what
it was in 1990 well in advance of the target set for 2015. It seems that target of 50% reduction in
disease set for 2015 appears possible in South East Asia and European regions due to comparatively
small acceleration in the present rate of progress.
According to WHO, there are an estimated 3.6% of new cases and 20.2% of old cases of
MDR-TB globally. The highest levels are seen in countries of Eastern Europe and Central Asia
where, more than 20% of new and 50% of previously treated cases have MDR-TB. Among new
cases, Belarus (34.8% in 2012), Estonia (19.7%in 2012), Kazakhstan (22.9% in 2012) and
Uzbekistan (23.2% in 2011) are the countries with highest MDR-TB. It has been documented that
92 countries have reported at least one case of XDR by September 2013. It has also been reported
that there were about 450,000 new cases of MDR-TB globally by the end of 2012. Almost half of
these total cases have been reported from three populous countries of China, India, and the Russian
Federation. Diagnosis of MDR-TB patients is also getting better and probably because of this reason
about 84,000 patients with MDR-TB were reported to WHO from all over the world in 2012
compared to 62,000 in 2011. (WHO, 2013).
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Pakistan is currently ranked fifth amongst high TB-burden countries globally. More than
60% of the TB burden in the WHO Eastern Mediterranean Region is from Pakistan. Around
420, 000 incident cases of TB emerge every year and majority of such cases turn out to be AFB
positive. It is also reported that overall Pakistan is ranked fourth highest in the prevalence of
MDR-TB worldwide. The number of TB cases diagnosed increased tremendously during the last
decade. The National Tuberculosis Control programme in Pakistan was revived in 2001 and due
to increased awareness and effective campaign launched, more than 1.5 million cases of
tuberculosis were treated free of cost. More than 5000 diagnostic and treatment centers which are
catering to provide free diagnostic and treatment services have been established in the public
sector (WHO/EMRO, 2011)
2.4 PATHOGENESIS OF TUBERCULOSIS
The human infections caused by M. tuberculosis usually starts with inhaling the aerosol droplets
containing the bacterium coughed out from patient with frank pulmonary disease. The dose required
to cause infection for a susceptible individual is considered to be as low as one bacterium to as high
as 200 bacilli. M. tuberculosis is carried in the form of airborne particles called droplet nuclei of size
varying in diameter from 1– 5 microns. Activities like coughing sneezing, shouting or singing from
a case of pulmonary or laryngeal tuberculosis lead to release of droplet nuclei containing infectious
particle. These released tiny droplets remain suspended in the environment for variable length of
time depending on the environmental conditions. Principal mean of transmission in M. tuberculosis
is through the air and not by surface contact. There are number of variables that decide the
likelihood of transmission of M. tuberculosis. These include the immune status of the susceptible
individual, degree of infectious nature of the person suffering from tuberculosis, environmental
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factors that affect the concentration of M. tuberculosis and proximity with duration of exposure
(CDC, 2013).
The bacilli then go to the alveolar spaces where they are instantly phagocytosed by alveolar
macrophages. These cells are then stimulated by ligation of toll like receptors (TLR’s) and other
protein recognition receptors (PRR’s) to produce proinflammatory response. This proinflammatory
response then drives the induction of more Neutrophils to the site of infection. Fundamental to this
process is the early arrival of both Neutrophils and Monocytes which are responsible to phagocytose
the surplus bacteria, then secrete more chemotactic factors and start the process of organization of
early granuloma. At the same time cells of dendritic system also phagocytose the bacterium and
move to nearby lymph nodes for the presentation of mycobacterial antigens to lymphocytes
(Hossain and Norazmi, 2013).
As an aftermath of proinflammatory cytokines and chemokines, there results the formation
of a well organized granuloma. This granuloma consists of central layer of infected
macrophages, which are surrounded by epetheliod macrophages, foam cells and sometimes
multinucleated giant cells. The outer layer of granuloma is composed of lymphocytes and fibrous
capsule. With the passage of time, the central portion of granuloma gets necrosed and there is
typical caseous necrosis of lesion due to high lipid and protein content of the dead macrophages.
The mycobacterium survives within the macrophages and extracellularly within the granuloma.
The presence of antigens and immunostimulatory lipids results in delayed type of
hypersensitivity response which is responsible for maintenance of granuloma
(Puissegur et al., 2004).
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Different chemokines are involved in granuloma formation, some of these are produced
by the epithelial cells of the respiratory tract, and others are produced by the immune cells
themselves. The chemokines binding to the CCR2 receptor (CCL2/MCP-1, CCL12, and CCL13)
are particularly important for the early recruitment of macrophages. In addition Osteopontin,
which is produced by macrophages and lymphocytes, promotes the adhesion and recruitment of
these cells (Co et al., 2004).
The lesion at primary site of implantation is termed the Ghon focus. The tubercle bacillus
may spread via lymphatics to regional draining lymph nodes before the formation of organized
granuloma consequently culminating in granulomatous form of lymphangitis and lymphadenitis.
This combination is also termed as the primary Ranke’s complex. During the early stages of
disease, hamatogenous dissemination can also occur within the lung or any other organ. As a
result of increased oxygen pressure and delayed immune response the upper lobes of lungs are
more likely sites for bacillary growth (Park et al., 1992).
Despite the fact that mycobacterium disseminates during primary infection, lesions in
most of infected persons resolve without becoming symptomatic. However, approximately 10%
of infected persons go on to develop severe manifestations of disease also called as primary
progressive disease. The likely reasons for this development include elevated bacterial load,
increased pathogenic potential of bacteria, and suppression in immune status of the affected
individual or genetic predisposition (Gomstock, 1982). In majority of individuals the preliminary
infection wanes and the contraction of collagen material results in a scar. Interestingly in most of
the cases, patients with pulmonary lesions have heterogeneous morphology which suggests that
each granuloma harbors the M. tuberculosis in multiple stages of latency and activation. When
the lesion becomes chronic in nature, the M. tuberculosis grows in outer part of this complex
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precisely within the foamy macrophages. In addition these bacilli survive also in chronic
granulomas which have resulted in cavitation and accessed in an airway (Fenhalls et al., 2000;
Kaplan et al., 2003).
If the progressive disease does not occur, an infected person may remain asymptomatic
for many years with M. tuberculosis surviving in latent state, most likely held in check by the
efficiency of immune system of the host. During subsequent years, reactivation of disease can
result due to any condition which can affect the immune system such as malnutrition,
malignancy, immunosuppressive medication or new bacterial or viral infection
(Verver et al., 2005). For reasons that are not clear, approximately 5-10% of latently infected
persons develop secondary disease during their life. Almost 15% of the reactivating cases of
tuberculosis occur at extra pulmonary sites such as the central nervous system, internal organs,
genitourinary system or skin (De-Becker et al., 2006).
Lung remains the most common site in case of secondary tuberculosis. The lesion usually
begins as exudative bronchopneumonia which progresses to typical caseous granuloma
formation which is followed by extensive necrosis and cavitary lesion. After sometime there is
collapse of the fibrous capsule which then makes connection and communication with airways
which results in intrapulmonary spread (Canetti, 1950).
2.5 ANTITUBERCULOSIS DRUGS
2.5.1 STREPTOMYCIN (SM)
The first isolation of this antituberculosis drug is attributed to Albert Schatz, who was a graduate
student at Rutgers University. The experiments of Schatz and his colleagues in the university
resulted in the discovery of many related antimicrobials. Of all those antibiotics only
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streptomycin and neomycin established widespread relevance and therapeutic effectiveness in
the treatment of many infectious diseases. In the cure against tuberculosis, streptomycin was the
first antimicrobial to be used in clinical practice. During the early days of manufacture, the
production of this drug was predominantly done by pharmaceutical company Merck. It was in
1946-47 that randomized trials of this drug were formally carried out against pulmonary
tuberculosis. These experiments which were both double blind as well as placebo controlled
were generally considered to have been the first such randomized curative trials for this deadly
disease (Comroe, 1978; Metcalfe, 2011).
Streptomycin acts by inhibiting protein. Its main action is to bind with 30S subunit of the
bacterial ribosome. This binding prevents the attachment of transfer RNA to the 30S
subunit. The end result of this action leads to ultimate inhibition of protein synthesis and finally
death of microbial cell. As human beings have structurally different ribosome from that of
M. tuberculosis, thus allowing this antibiotic to have its selective effect on the bacteria. This
antimicrobial can also have inhibitory effect on wide variety of both Gram-positive and Gram-
negative bacteria, and hence is also categorized to be a very a useful broad-spectrum antibiotic
(Sharma et al., 2007; Voet et al., 2004).
A committee was formulated by the medical research council of UK with the mandate to map
and carry out therapeutic trials of streptomycin for the cure of tuberculosis. As the quantity of the
antimicrobial available for the trials to be carried out on large scale was very limited, the
committee decided to restrict the testing for only those cases of tuberculosis which were acute in
nature. Hence trials were carried out on children suffering from tuberculous meningitis and acute
forms of military tuberculosis. These therapeutic trials were conducted in Hammersmith
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Hospital, Alder Hey Children Hospital Liverpool and the Royal Hospital for sick children,
Glasgow. The first groups of patients to be treated under these trials were hospitalized in January
1947 and total of 138 patients with acute manifestations of tuberculosis were admitted during
that year. The report was confined to 105 cases admitted till Aug 18 and those who survived
were observed for another about three months thus giving a minimal observation period of 120
days. After the encouraging results obtained from trials, the medical research council
consequently recommended to the ministry of health that this antimicrobial should be made
accessible for TB as extensively as supplies would permit. These recommendations paved the
way to the arrangement which was called as “Ministry of Health Scheme” under which the
treatment of severe forms of tuberculosis including tuberculous meningitis and acute military
tuberculosis was permitted to be undertaken in many hospitals throughout the country
(Marshall et al., 1948 ).
Consequently, controlled investigations into the effects of streptomycin in pulmonary
tuberculosis were carried out throughout England after the discovery of streptomycin. The results
of this trial were published in British Medical Journal in October 1948. A multicentre study was
carried out by renowned clinicians and pathologists belonging to Brompton hospital London,
Colindale Hospital London, Harefield Hospital Middlesex, Bangour Hospital West Lothian. The
investigations were based upon clinical observations on cases showing improvement with
streptomycin, radiological findings and microscopic details. The results of that multicentre
studies were very encouraging (Med Res Council, 1948).
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2.5.2 ISONIAZID (INH)
INH is a hydrazide of Isonicotinic acid. The two necessary components of its chemical structure
are the pyradine ring and the hydrazine group. In 1912, Isonicotinic acid hydrazide (INH) was
synthesized from ethyl isonicotinate and hydrazine by Meyer and Malley as part of their doctoral
work in Prague. Later in 1945, with the discovery of antituberculosis properties of Nicotinamide,
antituberculosis properties of isoniazid were discovered. The first clinical trial of the drug
commenced at one of the hospital in New York in 1951 and the results were reported to the
public in 1952. Prior to the discovery of INH, treatment of TB was dependant on streptomycin
only, but the use of this drug as monotherapy led to emergence of its resistance which lowered its
effectiveness. Therefore, a combination therapy was deemed necessary for the effective
treatment of tuberculosis, which became possible only after the discovery of INH. In 1967, the
American Thoracic Society recommended that everyone with a positive tuberculin skin test
should receive INH chemotherapy to prevent disease progression (CDC, 2000)
Although the precise mode of action of INH against M. tuberculosis is not yet completely
understood, but there are multiple ways in which this compound interacts with the bacteria and
several studies have been done to demonstrate these mechanisms. Inhibition of mycolic acid
synthesis is one of the mechanisms linked with the action of INH on M. tuberculosis. In 1952, it
was observed by Middlebrook that M. tuberculosis loses its acid fastness in the presence of INH.
This drug is also found to decrease the synthesis of those fatty acids which has chain lengths
longer than 16 carbons. Another mechanism of action of INH on M. tuberculosis was proposed
by Bekierkunst, who in 1966 found out a strong association and interaction of Nicotinamide-
adenine dinuceotide (NAD) concentration and M. tuberculosis. This drug was shown to increase
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the activity of NAD and hence Bekierkunst found out that this compound results in inactivation
of an inhibitor of NAD which enhances hydrolysis. Results of another study carried out by two
researchers Sriprakash and Ramakrishnan revealed that drug acts in two ways by affecting NAD.
On the one hand, INH increases the breakdown of NAD while on the other hand it decreases its
synthesis. The depletion of NAD is described as one of the reason as to why bacterium
accumulates much nicotinic acid. Lastly, it is also thought that mycobacterial catalase peroxidase
enzyme has a relation to the mechanism of action of INH. In 1954, it was discovered by
Middlebrook that the resistant strains of M. tuberculosis to INH showed little or no enzyme
activity. Similar to the mechanism of action, the precise mechanism of resistance to
M. tuberculosis is also unknown. Most of the studies conducted on the mechanism of action of
INH have also tried to incorporate possible reasons for resistance, but the primary mechanism of
resistance still remains unknown. In one of the study it was identified that the gene inhA, was
responsible for a substitution of Ser to Ala at position 94 in INH resistant strains of
M. smegmatis and M. bovis- BCG (Bannerjee et al., 2008). It suggests that InhA is a target of
mycolic acid synthesis. This result has also been observed in clinical isolates. Over expression of
the InhA protein shows that this finding may have clinical relevance as well. Similarly KatG
gene and catalase enzymes have also been implicated in the resistance of M. tuberculosis to INH
(Takayama et al., 1972; Davis, 1980; Scior et al., 2002).
Side effects occur in approximately 3.4 percent of persons using the drug. The most common
side effects are diarrhea, nausea, vomiting, light sensitivity and vision problems. INH may also
cause severe liver damage. Early indications of liver damage are eye pain, numbness or tingling
in the limbs, rash, fever, swollen glands, sore throat, bleeding, excessive bruising, or stomach
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pains. Clinically significant liver damage or hepatic injury by INH is seen in 1 percent of patients
but abnormal enzyme levels are usually observed in 10 to 20 percent of patients. Studies have
shown that INH can induce apoptosis by disrupting mitochondrial membrane potential and
breaks in DNA strands. INH also interferes with niacin metabolism, resulting in nervous system
toxicities. Peripheral neuropathy is the most common side effect, but toxic encephalopathy, optic
neuritis, cerebellar ataxia, and mild psychoses are also observed. Individuals hypersensitive to
isoniazid show symptoms of fever, pruritus, and rashes. In rare cases it can result in arthralgias,
anemia, or agrunulocytosis (Heemskerk et al., 2011).
2.5.3 RIFAMPIN (RMP)
This drug was developed in the Dow-Lepetit Research Laboratories (Milan, Italy) as part of an
extensive program of chemical modification of the rifamycins, which are the natural metabolites
of Nocardia mediterranei. As a result of these modifications, rifamycin SV was discovered and
this new compound was used in parenteral and topical form to treat multiple infections caused
due to Gram-positive bacteria. With the objective to find derivatives which can be administered
orally, a lot of experiments were performed. Those experiments which led to the detailed
understanding of structure activity relationship in the rifamycins resulted in the formation of
multiple hydrazones of 3-formylrifamycin SV. Out of these compounds, N-amino-N'-
methylpiperazine (rifampin) was found to be the most effective in the oral form for treating
infections in animals. Later on, after successful clinical trials, this compound was introduced into
clinical practice in 1968. In the subsequent years, lots of biologic and therapeutic studies have
established the significant role of RMP in treatment of M. tuberculosis and other selected
infectious diseases (Sensi, 1983).
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This antimicrobial exerts its mechanism of action by forming a stable complex with bacterial
RNA polymerase. As a result of this interaction, the enzyme activity is halted and as such
process of DNA transcription is severely jeopardized. While selectively inhibiting the bacterial
RNA polymerase the corresponding mammalian enzymes are not targeted by RMP.
Mycobacteria become resistant to RMP, when mutations cause change in the target enzyme RNA
polymerase. Interesting aspect of this resistance pattern is the fact that this is not an all-or-none
phenomenon. In fact, quite a large number of mycobacterial RNA polymerases with variable
degrees of susceptibility to RMP have been found. There exists no firm association between
sensitivity to enzyme and MIC values, as inhibition of RNA synthesis as result of enzyme
inactivity is not all the time demonstrated to the same degree in the two different methods used
for the determination of these values (Wehrli, 1983).
Although anti mycobacterial effects of RMP are well-known, but in addition RMP also has a
broad range of antimicrobial activity. It is bactericidal for gram-positive cocci such as
staphylococci (including methicillin-resistant strains), streptococci, and anaerobic cocci, with
MICs in the range of 0.01 to 0.5 mg/ml. However, it is bacteriostatic against enterococci.
N. gonorrhoeae, N. meningitidis, and H. influenzae, including b-lactamase-producing strains are
also susceptible to it, which is why it is used frequently in the prophylaxis of meningococcal and
H. influenza type b meningitis. This drug is also one of the most active agents against
L. pneumophila and other Legionella spp. Because of its ability to enterphagocytes, RMP inhibits
the intracellular growth of Brucella spp and C. burnetii, and it is used frequently in the
combination therapy of infections due to these organisms. Although Chlamydia spp are very
susceptible to RMP in vitro, resistance emerges rapidly when RMP is used alone
(Woodley et al., 1972).
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In addition to gastrointestinal discomfort, RMP is also associated with many other side effects
and hypersensitivity reactions, such as drug fever, skin rashes, and eosinophilia. It also produces
a harmless, orange-red coloration of saliva, tears, urine, and sweat. In up to 20% of patients, an
influenza-like syndrome with fever, chills, arthralgias, and myalgias may develop after several
months of intermittent therapy. This immunologic reaction may be associated with hemolytic
anemia, thrombocytopenia, and renal failure. Rifampin induced hepatitis occurs in 1% of patients
and is more frequent during concurrent INH therapy for tuberculosis. As a result of its ability to
induce human hepatic cytochrome P450 enzyme, RMP has clinically significant interactions with
many drugs, such as antagonizing the effect of oral contraceptives and diminishing the
anticoagulant activity of warfarin (CLSI, 2013).
2.5.4 ETHAMBUTOL (EMB)
It was in 1961 when the pharmaceutical company Lederle officially announced the
breakthrough of a new compound with activity against M. tuberculosis (Thomas et al., 1961).
During the process of choosing a synthetic compound randomly, it was revealed that N,N-
disopropylethylenediamine had protective effect on the mice from otherwise lethal infection with
strain H37RV of M. tuberculosis. With the use of in vitro concentrations of 1-4 μg/ml this
compound inhibited the growth of Mtb strain H37RV. This new antimicrobial was also found to
be effective against tuberculosis infected guinea pigs (Karlson, 1961). On the other hand it was
also found that this compound can cause toxic amblyopia. This side effect was seen in 44% of
those patients who received 60-100 mg/kg body weight of this compound daily (Carr &
Henkind, 1962). It was however heartening to note that these untoward effects on eyes improved
on stopping the drug.
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After now more than forty years of its discovery, this compound has established place as
first line antituberculosis agent. This drug has found its value for the shield it provides to other
antituberculosis drugs against the development and consequences of drug resistance. WHO has
recommended its use in adult age group with the caution that drug is immediately discontinued
in those patients who develop any deterioration of vision or change in perception of color
(WHO, 2003). Addressing at the concluding session of an international conference which was
held to discuss the use of this drug, Dr Aaron Chaves who was director of tuberculosis for the
department of health of the city of New York said “Enough has been said to suggest that EMB is
no competitor for isoniazid, but it might well be companion drug and replacement for PAS. Two
facts will determine this: cost and side reactions” (Anon, 1966).
In a diverse set of solid and liquid medias, the MIC of this drug varied from as low as 0.5
μg/ml to as high as 2.0μg/ml. The MIC range of this drug when tested in 7H12 BACTEC broth
was from 0.95 to 3.8 μg/ml and in 7H10 agent from 1.9 to 7.5 μg/ml (Suo et al., 1988). When the
trials of EMB were performed with guinea pigs, this drug alone was not successful in preventing
disease progression and also did not influence the bactericidal activity of INH and RMP
(Dickinson and Mitchison, 1976). It was thus concluded from these experiments that this drug
may not contribute to the sterilization of tuberculosis lesions but may help in the prevention of
drug resistance.
2.5.5 PYRAZINAMIDE (PZA)
Pyrazinamide is considered to be an important first line drug in treatment for tuberculosis.
Alongside INH and RMP, it is cornerstone in the modern Anti-TB regimen. It is one of the
important sterilizing drugs which shorten the duration of anti-tuberculous treatment, from
previously 9-12 months to 6 months. This is achieved as it kills those populations of
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M. tuberculosis which are in semi dormant state in acidic environment and notably these forms
are not killed by any other antituberculosis drug. This drug in not active against M. tuberculosis
under culture conditions as well as at neutral pH. In fact activity of the PZA under acidic pH is
also related to the acidity of the medium. Acid pH is produced during the active inflammatory
process by the Mycobacterium. It has been found that the low pH requirement of PZA action is
the result of large build up of pyrazinoic acid (POA), which is the active form of PZA in the
tubercle bacilli(Zhang & Mitchison, 2003).
Contrary to most antituberculosis drugs, which exert their action potently on more actively
growing bacteria than non-growing bacteria, the action of PZA is just the opposite. This drug is
less effective against young growing M. tuberculosis and more active against old non-growing
bacilli. PZA in itself is a pro-drug, whose active form is pyrazinoic acid (POA). PZA is
converted into PAO by bacterial enzyme nicotinamidase/pyrazinamidase (Zhang et al., 2003).
Those M. tuberculosis clinical isolates which are resistant to PZA lose this enzymatic activity
due to mutations in the pncA gene, which encodes the enzyme PZase (Scorpio & Zhang, 1996).
Another mechanism of resistance present in most PZA resistant mycobacteria is the presence of
pyrazinoic acid efflux mechanism. Naturally PZA resistant M. smegmatis has same mechanism
of resistance against this drug, i.e. they have extremely active pyrazinoic acid efflux mechanism
that quickly expels this out of the cell. M. tuberculosis is distinctively susceptible to PZA, and
this phenomenon correlates with a non active pyrazinoic acid efflux method in this organism
(Zhang et al., 1999).
The most common (approximately 1%) side effect of PZA is joint pains, but this is not usually
so severe that patients need to stop taking the PZA. This drug can precipitate gout flares by
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decreasing renal excretion of uric acid. The most dangerous side effect of PZA is hepatotoxicity,
which is dose related. The old dose for PZA was 40–70 mg/kg daily and the incidence of drug-
induced hepatitis has fallen significantly since the recommended dose has been reduced. In the
standard four-drug regimen (INH, RMP, PZA, EMB) PZA is the most common cause of drug-
induced hepatitis (Schaberg et al., 1996).
2.5.6 RIFABUTIN (RIF)
Rifabutin is one of the bactericidal anti-tuberculosis drugs. This can be used as a first line drug
for the treatment of tuberculosis. This drug is a semi-synthetic derivative of rifamycin S. This
drug is structurally similar to and shares many properties of RMP. This is also broad spectrum in
antimicrobial activity; In addition to its activity against mycobacteria, it is also effective against
large number of Gram-positive and Gram-negative bacteria, Chlamydia trachomatis and
Toxoplasma gondii. This drug is active against M. tuberculosis, M. leprae, and atypical
mycobacteria (Brogden & Fitton, 1994). This compound is a highly lipid-soluble and is
effectively absorbed when given orally. Rifabutin achieves high tissue concentrations in all
organs including lungs and liver. It has also shown to attain effective concentrations in
leukocytes, cells of mononuclear phagocyte system and the central nervous system. Dosage
adjustment of this antituberculosis drug is required as the pharmacodynamic properties of this
drug are highly influenced in patients suffering from renal or hepatic insufficiency. Simultaneous
administration of Clarithromycin can result in elevated plasma levels of drug due to inhibition of
liver microsomes by Clarithromycin resulting in altered metabolism
(Blaschke & Skinner, 1996).
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2.5.7 QUINOLONES
The first quinolone emerged in the early 1960s, with the isolation of 7-chloro-l-ethyl-1, 4-
dihydro-4-oxoquinoline-3-carboxylic acid, a by-product of the commercial preparation of
chloroquine. This compound was found to have anti-bacterial activity and was subsequently
modified to produce nalidixic acid, a 1, 8-naphthyridine, which was developed initially as a
urinary antiseptic (Gerard & Chew, 2003). Because of early emergence of resistance, Nalidixic
acid and its early analogs, oxolinic acid and cinoxacin, have limited clinical applications. Newer
quinolones have been synthesized by modifying the original two-ring quinolone (or
naphthyridone) nucleus with different side chain substitutions (Ball, 2000). Fluoroquinolones
(FQ), which are the new quinolones, contain a fluorine atom attached to the nucleus at position
6. The primary mechanism of actions of these FQ is their action on DNA gyrase, a type II DNA
topoisomerase enzyme essential for DNA replication, recombination, and repair (Hawkey, 2003).
Newer FQ also inhibit DNA topoisomerase IV. Inhibition of these bacterial enzyme targets
causes relaxation or decatenation of the supercoiled DNA, leading to termination of
chromosomal replication and interference with cell division and gene expression. By inhibiting
bacterial DNA synthesis, these agents are bactericidal. Bacterial resistance to quinolones can be
caused by several mechanisms. A single-step chromosomal mutations in the structural genes
(gyrA, gyrB, parC, and parE) encoding the DNA gyrase and topoisomerase IV can lead to
bacterial resistance. Mutations in the regulatory genes governing bacterial outer membrane
permeability to the drug can also cause resistance. Expression or over expression of energy-
dependent multidrug efflux pump AcrAB, are also a possible cause of bacterial resistance.
Acquisition of plasmid-mediated resistance genes (qnrA, qnrB, and qnrS) encoding proteins that
prevent the binding of quinolones to bacterial DNA gyrase and topoisomerase IV also lead to
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resistance (Morgan-Linell et al.,2003; Strahilevitz et al., 2009). Since 1980, FQ are under
investigation to assess their anti-tuberculosis activity. Although many FQ are active in vitro, but
only some like ofloxacin, levofloxacin, ciprofloxacin, sparfloxacin and lomefloxacin have been
tested clinically. They can be used in co-therapy with the available anti-TB drugs. However, the
choice of using a particular FQ should be based not only on the in vitro activity, but also on the
long-term tolerance of that particular drug (Bryskier & lowther, 2002). Ofloxacin (OFX) is one
of the potent FQ recommended to treat MDR-TB. Over a decade, the pre exposure of this drug
for the treatment of other bacterial infections has resulted in acquisition of FQ resistance among
M. tuberculosis strains (Verma et al., 2011). When MDR TB surfaced in the early 1990s,
physicians started looking for the popular FQ of that time, which was ciprofloxacin
(Sullivan et al., 1995). Unluckily strains of M. tuberculosis which were resistant to ciprofloxacin
were detected quickly; a fact which was also forewarned from in vitro studies
(Takiff et al., 1994). The ciprofloxacin resistance in fact caused problems on two accounts,
firstly it was ineffective in curing tuberculosis and secondly it also played its part in selecting out
the strains which were also non susceptible to other FQs (Shandil et al., 2005; Gumbo
et al., 2005). Shortly thereafter the clinicians treating MDR TB, embarked upon the journey to
start with newer FQs, OFX and its l-isomer, LEVO. These two drugs exhibited better cure rates
than ciprofloxacin. The clinical effectiveness of these drugs over CIPRO was most likely due to
better pharmacokinetics, and better intra macrophage penetration. In addition to these, other FQs
though have lower MICs for M. tuberculosis, but few of these were found to have adverse effects
for widespread use. To quote few for example sitafloxacin and sparfloxacin were found to be
phototoxic, and Gatifloxacin (GAT) in older patients resulted in both hypoglycemia and
hyperglycemia (Renau et al., 1996; Dawe et al., 2003; Yadav et al., 2006). Due to in vitro
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effectiveness of these FQ for studies done in animals and their clinical effectiveness in treatment
of drug resistant TB raised the hope that these compounds may be able to shorten the duration of
antituberculosis treatment if used as first line drugs (Pletz et al., 2004). Even if it is assumed that
some FQ like MOX can successfully cut down the duration of first-line antituberculosis
treatment, the looming risk of resistance is a legitimate basis for vigilance. The use of these
compounds as first-line of treatment may mean that most MDR strains would behave as FQ non
susceptible, thus diminishing the vital contribution of the FQs in treating MDR TB and possibly
promoting the progress of XDR- TB. In such scenario the isolate would be in addition resistant
to any FQ as well as to any second-line injectable antibiotic (Gandhi et al., 2006). The FQ act by
inhibiting the enzyme DNA gyrase, and ∼50 to 90% of FQ-resistant strains have shown
mutations in the gyrA gene which results in substitutions in different amino acids of the GyrA
subunit (Yin & Yu, 2010. Low level FQ resistance results due to amino acid substitutions in the
other gyrase subunit, GyrB in about 10% of cases. Such isolates of M. tuberculosis are generally
susceptible to treatment with high-dose MOX (Cui et al., 2011). It has also been documented
that’s some FQ-resistant strains exhibit no gyrase mutation, and are thus only intermediately
resistant to ciprofloxacin (MIC, 1 μg/ml) and sensitive to MOX. It is also interesting to note that
up to 50% or more of FQ-resistant strains don't show any gyrase mutations at all
(Huang et al., 2005). In vitro experiments have shown that low-level FQ resistance in
M. tuberculosis can be caused by multiple efflux pumps as well as the ATPase complex which
expels the drug out of the cell (Takiff et al., 1996; Pasca et al., 2004). It has been found that by
judiciously restricting the therapeutic use of FQ for nonspecific respiratory symptoms or
community-acquired pneumonia, chances of development of FQ resistant TB can be curtailed to
a significant effect. (Chen et al., 2011; Joen et al 2011).
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2.5.8 Oxazolidinones (Linezolid)
Linezolid (LZD) which belongs to Oxazolidinones class of antibiotics is one of only two new
antibiotics introduced into the market in the last almost four decades. This compound primarily
targets Gram positive bacteria including MRSA and vancomycin resistant enterococi. However
multiple in vitro and animal models have shown that this drug is also effective against
M. tuberculosis (Alcala et al., 2003; Cynamon et al., 1999). It is readily distributed to well
profused regions of body, penetrates well into bronchoalveolar tissues and acts by targeting the
ribosomes and inhibiting the protein synthesis at an early stage of translation
(Finch, 2003; Ford et al., 1997).
During the last decade number of clinical trials have been carried out to study the clinical
efficacy of this compound for the patients of MDR and XDR TB and was found to be effective in
multiple case series. (Park et al., 2006; Condos et al., 2008). However the toxicities and side
effects primarily bone marrow suppression and peripheral and optic neuropathy have also been
reported with long term use of this compound (Park et al., 2006).
The MIC reported by the pharmaceutical company (Pfizer) for MTB is 1 µg/ml. The
desirable cumulative weekly target area under curve (AUC) is 350-700µg per hr/ml. For the
treatment of M. tuberculosis it has been found that 7 daily doses of LZD per week achieves
AUC/MIC ratio of 999 per week, which is above the desired target. Based upon these findings it
has been postulated that LZD at a daily dosage of 600 mg once daily should be effective with
less toxicity (Schector et al., 2009).
Lately Oxazolidinones other than LZD has also been source of interest with PNU-100480
and AZD 4587 being widely studied along with LZD. The in vitro drug susceptibility results
have shown that these three Oxazolidinones has better bacteriostatic effect against actively
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growing bacilli but potent bactericidal activity against non replicating cells. It has also been
found that potency of PNU-100480 is better than that of LZD making it an attractive drug
candidate in the development of new combination therapies for latent tuberculosis
(Zhang et al., 2014).
2.5.9 Carbepenems & Clavulanic acid
Carbapenems are considered most potent β-lactams and were developed in the 1980s to combat
resistance to β-lactamases. Meropenem (MER) is a broad-spectrum carbapenem which is active
against several clinically relevant Gram-positive and Gram-negative aerobes and anaerobes. The
bactericidal activity of this compound results from the inhibition of cell wall synthesis through
the inactivation of penicillin-binding proteins (Edwards, 1995; Nicolau, 2008). Meropenem is
approved by food and drug administration authority (FDA) for the treatment of complicated skin
and soft tissue infections, intra-abdominal infections (appendicitis and peritonitis), and bacterial
meningitis (Baidwin et al., 2008). Clavulanic acid is food and drug administration (FDA)
approved as a β-lactamase inhibitor often administered with β-lactam to prevent hydrolysis of the
active compound (Finley et al., 2003)
Traditionally beta-lactam group of antimicrobials have not been used against M. tuberculosis
primarily due their lack of effectiveness. However, during recent times investigators around the
world have started to carry out in vitro experiments to reinvestigate this phenomenon. These
experiments have resulted in very important breakthroughs demonstrating that either deletion or
inhibition of the major b-lactamase of M. tuberculosis BlaC, can result in activity of these
antimicrobials against M. tuberculosis (Flores et al., 2005; Chambers et al., 2005). A
combination of clavulanic acid which is potent beta-lactamase inhibitor, and MER, a carbapenem
antibiotic, was shown to have effective activity in vitro killing XDR- MTB under aerobic as well
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as anaerobic conditions (Hugonnet et al., 2009). Meropenem has been found to be resistant to
M. tuberculosis BlaC beta-lactamase. Studies have shown that in vitro assays, combinations of
any of the carbapenems (imipenem, meropenem and ertapenem) with clavulanic acid reduce the
MIC of the carbapenem from 8–16 to 1–4 μg/ml. After the inclusion of Meropenem in group V
of the classification of anti tuberculosis drugs there has been plenty of clinical trials to treat drug
resistant tuberculosis cases with meropenem and clavulanic acid and the results to date have
been very encouraging (Dauby et al., 2011; De-Lorenzo et al., 2013).
2.6 Grouping of Antituberculosis agents:
According to WHO Guidelines for programmatic management of drug resistant tuberculosis
2008, following five groups of antituberculosis drugs have been outlined (WHO, 2008)
Group 1 drugs like INH and RMP are the first line oral agents. These are the most potent and
best tolerated drugs and these are recommended for use if the clinical history as well as
laboratory confirms that the antimicrobial from this group is effective.
Group 2 are the injectable agents which include AK and KAN. These antituberculosis drugs are
recommended if susceptibility to these drugs have been carried out and is documented. The
guidelines suggest the use of KAN or AK as the first option for injectable agent because of the
rising rate of streptomycin resistance in drug resistant patients. Both these antituberculosis drugs
have the advantage of being not very costly and having lesser side effects. Both AK and KAN
are considered to be very similar and have high frequency of cross resistance.
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Group 3 drugs include FQs. All patients should receive group 3 medication if the strain is
susceptible or if the agent is thought to have efficacy. Ciprofloxacin is no longer recommended
to treat susceptible or resistant tuberculosis. Currently the most effective available FQs in
descending order based on in vitro activity are MOX, LEVO and OFX. Among FQs, LEVO and
MOX are thought to be more effective against MTB than OFX based on animal and EBA data.
Until more data and evidence is available for MOX toxicity, LEVO is for the time being
considered the FQ of choice.
Group 4 includes the oral bacteriostatic second line agents. These agents are recommended to be
added based certain factors like estimated susceptibilities, drug history, efficacy, side effects and
cost.
Group 5 are agents with unclear efficacy. These drugs include LZD and Carbapenems. Lot of
clinical trials are being carried out to determine the in vitro efficacy of these compounds. The
drugs from this group should be used in consultation with an expert in the treatment of drug
resistant tuberculosis.
Following table summarizes the Groups of Antituberculosis drugs.
Table 1- Groups of Antituberculosis drugs (WHO, 2008)
GROUPING DRUGS
Group 1. First line oral agents INH, RMP, EMB, PZA, RIF
Group 2. Injectable agents KAN, AK, SM, CAPREO
Group 3. Fluoroquinolones MOX, LEVO, OFX
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Group 4. Oral bacteriostatic second line agents Ethionamide, Prctionamide, Cycloserine,
paraaminosalicylic acid (PAS) , Terizidone
Group 5. Agents with unclear efficacy. Not
recocommended by WHO for routine use in
MDR TB cases
Clofazimine, LZD, Amoxicillin/clavulanic
acid, Imipenem / cilastatin, Thiocetazone,
Clarithromycin
2.7 DEVELOPMENT OF DRUG RESISTANT TUBERCULOSIS.
The ability of the microorganism to survive the presence of a drug that normally kills or inhibits
growth is called resistance. If a microorganism lacks susceptibility target and is impermeable to
antituberculosis agent, this is known as innate resistance (Goering & Mins, 2007).
M. tuberculosis and other mycobacteria have certain traits of innate resistance which renders
these bacteria difficult to be treated. Some of these traits associated with such phenomenon
include waxy mycolic acid content, intracellular localization and efflux pumps
(Goering & Mins, 2007; Riccardi et al., 2009).
During 1940s, when monotherapy with streptomycin or PAS was used for the treatment of
tuberculosis, there were high rates of treatment failure. This phenomenon was however
countered by adding two or more drugs (Pyle, 1947). The studies performed at molecular
genetics level during the 1970s revealed that resistance against anti TB drugs are due to
mutations in the gene of Mtb (David, 1970). Studies performed subsequently demonstrated that
mutant pool exist within the population of M. tuberculosis, which arise as a result of spontaneous
point or deletion mutants (Zang & Talenti, 2000). As M. tuberculosis does not possess plasmids,
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mutations in the genome of M. tuberculosis results due to mutations in chromosomal DNA
(Riccardi et al., 2009; Pfyffer, 2000). Such mutations have been found for both first and second
line antituberculosis drugs as a result of genes encoding drug targets or drug inactivating
enzymes. (Ramaswamy & Mussser, 1998).
The resistant mutants of M. tuberculosis for any given drug take place approximately in every
107-10 cells (Parsons, 2004; Rattan et al., 1998). Antimicrobial treatment with a single
antituberculosis drug hence results into rapid selection of drug resistant mutants which keep on
dominating the lesions of patients. The chances of occurrence of one mutant M. tuberculosis
strain with resistance to two antituberculosis drugs at one time require a theoretical population of
1016 mycobacterial cells. The combination of two or more anti TB drugs in the treatment regimen
effectively reduces the chances of selection of resistant mutants in predominantly susceptible
population of M. tuberculosis. This fact was potentiated with the observation that combining
PAS and SM in the 1940s reduced the treatment failure by 9% (Med Res Council, 1949). Hence
for the last more than seventy years combination chemotherapy has remained as cornerstone for
the treatment of tuberculosis.
2.7.1 Molecular mechanism of resistance against INH
Soon after the introduction of INH it was frequently realized that INH resistant M. tuberculosis
strains lost catalase and peroxidase activity (Middlebrook, 1954). It has been documented now
that the main mycobacterial catalase peroxidase gene KatG has been cloned and sequenced. As a
result, mutations have been were detected in ˃ 50% of INH resistant clinical isolates of
M. tuberculosis proving the role of KatG enzyme in causing resistance against SM
(Somoskovi et al., 2001). Since then large number of different mutations have been found with
ser315Thr being the most common which occurs in approximately 40% of all INH resistant
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strains. This mutation results in a catalase enzyme that does not activate the ingredients of INH
(Marttila et al., 1998). M. tuberculosis with low level resistance to INH have also been found
which occurs due to mutations in the promoter regions or less commonly in the genes inhA,
acpM and kasA .These genes encode for mycolic acid synthesis and intracellular proteins (Goble
et al., 1993).
2.7.2 Molecular mechanisms of resistance against Rifampicin (RMP).
The rpoB gene is responsible for encoding the DNA dependent RNA polymerase (the target of
rifampicin). Spontaneous mutations comprising of either deletions, substitutions or insertions
taking place in the rpoB gene results into replacement of the aromatic and nonaromatic amino
acids in the target RNA polymerase preserving the activity of enzyme and hence resistance to
RMP(Telenti et al., 1993).
2.8 TB DRUG SUSCEPTIBILITIES
2.8.1 Over view
In order to find out susceptibility to any given antimicrobial drug, tests are carried out as in vitro
assay in the laboratory, a process termed as DST. In underdeveloped and resource limited
setting, the WHO recommends that DST must include at least RMP and INH- which are the two
most efficacious first line antituberculosis drugs which determine MDR-TB (Rich et al., 2006).
During the last more than fifty years, DST in the developing countries has been carried out
principally on conventional indirect susceptibility methods utilizing LJ solid medium (Canetti et
al., 1963). Indirect susceptibility testing is done by first isolating the pure culture colonies of
M. tuberculosis to be used as inoculums for DST. On the other hand direct DST is carried out by
inoculating smear positive samples instead of pure colonies of M. tuberculosis. Results obtained
with direct testing are much rapid and helps in segregating MDR from non MDR cases quickly.
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2.8.2 Conventional methods of DST
There are three conventional methods of performing DST. These include the proportion method,
the absolute concentration and resistance ratio method. These methods have undergone
standardization during the years and are extensively employed in many countries (Aziz et al.,
2003; Siddiqi et al., 2012).
2.8.2.1 Proportion method.
This is the oldest method and in this an equal quantity of standardized inoculums containing
M. tuberculosis is put in both a drug free and drug containing medium. The inoculum which has
been diluted 100 times is put in drug free medium while non diluted specimen is inoculated in
the drug containing medium. There should be discrete and large number of colony forming units
(CFU) present in the medium devoid of drug. On the other hand only preexisting resistant
mutants are expected to grow in medium containing drugs. The proportion of mutant population
based on the mutation rate of 1 in 107-10 is nevertheless much lower but for the sake of ease of
calculation and interpretation it is theoretically taken as 1% as this has been found to guess the
therapeutic outcome (Canetti et al.,1969).
Taking into account that 1% of the inoculums on drug containing medium are preexisting
mutants, and by dividing the number of CFU on medium containing drug by those on medium
devoid of drug, it can be assumed that isolate is susceptible ( ≤ 1%) or (˃1%) as resistant.
Hence in order to interpret the isolate to be susceptible, the principle is that the number of CFU
on the drug containing medium should not exceed those on medium devoid of drug
(Canetti et al., 1963 & 1969). The proportion method can be performed on both solid as well as
in liquid medium (Makinen et al., 2006).
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2.8.2.2. Absolute concentration method.
Absolute concentration method involves inoculating M. tuberculosis in either solid or liquid
medium containing several dilutions of each antituberculosis drug. Resistance is indicated when
by the end of four weeks the lowest concentration of the drug inhibits the growth (IUTLD, 1998)
2.8.2.3 Resistance ratio method (R/R)
This method is based upon the ratio of MIC of any antituberculosis drug for the patient’s strain
to the MIC of the drug susceptible reference strain H37Rv with both strains tested in the same
experimental conditions. (Heifets et al., 2000). After incubating for four weeks, growth on any
slope is defined as presence of 20 or more colonies and MIC is defined as the lowest
concentration of the drug where numbers of colonies are less than 20. A ratio of 2 or less
indicates sensitive strain and 8 or more indicates resistance.
2.8.3 Latest Phenotypic Methods.
These methods of susceptibility testing have been in vogue for about last two decades and have
been found to be extremely effective.
2.8.3.1 Mycobacterium growth indicator tube (MGIT)
The susceptibility testing on MGIT is based on the fluorescence detection of the mycobacterial
growth in the tube containing modified Middle brook 7H9 liquid medium along with
fluorescence quenching based oxygen sensor incorporated at the bottom of the tube
(Rusch- Gerdes et al., 1999). The growth of the microorganism leads to consumption of oxygen
in the medium which is detected by oxygen sensors and hence results in the fluorescence under
ultraviolet (UV) light. This method was initially introduced about twenty years ago as manual
system while automated version is also available. Different studies performed on this system for
first and second line antituberculosis drugs have revealed the sensitivity, specificity and
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concordance ranging from 90-100% compared to different conventional methods
( Johansen et al ., 2004; Grace Lin et al., 2009). The automated version has the benefit that more
than 900 samples can be tested at any given time. The results are rapid and easy to interpret. All
these attributes of the system are ideal for culture and DST in high TB burden areas.
2.8.3.2 Microscopic observation drug susceptibility assay (MODS)
This is a liquid based method for the indirect or direct detection of M. tuberculosis and MDR
from sputum. This method is based on the principles that M. tuberculosis grows faster in liquid
medium than in solid medium, there is characteristic cord formation which can be seen
microscopically in liquid medium in early stages and addition of antituberculosis drug allows
direct and rapid sensitivity testing (Moore et al., 2006). The presence of cord like structures in
liquid containing medium having the drug indicates resistance. The different studies done on
MODS have revealed sensitivities and specificities in the range of 86-100% for first and second
line drugs (Moore et al., 2006; Trollip et al., 2014).
MODS has been revised and reorganized to include a well with paranitrobenzoic acid (PNB) to
assist in identification of M. tuberculosis from atypical mycobacteria as PNB inhibits the growth
of M. tuberculosis but not that of atypical mycobacteria (Tran et al., 2013). This method is easy
to operate, requires minimal training and the results are available quickly. However microscopic
observation of cord like structures may be subjective.
2.8.3.3 Calorimetric methods
Calorimetric methods include nitrate reductase assay (NRA), the MTT (3-[4, 5-dimethythiazol-2-
yl]-2, 5-diphenyltetrazolium bromide) and alamar blue assay. For all these methods resistance is
seen by a change in color of the indicator after the detector reagent is added to the medium
containing viable mycobacterium. In NRA, resistance detection is based on visual observation of
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pink to purple color in a culture tube upon addition of the reagent called Griess reagent due to
nitroreductase enzyme in metabolically active mycobacterial cells converting nitrate to nitrite
(Angeby et al., 2002). This rapid and inexpensive test has the potential to be used for DST in
resource limited and underdeveloped countries. The sensitivities and specificities for first line
antituberculosis drugs range between 98-100% (Vayawahere et al., 2013).
2.8.3.4 E test .
In this method plastic strips are used that contain antibiotics in exponential gradients for
susceptibility testing of mycobacterium. The particular antibiotic diffuses into the medium and
thereby inhibits the growth of susceptible isolate. The MIC is noted and isolate is interpreted as
susceptible or resistant. Different studies done with this method has shown high accuracy
estimates close to 100% (Jaloba et al., 2000). One disadvantage associated with this method is
the fact that there is quick degradation of the diffused antimicrobial resulting in blurred cut off
point for MIC reading. The other disadvantage with this method is the need for heavy inoculums
(3 McFarland) which might not be possible with direct DST on sputum samples. It also poses
risk of aerosol production and chance inhalation by the staff in the biosafety level 2 laboratories
in developing countries. E test is also costly with each strip costing around six hundred rupees,
which may not be suitable in resource limited countries.
2.8.3.5 Phage based susceptibility tests.
These tests are based upon the ability of live and thus resistant M. tuberculosis preincubated with
the antituberculosis drug to sustain the growth of an infecting mycobacteriophage- a virus that
infects mycobacteria (Simboli et al., 2005). The commercially available fast plaque TB and the
in house types have been studied for the detection of RMP resistance of M. tuberculosis isolates
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and directly in clinical specimen with high quality and rapid results
( Simboli et al .,2005; Albert et al.,2004).
2.8.4 Molecular based tests.
WHO recommends molecular based assays for diagnosing drug resistance especially in high
burden & resource limited countries (WHO, 2008). New modalities for quick detection of drug
resistance have therefore become priority in TB research and development. Two techniques
involving line probe assays focused on rapid detection of RMP resistance alone as in ‘Genexpert’
or in combination with ‘Genotype MDTBDR plus’ have been evaluated and validated
( Huyen et al.,2010).
2.8.4.1 Genotype MTBDR plus
Genotype MTBDR plus (Hain lifesciences, Nehren, Germany) is commercially available line
probe assay that detects mutations in the inhA, KatG, rpoB genes that confer INH & RMP
resistance. The assay combines detection of M. tuberculosis complex with detection of mutations
in the 81-bp hot spot region of rpoB at codon 315 of KatG gene and in the inhA promoter region.
It performs well when applied directly to AFB smear positive sputum specimen (Ling et al.,
2008; Huyen et al., 2010)
2.8.4.2 GeneXpert.
The genexpert MTB / RMP assay is fully automated molecular diagnostic test for diagnosis of
tuberculosis and RMP susceptibility. It can simultaneously detect M. tuberculosis complex DNA
and mutations associated with RMP resistance which is considered reliable proxy for MDR-TB.
The advantage lies in the fact that direct sputum specimen can be dealt in less than two hours
with minimum staff manipulation and biosafety risk (Nelb et al., 2010). Genexpert is more
sensitive than sputum smear microscopy in detecting TB and has similar accuracy as culture
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(Boeheme et al., 2010 & 2011). The ability of system to detect RMP-resistant TB in less than
two hours significantly improves the chances of timely treatment options. Conventional culture
and DST are still required to complete the drug resistance profile and to monitor treatment
response. In December 2010, WHO endorsed the Genexpert for the rapid and accurate detection
of TB particularly suspected of having MDR-TB (WHO, 2011).
2.9 Building treatment regimen for MDR-TB
The development of treatment strategies and regimen for drug resistant tuberculosis vary
depending on access to drug susceptibility testing, the rates of drug resistant tuberculosis, HIV
prevalence, technical capacity and financial resources. It assumes that DST of first and second
line antituberculosis drugs comprising of isoniazid, rifampicin, the fluoroquinolones and the
injectable agents is quite reliable. It also stresses that DST of other agents is less reliable and that
individualized treatments should based on DST of these agents should be avoided (WHO,
2008). The first step involves the use of any first line oral agent like PZA and EMB. In the
second step in addition to the drugs mentioned in first step an injectable second line drug like
AK or KAN is recommended to be added. In the third step in addition to first two groups of
drugs, Fluoroquinolones are recommended to be added. In the fourth step oral bacteriostatic
agent like PAS is recommended to be added. In the step five, drugs are only recommended if the
options are not available from the first four groups. These drugs are to be added with caution
only with the recommendation of MDR TB expert. At least two drugs are recommended to be
added from this group if needed. The drugs included in this group include LZD, MER,
Amoxicillin / clavulanate, Imipenem / clavulanate, high dose INH & Clarithromycin.
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The WHO guidelines for programmatic management of drug resistant tuberculosis outlines
following stepwise treatment regimen for drug resistant tuberculosis (WHO, 2008)
Table II. Step wise building treatment regimen for MDR TB (Adopted from programmatic
management of drug resistant tuberculosis (WHO, 2013)
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CHAPTER-3
MATERIAL AND METHODS
3.1 Setting
The study was carried out at the Department of Microbiology, Armed Forces Institute of
Pathology Rawalpindi.
3.2 Study duration
The study was completed (including research work, data compilation, analysis and thesis
writing) in two years from the time of approval of synopsis.
3.3. Sample size
The sample size was 100.
3.4 Study design
Quantitative cross sectional study.
3.5 Sampling technique
Non-probability convenience
3.6 Inclusion criteria
All the MDR-TB isolates recovered from sputum, bronchoalveolar lavage (BAL), tissue, body
fluids and Cerebrospinal fluid (CSF) submitted at AFIP Rawalpindi during the study period. No
discrimination was made on age and gender.
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3.7 Exclusion criteria
Non MDR-TB clinical isolates were excluded from the study, similarly repeatedly cultured
clinical isolates of the same patient’s specimen.
3.8 Sequence of study
The study was done in two parts. The first part comprised of all the procedures leading to the
determination of MDR-TB, while the second part consisted of evaluation of newer compounds
(LZD & MER) and two of the classical second line antituberculosis drugs (AK & LEVO). Both
parts will be explained in the subsequent sections.
3.9 Determination of MDR-TB
For the determination of MDR-TB, following procedural details was adopted including specimen
collection.
3.9.1. Patient’s Profile
All the relevant information such as patient’s name, age, gender, Lab ID no, past history of TB,
family history of TB, past history of antituberculosis therapy taken and address were recorded in
the Performa.
3.9.2 Pulmonary Specimen collection
In case of sputum 2-10 ml of early morning expectorated sputum specimen was collected in a
leak proof, wide mouth and sterile container. BAL and endobronchial washings were treated as
sputum. For laryngeal swabs the swab was transferred in a sterile tube containing 2-3 ml of
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sterile water and then after thorough mixing this swab was removed, while the solution in the
tube was further processed.
3.9.3 Extra Pulmonary Specimen collection:
3.9.3.1 Pus Pus was collected as such in a sterile disposable syringe or in a sterile wide mouth
container. Specimens having volume of up to 10 ml were concentrated by centrifugation
(3000 × g) for 15-20 minutes.
3.9.3.2 Tissue About 1 Gm of the representative portion of the suspected infective tissue was
taken, which was then homogenized in a tissue grinder with a small quantity of sterile water (2-4
ml).
3.9.3.3 Other body fluids Other body fluids such as pleural fluid, peritoneal fluid, CSF and
synovial fluid were collected aseptically in sterile containers. Specimens up to 10 ml volume
were concentrated by centrifugation (3000 × g) for 15-20 minutes. After centrifugation, the
sediment of the specimen was suspended in 5 ml of sterile saline and then it was further
processed.
To reduce the chances of contamination from the transient and resident flora,
decontamination procedure of the specimens was performed by Digestion and Decontamination
technique (Petroff’s method) (Fig I). However this process was not applied on sterile body fluids.
Decontamination was followed by Ziehl-Neelsen (ZN) staining of all the specimens.
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3.9.4 Specimen processing
3.9.4.1 Digestion and decontamination of the specimen was done using following procedure.
Sample was taken in a 50 ml conical tube.
In case of tissue specimen, it was grinded thoroughly and then added along with its
original solution in conical tube.
Equal volume of decontamination fluid to that of specimen volume was added in
conical tube.
The contents in conical tube were mixed with the help of vortex for 10-15 sec and
then it was kept at room temperature for 15 minutes.
If solution in conical tube was 10 ml or less, phosphate (PO4) buffer was added to it
up to 25 ml mark in conical tube.
Whereas if the volume of solution in conical tube was > 10 ml, phosphate buffer was
added up to 45 ml mark in conical tube.
Contents of the conical tube were again mixed with the help of vortex.
All the tubes were centrifuged (3000 × g) for 20 minutes.
After centrifugation, the supernatant was discarded and deposit was used for ZN
staining and culture.
3.9.4.2 Ziehl-Neelsen (ZN) staining
As per CDC guidelines, 2-3 drops of the processed specimen were placed on a glass slide with
the help of a pipette to prepare a smear of about 1cm x 1½ cm before its inoculation into the
medium. The smear was then placed into an oven for about 5-7 minutes at a temperature of 56
ºC for drying. After drying, this smear was stained with ZN stain.
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The stained slides were then observed under microscope at (100X) oil immersion lens
(objective) with negative and positive control slides and the number of AFB present were
reported by Mycobacterial index as under:
No. of AFB seen Report
> 9/field 4 +
1-9/field 3+
1-9/10 fields 2+
1-9/100 fields 1+
1-2/300 fields doubtful
3.9.5 Culture & DST to first line anti-tuberculosis drugs
All the digested and decontaminated processed specimens along with MGIT growth supplement
Oleic acid, dextrose, albumen & catalase (OADC) and polymyxin B, amphotericin B, nalidixic
acid, trimethoprim, and azlocillin (PANTA) were inoculated in MGIT 960 TB system after
scanning the bar code and were incubated at a temperature of 37ºC ( Fig II & III). The tubes
were incubated till the instrument showed a tube positive or negative as per MGIT 960 system
manufacturer’s instructions. If there was no growth up to six weeks (42 days) of incubation, the
instrument showed such tube as negative. Once indicated by the system, the positive tube was
removed from the MGIT 960 system after bar code scanning and it was subjected to ZN staining
for the confirmation of AFB presence.
Now the confirmed M. tuberculosis isolates were subjected to DST by MGIT 960 system, as per
manual’s instructions (Siddiqi & Rusch-Gerdes, 2006) against INH, RMP, EMB, and SM. For
this purpose lyophilized drugs (MGIT 960 SIRE kit, Becton Dickinson, MD) were dissolved in
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diluents according to manufacturer’s instructions. From the dissolved drug solutions, 100μl was
pippeted into 7 ml MGIT 960 tube. The final drug concentrations used were 1.0 & 4μg/ml for
streptomycin (SM), 0.1 & 0.4 μg/ml for (INH), 5.0 & 7.5 μg/ml for (EMB) and 1.0 μg/ml for
(RMP).( Woods et al., 2007). Once the system indicated positive sign, the results for the DST
were interpreted as resistant or sensitive to respective drugs. Those 100 clinical isolates (MDR-
TB) showing resistance to INH and RMP were separated and were noticed for the presence or
absence of history of previous anti-tuberculosis treatment.
3.10 Culture & DST to second line anti-tuberculosis drugs
All MDR-TB isolates were subjected to susceptibility testing against two classical second line
drugs AK and LEVO. These drugs were obtained from chemically pure form from (Sigma,
Taufbirchen, Germany). Amikacin disulfate salt 710 μg/mg cat.N. A1774 with storage at 2-8 ͦ C
manufactured by Sigma and Levofloxacin > 98% HPLC cat.N. 28266 with storage at 2-8 ͦ C
manufactured by Sigma were used. These drugs were dissolved in deionized water. The stock
solutions of AK (84μg/l and LEVO (84μg/ml) were prepared in sterile water as per instructions
provided in leaflets of respective drugs as followed.
3.10.1 Amikacin (710 μg/mg)
The stock solution of AK was prepared as follows
- Dissolved 0.14 g of Amikacin powder in 10 ml sterile water = 10000 μg/ml (Solution A)
- 1 ml Solution A + 9 ml sterile H2O = 1000 μg/ml (Solution B)
- 4 ml Solution B + 1 ml sterile H2O = 800 μg/ml (Solution C)
- 1.05 ml Solution C + 8.95 ml sterile H2O = 84 μg/ml (STOCK SOLUTION)
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3.10.2 Levofloxacin
The stock solution of LEVO was prepared as follows
- Dissolved 0.10 g of Levofloxacin powder in 2.5 ml sterile NaOH 0.1 N Shake gently
- Add sterile water up to a final volume of 10 ml = 10000 μg/ml (Solution A)
- 1 ml Solution A + 9 ml sterile H2O = 1000 μg/ml (Solution B)
- 4 ml Solution B + 1 ml sterile H2O = 800 μg/ml (Solution C)
- 1.05 ml Solution C + 8.95 sterile H2O = 84 μg/ml (STOCK SOLUTION)
These stock solutions were filtered through 0.22 μm pore size Millex-GS filter units (Millipore,
Bedford, MA), aliquoted and stored at -70 oC. The working solutions of AK & LEVO were
diluted from the stock solution aliquoted and frozen for future use. The critical concentrations of
AK & LEVO used for BACTEC MGIT 960 system were 1.0μg/ml & 2.0μg/ml, (Rodrigues et
al., 2008; Sanders et al., 2004). Before subjecting the MDR-TB isolates to the test drugs, full
susceptible and quality control strain, American type culture collection (ATCC 27294) was
subjected to the critical concentration of drug used.
The drugs panel was consisted of three MGIT tubes, one for growth control and two for second
line drugs (Fig IV). Each 7ml MGIT tube was checked for any contamination or turbidity and
labelled properly. After mixing the growth supplement (OADC), 0.1 ml of each antibiotic stock
solution was added in respective MGIT tubes. 0.5ml of culture proven MDR-TB sample was
added to two MGIT tubes while 0.5ml of 1:100 diluted sample was added to control tube. After
bar code scanning all the inoculated tubes were entered in the instrument and incubated at a
temperature of 37oC. An un-inoculated MGIT tube was used as a negative control.
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3.10.3 Quality Control
A strain of M. tuberculosis, H37Rv (ATCC 27294), was used as a quality control (QC) strain and
was tested with each batch of DST at the critical concentration of each drug. This QC strain is
pan susceptible to the four drugs tested in the present study. If the QC strain did not yield the
expected results, the test with that batch had to be repeated.
3.11 Culture & DST to & Newer drugs (Linezolid & Meropenem):
All MDR-TB isolates were separately subjected to susceptibility testing against two newer
investigational drugs LZD and MER. Linezolid was provided by Continental pharmaceuticals
Karachi while Meropenem was provided by Musa Jee & Adam Karachi. Linezolid as pure
substance ca.100% Cat.N. 165800-03-3 with storage recommendation at room temperature and
manufactured by Pfizer was used. These drugs were dissolved in deionized water. The stock
solutions of LZD and MER were prepared in sterile water as per instructions provided in leaflets
of respective drugs as followed.
3.11.1 Linezolid
The stock solution of LZD was prepared as follows.
- Dissolve 0.01 g of Linezolid powder in 10 ml sterile water = 1000 μg/ml (Solution A)
- 4 ml Solution A + 1 ml sterile H2O = 800 μg/ml (Solution B)
- 1.05 Solution B + 8.95 ml sterile H2O = 84 μg/ml (STOCK SOLUTION)
3.11.2 Meropenem
- Dissolved 0.14 g of meropenem powder in 10 ml sterile water = 10000 μg/ml
(Solution A)
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- 1 ml Solution A + 9 ml sterile H2O = 1000 μg/ml (Solution B)
- 4 ml Solution B + 1 ml sterile H2O = 800 μg/ml (Solution C)
- 1.05 ml Solution C + 8.95 ml sterile H2O = 84 μg/ml (STOCK SOLUTION)
These stock solutions were filtered through 0.22 μm pore size Millex-GS filter units (Millipore,
Bedford, MA), aliquoted and stored at -70 oC. Three concentrations were used for both newer
drugs for BACTEC MGIT 960 system. For LZD (0.5, 1.0 & 2.0 μg/ml) and for MER (4.0, 8.0 &
16μg/ml) (Rusch-Gerdes et al., 2006). Before subjecting the MDR-TB isolates to the test drugs
full susceptible and quality control strain (ATCC 27294) was subjected to the three
concentrations of drugs used.
The drugs panel consisted of three MGIT tubes, one for growth control and two for each of the
three concentrations of investigational drugs. Each 7ml MGIT tube was checked for any
contamination or turbidity and labelled properly. After mixing the growth supplement (OADC),
0.1 ml of each antibiotic stock solution was added in respective MGIT tubes. 0.5ml of culture
proven MDR-TB sample was added to four MGIT tubes while 0.5ml of 1:100 diluted sample
was added to control tube. After bar code scanning all the inoculated tubes were entered in the
instrument and incubated at a temperature of 37oC. An un-inoculated MGIT tube was used as a
negative control.
3.12 Interpretation of Results
The MGIT 960 system supports the testing of various combinations of SIRE and PZA panels
configured by the manufacturer, but second-line drug panels are not available. Testing of second-
line drugs were registered in the MGIT 960 system as one of the SIRE panels, and we manually
entered the drug identification on the printout of the results. The MGIT 960 system flags the
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completion of a DST when the growth unit (GU) of the growth control reaches 400 and reports S
for susceptible or R for resistant, as well as a GU value for each drug-containing MGIT tube on
the printout. An isolate was interpreted to be susceptible when the GU of a drug-containing
MGIT tube was equal to or less than 100 or as resistant when the GU was greater than 100 (Fig
V & VI). If an isolate was interpreted to be resistant, a smear was made and stained to prove the
presence of AFB with morphology compatible with that of M. tuberculosis and the absence of
contaminants.
Following concentrations were used for the interpretations of MDR-TB isolates being
sensitive or resistant based on the multi centre validation studies to find out cut off
concentrations of second line and newer drugs for testing with MGIT 960 method (Rodrigues et
al., 2008; Sanders et al., 2004)
5. Amikacin (AK) 1.0 μg/ml
6. Levofloxacin (LEVO) 2.0 μg/ml
7. Linezolid ( LZD) 1.0 μg/ml
8. Meropenem(MER) 4.0 μg/ml
In case of LZD and MER additional concentrations of 0.5 & 2.0 μg/ml (LZD) and 8.0 &
16.0 μg/ml (MER) were also tested.
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Fig I. Decontamination of specimen before inoculation
Fig II. MGIT front view
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Fig III. Inner chamber with loaded DST tubes
Fig IV . Drug panel for second line drugs
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Fig V. MDR susceptible to second line drugs
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Fig VI. Resistant isolate to both Linezolid & Meropenem
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CHAPTER-6
RESULTS
Out of hundred MDR-TB isolates included in the study, 64 were from men and 36 from women (Fig VII).
The mean age was 34.9 ± 13.99 years ranging from 15 to 71 years. The maximum number of MDR TB
isolates (32%) were recovered from patients belonging to age group 15-25 , while only (11%) of the
isolates belonged to age group ˃ 56 years of age. (Fig VIII). A total of 100 MDR-TB isolates were tested
using AK, LEVO, LZD & MER. Out of these, 3 (3%) turned out to be XDR-TB based upon simultaneous
resistance to injectable second line antituberculosis drugs AK and one of the fluoroquinolones. (Table II).
These three XDR-TB isolates were recovered from two males and one female all with less than forty
years of age. Using 2.0 μg/ml as critical concentration of LVX, 76/100 (76%) of MDR-TB isolates were
found to be susceptible (Table-III). Based upon resistance to one of two second line drugs used (AK &
LEVO) 24% of MDR TB isolates were Pre XDR. Pre XDR isolates were recovered from sixteen
males and eight females with mean age of 34.08 ± 12.9 ranging from 15 to 63 years.
The association between susceptibilities of MDR TB isolates to classical second line drugs was
significant p- value of 0.002.Three concentrations of LZD 0.5, 1.0 & 2.0 μg/ml were tested.
Based on break point concentration of (0.5 μg/ml) used, 80/100 (80%) of the MDR-TB isolates
were sensitive while for breakpoint concentration of 1.0 μg/ml & 2.0 μg/ml, 96/100, (96%) of
MDR-TB isolates were susceptible (Table IV). As the number of MDR TB isolates susceptible
to LZD at breakpoint concentrations of 1.0 μg/ml & 2.0 μg/ml were equal, so the association of
susceptibilities of MDR-TB isolates to two concentrations i.e. 0.5 and 1.0 μg/ml were
statistically highly significant p-value .000 ( Table V).
For MER using breakpoint concentrations of 4.0 μg/ml no MDR-TB isolate was
susceptible, while at 8.0 μg/ml 3/100, (3%) and at 16.0 μg/ml 11/100, (11%) of MDR-TB
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isolates were susceptible. (Table VI). The association of susceptibility of MDR TB isolates to
different concentrations of meropenem was significant p.value 0 .002 (Table VII).
Fig VII. Gender distribution of the studied population
Fig VIII. Age distribution of the studied population
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Table III. Percentage of XDR-TB isolates
No of MDR-TB isolates AK (1.0 μg/ml) &
LEVO (2.0μg/ml) resistant
100 3 (3%)
Table IV. Susceptibility of MDR-TB isolates to LEVO
No of MDR-TB isolates Susceptible to LEVO
≤ 2.0μg/ml)
Percentage Susceptible
100 76 76%
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Fig IX. XDR and Pre XDR isolates in studied population (n=100)
Table V. Association between susceptibilities of MDR-TB isolates to
LEVO and AK
LEVO
Total
Sensitive resistant P value
AK Sensitive 76 21 97
Resistant 0 3 3 0.002
Total 76 24 100
(P value calculated by Pearson Chi square test)
0%
5%
10%
15%
20%
25%
3%
24%
XDR TB Pre XDR TB
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Table- VI. Susceptibility of MDR-TB isolates to different concentrations of LZD
Breakpoint concentration No of susceptible isolates Susceptible percentage
0.5 μg/ml 80 80%
1.0 μg/ml 96 96%
2.0 μg/ml 96 96%
Table-VII. Association of susceptibility of MDR-TB isolates to two different
Concentrations of LZD
LZD
(1.0 μg/ml)
Total
Sensitive Resistant P- value
LZD
(0.5 μg/ml)
Sensitive 80 0 80
Resistant 16 4 20 0 .000
Total
96
4
100
(P value calculated by Pearson Chi square test)
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Table- VIII. Susceptibility of MDR-TB isolates to different concentrations of MER
Breakpoint concentration No of susceptible isolates Susceptible percentage
4.0 μg/ml 0/100 0%
8.0 μg/ml 3/100 3%
16.0 μg/ml 11/100 11%
Table IX. Association of susceptibility of MDR-TB isolates to different concentrations of
MER
Concentration No of susceptible isolates p-value
4.0 μg/ml 0/100
.002 8.0 μg/ml 3/100
16.0 μg/ml 11/100
(P value calculated by Pearson Chi square test)
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CHAPTER-5
DISCUSSION
With the increase in MDR-TB strains around the globe, there has been an urgent need to carry
out drug susceptibility to first and second line antituberculosis drugs. It is imperative that
treatment of patients suffering from drug resistant TB should be carried out based on quick,
reliable and quantitative measure of susceptibility testing. This endeavor is a cornerstone for
prevention of resistance in treatment of TB and a way forward for optimal exploitation of the
available antituberculosis drugs (Mukherjee et al., 2004).
The reliable drug susceptibility testing method provides us with detailed knowledge on
quantitative drug resistance pattern which ultimately paves the way for empirical treatment of
drug resistant tuberculosis. During the last decade or so, MGIT 960 system has been extensively
studied and validated for susceptibility testing of first line antituberculosis drugs
(Bemer et al., 2002). The multicentre laboratory validation of the BACTEC MGIT 960 technique
for testing susceptibilities of M. tuberculosis to classical second line drugs and newer
antimicrobials (Rusch-Gerdes et al., 2006) has provided us with a guideline for resource poor
countries like Pakistan to endeavor testing such compounds against our local isolates.
According to WHO global report 2013, tuberculosis culture facility in Pakistan is
possible in only seven laboratories accounting to 0.2 laboratory per 5 million population while in
whole country only four laboratories can perform drug susceptibilities accounting to only 0.1
laboratory per 5 million population. To add fuel to the fire, Pakistan also could not achieve the
target of having at least one centre for carrying out smear microscopy under the WHO global
plan to stop TB 2011-2015 (WHO, 2013 ). In the backdrop of such sorry state of affairs our
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laboratory was one of the very few in Pakistan with capacity to carry out DST to first and second
line antituberculosis drugs (Ghafoor et al., 2014). With the aim of finding the susceptibility
pattern to classical second line and newer investigational drugs, it was challenge to embark upon
journey on the guidelines provided by validation studies.
According to WHO global report on tuberculosis 2013, an estimated 3.5% of new TB
cases are of MDR-TB in Pakistan whereas around 32% of retreatment TB cases fall in this
category. Similarly XDR-TB has been reported by 92 countries including Pakistan. Due to this
high burden of drug resistant TB cases, WHO has intensified its efforts to carry out drug resistant
surveys in Pakistan (WHO, 2013). Continuous surveillance of MDR-TB based on routine drug
susceptibility testing of the infected patients followed by systemic collection and compilation of
data remains the most effective approach to monitor trends in drug resistance over time.
It was found in this study that 3% of MDR-TB isolates turned out to be XDR-TB being
resistant to injectable second line drug AK and Fluoroquinolone LEVO. Since the paucity of
diagnostic facilities has already been documented by WHO, so the XDR cases found in this
study could just be the tip of an iceberg in our country. Although total numbers of LEVO
resistant MDR-TB cases were much higher (24%), hence the potential of further development of
XDR-TB in our isolates is much more. Cases with XDR-TB may become virtually untreatable
depending on the level of resistance to second line drugs and importantly the efficiency of health
system in each given setting. Incorrect treatment of tuberculosis is the primary risk factor for the
development of resistance among TB cases and usually is associated with intermittent use of the
drugs, errors in medical prescription, poor patient adherence and low quality of TB drugs
(Abubakar et al., 2013).
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The DST for fluoroquinolones LEVO and injectable drug AK was carried out by
automated MGIT 960 broth based technique which has been recommended as gold standard for
second line drugs (WHO, 2008). A cut off concentration of 1 μg/ml for AK and 2.0 μg/ml for
LEVO was used in this study following the recommendations of multicentre validation studies
carried out in Germany, Switzerland, Spain, UK, and Maryland USA (Rusch-Gerdes et al.,
2006). A cut off concentration of 1.5 μg/ml both for AK and LEVO has also been validated at
different research centers of USA (Grace et al., 2009). As 3% of our MDR-TB isolates were
XDR-TB with MIC’s of AK ˃ 1.0 μg/ml and LEVO ˃2 μg/ml, it is imperative that that we keep
a close eye on the MIC’s of our MDR-TB isolates for these second line drugs with ever
increasing threat of XDR-TB looming around us.
In one of the previous study done in Pakistan it was found that 2% of the MDR-TB
isolated from central part of Pakistan (Lahore) were XDR-TB (Iqbal et al., 2012). In Turkey
second line drug resistance rates of MDR-TB isolates to AK was 1.2% and 2.5 % with LEVO
(Bektöre et al., 2013). Our isolates definitely have much higher rates of resistance to these two
drugs. In India the percentage of XDR-TB cases among MDR-TB was reported to be 3.7%
(Porwal et al., 2013). The proportion of XDR-TB among MDR-TB cases was highest in
Azerbaijan (Baku city, 12.8%), Belarus (11.9%), Latvia (16%), Lithuania (24.8%) and Tajikistan
(Dushanbe city and Rudaki district (21%) (WHO, 2013). In one of the retrospective cohort study
carried out recently, 72% of XDR-TB patients been previously diagnosed as MDR-TB cases,
highlighting the role of ineffective TB programs in generating XDR-TB (Dheda et al., 2010).
If we consider LEVO resistance rate in isolation, we found that 24% of MDR-TB isolates
were resistant. Hence the frequency of Pre XDR among MDR-TB isolates in this study was 24%.
Pre XDR rates reported from other countries include 16.7% in Nigeria, 12.1% in Poland, 31% in
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China & USA and 28% in Taiwan. (Daniel et al., 2013; Kozińska et al., 2011; Qi et al., 2012;
Zhao et al., 2009; Banerjee et al., 2008 ; Wang et al., 2007). The high rate of this resistance
observed in my study is not surprising in a setting where majority of the antibiotics are freely
available over the counter (Butt et al., 2005). In addition the antimicrobial resistance among
other bacteria is very high in our community as well with 98 % of N. gonorrhea and 30% of
Salmonalla enterica serovar Typhi being resistant to fluoroquinolones (Jabeen et al., 2011;
Hassan et al., 2008).
Previous experiences with the use of FQs have shown that these compounds are
susceptible for indiscriminate use for bacterial infections (Adriaenssens et al., 2011). The results
of meta analysis carried out recently shows that patients who have been exposed to FQs prior to
diagnosis of tuberculosis are higher risk from FQs resistant tuberculosis than patients with
tuberculosis who have no previous exposure to the drug. Despite this documented risk little
effort is being done to restrict the use of FQs with only 7 of 15 European national guidelines
issued for respiratory tract infections cautioning against misuse (Migliori et al., 2012).
In Pakistan, low rate of literacy and tendency of patients to frequently change their
physicians and treatment facilities makes it practically impossible to find out as to who has not
received treatment in the past. There is every likelihood that patients who are bracketed in the
category of no history of treatment may actually have been partially or inadequately treated and
acquired drug resistance. As MDR & XDR tuberculosis in Pakistan are on the rise, it is
imperative to augment the awareness of local medical community as regards this emerging
health problem. There are studies which have already highlighted the gap in knowledge and
practice of physicians treating patients with tuberculosis (Marsh et al., 1996; Khan et al., 2003).
The quantum of knowledge regarding drug sensitive tuberculosis has been found to be
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inadequate among the community practitioners who are the first to encounter the TB patients
seeking treatment for their symptoms. In one study focusing general practitioners, it was found
that two third of the prescriptions written for 60 Kg man with newly diagnosed smear positive
pulmonary tuberculosis were not in accordance with national guidelines and only 3% of the GPs
knew all the five components of DOTS (Marsh et al., 1996). In another study conducted in
Pakistan, it was revealed that many doctors of various grades and seniority were not familiar
with correct and proper description of MDR & XDR –TB. This study involved three tertiary care
teaching hospitals in which medical specialists of different seniorities were surveyed and
questioned regarding the basic knowledge of MDR & XDR-TB. The results revealed
astonishingly lack of core knowledge about MDR & XDR tuberculosis. Only around 40 %
correct responses were received for definition of MDR-TB while only 3% correct answers were
recorded for XDR-TB (Ali et al., 2010).
As 24 % of our MDR-TB isolates were resistant to LEVO and this surely is a worrying
sign as cross-resistance among different members of FQs is of concern because they have a
mode of action different from that of the classical first-line anti-TB drugs. Having been used
widely for other infectious diseases, they are even available without prescription in several
countries, increasing the burden of selective pressure and compromising their efficacy in the
treatment of TB. The main target of FQ’s in M. tuberculosis is the DNA gyrase, encoded
by gyrA and gyrB and mutations in two short regions known as “quinolone resistance-
determining regions” have been associated with FQ resistance in M. tuberculosis
(Sun et al., 2008). Only very few centers reliably carry out testing for susceptibility to later-
generation FQs, consequently it is rarely checked and is known for very few of the isolates.
Multiple In vitro and In vivo studies have recognized differences in the efficacy of the various
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FQs in the treatment of TB. Therapeutic trials carried out in mice have revealed that MOX was
found to be the most bactericidal, followed by LEVO and OFX. Clinically significant resistance
(minimum inhibitory concentration 12 mg/ml) to CIP or OFX is conferred by a single gyrase
mutation, whereas for high-level resistance at least 2 mutations in gyrA or mutations in gyrA and
gyrB are required (Ginsburg et al., 2003). Even though cross-resistance among all FQ classes has
been documented in vitro (Devasia et al., 2009), the bactericidal activity exerted by later-
generation FQs may overcome low-level resistance. In addition to inadequate treatment of TB,
the challenging sociopolitical situation in Pakistan is likely to exacerbate this public health
problem. The isolation of XDR-TB strains is not cause of concern in this area but also to be
recognized as regional public health issue which requires national and international support.
Whereas we found 3% resistant isolates of AK and 24% of LEVO, nevertheless exact
susceptibility percentages of these two antimicrobials has become extremely important due to
WHO guidelines for the programmatic management of drug resistant tuberculosis
(Falzon et al., 2011). According to this, recommendation two of guidelines states ‘‘In the
treatment of patients with MDR-TB a FQ should be used”, whereas recommendation three states
‘‘in the treatment of patients with MDR-TB a later generation FQ should be used’’. One reason
of testing LEVO instead of CIP in this study was the fact that later generation FQs if
administered in dose of 750 mg/day have significantly better cure rate as compared to
ciprofloxacin (Ziganshina and Squire, 2008).
The second part of my study focused on finding the susceptibilities of LZD and MER
against MDR-TB isolates. The protocol and multicentre laboratory validation of the BACTEC
MGIT 960 technique for testing susceptibilities against LZD was first developed by
(Rusch-Gerdes et al., 2006). In this study which was conducted in three phases, multiple
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concentrations.0.5, 1.0 & 2.0 μg/ml of LZD were tested at three sites. The authors concluded that
MIC of 1.0 μg/ml be used as critical concentration of LZD for testing isolates by MGIT 960
technique. The results of this study serve as benchmark for carrying out the susceptibilities of
LZD by automated broth based MGIT 960 system. Multiple concentrations of the two
compounds were used in this study to find out the number and percentage of isolates susceptible
or resistant to the antimicrobial. The idea behind using multiple concentrations instead of one
breakpoint concentration was to firstly assess and compare our isolates with those reported
around the globe and secondly to make it as benchmark for further studies to be conducted in
Pakistan.
My study revealed that at breakpoint concentration of 0.5 μg/ml, 80% of MDR TB
isolates were susceptible, while at concentrations of 1.0 & 2 μg/ml, 96% of isolates were
susceptible. Hence only 4% of MDR-TB isolates from my study period were resistant at
breakpoint concentration of ˃1 μg/ml. Similar breakpoint concentration of 1.0 μg/ml has also
been followed and applied for checking the susceptibilities of 28 MDR-TB isolates recovered
from clinical samples of patients in Netherlands (Van-Ingen et al., 2010). The results of their
study carried out at National tuberculosis reference laboratory concluded that DST performed on
MGIT 960 method was advantageous over previous standard 7H10 agar dilution method on
account of shorter turnaround time.
It was first reported by (Richter et al., 2007) in Germany that 4 out of 210 (1.9%) strains
tested at German National Laboratory for Mycobacteria from 2003 to 2005 at MIC of ˃1 μg/ml
were resistant. In this evaluation study too, authors used BACTEC 460 and BACTEC 960
system for LZD keeping 1.0 μg/ml as breakpoint concentration.
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The in vitro susceptibility results of LZD have been found to have meaningful correlation
with the in vivo behavior of the drug as reported by (Zhang et al., 2014). According to their
results, those MDR-TB patients whose isolates had higher MIC range than the breakpoint
concentration had adverse clinical outcome compared to those patients who had MIC in the
susceptible range.
World Health Organization (WHO) has considered using LZD for the treatment of
multidrug resistant tuberculosis since 2006, but also recommending against its routine use (WHO
2006; WHO 2011). It was heartening to see the results of therapeutic trail conducted by
(Lee et al., 2012) as regards the efficacy and safety profile of this compound for salvage
treatment of MDR-TB. Another study performed by (Dalton et al., 2012) cautions the clinicians
managing the drug resistant patients who are unresponsive to treatment that up to 10.25%
resistance to LZD was detected in clinical trial conducted in eight countries. It was highlighted in
this multicentre study that true benefits of using LZD in the treatment of drug resistant
tuberculosis will ultimately depend on how judiciously the antimicrobial has been prescribed in
such patients. LZD has the potential to be misused by physicians and clinicians who prescribe it
for treatment of undiagnosed infections as has been reported by (Aubin et al., 2011). Some
countries have already started issuing guidelines on restricted use of LZD for the treatment of
pneumonia (Gupta et al., 2012). Such initiative if adopted by other countries will surely go a
long way in preserving this antimicrobial for the treatment of drug resistant tuberculosis.
In Pakistan, LZD is primarily used to treat infections caused by Gram positive organisms
including Methicillin resistant Staphylococcus aureus (MRSA). This antimicrobial has gained
importance due to its low MICs as compared to vancomycin against MRSA (Kaleem et al.,
2011). Hence there is likelihood that LZD due to its better efficacy, availability of oral
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preparation and low cost may be used for other bacterial infections. In my study only 4% of
MDR-TB isolates were non susceptible at break point concentration of ˃ 1.0 μg/ml. This level of
resistance can be explained on account of injudicious or uncontrolled usage as majority of the
antimicrobials are freely available over the counter. It is hoped that policy and decision makers
in Pakistan would realize the importance of this effect to formulate the guidelines to limit its
usage for the limited number of bacterial infections. Such steps would surely go a long way in
saving and protecting this antimicrobial for deadly infections like XDR-TB.
Beta lactam antibiotics have not been widely used against M. tuberculosis mainly due to
lack of efficacy. Recently there has been activity to reinvestigate this phenomenon and some
important development have taken place which indicates deletion or inhibition of major beta
lactamase enzyme of MTB, BLaC (Flores et al., 2005; Chambers et al., 2005). These studies
have created significant interest in the usage of beta lactam agents against M. tuberculosis. MER
which is a potent member of carbapenem group generated interest because it has low affinity
substrate for the enzyme with hydrolysis five times lower than ampicillin. Combination of this
compound with clavulanic acid has shown to have good in vitro activity against MDR-TB
including non replicative strains and is able to sterilize cultures in 14 days
(Hugonett et al., 2009).
MER was selected and was tested against QC strain at three breakpoint concentrations of
1.0, 2.0 and 4.0 µg/ml. The QC strain was susceptible at 4.0 µg/ml. hence three breakpoint
concentrations 4.0, 8.0 & 16 µg/ml of MER were prepared and tested against MTB. The results
revealed that none of the M. tuberculosis isolate was susceptible at 4.0 µg/ml while 3% of
isolates were susceptible at 8.0 µg/ml and 11% isolates at 16.0 µg/ml. As the reports of efficacy
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of MER clavulanic acid had started to pour in but lack of availability clavulanic acid base
powder hindered us to test this in combination.
There are plenty of reports in literature where combination of Meropenem & Clavulanic
acid has resulted in the cure of patients suffering from drug resistant tuberculosis
(Hugonnet et al., 2009; Dauby et al., 2011; De-Lorenzo et al., 2013). World Health organization
has already included MER in group V of their classification of drugs list with daily adult dosage
of 1000 mg twice or thrice daily to be administered by intravenous route( Lange et al., 2014).
It is high time that medical professionals dealing with cases of tuberculosis in Pakistan
are cognizant of the status of MDR, Pre XDR and XDR in our own population. It is also
extremely important that medical professionals should stress and strive to know the
susceptibilities of M. tuberculosis to first and second line drugs before initiating the definitive
therapy. Knowing the susceptibilities of M. tuberculosis against newer compounds like LZD and
MER would also provide them with a viable option, should they encounter cases of XDR. Last
but not the least is the issue of judicious use of antimicrobials specially Fluoroquinolones in
other infections to ward off the escalating resistance in M. tuberculosis.
Our study has definitely provided with platform for other researchers in Pakistan to test and try
newer antimicrobials for TB. As our study has revealed excellent results as regards the in vitro
susceptibility of M. tuberculosis against LZD so both the diagnosticians and clinicians can think
of trying this compound in their respective areas with sufficient level of confidence. On the other
hand MER has great potential and future specially when combined with Clavulanic acid for
managing cases of XDR TB.
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CHAPTER-6
CONCLUSION AND FUTURE PROSPECTS
In this study we evaluated one hundred MDR- TB isolates against two classical second line anti
tuberculosis drugs i.e. AK and LEVO in recommended cut off concentrations. The frequency of
XDR TB in our set up was 3% where as 24% of our isolates were in Pre XDR category. By
evaluating three concentrations of newer compounds, LZD & MER against MDR TB we found
that LZD revealed excellent in vitro susceptibility as 96% of our MDR TB isolates were
susceptible at concentrations of 1.0 μg/ml or more. In case of MER it was found that by serially
increasing the break point concentrations the more number of isolates became susceptible. This
finding thus strongly potentiates the fact that MER has potential to become effective
antituberculosis drug if it is combined with Beta lactamase inhibitor agent like clavulanic acid to
utilize its full potential.
These results have given us sufficient information about in vitro effectiveness of LZD and
potential of MER against MDR- TB isolates from Pakistan. It is hoped that relevant quarters in
National tuberculosis control programme in Pakistan would benefit from this study in
formulating the future guidelines as regards the treatment of drug resistant tuberculosis in
Pakistan is concerned. With 3% XDR-TB and 24% Pre XDR-TB isolates found in this study, it
should ring alarm bells because injudicious use of Quinolones without performing the DST can
be very detrimental in future. It is hoped that this study would serve as stepping stone for more
research in this area specially combining the Meropenem with clavulanic acid and determining
the break point concentrations then to compare it with the results obtained from this study.
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CHAPTER-7
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Annexure 1
Classical 2nd line drugs in MDR TB
S No. Age (years)
Gender 2nd line Drugs
Amika 1.0
µg/ml
Levo 2.0µg/ml
1 29 F R R
2 45 M S S
3 30 F S S
4 60 M S S
5 20 M S S
6 35 M S S
7 26 F S S
8 18 F S S
9 25 M S S
10 25 F S R
11 25 M S R
12 15 M S R
13 25 F S S
14 20 F S S
15 36 M S S
16 36 M S S
17 36 M S S
18 25 F S S
19 35 M S S
20 22 F S S
21 45 F S S
22 35 M S R
23 37 M S S
24 30 M S S
25 35 M S S
26 50 M S S
27 62 M S S
28 35 M S R
29 35 M S R
30 55 M S S
31 62 M S S
32 46 M S S
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33 49 M S S
34 49 M S S
35 23 M S S
36 40 M R R
37 15 M S S
38 24 M S S
39 42 M S S
40 25 M S R
41 43 M S R
42 27 F S S
43 27 F S S
44 48 M S S
45 17 F S S
46 53 F S R
47 50 F S S
48 30 F S S
49 34 M S R
50 26 F S R
51 23 M S S
52 33 M S S
53 41 M S S
54 65 M S S
55 25 F S S
56 46 M S S
57 32 F S S
58 63 M S S
59 57 M S S
60 15 F S S
61 71 M S S
62 63 M S R
63 45 F S S
64 35 M S S
65 43 F S S
66 29 M S S
67 22 F S S
68 56 F S S
69 31 M S S
70 19 F S R
71 30 F S S
72 44 F S R
73 52 M S S
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74 40 M S S
75 30 M S S
76 22 M S S
77 32 M S R
78 45 M S S
79 19 M S S
80 26 F S R
81 31 M S S
82 29 M S R
83 41 M S S
84 63 M S S
85 47 M S R
86 63 M S R
87 24 M S S
88 28 F S S
89 21 F S S
90 24 F S R
91 18 F S S
92 40 F S S
93 23 M S S
94 25 M S S
95 28 M S S
96 34 M R R
97 26 F S S
98 17 M S R
99 16 F S S
100 16 F S S
Total sensitive
97 76
XDR cases 3% on the basis of resistance to both AK & LEV
Pre XDR cases 24% on the basis of LEVO resistance
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Annexure 2
Linezolid susceptibility in MDR TB
S No. Age (years)
Gender Linezolid break point concentrations
0.5 µg/ml
1.0 µg/ml
2.0 µg/ ml
1 29 F R R R
2 45 M S S S
3 30 F S S S
4 60 M S S S
5 20 M S S S
6 35 M S S S
7 26 F S S S
8 18 F S S S
9 25 M S S S
10 25 F R S S
11 25 M R S S
12 15 M S S S
13 25 F S S S
14 20 F S S S
15 36 M R S S
16 36 M S S S
17 36 M R S S
18 25 F S S S
19 35 M S S S
20 22 F S S S
21 45 F S S S
22 35 M S S S
23 37 M R S S
24 30 M S S S
25 35 M S S S
26 50 M R S S
27 62 M S S S
28 35 M S S S
29 35 M R S S
30 55 M S S S
31 62 M S S S
32 46 M S S S
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33 49 M S S S
34 49 M S S S
35 23 M S S S
36 40 M R R R
37 15 M S S S
38 24 M R S S
39 42 M S S S
40 25 M S S S
41 43 M S S S
42 27 F S S S
43 27 F S S S
44 48 M R S S
45 17 F S S S
46 53 F S S S
47 50 F S S S
48 30 F S S S
49 34 M R S S
50 26 F R R R
51 23 M S S S
52 33 M S S S
53 41 M S S S
54 65 M S S S
55 25 F S S S
56 46 M S S S
57 32 F R S S
58 63 M S S S
59 57 M S S S
60 15 F S S S
61 71 M S S S
62 63 M R S S
63 45 F S S S
64 35 M S S S
65 43 F S S S
66 29 M S S S
67 22 F S S S
68 56 F S S S
69 31 M S S S
70 19 F S S S
71 30 F S S S
72 44 F S S S
73 52 M S S S
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74 40 M S S S
75 30 M S S S
76 22 M S S S
77 32 M R R R
78 45 M S S S
79 19 M S S S
80 26 F S S S
81 31 M S S S
82 29 M R S S
83 41 M S S S
84 63 M S S S
85 47 M R S S
86 63 M S S S
87 24 M S S S
88 28 F S S S
89 21 F S S S
90 24 F S S S
91 18 F S S S
92 40 F S S S
93 23 M S S S
94 25 M R S S
95 28 M S S S
96 34 M R S S
97 26 F S S S
98 17 M S S S
99 16 F S S S
100 16 F S S S
TOTAL Sensitive isolates
80 96 96
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Annexure 3
Meropenem susceptibility in MDR TB
S No. Age (years)
Gender Mereopenem break point concentrations
4.0 µg/ml
8.0 µg/ml
16.0 µg/ ml
1 29 F R R R
2 45 M R R R
3 30 F R R R
4 60 M R R R
5 20 M R R R
6 35 M R R R
7 26 F R R R
8 18 F R S S
9 25 M R R R
10 25 F R R R
11 25 M R R R
12 15 M R R R
13 25 F R R S
14 20 F R R R
15 36 M R R R
16 36 M R R R
17 36 M R R R
18 25 F R R R
19 35 M R R R
20 22 F R R S
21 45 F R R R
22 35 M R R R
23 37 M R R R
24 30 M R R R
25 35 M R R R
26 50 M R R R
27 62 M R R R
28 35 M R R R
29 35 M R R R
30 55 M R R S
31 62 M R R R
32 46 M R R R
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33 49 M R R R
34 49 M R R R
35 23 M R R S
36 40 M R R R
37 15 M R R R
38 24 M R R R
39 42 M R R R
40 25 M R R R
41 43 M R R R
42 27 F R R R
43 27 F R R S
44 48 M R R R
45 17 F R S R
46 53 F R R R
47 50 F R R R
48 30 F R R S
49 34 M R R R
50 26 F R R R
51 23 M R R R
52 33 M R R R
53 41 M R R R
54 65 M R R R
55 25 F R R R
56 46 M R R R
57 32 F R R R
58 63 M R R R
59 57 M R R R
60 15 F R R R
61 71 M R R S
62 63 M R R R
63 45 F R R R
64 35 M R R R
65 43 F R R R
66 29 M R R R
67 22 F R R R
68 56 F R R R
69 31 M R R R
70 19 F R R R
71 30 F R R R
72 44 F R R R
73 52 M R R S
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74 40 M R R R
75 30 M R S R
76 22 M R R R
77 32 M R R R
78 45 M R R R
79 19 M R R R
80 26 F R R R
81 31 M R R S
82 29 M R R R
83 41 M R R R
84 63 M R R R
85 47 M R R R
86 63 M R R R
87 24 M R R S
88 28 F R R R
89 21 F R R R
90 24 F R R R
91 18 F R R R
92 40 F R R R
93 23 M R R R
94 25 M R R R
95 28 M R R R
96 34 M R R R
97 26 F R R R
98 17 M R R R
99 16 F R R R
100 16 F R R R
Total NIL 03 11
