Study of isoniazid-resistant tuberculosis: mycobacterial genotypic characterization and clinical features in human subjects

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Content STUDY OF ISONIAZID-RESISTANT TUBERCULOSIS: MYCOBACTERIAL
GENOTYPIC CHARACTERIZATION AND CLINICAL FEATURES IN HUMAN
SUBJECTS
by
Patricio Escalante
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CLINICAL AND BIOMEDICAL INVESTIGATIONS)
August 2007
Copyright 2007 Patricio Escalante
ii
Table of Contents
Dedication iii
Acknowledgements iv
List of Tables v
Abbreviations vi
Abstract viii
Introduction 1
Methods 2
Design 2
Study Population 3
Bacterial Strains 5
Drug Susceptibility Testing 5
Bacterial Genotyping 6
Data Analysis 7
Results 8
Study Population and Characteristics of Patients 8
Genetic Characterization of MTB Isolates 10
Characteristics of Patients by Genotypic Groups 10
Characteristics of Contact Patients by Genotypic Groups 17
Discussion 17
Conclusion 22
Bibliography 23
iii
Dedication
This work is dedicated to all the people who helped to develop my academic interest in
tuberculosis, especially my wife Maria I. Rudis, my family, the patients I served, the late
Dr. Robert Awe and Dr. Michael D. Iseman.
iv
Acknowledgements
I thank Roberta McKean-Cowdin, Ph.D. for her great input during the data analysis and
her excellent assistance with the preparation of this manuscript. I also thank S.
Ramaswamy, Ph.D., N. Williams-Bouyer, PhD., L. Teeter, Ph.D., and especially Edward
A. Graviss, MPH., Ph.D. from the Houston Tuberculosis Initiative for their excellent
assistance with the isolates’ genotying analysis, data entry and project support. I also
thank Magdalena Arias-Arenas, M.D., Ravinder Bajwa, M.D., Carlos K. Wesley, M.D.
and Brenda E. Jones, M.D. for their assistance with information retrieval, data entry and
research project support. I also thank Mr. Samuel Sum for his excellent laboratory
assistance and all the support of the people of the Los Angeles Tuberculosis Control
Program, especially Director, Dr. Annette Nitta.
v
List of Tables
Table 1: Characteristics of Study Subjects 9
Table 2: Genetic Characteristics of INH resistance and MICs 11
of INHr-TB isolates
Table 3: Characteristics of Patients by Bacterial Genotypic Group 13
Table 4: Multivariate Analysis Between INHr Genotypes and 15
Sputum Conversion at 1 Month
Table 5: Multivariate Analysis between INHr Genotypes and 16
Unilateral Lung Involvement on Chest X-ray
vi
Abbreviations
TB = tuberculosis
MTB = Mycobacterium tuberculosis
INH = isonaizid
INHr = isoniazid-resistant
INHr-TB = isoniazid-resistant Mycobacterium tuberculosis
PS = pan-sensitive
Ser = Serine
Thr = Threonine
DNA = Deoxyribonucleic acid
HIV = Human Immunodeficiency virus
AIDS = Acquired immunodeficiency syndrome
COPD = chronic obstructive pulmonary disease
TST = Tuberculin skin test
PPD = Purified protein derivative
AFB = Acid-fast bacilli
USC = University of Southern California
vii
LAC = Los Angeles County
IRB = Institutional Review Board
HIPAA = Health Insurance Portability and Accountability Act
IATA/IACO = International Air Transport Association / International Civil Aeronautics
Organization
MIC = Minimal inhibitory concentration
RFLP = Restriction Fragment Length Polymorphism
IS6110 = Insertion sequence 6110
PCR = Polymerase chain reaction
viii
Abstract
Most isoniazid-resistant (INHr) M. tuberculosis (MTB) isolates have katG mutations.
KatG is a virulence factor in animal models. We aim to determine if MTB-INHr
genotype is associated with distinct clinical features. A retrospective case-control study
design was used to compare clinical characteristics of patients infected with INHr-MTB
(katG or non-katG mutations) to patients with pan-sensitive (PS) MTB isolates.
Univariate and multivariate analyses were used. Twenty-nine INHr-TB cases and 50 PS
controls were evaluated. INHr-MTB patients infected with non-katG mutants were
associated with a higher rate of sputum conversion at 1 month of treatment: Adjusted OR
= 6.03 (95%CI: 1.02-35.4; P = 0.04). Patients infected with katG mutants were associated
with a higher rate of unilateral lung disease: Adjusted OR = 10.7 (95%CI: 1.1-107; P =
0.04). Some MTB mutations resulting in INH resistance may be associated with a faster
response to treatment, and others with a more contained pulmonary involvement.
1
Introduction
Tuberculosis (TB) remains one of the most common causes of death in the world
1
despite recent global progress in TB control.
2
Isoniazid (INH) is one the first-line drug
agents against Mycobacterium tuberculosis (MTB) the causative agent of tuberculosis
(TB). INH resistance is frequently encountered in patients previously treated with this
agent in many countries in the world.
2,3
INH resistance in approximately 50-80% of
MTB strains is due to mutations in the katG gene that encodes a catalase-peroxidase
enzyme.
4-7
These mutations result in the production of an altered enzyme that is less able
to convert INH to its biologically active form, resulting in diminished mycobacterial
mycolic acid biosynthesis. Most resistant isolates with katG mutations have an amino
acid replacement at Ser315Thr.
5-8

Mutations that confer bacterial drug resistance are usually associated with a cost to
bacterial fitness.
9
The high prevalence of the Ser315Thr katG mutation in isolates of
MTB patients suggests that this genetic alteration is a convenient trade off mutation that
allows MTB to maintain important enzyme activity (catalase-peroxidase) necessary for
neutralizing external oxidant stress while also conferring INH resistance.
10-13
Strains that
lack catalase-peroxidase activity may be at a disadvantage in environments with
oxidative stress such as found in macrophage phagosomes, which could result in a less
virulent and perhaps less pathogenic bacteria.
14
Animal studies provide supportive
2
evidence that some INH resistant MTB (INHr-TB) genotypes cause less severe disease
and lower mortality in experimental models.
15, 16
It is not known whether some genetic types of INHr-TB cause a less severe and/or less
extensive TB disease in humans. We hypothesize that patients infected with some INH
resistance-conferring mutations may have milder forms of clinical presentation, an
insidious course and/or more rapid response to therapy. We attempted to assess whether
some MTB bacterial genotypes, including INHr katG and mutations other than katG
(non-katG mutations), are associated with distinct disease characteristics and response to
treatment in human subjects. Therefore, the main objective of the study is to characterize
the clinical characteristics and response to treatment in patients with katG and non-katG
INHr-TB genotypes, and to compare the same variables with frequency matched controls
infected with pan-sensitive MTB isolates.
Methods
Design
A retrospective case-control study of patients infected with katG and non-katG INHr-TB
and PS MTB controls.
3
Study Population
TB patients in the study were identified using existing medical records from all patients
diagnosed with INHr-TB (cases) at The Los Angeles County USC (LAC-USC) Medical
Center between 1997 and 2001. Pansensitive (PS) TB infected patients (controls) were
selected from a Los Angeles (LA) TB Control Program database by frequency matching
to INHr cases on age (within the same decade of age) and gender. A public health official
blinded to the study question assisted with the patient selection. All patients were treated
with effective TB therapy, including first-line 4-drugs (INH, rifampin, ethambutol and
pyrazinamide) treatment during the intensive 2-month phase, followed by a continuation
phase with rifampin, ethambutol and pyrazinamide for at least 4 months. Patients were
excluded from the study if they were infected with other types of drug-resistant strains
(including multidrug-resistant TB) or had HIV, concomitant immunosuppressive therapy,
history of non-adherence to treatment, or incomplete or unavailable medical records.
Data collected through chart review included demographic data, risk factors for TB
infection, co-morbidities, and clinical characteristics including patients’ symptoms at
presentation, radiological characteristics, microbiological information and response to
treatment. We also gathered information from contact investigations on individuals who
were believed to have been exposed to TB through the index cases by the TB public
health authorities. Study protocols were approved by the LAC Public Health and USC
Institutional Review Boards. The IRB approved waiver of informed consent and HIPAA
compliance.
4
Demographic and clinical characteristics included in the analysis are:
1. Demographic information (e.g. age, gender, ethnic group origin, country of origin)
2. Presence or absence of symptoms at onset of disease, and type of symptoms (e.g.
cough, fever, anorexia, weight loss, hemoptysis, pleuritic chest pain)
3. Prior history of tuberculosis as determined by medical records
4. Co-morbidities including history of diabetes mellitus, chronic obstructive pulmonary
disease (COPD), chronic renal failure, chronic liver disease, history of
gastrointestinal surgery and bronchiectasis
5. History of substance abuse including recreational drug use and significant alcohol
intake (significant alcohol intake was defined as at least one alcoholic drink daily)
6. Pulmonary/extra-pulmonary disease
7. Tuberculin skin test (TST): Purified protein derivative (PPD) positive vs. PPD
negative status
8. Initial sputum acid-fast bacilli (AFB) smear positive/negative and degree of positivity
(i.e. 1+ to 3+)
9. Sputum AFB culture conversion at 1 month and 2 months of treatment
10. Presence or absence of cavitary disease on initial chest x-ray
11. Radiographic unilateral and bilateral pulmonary disease involvement
12. General treatment outcomes: treatment completion and discharge, treatment failure,
relapses, and mortality related to tuberculosis
13. Contact cases’ information: Size of PPD reaction (in mm) and radiological findings
5
The study population was divided into three groups.
1. Patients infected with INHr-TB caused by any katG mutations (Case set 1).
2. Patients infected with INHr-TB caused by non-katG genetic alterations, including
isolates with not identifiable INH-conferring mutations with our genotyping
procedures (Case set 2).
3. Patients infected with PS MTB isolates (Controls).
Bacterial Strains
Isolates were obtained from patients at LAC-USC Medical Center between 1997 and
2001. The sputum samples were originally collected from patients suspected to have TB.
All sputum isolates were routinely decontaminated and the smears prepared with standard
methods of Auramine-Rhodamine fluorochrome and Ziehl-Neelsen acid-fast staining for
identification. Culture confirmation was performed with Lowenstein-Jensen solid media.
All isolates were submitted in 2ml cryovial containing 1ml of Middlebrook 7H-9 liquid
media, prior to transportation from LA to Houston for genotypic analysis. MTB isolates
were transported according to IATA/ICAO regulations of infectious materials.
Drug Susceptibility Testing
MTB complex identification was determined in all isolates by standard high
performance liquid chromatography and/or by positive hybridization with the
6
Accuprobe™ test (Gen Probe, San Diego, CA). Drug susceptibility testing was
determined by standard BACTEC methodology at the laboratory of the Department of
Public Health Sciences in LA, CA. INHr-TB strains were defined by a MIC 0.1 mcg/ml
using the BACTEC™ method (Becton, Dickinson and Co, Franklin Lakes, NJ).
Bacterial Genotyping
The Institute for the Study of Bacterial Pathogenesis at Baylor College of Medicine,
Houston, TX (Dr. Edward Graviss and colleagues) performed the DNA sequencing
analysis and genotyping of the MTB isolates. Chromosomal DNA extraction and IS6110
Restriction Fragment Length Polymorphism (RFLP) typing were performed by
internationally standardized methods.
18
We analyzed the IS6110 profiles by using the
BioImage Whole Band Analyzer™, version 3.2 software (BioImage, Ann Arbor, MI).
Spoligotyping was performed using the Isogen™ kit (Isogen, Bioscience, B.V. Maarseen,
Netherlands) for all isolates according to the manufacturer instructions. In addition, all
isolates also were assigned to 1 of 3 genetic group designation based on nucleotide
polymorphisms in katG463 and gyrA95 codons as previously described.
19
We used DNA sequencing of the putative regions of katG, mabA, inhA, kasA, ahpC, and
ndh genes to determine the genetic alterations associated with INH resistance. The
oligonucleotides primers and PCR conditions used to amplify all these genes as well as
the DNA sequencing methodology have been described previously.
5
7
Data Analysis
We compared the proportion of patients with select demographic, clinical, bacteriologic
and radiographic characteristics across the 3 MTB subtypes (katG, non-katG, PS).
Frequency of disease phenotypes by MTB subtype were compared using Chi-Square test
for categorical variables (Fisher’s exact test for cells with small numbers) and two-sided
t-tests for continuous variables. A P value of 0.05 was considered statistically
significant.
A multivariate analysis was completed to determine if patients with INHr-TB (katG or
non-katG) were more or less likely to have select clinical characteristics or response to
treatment than patients infected with PS TB. Multivariate analyses included modeling of
the clinical characteristic of disease and response to treatment as the outcome variables.
Clinical characteristics and response to treatment variables that were associated with
bacterial genotypes in univariate analyses were examined in the multivariate analyses.
Potential covariates included in the model were age (continuous variable), gender, ethnic
group (Hispanic, non Hispanic), co-morbid conditions (e.g. diabetes mellitus, significant
alcohol intake), initial sputum AFB smear results (positive/negative), presence of cavitary
disease (yes/no), and laterality of lung involvement on chest x-ray (unilateral or bilateral).
Variables that did not substantially change the effect estimates were excluded from the
final models.
17
We completed polytomous logistic regression to examine associations
with level of smear AFB positivity (as indicator of sputum smear bacillary load) on
8
presentation. Data were analyzed using SAS™ software, version 8.2 (SAS Institute,
Cary, NC).
Results
Study Population and Characteristics of Patients
The study population included a total 79 patients, 29 cases infected with INHr-TB and 50
patients infected with PS-TB isolates. Of the original 90 patients eligible for the study,
one patient with INHr-TB and 10 patients infected with PS-TB isolates were excluded
due to incomplete medical records. No significant differences in frequencies of
demographic or clinical characteristics were found by INHr status (Table 1).
Approximately 70% of the patients included in the study were male. A majority of the
patients were Hispanic (57% of INHr-TB cases and 64% of PS-TB controls) and
approximately one-third of the patients in each group had a history of significant alcohol
intake. The mean age of the INHr-TB and PS-TB groups were 51 (SD = 16.6) and 47 (SD
= 15.2), respectively (P > 0.05). History of homelessness (18% vs. 14%) and intravenous
drug abuse (7% vs. 4%), were not significantly different for the INHr-TB and PS-TB
groups.
9
Table 1: Characteristics of Study Subjects
INHr-TB Cases
(N=29)
PS-TB Cases
(N=50)
P *
Demographics
Male / Female 20 / 9 34 / 16 1.00
Age** (range in years) 23 - 87 23 - 81 0.33
Race (%): 0.40
Hispanics 16 (55.2) 32 (64.0)
Asians 11 (37.9) 13 (26.0)
African Americans 2 (6.9) 2 (4.0)
Caucasians 0 (0.0) 3 (6.0)
Selected Medical History (%)
Cough 25 (86.2) 36 (73.5) 0.26
Fever 13 (50.0) 26 (54.2) 0.81
Weight loss 19 (73.1) 32 (66.7) 0.61
Prior history of tuberculosis 8 (29.6) 7 (15.9) 0.23
Intravenous drug abuse 2 ( 7.1) 2 ( 4.2) 0.62
Alcohol abuse 10 (37.0) 15 (31.3) 0.62
Diabetes mellitus 5 (18.5) 10 (20.8) 1.00
System Involvement 1.00
Pulmonary only 24 (82.8) 40 (80.0)
Pulmonary and extrapulmonary 2 ( 6.9) 5 (10.0)
Extrapulmonary only 3 (10.4) 5 (10.0)
Radiographic Characteristics
Cavitary disease 8 (32.0) 17 (37.8) 0.79
Laterality 0.62
Bilateral 12 (48.0) 20 (45.6)
Unilateral 13 (52.0) 24 (54.6)
Positive Sputum AFB Smear 18 (62.1) 36 (72.0) 0.45
Response to Treatment
AFB Culture Negative at 1mo 15 (60.0) 12 (36.6) 0.11
AFB Culture Negative at 2mo 22 (91.7) 28 (87.5) 0.69
(*) Comparison by Fisher Exact test
(**) Comparison by T-test
PS-TB = Pansensitive TB cases. AFB = Acid-fast bacilli
10
Genetic Characterization of MTB Isolates
Bacterial genotyping was completed for 28 of 29 cases. One INHr-TB isolate did not
have enough DNA for genotyping. Genotypic MTB strain analysis using standard
genotyping techniques (IS6110 fingerprinting, spoligotyping and group designation)
showed no clonality among the isolates. DNA sequencing of the 7 INHr-conferring genes
identified 13 (46.4%) isolates having katG mutations. The most frequent katG mutation
was the Ser315Thr (S315T) mutation in 8 (28.5%) cases, followed by 5 cases of katG
mutations other than katG315. Nine patients (32.2%) infected with INHr-TB isolates had
mutations in other genes besides katG, including genetic alterations in mabA, inhA, kasA,
and ahpC. None of the INHr-TB isolates showed alterations in the ndh gene. Eight
(28.6%) INHr-TB isolates had 2 or more genetic mutations. Six (21.4%) of the INHr-TB
isolates showed not detectable alterations in all the analyzed genes (Table 2).
Characteristics of Patients by Genotypic Groups
The frequency of patients’ demographics, clinical characteristics and response to
treatment stratified by the 3 genotypic groups (katG, non-katG and PS) are shown in
Table 3. There were no statistically significant differences for the proportions of these
patients’ characteristics across the 3 bacterial subtypes with the exception of history of
11
Table 2: Genetic Characteristics of INH resistance and MICs of INHr-TB isolates
Case #
KatG MabA InhA KasA AhpC Ndh MIC
(μg/ml)
1 315; AGC->ACC, Ser->Thr WT WT 6; ACC->ACT, Thr->Thr Nt.100 ups; G->A WT 1
2 315; AGC->ACC, Ser->Thr WT WT 6; ACC->ACT, Thr->Thr;
312; GGC->AGC, Gly->Ser
Nt.100 ups; G->A WT 1
3 315; AGC->ACC, Ser->Thr WT WT WT WT WT 1
4 315; AGC->ACC, Ser->Thr WT WT WT WT WT 1
5 315; AGC->ACC, Ser->Thr WT WT WT WT WT 1
6 315; AGC->ACC, Ser->Thr WT WT WT WT WT 1
7 315; AGC->ACC, Ser->Thr WT WT WT WT WT 1
8 315; AGC->ACC, Ser->Thr WT WT WT WT WT 1
9 498; CGC->CAC, Arg->His WT WT WT WT WT 0.2
10 155; TAC->TCC, Tyr->Ser WT WT WT WT WT 0.2
11 280; CCG->TCG, Pro->Ser 15 ups; C->T MabA241;
GGC->GGT, Gly->Gly
6; ACC->ACT, Thr->Thr;
312; GGC->AGC, Gly->Ser
Nt.100 ups; G->A WT 0.1
12 146; CGG->TGG, Arg->Trp WT WT WT WT WT 0.1
13 105; ATG->ATA, Met->Ile WT WT WT Nt.12 ups; C->T WT 0.2
14 WT 15 ups; C->T WT WT WT WT 0.2
15 WT WT WT WT WT WT 0.1
16 WT WT WT WT WT WT 0.2
17 WT 15 ups; C->T WT WT WT WT 0.2
18 WT 15 ups; C->T WT 6; ACC->ACT, Thr->Thr Nt.100 ups; G->A WT 0.2
19 WT WT WT WT Nt.10 ups; C->T WT 5
20 WT 15 ups; C->T WT 6; ACC->ACT, Thr->Thr;
312; GGC->AGC, Gly->Ser
Nt.100 ups; G->A WT 0.1
21 WT 15 ups; C->T WT 6; ACC->ACT, Thr->Thr;
312; GGC->AGC, Gly->Ser
Nt.100 ups; G->A WT 1
22 WT 15 ups; C->T WT 6; ACC->ACT, Thr->Thr Nt.100 ups; G->A WT 0.1
12
Table 2, Continued: Genetic Characteristics of INH resistance and MICs of INHr-TB isolates
23 WT 15 ups; C->T WT WT WT WT 0.2
24 WT WT WT WT WT WT 0.1
25 WT WT WT WT WT WT 0.1
26 WT WT WT WT WT WT 5
27 WT WT WT WT WT WT 0.1
28 WT 15 ups; C->T WT WT WT WT 0.2
WT = wild type genetic sequence; ups =upstream; MIC = Minimal inhibitory concentration: < 1 (Low), 1 (High); KatG315 mutation
cases = #1 to 8; KatG other than KatG315 cases = #10 to 13; not-KatG INHr-TB cases = #14 to 28
13
Table 3: Characteristics of Patients by Bacterial Genotypic Group
Characteristics (%) All KatG Non-KatG* Pansensitive-TB P **
Mutations Mutations Mutations
(N = 13) (N = 15) (N = 50)
Hispanic Race 8 (61.5) 8 (53.3) 32 (64.0) 0.75
Prior History of TB 2 (15.4) 5 (33.3) 7 (15.9) 0.42
Cough 11 (84.6) 13 (86.7) 36 (73.5) 0.57
Fever 5 (38.5) 7 (46.7) 26 (54.2) 0.74
Weight loss 8 (61.5) 11 (73.3) 32 (66.7) 0.48
Diabetes Mellitus 5 (38.5) 0 ( 0.0) 10 (20.8) 0.05
Alcohol intake 4 (30.8) 6 (40.0) 15 (31.3) 0.77
Pulmonary Involvement 11 (84.6) 12 (80.0) 40 (80.0) 0.98
Cavitary Disease 4 (30.8) 3 (20.0) 17 (34.0) 0.81
Unilateral Lung Involvement 9 (69.2) 3 (23.1) 20 (45.6) 0.02
Positive Sputum AFB Smear 8 (61.5) 9 (60.0) 36 (72.0) 0.60
Culture Negative at 1 mo 6 (46.2) 9 (60.0) 12 (36.6) 0.08
Culture Negative at 2 mo 8 (61.5) 13 (86.7) 28 (57.8) 0.29
(*) Includes INHr-TB cases without identified genetic-conferring mutation. One INHr-TB isolated excluded because of no available
DNA for genotyping
(**) Comparison by Fisher Exact test
14
diabetes mellitus and unilateral lung disease (P < 0.05). There was a marginally
significant difference for sputum conversion at 1 month of treatment across the 3
bacterial genotypes (P = 0.08). Furthermore, there was no statistically significant
difference in the proportion of TST positive (TST induration 10 mm) vs. TST negative
for patients infected with MTB isolates from the 3 bacterial genotypes (P > 0.05) (data
not shown).
Using multivariate analysis, we found INHr-TB patients infected with non-katG mutants
were more likely to have AFB culture conversion at 1 month of treatment than patients
infected with PS isolates, after adjusting for age, ethnicity, sputum AFB smear results,
presence of cavitary disease and unilateral lung involvement on chest x-ray: Adjusted OR
= 6.03 (95%CI: 1.02-35.4; P = 0.04) (Table 4). INHr-TB patients infected with katG
mutants were more likely to have unilateral disease on chest X-rays than patients with
PS TB after adjustment for age, history of diabetes mellitus, history of daily alcohol
intake and sputum AFB smear results: Adjusted OR = 10.7 (95%CI: 1.1-107; P = 0.04)
(Table 5). Multivariate analysis found no other statistically significant association
between clinical characteristics of TB patients and bacterial genotypes of INHr-TB cases.
We also found no association between the genotypic groups and the level of smear AFB
positivity (sputum smear bacillary load) on presentation based on polytomous logistic
regression analysis (data not shown).
15
Table 4: Multivariate Analysis Between INHr Genotypes and Sputum Conversion at 1 Month
Sputum Conversion at 1 mo.
Yes No Crude Adjusted*
N (%) N (%) OR (95% CI) P OR (95% CI) P
All KatG Mut. 6 (50.0) 6 (50.0) 1.8 (0.46-6.7) 0.41 1.33 (0.22-8.2) 0.75
Pansensitive-TB 12 (36.4) 21 (63.6)
Non-KatG Mut. 9 (75.0) 3 (25.0) 5.2 (1.19-23) 0.04 6.02 (1.02-35) 0.04
Pansensitive-TB 12 (36.4) 21 (63.6)
All INHr 15 (60.0) 10 (40.0) 2.6 (0.90-7.6) 0.08 2.31 (0.69-7.8) 0.17
Pansensitive-TB 12 (36.4) 21 (63.6)
(*) Adjusted by Age, Hispanic ethnicity, sputum AFB smear results, and presence of cavitary disease and unilateral lung involvement
on chest x-ray.
16
Table 5: Multivariate Analysis between INHr Genotypes and Unilateral Lung Involvement on Chest X-ray
Unilateral Lung Disease
Yes No Crude Adjusted*
N (%) N (%) OR (95% CI) P OR (95% CI) P
All KatG Mut. 9 (81.8) 2 (18.2) 5.4 (1.0-28) 0.04 10.7 (1.1-107) 0.04
Pansensitive-TB 20 (45.5) 24 (54.5)
Non-KatG Mut. 3 (23.1) 10 (76.9) 0.36 (0.09-1.5) 0.16 0.35 (0.07-1.9) 0.22
Pansensitive-TB 20 (45.5) 24 (54.5)
All INHr 13 (52.0) 12 (48.0) 1.30 (0.48-3.5) 0.60 1.45 (0.48-4.4) 0.51
Pansensitive-TB 20 (45.5) 24 (54.5)
(*) Adjusted by age, history of diabetes mellitus, history of daily alcohol intake and sputum AFB smear results.
17
Characteristics of Contact Patients by Genotypic Groups
We assessed the proportion of TST positive (TST induration 5mm) vs. TST negative
cases, and rates of new TST conversion from contact cases of patients infected with MTB
isolates from the 3 bacterial subtypes. Comparison of the rate of TST positivity and new
TST conversion showed no statistical significant differences among contact subjects of
patients infected with the 3 bacterial genotypes (P > 0.05) (data not shown).
Discussion
Pathogenicity is defined by the capacity of the bacterium to produce disease. The clinical
consequences of infection with MTB are related to a complex interplay of host,
environment, and bacterial factors.
20
It is well known that host factors that affect the
integrity of the cell-mediated immune response, influence TB pathogenicity and TB
disease characteristics in humans. The best example is an increase in extrapulmonary and
disseminated forms of TB disease among AIDS and HIV co-infected patients.
21-23
Environmental factors influence TB transmission, but it is less clear how environmental
and life style factors could influence TB pathogenicity and TB disease characteristics in
humans. Perhaps the one rare exception is TB transmission through TB infected organs in
transplant patients resulting in extrapulmonary TB.
24, 25
Little is known about the
18
bacterial factors that affect TB pathogenicity in humans. In contrast, many bacterial
“virulence” factors have been identified to alter the organisms’ behavior in vitro and
animal studies.
26
However, it is challenging to correlate the role of these bacterial factors
in laboratory and animal models with actual TB pathogenesis and disease characteristics
in humans.
26
Our study takes advantage of a natural model of TB infection to test the hypothesis that
bacterial genetic factors can influence TB disease characteristics in humans. We used a
well-recognized bacterial fitness factor (KatG) that can alter the organism’s behavior
depending on the type of katG genetic alteration.
15, 16
We tested whether this theoretical
differential bacterial behavior can translate into distinct TB disease characteristics and
response to treatment in human subjects using comparative genomics and clinical data.
We found that INHr-TB patients infected with non-katG mutants were significantly more
likely to have AFB culture conversion at 1month of treatment after adjusting for potential
confounding variables such as age, ethnicity, sputum AFB smear results, and presence of
cavitary disease and unilateral lung involvement on chest x-ray. The fact that we only
included patients without treatment adherence problems and with the same type of TB
treatment excludes differential treatment effect among the study subjects. Interestingly,
the higher rate of sputum conversion might suggest a faster sterilizing time and perhaps a
lower degree of pathogenicity among these types of MTB strains. However, the sputum
conversion at 2 months was not statistically different for patients infected with the two
INHr-TB bacterial genotypes compared to patients with PS-TB, suggesting a weak
19
association or subclinical differential pathogenicity for patients with the non-katG
genotype compared to the PS group. Moreover, the 1-month sputum conversion
association was independent of the presence of sputum smear positivity and cavitary
disease on chest x-ray, which suggest that number of infectious bacilli at disease onset
was similar among the study groups, and less likely a factor for a faster treatment
response. Of note, sputum AFB culture conversion at 2 month is a well-known surrogate
marker for severity of pulmonary TB disease, lack of sputum culture conversion at 2
month along with presence of cavitary disease predicts a higher risk for relapse in clinical
trials.
27
It is postulated that the lack of catalase activity phenotype is associated with a fitness
disadvantage for MTB with the INHr genotypes in animal models.
15, 16
However, there is
no reason to suspect low or absence of catalase activity in our group of MTB isolates
with wild-type KatG genotypes. While we did not have catalase activity information from
our study of MTB isolates, we have an expected degree of catalase activity for most MTB
isolates based on prior publications that describe the association between the type of
INHr genetic alterations, MICs and catalase activity.
5, 7
Most of our non-katG mutants
had a common mutation in the mabA-inhA promoter (8 out of 15 isolates), which is
associated with low-intermediate MIC level for INH resistance and a preserved catalase
activity,
7
suggesting that the faster sputum conversion response observed in our non-
katG group would have been independent of the presence of a dysfunctioning catalase
among those INHr-TB isolates. Most katG mutants in our study had the common S315T
20
katG genetic alteration (8 out of 13), which is also associated with preserved levels of
catalase activity.
5, 7, 13, 16
The rest of INHr-TB isolates with mutations in katG other than
S315T had a low MIC, which is also associated with a preserved catalase activity.
7, 13
Interestingly, the OR for sputum culture conversion for the patients infected katG
mutants was not statistically different compared to patients infected with PS isolates,
suggesting no difference in pathogenicity. A recent molecular epidemiological study
from San Francisco suggests that secondary transmission of INHr-TB S315T katG
genotypes is preserved compared with genotypes with katG mutations other than S315T,
none of which were found to be clustering in the community.
28
Of note, we found no
INHr-TB isolates with mutations in katG other than S315T with a high MIC, which are
the bacterial genotypes associated with a low catalase activity
13
and low virulence in
animal models,
15, 16
suggesting that this type of INHr-TB isolates are less pathogenic and
probably uncommon among clinical isolates.
5
Another interesting observation from our study is that INHr-TB patients infected with
katG mutants were associated with a higher rate of unilateral disease on chest x-rays after
adjusting for potential confounding factors such as age, history of diabetes mellitus,
history of daily alcohol intake and initial sputum AFB smear results. It is unclear the
significance of unilateral vs. bilateral pulmonary disease in TB pathogenicity; however,
we could postulate that unilateral pulmonary disease involvement might be a marker of
lesser disease severity and/or lesser tendency for disease dissemination compared with
bilateral pulmonary disease. Another factor to consider is whether those findings
represent a differential degree or type of immune response, in which unilateral disease is
21
better contained or less likely to reactive in more than one area of the lungs. Highly
virulent clinical MTB isolates trigger a diminished Th1 response and an upregulation of
interferon alpha and beta production in animal models, suggesting that some MTB
virulence factors are associated with the bacterial ability to impair cell-mediated immune
response rather than actual increase in bacterial virulence.
29
Of note, none of the 2
groups of INHr-TB bacterial genotypes were associated with a higher rate of cavitary
disease, which is a marker of disease severity,
27
which in turn is associated with the
presence of Th2 lymphocyte subsets in human broncholaveolar lavages.
30
It would be
interesting to assess the immunological profile triggered by this type of INHr-TB katG
bacterial genotypes in animal models. Perhaps, a well-contained unilateral lesion is a
marker of an adequate Th1 immune response for these types of isolates.
To our knowledge, only one study suggested a relationship between specific
mycobacterial genetic alterations (phospholipase-C gene D; plcD) and an increased risk
of extrapulmonary TB. The study used comparative genomics and statistical analyses of
data from a population of TB infected patients. The study did not include HIV status
information in about half of the study subjects, and therefore the authors were unable to
completely adjust for this well-known confounding factor for extrapulmonary TB.
31
Moreover, infection with Beijing/W lineage MTB isolates (bacteria-related factor) and
polymorphism in the P2X7 gene (host-related factor) were also found to increase the risk
for extrapulmonary TB,
32, 33
suggesting that the presence of extrapulmonary TB is
determinate by more than one type of factor.
22
Conclusion
In conclusion, patients with not-katG INHr-TB genotypes showed a faster bacteriologic
response to anti-tuberculous treatment in comparison to those with PS-TB. Patients
infected with INHr-TB with katG genotypes were associated with more unilateral lung
disease on presentation. These results suggest bacterial genetic factors may influence
disease presentation and response to treatment in human subjects.
23
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Abstract (if available)

Abstract Most isoniazid-resistant (INHr) M. tuberculosis (MTB) isolates have katG mutations. KatG is a virulence factor in animal models. We aim to determine if MTB-INHr genotype is associated with distinct clinical features. A retrospective case-control study design was used to compare clinical characteristics of patients infected with INHr-MTB (katG or non-katG mutations) to patients with pan-sensitive (PS) MTB isolates. Univariate and multivariate analyses were used. Twenty-nine INHr-TB cases and 50 PS controls were evaluated. INHr-MTB patients infected with non-katG mutants were associated with a higher rate of sputum conversion at 1 month of treatment: Adjusted OR = 6.03 (95%CI: 1.02-35.4

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Creator Escalante, Patricio (author)

Core Title Study of isoniazid-resistant tuberculosis: mycobacterial genotypic characterization and clinical features in human subjects

School Keck School of Medicine

Degree Master of Science

Degree Program Clinical and Biomedical Investigations

Publication Date 08/07/2007

Defense Date 07/30/2007

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag clinical presentation,drug-resistance,isoniazid,katG,OAI-PMH Harvest,Tuberculosis,virulence

Language English

Advisor McKean-Cowdin, Roberta (committee chair), Azen, Stanley Paul (committee member), Larsen, Robert (committee member)

Creator Email escalant@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m755

Unique identifier UC1448585

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Legacy Identifier etd-Escalante-20070807.pdf

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Document Type Thesis

Rights Escalante, Patricio

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

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