Current Prespectives In Diagnosis Of Tuberculosis

Current Perspective in Diagnosis of Tuberculosis

Rajiv Singla

Moderator: Dr. M.K.Daga

WHO TB fact sheet 2005      

2 billion people ,equal to a third of world's population, are infected with TB bacilli. 2 million people die every year due to TUBERCULOSIS TB is curable but kills 5000 people everyday. 98% of TB deaths are in developing world affecting mostly young adults in their most productive years Global TB incidence is still growing at 1% a year . TB is a worldwide pandemic; though the highest rates per capita are in Africa, half of all new cases are in 6 asian countries(Bangladesh, China, India, Indonesia, Pakistan, Philippines)

Tuberculosis: Transmission and Natural History Self-Cure – 90%

Infection

Initial containment – 95%

Early Progression - 5%

Late Progression - 5%

Tuberculosis: Global epidemiology

1.7 billion people

8.4 million cases, 1.9 million deaths each year

Latent TB Infection (LTBI) • Occurs when person breathes in bacteria

and it reaches the air sacs (alveoli) of lung • Immune system keeps bacilli contained and under control • Person is not infectious and has no symptoms

LTBI vs. TB Disease LTBI

TB Disease Tubercle bacilli in the body

Tuberculin skin test reaction usually positive Chest x-ray usually normal

Chest x-ray usually abnormal

Sputum smears and cultures negative

Symptoms smears and cultures positive

No symptoms

Symptoms such as cough, fever, weight loss

Not infectious

Often infectious before treatment

Not a case of TB

A case of TB

Groups That Should Be Tested for LTBI Persons at higher risk for exposure to TB Close contacts of a person known or suspected to have TB Residents and employees of high-risk congregate settings Health care workers (HCWs) who serve highrisk clients

Groups That Should Be Tested for LTBI (Cont.)

Persons at higher risk for TB disease once infected Persons with HIV infection Persons with certain medical conditions Persons with a history of inadequately treated TB

Testing for M. tuberculosis Infection Mantoux tuberculin skin test (TST) QuantiFERON® -TB test QuantiFERON® - Gold T Spot-TB test

Montoux 

The TST functions by eliciting cell mediated immune response in previously sensitized individuals.



An intradermal injection of PPD evokes a delayed-type hypersensitivity response mediated by sensitized T cells and results in cutaneous induration.



PPD is a precipitate of M. tuberculosis culture supernatant which contains roughly 200 antigens, many of which are shared by other mycobacteria including many NTM and M bovis BCG



This cross-reactivity seriously limits the specificity of the TST.

Sensitivity of the Montoux 

On average, 10%-30% of patients do not react to tuberculin (sensitivity ~75-90%)  False negative rates among persons with confirmed TB vary across studies: 4%, 17%, 20%, 21%



False negative rates can exceed 50% among patients with disseminated TB, due to a combination of poor nutrition, poor general health, acute illness, and immunosuppression.



HIV+ persons may have a limited ability to react to tuberculin, even if TB-infected. Reaction is inversely related to immune function.

Factors that affect the PPD Reaction Type of Reaction False-positive False-negative

Possible Cause

Nontuberculous mycobacteria BCG vaccination Anergy Recent TB infection Very young age (< 6 months old) Live-virus vaccination Overwhelming TB disease

Montoux – contd. 

Studies of the prevalence of LTBI in India have yielded prevalence rates ranging from 9% to more than 80% in various populations.



In India guidelines being followed are: >10 positive, <6 negative, 6-9 doubtful.



Strong reactors(i.e. >20 mm reaction) have greater chance of developing tuberculosis than those showing 10 mm induration.



Those with less than 5 mm induration have more risk of developing tuberculosis than those with 6-9 mm induration.

Markowitz N, Hansen NI, Wilcosky TC, et al. Ann Intern Med 1993. Mayurnath Seth al. Indian J Med Res 1991.

Inability of the PPD in distinguishing active TB from inactive infection TB contacts Active TB Inactive TB infection

BCG Vaccination and Tuberculin Skin Test  No

reliable way to distinguish tuberculin skin test reactions caused by bacille Calmette-Guérin (BCG) vaccine from TB infection

 Evaluate

all BCG-vaccinated persons who have a positive skin test result for treatment of LTBI

Need for a New Test  Tuberculin  Was

skin test (TST)

only test for latent TB infection  Wide variation in applying and reading.  False-positive from BCG and nontuberculous mycobacteria (NTM)  Boosting from prior TST  False negative results

Recent advances  Detection

of gamma interferon produced by T lymphocytes in response to stimulation with M. tuberculosis antigen

 “Like

the TST, the IFN-γ assay detects cell-mediated immunity to tuberculin.”  TST

and QFT are not independent.

Rationale for T-Cell based approach  M.tuberculosis:

intracellular pathogen, difficult to recover from infected subjects

 Humoral

responses in M.tuberculosis are

weak  Infection

evokes a strong Th1-type cell mediated immune response (IFN-γ CMI)

IFN-γ assays - mechanism

QFT  in

vs.

vitro  multiple antigens  no boosting  1 patient visit  minimal inter-reader variability  results in 1 day

TST  in

vivo  single antigen  boosting  2 patient visits  inter-reader variability  results

in 2 - 3 days

Antigens used o

QuantiFERON® -TB test PPD is used as antigen

o

QuantiFERON® - Gold ESAT-6 and CFP-10 are antigen used

o

T Spot-TB test identifies ESAT-6- or CFP-IO-specific IFN-γ secreting CD4+ T cells.

M. tuberculosis-specific antigen 

In 1999, by comparative hybridization experiments on a DNA microarray led to the identification of a genomic region known as Region of Difference (RD I).



The gene products of RD I are found only in M. tuberculosis, in pathogenic M bovis strains and in four NTM (M kansasii, M szulgai, M.flavescens, and M marinum).



Only M kansasii overlaps clinically with M tuberculosis, and because M. kansasii infection is uncommon, the RD I region antigens are essentially specific to M tuberculosis.



Among these antigens are, of course, ESAT-6 and CFP-IO, as well as MPT-64. ESAT-6

Behr MA et al. Science 1999., Mahairas GG et al. J Bacteriol 1996 Harboe M et al Infect Immun 1996 , Philipp WJ et al. . Microbiol 1996 .

QFT-Gold 

The improved specificity over the Quanti-FERON-TB assay for PPD and over the TST was confirmed in a study by Johnson et al, Clin Diagn Lab Immunol. 1999



Of 60 medical students, 0-mm tuberculin skin tests (TSTs) at study entry



58 (97%) were initially classified as negative for M. tuberculosis infection by PPD QIFN.



Five months after BCG immunization, 7 of 54 students (13%) had a TST result of >/=10 mm and 11 of 54 students (20%) tested positive by PPD QIFN.



ESAT-6- and MPT-64-stimulated IFN-gamma responses in the medical students were negative prior to and after BCG immunization.

Johnson et al, Clin Diagn Lab Immunol. 1999

Johnson et al, Clin Diagn Lab Immunol. 1999

Johnson et al, Clin Diagn Lab Immunol. 1999

Mori T et al. . Am J Respir Crit Care Med 2004 

Mori and colleagues studied a group of 216 Japanese student nurses who had no identified risk for M. tuberculosis exposure



All vaccinated with BCG.



In this group 64.6% of the subjects had a TST response measuring 10 mm or more, yielding a specificity of 35.4% for the TST



The QuantiFERON--TB ESAT-6/CFP-l 0 assay, on the other hand, yielded a specificity of 98.1 % in this group, far superior to the TST.



The sensitivity of the assay, 89.0%, was determined in a separate group of patients who had culture-proven active disease.

T SPOT-TB test  Identifies

ESAT-6- or CFP-IO-specific IFN-γ secreting CD4+ T cells

 Projected

as much more sensitive test as compared to all other methods

 Approved  Not

by European Union

approved by FDA as yet

T SPOT-TB test

Summary- so far……  The

improved specificity would decrease unnecessary treatment in those who are not truly infected.  The improved sensitivity of these tests over the TST would capture a cohort of patients who other-wise would go without treatment of LTBI  Esp. important in those who are most likely to have false-negative TST results, e.g. immunosuppressed individuals.  Longitudinal studies linking positive assays with risk for development of active disease are ongoing and are crucial to demonstrating the true role of these tests.

An ideal test for active tuberculosis

 rapid

results (available within I day),  high sensitivity and specificity,  low cost, and robustness,  highly automated or easily performed without the need for excessive sample preparation or technical expertise,  drug-susceptibility data.  distinguish between LTBI and active disease.

Sputum-based diagnosis  5000

to 10,000 bacilli per milliliter are required for sputum to be AFB positive  Expectorated sputum is generally the starting point.  The sensitivity of expectorated sputum ranges from 34% to 80%  In no way does a negative sputum smear eliminate the diagnosis of active tuberculosis Murray PR et al. Ann Intern Med 1980

Sputum Induction  Nebulisation

with hypertonic saline  Described in 1961 by Hensler and colleagues  In patients who are unable to expectorate or who had smear-negative sputum samples  Adequate sputum sample in 60-80%  25-45% of which are sputum positive Parry CM et al. Tuber Lung Dis 1995 Hartung TK et al. S Afr Med J 2002

Fibro-optic Bronchoscopy  Should

be done in patients who are negative on

SI A

study by McWilliams and colleagues

 SI

had an overall yield of 96.3% after three tests, confirming the utility of repeated Sls.

 The

yield of FOB was only 51.9% McWilliams T et al. Thorax 2002

FOB and SI - precaution  SI

and bronchoscopy are cough-inducing procedures and generate infectious droplet nuclei, causing increased exposure to M. tuberculosis.  Ideally, both procedures should be performed using local exhaust ventilation devices or rooms that meet the ventilation requirements for TB isolation and those in attendance should wear respiratory protection.

Diagnosis of active tuberculosis  Microscopy:   

 

Easiest & quickest test Limited sensitivity(46-78%) but specificity is virtually 100%* Centrifugation & fluorochrome staining(auramineO) with UV microscopy markedly increase the sensitivity ZN staining – 10000 bacilli/ml Fluorochrome staining – 1000 bacilli/ml

*Chest 1989; 95: 1193

Traditional Culture 

The gold standard method for TB diagnosis



More sensitive & can be positive even when bacterial load is low(10-100 bacilli/ml)



Required for precise identification of causative organism



Two types of media are used: 

Egg based: LJ, Petragnani and ATS



Agar based: Middlebrook 7H10 or 7H11

Growth is slow & takes 6-8 weeks. Thereafter the same length of time is required for complete identification & sensitivity testing

Cultures Colonies of M. tuberculosis growing on media

Broth Based Culture Methods  BACTEC  Septicheck

AFB  Mycobacterial growth indicator tubes  MB/Bac T  Myco-ESPculture System II  BacT/ALERT MB Susceptibility Kit

BACTEC  Bactec

460 TB is an automated system that measures the specific metabolic activity of TB bacilli using the radiometric method.

 Bactec

system requires an average of only 8 18 days for culture

5

to 8 days for determining drug susceptibility.

BACTEC Mean time to detection of mycobacteria in clinical specimens Average no. of days (range) to detection of: Culture method

M. tuberculosis complex All isolates Smear positive

Smear negative

BACTEC 460 TB

11.2 (2–53)

8.0 (3–18)

18 (9–30)

LJ medium

26.8 (7–47)

28.5 (16–29)

36.2 (28–41)

Francesca Brunello, Flavio Favari, Roberta Fontana Journal of Clinical Microbiology, April 1999

BACTEC Detection of mycobacteria from clinical specimens according to initial smear results No. (%) of isolates detected by: Isolate (no. of specimens) BACTEC 460 TB

LJ medium

All smear-positive specimens (107)

107 (100)

107 (100)

All smear-negative specimens (66)

65 (98.4)

59 (89.3)

Smear-positive M. tuberculosis (96)

96 (100)

95 (98.9)

Smear-negative M. tuberculosis (18) 18 (100)

16 (88.8)

Smear-positive NTM (11)

11 (100)

11 (100)

Smear-negative NTM (48)

47 (97.9)

43 (89.5)

Francesca Brunello, Flavio Favari, Roberta Fontana Journal of Clinical Microbiology, April 1999

Mycobacterial Growth Indicator Tube  Rapid

method

 Consists

of round bottom tubes containing 4 ml of modified Middlebrook 7H9 broth which has an oxygen sensitive fluorescent sensor at the bottom

 When

mycobacteria grow, they deplete the dissolved oxygen in the broth & allow the indicator to fluoresce brightly in a 365nm UV light J Clin Microbiol 1999; 37: 748-752

Mycobacterial Growth Indicator Tube  Positive

signals are obtained in 10-12 days

 MGIT

can also be used as a rapid method for the detection of drug resistant strains of M.tb directly from acid-fast smear positive samples, as well as from indirect drug susceptibility studies.

J Clin Microbiol 1999; 37: 45-48

The ESP culture system II  Detection

of pressure changes within the headspace above the broth culture medium in a sealed bottle  The mean time for recovery of M.tuberculosis complex 15.5  Reliable nonradiometric less labourintensive alternative to BACTEC 460 system for the growth and detection of mycobacteria.

The MB/BacT system  Non-radiometric

continuous monitoring system  The system is based on colorimetric detection of CO2.  Mean time for detection of M.tuberculosis was 13.7 days by the MB/BacT system.  Acceptable alternative for BACTEC 460 method despite some minor disadvantages such as increased contamination and slightly longer time for detection of growth.

The septi-check AFB system  Consists

of a paddle enclosed in a plastic tube,  One side of the paddle is covered with nonselective Middlebrook 7H11 agar  The reverse side is divided into two sections: 



one contains 7H11 agar with para-nitro-á-acetylaminoß-hydroxypropiophenone (NAP) for differentiation of M.tuberculosis from other mycobacteria, the other section contains chocolate agar for detection of contaminants.

The septi-check AFB system  This

method requires about 3 weeks of incubation.  The unique advantage of this technique is the simultaneous detection of M.tuberculosis, non-tuberculous mycobacteria (NTM), other respiratory pathogens and even contaminants.

TK Medium  The

color change in TK Medium is based on multiple dye indicators  Depends on the metabolites and enzymes produced by different species of microorganisms.  The color change occurs long before the colonies become visible.  It can also be used for drug-susceptibility testing  Can differentiate a contaminated specimen.

TK Medium

Nucleic acid amplification assays  NAA

assays amplify M. tuberculosis-specific nucleic acid sequences using a nucleic acid probe.  The sensitivity of the NAA assays currently in commercial use is at least 80% in most studies  Require as few as IO bacilli from a given sample  NAA assays are also quite specific for M. tuberculosis, with specificity in the range of 98% to 99%. Official statement of ATS and CDC, July 1999

NAAs- various types  AMPLICOR  Amplified  LCx  BD

M. TUBERCULOSIS assay

M.tuberculosis Direct (AMTD2) assay

MTB assay, ABBOTT LCx probe system

ProbeTec energy transfer (ET) system (DTB)

 INNO-LiPA

RIF.TB assay

NAAs- various types

AMPLICOR M. TUBERCULOSIS assay

Cohen, R. A., 1998. Am. J. Respir. Crit. Care Med. 156:156–161. Bonington, A., 1998. J. Clin. Microbiol. 36:1251–1254. Al Zahrani, 2000. Am. J. Respir. Crit. Care Med. 162:1323–1329.

Amplified M.tuberculosis Direct (AMTD2) assay

Catanzaro et al.. JAMA 2000.283:639–645. Bergmann, J. S.1999 J. Clin.Microbiol. 37:1419–1425.

NAA- summary 

Useful technology for rapid diagnosis of smear negative cases of active TB



Able to identify 50-60% of smear -ve culture +ve cases



Distinguish M.tb from NTM in smear +ve cases



Should not be used to replace sputum microscopy as an initial screen & culture remains the gold standard



Very high degree of quality control required

NAA- summary  They

are able to detect nucleic acids from both living and dead organisms so in pts on ATT, PCR should not be used as an indicator of infectivity as this assay remains positive for a greater time than do cultures

A

major limitation of NAA tests is that they give no drug-susceptibility information.

 NAA

should always be performed conjunction with microscopy and culture

in

Extra pulmonary tuberculosis  In

the case of miliary tuberculosis FOB may play a significant role  There is clearly a role for NAA assays  The overall sensitivity in nonrespiratory specimens for the MTD or E-MTD ranges from 67% to 100%  Much more sensitive in cerebrospinal fluid than in pleural fluid

Adenosine Deaminase Levels 

In a recent meta-analysis of 40 studies investigating ADA for the diagnosis of tuberculous pleuritis sensitivity equaled specificity at 92.2%



In one study, the sensitivity and specificity were both 55%



Trajman A et al. argue that ADA alone is superior to ADA combined with PCR

1.Goto M et al. Diagnostic value of adenosine deaminase in tuberculous pleural effusion: a meta-analysis. Ann Clin Biochem 2003; 40(Pt 4):374-81. 2.Nagesh BS et al. Chest 2001

Adenosine Deaminase  T.

Söderblom, P. Nyberg, A.M. Teppo, (Eur Respir J, 1996, 9, 1652–1655) on pleural ADA and IFN-γ, showed :

ADA in CSF   

 

ADA may be of limited value in diagnosing tuberculous meningitis. There is consensus neither regarding ‘cut-off value’ nor about sensitivity or specificity. In one study, Ribera E et al analyzed that the mean enzyme value was clearly higher for the patients with tuberculous meningitis (15.7 +/- 4.3 U/liter) than for the other patients (1.4 +/- 1.5 U/liter). The sensitivity of the test for diagnosing tuberculous meningitis was 100% and specificity, 99%. Rohani MY et al reported specificity of 87.6% and a cutoff value of 9

Ribera E et al .Activity of adenosine deaminase in cerebrospinal fluid, J Infect Dis.

ADA in CSF  N. Selvakumar, CEREBROSPINAL FLUID ADENOSINE DEAMINASE AND LYSOZYME LEVELS IN THE DIAGNOSIS OF TUBERCULOUS MENINGITIS, in Ind. J. Tub.:

IFN-γ levels 

Another test that has received some attention for the diagnosis of pleural and pericardial tuberculosis is pleural or pericardial fluid IFN-γ

To Conclude….. 







Diagnostic testing for tuberculosis remained unchanged for nearly a century, but newer technologies hold the promise of a time revolution in tuberculosis diagnostics. The IFN-γ release and T-cell-based assays may well supplant the TST in diagnosing LTBI in much of the world. NAA assays are proving their worth in more rapidly diagnosing both pulmonary and extrapulmonary tuberculosis with great sensitivity and specificity. These tests are likely to play an ever-increasing role in the coming years.

THANK YOU

How QFT Is Performed Stage 1 Whole Blood Culture Nil Avian Tb Mitogen Control PPD PPD Control

Stage 2 IFN-γ ELISA

Transfer undiluted whole blood into wells of a culture plate and add antigens

COLOR TMB

Harvest plasma from above settled cells and incubate 60 min in ‘Sandwich’ ELISA

Wash, add substrate, incubate 30 min then stop reaction

Culture overnight at 37oC TB infected individuals respond by secreting IFN-γ

Standard Curve OD 450nm

Heparinized whole blood

IFN-γ IU/ml

Measure OD, determine IFN-γ levels and interpret test

M. tuberculosis-specific antigen 

Harboe and colleagues [1986] reported the first M. tuberculosisspecific antigen, MPB-64 (later known as MPT-64).



Andersen and colleagues [1995] reported the highly immunogenic antigen target of the cellular immune response to tuberculosis in mice, known as the early secreted antigenic target 6 (ESAT-6).



Berthet and colleagues [1998] described culture filtrate protein (CFP-IO)



In 1998, the complete genome sequence of M tuberculosis was determined

Harboe M et al. Infect Immun 1986.,Andersen P, et al. J Immunol 1995 Berthet FXet al. Microbiol 1998