Tuberculosis diagnostics

ปลอด TB ชีวีมีสุข
แนวทางเพื่อการวินิจฉัยที่แม่นยำ รักษาได้ตรงจุด และได้ผลการรักษาที่ดี
S a n t i S i l a i r a t a n a , M D
Division of Pulmonary Medicine, Department of Medicine,
Facul ty of Medicine Vaj i ra Hospi tal
Navamindradhiraj Unive r s i ty

Tuberculosis
ESTIMATED
INCIDENCE
1,788,043
1,334,066
627,047
362,819
360,767
278,392
251,685
241,537
236,885
195,207
194,627
160,688
144,942
137,260
110,319
106,201
89,351
86,130
85,015
84,546
79,656
WHO. Global tuberculosis control: surveillance, planning, financing: WHO report 2005. Geneva: WHO, 2005.
2.
More people die from TB than from any
Poverty
Congregation
HIV pandemic

Tuberculosis-HIV Coinfection
resistant, it is estimated that over
countries) in active TB cases.
countries or the volume of testing.
Figure 5
PREVALENT ADULT TB CASES COINFECTED WITH HIV, 2004
Source: reference 3.
Dye C, Watt CJ, Bleed DM et al. Journal of American Medical Association 2005; 293:2767-75.

The Gap between Estimated and Notified Cases
Estimated TB cases
8.8 Million
4.1 Million cases
Recorded & reported
Health
facility
TB cases
Diagnostic
tests
reported
Detected but
not notified
private sector
military
prisons
⊕
⊖
WHO. Global tuberculosis control: surveillance, planning, financing: WHO report 2005. Geneva: WHO, 2005.

Multidrug-resistant and Extensively drug-resistant TB
Multidrug-resistant (MDR) TB
Resistance against at least
rifampicin and isoniazid
Extensively drug-resistant (XDR) TB
MDR-TB PLUS
Resistance to any fluoroquinolones
AND
≥1 injectable second-line agents
O’Grady J, Maeurer M, Mwaba P et al. Current Opinion in Pulmonary Medicine 2011, 17; 134-141.
(ethionamide, prothionamide, cycloserine, terizidone,
para-aminosalicylic acid, clofazimine, amoxicillin-clavula-nate,
are those used directly on patient samples where a set
of drug-containing and drug-free media is inoculated
136 Infectious diseases
Figure 2 Estimated percentage of multiple drug resistant tuberculosis among new tuberculosis cases, 2008a
, 0 to <3; , 3 to <6; , 6 to <12; , 12 to <18; , "18;‘, no data available; , subnational data only. Reproduced with
permission from [2].

Establishing Tuberculosis: Pulmonary TB
AFB stain
Myc Culture
Drug susceptibility
Chest radiography
CT scan
History
Chronic productive cough*
Sputum production*
Prolonged low grade fever
Night sweats
Weight loss
Physical examination
Bronchial breath sound
Crepitation
Digital clubbing
Imaging
Additional test(s)
Clinical features
suggestive for
tuberculosis
Microbiology

Diagnostic Algorithm: Clinically-suggestive
Patient with clinical features suggestive
for pulmonary tuberculosis
Sputum examination for acid-fast bacilli
Chest radiograph
AFB - positive
CXR - compatible with TB
AFB - negative
AFB - negative
CXR - incompatible with TB
Sputum culture and drug susceptibility testing for mycobacteria
Treatment for pulmonary tuberculosis
Look for
alternative diagnosis
แนวทางเวชปฏิบัติการรักษาวัณโรคในผู้ใหญ่ พ.ศ. 2556 (ฉบับร่าง). สำนักวัณโรค กรมควบคุมโรค สมาคมอุรเวชช์แห่งประเทศไทย

Diagnostic Algorithm: Radiographically-suggestive
Asymptomatic patient with
radiographically suggestive tuberculosis
Sputum examination for acid-fast bacilli
Review previous chest radiograph
AFB - positive
AFB - negative
CXR - unavailable
AFB - negative
CXR - unchanged
Sputum culture and drug susceptibility testing for mycobacteria
Treatment for pulmonary tuberculosis
Re-evaluation
and repeat CXR in 3 months
AFB - negative
CXR - active TB
AFB - negative
CXR - old lesion
แนวทางเวชปฏิบัติการรักษาวัณโรคในผู้ใหญ่ พ.ศ. 2556 (ฉบับร่าง). สำนักวัณโรค กรมควบคุมโรค สมาคมอุรเวชช์แห่งประเทศไทย

Methods to Detect TB infection

Detecting TB Infection
Microscopic
examination
Gene/molecular-based
techniques
Mycobacterial
culture
Immune reactivity
detection
M.
tuberculosis
detection

Sputum Microscopy for Acid-fast Bacilli
Friedrich Carl Adolf Neelsen
(1854-1898)
Franz Ziehl
(1857-1926)
Neelsen-Ziehl (Acid fast bacilli) Staining
Acid-fast bacilli appear pink
in a contrasting methylene blue background

Diagnostic Threshold underly Light Microscopy
system
may
TB
appropri-ate
Threshold for visibility of AFB by smear microscopy
10,000
Number of TB bacilli per millilitre
(ml) of sputum
Cough worsens:
patient returns
to clinic
Blood appears
in sputum;
infant daughter
infected
with TB
Too weak
to work
AFB+:
TB diagnosis
made
Patient
visits clinic:
no diagnosis
made
First smear:
AFB negative
Patient
returns
to clinic
Patient visits
pharmacy
Night cough
begins
Infection of
healthy patient
Patient feels
unwell
first month second month third month fourth month fifth month
AFB = acid-fast bacilli = smear+
Figure 6
A TB PATIENT’S JOURNEY FROM SYMPTOMS TO DIAGNOSIS
WHO. Diagnostics for Tuberculosis: Global Demand and Market Potential. Geneva: WHO 2006.

Fluorescence Microscopy: Mercury Vapor Lamp
Use Mercury Vapor as a light source
Staining of specimens with Auramine-O
Higher sensitivity than light microscopy,
comparable specificity
Requires a dark room for examination
WHO. Fluorescent light-emitting diode (LED) microscopy for diagnosis of tuberculosis: policy statement. Geneva: WHO 2011.

Light Emitting Diode (LED) Fluorescence Microscopy
Same (or slightly more) sensitivity
Cheaper and longer life duration of bulb (10,000 hr)
Cheaper microscopy
A dark room is not required
WHO recommended to use LED fluorescence
microscope as a standard technique
WHO. Fluorescent light-emitting diode (LED) microscopy for diagnosis of tuberculosis: policy statement. Geneva: WHO 2011.

Methods Sensitivity and Specificity
Method Sensitivity (%) Specificity (%)
Light microscopy 32-94 94
Mercury vapor
fluorescence
microscopy
52-97 94
LED fluorescence
microscopy 58-97 95
Steingart KR, Ng V, Henry M, et al. Lancet Infect Dis. 2006

Detecting TB Infection
Immune reactivity
detection
Microscopic
examination
Gene/molecular-based
techniques
Mycobacterial
culture
M.
tuberculosis
detection

Mycobacterial Culture
Richter E, et al. Exper Rev Resp Med. 2009; 3 (5): 497-510.
Minion J, et al. The Lancet Infectious Disease. 2010; 10 (10): 688-698.
Conventional
TB culture
system
Rapid colorimetric drug susceptibility test
20-30 days
Liquid culture-based technique
Mycobacterial growth indicator tube (MGIT)
7-10 days

Gene Xpert MTB/RIF: Features
Integrated sample processing
and PCR in a disposable plastic cartridge
All automatic
Bacterial lysis
Nucleic acid extraction
Amlification
Amplicon detection
Boehme CC, Nabeta P, Hillermann D, et al. N Engl J Med 2010; 363:1005-1015.

Gene Xpert MTB/RIF
The new england journal o f medicine


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Figure 2. Assay Procedure for the MTB/RIF Test.
Two volumes of sample treatment reagent are added to each volume of sputum. The mixture is shaken, incubated at room temperature
for 15 minutes, and shaken again. Next, a sample of 2 to 3 ml is transferred to the test cartridge, which is then loaded into the instru-ment.
All subsequent steps occur automatically. The user is provided with a printable test result, such as “MTB detected; RIF resistance

Gene Xpert MTB/RIF: Performance
98.2% 72.5%
Sensitivity 98%
Smear-positive
specimens
Smear-negative
specimens
Sensitivity
compared with culture
99.2%
Specificity
Specificity 99%
MTB
detection
Rifampin
resistance
detection

Gene Xpert MTB/RIF: Pros and Cons
Easy preparation and processing
Almost all steps run automatically
Test results can be reported
within 2 hours
Can be used both for TB identification
and Rifampin susceptibility test
Adventages
Disadventages
High cost
High maintenance cost
Rifampin resistance detection only

Line Probe Assay (LPA)
Rapid molecular drug resistance detection
Reverse line blot hybridization
!
INNO-LiPA Rif.TB Test
Hain test: MDRTBplus, MDRTBsl

Line Probe Assay (LPA): MDRTBplus and MDRTBsl
First-line drugs Second-line drugs

Line Probe Assay (LPA)
≥97% ≥99%
Sensitivity Specificity
for detection of rifampin resistance
≥90% ≥99%
Sensitivity Specificity
for detection of
combined INH-RIF resistance

LPA vs Conventional DST
Parsons LM, Somoskövi A, Gutierrez C et al. Clin Microbiol Rev. 24 (2). 2011; 314-350.

Line Probe Assay (LPA): Pros and Cons
Rapid processing and reporting (2-7 days)
Drug susceptibility testing to INH and RIF
(INNO-LiPA Rif.TB and MTBDRplus)
Drug susceptibility testing to second-line
agents (MTBDRsl)
NTM species identification
Adventages
Disadventages
Labour intensive
Requires highly trained personnel
Requires dedicated laboratory
space and equipment
Expensive (but cheaper than Xpert)

Indications for Rapid Drug Susceptibility Test
Risk factor(s) to carry drug resistant strains
Tuberculosis in the setting of close contact to MDR-TB
patient
Positive smear at 3 months after treatment
Positive smear at 5 months after treatment
Before changing regimen or adding any drugs to
the treatment regimen
Suspected NTM infection in smear positive patient

The Mantoux Tuberculin Skin Test
Injecting 0.1 mL of tuberculin purified protein
derivative (PPD) into the inner surface of the
forearm (intradermal injection)
Injection should be made with a tuberculin syringe
The needle bevel facing upward
The injection should produce a pale elevation of the
skin 6-10 mm in diameter
CDC. MMWR 2005; 54 (RR-17).
American Thoracic Society and CDC. Am J Respir Crit Care Med. 2000; 161.

Tuberculin Skin Test: Reading and Interpretation
An induration of ≥5 mm
!
HIV infected persons
A recent contact
Persons with fibrotic changes on chest radiograph consistent with prior TB
Patients with organ transplants
Immunosuppressed patients (e.g., 15 mg/day of prednisolone for ≥1 mo)
An induration of ≥10 mm
!
Recent immigrants (5 years) from high prevalence countries
Injection drug users
Residents and employees of high-risk congregate setting
Mycobacteriology laboratory personnel
Patient with clinical conditions that place them at high risk
Children 4 years of age
CDC. MMWR 2005; 54 (RR-17).
American Thoracic Society and CDC. Am J Respir Crit Care Med. 2000; 161.
POSITIVE
an induration
≥15 mm
48-72 hr after injection

Interferon-Gamma Release Assays (IGRAs)
QuantiFERON-TB Gold in-Tube T SPOT.TB
Measurement of a person’s immune
reactivity to M. tuberculosis
Do NOT help differentiate latent
tuberculosis (LTBI) from
tuberculosis disease
Routine testing with IGRA is NOT
recommended
Centers for Disease Control and Prevention. MMWR 2010; 59 (No.RR-5).

Characteristics of Commercially Available IGRAs
National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention
Division of Tuberculosis Elimination
(Page 1 of 3)
To conduct the tests, fresh blood samples are mixed
with antigens and controls. The antigens, testing
methods, and interpretation criteria for IGRAs differ
(see Table 1).
assay can decrease the accuracy of IGRAs.
t Limited data on the use of IGRAs to predict
who will progress to TB disease in the future.
Table1: Differences in Currently Available IGRAs
QFT–GIT T–Spot
Initial Process Process whole blood within 16 hours Process peripheral blood mononuclear
cells (PBMCs) within 8 hours, or if T-Cell
Xtend® is used, within 30 hours.
M. tuberculosis Antigen Single mixture of synthetic peptides
representing ESAT-6, CFP-10 and TB7.7
Separate mixtures of synthetic peptides
representing ESAT–6 and CFP-10
Measurement IFN-g concentration Number of IFN-g producing cells (spots)
Possible Results Positive, negative, indeterminate Positive, negative, indeterminate,
borderline
Centers for Disease Control and Prevention. MMWR 2010; 59 (No.RR-5).

Tuberculosis diagnostics

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