Diagnosis of Diagnosis of tuberculosistuberculosis

Diagnosis of tuberculosisDiagnosis of tuberculosis

Active disease Latent tuberculosisActive disease Latent tuberculosis

1. Clinical suspicion, 2. Response to treatment, 3. Chest radiographs,4. Staining for acid-fast bacilli

(AFB), 5. Culture for mycobacteria, 6. Nucleic acid amplification

(NAA) assays

1. Tuberculin skin test

2. Interferon γ

Active tuberculosisActive tuberculosisAn ideal test for active tuberculosis :An ideal test for active tuberculosis :1. Rapid results (available within 1 day), 2. High sensitivity and specificity, 3. Low cost, 4. Robustness (ability to provide reproducible results

in a variety of settings), 5. Highly automated or easily performed without the

need for excessive sample preparation or technical expertise,

6. Provide drug-susceptibility data.

6. The ideal test would be able to distinguish between LTBI and active disease:

--For LTBI, such a test would distinguish true infection from bacille Calmette-Guerin (BCG) vaccination and infection with nontuberculous mycobacteria (NTM).

--For active disease, it would be valuable to be able to determine infectiousness, follow response to therapy, distinguish Mycobacterium tuberculosis from NTM in AFB positive specimens and obtain drug-susceptibility information.

No ideal test for active No ideal test for active TB diagnosisTB diagnosis

Clinical algorithm for the laboratory Clinical algorithm for the laboratory diagnosis of tuberculosisdiagnosis of tuberculosis..

Infection with TB generally requires prolonged and close contact with a smear-positive individual. Infection is influenced by a variety of factors, including the number of bacilli present in the sputum of the smear-positive individual, the closeness of the relationship, the duration of exposure, the age of the contact (the risk is higher in children), and the immunological status of the contact.

TB cannot be distinguished from other infections on TB cannot be distinguished from other infections on the basis of clinical signs and symptoms alone.the basis of clinical signs and symptoms alone.

Most cases are characterized by an insidious, not very alarming, and quite variable onset that depends on the virulence of the causal pathogen, the patient’s age, the organ infected, and the host’s immune status. Symptoms can be classified into 2 groups:

1. Systemic symptoms. The most common systemic symptoms are fever, loss of appetite, weight loss, asthenia, profuse nocturnal sweating, and general malaise.

2. Organ specific symptoms.

Pulmonary tuberculosisPulmonary tuberculosis To establish a diagnosis of pulmonary

tuberculosis, respiratory samples are submitted to the laboratory for microscopy (AFB smear) and for mycobacterial culture.

Expectorated sputum is generally the starting point. Three samples are collected on three separate days and stained for AFB.

Samples are generally sent simultaneously for smear and culture, because culture data are essential for confirmation of the diagnosis.

AcidAcid--fast stainingfast staining:: The material to be examined on a glass slide and The material to be examined on a glass slide and

staining with carbolstaining with carbol--fuchsin by means of either fuchsin by means of either the Ziehlthe Ziehl--Neelsen or Kinyoun techniqueNeelsen or Kinyoun technique. .

The sensitivity of detection of organisms by acidThe sensitivity of detection of organisms by acid--fast smears is increased if the specimen is fast smears is increased if the specimen is digested and centrifuged and the concentrated digested and centrifuged and the concentrated sediment is stainedsediment is stained. .

Stained smears are usually examined under Stained smears are usually examined under standard brightstandard bright--light microscopy with oil light microscopy with oil immersion, a technique that can be performed immersion, a technique that can be performed under very primitive conditionsunder very primitive conditions..

The sensitivity of microscopic The sensitivity of microscopic examination is low; the level of examination is low; the level of detection is approximately 5000-detection is approximately 5000-10,000 bacilli per milliliter of 10,000 bacilli per milliliter of secretions, if 100 oil immersion secretions, if 100 oil immersion microscopic fields are examinedmicroscopic fields are examined..

In practice, 40% to 70% of patients In practice, 40% to 70% of patients with with M. tuberculosisM. tuberculosis isolated in isolated in culture have positive smears. culture have positive smears.

The sensitivity tends to be highest in patients who have cavitary disease and lowest in patients who have weak cough or less advanced disease.

Fluorochrome staining Fluorochrome staining procedureprocedure :: Sensitivity of detection of acidSensitivity of detection of acid--fast fast

organisms is increased by a organisms is increased by a fluorochrome staining procedure with fluorochrome staining procedure with auramine Oauramine O, a fluorescent stain, a fluorescent stain. .

This procedure requires use of a This procedure requires use of a fluorescent microscope but is faster than fluorescent microscope but is faster than acidacid--fast staining because the intensity fast staining because the intensity of the fluorescent signal enables slides of the fluorescent signal enables slides to be scanned at lower magnificationto be scanned at lower magnification..

In most situations in which an In most situations in which an acidacid--fast organism is detected by fast organism is detected by

microscopy, it should be microscopy, it should be assumed to beassumed to be MM . . tuberculosistuberculosis

until proven otherwise.until proven otherwise.

If a patient is suspected of having pulmonary tuberculosis but is smear negative on expectorated sputum or is unable to produce sputum for testing (30% of patients in one series), further diagnostic testing may be warranted.

The options include:1. Sputum induction (SI), 2. Fiberoptic bronchoscopy (FOB),3. Gastric washings (GW) “limited role in

adults”.

The utility of FOB (or SI):1. Generating a sample in patients who do

not have spontaneous sputum creates the potential for making a diagnosis.

2. Rapid diagnosis (by positive smear or histopathology) in either subset of patients provides the potential for earlier intervention and treatment while awaiting culture results.

Sputum induction Sputum induction provided

appropriate samples for diagnosis and increased the early diagnostic yield significantly. Sputum induction also seems to be cost-effective in the resource-poor setting.

Sputum induction Each successive sputum induction ,

up to three in total, increased the yield for culture-positive samples significantly. Sputum induction was performed without difficulty in 96% of patients and had an overall yield of 96.3% after three tests, confirming the utility of repeated sputum induction .

By contrast, the yield of fibrooptic bronchoscopy was only 51.9%,

making sputum induction significantly more sensitive.

Fibrooptic bronchoscopyFibrooptic bronchoscopy FOB encompasses BAL, bronchial

brushings, transbronchial biopsy, and postbronchoscopy sputum collection.

The most productive use of FOB is in pulmonary The most productive use of FOB is in pulmonary tuberculosis suspects who produce no sputum or tuberculosis suspects who produce no sputum or who are smear negative and in patients in whom who are smear negative and in patients in whom

there is considerable diagnostic uncertainty, there is considerable diagnostic uncertainty, where lung biopsy may produce an alternative where lung biopsy may produce an alternative

diagnosis.diagnosis.

Fibrooptic bronchoscopyFibrooptic bronchoscopy Overall, cultures of M. tuberculosis

from FOB were positive in 90%. More significantly, a rapid diagnosis was made in fully 72% of cases.

The bronchial brushing smears was a result of brushing caseous material in the bronchi when visible and had a high diagnostic yield.

CulturesCultures Cultures of mycobacteria require only 10

to 100 organisms to detect M. tuberculosis. the sensitivity of culture is excellent,

ranging from 80% to 93%. the specificity is quite high, at 98%. Cultures increase the sensitivity for

diagnosis of M. tuberculosis and allow speciation, drug-susceptibility testing, and, if needed, genotyping for epidemiologic purposes

There are three types of culture media:

1. Solid media, which includes A. egg-based media (Lowenstein-Jensen) B. agar-based media (Middlebrook 7H10

and 7H11), 2. Liquid media (Middlebrook 7H12

and other broths).

Solid media: the standard.the standard. slower than liquid media.slower than liquid media. widely used alongside solid media to widely used alongside solid media to

increase sensitivity and decrease increase sensitivity and decrease recovery time.recovery time.

Lowenstein-Jensen 7H10 and 7H11 Lowenstein-Jensen 7H10 and 7H11 media may detect mycobacteria in less media may detect mycobacteria in less than 4 weeks but they require incubation than 4 weeks but they require incubation for as long as 6 to 8 weeks before they for as long as 6 to 8 weeks before they can be classified as negative.can be classified as negative.

Broth media:Broth media: broth media combined with DNA broth media combined with DNA

probes for rapid species identification probes for rapid species identification typically provide results in less than 2 typically provide results in less than 2 weeks with smear positive samples weeks with smear positive samples and somewhat longer with smear and somewhat longer with smear negative samples.negative samples.

They allow more rapid determination They allow more rapid determination of drug susceptibilities.of drug susceptibilities.

Newer culture Newer culture technologies:technologies: TK MediumTK Medium uses multiple-color dye uses multiple-color dye

indicators to identify M. tuberculosis indicators to identify M. tuberculosis rapidly.rapidly.

It can also be used for drug-It can also be used for drug-susceptibility testing and can susceptibility testing and can differentiate a contaminated differentiate a contaminated specimen.specimen.

Radiometric and Colorimetric Radiometric and Colorimetric Detection SystemsDetection Systems

Radiometric culture systems (Radiometric culture systems (the BACTEC)the BACTEC) incorporate incorporate 1414C-labeled palmitic acid into a C-labeled palmitic acid into a liquid culture medium.liquid culture medium.

Growth of mycobacteria results in liberation Growth of mycobacteria results in liberation of of 1414CO2 that can be measured by the CO2 that can be measured by the detection device. The increased sensitivity detection device. The increased sensitivity of the system enables growth to be detected of the system enables growth to be detected sooner, usually in 10 to 14 days. sooner, usually in 10 to 14 days.

A positive niacin production test or DNA A positive niacin production test or DNA probe testing confirms an isolate as being probe testing confirms an isolate as being M. tuberculosisM. tuberculosis. .

ChromatographyChromatography High-performance liquid chromatography High-performance liquid chromatography

(HPLC) is a rapid and highly specific (HPLC) is a rapid and highly specific method for detecting the unique pattern method for detecting the unique pattern of mycolic acids for identifying of mycolic acids for identifying mycobacterial species.mycobacterial species.

Tuberculostearic acidTuberculostearic acid, a component of , a component of M. tuberculosis, can be easily detected M. tuberculosis, can be easily detected even in infinitesimal (femtomole) even in infinitesimal (femtomole) quantities by gas–liquid chromatography quantities by gas–liquid chromatography (GLC).(GLC).

Nucleic Acid Amplification Nucleic Acid Amplification TestsTests

In each test, either DNA or ribonucleic In each test, either DNA or ribonucleic acid (RNA) is amplified to detectable acid (RNA) is amplified to detectable levels. levels.

Nucleic acid amplification tests include Nucleic acid amplification tests include those that involve PCR amplification, those that involve PCR amplification, transcriptiontranscription--mediated amplification, mediated amplification, strandstrand--displacement amplification, ligase displacement amplification, ligase chain reaction, and Q Beta replicase chain reaction, and Q Beta replicase amplification.amplification.

NAA assays are also quite specific for M. tuberculosis, with specificities in the range of 98% to 99%.

Two FDA-approved NAA assays are widely available for commercial use:

1. The AMPLICOR M. tuberculosis uses DNA polymerase chain reaction (PCR) to amplify nucleic acid targets.

2. The Amplified Mycobaterium Tuberculosis Direct (MTD) Test is an isothermal strategy for detection of M. tuberculosis rRNA.

Both used on smear positive sample with a sensitivity of 70-90% and specificity of 93-99% (up to 100% in some studies).

The E-MTD (enhanced MTD test) brings with it an improved sensitivity , especially in smear-negative specimens.

In smear negative patients, the sensitivity was 90.2%, and the specificity was 99.1%

Amplification systems can detect as few as Amplification systems can detect as few as 1–101–10 organismsorganisms in clinical specimens and in clinical specimens and clearly distinguishes M. Tuberculosis in the clearly distinguishes M. Tuberculosis in the specimens containing nucleic acids from specimens containing nucleic acids from human cells, other mycobacterial species human cells, other mycobacterial species and common contaminating organisms.and common contaminating organisms.

These amplification techniques provide These amplification techniques provide results in less than a day and have been used results in less than a day and have been used for screening a variety of clinical specimens.for screening a variety of clinical specimens.

The major limitation of NAA tests, they can The major limitation of NAA tests, they can not detect drug sensitivity.not detect drug sensitivity.

Polymerase chain Polymerase chain reaction reaction ((PCRPCR))

PCR targets DNA, rRNA, insertion PCR targets DNA, rRNA, insertion and repetitive elements, and various and repetitive elements, and various protein-encoding genes.protein-encoding genes.

The PCR amplification process can The PCR amplification process can be completed in 2–4 h after be completed in 2–4 h after obtaining the processed clinical obtaining the processed clinical sample. The detection assay requires sample. The detection assay requires an additional 2–24 h.an additional 2–24 h.

Polymerase chain Polymerase chain reaction reaction ((PCRPCR))

Disadvantages: Disadvantages: 1.1. PCR tests are currently expensive.PCR tests are currently expensive.2.2. the PCR tests are unable to the PCR tests are unable to

differentiate between viable and the differentiate between viable and the dead AFB, which may lead to dead AFB, which may lead to disagreement between the sputum disagreement between the sputum smear and PCR results.smear and PCR results.

3.3. PCR studies show unacceptable level PCR studies show unacceptable level of sensitivity in smear negative but of sensitivity in smear negative but culture-positive cases.culture-positive cases.

False positive PCR results, usually due to False positive PCR results, usually due to laboratory introduced contamination.laboratory introduced contamination.

Polymerase chain Polymerase chain reaction reaction ((PCRPCR))

Effectiveness of the PCR technique in Effectiveness of the PCR technique in diagnosing tuberculosis is related to: diagnosing tuberculosis is related to:

(a) DNA concentration in the clinical sample(a) DNA concentration in the clinical sample(b) size of the target DNA sequence, (b) size of the target DNA sequence, (c) repetitiveness of the amplified sequence, (c) repetitiveness of the amplified sequence, (d) choice of primers, and (d) choice of primers, and (e) expertise of the personnel conducting (e) expertise of the personnel conducting

the assay.the assay.

TranscriptionTranscription--mediated mediated amplification amplification ((TMATMA)) “MTD “MTD

test”test” Isothermal, target-based Isothermal, target-based

amplification system is based on amplification system is based on amplification of ribosomal RNA amplification of ribosomal RNA (rRNA) unlike PCR, which is based (rRNA) unlike PCR, which is based on amplification of DNA.on amplification of DNA.

Probes that target rRNA are 10- to Probes that target rRNA are 10- to 100 fold more sensitive than those 100 fold more sensitive than those that target DNA.that target DNA.

TranscriptionTranscription--mediated mediated amplification amplification ((TMATMA))

the amplified M. tuberculosis direct (MTD) the amplified M. tuberculosis direct (MTD) test is entirely done entirely in one test test is entirely done entirely in one test tube, which minimizes laboratory-tube, which minimizes laboratory-introduced contamination. introduced contamination.

The entire test may be completed in 3–4 h The entire test may be completed in 3–4 h after obtaining the processed clinical after obtaining the processed clinical specimen. specimen.

But like PCR, the MTD test lacks But like PCR, the MTD test lacks sensitivity in the smear-negative but sensitivity in the smear-negative but culture positive cases.culture positive cases.

A reasonable use of NAA assays for rapidA reasonable use of NAA assays for rapiddiagnosis of pulmonary tuberculosis is as followsdiagnosis of pulmonary tuberculosis is as follows::

NAA assays should be used to confirm that a positive AFB smear does indeed represent M. tuberculosis.

1. If both smear and NAA are positive, pulmonary tuberculosis is diagnosed with near certainty.

2. If the smear is positive and the NAA is negative, testing the sputum for inhibitors and repeating the assay is warranted. If inhibitors are not detected, and the process is repeated on additional specimens and is negative, the patient can be presumed to have NTM

If a sputum sample is smear negative (with clinical suspicion is intermediate or high)) but E-MTD “ enhanced Mycobaterium Tuberculosis Direct” positive (only the E-MTD is approved for smearnegative specimens), it is recommended testing additional samples. If further samples are E-MTD positive, the patient can be assumed to have pulmonary tuberculosis.

4. If both the smear and E-MTD are negative, an additional specimen should be tested by E-MTD. If negative, the patient can be assumed not to have infectious pulmonary tuberculosis.

NAA should not be performed on sputum samples from cases in which the AFB smear is negative and the clinical index of suspicion is low.

Testing should also be limited to those who have not been treated recently for active disease. ((NAA tests are able to NAA tests are able to detect nucleic acids from both living and detect nucleic acids from both living and dead organisms and may be falsely positive).dead organisms and may be falsely positive).

Other NAA testsOther NAA testsStrand displacement Strand displacement amplification amplification ((SDASDA))

SemiSemi--quantitative isothermal in vitro nucleic quantitative isothermal in vitro nucleic acid amplification technique.acid amplification technique.

The entire assay may be completed within four The entire assay may be completed within four hours after the receipt of the processed clinical hours after the receipt of the processed clinical specimen. specimen.

The amplification is carried out in a single tube The amplification is carried out in a single tube (multiplexing), and the products can be (multiplexing), and the products can be differentiated by the detection system, without differentiated by the detection system, without significant loss of sensitivity.significant loss of sensitivity.

Species-specific SDA assays (for M. Species-specific SDA assays (for M. tuberculosis, M. avium, and M. kansasii) and a tuberculosis, M. avium, and M. kansasii) and a genus-specific assay have also been developed.genus-specific assay have also been developed.

QQ--beta replicase beta replicase amplificationamplification

Q-Beta replicase enzyme is used for Q-Beta replicase enzyme is used for producing RNA in the amplification producing RNA in the amplification reaction at a fixed temperature.reaction at a fixed temperature.

M. tuberculosis is detected with a M. tuberculosis is detected with a sensitivity of up to one colony-sensitivity of up to one colony-forming unit.forming unit.

Ligase chain reaction Ligase chain reaction ((LCRLCR))

LCR, a variant of PCR, is potentially useful for LCR, a variant of PCR, is potentially useful for screening persons at high risk for developing screening persons at high risk for developing tuberculosis and extrapulmonary tuberculosis.tuberculosis and extrapulmonary tuberculosis.

The LCx M. tuberculosis assay (Abbot), The LCx M. tuberculosis assay (Abbot), primarily makes use of the respiratory primarily makes use of the respiratory specimens. It has a high sensitivity and specimens. It has a high sensitivity and specificity for smear-positive and -negative specificity for smear-positive and -negative specimens.specimens.

Currently, the use of this technique is limited Currently, the use of this technique is limited by its high cost and lack of skilled personnel.by its high cost and lack of skilled personnel.

Rapid detection of drug resistance

1.1. Line probe assays.Line probe assays.2.2. Molecular beacons.Molecular beacons.3.3. Phage amplification.Phage amplification.4.4. Luciferase reporter phages.Luciferase reporter phages.

Pulmonary disease is suspectedPulmonary disease is suspected 1. Three induced or spontaneous (respiratory isolation) a.m. 1. Three induced or spontaneous (respiratory isolation) a.m.

sputum samples for AFB staining and microscopy;sputum samples for AFB staining and microscopy;- 5 to 10 mL each, accept watery specimens- 5 to 10 mL each, accept watery specimens

2. One sputum sample for MTb complex PCR;2. One sputum sample for MTb complex PCR;3. Same three sputum samples for culture;3. Same three sputum samples for culture;4. If effusion is present, consider sending diagnostic thoracentesis 4. If effusion is present, consider sending diagnostic thoracentesis

sample for smear, culture, PCR, and adenosine deaminase sample for smear, culture, PCR, and adenosine deaminase (ADA) testing- pleural biopsy has higher culture sensitivity;(ADA) testing- pleural biopsy has higher culture sensitivity;

5.Consider early morning gastric aspiration of 50 mL in children 5.Consider early morning gastric aspiration of 50 mL in children or failed sputum inductions; and,or failed sputum inductions; and,

6.Consider an IFN based assay on serum or Tuberculin Skin 6.Consider an IFN based assay on serum or Tuberculin Skin Test- negative test does not exclude disease.Test- negative test does not exclude disease.

Other than pulmonary disease Other than pulmonary disease suspectedsuspected

1.Samples as you would for suspected pulmonary 1.Samples as you would for suspected pulmonary disease; and,disease; and,

2.Sample from suspected site. For tissue collections, 2.Sample from suspected site. For tissue collections, collect both with and without formalin.collect both with and without formalin.

a. a. Lymph nodeLymph node-excisional biopsy preferred-excisional biopsy preferred-fine needle aspiration if unable to obtain excisional -fine needle aspiration if unable to obtain excisional

biopsy, e.g. CT guided biopsy in setting of mesenteric biopsy, e.g. CT guided biopsy in setting of mesenteric adenitisadenitis

-AFB smear and culture-AFB smear and culture-histopathologic analysis-histopathologic analysis

Other than pulmonary disease Other than pulmonary disease suspected (cont.)suspected (cont.)

b. b. CSFCSF-cell count and differential-cell count and differential- chemistries to include glucose and protein- chemistries to include glucose and protein- AFB smear (typically negative) and culture (10 mL often - AFB smear (typically negative) and culture (10 mL often

required)required)- consider PCR- consider PCRc. c. Pleural, pericardial, or ascitic fluidPleural, pericardial, or ascitic fluid-cell count and differential-cell count and differential- chemistries to include glucose and protein- chemistries to include glucose and protein-AFB smear and culture (lining biopsies have higher yield)-AFB smear and culture (lining biopsies have higher yield)-consider ADA analysis-consider ADA analysis-PCR-PCR

Other than pulmonary disease Other than pulmonary disease suspected (cont.)suspected (cont.)

d. d. Other tissues (bone marrow, skin, liver, spleen, ..., kidney)Other tissues (bone marrow, skin, liver, spleen, ..., kidney)- AFB smear and culture- AFB smear and culture- histopathologic analysis, may consider specialized AFB staining, and subsequent - histopathologic analysis, may consider specialized AFB staining, and subsequent

targeted PCRtargeted PCR-PCR-PCRee. . BloodBlood- Culture - PCR- Culture - PCRff. . StoolStool, consider intestinal biopsy, consider intestinal biopsy- Culture - PCR- Culture - PCRg. g. Urine (first morning mid-void stream collection), three daily samplesUrine (first morning mid-void stream collection), three daily samples- urinalysis and conventional culture- urinalysis and conventional culture- Culture - PCR- Culture - PCR

Latent Latent tuberculosistuberculosis

11 . .Tuberculin skin testTuberculin skin test Latent TB until very recently, has been diagnosed

exclusively by the tuberculin skin test (TST). The tuberculin skin test has both poor sensitivity

and specificity. The standard test: “the intracutaneous injection

(Mantoux's method) of 0.1 mL (5 TU) of PPD”

Tuberculin purified protein derivative (PPD)-RT23 with Tween 80 administered at a dose of 2 tuberculin units (TU) per 0.1 mL, which is biologically equivalent to the recommended dose (5 TU) of the international standard tuberculin (PPD-S).

Interpretation of Tuberculin Interpretation of Tuberculin Skin Test Reactions:Skin Test Reactions: ≥≥5 mm: 5 mm: Known or suspected HIV infection or Known or suspected HIV infection or

other immunosuppressed condition other immunosuppressed condition  Recent close contact with infectious Recent close contact with infectious

tuberculosis tuberculosis  Chest film abnormalities suggestive Chest film abnormalities suggestive

of old tuberculosisof old tuberculosis

Interpretation of Tuberculin Skin Interpretation of Tuberculin Skin Test Reactions:Test Reactions:≥≥10 mm10 mm Persons born in high-prevalence areasPersons born in high-prevalence areas Injection drug users Injection drug users  Medically underserved, low-income Medically underserved, low-income

populations: high-risk minority populations populations: high-risk minority populations  Residents of long-term care facilities Residents of long-term care facilities

(nursing homes, correctional facilities, etc.) (nursing homes, correctional facilities, etc.)  Persons with medical conditions that Persons with medical conditions that

increase the risk of tuberculosisincrease the risk of tuberculosis≥≥15 mm15 mm All others All others

Potential Causes of FalsePotential Causes of False--Negative Negative Tuberculin Skin TestTuberculin Skin Test

Factors Releated to the Person Being TestedFactors Releated to the Person Being Tested Concurrent infections Concurrent infections  Human immunodeficiency virus Human immunodeficiency virus  Other viral infections (measles, mumps, chickenpox)  Other viral infections (measles, mumps, chickenpox)   Bacterial diseases (typhoid fever, brucellosis, typhus, Bacterial diseases (typhoid fever, brucellosis, typhus,

leprosy, pertussis, overwheiming tuberculosis)  leprosy, pertussis, overwheiming tuberculosis)   Live virus vaccination (measles, mumps, polio) Live virus vaccination (measles, mumps, polio) Diseases affecting lymphoid organs (Hodgkin's disease, Diseases affecting lymphoid organs (Hodgkin's disease,

lymphoma, chronic lymphocytic leukemia, sarcoidosis) lymphoma, chronic lymphocytic leukemia, sarcoidosis) Immunosuppressive drugs (corticosteroids and many Immunosuppressive drugs (corticosteroids and many

others) others) Age (newborns, elderly) Age (newborns, elderly) Metabolic conditions (advanced renal failure, Metabolic conditions (advanced renal failure,

malnutrition)malnutrition) Recent surgeryRecent surgery BurnsBurns

Factors Related to the TestFactors Related to the Test Loss of antigen potency (exposure to Loss of antigen potency (exposure to

heat and/or light)heat and/or light) Incorrect administration (too little Incorrect administration (too little

antigen, deep injection)antigen, deep injection) Incorrect reading (inexperienced Incorrect reading (inexperienced

reader, reader bias, recording error)reader, reader bias, recording error)

2. Interferon 2. Interferon γγ They include : They include : 1.1. QuantiFERONQuantiFERON--TB, TB, 2.2. QuantiFERONQuantiFERON--TB Gold, TB Gold, 3.3. T SPOTT SPOT--TB test.TB test. The basis of these tests is the detection in serum of The basis of these tests is the detection in serum of

either the release of IFN-either the release of IFN-γγ on stimulation of on stimulation of sensitized T cells by M. tuberculosis antigens in sensitized T cells by M. tuberculosis antigens in vitro (QuantiFERON) or detection of the T cells vitro (QuantiFERON) or detection of the T cells themselves (T SPOT-TB).themselves (T SPOT-TB).

QuantiFERONQuantiFERON--TB Gold:TB Gold: The ELISA-based QuantiFERON TB

test and the RD1 antigens, ESAT-6 (the early secreted antigenic target 6) and CFP-10 (culture filtrate protein-10), led to a more specific test.

T SPOTT SPOT--TB test: “TB test: “ELISPOT technique” In healthy BCG-vaccinated controls,

the ELISPOT was not confounded by BCG. In TST-positive household contacts of a case of active disease (expected cases of LTBI), 85% were ELISPOT positive, suggesting a sensitivity for LTBI of 85% if the TST is taken to be the reference standard.