TB diagnostic tests--history
• Microscopy (1880s)• Culture (1880s)• Chest x-ray (1930s)• Tuberculin skin test (Mantoux-1907; PPD- 1939))• Nucleic acid amplification tests (1990s)• Interferon release assays (2000Diagnosis of TB
The key to the diagnosis of tuberculosis is a high index of suspicion. X-RaySkin TestDirect demonstration of AFB in sample• Growth of TB bacilli in cultureRole of Chest X-ray
No chest X-ray pattern is absolutely typical of TB. 10-15% of culture-positive TB patients not diagnosed by X-ray 40% of patients diagnosed as having TB on the basis of x-ray alone do not have active TBAdministering Tuberculin Skin Test
Purified protein derivative (PPD) TU PPD tuberculin. Read reaction 48-72 hours after injection Measure only induration Record reaction in millimetFactors that affect the PPD Reaction
Type of Reaction Possible Cause False-positive Nontuberculous mycobacteria BCG vaccination Anergy False-negative Recent TB infection Very young age (< 6 months old) Live-virus vaccination Overwhelming TB diseaseAFB Smear Microscopy
Microscopy is a simple convenient test Requires minimal infrastructure and equipment• Highly accurate, inexpensive and fast .• Accessible to the majority of patients Prioritizes infectious casesAFB Smear Microscopy
Fluorescence acid-fast staining is more expensive than conventional Ziehl–Neelsen staining but is associated with a higher rate of detection because the slides can be examined faster at lower magnifications.
M. Tuberculosis – the organism Acid fast sputum
Acid fast tissueAurimine fluorescence
Scanning electron micrograph of Mycobacterium tuberculosis
Limitations of Microscopy Limitations of Microscopy
Can not distinguish between dead or live bacteria .• High bacterial load >3000–5000 AFB 5000 AFB/mL is required for detection • Can not do species identification • Can not perform Drug Sensitivity Test.Culture Media main types
Egg = LJG, LJP, Stone brink, Ogawa Agar = 7H10, 7H11, Blood Liquid = Kirchner, 7H9, 7H12, Dubos New Types= Bactec 460, MGIT, MB BacT . BACTEC 9000 MB system Septi-Chek AFB system (Becton Dickinson)Radiometric Technology
The only well established rapid radiometric method for detecting mycobacteria in clinical specimens is the BACTEC 460TB system (Becton-Dickinson Diagnostic Instruments Systems, Maryland). This system is based on the detection of radioactive carbon-dioxide produced by bacterial metabolism of palmitic acid labelled with carbon 14. Growth of the mycobacteria can be detected within as few as 3 days, and the mean time to detect the M. tuberculosis complex is about 14 days (87-96%)Radiometric Technology
BACTEC system, which employs a superscript 14C-labeled substrate medium that is almost specific for mycobacteria. Instrument Systems, Sparks, Md. has been reported to significantly decrease the time required for detection of mycobacterial TB BACTEC method has provided more rapid growth (average, 9 -14days), specific identification of M. tuberculosis (5 days), and rapid drug susceptibility testing (6 days).Non-Radiometric Technology
BACTEC 9000 MB system (Becton Dickinson).This system uses MYCO/F medium, a modified Middlebrook 7H9 broth.(8-13 days) The system responds to changes in oxygen concentration. Each vial contains a silicon rubber disk, impregnated with a ruthenium metal complex, which serves as an oxygen-specific sensor. Oxygen quenches the fluorescent output of the sensor. Oxygen consumption by microorganisms can be detected by the increase in fluorescence.
Non-Radiometric Technology
BACTEC 960 MGIT ,MGITstands for Mycobacteria Growth Indicator Tube, and 960 indicates the total number of culture tubes it can hold at any given time Evaluation of mycobacteria recovery from the fluorometric BACTEC 960 and the radiometric BACTEC 460 TB system have shown that they are more sensitive in recovery of mycobacteria than the conventional L-J and smear microscopy. There is no significant difference between the radiometric BACTEC 460 TB and the fluorometric BACTEC 960 with 91.9% positivity and 95.1% positivity respectively. Results available in 7-14 daysCytokine Release Assays
QuantiFERON-TB GOLD test . Blood samples must be processed within 12 hours after collection while white blood cells are still viable. followed by measurement of Interferon-gamma Assays released by sensitized lymphocytes in an enzyme-linked immunosorbent assay (ELISA). At present, the QuantiFERON-Gold TB test is recommended for screening for latent tuberculosis infection . After incubation of the blood with antigens for 16 to 24 hours, The white blood cells will release IFN-gamma in response to contact with the TB antigens ,the amount of interferon-gamma (IFN-gamma) is measured.Cytokine Release Assays
QuantiFERON-TB GOLD testShould not give false-positive result due to:BCG vaccinationNontuberculous mycobacteria.The test’s performance may be enhanced by the use the Early Secreted Antigen Target -6 (ESAT-6 ) and Culture Filtrate Protien-10 (CPF-10).The Xpert MTB/RIF TB Test
Mean time for Detection of MTB GeneXpert = < day, Microscopy = 1 day, Liquid culture - MGIT = 17 days, Solid Culture = > 30 days Mean time for Detection of Rifampicin Resistance GeneXpert = < 1day Liquid DST = 30 days Conventional DST ( Solid proportional Method) = 75 daysHow does the test work?
The Xpert MTB/RIF TB TestThe Xpert MTB/RIF TB Test
Rapid tests immunochromatographic assays
Rapid tests immunochromatographic assays
Results are then read in as little as 20 minutes.lateral-flow tests (strip tests)
ESAT-6 and CFP10Mycobacterium tuberculosis-specific antigens (ESAT-6 and CFP10) in experimental animals as well as during natural infection in humans and cattle. combination of ESAT-6 and CFP10 was found to be highly sensitive and specific for both in vivo and in vitro diagnosis. In humans, the combination had a high sensitivity (73%) and a much higher specificity (93%) for active tuberculosis than PPD (7%).
Enzyme-linked immunospot assay
T-cell–based interferon-γ release assayThe ELISpotPLUS assay incorporates a novel region of difference-1 encoded antigen, Rv3879c, alongside the ESAT-6 and CFP10.ELISpotPLUS sensitivity is 89% higher than that of the standard ELISpot. The combined sensitivity of ELISpotPLUS and tuberculin skin testing in confirmed and highly probable cases of TB was 99%.Serologic Diagnosis of Tuberculosis
Serologic Diagnosis of TuberculosisAntituberculous glycolipid antigen TBGL. The lipoarabinomannan (LAM) polysaccharide antigen. Antigen 60 (A60), which is derived from purified protein derivatives. The combination of LAM, A60, and TBGL appears to be the best choice of antigens for the serodiagnosis of TB
Chemical Detection of Biologic Compounds
Adenosine deaminase, a host enzyme produced by activated T cells and easily detected by a colorimetric procedure, was shown to increase in concentration during the active stages of tuberculous meningitis and to decrease to normal levels after effective antituberculosis therapy. A more complicated technology detects the presence of tuberculostatic acid in the spinal fluid or serum of patients.Gen-Probe AMPLIFIED TM
The MTT&MTD are chemical tests, the amplification is to produce sufficient nucleic acid, within a few hours, these tests can recognize MTC in an AFB-positive specimen, with nearly 96% sensitivity and 100% specificity. The NAA result can be falsely negative if there are very few tubercle bacilli, NAA test can amplify DNA from both viable and non-viable organisms.