TB diagnostic tests—history v Microscopy 1880 s

TB diagnostic tests--history v • Microscopy (1880 s) v • Culture (1880 s) v • Chest x-ray (1930 s) v • Tuberculin skin test (Mantoux-1907; PPD- (1939) v • Nucleic acid amplification tests (1990 s) v • Interferon release assays (2000

Diagnosis of TB v The key to the diagnosis of tuberculosis is a high index of suspicion. v X-Ray v Skin Test v Direct demonstration of AFB in sample v • Growth of TB bacilli in culture

Role of Chest X-ray v No chest X-ray pattern is absolutely typical of TB. v 10 -15% of culture-positive TB patients not diagnosed by X-ray v 40% of patients diagnosed as having TB on the basis of x-ray alone do not have active TB

Administering Tuberculin Skin Test • • Purified protein derivative (PPD) TU PPD tuberculin. • Read reaction 48 -72 hours after injection • Measure only induration • Record reaction in millimet

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

AFB Smear Microscopy v Microscopy is a simple convenient test Requires minimal infrastructure and equipment v • Highly accurate, inexpensive and fast. v • Accessible to the majority of patients Prioritizes infectious cases

AFB Smear Microscopy Ø 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 Aurimine fluorescence Acid fast tissue Scanning electron micrograph of Mycobacterium tuberculosis

Limitations of Microscopy v Can not distinguish between dead or live bacteria. v • High bacterial load >3000– 5000 AFB/m. L is required for detection • Can not do species identification • v Can not perform Drug Sensitivity Test.

Culture Media main types v Egg = LJG, LJP, Stone brink, Ogawa v Agar = 7 H 10, 7 H 11, Blood v Liquid = Kirchner, 7 H 9, 7 H 12, Dubos v New Types= Bactec 460, MGIT, MB Bac. T. BACTEC 9000 MB system v Septi-Chek AFB system (Becton Dickinson)

Radiometric Technology The only well established rapid radiometric method for detecting mycobacteria in clinical specimens is the BACTEC 460 TB system (Becton-Dickinson Diagnostic Instruments Systems, Maryland). v This system is based on the detection of radioactive carbon-dioxide produced by bacterial metabolism of palmitic acid labelled with carbon 14. v 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%) v

Radiometric Technology v BACTEC system, which employs a superscript 14 C-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 v BACTEC method has provided more rapid growth (average, 9 -14 days), 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 7 H 9 broth. (8 -13 days) v 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. v Oxygen consumption by microorganisms can be detected by the increase in fluorescence. v

Non-Radiometric Technology § § § v 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 days

Cytokine Release Assays Quanti. FERON-TB GOLD test. Blood samples must be processed within 12 hours after collection while white blood cells are still viable. v followed by measurement of Interferon-gamma Assays released by sensitized lymphocytes in an enzyme-linked immunosorbent assay (ELISA). At present, the Quanti. FERON-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. v

Cytokine Release Assays v Quanti. FERON-TB GOLD test v Should not give false-positive result due to: v. BCG vaccination v. Nontuberculous mycobacteria. v. 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 v Mean time for Detection of MTB • Gene. Xpert = < day, Microscopy = 1 day, Liquid culture - MGIT = 17 days, Solid Culture = > 30 days • • • v • • • Mean time for Detection of Rifampicin Resistance Gene. Xpert = < 1 day Liquid DST = 30 days Conventional DST ( Solid proportional Method) = 75 days

How does the test work? • Detects DNA sequences specific for Mycobacterium Tuberculosis and Rifampicin resistance by PCR • Based on Nucleic Acid Amplification Test (NAAT). The Xpert® MTB/RIF v v purifies concentrates amplifies (by real-time PCR) and identifies targeted nucleic acid sequences in the Mycobacterium tuberculosis genome,

The Xpert MTB/RIF TB Test The Xpert MTB/RIF is a cartridge-based, automated diagnostic test that can identify Mycobacterium tuberculosis (MTB) and resistance to rifampicin (RIF). In December 2010 WHO endorsed the Xpert MTB/RIF technology and released a recommendation and guidance

Rapid tests immunochromatographic assays lateral-flow tests or simply strip tests v 1 - Add 40 µl of serum or plasma sample to the T (Test) area of the test card add 1 drop of sample to the T (Test) area. v 2 - Follow sample addition with 2 drops of the diluent provided in the dropper v bottle by holding the bottle vertically over the T (Test) Area. v 3 - Results are then read in as little as 20 minutes.

Rapid tests immunochromatographic assays lateral-flow tests (strip tests) tests 1 - Add 40 µl of serum or plasma sample to the T (Test) area of the test card add 1 drop of sample to the T (Test) area. 2 - Follow sample addition with 2 drops of the diluent provided in the Results are then read in as little as 20 minutes dropper

ESAT-6 and CFP 10 Mycobacterium tuberculosis-specific antigens (ESAT -6 and CFP 10) in experimental animals as well as during natural infection in humans and cattle. v combination of ESAT-6 and CFP 10 was found to be highly sensitive and specific for both in vivo and in vitro diagnosis. v In humans, the combination had a high sensitivity (73%) and a much higher specificity (93%) for active tuberculosis than PPD (7%). v

Enzyme-linked immunospot assay T-cell–based interferon-γ release assay The ELISpot. PLUS assay incorporates a novel region of difference-1 encoded antigen, Rv 3879 c, alongside the ESAT-6 and CFP 10. v ELISpot. PLUS sensitivity is 89% higher than that of the standard ELISpot. v The combined sensitivity of ELISpot. PLUS and tuberculin skin testing in confirmed and highly probable cases of TB was 99%. v

Serologic Diagnosis of Tuberculosis ELISA measurement of Ig antibody to mycobacterial antigens v Antigen 60 Ig. G measurement v Antigen 38 kda Ig. G Antigen Kp 90 Ig. A &measurement v Antigen 60 Ig. G seemed to be superior to the others (i. e. , the cutoff value was justified by both the sensitivity and the specificity. v Am. J. Respir. Crit. Care Med. , Volume 156, Number 3, September 1997, 906 -911

Serologic Diagnosis of Tuberculosis Antituberculous glycolipid antigen TBGL. v The lipoarabinomannan (LAM) polysaccharide antigen. v Antigen 60 (A 60), which is derived from purified protein derivatives. v The combination of LAM, A 60, and TBGL appears to be the best choice of antigens for the serodiagnosis of TB v

Chemical Detection of Biologic Compounds v Adenosine deaminase, a host enzyme Adenosine deaminase 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. v A more complicated technology detects the presence of tuberculostatic acid in the spinal fluid or serum of patients.

Gen-Probe AMPLIFIED TM v 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. v The NAA result can be falsely negative if there are very few tubercle bacilli, v NAA test can amplify DNA from both viable and non-viable organisms.

Gen-Probe AMPLIFIED TM v Polymerase chain reaction (PCR) v v v Yield 95% of smear+ and only 50% of smear negatives. main advantages: speed + sensitivity : v in principle able to pick up 1 TB bacillus v in practice : less sensitive than culture v v Serve only the diagnosis not monitor the treatment outcome Cannot replace culture Not able to determine infectiousness. Very expensive ($50 -$100 per assay)

Real-time Polymerase Chain Reaction Techniques v Real-time PCR methods are based on hybridization of amplified nucleic acids with fluorescent-labelled probes spanning DNA regions of interest and monitored inside thermal cyclers. v The main advantage of real-time PCR methods is its speed in giving results, 1. 5 -2. 0 h after DNA extraction.

Non-molecular Techniques The Fast. Plaque Tuberculosis Assay v The Fast. Plaque TB assay , relies on the ability of M. tuberculosis to support the growth of an infecting mycobacteriophage. The assay have shown a sensitivity of 50 -65% in smearnegative specimens with specificity of 98%. v It is a rapid, manual test, easy to perform and has a higher sensitivity than microscopy, in newly diagnosed smear +ve pts. Int J Tuberc Lung Dis 1998; 2: 160