Testing for TB Infection * Tuberculosis (TB) is a disease that is spread through the air from one person to another. There are two kinds of tests that are used to determine if a person has been infected with TB bacteria: the tuberculin skin test and other direct and indirect Tests .Diagnosis of TB Disease * People suspected of having TB disease should be referred for a medical evaluation, which will include Medical history, Physical examination, Test for TB infection (TB skin test or TB blood test), * Chest radiograph (X-ray), and * Appropriate laboratory testsTypes of specimens: -Sputum. - BAL. -Pleural effusions - Urine - Stool -CSF -Aspiration ( gastric — cold abscess) - Blood in case of haematogenous TBLaboratory Diagnosis 1- Sputum smears stained by Z-N stain Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB. Advantage: - cheap — rapid - Easy to perform ~ High predictive value > 90% - Specificity of 98% Disadvantages: - sputum ( need to contain 5000-10000 AFB/ ml.) - Young children, elderly & HIV infected persons may not produce cavities & sputum containing AFB.* Sputum specimens are essential to confirm TB — Specimens should be from lung secretions, not saliva * Collect 3 specimens on 3 different days * Spontaneous morning sputum more desirable than induced specimens * Collect sputum before treatment is initiatedInterpretation of sputum stained by ZN Stain (WHO ) More than 10 bacilli / field ------- +4++ From 1 — 10 bacilli / field ------- ++ From 10 — 99 bacilli / 100 fields ----- + From 1 -9 bacilli/100 fields ------ write the no. No bacilli seen ---------- negative2- Detecting AFB by fluorochrome stain using fluorescence microscopy: The smear may be stained by auramine-O dye. In this method the TB bacilli are stained yellow against dark background & easily visualized using florescent microscope. Advantages: - More sensitive - Rapid Disadvantages: - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stainCulture * Used to confirm diagnosis of TB * Culture all specimens, even if smear is negative * Initial drug isolate should be used to determine drug susceptibilityLaboratory Diagnosis 1- Sputum smears stained by Z-N stain Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB. Advantage: - cheap — rapid - Easy to perform - High predictive value > 90% - Specificity of 98% Disadvantages: - sputum ( need to contain 5000-10000 AFB/ ml.) - Young children, elderly & HIV infected persons may not produce cavities & sputum containing AFB.2- Detecting AFB by fluorochrome stain using fluorescence microscopy: The smear may be stained by aura mine-O dye. In this method the TB bacilli are stained yellow against dark background & easily visualized using florescent microscope. Advantages: - More sensitive - Rapid Disadvantages: - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stainLowenstein—Jensen medium * When grown on LJ medium, M. tuberculosis appears as brown, granular colonies (sometimes called “buff, rough and tough"). The media must be incubated for a significant length of time, usually four weeks, due to the slow doubling time of M. tuberculosis3- Cultures on L J media Lowenstein —Jensen medium is an egg based media with addition of salts, 5 % glycerol, Malachite green. Advantages: - Specificity about 99 % - More sensitive (need lower no. of bacilli 10-100 / ml) - Can differentiate between TB complex & NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St, INH, Rif., E) Disadvantages: Slowly growing ( up to 8 weeks )Detection and identification of mycobacteria directly from clinical samples Genotypic Methods : PCR LAMP TMA / NAA Ligase chain reaction Phenotypic Methods : FAST Plaque TB Boa 8 8 a 8Polymerase Chain Reaction (PCR) * Essentially PCR is a way to make millions of identical copies of a specific DNA sequence , which may be a gene, or a part of a gene, or simply a stretch of nucleotides with a known DNA sequence, the function of which may be unknownPolymerase Chain Reaction (PCR) & Gene probe Nucleic acid probes & nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells. Advantages: - Rapid procedure _—- High sensitivity (1-10 (3 —4 hours) bacilli / ml sputum)TB blood tests TB blood tests (also called interferon-gamma release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB. An IGRA measures how strong a person’s immune system reacts to TB bacteria by testing the person’s blood in a laboratory. Two IGRAs are approved by the U.S. Food and Drug Administration (FDA) and are available in the United StatesPositive IGRA * This means that the person has been infected with TB bacteria. Additional tests are needed to determine if the person has latent TB infection or TB disease. A health care worker will then provide treatment as needed. Qpmcone FY) seem Interferon Gamma Release Assays (|GRA) An Alternative to TST? + Princple: Measure interferon gama (FN) produced by sensitized T ctf slimulated by TB antigensNegative IGRA: * This means that the person’s blood Ps did not react to cea om -3- » the test and that eo = @= latent TB infection re he or TB disease is - @ not likely.