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    Lab Diagnosis New

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    devikarenjith.12a2
    This document provides information on the diagnosis of tuberculosis (TB). It discusses specimen collection methods, staining and microscopy techniques for direct visualization of acid-fast bacilli, conventional and automated culture methods considered the gold standard for diagnosis, and molecular diagnostic methods like PCR and gene sequencing. Newer techniques like interferon-gamma release assays and gene sequencing are more sensitive and specific than older tests like tuberculin skin tests for diagnosis of latent TB infection. Proper specimen collection and processing are important for maximizing diagnostic yield across these methods.

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    Lab Diagnosis New

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    devikarenjith.12a2
    17 views42 pages
    This document provides information on the diagnosis of tuberculosis (TB). It discusses specimen collection methods, staining and microscopy techniques for direct visualization of acid-fast bacilli, conventional and automated culture methods considered the gold standard for diagnosis, and molecular diagnostic methods like PCR and gene sequencing. Newer techniques like interferon-gamma release assays and gene sequencing are more sensitive and specific than older tests like tuberculin skin tests for diagnosis of latent TB infection. Proper specimen collection and processing are important for maximizing diagnostic yield across these methods.

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    This document provides information on the diagnosis of tuberculosis (TB). It discusses specimen collection methods, staining and microscopy techniques for direct visualization of acid-fast bacilli, conventional and automated culture methods considered the gold standard for diagnosis, and molecular diagnostic methods like PCR and gene sequencing. Newer techniques like interferon-gamma release assays and gene sequencing are more sensitive and specific than older tests like tuberculin skin tests for diagnosis of latent TB infection. Proper specimen collection and processing are important for maximizing diagnostic yield across these methods.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    17 views42 pages

    Lab Diagnosis New

    Uploaded by

    devikarenjith.12a2
    This document provides information on the diagnosis of tuberculosis (TB). It discusses specimen collection methods, staining and microscopy techniques for direct visualization of acid-fast bacilli, conventional and automated culture methods considered the gold standard for diagnosis, and molecular diagnostic methods like PCR and gene sequencing. Newer techniques like interferon-gamma release assays and gene sequencing are more sensitive and specific than older tests like tuberculin skin tests for diagnosis of latent TB infection. Proper specimen collection and processing are important for maximizing diagnostic yield across these methods.

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    of 42

    LAB

    DIAGNOSIS
    TUBERCULOSIS
    DIAGNOSIS
    ● Specimen Collection
    ● Digestion, Decontamination & Concentration
    ● Direct Microscopy
    ● Conventional Culture Media
    ● Automated Culture Methods
    ● Culture Identification
    ● Molecular methods
    ● Diagnosis of latent TB
    SPECIMEN COLLECTION
    ● In Pulmonary TB, two sputum samples are
    recommended—spot sample and early morning
    sample.

    ● Precautions:
    ○ Collection should be done open well ventilated
    space
    ○ Early morning sputum should be collected in empty
    stomach, after rinsing the mouth well
    ○ Patient should be advised to inhale deeply (2-3
    times) and cough out deep from the chest

    ● Quality:
    ○ Sputum should be at least 3-5 mL in quantity
    ○ Thick and purulent (yellowish mucus)
    CSF

    Pleural Fluid

    Synovial Fluid
    Urine
    DIGESTION, DECONTAMINATION &
    CONCENTRATION

    ● Digestion (to liquefy the thick pus cells and


    homogenization)
    ● Decontamination (to inhibit the normal flora)
    ● Concentration (to increase the yield)
    ● Not required for molecular methods
    ● Not required for processing of extrapulmonary
    specimens collected aseptically from sterile sites
    ● Modified Petroff’s method (4% NaOH)
    (recommended for LJ culture)

    ● NALC (N-acetyl-L-cysteine) + 2% NaOH


    DIRECT MICROSCOPY
    Ziehl Neelsen - Acid Fast Staining

    M. tuberculosis appears as long slender, beaded, less


    uniformly stained red colored acid-fast bacilli
    ● Microscopy- Presumptive diagnosis
    ● Advantages: Smear microscopy is rapid, easy to
    perform at peripheral laboratories and is cheaper
    ● Disadvantages:
    (i) Smear microscopy is less sensitive than culture
    (ii) Low sensitivity with a detection limit of 10,000
    bacilli/mL of sputum
    (iii) It cannot determine the viability of bacilli
    NTEP Grading

    ●
    .
    ● NTEP grading is useful for:
    ○ Monitoring the treatment response of the patients
    ○ Assessing the severity of disease
    ○ Assessing the infectiousness of the patient: Higher
    the grade more is the infectiousness. Smear
    negative patients (<10,000 bacilli/mL of sputum) are
    less infectious
    Kinyoun’s Cold Acid-fast Staining

    ● It differs from ZN stain in that


    (i) heating is not required
    (ii) phenol concentration in carbol fuchsin is increased
    (iii) duration of carbol fuchsin staining is more
    Fluorescence (Auramine) Staining

    The bacilli appear brilliant yellow against the dark


    background
    ● Advantages-
    ○ Smears are screened by using 20× or 25×
    objective, hence can be screened faster
    ○ It is more sensitive than ZN staining and has been
    the recommended screening method by NTEP

    ● Disadvantages-
    ○ Artifacts may confound with the interpretation.
    CULTURE METHODS

    ● Culture is traditionally considered as the gold standard


    method of diagnosis of TB.
    ● Advantages:
    ○ It is more sensitive than microscopy with the
    detection limit of 10–100 viable bacilli
    ○ Indicates viability
    Conventional Solid Media
    (Lowenstein-Jensen Medium)

    Colonies: M. tuberculosis produces typical


    rough, tough and buff-colored colonies
    ● It is composed of coagulated hen’s eggs, mineral salt
    solution, asparagine and malachite green (as a
    selective agent)
    ● Inoculated media are incubated for a prolonged duration
    of 6–8 weeks.
    ● M. bovis produces smooth, moist and white colored
    colonies that break up easily when touched
    Automated Liquid Culture

    Middlebrook 7H9 Medium


    ● Offer a faster turnaround time compared to
    conventional culture.
    ● Positive growth (99%) gets detected within 3–4 weeks.
    ● However, the negative result is reported after 6 weeks
    of incubation
    ● Medium is supplemented with enriched growth media
    and antibiotic mixture (to inhibit other organisms).
    Automated systems
    -

    MGIT liquid BACTEC MGIT BacT/ALERT


    culture
    ● BACTEC MGIT (Mycobacteria growth indicator tube):
    ○ This is the automated system endorsed by WHO and
    NTEP
    ○ It detects growth of mycobacteria
    ○ Also performs the drug susceptibility testing
    against first-line and second-line antitubercular drugs
    ● Other automated systems include BacT/ALERT systems
    CULTURE IDENTIFICATION

    MALDI-TOF
    ● The colonies grown on LJ media and the broth from a
    positively flagged automated culture bottle are first
    subjected to acid-fast stain. If found AFB positive,
    then further tests are done for species identification
    ● MPT 64 antigen detection by rapid
    immunochromatographic test
    ● Automated identification system such as MALDI-TOF
    MOLECULAR METHODS

    ● Advantages:
    ○ They take less time than culture
    ○ They are more sensitive than culture.
    ○ They can also detect the genes coding for drug
    resistance
    ○ Used for epidemiological typing of strains
    ● Polymerase Chain Reaction (PCR)
    Nested PCR targeting IS6110 gene was the most
    common molecular test used earlier.
    ● Automated Real Time PCR
    1. Cartridge-based nucleic acid amplification test
    (CBNAAT): e.g. GeneXpert
    2. Chip-based real-time PCR: e.g. Truenat.
    GeneXpert (CBNAAT)
    ● Rapid: It has the lowest turnaround time (2 hours)
    ● Principle: It is based on real-time PCR technique, it uses
    five probes targeting various sequences of rpoB gene
    ● Diagnostic utility: The detection limit of GeneXpert is
    about 131 bacilli/mL of specimen
    ● Disadvantages:
    ○ Very expensive
    ○ Cannot further speciate MTB complex
    ● Compared to culture, the sensitivity and specificity are as
    follows:
    ○ For detection of TB bacilli: It is 88% sensitive and
    99% specific
    ○ For detection of rifampicin resistance: It is 95%
    sensitive and 98% specific.
    ● EPTB: WHO recommends GeneXpert as the initial test
    for diagnosis of EPTB
    ● Next generation GeneXpert (Xpert Ultra): It was
    endorsed by WHO in 2017
    ○ It contains two additional molecular targets to detect
    TB (IS6110, IS1081)
    ○ It is more sensitive and specific than GeneXpert, with
    a detection limit of 16 bacilli/mL
    Chip Based NAAT (Truenat)
    ● Advantages:
    ○ It is an automated battery operated device; can be
    used at level of primary health center where
    GeneXpert cannot be used as it needs
    uninterrupted power supply and air conditioning
    ○ The turnaround time is around one hour
    ● Disadvantages:
    ○ Very expensive
    ○ Cannot further speciate MTB complex
    ○ It tests only limited samples (1-4) at a time
    Line Probe Assay (LPA)
    ●
    ● Line probe assay involves probe-based detection of
    amplified DNA in the specimen.
    ● Uses:
    ○ Identification of MTB complex
    ○ Detection of resistance to first-line and second line
    antitubercular drugs
    ○ Speciation of MTB complex and NTM
    ● Limitation: LPA can be performed only on positive
    cultures or smear positive clinical specimens.
    ● LPA is useful particularly in isoniazid mono-resistant
    cases of TB
    DIAGNOSIS OF LATENT TB
    ● Latent tuberculosis is diagnosed by demonstration of
    delayed or type IV hypersensitivity reaction against
    the tubercle bacilli antigens.
    ● Two methods are available-
    (1) Tuberculin skin test
    (2) IFN-γ release assay
    Tuberculin Skin Test (TST)
    ● Antigens: PPD-23
    ● Mantoux test is the most commonly employed method. 0.1 mL of
    PPD containing 1 TU is injected intradermally into flexor surface
    of forearm
    ● Reading: It is taken after 48–72 hours. At the site of inoculation,
    an induration surrounded by erythema is produced. If the width of
    the induration is:
    ○ ≥10 mm: Positive (tuberculin reactors)
    ○ 6–9 mm: Equivocal/doubtful reaction
    ○ <5 mm: Negative reaction
    ● Adults: Indicates present or past exposure with tubercle
    bacilli
    ● Children: A positive test indicates active infection and
    used as a diagnostic marker

    ● Prevalence of tuberculosis is calculated by counting all


    tuberculin reactors in a community

    ● Incidence of tuberculosis is calculated by counting new


    converters to TST in a community
    ● False-positive:
    ○ BCG vaccination (after 8–14 weeks)
    ○ Nontuberculous mycobacteria infection

    ● False-negative:
    ○ Early or advanced TB
    ○ Miliary TB
    ○ Decreased immunity (HIV-infected people)
    Interferon Gamma Release Assay (IGRA)
    ● This uses highly specific M. tuberculosis antigens such
    as CFP10 (culture filtrate protein) and ESAT6 (early
    secreted antigenic target-6); both coded by RD1 genes

    ● Advantage: It is highly specific; there are no false


    positive results
    THANK YOU

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