Changing approaches to the diagnosis of tuberculosis

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Changing approaches to the diagnosis of
tuberculosis

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The need for new diagnostic tests for tuberculosis

• The value of the the sputum smear exam for AFS
  has been deminished by the increasingly common
  phenomenon of Mycobacterium avium complex in
  elderly persons with cough and abnormal CXR, or
  with HIV.
• This has resulted in a marked decrease in the
  specificity and PPN of the smear, in some cases
  to as low as 50.

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The need for new diagnostic tests for tuberculosis

• A substantial percentage of TB cases, even in
  poor countries, are smear negative, and delayed
  diagnosis of such cases undoubtedly has a harmful
  effect on individual pts.
• The threshold for detecting bacilli on light
  microscopy is about 5000-10000 bacilli/mL, while
  the infecting dose of TB is estimated to be fewer
  than 10 organisms.

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The need for new diagnostic tests for tuberculosis

• Recent studies using restriction fragment length
  polymorphism (RLFP) analysis indicate that smear
  negative cases of TB contribute much more to
  ongoing transmission than has previously been
  believed. (17)

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New diagnostic tests- tests for active TB

• Broth-based culture systems.
• When combined with DNA probes for rapid species
  identification, are capable of producing positive
  results in 2 wks or less.
• Nucleic acid amplification (NAA) assays.
• There are currently 2 NAA assays available for
  commercial use in USA MTD (M. tuberculosis
  Direct) test, and Amplicor.

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Nucleic acid amplification assay

• With the use of amplificaiton systems, nucleic
  acid sequences unique to M tuberculosis can be
  detected directly in clinical specimens, offering
  better accuracy than AFB smear and greater speed
  than culture.

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Nucleic acid amplification assay

• Despite these different approaches to
  amplification of target DNA regions of interest,
  substantial published experience indicates that
  the tests are roughly equivalent in clinical use.
• Each test accurately diagnoses nearly every case
  of sputum smear() TB, and each will also
  diagnosis about half the cases of smear(-),
  culture() TB.

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Nucleic acid amplification assay

• The overall accuracy of the NAA assays was much
  higher than that of smear and not very much lower
  than that of culture.
• Newer versions of these tests also appear to have
  a significantly improved ability to detect
  smear(-) cases as well.
• In other words, in diagnosis of active TB, the
  answer will be correct 92-95 of the time using
  NAA, as compared with 80 of the time if smears
  are used.

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When to use NAA?

• It should be used to confirm a positive AFS.
• If smears (-), but clinical suspicion is high,
  NAA should be done on a sputum sample, either
  expectorated or induced.
• NAA should not be done on sputum samples from
  cases in which the clinical suspicion is low and
  the AFS is negative.

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CDC recommendation

• Smear () NAA ()
• TB is diagnosed with near total certainty.
• Smear () NAA (-)
• Presumed to have non-TB mycobacteria.
• Smear (-) NAA ()
• Additional sample. If ()? TB
• Smear (-) NAA (-)
• Additional NAA, if (-), the pt can be presumed
  not to have infectious TB.

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Rapid detection of drug resistance

• Rapid detection of rifampin resistance is
  technologically feasible by several approaches
  that examine either genotypic abnormalities or
  actual phenotypic resistance.

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Luciferase reporter gene assay

• In this assay, a sample is placed into medium and
  is then transfected with a lucerifase-containing
  mycobacterial phage. If viable M. tuberculosis is
  present in the sample, it will take up the phage
  and the luciferase gene with function, producing
  visible light when luciferin is added to the
  assay.

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Luciferase reporter gene assay

• Drug susceptibility test- inoculating the
  clinical sample into antibiotic containing
  medium.
• The current version of the assay, the Bronx
  Box, may be capable of detecting viable
  M.tuberculosis in as few as 2 days.

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Molecular beacons

• Molecular beacons are molecules that emit light
  when a chemical reaction occurs. This reaction
  will occur only when primers with DNA specificity
  bind their appropriate target region in PCR
  amplicons. In this way, rapid and sensitive
  diagnosis can be established.

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Molecular beacons

• Piatek and colleagues demonstrated both the
  sensitivity and specificity of this assay not
  only in making a diagnosis of TB, but also in
  rapidly identifying mutations associated with
  antibiotic resistance.

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Tests for latent tuberculosis infection

• Assays of interferon production by peripheral
  blood mononuclear cell
• T lymphocytes, both CD4 and CD8, are capable of
  producing the proinflammatory cytokine
  interferon-? in response to stimulation with M.
  tuberculosis.
• Peripheral blood mononuclear cells (PBMCs)
  separated from blood samples drawn from pts with
  known infection with M. tuberculosis can be
  simulated in vitro with PPD.

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Latent tuberculosis infection

• Production of interferon-? by PBMCs can then be
  measured by ELISA.
• Commercialy sold of the assay in Australia and
  elsewhere as Quantiferon indicates that this may
  be an accuratre method of detection of latent
  tuberculosis infection.

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Latent tuberculosis infection

• Compare tuberculin skin test (TST) and interferon
  release in populations well characterized for
  actual risk of TB, history of BCG vaccination and
  likelihood of exposure to M. avium.
• Result a total of 1226pts were included. Overall
  390 TST(), 349 interferon-? ().
• 83.1 of agreement and a ? statistic of 0.6.

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Latent tuberculosis infection

• TST (), interferon-? (-) were 6 times more
  likely to have received BCG vaccine than TST(),
  interferon-? (), suggesting that this assay may
  be able to discriminate between true infection
  and BCG vaccination.
• In addition, the assay displayed some ability to
  distinguish between infection with M.
  tuberculosis and NTM.

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Conclusion

• Although some tests may represent a significant
  advance in overall accuracy compared with AFS,
  and are much faster than cultures, cost and
  technician requirements have limited their
  adoption in many clinical settings.
• Future trials of novel diagnostics for latent
  infection and active disease should incorporate
  evaluation of clinical practice algorithms and
  assessments of cost effectiveness.