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Laboratory Diagnostic

Techniques

Hugo Donaldson

Consultant Microbiologist

Imperial College Healthcare NHS Trust

• 1) When to consider a diagnosis of TB

• 2) When to consider a referral to TB services

• 3) Review the initial laboratory investigations

Learning Objectives

Laboratory Diagnostic

Techniques

Microscopy

Culture

Identification

• Specimens should be fresh, transported and received in the laboratory within one working day of collection and processed as soon as possible.

• Collect specimens before antimicrobial therapy where possible.

• Antimicrobials used for other indications may also have significant anti-mycobacterial activity, notably the fluoroquinolones and macrolides.

Specimens

Health Protection Agency. (2012). Investigation of Specimens for Mycobacterium species. UK Standards for Microbiology Investigations. B 40 Issue 6. http://www.hpa.org.uk/SMI/pdf.

• Sputum specimens should be relatively fresh (less than 1 day old) to minimise contamination.

• Purulent specimens are best.

• Three samples of ≥ 5 mL should be collected approximately 8-24 hours apart with at least one from early morning

Sputum specimens

Health Protection Agency. (2012). Investigation of Specimens for Mycobacterium species. UK Standards for Microbiology Investigations. B 40 Issue 6. http://www.hpa.org.uk/SMI/pdf.

A Minimum 5.0 ml of Sputum Improves the Sensitivity of Acid-fast Smear for Mycobacterium tuberculosis Warren, et al. Am J Respir Crit Care Med Vol 161. pp 1559–1562, 2000

• A minimum volume of 5.0 ml was required before sputum processing was performed.

• When necessary, additional sputum was requested during period 2 for specimens with a volume less than 5.0 ml.

• Sputum specimens <5.0 ml were kept at 4°C for up to 48 h, and pooled for processing as a single specimen when a total volume of at least 5.0 ml was obtained.

Period 2

A Minimum 5.0 ml of Sputum Improves the Sensitivity of Acid-fast Smear for Mycobacterium tuberculosis Warren, et al. Am J Respir Crit Care Med Vol 161. pp 1559–1562, 2000

Volume of CSF

Thwaites GE, Chau TT, Farrar JJ. Improving the bacteriological diagnosis of tuberculous meningitis. J Clin Microbiol 2004; 42(1):378e9.

• Multivariate analysis showed that increasing the volume of CSF increased the likelihood of confirming TBM in HIV negative patients

Auramine Stain

Auramine-phenol staining is more sensitive than that by the Ziehl-Neelsen method andis therefore more suitable for assessment of smears from clinical specimens

Ziehl-Neelsen (Kinyoun) Stain

Ziehl-Neelsen staining provides morphological details and is useful for the examination of AFB in positive cultures, and may be used to review results from clinical specimens that are positive with auramine-phenol.

• Concentration of sputum samples, eg by centrifugation, increases the sensitivity of initial microscopy (15 mins)

• If sample volume is insufficient for both, culture is usually preferred to microscopy due to greater sensitivity.

Centrifugation

Health Protection Agency. (2012). Investigation of Specimens for Mycobacterium species. UK Standards for Microbiology Investigations. B 40 Issue 6. http://www.hpa.org.uk/SMI/pdf.

• Automated culture systems are recommended for faster and easier detection of growth of mycobacteria.

• Their limitations lie in a single incubation temperature and the difficulty of providing the growth additives necessary for certain very fastidious species.

Culture

Health Protection Agency. (2012). Investigation of Specimens for Mycobacterium species. UK Standards for Microbiology Investigations. B 40 Issue 6. http://www.hpa.org.uk/SMI/pdf.

• Solid media are used in addition

• Rare isolates of M. tuberculosis are recovered only on egg-based media, such as a Löwenstein Jensen (LJ) slope

Culture

Health Protection Agency. (2012). Investigation of Specimens for Mycobacterium species. UK Standards for Microbiology Investigations. B 40 Issue 6. http://www.hpa.org.uk/SMI/pdf.

MTB complex on Lowenstein-

Jensen agar

• A fluorescent compound is embedded in silicone on the bottom of the tube.

• It is sensitive to the presence of oxygen dissolved in the broth.

• The MGIT 960 system monitors tubes every 60 minutes for increasing fluorescence.

The Mycobacteria Growth

Indicator Tube (MGIT) System

Mean Time to Detection

(Days)

Yan JJ, Huang AH, Tsai SH, Ko WC, Jin YT, Wu JJ. Comparison of the MB/BacT and BACTEC MGIT 960 system for recovery of mycobacteria from clinical specimens. Diagn Microbiol Infect Dis 2000;37:25-30

• At least one AFB isolate from each new patient should be identified to complex/species level, and suitable susceptibility tests performed if identified as MTB Complex.

• Such tests are usually performed at a mycobacterial reference laboratory

Minimum level of

identification in the laboratory

Health Protection Agency. (2012). Investigation of Specimens for Mycobacterium species. UK Standards for Microbiology Investigations. B 40 Issue 6. http://www.hpa.org.uk/SMI/pdf.