Laboratory Diagnostic
Techniques
Hugo Donaldson
Consultant Microbiologist
Imperial College Healthcare NHS Trust
Laboratory Diagnostic Techniques
• Microscopy
• Culture
• Identification
Specimens
• 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.
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
• 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
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
Period 2
• 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.
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
CSF
• Collect aseptically as much (eg >6 mL in
adults) CSF sample as possible
• If only a small volume is available after initial
lumbar puncture and the findings of cell
counts and protein suggest TB meningitis, a
second procedure should be considered to
obtain a larger volume to improve chances of
achieving positive cultures
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.
Volume of CSF
• Multivariate analysis showed
that increasing the volume of
CSF increased the likelihood of
confirming TBM in HIV negative
patients
Thwaites GE, Chau TT, Farrar JJ. Improving the bacteriological diagnosis of tuberculous
meningitis. J Clin Microbiol 2004; 42(1):378e9.
Pleural and Pericardial Fluid
• Pleural or pericardial fluids are not very
sensitive samples for the detection of M.
tuberculosis.
• A concurrent pleural or pericardial biopsy
taken with the fluid is more useful.
• A negative result on these fluids does not rule
out the diagnosis.
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.
Auramine Stain
Auramine-phenol staining is more sensitive than that by the ZiehlNeelsen method and is 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.
Cording
Centrifugation
• 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.
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.
Urgent Requests
Requests are frequently made for urgent
Auramine. This must be carried out as soon as
possible by making a direct smear of the sample
(usually sputum or BAL/Pleural fluid) without
waiting for routine processing. If the specimen
would have been processed in the next batch on
the same day as the urgent request, check if this
is acceptable. A processed sample will always
give a more reliable microscopy result than a
blind urgent auramine.
Specimens
• Specimens submitted for mycobacterial
culture fall into 2 categories:
• specimens normally contaminated with
resident flora
• specimens from normally sterile sites.
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.
Specimens
• Contaminated specimens require a
decontamination step before culture to reduce
the likelihood of overgrowth by organisms other
than mycobacteria.
• Excessive decontamination of specimens should
be avoided as, although mycobacteria are more
resilient than other bacteria to the
decontaminating agents used, they are not
entirely so and hence this can produce false
negative results.
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.
Decontamination and neutralisation:
• Removes contamination and balances pH. The
timing of the various stages should be
reviewed in light of individual laboratory
contamination rates.
• Laboratories using automated culture systems
should refer to manufacturer’s
recommendations for compatible
decontamination methods.
• We use Sodium hydroxide (NaOH) for
decontamination of specimens
SAMPLE
BEFORE PROCESSING
NaOH DECONT.
SPUTUM
Transfer to Universal
YES
MGIT
AURAMINE
KIRSHNER
YES
YES
NO
NO
NO
YES
NO
YES
NO
YES
NO
YES
NO
YES
YES – layer slide if
NO
6 weeks
sufficient sample
6 weeks
URINE
Add 1 drop of tannic acid per 50ml urine, leave to settle in fridge
YES
overnight, decant supernatant and centrifuge remainder in a
YES
6 weeks
labelled sterile universal
BAL
Centrifuge if >5ml received
YES
EBUS
Centrifuge, process
YES
YES
6 weeks
PCR first, then process remainder as routine
PUS
YES
12 weeks
Transfer to Universal
YES
YES
6 weeks
FLUIDS
Centrifuge if >5ml received
If heavily bloodstained
or MC&S has grown
YES
6 weeks
bacteria.
CSF
Note appearance and look for spider web clot
NO
(can use <0.5ml if
insuff)
SWABS
Swab: Check if pus or tissue also received
&
Brush: break off into Selective Kirschner only
NO
NO
NO
SELECTIVE
FOR 8 WEEKS
BRUSHES
TISSUE
Cut and into several pieces if large enough place some in a 3ml
YES if from unsterile site
ballotini bead saline, vortex.
YES
YES from the Ballotini
NO
6 weeks
bead / saline mix
YES
YES
NO
NO
8 weeks
Retain some sample in the original pot if poss
GASTRIC WASH.
Neutralise immediately on receipt by adding equal volumes of
YES
PBS.
6 weeks
Pool if multiple samples received
SKIN BIOPSIES
Check for areas of necrosis or obvious infection.
NO
Cut into several pieces if large enough.
YES*
1 X37OC SELECTIVE
8 weeks
1 X30OC SELECTIVE
*If only a small piece is received, put into MGIT only.
For 8 weeks
BONE MARROW
Inoculate TB BACTEC bottle and put onto BACTEC FX
NO
NO
YES
(if slide rec’d)
NO
Culture
• 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.
• 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
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.
Culture Guidance
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.
Culture Guidance
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
Mean Time to Detection (Days)
solid media
MGIT 960
MB/BacT
all mycobacteria
31.7
8.7
13. 9
M. tuberculosis
32.9
9.3
13.9
nontuberculous
mycobacteria
27.2
8.1
14. 1
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
The Mycobacteria Growth Indicator
Tube (MGIT) System
• 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.
• Initially the large amount of dissolved oxygen
quenches emissions from the compound and
little fluorescence can be detected.
• Later, actively respiring micro-organisms consume
the oxygen and allow the fluorescence to be
detected.
• The MGIT 960 system monitors tubes every 60
minutes for increasing fluorescence.
The Mycobacteria Growth Indicator
Tube (MGIT) System
• The MGIT tube contains 7 mls of modified
Middlebrook 7H9 Broth base.
• This is supplemented with MGIT OADC
enrichment/MGIT PANTA antibiotic mixture
prior to inoculation with a clinical specimen.
• MGIT OADC enrichment contains Oleic acid,
Albumin (bovine), Dextrose and Catalase
MGIT PANTA antibiotic mixture
•
•
•
•
•
Polymixin B,
Amphotericin B,
Naladixic acid,
Trimethoprim,
Azlocillin.
BACTEC FX
• Modified Middlebrook 7H9 and Brain Heart Infusion
broth formulated for the recovery of Mycobacteria,
yeast and fungi from blood and sterile body fluids.
• Mycobacteria metabolise nutrients in the culture
medium.
• A dye in the sensor reacts with the CO2 and modulates
the amount of light that is absorbed by a fluorescent
material in the sensor.
• The instrument’s photo detectors measure the level of
fluorescence, which corresponds to the amount of CO2
released by the organisms.
Minimum level of identification in the
laboratory
• Mycobacterium genus level (based on Ziehl-Neelsen or
auramine-phenol stain from culture).
• At least one AFB isolate from each new patient should
be identified to complex/species level, and suitable
susceptibility tests performed if identified as MTBC.
• Such tests are usually performed at a mycobacterial
reference laboratory
• Repeat AFB isolates from the same patient should
usually be identified again and susceptibility tests
performed for MTBC, if cultured from a sample
collected 3 months or more after a previously referred
MTBC isolate.
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.
MGIT TBc Identification Test
• Rapid MPT64-based immunochromatographic
tests have been developed to detect
Mycobacterium tuberculosis complex (MTBC)
in culture.
• MPT64 is a 23-kD secreted protein restricted
to members of the Mycobacterium
tuberculosis complex .
Kirchner’s Liquid Medium
• Liquid culture for non-sputum specimens
• A positive Kirschner vial will typically have the
appearance of ‘snow flakes’ or a waxy layer on
or near the surface.
Kirchner’s Liquid Medium
MPT64 immunochromatographic tests
Brent et al. Performance of the MGIT TBc Identification Test and Meta-Analysis of MPT64 Assays
for Identification of the Mycobacterium tuberculosis Complex in Liquid Culture J Clin Microbiol.
2011 December; 49(12): 4343–4346
MGIT TBc Identification Test
• A small minority of MTBC isolates are not
detected by MPT64 assays due to deletion or
mutation of the mpb64 gene (absent from
some M. bovis BCG strains) or to low MPT64
concentrations in early cultures or mixed
cultures.
Brent et al. Performance of the MGIT TBc Identification Test and Meta-Analysis of MPT64 Assays
for Identification of the Mycobacterium tuberculosis Complex in Liquid Culture J Clin Microbiol.
2011 December; 49(12): 4343–4346
MALDI-TOF
• Matrix-assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectroscopy
analyses 16s ribosomal proteins.
• It compares the mass peaks achieved by the
test strains against those of known reference
strains.
Figure 1. MALDI-TOF MS spectra obtained from the M. tuberculosis H37Rv strain using the protocols tested
herein.
El Khéchine A, Couderc C, Flaudrops C, Raoult D, et al. (2011) Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Identification of Mycobacteria in Routine Clinical Practice. PLoS ONE 6(9): e24720. doi:10.1371/journal.pone.0024720
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024720
MALDI-TOF
• MALDI-TOF provides a rapid means of
identification for this important group of
pathogens, potentially allowing accurate
treatment regimens to be started earlier.
• Further validation is required and it is not yet
widely available.
Thank you
Any questions?