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.

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.

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.

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.

Auramine Stain Auramine-phenol staining is more sensitive than that by the Ziehl- Neelsen 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.

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.

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.

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 PROCESSINGNaOH DECONT.MGITAURAMINEKIRSHNER SPUTUM Transfer to Universal YES 6 weeks YES NO URINE Add 1 drop of tannic acid per 50ml urine, leave to settle in fridge overnight, decant supernatant and centrifuge remainder in a labelled sterile universal YES 6 weeks NO BALCentrifuge if >5ml receivedYES 6 weeks YES NO EBUS Centrifuge, process PCR first, then process remainder as routine YES 12 weeks YES NO PUS Transfer to Universal YES 6 weeks YES NO FLUIDS Centrifuge if >5ml received If heavily bloodstained or MC&S has grown bacteria. YES 6 weeks YES NO CSFNote appearance and look for spider web clotNO YES 6 weeks (can use <0.5ml if insuff) YES – layer slide if sufficient sample NO SWABS & BRUSHES Swab: Check if pus or tissue also received Brush: break off into Selective Kirschner only NO SELECTIVE FOR 8 WEEKS TISSUE Cut and into several pieces if large enough place some in a 3ml ballotini bead saline, vortex. Retain some sample in the original pot if poss YES if from unsterile site YES 6 weeks YES from the Ballotini bead / saline mix NO GASTRIC WASH. Neutralise immediately on receipt by adding equal volumes of PBS. Pool if multiple samples received YES 6 weeks YES NO SKIN BIOPSIES Check for areas of necrosis or obvious infection. Cut into several pieces if large enough. *If only a small piece is received, put into MGIT only. NO YES* 8 weeks NO 8 weeks 1 X37 O C SELECTIVE 1 X30 O C SELECTIVE For 8 weeks BONE MARROWInoculate TB BACTEC bottle and put onto BACTEC FXNO 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.

Culture Guidance Health Protection Agency. (2012). Investigation of Specimens for Mycobacterium species. UK Standards for Microbiology Investigations. B 40 Issue 6.

Culture Guidance Health Protection Agency. (2012). Investigation of Specimens for Mycobacterium species. UK Standards for Microbiology Investigations. B 40 Issue 6.

MTB complex on Lowenstein-Jensen agar

Mean Time to Detection (Days) solid mediaMGIT 960MB/BacT all mycobacteria M. tuberculosis nontuberculous mycobacteria 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 CO 2 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 CO 2 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.

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 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 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): e doi: /journal.pone

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?