Specialist reference laboratory services for mycobacteriology are provided in the UK by:

• HPA National Mycobacterium Reference Laboratory (NMRL), London (contact details)
• HPA Regional Centres for Mycobacteriology, Birmingham and Newcastle
• Royal Brompton Hospital Microbiology Department, London
• Wales Centre for Mycobacteriology, Cardiff
• Scottish Mycobacteria Reference Laboratory, Edinburgh ( further information)
• Northern Ireland Public Health Laboratory, Belfast

Mycobacteriology reference services have been concentrated at these sites to maintain expertise and to develop new diagnostic procedures in response to an increasing threat from tuberculosis.

Principal services offered by the reference laboratories may include:

• Detection of mycobacteria
• Isolation of mycobacteria
• Identification of mycobacteria
• Susceptibility testing of clinically relevant isolates
• Training and education
• Surveillance and epidemiology of tuberculosis
• Molecular epidemiological typing of mycobacterial isolates

Detection of mycobacteria

Mycobacteria are usually detected by auramine fluorescent staining, and the results are available within one working day. Laboratories may also offer molecular amplification tests for the detection of Mycobacterium tuberculosis in smear positive pulmonary specimens and in possible cases of tuberculous meningitis. Two specimens of cerebrospinal fluid (CSF) are usually needed to enhance sensitivity. In all cases, the specimens are usually processed using rapid culture systems - for example, MB/BacT or BacT/ALERT/3D or MGIT. Molecular amplification techniques, such as polymerase chain reaction (PCR), are less sensitive than culture but faster; in numerous studies, PCR-based detection systems using various gene targets have detected 90 to 95% of smear positive, culture positive isolates, but only between 60 to 70% of all smear negative, culture positive isolates.

Isolation of mycobacteria

Mycobacteria should be isolated by rapid automated liquid culture and by conventional solid culture. Local laboratories that handle few specimens should consider referring primary specimens to a reference laboratory for mycobacterial culture. All reference laboratories are able to advise on and perform rapid culture.

Specimens can be referred to the reference laboratories as original specimens. The processing time will depend on the species and smear positivity of the specimen. M. tuberculosis is usually detected and identified within 7 to 21 days depending upon biomass, which is also reflected in the smear result. Positive cultures are reported as they are detected and all negative cultures will normally be incubated for at least eight weeks before a negative report is sent out. When unusual mycobacterial species are suspected then incubation will be further prolonged.

Identification of mycobacteria

Isolates may be submitted to the reference laboratories by courier as culture slopes or as growths in liquid medium. Such cultures are assigned as UN 3373 (Diagnostic specimens) and referring laboratories should ensure that all specimens are packed and marked according to Packing Instructions P650. In particular, mycobacterial cultures should be sent in screw-top, preferably plastic or thick-walled glass, bijou or universal bottles packed inside a rigid container with enough absorbent material to absorb the contents if the bottle breaks.

Identification normally includes molecular DNA analysis and/or standard numerical taxonomy procedures based on macroscopic and microscopic appearances, growth, biochemical characteristics and drug resistance. All reference laboratories report receipt of the isolate and initial identification results to the source laboratory within one working day.

Susceptibility testing of clinically relevant isolates

Drug susceptibility testing has three main goals:

• To facilitate the management of individual patients, particularly if drug resistance is likely
• To provide data on which to plan drug combinations for treatment
• To provide a surrogate measure of the relative effectiveness of tuberculosis control programmes

Drug susceptibility testing is performed using one of three main methods: absolute concentration method, the resistance ratio method, or the proportion method. All of these methods are acceptable if their quality is adequately controlled and validated. All reference laboratories employ the resistance ratio method using solid media and/or the proportional method using the automated liquid culture systems. Results of tests for primary therapeutic agents should be completed within 14 days of receipt of the isolate by the reference laboratory. However, for some atypical mycobacteria, this may be longer.

All reference laboratories perform drug susceptibility testing for first line and (with the exception of Northern Ireland) most second line chemotherapeutic agents. Susceptibility testing for second line drugs is performed whenever resistance to more than one first line drug is identified, if the patient has been in contact with drug resistant cases, or if multi-drug resistant tuberculosis is more likely (for example, in patients who are poorly compliant with treatment, have had treatment for a prior infection, or on request, such as when a patient has impaired absorption with or without vomiting). Molecular assays are also available to detect rifampicin resistance from cultures and primary specimens.

The MRU performs tests for third line drugs such as ethionamide, cycloserine, capreomycin and para-aminosalicylic acid (PAS); other agents may be tested as needed. The MRU is also a WHO SupraNational Reference Laboratory and European Co-ordinating Centre within the Global Programme on Drug Resistance and operates an EQA scheme for drug resistance on behalf of the WHO.

Atypical mycobacteria are commonly found in the environment and their clinical significance depends on the body site from which the organism is isolated and the frequency of isolation. The likely clinical significance is a criterion used to decide whether drug susceptibility tests will be performed. Clinical details must therefore accompany the specimen as results can be interpreted only in clinical context.

Training and education

The HPA provides training courses and technical support for basic techniques in the laboratory diagnosis of tuberculosis. Trainee staff who need experience in identification and susceptibility procedures will be welcome in any of the reference laboratories by prior arrangement and subject to local safety guidelines. The reference laboratories may also offer formal short courses of basic training in mycobacteriology.

Surveillance and epidemiology of tuberculosis

The reference laboratories contribute to the surveillance of tuberculosis in the UK in collaboration with the HPA Centre for Infections. Full details of the patients for whom specimens are submitted are needed to meet this surveillance function. The UK Mycobacterial Surveillance Network (MycobNet) is a system used to collate and monitor drug susceptibility information on all cases of tuberculosis confirmed by culture at the reference laboratories.

Molecular epidemiological typing of mycobacterial isolates

M. tuberculosis isolates may undergo molecular strain typing if a clear clinical or epidemiological benefit is likely - for example, in the event of an outbreak of tuberculosis in a hospital or the community where common strain typing data between isolates can indicate transmission. Several outbreaks of drug sensitive and resistant tuberculosis have been investigated using molecular strain typing data. The HPA collaborates nationally and internationally in the development of molecular epidemiological techniques and their application in the recognition of outbreaks and for population studies. Prospective typing of all isolates of M. tuberculosis complex is carried out in some reference laboratories, and is planned to be done throughout the UK in the near future.

• The HPA National Tuberculosis Strain Typing Project (PDF, 414 KB)
  What it involves and how it affects you
  Added/updated: 25 March 2011
  
• Tuberculosis UK-wide diagnostics initiative
  The Health Protection Agency, Royal College of Pathologists and Association of Clinical Pathologists, in association with Keele University, Health Protection Scotland and Public Health Wales are launching a UK-wide project looking at tuberculosis diagnostic services in the UK. The aim of this work is to describe and audit the current diagnostic service in the UK, to evaluate new technologies, and to use this information to support both laboratories and commissioners.
  Added/updated: 3 November 2011