Tuberculosis Reference Laboratory
An introduction to the Work of the Tuberculosis Reference Laboratory
in the Department of Health
As a part of the Public Health Laboratories in the
Department of Health, HKSAR, the Tuberculosis (TB)
Reference Laboratory (the Laboratory) supports the TB &
Chest Service in the Department in providing a range of
laboratory services for the clinical management of tuberculosis
(TB) and other mycobacterial diseases, as well as organizing
and participating in TB surveillance and epidemiological studies.
The Laboratory also takes part in the TB Notification System
by submitting demographic information of patients with
positive Mycobacterium tuberculosis (MTB) culture isolation
to the TB & Chest Service. The Laboratory is now receiving
patients' specimens, mainly sputum, from all Government
Chest Clinics, some hospitals under the Hospital Authority,
private laboratories and practitioners for routine microscopy
examination and mycobacterial culture. Moreover, positive
mycobacterial cultures from most hospitals under the Hospital
Authority and also from private laboratories are also sent to the
Laboratory for bacterial identification and drug susceptibility
tests.
In order to maintain high accuracy of the laboratory
testing, the Laboratory organizes as well as participates in
various quality assurance programmes. In collaboration with
Hong Kong Medical Technology Association, it has organized
an "Acid Fast Bacilli (AFB) smear Quality Assurance
Programme" for most clinical bacteriology laboratories in Hong
Kong. Moreover, the Laboratory also joins the External
Quality Assurance Programme organized by the United
Kingdom NEQAS (Microbiology Quality Assessment) and
World Health Organization Supranational Reference
Laboratory for mycobacteria smear and culture, and drug
susceptibilities testing respectively.
Following is a list of services presently provided by the
Laboratory. In general, these can be divided into eight major
categories:
1. Direct microscopy for the detection of AFB
2. Direct detection of MTB from sputum by nucleic acid
amplification method
3. Primary isolation of mycobacteria from clinical
specimen
4. Positive culture identification
5. Drug susceptibility tests
6. MTB strain typing for outbreak investigation
7. Urine isoniazid metabolite tests
8. Collaborative studies and training
1. Direct microscopy for the detection of acid-fast
bacilli
Direct microscopy is used for the rapid
diagnosis of TB and other mycobacterial diseases, as a
relatively long period of time is required for
mycobacteria to be detected by culture methods.
Patients with positive smears are considered to have
higher bacterial load, and thus more likely to spread TB.
Results from direct microscopy can be available within
24 hours. Clinically, it is important to detect the
presence of mycobacteria as rapidly as possible for
implementation of appropriate patient care and public
health measure. Moreover, direct microscopy on
successive specimen can also be used to monitor the
success of chemotherapy for smear positive patients.
In the Laboratory, fluorochrome-staining
method, which is found to be more sensitive, is used
for the detection of acid-fast bacilli. Using this staining
method, a minimum concentration of 5,000 to 10,000
bacilli per mL of specimen is required for the detection
of positive smear. When compared with culture, the
overall sensitivity of the direct smear has been reported
to range from 22 to 43%. Moreover, despite the high
specificity of direct smear for detection of mycobacteria,
this test cannot be used to differentiate between various
mycobacterial species.
2. Direct detection of Mycobacterium tuberculosis from
sputum by nucleic acid amplification method.
Due to the slow growth of Mycobacterium
tuberculosis (MTB), a usual period of 3 to 8 weeks is
required to obtain a definite TB diagnosis by culture
examination. In this connection, the Laboratory
provides another option for TB diagnosis by using a
Food and Drug Agency, U. S. A. (FDA) approved
nucleic acid amplification (NAA) assay to detect and
identify MTB directly from smear-positive clinical
respiratory specimen. This test was found to have a
sensitivity of >95% and a specificity of >98%, and the
result can be obtained within 2 days. This NAA assay is
particularly useful when rapid differentiation between
MTB infection and non-tuberculosis mycobacteria
infection is required, such as for AIDS patients.
Despite the rapidity of this test, sensitivity for
this NAA assay against smear negative specimen is
about 50% and only limited data are available for
guiding the interpretation of the test for non-respiratory
specimen. In this connection, discussion with the
Laboratory's medical microbiologists can be arranged
for individual cases on the usefulness and limitation of
using this relatively expensive NAA assay for primary
TB diagnosis.
3. Primary isolation of mycobacteria from clinical specimen.
Isolation of definite pathogen e.g. MTB or
repeated isolation of opportunistic pathogens from
clinical specimen may confirm a definitive diagnosis of
TB or other mycobacterial infections. Due to the slow
growth of most mycobacteria including MTB, 3 to 8
weeks are required for conventional cultures. When
specially requested for individual cases, rapid culture
using broth medium can be arranged, with which about
90% of positive culture can be obtained within 3 weeks.
Since a long incubation period is required for
performing mycobacteria culture, it is inevitable to have
contamination due to growth of other bacteria and
fungi. In general, a 5% level of contamination should
be expected.
4. Positive culture identification.
All positive cultures have to be identified for the
confirmation of pathogen. In the Laboratory,
mycobacteria will always be identified to the species
level if possible. Batches of mycobacteria identification
methods are available in the Laboratory. These
identification methods include conventional
biochemical tests, analysis of mycobacterial fatty acid
or mycolic acid by chromatography, and genetic
investigation through the use of nucleic acid probes,
restriction fragment analysis of DNA amplification
products, and DNA sequencing.
Since the amount of mycobacteria isolates is
always limited for performing biochemical identification
tests, additional time is required for performing sub-
culture in order to obtain sufficient growth in order to
do the identification tests. However, for the
identification of positive cultures as MTB and
Mycobacterium avium-intracellulare complex, rapid
identification by using the DNA probes can be arranged
when specially requested, and in most cases, results can
be available within 2 working days.
5. Drug susceptibility tests (DST)
With the emergence of drug resistant MTB
isolates, DST for MTB become a very important tool
for revealing the cause of treatment failure and
providing a guide on the choice of anti-TB drugs. The
Laboratory routinely performs DST against all clinical
MTB isolates for the 4 first line anti-TB drugs
(streptomycin, isoniazid, rifampicin and ethambutol).
For MTB isolated from re-treatment, relapse or failure
cases, drug susceptibilities on ethionamide, amikacin
and ofloxacin will also be included. Moreover, tests on
kanamycin, capreomycin, cycloserine and pyrazinamide
will also be added if required.
The Laboratory is using a well calibrated,
standardized absolute concentration method to perform
the DST with results available usually in 4 weeks.
Moreover, DST will also be conducted in broth
medium for those MTB isolates obtained by broth
culture. In these cases, results can be ready in 2 to 3
weeks.
Apart from MTB, the Laboratory also conducts
DST for M. avium-intracellulare complex (MAC) by
Bactec macrobroth dilution method. For performing
DST on other anti-mycobacterial agents or against
other mycobacteria, our medical officers or
microbiologist can be consulted for further discussion.
6. MTB strain typing for outbreak investigation.
Epidemiological studies of tuberculosis is
essential in the prevention and control of TB. DNA
fingerprinting (restriction fragment length
polymorphism (RFLP) using the insertion sequence
IS6110 as standard probe) has proven to be a powerful
epidemiological tool for this purpose. In the Laboratory,
DNA fingerprinting can be arranged for suspected TB
outbreaks.
7. Urine isoniazid metabolite test
The method facilitates the monitoring of isoniazid
drug uptake by TB patients and also the use of isoniazid
as a marker for drug compliance by assessing the
regularity with which other drugs prescribed for self-
administration are actually ingested. Test results usually
can be available within a week.
8. Collaborative studies and training
In collaboration with the TB & Chest Service,
the Laboratory is actively involved in a number of anti-TB
drug trials, as well as epidemiological studies. With
approval from the Department of Health Headquarters,
we also provide short term training for visiting laboratory
officers and technical staff from this region.
From the Tuberculosis Reference Laboratory,
Department of Health, Hong Kong SAR.
Dated 17th December, 2001.
Biological safety cabinet
Inoculation
Specimen decontamination for TB culture
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