Tuberculosis
(TB) is a bacterial infection that causes a major illness and death with
approximately 9 million new cases and 1.3 million deaths annually throughout
the world caused by members of Mycobacterium
tuberculosis complex (MTBC) which includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium
africanum, Mycobacterium pinnipedii, Mycobacterium microti, Mycobacterium
caprae and Mycobacterium canettii.
However most human cases of TB are caused by the first three organisms in the
complex mentioned above. Respiratory infections can also be caused by members
of non-tuberculosis mycobacteria called Mycobacterium
avium complex (MAC) and includes Mycobacterium avium and Mycobacterium intracellulare.
Mycobacterium tuberculosis is a small, aerobic, nonmotile bacillus that causes TB. TB
is primarily a disease of the lung but may spread to virtually any organ of the
body or proceed to a generalised infection (miliary tuberculosis) and is
characterised by granuloma formation. It contains distinctive cell walls with
high concentration of lipids, notably mycolic acids which offers a high degree
of protection to the cells and accounts for other properties which include
resistance to acids and alkalis, resistance to antibiotics and disinfectants,
resistance to drying and osmotic lysis, impermeability to stains and survival
within macrophages. It divides every
16 to 20 hours, which is an extremely slow rate compared with other bacteria,
which usually divide in less than an hour. General signs and symptoms include fever, chills, night sweats, loss of appetite, weight loss, and fatigue, and significant finger clubbing may also occur.
Chest x-ray, tuberculin skin test, acid-fast bacilli stain and culture are the
diagnostic methods for TB.
UK
standard of Microbiology investigations under the investigation of specimen for
Mycobacterium species are a
collection of recommended algorithms and procedures covering all stages of the
investigative process in Microbiology. These standards are developed under the
auspices of the Health Protection Agency (HPA), working in partnership with
National Health Services (NHS), Public Health Wales and other professional
organisation.
Specimens
for the diagnosis of TB and available standard techniques for the detection of Mycobacteria
in patient samples
Sputum, Bronchoalveolar lavage (BAL), pleural
fluid and tissues samples from any site of the body. Detection of TB includes
the initial AAFB staining and microscopy which is vital because results can be
available within one hour of receipt of the specimen in the laboratory and
several weeks before culture results because of the slow growth of the
organism. Therefore, microscopy plays a major role in the patient treatment and
management especially in positive cases. However, TB microscopy despite the
simplicity and rapid results, lack sensitivity and does not identify drug
resistant strains thus if the clinical details suggests TB, then treatment will
be given to the patient regardless.
Staining
techniques used in TB microscopy include Ziehl-Neelsen (ZN) and auramine stain
(AP). Both stains use phenol which acts as a detergent and reduces the
hydrophobic effect of the lipids and thus enables the dye to penetrate the cell
wall. The stained isolate was then viewed under the fluorescence microscope the
bacteria appear brilliant greenish yellow against dark background.
Pre-treatment - Non-purulent
liquid specimens are spun down in a centrifuge at 2500 rpm for 10 minutes to
concentrate them. The supernatant is then separated into a sterile universal
bottle leaving 1ml to resuspend the pellet.
Homogenisation of
specimen improves the sensitivity of the culture by permitting the bacteria to
be released from the thick sputum and can be achieved by the following methods
(a)
Repeatedly vortexing during decontamination process until suspension is fully
homogenised.
(b)
Treatment with Sputasol (Oxoid Ltd, Basingstoke, UK; containing 100µg/ml
dithiothreitol) is used to homogenise the specimen by adding equal volume of
0.1% solution of Sputasol to the sample and vortex intermittently and leave for
15 minutes at room temperature, followed by gentle vortex to assist
homogenisation and
(c)
Treatment with N-acetyl-L-cysteine (NALC) during decontamination.
Decontamination can
be achieved by either by the use of sodium hydroxide (NaOH) or NALC-NaOH. The contaminating
normal flora is preferentially killed at this stage (decontamination). Specimens
that require decontamination include sputum, bronchial secretions, washings, or
biopsies, urines and all other specimens from sites contaminated with normal
microbial flora. However, contaminating organisms should not be present in
samples obtained by bronchoscopy such as bronchoalveolar lavage (BAL) and any
pathogens present will have been diluted by the saline used in bronchoscopy.
(a)
0.7ml of NaOH (0.5N) is added to the specimen and allows to act for 30 minutes
at room temperature and vortexing at regular intervals. The specimen is then
neutralised with 14ml of sterile 0.067 M phosphate buffer (pH 6.8).
Alternatively, follow the above procedure but add 2ml of 1N NaOH (4%w/v) to 2ml
of specimen instead of 0.7ml of NaOH (0.5N) and neutralise with 3ml of sterile
0.067 M phosphate buffer (pH 6.8) instead of 14ml.
(b)
Add equal volume of working NALC-NaOH solution (2% NALC and 0.5N NaOH, no more
than 48 hours old) to the specimen and vortex for approximately 20 seconds.
Allow to stand for 30 minutes at room temperature to decontaminate the specimen
and dilute the mixture to a minimum of 20 ml with 0.067 M phosphate buffer (pH
6.8). Invert several times to ensure that the content is mixed.
Concentration: Specimens
are spun down in a centrifuge at 3000 rpm for 15 minutes to concentrate them.
The supernatant is then discarded into a disinfectant leaving 1ml to resuspend
the pellet or resuspend in 0.067 M phosphate buffer (pH 6.8).
Specialised
techniques available at reference laboratories
TB staining,
culture, identification, sensitivity and typing are done at a specialised TB
laboratory and the procedures and techniques used are described below:
Culture
Culture
of Mycobacterium tuberculosis is an
important part of the laboratory investigation of TB. Specimens undergo the
above pre-treatment processes before culture to eliminate contaminants and
concentrate the specimen before culture. There are three types of media used
for conventional Mycobacterial culture which includes egg based solid media
(Lowenstein-Jensen medium), Agar based solid media (Middlebrook agar) and
liquid media.
Lowenstein-Jensen
medium (LJ) is the most commonly used media in United Kingdom and is prepared
in bottles which are heated while tilted to make slopes. The heat dehydrates
the egg proteins so the medium solidifies. Malachite green dye which is
inhibitory to most bacteria but not to Mycobacteria is incorporated in the
culture medium to prevent the growth of organisms that survived decontamination
process. Typical Mycobacterium
tuberculosis appears on LJ medium after a couple of weeks of incubation at
35-37 oC as irregular, dry colonies that are beige or buff in
colour. The culture is considered negative if no growth after 10-12 weeks of
incubation with checks every week for possible acid-fast growth. Presence of
Acid-fast Bacilli in positive cultures is confirmed with ZN or AP stain and
aliquots are then sent for susceptibility test.
Growth
of organisms consumes oxygen and produces carbon dioxide and may be detected by
changes in radioactivity, fluorescence, reflectance and pressure. Automated
Mycobacterial culture methods such as BACTEC MGIT 960 using the Mycobacterial
Growth Index Tube (MGIT) system is based on liquid culture and detects the growth
of Mycobacteria faster than conventional culture. Fully automated systems capable
of holding up to 960 patient samples continuously (every 60 minutes) monitor
the culture bottles and flag new positives cultures usually within 10 – 12 days.
It utilises fluorescence technology (O2 reduction). In MGIT, a fluorescent
oxygen sensor is embedded in the base of the tube that detects any decrease in
O2 dissolved in broth. Oxygen sensor will emit light when exposed to UV with actively
respiring organisms consume O2 and reduction in O2 is detected by machine thus
the machine flags tube as positive. Positive tubes flagged by machine are then
removed, centrifuged for 15 minutes and stained using an AFB stain. Another
automated analyser used for Mycobacterial culture is the Biomerieux BacT/ALERT 3D
MP which monitors the production and presence of carbon dioxide (CO2)
produced by the organism by using a colorimetric sensor and reflected light. As
the organisms grow and metabolise substrates in the culture medium, CO2 is
produced and is detected by the analyser when the level of CO2 produced
reaches a certain threshold. This threshold is determined by the colour change to
lighter green or yellow at the bottom of the culture bottle which has an
in-built gas permeable sensor. The reflectance units monitored by the analyser
increases as a result of the lighter colour and is then recorded every 10
minutes. The colony forming unit (CFU) at the time of detection is
approximately 106 – 107 per ml.
Identification
Identification
of Mycobacterium tuberculosis
following isolation is usually done at a National Mycobacterial reference laboratory. It is identified
to complex/species level and follows the use of AFB stains (ZN and AP),
biochemical, hybridization gene probe and nucleic acid amplification tests
(NAATs). Current UK guidelines recommend that a NAATs or hybridization gene
probe test which may allow rapid diagnosis of TB
should be performed within one working day of isolation of Mycobacterium tuberculosis (National
Institute for Health and Care Excellence –NICE and HPA UK Standard for
investigation of specimens for Mycobacterium species). NAATs (PCR) analyser
used at HPA Freeman Hospital, Newcastle is the Cepheid GeneXpert using the
Xpert MTB/RIF cartridge.
Matrix-assisted
laser desorption ionisation – time of flight (MALDI-TOF) mass spectroscopy is
another automated Mycobacterial identification method and analyses 16s
ribosomal proteins and can identify Mycobacterium
species within 20 minutes.
Typing
MTBC
isolates need to be typed which simply means the use of further tests that can
discriminate between multiple isolates of the same species. The detection of
genomic differences between isolates (genotyping) is the preferred method of
typing rather than typing based on the differences in their behaviour (phenotyping).
In
UK, the current recommended typing method enables comparisons to be made
nationally or internationally. This is known as mycobacteria interspersed
repetitive units-variable number tandem repeats (MIRU-VNTR) typing. It is
recommended that an MIRU-VNTR genotype for each new MTBC isolate should be
available and entered on the national database within 21 days of mycobacterial
reference laboratory receipt for ≥95% isolates.
Diagnosis of latent TB
Diagnosis
of latent TB infection involves assessing the host’s cell-mediated immune
response by detecting a cytokine called interferon- gamma (IFN-γ). This test
does not involve the detection of mycobacteria. Tuberculin skin test or Mantoux
test is a screening test for TB used in the detection of latent TB, detection
of recent infection and as part of the diagnosis.
The
standard Mantoux test in the UK consists of an intradermal injection of two
tuberculin units (2TU) of Statens Serum Institute (SSI) tuberculin RT23 in
0.1ml solution for injection and read 48 to 72 hours after administration. A
reading is then obtained by measuring and recording the presence or absence of
induration. The diameter of the induration which is a hard, dense, raised formation
is measured. In the absence of specific clinical details of risk factors for
TB, a reading of 6-15mm is more likely to be due to previous BCG vaccination or
infection with environmental mycobacteria than TB infection.
Other
tests used in diagnosis of latent TB include the Interferon-γ release assays
(IGRAs) on a blood sample test called QuantiFERON-TB Gold in-tube. This
analysis involves the in vitro
stimulation of cells in blood using peptide stimulating antigens (ESAT-6,
CFP-10 and TB7.7 (p4)). Enzyme-Linked Immunosorbent Assay (ELISA) detect the
production of Interferon-γ is then used to identify responses to these peptide
antigens in vitro that are linked to Mycobacterium tuberculosis.
Treatment options for TB
TB
can be treated with antibiotics to kill the bacteria. Effective TB treatment is difficult, due to the unusual structure and
chemical composition of the mycobacterial cell wall described above, which
hinders the entry of drugs and makes many antibiotics ineffective. Antimicrobacterial
susceptibility testing can be based on inhibition of growth or detection of
generic mutations. Automated liquid systems detect antimicrobial resistance by
adding antimicrobial substances to the culture while conventional methods
detect resistance by relying on growth, or inhibition of growth of the
organism. The
treatment of latent TB usually involves
the use of a single antibiotic of either Isoniazid or Rifampicin, while active TB disease is best treated with
combinations of a few antibiotics. Latent TB is usually treated to prevent the
infection progressing to active state while active TB is treated with a few
antibiotics to reduce the risk of the bacteria developing antibiotic resistance. Antibiotic treatment of Mycobacterium tuberculosis includes long term administration (6
months) of multiple antimicrobial agents. Sensitivity testing is performed at
the National Mycobacterial reference
laboratory. In the UK, the antimicrobial treatment of active TB comprises two
stages:
(1)
Initial stage of Isoniazid, Rifampicin and Pyrazinamide for two months.
(2)
Continuation stage of Isoniazid and Rifampicin for further four months.
Multidrug
resistant strains TB (MDR-TB) develops in
otherwise treatable TB when the course of antibiotics is
interrupted and the levels of drug in the body are insufficient to kill 100
percent of the bacteria. This can happen for a number of reasons such as patients
may feel better and halt their antibiotic course, drug supplies may run out or
become scarce, patients may forget to take their medication from time to time
or patients do not receive effective therapy. Secondly, MDR-TB can become
resistant to the major second-line drug groups such as fluoroquinolones and
injectable drugs. When MDR-TB is resistant to at least one drug from each
group, it's defined as extensively
drug-resistant tuberculosis (XDR-TB).
Recent
statistics show that approximately 1% of UK isolates of Mycobacterium tuberculosis are MDR with Rifampicin resistance used
as a marker for possible MDRTB. MDR are usually resistant to Isoniazid and
Rifampicin and are treated with five drugs. The five drugs should be chosen in
the following order (based on known sensitivities): Aminoglycoside (such as
Amikacin, Kanamycin) or polypeptide antibiotic (such as Capreomycin),
Pyrazinamide, Ethambutol, a fluoroquinolone such as Moxifloxacin (Ciprofloxacin
should no longer be used), Rifabutin, Cycloserine, a thioamide such as
Prothionamide or Ethionamide, 4-aminosalicyclic acid (PAS), a macrolide such as
Clarithromycin, Linezolid, high dose INH (if low level resistance), Interferon-γ,
Thioridazine and Ampicillin.
Drugs placed nearer the top of the list are more effective
and less toxic; drugs placed nearer the bottom of the list are less effective,
more toxic, or more difficult to obtain. The recommended treatment of new-onset
pulmonary tuberculosis, as of 2010, is six months of a combination of
antibiotics containing rifampicin, isoniazid, pyrazinamide and ethambutol for
the first two months, and only rifampicin and isoniazid for the last four
months and where resistance to isoniazid is high, ethambutol may be added for
the last four months as an alternative.
Response to treatment must be obtained by repeated sputum
cultures (monthly if possible). Treatment for MDR-TB must be given for a
minimum of 18 months and cannot be stopped until the patient has been
culture-negative for a minimum of nine months. It is not unusual for patients
with MDR-TB to be on treatment for two years or more. Patients with MDR-TB
should be isolated in negative-pressure rooms, if possible. Patients with
MDR-TB should not be accommodated on the same ward as immunosuppressed patients
(HIV-infected patients, or patients on immunosuppressive drugs).
