Monday, 19 October 2015

Diagnosis of Mycobacterium tuberculosis (TB)

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 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 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.
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).

WBCs and RBCs count in sterile body fluids (such as CSF)

The quantity of white blood cells and red blood cells in sterile body fluids (such as CSF) can be obtained by performing a cell count on the uncentrifuged specimen, preferably the last specimen taken, using a modified mirrored Fuchs Rosenthal counting chamber. The cell count of sterile body fluids specimen such as cerebrospinal fluids (CSF) usually gives important clues of presence of infection or not and if present, the infecting organism. Sequential specimens 1 to 4 are usually obtained from one lumbar puncture and specimen 1 is sent to Clinical Biochemistry laboratory while 2 – 4 are sent to Microbiology laboratory. Red cell count is done on the entire 3 Specimen sent to Microbiology laboratory and both RBC and WBC count is performed on specimen 4. Uniform blood staining of all samples suggests previous haemorrhage into the subarachnoid space, whereas reducing counts in sequentially obtained samples suggest bleeding induced by the tap procedure.  CSF obtained more than 12 hours post intra-cranial haemorrhage may show raised WBC counts of up to 500 x 106/l as a result of an inflammatory response.
The procedure for performing a cell count starts with drawing a line with a chinagraph pencil on the external supports of the clean and dry counting chamber and then gently pushes the cover glass onto the counting chamber from the front. The formation of interference lines (Newton rings) between the external support and the cover glass shows that the cover glass is correctly positioned. Fill the counting chamber with the specimen using a fine tip sterile pipette and allows to settle for 5 minutes. Observe under x10 objective lens microscope to focus and the x10 or x40 to count cells.
The modified Fuchs Rosenthal counting chamber has nine (9) large triple lined squares; each divided in 16 small squares and has a depth of 0.2 mm. as shown in Figure below.  Each large square is 1 mm2; therefore 5 large triple lined squares are counted to get the count /mm3. The four corner squares and the middle are also counted.  If cells are lying on the triple lines between squares count only the cells lying on the inner two lines of the top and the left side lines.
The ratio of WBC and RBC in a normal blood in WBC1-2: 1000 RBC and under normal conditions the number of white cells in a CSF is <5/mm3 in children and <20/mm3 in adults. In some situations where the sample is turbid or blood stained, with a high expectation of RBC count of >200 mm3, dilution is often performed in sterile saline before loading on the counting chamber. The cell count result must then be multiplied by the dilution factor.

Normal CSF values

Normal CSF values              Neonates                   Adults

Leucocytes                         Neonates              0-30  cells / cu mm   ( x 106/l)
                                           1-4yr old                0-20  cells / cu mm   ( x 106/l)
                                           5yr-puberty            0-10  cells / cu mm   ( x 106/l)
                                           Adults                     0-5   cells / cu mm   ( x 106/l)
Erythrocytes                Newborn                     0-675 cells / cu mm   ( x 106/l)
                                      Adults                        0-10  cells / cu mm   ( x 106/l)

The counting chamber grid