Terry's case: A Youth at Risk

Tuberculin Skin Testing

(Source: Canadian Tuberculosis Standards, 5^th Edition, 2000. The Canadian Lung Association, pages 45-65.)

Diagnosis of Tuberculosis Infection

The major tool to diagnose tuberculosis infection is the tuberculin skin test. This test consists of the intradermal injection of a small amount of purified protein derived from M. tuberculosis bacilli. In a person who has previously been exposed and has developed cell-mediated immunity to these tuberculin antigens, a delayed cell-mediated reaction (delayed hypersensitivity type) will occur within 48-72 hours. The reaction will cause localized swelling and manifest as induration of the skin at the injection site. In persons who are newly exposed and become infected with tubercle bacilli, this cell-mediated reaction to tuberculin will not be manifested immediately. It will develop between 3 and 8 weeks after the acquisition of infection.¹

Indications for Tuberculin Testing

In general, tuberculin skin testing should be performed to diagnose TB infection in persons at increased risk of developing the disease. There are two general situations when risk of disease is increased:

1) Recent infection - most commonly contacts of a recently diagnosed patient with active contagious pulmonary TB, or immigrants within 5 years of their arrival in Canada from countries where TB is still common,

2) Increased risk of reactivation due to impaired immunity. This includes HIV infection, diabetes, renal failure, corticosteroids or other immunosuppressant medication and pulmonary silicosis.

In addition, the tuberculin skin test is useful in epidemiologic surveys to define the prevalence of infection in population groups or to estimate prevalence or risk of infection in certain population groups.

Note: Tuberculin screening of low risk populations is generally discouraged, although testing may be performed for individuals.

Contraindications

The following persons should not undergo tuberculin testing:

1. Patients with severe blistering tuberculin reactions in the past.

2. Patients with documented active tuberculosis or a clear history of treatment for TB infection or disease in the past.

3. Patients with extensive burns or eczema.

4. Patients with major viral infections or live-virus vaccinations in the past month, for example vaccination against mumps or measles. Patients with a common cold may be tuberculin tested.

Note: Patients can be tuberculin tested even under the following circumstances:

• They have recently been vaccinated but with non-live virus vaccines.
• They are pregnant.
• They have received BCG vaccination in the past.
• They give a history of a positive tuberculin skin test but this is not documented.

Types of Tuberculin Tests

1. Mantoux: this is an intradermal test and is the most accurate, consistent and reliable.

2. Multi-puncture tests, such as the Tine test. These tests may have significant false-negative rates, readings are very difficult to standardize, and they are not recommended.^2,3

Technique of Mantoux Test

Administration: 0.1 mL of 5 tuberculin units (5-TU) of purified protein derivative (PPD), bio-equivalent to 5 tuberculin units of PPD-S (standard), is injected intradermally on the volar aspect of the forearm, where it is easiest to administer and where the reaction is easiest to read. Tubersol (manufactured by Connaught Laboratories, Toronto) is recommended throughout North America.⁴ If given correctly, the injection should raise a small weal of 5 mm diameter, which will disappear in 10 to 15 minutes. Use of one tuberculin unit (1-TU) is not recommended, because there are too many false-negative reactions. Similarly, use of 250-TU is not recommended, as this is associated with a very high rate of false-positive reactions.⁵

Reading: Should be performed 48 to 72 hours after administration. The tuberculin test must be read by a trained health professional. Self-reading is very inaccurate and is strongly discouraged.⁶ Reactions may persist for up to 1 week, but as many as 21% of individuals positive at 48 to 72 hours will be negative by 1 week.⁷

1. Induration, not redness should be measured. Blistering, which can occur in 3% to 4% of subjects with positive tests, should be noted.

2. The tip of a ballpoint pen is pushed at a 45° angle toward the site of injection. The tip will stop at the edge of the induration.⁸

3. The transverse diameter (to the long axis of the forearm) should be measured and recorded in millimetres. Recordings of "negative", "doubtful", or "positive" are not recommended.

4. Approximately 2% to 3% of persons tested will have localized redness or rash (without induration), which occurs within the first 12 hours. These are allergic reactions, are not serious and do not indicate tuberculosis infection.⁹

Interpretation

When interpreting the tuberculin test, one must not think simply in terms of one dimension - that of size - but, rather, in three dimensions. The three dimensions of tuberculin test interpretation are:

(1) size of the reaction; (2) predictive value of the test based on possible causes of false-negative and false-positive reactions; and (3) risk of development of active tuberculosis.

Causes of false-negative reactions

1. Poor injection technique.¹⁰

2. Immune suppression due to advanced age, corticosteroids, cancer therapy agents, or HIV infection, especially if advanced (CD4 count < 500).¹¹

3. Malnutrition, particularly when there has been recent weight loss.¹²

4. Severe illness, which can include tuberculosis.¹³

5. Viral illness or vaccination with live virus vaccine such as mumps or measles vaccine. If vaccination or viral illness has occurred recently, tuberculin testing should be delayed by at least 1 month.

Causes of false-positive reactions

i. Nontuberculous mycobacteria (NTM or environmental)
In most of Canada, the prevalence of reactions to antigens derived from NTM (such as PPD-B for M. avium-intracellulare) is very low. A recent Canadian study demonstrated that less than 5% of Canadian-born young adults had reactions to PPD-B, and these were responsible for less than 5% of all reactions of 10 mm or greater to standard tuberculin tests.^14,15 An earlier study in British Columbia gave different results. In most of Canada, sensitivity to nontuberculous mycobacterial antigens is uncommon and is not an important cause of tuberculin reactions of 10 mm or greater.¹⁶ Therefore, in Canada, 10 mm remains the standard cut point to determine whether tuberculosis infection is present.

ii. BCG vaccination
BCG vaccination may have been received by several population groups, including immigrants from many European countries and most developing countries. In Canada, many Aboriginal Canadians and persons born in Quebec and Newfoundland from the 1940s until the early 1980s have been vaccinated.

From studies conducted in Canada and in several other countries, if BCG is received in infancy (the first year of life), it is very unlikely to cause tuberculin reactions of 10 mm or more after the age of 2 or 3. Therefore, a history of BCG vaccination received in infancy can be ignored in all population groups when interpreting a tuberculin reaction of 10 mm or greater.^17-20

If the BCG vaccination was received between the ages of 2 and 5, persistently positive tuberculin reactions will be seen in 10% to 15% of subjects even 20 to 25 years later.²⁰ Among subjects vaccinated at the age of 6 or older (i.e. during school age years), up to 25% will have persistent positive reactions. BCG-related reactions may be as large as 25 mm or even greater.^18,21,22 Therefore, if BCG vaccination was received after the first year of life, it can be an important cause of false-positive tuberculin reactions, particularly in populations in which the expected prevalence of TB infection (i.e. true-positive reactions) is less than 10%. This means that in the general population of non-Aboriginal Canadians or immigrants from industrialized countries who received BCG vaccinations after the age of 2, this would be the more likely cause of a positive test than true infection.

On the other hand, in populations with a high prevalence of TB infection, such as immigrants from TB-endemic countries, Aboriginal Canadians, or close contacts of an active case, the likelihood of true infection would be greater than the likelihood of a false-positive reaction, and BCG vaccination should be ignored. An additional group in whom the history of BCG vaccination should be ignored are those with high risk of development of active disease if infected, such as immunocompromised individuals, those with renal failure, diabetes, or HIV, or patients with abnormal chest radiograph consistent with inactive TB.

Interpretation when Sequential Tuberculin Testing is Performed

Non-specific variation

Because of differences of technique of administration or reading, or because of biologic differences in response, there may be differences in the same individual from test to test of as much as 5 mm in reaction size. Therefore, 6 mm has been selected as the criterion to distinguish a real increase from non-specific variation.¹

Conversion

The clinical situation is the most helpful in distinguishing conversion from boosting. If there has been recent exposure such as close contact with an active case, or a worker with occupational TB exposure, then conversion will be more likely than in a situation where there has been no exposure. Conversion is defined as a tuberculin reaction of 10 mm or greater when an earlier test resulted in a reaction of less than 5 mm. If the earlier result was between 5 and 9 mm, the definition of conversion is more controversial. There are at least two criteria in use, although neither have strong supportive evidence:

1. An increase of 6 mm or more - this is a more sensitive criterion.

2. An increase of 10 mm or more - less sensitive, but a more specific criterion. In general, the larger the increase, the more likely that this is due to true conversion.¹

Booster effect (from 2-step tuberculin testing)

A single tuberculin test may elicit little response yet stimulate an anamnestic immune response, so that a second tuberculin test at any time from 1 week to 1 year later will elicit a much greater response. This phenomenon is important to detect as it could be confused with tuberculin conversion. The booster effect was first described in older populations in whom it was felt to represent remote tuberculosis infection when immunity had waned.²³ It has also been described in persons with prior BCG vaccination^15,24 or sensitivity to nontuberculous mycobacterial antigens, such as PPD-B.^15,25

Indications for 2-step Testing.

Two-step skin testing should be performed if tuberculin skin testing will subsequently be conducted at regular intervals or after exposure, for instance, among health care or prison workers. It could be considered for travellers to high prevalence areas for prolonged visits.

Technique.

The same material and techniques of administration and reading should be used. The second test should be performed 1 to 4 weeks later: less than 1 week does not allow enough time to elicit the phenomenon, more than 4 weeks allows the possibility of true tuberculin conversion to occur. Both tests should be read and recorded at 48 to 72 hours. In some centres, to reduce the total number of visits required to three, the first test is read at 1 week, and those persons whose results are not significant can have the second tuberculin test performed immediately. However, reading performed at 1 week is less accurate and is not recommended.

Interpretation.

The only two longitudinal studies of the risk of TB following a booster reaction defined the reaction simply as a second test result of 10 mm or more induration.^13,26 Therefore, it is recommended that a second tuberculin test result of 10 mm or more should be considered significant, and the patient referred for medical evaluation and chest radiography. In the elderly, a significant booster reaction most likely represents remote tuberculous infection. In longitudinal studies, subjects with a second tuberculin test (booster) response of 10 mm or more had a risk of TB that was approximately half that of subjects whose first test response was 10 mm or more.²⁶ In general, those with a significant reaction on two-step testing should be considered to have a risk of TB disease that is intermediate between individuals with positive and individuals with negative initial tuberculin tests from the same population group.

Management.

When subjects have a significant PPD reaction after two-step testing they should be referred for medical evaluation regarding risk factors for tuberculosis, such as contact history or other medical illnesses as already outlined. All subjects should have chest radiography, and in the presence of symptoms, sputum for acid-fast bacilli smear and culture should be obtained. Those with a normal chest radiograph and without risk factors have a low risk of tuberculosis. Since the risk of tuberculosis is not absent but is about half that of patients whose initial PPD reaction is significant, the decision to give INH should be individualized. No further tuberculin tests should be performed, as even years later the results will be impossible to interpret.

Chest Radiography

The chest radiograph is not considered a tool to diagnose tuberculosis infection. However, it is quite commonly carried out for some other reason and radiographic abnormalities consistent with previous TB infection are detected. As well, all persons aged over 11 who apply for permanent residence in Canada undergo screening with chest radiography. Some of these may have radiographic abnormalities (see Chapter III-C, Surveillance and Screening in Tuberculosis Control). Individuals are considered to have "inactive TB" when the chest radiograph shows certain abnormalities consistent with TB infection and a tuberculin test reaction of at least 5 mm. These individuals have increased risk of reactivation and may be considered for treatment of latent tuberculous infection (LTBI) (see again Table 1 and Chapter II-E, Treatment of Tuberculosis Disease and Infection).

The following radiographic findings are commonly believed to represent inactive TB. Some are associated with increased risk of reactivation of active TB disease in future, others are not:

1. Granulomas, which may be calcified or not. This gives the subject approximately double the risk of reactivation of active disease.

2. Calcified hilar lymph nodes. If there are no parenchymal lesions, this group does not appear to have increased risk relative to tuberculin reactors with normal radiographs.

3. Costophrenic angle blunting. This is due to past pleural effusion or pleurisy, which can have many causes. The most common cause in individuals from TB-endemic areas is previous primary tuberculosis; such individuals have increased risk of reactivation of TB disease.

4. Apical pleural capping. This is not felt to be related to TB infection and is a non-specific finding that is more common in older individuals.

5. Apical fibronodular disease. This is associated with increased risk ranging from six to 19 times that of tuberculin reactors with normal radiographs. Individuals with more extensive abnormalities have greater risk of disease.

Diagnosis of Pulmonary TB Disease
(for Diagnosis of Extrapulmonary Disease, see Chapter II-D) Clinical picture

1. Epidemiologic risk group: As summarized in Chapter I-A, foreign-born individuals, particularly those from TB-endemic countries, Aboriginal Canadians, elderly people, particularly elderly males, and close contacts are at increased risk.

2. Symptoms: The classic symptom of pulmonary TB disease is a chronic cough of at least 3 weeks' duration. This cough is initially dry although after 2 to 3 months will become productive. Fever and sweats are common but may be absent in the very young and the elderly. Hemoptysis, anorexia, weight loss, chest pain and other symptoms are generally manifestations of more advanced disease.

3. Signs: The most common physical finding in pulmonary tuberculosis is a totally normal examination, even in relatively advanced cases. Bronchial breathing, rales or crepitations will be found in more advanced cases. It is important to examine for signs of extrapulmonary disease such as lymphadenopathy, pleural effusion, abdominal or bone and joint involvement, as these are often found concomitantly, particularly in HIV-infected individuals.

Tuberculin skin testing

Tuberculin skin testing is not recommended for diagnosis of disease in adults. There may be a limited role for tuberculin testing in diagnosis of pediatric TB (see Chapter II-G, Pediatric TB). Tuberculin tests will give false-negative results in 20% to 30% of patients with active TB at the time of initial diagnosis.¹³ In addition, because TB disease occurs in epidemiologic groups with high prevalence of TB infection, tuberculin tests will often be positive even when TB disease is not present - i.e. the predictive value of a positive test is very low.

Chest radiography

The chest radiograph is the usual first step in evaluation of an individual with pulmonary symptoms. However, it is important to be aware that chest radiography has substantial limitations for the diagnosis of pulmonary TB disease.

1. Typical findings. There is a triad of classic findings that will be seen in non-immunocompromised adults.

• position - apical posterior or superior segment in 90%.

• volume loss - this is a hallmark of TB disease because of its destructive and fibrotic nature.

• cavitation - this is seen at a later stage and depends upon a vigorous immune response. Therefore, it will not be seen in severely immunocompromised individuals.

2. Atypical features: hilar and mediastinal lymphadenopathy will be seen, particularly in HIV infected individuals.

• non-cavitary infiltrates and lower lobe involvement will be seen in the immunocompromised, such as patients with diabetes, renal failure, or HIV infection.

3. Radiographic signs of complications:

• endobronchial spread - TB may spread endobronchially to the ipsilateral and contralateral lower lobes. This results in irregular, poorly defined, small nodular shadows (acinar shadows).

• pleural effusion can be seen concomitant with pulmonary disease and may represent TB empyema.

• pneumothorax can rarely occur due to erosion of caseous focus into a bronchus and simultaneously into the pleural space causing a broncho-pleural fistula.

Limitations of the chest radiograph

1. Sensitivity. Chest radiographs will have a sensitivity of only 70% to 80% for diagnosis of active TB. If any abnormality is considered the sensitivity will be more than 95%.²⁷ Approximately 10% of HIV-positive individuals and close contacts of active pulmonary disease will have normal radiographs.

2. Specificity is relatively poor, in the range of 60% to 70%.²⁷ If sensitivity is improved (any abnormality is considered possible TB) then specificity will be much lower.

3. Interreader variability. One of the greatest problems of chest radiograph reading is that the interpretation is highly variable. There is very poor agreement among readers regarding the presence of cavitation, hilar lymphadenopathy, and likelihood of active disease.²⁷

In summary, the chest radiograph is not considered the gold standard for diagnosis of pulmonary TB.

Microbiology
(see also Chapter II-A, Bacteriologic Aspects of Tuberculosis and Mycobacterial Infection)

The role of the mycobacteriology laboratory is to isolate, identify and perform susceptibility tests on clinically significant mycobacteria. Examination of stained smears of appropriate clinical material and culture, using both solid and liquid media, continues to be the most widespread method for detection. However, new molecular-based techniques for the detection and identification of Mycobacterium species are being introduced, allowing more rapid identification of individuals with disease due to MTB.²⁸

Concerning the detection of MTB, the Centers for Disease Control and Prevention (CDC) in Atlanta, Ga., has recommended that acid-fast examination of specimens should be reported within 24 hours of specimen receipt by the laboratory. Identification of M. tuberculosis should be made within an average of 14 to 21 days, and results of drug susceptibility testing should be reported within 30 days of specimen receipt.^29,30 The time between specimen collection and receipt in the laboratory should be a matter of at most a few hours.

Mycobacteria can be cultured on egg-based media, such as Lowenstein-Jensen medium, or on certain semisynthetic solid or liquid media. The "time-topositivity" of cultures depends on a number of factors, but particularly the culture medium, the metabolic state and the number of mycobacteria in the original specimen. Most isolates of MTB are detected on solid media after 12 to 28 days of incubation. With use of broth culture media, such as the BACTEC 460 system (Becton Dickinson, Sparks, MD), most mycobacteria, including MTB, can now be detected within an average of 9 to 14 days.^31-34 Therefore, in order to achieve the goals described above, it is necessary that specimens be processed daily, broth-based or liquid media used for culture, and a rapid technique used for identification - either chromatography or a molecular-based method, such as polymerase chain reaction (PCR). Besides work load, expertise and biosafety requirements, the high cost of these new techniques justifies greater centralization of mycobacteriology services. Each health care facility must decide which procedures will be performed on site and which will be referred to a reference laboratory.^30,35

Specimen Collection and Transport

All specimens should be collected in sterile leak-proof, laboratory-approved containers and accompanied by a carefully completed requisition form, providing the patient's demographic data, the physician's name, the date and time of collection, and the specimen type and site. As much as possible, specimens collected for initial diagnosis should be obtained before the initiation of anti-TB therapy.

Most specimens submitted for mycobacterial culture originate from the respiratory tract, but tissue, sterile body fluids, urine and gastric aspirates are also commonly submitted. Aspirates of pus are acceptable, but swabs of exudate should be rejected because of the limited material obtained and because the hydrophobic nature of the cell wall inhibits the transfer of the organisms from the swab to the culture medium.³³

After collection, specimens should be processed as soon as possible to avoid overgrowth by bacteria and fungi. If transportation or processing is delayed more than 1 hour, all specimens except blood should be refrigerated at 4°C to slow the multiplication of the normal flora. Most mycobacteria, including MTB, are sensitive to heat and light, especially ultraviolet light. This is another reason why all specimens that are not being transported or processed immediately should be refrigerated and protected from light. Specimens should not be frozen. MTB is resistant to many disinfectants. The most effective disinfectants for mycobacteria are phenol (5%), glutaraldehyde (2%) and formaldehyde (5 to 8%).^31-34,36

Respiratory specimens

Sputum

Sputum specimens of 5 to 10 mL should be collected, preferably in the early morning. A series of three specimens is strongly recommended.²⁷ Twenty-four hours collection is unacceptable because of the lower sensitivity and significantly increased bacterial contamination.

Induced sputum

This technique was first introduced for the diagnosis of tuberculosis more than 35 years ago. Induced sputum has a sensitivity of 90%³⁷ - better than gastric aspirate (77%)^38-40 or bronchoscopy (also 77%). It is important that induced sputum is obtained with large volumes of 3% hypertonic saline. For best results, an ultrasonic nebulizer should be used that can administer 5 to 6 mL per minute over 15 minutes. Using this, virtually all patients will produce sputum and a single sputum induction will have equivalent or better yield than fibre optic bronchoscopy.³⁷ It is important to indicate on the requisition that the sputum is induced, because the resulting specimen often appears watery. However, it can be handled in the laboratory in the same way as spontaneously expectorated sputum.

Bronchoscopy

Bronchoscopy may be used to confirm the diagnosis of tuberculosis when spontaneous sputum and induced sputum are unavailable, or all samples are smear negative. Bronchoscopy is very useful if other pulmonary diseases such as lung cancer are also suspected. However, bronchoscopy for the diagnosis of active TB entails risk and discomfort for the patient, is expensive, and can contribute to nosocomial spread of TB. In addition, the overall yield of bronchoscopy in prospective series of patients is only 77%.^41-44 If bronchoscopy is done, post-bronchoscopy sputum should be sent for AFB, as this has a similar yield as bronchial aspirate.

Gastric aspirate

This technique was introduced more than 70 years ago and is still used in some centres.⁴⁵ The primary indications are investigation of possible tuberculosis in children or elderly, demented patients who cannot expectorate sputum.

The technique is relatively simple. When the patient first awakens, a nasogastric tube is introduced to the stomach and the contents are aspirated. If nothing is obtained, small quantities (20 to 50 mL) of sterile saline can be instilled and aspirated. The fluid has to be adjusted to neutral pH within 4 hours of collection because acid is detrimental to mycobacteria. If the sample cannot be processed rapidly, it should be placed in a container with 100 mg of sodium carbonate within 4 hours, until processed. Problems with this technique include the discomfort and unpleasantness for patients, and the need to perform the procedure immediately upon the patient awakening. This often means that the patient must be kept overnight in hospital, although it can be done in the home (before 7 a.m.). In children less than 2 years old, 70% sensitivity for gastric washings has been reported, compared with a sensitivity of 30% to 40% in children between the ages of 2 and 12 years.⁴⁶

Urine

Since microorganisms accumulate in the bladder during the night, a first morning, clean-voided urine is the preferred specimen. This should be obtained on at least 3 consecutive days, and a minimum of 40 mL of urine per specimen is required for culture. Twenty-four hour urine collections are not acceptable for mycobacterial culture. Overgrowth by contaminated flora and prolonged exposure to the acidity of urine will reduce the viability of mycobacteria that might be present. Collection of specimens is performed in the same way as a midstream urine specimen. The urethral area should be cleaned carefully to reduce contamination with flora, which may include nontuberculous mycobacteria.

Body fluids

Even with symptomatic disease, most normally sterile body fluids (cerebrospinal, pleural, peritoneal, pericardial) contain only a small number of mycobacteria. Therefore, collection of as much fluid as possible is recommended, because large volumes of specimens are easier to work with, less subject to sample variation, provide increased probability of detection, and reduce the time to positivity. For CSF, at least 2 mL must be submitted. If a "spider web" is seen, consider the possibility of TB meningitis and send a portion of CSF for TB culture. Multiple samples of spinal fluid will increase the yield on smear and culture.

Biopsies

Biopsy of tissue is often the most sensitive diagnostic procedure in extrapulmonary disease. For example, lymph node excision has a sensitivity of 80% for detection of tuberculosis adenitis. Specimens must not be placed in formalin. Tissue for biopsy should be sent in a dry, sterile container without saline or with very small amounts of saline (less than 5 mL). Large volumes of saline dilute the sample and may make recovery of mycobacteria more difficult.

Shipping and Transport

Specimens should be transported to the laboratory promptly. If processing within 1 hour is not possible, samples should be refrigerated at 4° C (not frozen) and protected from light. Clinical specimens, such as sputum, are not more contagious than any other clinical specimens and, therefore, can be handled according to the same procedures. Cultures of MTB are much more hazardous. Therefore, careful protocols for packaging and shipment are required for mycobacterial cultures.³⁶

Acid-Fast Staining and Microscopic Examination
(AFB Smear)

The fluorochrome stain is the most widely used staining method for initial examination of clinical specimens because it can be read at a lower magnification than the more classical Ziehl-Neelsen stain and thus more material can be examined in a given period. However, the sensitivity of all staining methods is inferior to culture: a minimum of 5 x 103-104 bacilli per mL of sputum is required for detection by microscopy using the auramine stain, and 10⁵ organisms per mL are required using the Ziehl-Neelsen stain. However, as few as 10 to 100 viable organisms can be detected by culture.²⁷

The overall sensitivity of the direct AFB smear varies from 22% to 80% ^47-49 depending on the type of specimen, patient population, staining technique, concentration technique and experience of the technologist. The sensitivity is higher for respiratory than for nonrespiratory specimens, particularly body fluids.

The specificity of the direct smear is high for mycobacteria, although other genera of bacteria can occasionally contain acid-fast organisms. It is important to remember that all nontuberculous mycobacteria (NTM) will be AFB positive. Therefore, a positive AFB smear almost always indicates mycobacterial disease, but not necessarily because of MTB.

When acid-fast organisms are seen, the number of bacilli are reported semiquantitatively, as shown in Table 3.

Table 3
Number of bacilli seen on microscopy and laboratory interpretation³³

(Number of AFB seen by staining methods)
Fuchsin stain Ziehl-Neelson (1000 fold magnifi- cation)	Fluorochrome (250 fold magnifi- cation)	Laboratory report
0	0	Negative
1-2 per 300 fields (3 sweeps)	1-2 per 30 fields (1 sweep)	Indeterminate, repeat
1-9 per 100 fields (1 sweep)	1-9 per 10 fields	1+
1-9 per 10 fields	1-9 per field	2+
1-9 per field	10-90 per field	3+
>9 per field	>90 per field	4+

 

Mycobacterial Culture

Culture for MTB is considered the gold standard in diagnosis. For pulmonary tuberculosis, the sensitivity of three sputum cultures exceeds 90%, although six specimens are required to achieve a sensitivity of 100%.²⁷ It is for this reason that three sputum are recommended, as this represents the best balance between high sensitivity and efficiency. A single positive culture for M. tuberculosis, in general, is considered to define active disease. However, it is important to remember that occasionally cultures can be false positive, largely because of cross-contamination within the laboratory.^50,51 Therefore, if there is a report of a single positive culture, especially with a long detection time and/or few colonies, and clinical suspicion is very low, then the laboratory should be alerted to the possibility of a false-positive culture and appropriate verification conducted (generally this will mean use of DNA fingerprinting studies).

Identification of Mycobacterial Species^32-34

1. Biochemical tests. Historically, mycobacteria were identified on the basis of their rate of growth and pigmentation as well as biochemical tests. For example, M. tuberculosis is a non-chromogenic mycobacterium. These tests were well standardized and inexpensive, but results were available only 2 to 4 weeks after growth was first detected. Therefore, they are rarely performed in Canadian laboratories nowadays.

2. NAP test. The NAP is carried out in laboratories using the BACTEC 460 system to identify bacteria of the M. tuberculosis complex. The NAP inhibits growth of mycobacteria belonging to the MTB complex, but does not inhibit growth of other mycobacteria.

3. HPLC. High performance liquid chromatography (HPLC) analyzes the cell wall lipids of mycobacteria. This is technically complex and requires expensive equipment, so it is available in only a few reference laboratories in Canada.

4. DNA probes. DNA probes are now available for the identification of many mycobacterial species, including M. tuberculosis complex, M. avium complex, M. avium, M. intracellulare, M. gordonae, and M. kansasii (Accuprobe; Gen-Probe, San Diego, Calif.). These results are available within 2 hours, but can be used only to test positive cultures as they are not sensitive enough to detect mycobacteria in clinical specimens. The probes for mycobacteria other than MTB are rarely available in Canadian laboratories and are very expensive.

Amplification Tests
(see also Chapter II-A, Bacteriologic Aspects of Mycobacterial Infection)

Amplification tests are now commercially available to detect M. tuberculosis in clinical specimens. These tests involve two steps. First, specific segments of MTB DNA are amplified, then the amplified DNA is detected with DNA probes. Most of the commercially available assays can be completed in 4 to 6 hours. The sensitivity of these assays when used with clinical specimens of sputum is approximately 95% for specimens that are smear positive, and 50% to 60% for smear-negative specimens.^52-54 The specificity is excellent - 98%. At present these tests are recommended only for smear-positive respiratory specimens. Their usefulness could increase in the near future if the sensitivity is improved and the cost is lowered.

DNA Fingerprinting of Strains

A standardized technique for restriction-fragment-length polymorphism (RFLP) analysis has been developed based on differences in the number and sites of a chromosomal insertion sequence, IS6110, which is found in the vast majority of M. tuberculosis strains. This method provides a very powerful tool to demonstrate which isolates are the same or different strains.^55-57 It has been used for outbreak investigations to distinguish reactivation from reinfection, or for the investigation of cross-contamination of specimens in the laboratory. However, the technique is available only in a few reference laboratories in Canada and, to date, has been mainly used for epidemiologic research.

Serology

A serologic test for the diagnosis of tuberculosis was first described in 1898. However, after a century of efforts, there is still no accurate serologic test for the diagnosis of active TB. Earlier assays were quite crude and unstandardized, resulting in poor sensitivity and specificity.⁵⁸ Newer antigens using ELISA (enzyme-linked immunosorbent assay) techniques have better specificity.

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