TB Manual

3.2.1. Smear microscopy

Sputum smear microscopy is the most widely used test for TB disease.²⁶ Two stains are widely used: 1) the traditional Ziehl-Neelsen or Kinyoun staining, which requires a light or bright field microscopy and 2) the auramine-rhodamine stain, which requires fluorescence microscopy (see Appendix 1). In most high-income countries (including Canada), fluorescence microscopy is standard practice because it can be read at a lower magnification than the classic Ziehl-Neelsen or Kinyoun stain, thus allowing slides to be read more quickly.²⁸ The sensitivity of all staining methods, however, is inferior to that of culture. The threshold of detection of AFB in concentrated specimens using a fluorochrome stain is 5,000-10,000^45,46 bacteria/mL of sputum and is 100,000 bacteria/mL using the Ziehl-Neelsen stain. The threshold of detection in unconcentrated smears is 10-fold higher, resulting in much lower sensitivity. This is important to remember, since often “stat” smears are unconcentrated. In contrast, as few as 10 viable bacteria can be detected by culture.

The specificity of the AFB smear is high for mycobacteria, but it is important to remember that all nontuberculous mycobacteria (NTM) will be AFB-positive. Other organisms, such as Nocardia and other actinomycetes, can be weakly acid-fast, but these are less common. Therefore, a positive AFB smear almost always indicates the presence of mycobacteria, but not necessarily M. tuberculosis.²⁸

When acid-fast organisms are seen, the number of bacteria is reported semi-quantitatively, from 0 to 4+ as detailed in Appendix 1.

Smear microscopy is both rapid and inexpensive and identifies the most infectious TB patients (see Chapter 11: Tuberculosis Contact Investigation and Outbreak Management).⁴⁷ Although it has long been used to help assess contagiousness and manage isolation, the test has well-known limitations:

• Sensitivity is modest and variable (20-80%) depending upon the type of specimen, patient population, stain used, time used to examine and the experience of the microscopist. Thus, multiple sputum smears are recommended to increase the overall sensitivity. Sensitivity is higher for respiratory than for non-respiratory specimens, particularly body fluids.

• In low TB-incidence settings, smear microscopy has lower specificity — a positive smear could be due to NTM.

• Smear microscopy has lower sensitivity in childhood TB and extra-pulmonary disease, especially in HIV-infected people.

• Smear microscopy cannot be used to determine drug resistance.

Recommendations

• We strongly recommend that smear microscopy should be performed on concentrated samples where technically feasible (good evidence).

• We strongly recommend fluorescent microscopy to maximize the sensitivity of smear microscopy (good evidence).

3.2.2. Mycobacterial culture

Mycobacterial culture can be performed on all specimen types so long as it is received in appropriate condition.²⁶ Culture for M. tuberculosis is considered the gold standard in diagnosis, as it is more sensitive than microscopy or currently available NAAT tests. Culture allows for the identification of the pathogen, serves as the basis for DST and can provide isolates for molecular epidemiology using deoxyribonucleic acid (DNA) fingerprinting or genome sequencing. Standards and technical details for mycobacterial culture are described in Appendix 1.

A single positive culture for M. tuberculosis, in general, is considered definitive for active disease. However, it is important to remember that cultures occasionally can be falsely positive due to cross-contamination within the laboratory. When clinical suspicion is low, a report of a single positive culture, especially with a negative smear and a long detection time, should raise the possibility of a false-positive result. If this is a clinical possibility, the lab should be contacted to do further investigations.

Culture results typically take 2-to-8 weeks, depending on the culture method used and the number of bacteria in the inoculum. Once there is evidence of growth, labs will assign a presumptive identity of M. tuberculosis complex or non-tuberculous mycobacterium before further testing is done to provide a species name (see Appendix 1 for details).

Recommendations

• We strongly recommend that every specimen of sufficient volume from patients with suspected TB undergo testing with both smear microscopy and culture; for very low volumes (< 2 mL),⁴⁸ culture should take precedence over smear microscopy (good evidence).

• We strongly recommend that mycobacteria culture testing should include liquid medium culture at a minimum and both liquid and solid culture whenever possible (good evidence).

3.2.3. Nucleic acid amplification tests

The amplification of nucleic acids for the diagnosis of TB from specimens produces a faster result than conventional culture methods.^49,50 Some of these assays also provide predictions about drug resistance, discussed further in the following section on drug-resistance testing.

On the one hand, the sensitivity of NAATs to detect TB is high (>95%) in sputum smear-positive samples and they are, therefore, used to provide a rapid presumptive diagnosis of TB while awaiting culture results.^49,51 On the other hand, the sensitivity of NAATs is lower (50-70%) when smear-negative/culture-positive specimens are tested.⁵¹ The difference in sensitivity is because the analytic sensitivity of NAATs is around 100 bacteria per mL – this is lower than the analytic sensitivity of AFB smear (5000-10,000 bacteria per mL) but higher than the analytic sensitivity of culture (fewer than 10 bacteria per mL).^49–52 The sensitivity of NAATs is also lower in extra-pulmonary specimens.⁵² Negative NAAT results alone should not be used to rule out TB and considerations for patient management should be informed by other factors, such as the epidemiologic risk factors, clinical suspicion and results of culture.

Because certain cartridge-based NAAT technologies (eg, the Xpert, by Cepheid) are simple and can be implemented in peripheral laboratories, NAATs may be potentially useful in remote settings, where there is no on-site capacity for routine smear microscopy and cultures. In such settings, NAAT results can be available within hours and could potentially help reduce diagnostic delays. However, it is important to note that the use of Xpert in these settings should not replace conventional smears and cultures.

NAAT assays are continuously evolving. Front-line healthcare providers should be aware of when a test is replaced, as this could affect the negative or positive predictive values of the results. For example, the widely useful GeneXpert platform has recently introduced the Xpert MTB/RIF Ultra, which has greater sensitivity but reduced specificity compared to the first-generation assay.⁵² The greater sensitivity of the Ultra (91%) might guide a clinician to consider another diagnosis if the test is negative in a patient with a low pretest probability. In high-burden countries the Ultra had a lower specificity of 96%, whereas retrospective studies demonstrated that in low-burden settings where there is limited TB transmission, the specificity of Ultra is high (99.3%, 95%CI 96-99).⁵³ In the absence of other corroborating information supporting a diagnosis of TB, one might consider the possibility of a false-positive. Further details on these assays are provided in Appendix 1. If in doubt, providers should contact the laboratory to ask more details on the NAAT test in use and its limitations.

Because NAATs can amplify nonviable AFB, they are not recommended for use in monitoring TB treatment response or to assess patients’ contagiousness after treatment initiation.

Recommendations

• We strongly recommend that in all new smear-positive patients, at least one acid-fast bacilli positive respiratory sample should be tested with a Health Canada-approved or -validated laboratory-developed nucleic acid amplification test (good evidence).

• We conditionally recommend that in smear-negative patients suspected of having TB, a nucleic acid amplification test may be performed on one acid-fast bacilli negative sample upon request by the physician or public health (poor evidence).

• We strongly recommend against using nucleic acid amplification test results for monitoring TB treatment response or patient contagiousness after start of therapy (good evidence).

• We conditionally recommend that, in remote settings where there is currently no on-site capacity for routine smear microscopy and culture, an automated cartridge-based nucleic acid amplification test can be used to make rapid decisions on TB treatment and isolation. However, specimens should also be sent to clinical/reference laboratories for smear microscopy and culture in such contexts (poor evidence).