3.1. Clinical samples

Given the critical importance of microbiology for TB diagnosis, it is important to ensure that specimens are correctly collected and processed to achieve valid results. All specimens should be collected in sterile, leak-proof, laboratory-approved containers and accompanied by a completed requisition form providing the patient identifier, the ordering physician’s name, the date and time of collection and the specimen type and collection site. As much as possible, specimens collected for initial diagnosis should be obtained before the initiation of anti-TB therapy.²⁶^,²⁹

Once collected, specimens should be transported to the laboratory promptly. If transport or processing within 1 hour is not possible, samples should be kept at 2-8 °C (not frozen) and protected from light until transport.

3.1.1. Sputum

At least 3 sputum specimens, optimally 5-10mL each, should be collected and tested with microscopy as well as culture. While available evidence shows that the incremental yield of the third sputum smear is only an additional 2-5%,³⁰^,³¹ the incremental yield of the third culture may be as high as 5-10%, especially in HIV-infected people.³¹^,³²

While it is conventional to collect three separate morning sputum specimens, it is well known that this scheme is inconvenient to patients, making drop-outs during diagnosis common. Published research has demonstrated the feasibility of “frontloaded” diagnosis of TB using specimens collected on the same day and shown that the diagnostic yield is undiminished.³³

3.1.2. Induced sputum

A systematic review of 23 studies reported that the overall success rate of sputum induction was high, ranging from 76.4 to 100%, while adverse events associated with sputum induction were infrequent and mild.³⁴ The sensitivity of microscopy is variable, presumably because the bacteria are diluted by the inhaled saline. Yield of induced sputum is generally higher than nasopharyngeal aspiration and gastric lavage,³⁴ or stool samples.

It is important that sputum induction be performed 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.³⁵ With this use, virtually all patients will produce sputum, and a single sputum induction will have equivalent or better yield than fiberoptic bronchoscopy.³⁶ Sputum induction has also been performed successfully in very young children³⁷ (see Chapter 9: Pediatric Tuberculosis). Although the specimen often appears watery, it can be handled in the laboratory in the same way as spontaneously expectorated sputum.

3.1.3. Bronchoscopy

Bronchoscopy may be used to facilitate the diagnosis of TB when spontaneous and induced sputum are unavailable, or if another disease, such as lung cancer, is suspected.³⁸ Used solely for the diagnosis of active TB, however, it entails risk and discomfort for the patient, is expensive and can contribute to nosocomial spread of TB if not performed in an appropriate environment with protection of staff. In addition, the overall yield of bronchoscopy in prospective series of patients is only 77%.^39–42 If bronchoscopy is done, post-bronchoscopy sputum should be sent for AFB testing, as this has a yield similar to that of bronchial washings and lavage.

3.1.4. Gastric aspirate

This technique was introduced in the early 20th century and is still used in some centers.⁴³ The primary indications are investigation of possible TB in children who cannot expectorate sputum or, for the same reason, elderly patients with dementia. The technique is relatively simple and is described in Chapter 9: Pediatric Tuberculosis.

3.1.5. Stool

As young children swallow their sputum, recovery of M. tuberculosis from stool samples may be a way to diagnose TB disease. Collection of stool samples is noninvasive and doesn’t require specialized equipment/expertise. However, there are no standardized recommendations for stool processing for culture and NAAT testing and various studies have demonstrated relatively lower sensitivity of stool specimens vs sputa or gastric aspirates, as well as higher potential for culture contamination.⁴⁴ As such, stool cultures are currently not recommended in Canada for the purposes of PTB diagnosis.

3.1.6. Other specimen types

A variety of specimen types may be collected for diagnosing extra-pulmonary TB. In general, tissue has a higher yield than liquid/biologic fluids. The general best practices for collection and transport of these specimens are the same as for those routinely used for diagnosis of pulmonary TB. Handling of these specimens in the laboratory may differ with respect to the ability to perform AFB smears and/or molecular testing, as well as full culture set-up. Given that extra-pulmonary samples can have a smaller number of bacteria and there is often a small volume of sample, priority should be given to culture over other assays, such as microscopy or NAAT testing. Additional considerations specific to individual specimen types are described in Appendix 1 of this chapter and in Chapter 7: Extra-pulmonary Tuberculosis.

Recommendations

We strongly recommend that in all persons with suspected pulmonary TB, at least three (either spontaneous or induced) sputum specimens should be collected and tested with microscopy and culture (good evidence).
We strongly recommend that three sputum specimens (either spontaneous or induced) should be collected on the same day, at least 1 hour apart (good evidence).
We conditionally recommend that sputum samples should be collected in sterile containers without any transport medium and transported to the mycobacteriology laboratory within 1 day or stored at 2-8 °C until transport (poor evidence).

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