3.1 Sputum smear microscopy

Sputum smear microscopy allows a rapid and reliable identification of patients with pulmonary tuberculosis (PTB) where there are more than 5000 bacilli/ml of sputum.
If the sputum has less than 5000 bacilli/ml, smear microscopy is highly unlikely to diagnose PTB, thus has an overall low sensitivity for PTB1,2,3.

Another shortcoming of smear microscopy is its non-specificity, such that M. tuberculosis appears the same as non tuberculous mycobacteria (NTM). However, in areas of high TB prevalence, positive smears have a very high probability of being M. tuberculosis.

The reliability of sputum microscopy depends on the quality of sputum collection. Sputum produced on early morning often shows a higher concentration of M. tuberculosis. Importantly, the reliability of sputum microscopy depends on the proper preparation and interpretation of slides. Thus, laboratory technicians must be properly trained and quality control checks must be regularly carried out in a supervising laboratory.

It is recommended that all patients suspected of PTB should submit at least two sputum specimens. Studies have shown that, when collection and examination techniques are correctly conducted, about 80% of sputum smear-positive patients are found during the first sputum examination and over 15% more during the second. Successive, repeated examinations yield fewer positives4.

Usually, a first sample is collected at the time of the consultation when the patient is identified as a suspected TB case. A second sample is collected in the early morning the day after the initial consultation (and the patient brings the sample to the health facility if it is collected at home).

In order to limit the number of visits to the health facility, “frontloaded microscopy” (also referred to as 'same day' or 'spot-spot' microscopy) can be performed. Two sputum specimens are collected one hour apart. This strategy has shown similar results to the standard strategy over two days (spot-morning-spot) in terms of diagnostic yield5.

See Appendix 1 for sputum specimen collection, storage and shipment.

The staining methods uses a technique where the mycobacteria retain a primary stain after exposure to decolourising acid-alcohol, hence the term “acid-fast bacilli” (AFB). The two most common methods of staining, which determine the acid-fast nature of the mycobacteria, are Ziehl-Neelsen staining and auramine staining (Appendix 2)6.

Auramine staining has the advantage of permitting a more rapid slide reading. It is recommended in laboratories with a high workload defined as ≥ 20 slides per reader per day. It requires trained, experienced technicians and a fluorescent microscope. LED (lightemitting-diodes) modules that can be adapted to ordinary microscopes or new LED microscopes are simpler, cheaper and safer alternatives to traditional mercury vapor lamp microscopes and do not require dark room.

Concentration techniques (Appendix 4) increase the sensitivity of sputum smear microscopy and fluorescence and have also been shown to increase the detection up to 20% in some settings with high HIV prevalence7.