Sputum smear microscopy

Clinical description

Microscopy of sputum smears is simple and inexpensive and allows rapid detection of the most infectious cases of pulmonary TB. It is cheap and simple and produces rapid and reliable results.

Two staining methods can be used to identify acid-fast bacilli: Ziel-Neelsen (ZN) staining or fluorescent auramine staining (LED FM)4 Advantages

• Sputum specimens from patients with pulmonary TB, especially those with cavitary

disease, often contain sufficiently large numbers of AFB to be detected by microscopy.

• Microscopy is suitable for peripheral-level and higher-level laboratories.
• Microscopy can be done safely in a laboratory that has implemented only a low level of

precautions to mitigate the risk of laboratory acquired TB infection.

• It is essential for treatment monitoring of patients with susceptible TB.

Disadvantages

• Direct sputum-smear microscopy is relatively insensitive: at least 5000 bacilli per

ml of sputum are required for a positive result. The sensitivity is further reduced in patients with extrapulmonary TB, children and in those who are coinfected with HIV.

• A comprehensive quality assurance programme is necessary; although this may be

challenging to implement, it is necessary to ensure high-quality test results.

Limitations

• Microscopy for AFB cannot distinguish Mycobacterium tuberculosis complex (MTB)

from non-tuberculous mycobacteria;

• Microscopy cannot distinguish viable from non-viable organisms;
• Microscopy cannot distinguish drug-susceptible strains from drug-resistant strains;
• Smears that have been stained with auramine will need to be stained again if they are

to be rechecked as part of an external quality assessment programme.

4 WHO. Implementing Tuberculosis Diagnostics framework. 2015 http://apps.who.int/iris/bitstream/10665/162712/1/9789241508612_eng.

pdf?ua=1&ua=1. Accessed 1 April 2017.

34 National Tuberculosis Control Programme

• ZN microscopy has low sensitivity (40-60%) and requires at least 5000-10,000 bacilli

per ml of sputum to give positive results.

• LED FM provides additional advantages over ZN microscopy, with less time required

for slide preparation and reading. It has better sensitivity (around additional 10%) over ZN microscopy to identify bacillus.

Laboratory personnel report results based on the number of TB bacilli seen when evaluating areas visualized under the microscope called “fields” (see Table 4.2-1).

Table 4.2-1: Reporting laboratory results Result Reported Interpretation Negative No bacilli seen in 100 fields Scant or actual number counted 1 to 9 bacilli seen (either acid-fast or fluorescent) in 100 fields* 1+ 10 to 99 bacilli seen (either acid-fast or fluorescent) in 100 fields 2+ 1 to 10 bacilli seen (either acid-fast or fluorescent) per 1 field 3+ > 10 bacilli seen (either acid-fast or fluorescent) per 1 field *A finding of <3 bacilli in 100 fields does not correlate well with culture positivity

• Sputum smear result is positive if at least one tubercle bacillus (acid-fast OR fluorescent)

is detected on one or more sputum smears.