Before rapid and molecular diagnostic tests were developed for the identification of Mycobacterium tuberculosis, the primary causative agent for tuberculosis (TB), the screening, diagnosis, and monitoring of treatment success and cure of patients undergoing anti-TB therapy mainly relied on microscopy. Acid-fast bacteria (also known as acid-fast bacilli or AFB) are microorganisms resistant to decolorization by an acid, hence, the term acid-fast. Acid fastness is a unique characteristic of M. tuberculosis. However, other mycobacterial species may exhibit the same feature. This makes the test sensitive but not specific. Advances in technology optimized the use of TB microscopy. Similar terms include AFB smear microscopy, direct sputum smear microscopy. While the use of highly advanced molecular diagnostic tests has come into play, the value of microscopy cannot be underestimated especially in low and middle-income countries.
The primary specimen required in TB microscopy is sputum. Patients are advised to follow the correct procedure in specimen collection because it is critical in the generation of a quality microscopy result. The specimen collection areas are dedicated sites which must be well ventilated to prevent inhalation of aerosols by other persons. Producing an optimum quality of sputum reflects the lung pathology, thus, revealing the correct disease status of each patient. Purulent and mucopurulent sputum are the best specimens for microscopy. Suboptimal specimens include mucoid, mucosalivary and salivary specimens. It is important to differentiate salivary sputum specimens from saliva from the mouth and mucus from the nose. Saliva and mucus are not acceptable specimens because they are not representative of lung status and may give false-negative results. The presence of food debris and contaminants is not advised. Blood tinged or bloodstained specimens may be accepted but ‘bloody’ specimens should be rejected.
Medical laboratory scientists in the laboratory should regularly evaluate for specimen quality before performing the test. Patients are requested to repeat the collection until an acceptable specimen is achieved. Other than the quality, the quantity of the specimen is also important which should be within a minimum of 5 milliliters. The inadequate volume will affect the sensitivity of the laboratory test.
Specimens must be collected in appropriate sputum containers. Tuberculosis programs usually use a 50-ml capacity plastic, screw-capped, and transparent container to foster a secure containment and visual inspection of the specimen. Appropriate labeling with the patient’s name and date of collection must be observed. In recent years, submission of sputum specimens for microscopy requires three (3) collections. In those countries with an established and well implemented external quality assessment (EQA) program but with limited human resources, the WHO recommends shifting to two (2) specimen collections. This is to facilitate early or “same day” diagnosis of TB patients from the community.
Theoretically, there are 2 staining methods of TB microscopy that were developed in the past – the Ziehl-Neelsen method, often termed as the “hot method” and the Kinyoun method which refers to the “cold method.” However, this classification is not applicable at present. The current classification of staining procedures for TB microscopy was made based on the technology used, which therefore includes: (1) the Ziehl-Neelsen method which is done using brightfield microscopy, and, (2) the auramine method which requires the use of fluorescence microscopy. Despite the differences in the type of microscope, the use of special stains makes one distinct from the other. The principle of acid fastness remains the foundation of these staining methods.
Procedures of TB microscopy begin with smearing, followed by staining, and lastly, reading the smeared slides. Smear sizes can either be as large as 3 cm x 2 cm (length x width) or as small as 2 cm x 1 cm depending on the TB burden (i.e., prevalence of TB patients and other context-specific factors) of specific countries, which are defined by national TB program managers, laboratory network managers, and technical advisors. Smearing must be done correctly, pressing and spreading the sputum evenly within the whole smear area. Ideally, the smear should be made at the center of the slide to facilitate reading in the microscope. Smear too small or too large affect the quality of microscopy result. Using a standard template for the smear size may be helpful in maintaining the correct size, but caution is warranted in the development of such templates or patterns. Training of staff in smearing is necessary before performing the test. The smeared slides should be adequately heat-fixed before proceeding to the staining procedure.
In Ziehl-Neelsen procedure, smeared slides are primarily stained with carbol fuchsin (CF). This stain makes the AFB red. Slides with CF are then heated using an alcohol lamp until steaming, but not boiling so that the stain penetrates well inside each bacterium. The stain and smear contact should remain for approximately 10 minutes to allow subsequent cooling and trapping of the stain within the bacterial cell wall. Then, drain the slides to remove excess CF and wash with a gentle stream of water. Cover the whole slide with acid alcohol and wait for approximately 2 to 3 minutes or until the smear is completely decolorized. No obvious red stain should be seen. The decolorization step makes the non-acid-fast organisms, and other components lose its red stain.
In contrast, AFB keeps the red stain of CF because of its acid fastness. Then, washed slides are stained with methylene blue that serves as a counterstain. The recommended time is 1 minute (60 seconds) but will still depend on the quality of methylene blue. Counterstaining fosters an effective visual differentiation of AFB during microscopy.
In the auramine staining procedure, the primary stain used is auramine (chemical name, auramine O), a hydrochloride dye which enables the stained AFB to emit fluorescence (light green or yellow) when viewed under a fluorescence microscope. Unlike in CF, no steaming via heat is required. The contact time, however, is prolonged up to about a minimum of 20 minutes before washing with water. For 1 to 2 minutes, decolorize each slide with acid alcohol. Rinse with water and counterstain. Methylene blue can still be used, but in more settings, the use of potassium permanganate as a counterstain provides a darker background, thus, giving better contrast and improving sensitivity to fluorescing AFB. Finally, wash the smeared slides with water and allow to air dry. Blotting slides will not be necessary since it may damage the stained smears.
TB microscopy is generally indicated for presumptive TB patients. Patients should always receive detailed instruction for specimen collection from physicians, nurses, laboratory professionals, and other authorized staff. One positive smear out of the 2 smears submitted means the patient is positive for the presence of AFB, not for M. tuberculosis. Patients should be guided accordingly regarding the course of the diagnostic algorithm whatever result they get. Result interpretation should be sought only from their attending physicians or other authorized healthcare personnel to avoid misinterpretation and confusion. Nebulization may help enhance sputum induction but is not required if appropriate supervision is given. Children may have difficulty producing sputum.
Results of TB microscopy must always be clinically correlated with the patient’s history, physical examination, and other diagnostic tools. A TB patient shall undergo treatment within 6 months comprising the initial and the continuation phase. TB microscopy is found effective and reliable in monitoring the patient's response to therapy. For instance, a cured TB patient is confirmed by a negative microscopy result at the end of the treatment regimen.
Multidrug-resistant (MDR TB) and extremely drug resistant (XDR TB) tuberculosis is not detected via TB microscopy and would need a more specific and sensitive test such as TB culture and drug susceptibility test (DST) either by phenotypic or genotypic methods. The Xpert MTB/Rif assay is an example of a nucleic acid amplification test (NAAT) which provided a diagnostic breakthrough for TB and has replaced TB microscopy as a primary diagnostic tool in some settings. However, monitoring the progression of patients on chemotherapy using this molecular technology has not found to be effective, thus, the need to utilize TB microscopy is still significant.
Reading smears is a critical step in TB microscopy. Recently, the Global Laboratory Initiative (GLI) developed a handbook for TB microscopy which specifies guidelines for reading, recording, and reporting of microscopy results both for Ziehl-Neelsen and auramine methods. Reporting can vary from 0 if no acid-fast bacilli were seen; +n for scanty AFBs and 1+, 2+ and 3+ for increasing number of AFBs (or its average) examined per field. The recommended number of visual fields examined is 150 for 3 cm x 2 cm smear size and 100 for 2 cm x 1 cm smear size. Remember that countries should institutionalize national policies and guidelines, especially the recording and reporting of TB microscopy results to establish standards that will be followed by the laboratory network. Reports generated from the peripheral, provincial, regional and national levels will be useful for monitoring the TB program. Information from TB microscopy will further inform policy makers and health authorities on how to strengthen laboratory capacity and improve health service delivery.
Several factors can affect the result of TB microscopy. Such include but not limited to the pre-analytical factors from specimen collection (as previously stated), inappropriate labeling (patient’s name), specimen storage (exposing specimens in direct sunlight may destroy a significant amount of AFB from the sample). Analytical factors include poor performance of smearing (inappropriate selection sample to be smeared, poor smear size and technique, unfixed smears), staining (contamination of slides, incorrect staining time and procedure, blotting smears, fading of auramine-stained smears) and microscopy reading (incorrect recording and reporting of results). Post-analytical factors may involve mix-up and release of mismatched patient results and other clerical errors. Long turnaround time and an incorrect result interpretation could also interfere and might happen across healthcare settings.
When the use of fluorescence microscopy was introduced several years ago in the Philippines, TB laboratory scientists and experts at the national reference laboratory have performed validity checks on the results yielded by auramine method. Auramine-stained smears were directly restained with Ziehl-Neelsen and examined under brightfield microscopy. The experiment revealed that the comparison of the two results did not yield similar results. Restaining previously stained smears with a different method, as seen in this case, turned out a lower number of AFB in contrast to its original result, hence, making this approach not recommended for future quality assurance activities. Moreover, scientists observed the presence of “bacilli-shaped figures” without color or stain, which were initially referred to as “ghost AFB.” It was hypothesized that these figures were initially the AFB that could previously absorb and keep the auramine which later lost its capacity to absorb carbol fuchsin during restaining. This is an important finding for countries and laboratories who intend to implement fluorescence microscopy for tuberculosis in their areas.
Complications may arise from releasing false-positive and false-negative results of TB microscopy. False-positive results mean the patient did not have the AFB but is given the anti-TB drugs such as rifampicin, isoniazid, pyrazinamide, and ethambutol, making the patient at risk of drug-induced diseases and at the same time, wasting significant resources. On the other hand, false-negative results mean that the patient had AFB; however, they were not given appropriate chemotherapy. The patient will continue to develop the disease and consequently, spread the bacteria, making communities at risk of developing tuberculosis. Pulmonary tuberculosis may progress into extrapulmonary tuberculosis affecting other organs if no treatment is immediately started. During the treatment, the disease may worsen and become MDR TB if the patient is not well monitored.
The stigma of TB still exists. The value of patient awareness about the disease and the possibility of treatment and cure is critical so that notification rates would increase and more patients will then be initiated on treatment, further controlling its incidence. Knowledge of the availability and access to an accurate and reliable diagnostic tool such as TB microscopy can build patient trust and confidence to healthcare providers.
TB microscopy remains a valuable laboratory test for case finding and case holding activities especially in low and middle-income countries with high TB burden. Public and private sectors collaboration was found to be effective in unifying the approach of TB control efforts. Health authorities, clinicians and specialists, and technical experts measure the attainment of program objectives with the use of TB microscopy results. Provision of high-quality TB microscopy is and will always be one component of laboratory and health service for patients as stated in the International Standards for TB care (ISTC). From this, continuous monitoring of TB microscopy laboratories becomes essential for both clinical and public health outcomes.