The smear is a diagnostic tool for active pulmonary tuberculosis. It is a laboratory technique that allows the detection and measurement of acid-fast bacilli such as Mycobacterium tuberculosis. It is the most widely used method worldwide for making the diagnosis of active pulmonary tuberculosis in the community and for evaluating the benefits of treatment.
Pulmonary tuberculosis is an infectious disease caused by Mycobacterium tuberculosis that infects the lungs but can affect other organs and systems. Worldwide, more than 6 million people suffer from pulmonary tuberculosis (TB).
M. tuberculosis cells in a sputum smear (Source: Microrao / CC BY-SA (https://creativecommons.org/licenses/by-sa/4.0) via Wikimedia Commons)
Mycobacterium tuberculosis and other non-tuberculous Mycobacterium species possess a lipid coating on the cell wall that is rich in mycolic acid, which is resistant to acid alcohol discoloration after staining with basic dyes.
Therefore, special staining methods such as the Ziehl-Neelsen method are used. These simple and inexpensive methods allow the wall to retain the stain when the sample is rinsed with an acid solution, and the Mycobacterium wall turns red in contrast to other bacteria that turn blue.
To do a smear microscopy, a sputum sample is required, preferably taken in the morning when you get up. Tissue samples or body fluid samples can also be used.
Process
The samples to be processed for smear microscopy are sputum samples, tissue samples, or samples of body fluids. Tissue samples can be extracted by bronchoscopy or by biopsies of lymph nodes or other tissues.
As tuberculosis can affect any organ or system, the samples can be very varied, including: urine samples, cerebrospinal fluid, pleural fluid, ascitic fluid, blood, pus from open cavities, biopsies, etc.
Extrapulmonary lesion specimens should also be processed for culture. Sputum samples are collected in the morning hours upon rising.
Several samples can be collected in a row or on consecutive days. Samples should be labeled in closed, wide-mouth bottles. They should have the patient's data, the type of sample and the date of the sample collection.
Technical procedure
- Once the sample is obtained, it can be centrifuged to concentrate or used directly. The initial procedure consists of placing a few drops of the sample and spreading them on a slide.
- The sample is covered with filtered fuchsin (staining solution).
- Then it is heated until obtaining about three emissions of whitish vapor.
- Wait five minutes for the sample to cool down and wash it with water.
- It is covered with bleaching solution (acid alcohol) for a period of 2 minutes.
- It is washed again with water.
- The sample is covered with methylene blue and allowed to stand for one minute.
- Washes off with water.
- Let it dry in the air and proceed to the observation under the microscope.
Photograph from a microscope (Image by Konstantin Kolosov at www.pixabay.com)
This procedure is quick, simple, inexpensive, and allows Mycobacterium tuberculosis cells to stain red. This technique is called the Ziehl-Neelsen staining technique.
Microscopic observation consists of identifying the presence of acid-fast bacilli and their quantification by field.
Mycobacterium tuberculosis cells appear as red, granular, curved rods on a blue background. They can be isolated, paired or grouped. Observation should be done by field and at least 100 fields should be examined for each smear.
The result is considered positive when there are, in each field, more than 10 M. tuberculosis cells. This corresponds to 5,000 to 100,000 bacilli for each milliliter of sample.
In these cases, it is considered positive for Mycobacterium tuberculosis, since nontuberculous Mycobacterium or other acid-resistant bacteria have different forms and generally the amount observed per field is less than 10.
The person examining the sample must have experience or expertise in the detection and recognition of these microorganisms, since false negatives or false positives may exist in the results.
There are other fluoroscopic techniques that are more sensitive, but much more expensive. One such technique uses Auramine-O staining to obtain green fluorescence or Auramine O / Rhodamine B staining to observe yellow / orange fluorescence.
Positive cases are generally carried out for culture and antibiogram to confirm the diagnosis and the appropriate selection of treatment.
What is smear microscopy for?
Tuberculosis is an infectious disease that affects the lung and other organs and organ systems of the body. It is a contagious disease that produces serious injuries to the respiratory system that, if not treated properly, can cause the death of the patient.
Tuberculosis is transmitted almost exclusively through droplets suspended in the air from the expectoration of a patient infected with pulmonary tuberculosis. These small droplets can remain in the air and have the ability to infect the person who inhales them.
M. tuberculosis cells (Source: NIAID / CC BY (https://creativecommons.org/licenses/by/2.0) via Wikimedia Commons)
Infection requires prolonged contact time with an infected patient who continually spits bacilli in a poorly ventilated environment. People with immunodeficiencies are more likely to become infected.
HIV-positive patients are more likely than the rest of the population to develop serious respiratory infections, including pulmonary tuberculosis. Hence the importance of smear microscopy as a rapid and inexpensive diagnostic method for tuberculosis.
Smear microscopy allows a bacteriological diagnosis of tuberculosis to be made, however, a positive result must be confirmed with other studies.
Studies that confirm the diagnosis of tuberculosis include radiological examinations that show characteristic images in the upper lobes of the lung, positive tuberculin tests, and positive sample cultures.
Smear negative
A negative smear microscopy does not necessarily rule out the diagnosis of tuberculosis, since especially when the sample is sputum, the elimination of bacilli is not constant. Therefore, in these cases, when the diagnosis is suspected, serial samples are necessary.
Positive smear
A positive smear microscopy is indicative of tuberculosis and must be confirmed with other complementary studies. Whenever the result is positive, a culture and antibiogram should be done.
Once the treatment is established, a sustained decrease in the positivity scale of the smear microscopy until obtaining negative results, allows to control the benefits of the treatment.
References
- Aziz, MA (2002). External quality assessment for AFB smear microscopy.
- Desikan, P. (2013). Sputum smear microscopy in tuberculosis: is it still relevant ?. The Indian journal of medical research, 137 (3), 442.
- Fauci, AS, Kasper, DL, Hauser, SL, Jameson, JL, & Loscalzo, J. (2012). Harrison's principles of internal medicine (Vol. 2012). DL Longo (Ed.). New York: Mcgraw-hill.
- Ngabonziza, JCS, Ssengooba, W., Mutua, F., Torrea, G., Dushime, A., Gasana, M.,… & Muvunyi, CM (2016). Diagnostic performance of smear microscopy and incremental yield of Xpert in detection of pulmonary tuberculosis in Rwanda. BMC infectious diseases, 16 (1), 660.
- Sardiñas, M., García, G., Rosarys, MM, Díaz, R., & Mederos, LM (2016). Importance of quality control of bacilloscopy in laboratories that perform diagnosis of tuberculosis. Chilean Journal of Infectology: Official Organ of the Chilean Society of Infectology, 33 (3), 282-286.
- Sequeira de Latini, MD, & Barrera, L. (2008). Manual for the bacteriological diagnosis of tuberculosis: standards and technical guide: part 1 smear microscopy. In Manual for the bacteriological diagnosis of tuberculosis: standards and technical guide: part 1 smear microscopy (pp. 64-64).