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What is the rationale of collecting three specimens for sputum microscopy?

Of the total smear positive cases presenting at health Institutions, about 69% can be detected by smear examination of first sputum specimen and another 20% by examination of second specimen. The additional case yield by subsequent specimen is minimal. Therefore, a minimum of two specimens should be examined for obtaining acceptable sensitivity of smear microscopy as a case finding tool. Further, there is likelihood albeit small of single positive sputum smear result to be false positive. However, the chances of two positive results to be false positive are practically non-existent. Therefore, the criteria of at least two smear positive results to label a case as smear positive increases the specificity of the test. Since the bacilli are not excreted consistently in all specimens, at least three specimens should be examined to satisfy the above criterion.

Why should we examine at least 200 oil-immersion fields to declare a sputum smear as negative?

A sputum smear is spread over an area of 200 mm2 and contains about 0.01 ml of the specimen. Each oil-immersion field has an area of 0.02 mm2. Therefore, there are 10,000 fields on the slide. If a specimen contains 5000 bacilli/ml, there will be 50 bacilli distributed over entire smear and 1 bacilli over 200 fields. Considering that the bacilli are not distributed evenly over the smear, there must be at least 10,000 bacilli/ml to detect 1 bacillus over 200 fields.

What precautions are needed to reduce false positive sputum smear results?

Rinse mouth before sputum collection or collect specimen before food. Use separate wooden stick for each smear preparation and stain each slide separately on a rack. Use new slides. Nevertheless, the scratches on the slide can be differentiated as these occur in parallel rows and are found deeper. Use fresh stains and preferably filter the stains before use. Preferably, use distilled water to avoid false positive result from environmental mycobacteria. Do not touch the slide with the objective of the microscope or the oil dropper. Clean lens with a lens cleaning paper (not cotton) after exam of each slide.

What are common reasons of false negative results?

Improper guidance to the patient resulting in poor quality of the sputum sample is the most common reason of a false negative result. Bacilli may loose acid fastness if exposed to excessive heat (over heating while fixing the slide), direct sunlight, or long period of storage in hot and humid conditions. Smear not prepared from blobs that contain dead caseous tissue discharged from cavity. Too thin or too thick smear Improper fixing To short carbol-fuchsin staining or boiling it Intensive counter staining Erratic and brief examination Administrative errors

What is the role of X-ray in RNTCP?

Chest X-ray has a role in differential diagnosis of pulmonary disease among chest symptomatic patients whose sputa are consistently negative on smear microscopy.

Is it possible to detect cases early by X-ray or culture?

Smear positive cases do not necessarily pass through an early smear negative stage and only a few smear positive cases would be prevented by use of additional more sensitive methods for detecting pulmonary TB cases, which will also shift priority from detecting and treating most infectious cases.

What is the role of culture?

A cavity of 2-cm diameter contains about 100 million organisms, easily detected by sputum smear microscopy. Almost the same number of new cases can be detected by the first two smears as by first 3-culture examination. Therefore, the additional yield of cases by culture is small. Only nodular lesions discharging small amounts of bacilli are negative by microscopy. Moreover, only culture positive TB cases (negative on microscopy) discharge bacilli intermittently, compared to smear positive cases, which discharge bacilli more consistently. Nevertheless, culture has a role in drug sensitivity testing, diagnosis of extra-pulmonary TB like lymph node TB and for differential diagnosis in a few cases after carefully evaluating microscopy and radiology.

Why are there so many grades of microscopy while treatment does not change with grading?

Grading assists in quality control and also saves time since fewer fields have to be examined for higher grades. It also assists in monitoring prognosis during course of treatment. A higher proportion of 2+ and 3+ smears are likely to remain positive at the end of intensive phase and may require additional one month of intensive Phase.

Why should the grading vary from one sample to another in the same patient?

Bacilli are not evenly distributed in a specimen but are found in clumps. (Specimens consistently positive contain at least 105 to 106 bacilli per ml.

What should be done if scanty positive is the result of smear microscopy among chest symptomatics?

A scanty positive smear result should be supported by another positive smear (more than scanty positive) or by suggestive chest X-ray. Otherwise, repeat sputum collection and smear examination is preferable.

Why is it important to exam smear within a week of its preparation?

In hot & humid climates, the bacilli seem to loose their acid-fatness. Even the stained bacilli may also loose the stain by osmosis in such climates. Therefore, slides labeled positive for Acid fast Bacilli (AFB) by lab technician (L.T.) but negative by senior lab technician (STLS) should be re-stained.

It has been observed that false negative errors are more common in sputum microscopy. On the other hand, only 10% random sample of negative slides is cross-checked in RNTCP?

The purpose of cross checking is to identify lab technicians that require retraining rather than identification of individual slide errors.

What is the role of newer diagnostic tests?

(i) Serological tests have poor sensitivity and specificity. Their sensitivity is highest in patients with smear positive disease and much lower among TB cases not detected by smear microscopy viz. sputum smear negative cases of pulmonary TB, extra-pulmonary TB, children diagnosed with TB and HIV infected TB cases. Also, these tests do not reliably distinguish active TB from dormant infection. (ii) Amplification tests e.g. polymerase chain reaction (PCR) are 95% sensitive in smear positive cases and only 50% sensitive in smear negative and culture positive cases. Though specificity is high, it is lower under field conditions. In addition, inability to distinguish viable from dead bacilli and high cost dissuade their use in developing countries. (iii) The Assay of cell-mediated immunity is highly complex.

What are the causes of smear positive specimen negative on culture?

(i) False positive smear result

(ii) Bacilli might have lost ability to grow in culture due to the following reasons:

- Bacilli killed or harmed by treatment

- Exposure to heat and sunlight

- Long storage of sputum specimen

- Excessive decontamination procedures while processing the specimen for culture

Why a cut off point of one month is considered for labeling a patient as a new case?

Patients with history of treatment of less than one month have been found to respond similarly, as those never treated before. Also, the chances of development of drug resistance with less than one month therapy are remote.

Is there a higher incidence of TB among contacts? What is the role of contact tracing under RNTCP?

Though relative risk of acquiring infection and developing TB is higher among contacts, the case yield from contact tracing is low. However, the risk of breakdown is maximum during the period immediately following infection, especially among children. Therefore, all child contacts and symptomatic adult contacts of smear positive cases, irrespective of the duration of symptoms should be examined at the health centre, to identify and treat TB cases and to provide preventive treatment to children.