Abstract
Introduction: Reports indicate that fluorescent staining of smears increases sensitivity of direct microscopy; so ZN staining is being replaced with fluorescent microscopy in RNTCP in India. Chemical processing and sputum concentration may also improve sensitivity of microscopy. Objective: To compare the sensitivity and specificity of microscopy for AFB using ZN and fluorescent stains in direct and concentrated specimen with culture as gold standard. Methods: Morning sputum specimen of patients, suspected of having pulmonary tuberculosis, over a period of 6 months was subjected to direct microscopy using fluorescent stain; the same slide was over-stained with ZN stain. Same sputum sample was concentrated by Petroff’s method and subjected to fluorescent microscopy followed by ZN microscopy and finally to culture for AFB. Results: Sensitivity of fluorescent stained concentrated sputum samples was maximum and of ZN stained unprocessed sputum samples was minimum. Specificity of three of the methods was equal at 0.96 but of ZN stained concentrated sputum smears was 0.97. Sensitivity of total fluorescent stains was 0.85 (Specificity 0.96) and sensitivity of total ZN stained smears was 0.80 (Specificity 0.96). Discussion: We used same smear for fluorescent and ZN stains, so smear related variability is decreased. Blinding for microscopy was practically complete. Conclusion: The sensitivity of sputum microscopy for AFB can be increased by concentrating the sputum and using fluorescent microscopy. The specificity remains high in all the methods.
Highlights
Reports indicate that fluorescent staining of smears increases sensitivity of direct microscopy; so ZN staining is being replaced with fluorescent microscopy in RNTCP in India
Fluorescent microscopy has been proposed by some experts for using in countries with a high prevalence of HIV infection [7] [8] and ZN staining is being replaced with fluorescent microscopy in RNTCP in India
Inclusion and exclusion criteria All the patients who were suspected of having pulmonary tuberculosis by the principal investigator based on history, clinical examination and x-ray, requiring microscopy for acid-fast bacilli (AFB) were included in the study after getting their written informed consent on proforma approved by Institutional Ethics Committee
Summary
Reports indicate that fluorescent staining of smears increases sensitivity of direct microscopy; so ZN staining is being replaced with fluorescent microscopy in RNTCP in India. Different factors that affect ZN sensitivity, smears and smear staining may be of poor quality, and it may not be possible to examine the recommended number of fields per slide due to fatigue and excessive workload [5] These factors diminish the sensitivity of ZN microscopy and tuberculosis cases may be missed. The examination time is reduced about 10-fold with fluorescence compared to bright-field microscopy using a four-fold different magnification (250 vs 1000) [6] For this reason, fluorescent microscopy has been proposed by some experts for using in countries with a high prevalence of HIV infection [7] [8] and ZN staining is being replaced with fluorescent microscopy in RNTCP in India. A potential shortcoming of fluorescence microscopy is the possibility of false-positive results because inorganic objects may incorporate fluorochrome dyes [9] [10]
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