A Comparative Study on Ziehl-Neelsen Staining (Light Microscopy), Auramine O Staining (Iled- Fluorescent Microscopy) and Culture on LJ Media of Sputum Samples for the Diagnosis of Pulmonary Tuberculosis
Authors: Bivek Timalsina1*, Bidur Kutu1, Rusna Pradhan1, Bhagwan Maharjan2
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Background: Tuberculosis remains a major public health threat in Nepal and one of the leading causes of death from communicable diseases among adults. The majority of tuberculosis is pulmonary tuberculosis infecting lungs and sputum examination is important for this as it is the major way of transmission of disease. The diagnostic procedures rely on simple and inexpensive methods mostly of microscopy and culture examination. Therefore the evaluation of these diagnostic approaches has great importance.
Objectives: To evaluate and compare the efficacy of Ziehl-Neelsen staining, Auramine O staining and culture of sputum samples for the diagnosis of pulmonary tuberculosis.
Materials and methods: Total 299 sputum samples (170 samples from 78 Group I suspected cases with no treatment; 42 samples from 22 Group II DOTS follow-up cases; and 87 samples from 87 Group III MDR follow-up cases) were subjected to direct smear preparation each for ZN and AO staining for 1000x light microscopy and 400x fluorescent microscopy examination respectively and the remaining sample were further processed with NALC-NaOH method for culture on modified Lowenstein-Jensen Media for detection of Mycobacterium tuberculosis. Positive smears were graded according to IUATLD/WHO guideline.
Result: Out of total 299 sputum samples of all types of cases, 19.06%, 29.1% and 24.41% were found pulmonary tuberculosis positive by ZN, AO and culture respectively. The case detection rates for suspected patients with no treatment were 20%, 25.88% & 28.24%; for DOTS follow-up patients were 30.95%, 57.14% & 19.05%; and for MDR follow-up patients were 11.49%, 21.84% & 19.54% for ZN, AO and culture respectively. The difference in their case detection rates was statistically significant (p < 0.01). No AO negative result with ZN positive samples was found. More number of paucibacillary cases was detected by AO method than ZN. There were 25 cultural contaminated samples. Removing contaminated cultural samples and taking culture as gold standard, the sensitivity and specificity of direct microscopic examination were found to be 60.03% and 98.51% for ZN method; and 83.56% and 94.53% for fluorescent AO method respectively. The percentage of false negative by AO staining was only 16.44% which was in sharp contrast to that of ZN (39.73%).
Conclusion: This comparative study proves that AO staining (Fluorescent microscopy) is superior to ZN staining (Light microscopy) in several aspects as efficacy, sensitivity, false negativity. Thus the AO staining aided with culture can prove to be important tool for the effective and reliable diagnosis and screening of pulmonary tuberculosis.