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Tuberculosis Research Centre (ICMR),1 Government Hospital for Thoracic Medicine, Tambaram Sanatorium, Chennai,3 South East Asia Regional Office, Stop Tuberculosis Unit, World Health Organization, New Delhi, India2
Received 3 March 2002/ Returned for modification 11 April 2002/ Accepted 5 May 2002
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ABSTRACT |
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The study was conducted at a large tertiary-care hospital in Chennai, Tamil Nadu, South India. Each patient attending the outpatient department of the hospital was screened for chest symptoms. One spot sputum specimen was collected in a McCartney bottle from each patient who reported having a cough for 3 weeks or longer. Patients who were on antituberculosis treatment at the time were excluded from the study. All samples were collected during the months of August and September 2001.
Processing of sputum samples. Sputum specimens were transported and processed within 4 h of collection. Each sample was divided into two portions of 3 to 5 ml each by pouring the sample from one McCartney bottle to another, such that both portions were approximately equal in volume and appeared similar in quality. The two portions were randomly allocated; one to the modified Petroff method for culture of Mycobacterium tuberculosis (10) and the other to the PhAS sediment smear method. For the Petroff method, sputum was homogenized for 15 min in a shaker by using an equal volume of 4% sodium hydroxide. After centrifugation at 3,000 rpm for 15 min in a Megafuge 1.0 (Heraeus), the deposit was neutralized with about 20 ml of sterile distilled water. Samples were again centrifuged, and the deposit was inoculated into Lowenstein-Jensen medium and incubated for 8 weeks at 37°C. The isolated cultures were confirmed for M. tuberculosis by a niacin test, a 68°C catalase test, and growth on para-nitrobenzoic acid.
The PhAS reagent was prepared by dissolving 50 g of phenol crystals (Qualigens, Chennai, India) and 40 g of ammonium sulfate (E. Merck, Mumbai, India) in 950 ml of distilled water. The reagent was prepared and kept in the laboratory until use (3 to 5 days). The portion allocated to the PhAS sediment smear method was used to prepare a direct smear and a PhAS sediment smear. First, a direct smear was prepared by using a wire loop; the smear was left to dry for 10 to 15 min and was then heat fixed and stained by the Ziehl-Neelsen method (8). To the remaining sputum sample an equal volume of PhAS reagent was added. The sample was mixed well and left to stand overnight at room temperature. Next morning, a PhAS sediment smear was prepared after the clear supernatant was discarded and a drop of the sediment was placed on a glass slide by using a wire loop. Sputum samples were not centrifuged to obtain the sediment. The direct and PhAS sediment smears were prepared by experienced technicians.
Reading of smears. All smears were read within 1 to 2 days after sputum collection. For each patient, the direct and PhAS sediment smears were read by the same technician; however, the smears were coded such that the technician reading them was unable to identify which PhAS smear and direct smear came from the same patient. Because the PhAS and direct smears are distinct in appearance, it was not possible to blind the reader to the type of smear. To minimize potential bias in reporting, a duplicate PhAS sediment smear was prepared from every fifth sputum sample. The sensitivity and specificity of the PhAS sediment smear method were determined by using the culture results as the "gold standard."
Sterilizing activity of PhAS. To ascertain the sterilizing activity of PhAS, 20 smear-positive sputum samples were aliquoted into two portions. One portion was processed for culture on Lowenstein-Jensen medium; the other portion was treated with PhAS reagent, and the sediment was then cultured for M. tuberculosis.
Acceptability of the methods. To assess the acceptability of each method, a structured questionnaire was given to seven laboratory technicians who had used both methods. Information collected from respondents included the safety of the method, the time taken for making and reading each type of smear, advantages and disadvantages of each method, and overall preference.
Data were entered and processed by using Microsoft Excel and were analyzed by using SPSS (version 4.0). The chi-square test with the Yates correction and a 5% level of significance was used to determine the statistical significance of the difference observed between the direct smear and PhAS sediment smear methods. The kappa value was calculated in order to determine the agreement between the results of each smear method and the culture results.
The results of the duplicate PhAS smears were used to measure the magnitude of bias in reporting. The discordance between the original and the duplicate PhAS smear was 2.7%, indicating insignificant bias in reporting the results of PhAS sediment smears.
Table 1 presents the results of comparison of the PhAS sediment smear method and the direct smear method with the bacteriologic culture method. M. tuberculosis was isolated from 547 of 2,400 samples. Fifty-nine samples were found to be contaminated and were excluded from further analysis. Of the culture-positive samples, 465 (85%) and 454 (83%) were positive for AFB by the PhAS sediment smear method and direct smear method, respectively. The observed difference in sensitivities was not statistically significant. The specificity of each method was 97%.
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Treatment with the PhAS reagent sterilized all the 20 smear-positive sputum samples that were used to test the sterilizing activity of the reagent. While the aliquots from the above 20 samples which were not treated with PhAS grew M. tuberculosis in culture, all the aliquots that were treated with PhAS were culture negative.
All seven of the laboratory technicians interviewed preferred the PhAS method over the direct smear method. Reasons stated for preferring the PhAS method included ease of specimen handling, ease of making smears, ease of reading and grading smears, and ease of disposal of specimens.
Although the sensitivity of the PhAS sediment smear method was not significantly higher than that of the direct smear method, the PhAS method has several advantages. Because PhAS-treated samples lose resemblance to sputum samples (Fig. 1), they are less aesthetically offensive to laboratory technicians. Additionally, the PhAS sediment smear method is safer, since the sputum is rendered sterile and PhAS-treated samples can be easily disposed of along with other hospital wastes. The time taken to read PhAS sediment smears, especially the high-grade smears, can be reduced. It has been reported previously that the concentrated smears were washed off from the slides during processing (2). In the present study, all the smears were found to be intact; it is likely that ammonium sulfate precipitated the mucus component of the sputum, allowing firm fixation of the smears on the slides. PhAS sediment smears were reported to be easy to read, with well-defined margins (Fig. 2) and with distinct AFB against a clear background. In large laboratories where several samples are processed, the PhAS method can substantially increase the efficiency of sputum smear microscopy. Other advantages of the method are that phenol is inexpensive, is stable at room temperature, and can be prepared at reference laboratories and supplied to peripheral health centers.
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In conclusion, the PhAS sediment smear method is as sensitive and specific as the direct smear method in the diagnosis of smear-positive pulmonary tuberculosis and would be safe and suitable for use in peripheral microscopy centers.
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ACKNOWLEDGMENTS |
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We thank the laboratory technicians in the Model DOTS Project and in the Department of Bacteriology for excellent technical support, the field staff for assisting in sputum collection and transportation, V. R. Vijayalakshmi for secretarial assistance, and Jaya Shreedhar for editorial assistance. Lastly, we are grateful to all the patients for their cooperation.
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FOOTNOTES |
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