A controlled evaluation of filter paper use during staining of sputum smears for tuberculosis microscopy

Bailon, Nataly, Ramos, Eric, Alvarado, Keren, Bernaola, Lenin, Wilson, James, Montoya, Rosario, Valencia, Teresa, Evans, Carlton A and Datta, Sumona (2023) 'A controlled evaluation of filter paper use during staining of sputum smears for tuberculosis microscopy'. Wellcome Open Research, Vol 8, e171.

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Abstract

Background:
Some sputum smear microscopy protocols recommend placing filter paper over sputum smears during staining for Mycobacterium tuberculosis (TB) . We found no published evidence assessing whether this is beneficial. We aimed to evaluate the effect of filter paper on sputum smear microscopy results.

Methods:
Sputum samples were collected from 30 patients with confirmed pulmonary TB and 4 healthy control participants. From each sputum sample, six smears (204 smears in total) were prepared for staining with Ziehl-Neelsen (ZN), auramine or viability staining with fluorescein diacetate (FDA). Half of the slides subjected to each staining protocol were randomly selected to have Whatman grade 3 filter paper placed over the dried smears prior to stain application and removed prior to stain washing. The counts of acid-fast bacilli (AFB) and precipitates per 100 high-power microscopy fields of view, and the proportion of smear that appeared to have been washed away were recorded. Statistical analysis used a linear regression model adjusted by staining technique with a random effects term to correct for between-sample variability.

Results:
The inclusion of filter paper in the staining protocol significantly decreased microscopy positivity independent of staining with ZN, auramine or FDA (p=0.01). Consistent with this finding, there were lower smear grades in slides stained using filter paper versus without (p=0.04), and filter paper use reduced AFB counts by 0.28 logarithms (95% confidence intervals, CI=0.018, 0.54, p=0.04) independent of staining technique. In all analyses, auramine was consistently more sensitive with higher AFB counts versus ZN (p=0.001), whereas FDA had lower sensitivity and lower AFB counts (p<0.0001). Filter paper use was not associated with the presence of any precipitate (p=0.5) or the probability of any smear washing away (p=0.6) during the staining process.

Conclusions:
Filter paper reduced the sensitivity of AFB microscopy and had no detectable beneficial effects so is not recommended.

Item Type:	Article
Subjects:	QY Clinical Pathology > Diagnostic Tests > QY 120 Sputum WF Respiratory System > Tuberculosis > WF 200 Tuberculosis (General)
Faculty: Department:	Clinical Sciences & International Health > Clinical Sciences Department
Digital Object Identifer (DOI):	https://doi.org/10.12688/wellcomeopenres.18827.1
SWORD Depositor:	JISC Pubrouter
Depositing User:	JISC Pubrouter
Date Deposited:	09 Oct 2023 10:46
Last Modified:	09 Oct 2023 10:46
URI:	https://archive.lstmed.ac.uk/id/eprint/23292

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