Fluorescein Diacetate Vital staining for detecting viability of acid-fast bacilli in sputum of pulmonary tuberculosis patients

Abstract

Objective: To compare the performance of the Fluorescein diacetate (FDA) vital staining method with Ziehl-Neelsen staining method in detecting the viability of acid-fast bacilli using MGIT culture as "reference standard".

Methods: This was a single centre prospective observational study conducted from October 2015 to November 2016. Microbiologically confirmed ZN-Smear positive (3+) sputum specimens were obtained from 30 pulmonary tuberculosis patients taking anti-tuberculosis treatment at DOTS centre of NITRD, New Delhi. Patients were made available to collect the first baseline sputum sample before commencing treatment, and an early morning sputum sample was collected as per RNTCP guidelines. After starting treatment, sputum specimens were collected weekly in the first month and thereafter twice-weekly until 18th week. All sputum specimens from patients receiving anti-tuberculosis treatment were examined using Ziehl-Neelsen (ZN) smear microscopy, FDA vital staining, and MGIT culture.

Result: Out of 360 follow up sputum specimens collected from 30 adult microbiologically confirmed ZN- Smear (3+) pulmonary tuberculosis patients, 146 were ZN-positive and 130 FDA-positive. Of 130 FDA-positive sputum samples, mycobacteria tuberculosis (MTB) growth was found in 116 sputum samples, of which 116 sputum specimens were positive for FDA. Additionally, 14 culture-negative specimens were FDA positive. No FDA-negative sputum samples were positive for MGIT culture. Among ZN positive specimens, FDA had 100% sensitivity and 85.3% specificity with an accuracy of 96.58% for the detection of viable mycobacteria. Among ZN negative sputum specimens, FDA had comparatively high specificity (95.7%). Using positive MGIT culture as a reference for viability, negative predictive value (NPV) and positive predictive value (PPV) from FDA vital staining method were found to be 100 and 89% respectively.

Conclusion: FDA staining is a simple and rapid tool for identifying viable MTB bacilli. Because of its excellent NPV and encouraging specificity, FDA staining is useful to identify patients with non-viable bacilli (FDA negative) among retreatment cases at diagnosis and patients on anti-tuberculosis treatment for both drug-sensitive and drug-resistant tuberculosis for follow up for the response of treatment.

Keywords: Fluorescein diacetate (FDA); MGIT Culture; Tuberculosis; Vital staining; Ziehl-Neelsen (ZN) smear microscopy.