Sputum Microscopy With Fluorescein Diacetate Predicts Tuberculosis Infectiousness

Abstract

Background: Sputum from patients with tuberculosis contains subpopulations of metabolically active and inactive Mycobacterium tuberculosis with unknown implications for infectiousness.

Methods: We assessed sputum microscopy with fluorescein diacetate (FDA, evaluating M. tuberculosis metabolic activity) for predicting infectiousness. Mycobacterium tuberculosis was quantified in pretreatment sputum of patients with pulmonary tuberculosis using FDA microscopy, culture, and acid-fast microscopy. These 35 patients' 209 household contacts were followed with prevalence surveys for tuberculosis disease for 6 years.

Results: FDA microscopy was positive for a median of 119 (interquartile range [IQR], 47-386) bacteria/µL sputum, which was 5.1% (IQR, 2.4%-11%) the concentration of acid-fast microscopy-positive bacteria (2069 [IQR, 1358-3734] bacteria/μL). Tuberculosis was diagnosed during follow-up in 6.4% (13/209) of contacts. For patients with lower than median concentration of FDA microscopy-positive M. tuberculosis, 10% of their contacts developed tuberculosis. This was significantly more than 2.7% of the contacts of patients with higher than median FDA microscopy results (crude hazard ratio [HR], 3.8; P = .03). This association maintained statistical significance after adjusting for disease severity, chemoprophylaxis, drug resistance, and social determinants (adjusted HR, 3.9; P = .02).

Conclusions: Mycobacterium tuberculosis that was FDA microscopy negative was paradoxically associated with greater infectiousness. FDA microscopy-negative bacteria in these pretreatment samples may be a nonstaining, slowly metabolizing phenotype better adapted to airborne transmission.

Keywords: TB; fluorescein diacetate; infectiousness; microscopy; tuberculosis.

Figure 1.

Study flowchart.

Figure 2.

Patients’ pretreatment sputum results. A, Distribution of concentrations in sputum of bacilli staining with acid-fast auramine microscopy, bacilli staining with fluorescein diacetate (FDA) microscopy, and colony-forming units (CFU) in quantitative culture. B, Distribution of FDA microscopy–positive bacterial concentration as a percentage of auramine microscopy–positive bacteria per microliter.

Figure 3.

Figure 4.

Kaplan-Meier time-to-event curves for patients’ household contacts being diagnosed with tuberculosis (TB) disease analyzed according to whether the study patients’ pretreatment sputum fluorescein diacetate (FDA) microscopy result was above (high) or below (low) the median concentration. A, Patients with quantitative culture results (in colony-forming units [CFU]) below the median concentration. B, Patients with quantitative culture results above the median concentration. P values indicate the results of Cox regression analysis.