Fluorescent antibody-based detection and ultrastructural analysis of Streptococcus pneumoniae in human sputum

Pneumococcal pneumonia continues to be a significant global health burden, affecting both children and adults. Traditional diagnostic methods for sputum analysis remain challenging. The objective of this study was twofold: to develop a rapid and easy-to-perform assay for the identification of Streptococcus pneumoniae (Spn) directly in sputum specimens using fluorescence microscopy, and to characterize with high-resolution confocal microscopy the ultrastructure of pneumococci residing in human sputum.

We fluorescently labeled antibodies against the pneumococcal capsule (Spn-FLUO). The specificity and sensitivity of Spn-FLUO for detecting Spn was evaluated in vitro and in vivo using mouse models of carriage and disease, human nasopharyngeal specimens, and sputum from patients with pneumococcal pneumonia. Spn was confirmed in the specimens using culture and a species-specific qPCR assay. Spn strains were serotyped by Quellung. Confocal microscopy and Imaris software analysis were utilized to resolve the ultrastructure of pneumococci in human sputum.

Compared with cultures and qPCR, Spn-FLUO demonstrated high sensitivity (78-96%) in nasopharyngeal samples from mice and humans. The limit of detection (LOD) in nasopharyngeal samples was ≥ 1.6 × 10⁴ GenEq/ml. The specificity in human nasopharyngeal specimens was 100%. In lung specimens from mice infected with pneumococci, Spn-FLUO reached 100% sensitivity with a LOD of ≥ 1.39 × 10⁴ GenEq/ml. In human sputum, the sensitivity for detecting Spn was 92.7% with a LOD of 3.6 × 10³ GenEq/ml. Ultrastructural studies revealed that pneumococci are expectorated as large aggregates with a median size of 1336 μm².

Spn-FLUO is a rapid and sensitive assay for detecting Spn in human sputum within 30 min, encompassing a range of both vaccine and non-vaccine serotypes associated with pneumococcal pneumonia. The study highlights that most pneumococci form aggregates in human sputum.