Accuracy of High-Throughput Nanofluidic PCR-Based Pneumococcal Serotyping and Quantification Assays Using Sputum Samples for Diagnosing Vaccine Serotype Pneumococcal Pneumonia: Analyses by Composite Diagnostic Standards and Bayesian Latent Class Models

Abstract

The lack of reliable diagnostic tests for detecting vaccine serotype pneumococcal pneumonia (VTPP) remains a challenging issue in pneumococcal vaccine studies. This study assessed the performances of high-throughput nanofluidic PCR-based pneumococcal serotyping and quantification assay methods using sputum samples (the nanofluidic sputum quantitative PCR [Sp-qPCR] assay) to diagnose 13-valent pneumococcal conjugate VTPP compared with the performance of the serotype-specific urinary antigen detection (UAD) assay using urine samples. Adult pneumonia patients from Japan were enrolled in this study between September 2012 and August 2014. Sputum samples were subjected to the nanofluidic Sp-qPCR assay, quantitatively cultured, and serotyped by the Quellung reaction (SpQt). Urine samples were tested by the UAD method. The diagnostic performances of these tests were assessed using composite reference standards and Bayesian latent class models (BLCMs). Among 244 total patients, 27 (11.1%) tested positive with the UAD assay, while 16 (6.6%) and 34 (13.9%) tested positive with the SpQt and nanofluidic Sp-qPCR assays, respectively, with a cutoff value of ≥104 DNA copies/ml, which showed the maximum value of the Youden index. Using BLCMs, the estimated prevalence for VTPP was 12.9%, and the nanofluidic Sp-qPCR assay demonstrated the best performance (sensitivity, 90.2%; specificity, 96.9%), followed by UAD (sensitivity, 75.6%; specificity, 97.9%) and SpQt (sensitivity, 45.8%; specificity, 99.5%). However, when a higher cutoff value of ≥107 DNA copies/ml was applied, the performance of UAD became comparable to that of Sp-qPCR. The vaccine serotype-specific pneumococcal DNA load in sputum among UAD-positive patients was 3 logs higher than that among UAD-negative patients (P = 0.036). The nanofluidic Sp-qPCR assay may be accurate and useful for detecting VTPP among adults.

Usefulness of sputum gram stain for etiologic diagnosis in community-acquired pneumonia: a systematic review and meta-analysis

“…In addition, other studies have found that PCR did not show significant increase in either sensitivity or specificity in pneumococcal pneumonia, even when quantification was included [38–41]. In this regard, Stralin et al [42] and Kakiuchi et al [43] obtained high sensitivity and specificity using PCR assays for S. pneumoniae applied to sputum and nasopharyngeal aspirate samples in patients with CAP. Moreover, protected specimen brush and bronchoalveolar lavage quantitative cultures added nearly 30% more microbiological documentation for CAP compared to sputum cultures.…”

Current situation and need for prevention of invasive pneumococcal disease and pneumococcal pneumonia in 6- to 64-year-olds in Japan

“…PPSV23 coverage in patients with PP was reported in 5 studies [34,35,54,56,57]. In these studies, the coverage decreased from 63.7% to 54.1% during the study period (2008e2016) in patients aged 16 years [57]; was 68.2% among those aged 61e79 years [56]; and was the lowest (8.2%) among those aged 22e99 years [35].…”

“…Mortality rate was in the range of 4.5%e25.0% in 4 studies in patients with PP [22,31,34,35]. Among patients aged 70.4 ± 14.6 years and 22e99 years, death was reported in 34/ 135 (25%) patients [22] and 11/244 (4.5%) patients, respectively [35].…”

“…Comorbidities were reported in 7 studies in patients with PP and were presented as individual disease names rather than organ system involvements [13,19,31e35]. Among them 2 studies have comorbidities of CAP patients including 21.7% and 22% PP patients [33,35]. Among pneumonia patients aged 1 month to 14 years 6 months including S. pneumoniae, a higher proportion of comorbidities, including chromosomal abnormality (15.15% vs 0.07%), cerebral palsy (12.12% vs 0%), epilepsy (10.1% vs 0.29%), neuromuscular disease (5.1% vs 0%), and sequelae of hypoxic encephalopathy (4% vs 0%), were reported at a tertiary facility vs at a secondary facility [13].…”

A digital PCR system based on the thermal cycled chip with multi helix winding capillary

“…It can amplify nucleic acid molecules from one to several million in a very short period of time (usually in tens of minutes), so it is regarded as the most important molecular biology technology today. It plays an important role in the fields including next-generation high-throughput sequencing and clinical nucleic acid marker detection [1][2][3][4][5][6][7][8][9][10][11][12][13][14] .…”