Utility of the real-time PCR (fluidigm) for serotype-specific characterisation of streptococcus pneumoniae in adults hospitalised with pneumonia
Date
2022
Authors
Jeche, Tariro Ruth
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Streptococcus pneumoniae has been identified as the most common cause of bacterial pneumonia; however, the classical tests and sampling methods used to detect and serotype S. pneumoniae have limitations. The primary aim of the study was to optimise the Biomark HD real-time qPCR system (Fluidigm) to simultaneously detect and serotype S. pneumoniae in urine samples. A secondary objective was to evaluate the carriage prevalence of vaccine-type S. pneumoniae in adults hospitalised with pneumonia as diagnosed by the attending physician. Urine samples were collected as part of the ―Hospital surveillance for respiratory illness” study from April 2018 to August 2019.
Streptococcus pneumoniae has been identified as the most common cause of bacterial pneumonia; however, the classical tests and sampling methods used to detect and serotype S. pneumoniae have limitations. The primary aim of the study was to optimise the Biomark HD real-time qPCR system (Fluidigm) to simultaneously detect and serotype S. pneumoniae in urine samples. A secondary objective was to evaluate the carriage prevalence of vaccine-type S. pneumoniae in adults hospitalised with pneumonia as diagnosed by the attending physician. Urine samples were collected as part of the ―Hospital surveillance for respiratory illness” study from April 2018 to August 2019.
The efficiency of the reactions ranged from 92% to 105%. Within the linear dynamic range, the correlation coefficients (r2 ) of the reactions were >0.95. Further, all assays showed a good analytical sensitivity within their respective primer and probe pairs with the limit of detection (LLD) equivalent to >1 000 copies per PCR for all reactions. The intra-assay variability (repeatability) and inter-assay variability (reproducibility) for all assays were within the acceptable range of ± 0.167 (0-0.15) standard deviation (SD) and (0.02-0.16) SD respectively. The positivity for S. pneumoniae detection on urine samples in pneumonia cases was 19% (18/96) using the BinaxNOW™ Streptococcus pneumoniae antigen card and 7% (7/96) on LytA qPCR. None of the urine samples tested positive on the Fluidigm assay.
In conclusion, Fluidigm was optimised successfully to detect and simultaneously serotype S. pneumoniae in spiked urine samples. Nevertheless, the assay failed to detect or serotype S. pneumoniae in clinical samples, which could be due to very low bacterial loads. In comparison, testing using the BinaxNOW™ Streptococcus pneumoniae antigen card indicated that 19% of the pneumonia cases were attributable to being associated with S. pneumoniae. Thus, BinaxNOW, or alternative methods that rely on antigen testing remain the most effective method for detecting S. pneumoniae related pneumonia cases.
Description
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine (Clinical Microbiology and Infectious Diseases) to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2022