Diagnostic yield of broad-range 16s rRNA gene PCR varies by sample type and is improved by the addition of qPCR panels targeting the most common causative organisms

Abstract

Introduction. Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or following empirical antimicrobial treatment. Hypothesis/Gap Statement. Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens. Aim. To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm. Methodology. In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS). Results. Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species. Streptococcus spp., Staphylococcu s spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9 % were positive by 16S and 61 % were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3 % of samples positive by 16S and CSF with 4.8 % of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types. Conclusion. Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.

Untargeted metagenomics protocol for the diagnosis of infection from CSF and tissue from sterile sites

“…For this reason we recommend interrogating the metagenomics results in tissue for viruses with <5 reads, which should be confirmed with a targeted PCR prior to clinical reporting. It is worthwhile noting that whilst the sensitivity of metagenomics in tissue for DNA organisms is suboptimal, it is equivalent to the sensitivity of broad-range bacterial PCR widely used in clinical practice [ 8 ] and has been used successfully by our laboratory to diagnose multiple case of encephalitis [ [9] , [10] , [11] , [12] , [13] , [14] ] and hepatitis [ [2] , [15] ].…”