Evaluation of a Commercially Developed Semiautomated PCR–Surface-Enhanced Raman Scattering Assay for Diagnosis of Invasive Fungal Disease

Abstract

c Nonculture-based tests are gaining popularity in the diagnosis of invasive fungal disease (IFD), but PCR is excluded from disease-defining criteria because of limited standardization and a lack of commercial assays. Commercial PCR assays may have a standardized methodology while providing quality assurance. The detection of PCR products by a surface-enhanced Raman scattering (SERS) assay potentially provides superior analytical sensitivity and multiplexing capacity compared to that of realtime PCR. Using this approach, the RenDx Fungiplex assay was developed to detect Candida and Aspergillus. Analytical and clinical evaluations of the assay were undertaken using extraction methods according to European Aspergillus PCR Initiative (EAPCRI) recommendations. A total of 195 previously extracted samples (133 plasma, 49 serum, and 13 whole blood) from 112 patients (29 with proven/probable IFD) were tested. The 95% limit of detection of Candida and Aspergillus was 200 copies per reaction, with an overall reproducibility of 92.1% for detecting 20 input copies per PCR, and 89.8% for the nucleic acid extraction-PCR-SERS process for detecting fungal burdens of <20 genome equivalents per sample. A clinical evaluation showed that assay positivity significantly correlated with IFD (P < 0.0001). The sensitivity of the assay was 82.8% and was similar for both Candida (80.0%) and Aspergillus (85.7%). The specificity was 87.5% and was increased (97.5%) by using a multiple (>2 samples) PCR-positive threshold. In summary, the RenDx Fungiplex assay is a PCR-SERS assay for diagnosing IFD and demonstrates promising clinical performance on a variety of samples. This was a retrospective clinical evaluation, and performance is likely to be enhanced through a prospective analysis of clinical validity and by determining clinical utility.

Diagnostic accuracy of fungal PCR and β-d-glucan for detection of candidaemia: a preliminary evaluation

“…Meta-analysis of results for various PCR-based methods for detection of candidaemia has concluded that PCR has very high sensitivity of 95% 6. We found the test’s specificity to be high, similar to that reported previously 12. The relatively lower sensitivity in this study may be explained by both the small case number and retrospective testing.…”

“…The RenDx PCR test has previously been validated in a study of 93 blood specimens spiked with Aspergillus or Candida as well as blood samples from 112 high-risk patients, of whom seven had candidaemia; all seven were positive with the RenDx test 12. By comparison, another commercially available Candida PCR test (from T2 Biosystems) has been validated using 300 spiked blood specimens as well as blood samples from a large patient cohort, of which six had documented candidaemia; four of these were detected by the T2 assay 14…”

Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer?

“…163 SERRS uses sensors that detect light scattered through the production of PCR-amplified DNA, in conjunction with a specific dye placed against a roughened surface such as gold or silver. 164 There was a 95% limit of detection of Candida and Aspergillus of 200 copies/reaction with overall reproducibility of 92.1%. A new platform, the RenDx (Renishaw Diagnostics, UK), was developed for detection of nucleic acid targets by Raman spectroscopy, including multiplex detection of up to 10 targets per reaction.…”

Bioanalytical applications of surface-enhanced Raman spectroscopy: de novo molecular identification

“…While SERS-tags have evolved into regulatory approved clinical assays, 178 the de novo identification of biomolecules is quickly catching up. De novo SERS detection described above indicates that a number of factors are important for SERS identification and quantification.…”