Diagnostic microbiology and infectious disease · 2025
Optimizing a fully automated real-time PCR assay on the cobas® 6800 Platform for detection of Candida auris in surveillance samples pre-treated with sonication.
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Lui Eric Tin Long, Ho Alex Yat Man, Chan Janice Sze Wing, Lee Kin Ping, To Wing Kin, Cheung Wing Sze, Chow Viola Chi Ying, Luk Shik
Аннотация
BACKGROUND: Candida auris is a threat to public health globally. The goal of this study is to validate the real-time PCR assay performed on the fully automated cobas® 6800 system, using sonication to augment DNA extraction from surveillance clinical samples. Pooled sample testing was also evaluated.
METHODS: Analytical aspects of the assay were assessed by testing on a series of dilutions of a C. auris standardized suspension. Sensitivity was tested using 235 dual swabs with and without prior sonication. Enrichment culture was the gold standard. Fifty-nine RT-PCR positive samples were subjected to 4-in-1 pooled PCR testing after sonication pre-treatment.
RESULTS: The limit of detection of the cobas® 6800 PCR was 7.51 CFU/mL (95 % CI: 5.27- 20.22 CFU/mL), without cross-reactivity to other microorganisms. Out of 48 culture positive samples, 46 (95.8 %) and 44 (91.7 %) samples were RT-PCR positive with and without the sonication step respectively. Sonicated samples had a lower median Ct value by 2.07 (p < 0.05). Pooled testing of sonicated samples had a positive agreement of 79.7 % (95 %CI: 67.7 %-88.0 %). At a positive rate of 0.53 %, pooled testing could reduce the cost by three-fold; and the incremental cost would be US$ 242,995 for further identification of 14 positive samples among 12978 samples using individual testing.
CONCLUSIONS: The cobas® 6800 fully automated system is a reliable platform for C. auris PCR testing. Pre-sonication enhances PCR sensitivity. Pooled PCR testing is a cost-effective approach to identify C. auris carriers with high fungal load in low prevalence settings.
METHODS: Analytical aspects of the assay were assessed by testing on a series of dilutions of a C. auris standardized suspension. Sensitivity was tested using 235 dual swabs with and without prior sonication. Enrichment culture was the gold standard. Fifty-nine RT-PCR positive samples were subjected to 4-in-1 pooled PCR testing after sonication pre-treatment.
RESULTS: The limit of detection of the cobas® 6800 PCR was 7.51 CFU/mL (95 % CI: 5.27- 20.22 CFU/mL), without cross-reactivity to other microorganisms. Out of 48 culture positive samples, 46 (95.8 %) and 44 (91.7 %) samples were RT-PCR positive with and without the sonication step respectively. Sonicated samples had a lower median Ct value by 2.07 (p < 0.05). Pooled testing of sonicated samples had a positive agreement of 79.7 % (95 %CI: 67.7 %-88.0 %). At a positive rate of 0.53 %, pooled testing could reduce the cost by three-fold; and the incremental cost would be US$ 242,995 for further identification of 14 positive samples among 12978 samples using individual testing.
CONCLUSIONS: The cobas® 6800 fully automated system is a reliable platform for C. auris PCR testing. Pre-sonication enhances PCR sensitivity. Pooled PCR testing is a cost-effective approach to identify C. auris carriers with high fungal load in low prevalence settings.