Key result
In-house assays achieve ~25 percentage points higher low-copy enterovirus detection than commercial tests.
Why the study?
PCR effectiveness for enterovirus and parechovirus depends critically on high assay sensitivity given extremely low viral loads in cerebrospinal fluid, prompting an evaluation of sensitivity and specificity across diagnostic and typing assays.
Do in-house PCR assays have different sensitivity compared to commercial assays for detecting enterovirus and parechovirus RNA transcripts?
Cross-Sectional (n=39)
Blinded testing
Yes
Do in-house PCR assays have different sensitivity compared to commercial assays for detecting enterovirus and parechovirus RNA transcripts?
Absolute Event Rate: 81% vs 56%
p-value: p=7 × 10^-5
In-house PCR assays demonstrate significantly higher sensitivity for detecting low-copy enterovirus and parechovirus transcripts compared to commercial assays, which are often used as point-of-care tests.
Commercial assays may miss low-copy transcripts; leaves open need for clinical validation before changing diagnostic practice.
Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in‐house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV‐A71, echovirus 30, coxsackie A virus 21, and EV‐D68), HPeV3, and specificity controls. Reported results from 48 in‐house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In‐house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10 −5 ). EV‐specific PCRs showed low cross‐reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in‐house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point‐of‐care tests.
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Hayes et al. (2019) conducted a cross-sectional in Enterovirus and human parechovirus infections (n=39). In-house diagnostic assays vs. Commercial diagnostic assays was evaluated on Detection frequency of low copy (10 copies/5 µL) enterovirus transcripts (p=7 × 10^-5). In-house diagnostic assays demonstrated significantly higher detection frequencies for low-copy enterovirus and human parechovirus transcripts compared to commercial point-of-care assays (81% vs 56% for enterovirus; P=7×10^-5).
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