Diagnostic performance of different specimens in detecting enterovirus A71 in children with hand, foot and mouth disease

Abstract

Hand, foot and mouth disease (HFMD) is a major public health problem among children in the Asia-Pacific region. The optimal specimen for HFMD virological diagnosis remains unclear. Enterovirus A71 (EV-A71) neutralizing antibody titres detected in paired sera were considered the reference standard for calculating the sensitivity, specificity, positive and negative predictive value of throat swabs, rectal swabs, stool, blood samples and cerebrospinal fluid (CSF) by RT-PCR or ELISA assay. In this study, clinical samples from 276 HFMD patients were collected for analysing the sensitivity of different kind of specimens. Our results showed that stool had the highest sensitivity (88%, 95% CI: 74%-96%) and agreement with the reference standard (91%). The order of diagnostic yield for EV-A71 infection was stool sample ​≥ ​rectal swab ​> ​throat swab ​> ​blood sample ​> ​CSF sample, and using a combination of clinical samples improved sensitivity for enterovirus detection. The sensitivity of ELISA for IgM antibody detection in sterile-site specimens was significantly higher than that of RT-PCR (serum/plasma: 62% vs. 2%, CSF: 47% vs. 0%) (P ​< ​0.002). In conclusion, our results suggest that stool has the highest diagnostic yield for EV-A71-infected HFMD. If stool is unavailable, rectal swabs can be collected to achieve a similar diagnostic yield. Otherwise, throat swabs may be useful in detecting positive samples. Although IgM in blood or CSF is diagnostically accurate, it lacks sensitivity, missing 40%-50% of cases. The higher proportion of severe cases and shorter interval between onset and sampling contributed to the increase in congruency between clinical testing and the serological reference standard.

Keywords: Enterovirus A71 (EV-A71); Evaluation of diagnostic methods; Hand, foot and mouth disease (HFMD); Sensitivity; Specificity.

Fig. 1

Flowchart of patient enrolment. “∗” indicates that the patient met one of the following criteria: previous paediatric intensive care unit (PICU) admission/ventilation (123, 54%); premature birth (before 37 weeks) (91, 40%); any prior chronic respiratory, cardiac, or other illness (i.e., congenital hypothyroidism, congenital epilepsy, asthma) (37, 16%); prior delayed development or neurodevelopment (34, 15%); or prior learning disability or neurological regression (8, 4%). In addition, some patients met more than 1 criterion.

Fig. 2

Interval between onset and sampling (days) by sample type. A The distribution of intervals between onset and sampling (days) among the different sample types. B The cumulative probability of interval time between onset and sampling (day) among the different sample types. Acute-phase serum samples (NT) were tested by the NT assay. CSF (IgM) and acute-phase serum samples (IgM) were tested by ELISA. Other five samples were tested by RT-PCR.

Fig. 3

Overview of patient enrolment and detection results. “∗” indicates the 1564 excluded patients, as shown in Fig. 1. Non-sterile samples included throat swabs, rectal swabs and stool samples. Sterile samples included CSF, plasma and vesicle fluid. Sterile sites (266 cases) and non-sterile sites (274 cases) were both classified based on the following: EV-A71 positivity was defined as EV-A71 detection in at least one sample, and EV-A71 negativity was defined as no EV-A71 detection in all samples for which EVs were or were not detected.

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