Performance of Detecting IgM Antibodies against Enterovirus 71 for Early Diagnosis

Enterovirus 71 (EV71) infection is more likely to induce severe complications and mortality than other enteroviruses. Methods for detection of IgM antibody against EV71 had been established for years, however, the performance of the methods in the very early diagnosis of EV71 infection had not been fully evaluated, which is especially meaningful because of the short incubation period of EV71 infection. In this report, the performance of an IgM anti-EV71 assay was evaluated using acute sera collected from 165 EV71 infected patients, 165 patients infected with other enteroviruses, and more than 2,000 sera from healthy children or children with other infected diseases. The results showed a 90% sensitivity in 20 patients who were in their first illness day, and similar sensitivity remained till 4 days after onset. After then the sensitivity increased to 95% to 100% for more than one month. The specificity of the assay in non-HFMD children is 99.1% (95% CI: 98.6–99.4), similar as the 99.9% specificity in healthy adults. The cross-reaction rate in patients infected with other non-EV71 enteroviruses was 11.4%. In conclusion, the data here presented show that the detection of IgM anti-EV71 by ELISA affords a reliable, convenient, and prompt diagnosis of EV71 infection

A one-step, triplex, real-time RT-PCR assay for the simultaneous detection of enterovirus 71, coxsackie A16 and pan-enterovirus in a single tube.

The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD), which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71) and coxsackie A16 (CA16) are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs) in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001-0.04 TCID50/ml, 0.02 TCID50/ml, and 0.001 TCID50/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD

Serial foodborne norovirus outbreaks associated with multiple genotypes.

Noroviruses (NoV) have been recognized as an important pathogen associated with acute gastroenteritis worldwide during the past three decades. In the spring of 2012, a series of foodborne outbreaks in tourist groups were reported to Xiamen Center for Disease Control and Prevention, Xiamen, Fujian province, China. Among a total of 268 tourists in 7 groups, the prevalence rate of acute gastroenteritis was 16.0% (43/268). Twenty-three feces or anal swabs were collected for laboratory tests of causative agents, no bacterial pathogen was identified, while 22 of them were positive for NoV RNA. In addition, thirteen NoV fragments were recovered from positive specimens and sequenced, belonging to five genotypes such as GI.3, GI.4, GII.4, GII.6, and GII.14, respectively. However, NoV fragments obtained from locally infected patients showed distinct genotypes. Therefore, epidemiological investigation and laboratory analyses demonstrated that the serial foodborne NoV outbreaks in tourists were co-infection of multiple genotypes induced acute gastroenteritis linked to a restaurant

Specific primer amplification of the VP1 region directed by 5 ' UTR sequence analysis: Enterovirus testing and identification in clinical samples from hand-foot-and-mouth disease patients

Science and Technology Project of China [2008BAI70B01, 2011AA02A114]; National Natural Science Foundation of China [30972826]Many genotypes of the enterovirus (EV) pathogens can cause clinical hand-foot-and-mouth disease (HFMD). Therefore, rapid identification and monitoring of HFMD pathogens can be difficult, especially from the original clinical specimens. In this study, both universal pan-enterovirus and EV71/CA16 VP1-specific primer sets were designed and used to examine clinical specimens from HFMD patients. Based on the initial sequence analysis of the 5'-untanslated region (5'-UTR) and VP1 amplification products, additional primers for the VP1 region were redesigned for further genotyping of the remaining small portion non-EV71/non-CA16 specimens. With a known panel, it was possible to identify 15 out of 16 members using 5'-UTR sequence typing and VP1 typing, suggesting good detectability and genotyping of this method. One strain that was not typed by 5'-UTR was shown to be a recombinant virus. When this method was applied to examine clinical specimens from 44 suspected HFMD patients, 41 were detected as EV positive. In only one case, the VP1 sequence could not be identified. Four types of EVs, including CA16 (26/41, 63.4%), EV71-C4 (6/41, 14.6%), CA6 (5/41, 12.2%) and CA10 (3/41, 7.3%), were detected. In conclusion, 5' UTR amplification sequencing and subsequent VP1 specific primer amplification ensures a high detection rate and good genotyping accuracy in the examination of clinical samples. This detection strategy can be used for routine evaluation and monitoring of HFMD to follow local trends of EV infection. (C) 2013 Published by Elsevier B.V

The results from triplex RT-PCR.

<p>A. Detection of mixed samples of EV71 and CA16 in one tube. B. Detection of the negative control.</p

The triplex RT-PCR results for clinical samples from HFMD patients.

<p>The triplex RT-PCR results for clinical samples from HFMD patients.</p

Detection results for the HFMD viral stocks.

<p>Detection results for the HFMD viral stocks.</p

Quantitative analysis of the triplex RT-PCR for detecting EV71, CA16 and EVs- CB2.

<p>Quantitative analysis of the triplex RT-PCR for detecting EV71, CA16 and EVs- CB2.</p

Summary of investigation on NoV acute gastroenteritis outbreaks in tourist groups.

*<p>The exposure date for patients in Event H was between April 15 to April 17, 2012.</p>**<p>indicate anal swabs from employees of the restaurant under investigation.</p

List of positive specimens in NoV RNA detection and their genotypes.

<p>NT, not tested; NA, not available.</p