Direct Detection of Enterovirus 71 (EV71) in Clinical Specimens from a Hand, Foot, and Mouth Disease Outbreak in Singapore by Reverse Transcription-PCR with Universal Enterovirus and EV71-Specific Primers

Abstract

A recent outbreak of hand, foot, and mouth disease in Singapore in 2000 affected several thousand children and resulted in four deaths. The aim of this study was to determine the applicability of reverse transcription-PCR (RT-PCR) with universal pan-enterovirus primers and enterovirus 71 (EV71) type-specific primers for the direct detection of enteroviruses in clinical specimens derived from this outbreak. With the universal primers, EV71 RNA sequences were successfully detected by RT-PCR and direct sequencing in 71% of positive specimens. Three pairs of EV71 type-specific primers were evaluated for rapid detection of EV71 directly from clinical specimens and cell culture isolates. By using a seminested RT-PCR strategy, specific identification of EV71 sequences directly in clinical specimens was achieved, with a detection rate of 53%. In contrast, cell culture could isolate EV71 in only 20% of positive specimens. EV71 was detected directly from brain, heart, and lung specimens of two deceased siblings. Although more than one type of enterovirus was identified in clinical specimens from this outbreak, 90% of the enteroviruses were confirmed as EV71. The data demonstrate the clinical applicability of pan-enterovirus and seminested RT-PCR for the detection of EV71 RNA directly from clinical specimens in an outbreak situation.Hand, foot, and mouth disease (HFMD) is of serious concern in the Asia-Pacific region as a result of two major epidemics with high fatalities in Malaysia and Taiwan in recent years (6,7,11). The recent HFMD outbreak in Singapore in October 2000 affected several thousand children and resulted in four deaths (1). In Singapore, HFMD is endemic and the disease manifests mainly as febrile upper respiratory illness and herpangina. There have been reports of some children with acute flaccid paralysis who fully recovered (12). The classical diagnostic method for enteroviruses is propagation by cell culture followed by neutralization with specific antisera to confirm the serotype. Cell culture from clinical specimens is considered the "gold standard," but the procedure is time-consuming and may take a few weeks. Furthermore, some enteroviruses replicate poorly in cell cultures, require several passages, or may be untypeable (9).In view of its high sensitivity and specificity, reverse transcription-PCR (RT-PCR) is beneficial for the rapid detection of enterovirus 71 (EV71) in outbreak situations. The rapid establishment of enteroviral etiology would eliminate the unnecessary use of antibiotics and lead to better patient management. Early diagnosis can help to limit the spread of the disease in the community during an outbreak. The aim of this study was to identify the etiological agents involved in the HFMD outbreak. We investigated the applicability of RT-PCR by using universal pan-enterovirus and type-specific primers for the direct detection of EV71 in clinical specimens. Direct sequencing of RT-PCR products amplified from the 5Ј untranslated region (5Ј UTR) by using universal primers (10, 16) was com...

Frequent Importation of Enterovirus 71 from Surrounding Countries into the Local Community of Yamagata, Japan, between 1998 and 2003

“…Because fatal cases have been reported in Asian countries, such as Malaysia (1997), Taiwan (1998), Singapore (2000), and Japan (2000), careful monitoring of the incidence of HFMD and a plan for dealing with severe outbreaks of HFMD are considered to be of great importance (3,5,16,18,20). To realize this, understanding the epidemiology and pathogenesis of EV71 is primary.…”

Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay

“…It is difficult to achieve rapid detection using traditional virus isolation and neutralization because of their low specificities and detection limits, as well as the long time that is spent obtaining results (4)(5)(6). Nucleic acid amplification techniques, such as reverse transcription-PCR (RT-PCR) (7)(8)(9) and reverse transcription-quantitative PCR (qRT-PCR) (10, 11) take less time (2 to 3 h) and are generally used for making a standard diagnosis due to a high detection limit. In China, a few commercial qRT-PCR-based diagnostic kits for EV71 and CVA16 are available and have been approved by the State Food and Drug Administration for HFMD pathogen surveillance.…”

How to understand the efficacy measurements for enterovirus type 71 vaccine?

“…Even combined throat plus rectal swabs enabled an increase probability of virus isolation, but still imperfect. 26,27 By contrast, molecular methods such as reverse transcription polymerase chain reaction (RT-PCR) have been proved to be more sensitive for EV71 detection than the virus isolation and cell culture method. 28 Among 3 common used RT-PCR tests (conventional RT-PCR, two-step semi-nested RT-PCR, and realtime RT-PCR), real-time RT-PCR was priority because it's more rapid and with lower risk of cross-contamination, although the cost was a little higher.…”

“…For severe cases the shedding duration of EV71 could be even longer. 24,27 However the sampling time is still essential for the sensitivity of diagnosis. It has been reported that most specimens collected from EV71-associated cases within 0-8 d after onset could be positive in the laboratory detection, but then the positive rate decreased significantly.…”