Hsiao Chen Ning

Clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease

BACKGROUND In Taiwan, from April to July, 1998, an epidemic of hand, foot, and mouth disease associated with enterovirus 71 (EV71) occurred with fatal complications. We did a clinical study of EV71-related diseases in Taiwan. METHODS We studied 154 children with virus-culture confirmed EV71 infection. Children were divided into three groups: 11 patients with pulmonary oedema; 38 patients with central nervous system (CNS) involvement and no pulmonary oedema; and 105 children without complications. We compared the clinical features, laboratory findings, risk factors, and outcome among these three groups. FINDINGS Nine children with pulmonary oedema had hand, foot, and mouth disease, one had herpangina, and one had febrile illness with eight children with limb weakness and one with limb hypesthesia. All children had had sudden onset of tachycardia, tachypnoea, and cyanosis 1-3 days after onset of the disease. Nine of 11 children died within 12 h of intubation; one child was braindead within 15 h and died 17 days after intubation; one child was in deep coma and died 3 months later. In children with CNS complication and no pulmonary oedema, one child died of pneumonia after 4 months of ventilator support and four children had sequelae. All 105 children without complications recovered. There was a significant association between CNS involvement and pulmonary oedema (odds ratio 12.4 [95% CI 2.6-60.1], p=0.001). Risk factors for pulmonary oedema after CNS involvement were hyperglycaemia, leucocytosis, and limb weakness. Hyperglycaemia was the most significant prognostic factor for pulmonary oedema (odds ratio 21.5 [3-159], p=0.003). INTERPRETATION EV71 can cause hand, foot, and mouth disease, CNS involvement with severe sequelae, and fatal pulmonary oedema. Hyperglycaemia is the most important prognostic factor.

Comparison of enterovirus 71 and coxsackievirus A16 clinical illnesses during the Taiwan enterovirus epidemic, 1998

OBJECTIVES To compare enterovirus 71 (EV 71) with coxsackievirus A16 (Cox A16) clinical illness in patients at Chang Gung Childrens Hospital during Taiwans enterovirus epidemic of 1998. METHODS With the use of the immunofluorescence assay and neutralization test, 177 cases of EV 71 and 64 cases of Cox A16 illness were confirmed from April to September, 1998. The clinical signs and symptoms, complications and case fatality rates were compared. RESULTS Three-fourths of the cases were younger than 3 years of age, and the ratio of males to females was 1.3 in the EV 71 group and 1.2 in the Cox A16 group. In the EV 71 group 120 (68%) cases were uncomplicated, including 94 cases of hand, foot and mouth disease and 15 cases of herpangina, and 57 (32%) cases had complications, including 13 (7.3%) cases of aseptic meningitis, 18 (10%) cases of encephalitis, 4 (2.3%) cases of polio-like syndrome, 8 (4.5%) cases of encephalomyelitis and 12 (6.8%) cases of fatal pulmonary edema. Fourteen (7.9%) patients died, including 12 cases of pulmonary edema and 2 cases of encephalitis; seven (4%) patients had sequelae. By contrast, 60 (94%) of the 64 cases of Cox A16 infection were uncomplicated and only 4 (6.3%) cases were complicated by aseptic meningitis; no fatalities or sequelae were observed. By multivariate analysis vomiting (P = 0.01) and fever higher than 39 degrees C plus lasting longer than 3 days (P = 0.02) were significantly more frequent in the EV 71 group. CONCLUSION EV 71 illness is more severe with significantly greater frequency of serious complications and fatality than is illness caused by Cox A16.

Acute Encephalomyelitis during an Outbreak of Enterovirus Type 71 Infection in Taiwan: Report of an Autopsy Case with Pathologic, Immunofluorescence, and Molecular Studies

We report a fatal case of enterovirus type 71 (EV 71) infection in an 8-year-old girl during a summer outbreak of hand, foot, and mouth disease in 1998 in Taiwan. The clinical course was rapidly progressive, with manifestations of hand, foot, and mouth disease, aseptic meningitis, encephalomyelitis, and pulmonary edema. The patient died 24 hours after admission. Postmortem study revealed extensive inflammation in the meninges and central nervous system and marked pulmonary edema with focal hemorrhage. Brain stem and spinal cord were most severely involved. The inflammatory infiltrates consisted largely of neutrophils involving primarily the gray matter with perivascular lymphocytic cuffing, and neuronophagia. The lungs and heart showed no evidence of inflammation. EV 71 was isolated from the fresh brain tissues and identified by immunofluorescence method with type-specific EV 71 monoclonal antibody. It was also confirmed by neutralization test and reverse-transcriptase polymerase chain reaction with sequence analysis. The present case was the first example in which EV 71 was demonstrated to be the causative agent of fatal encephalomyelitis during its epidemic in Taiwan.

Oral acyclovir prophylaxis of varicella after intimate contact

OBJECTIVES Whether oral acyclovir (ACV) given in late incubation can prevent clinical varicella or not. MATERIALS AND METHODS Twenty-seven healthy infants and children susceptible to varicella received oral ACV (40 mg/kg daily in four divided doses) for 5 days, starting 9 or 11 days after exposure from the index case in the family (2 in the classroom). The clinical features were compared with 13 control children who did not receive ACV. Enzyme-linked immunoassay was used to detect varicella-zoster virus (VZV) antibody and, in follow-up immunologic studies, lymphocyte proliferative response was added. In some cases, blood culture and polymerase chain reaction with Southern hybridization were used for detection of viremia. RESULTS Among the 27 children in the treatment group, two (7.4%) developed the disease and seroconversion was observed in 17 subjects (63%). Follow-up immunologic studies in 12 of these 17 seroconverted subjects 30 months later showed persistent cellular and/or humoral immunity to VZV. Only one subject, bled 11 days after exposure, had positive VZV DNA and blood culture for VZV. On the other hand 10 of 13 (77%) control subjects developed clinical varicella. CONCLUSIONS Oral ACV administration to healthy susceptible subjects at the beginning of secondary viremia in the late incubation period (9 days after exposure) can effectively prevent or modify clinical varicella.

Clinical Manifestations and Laboratory Assessment in an Enterovirus 71 Outbreak in Southern Taiwan

An epidemic of enterovirus 71 (EV71) infection compatible with hand, foot and mouth disease and associated with high morbidity and mortality occurred in Taiwan in 1998. We recruited 90 patients (50 males, 40 females) with definite EV71 infections for clinical and laboratory analysis. The neurological signs and symptoms, all of which occurred during the febrile period, in patients with central nervous system (CNS) involvement (aseptic meningitis, encephalitis or myelitis) were myoclonic jerks (23/33), vomiting (10/33), ataxia (7/33), lethargy (6/33), seizure (4/33) and tremor (2/33). Patients with CNS involvement had longer durations of fever (4.6 ± 0.2 vs. 3.1 ± 0.3 d; p< 0.01) and a higher white blood cell count (12,512 ± 658 vs. 10,607 ± 409 cells/mm3; p= 0.01) than patients without CNS involvement. The case fatality rate in patients with CNS involvement was 4/33 (12%), whereas no fatalities (0/57) occurred in patients without CNS involvement. Six of 11 patients subjected to MRI showed a high intensity T2-weighted signal in the brainstem. A nested fluorescent RT-PCR for detection of virus in throat and stool specimens showed higher sensitivity than viral culture. Viremia was detectable using RT-PCR in 20% of cases (3/15), whereas no virus was isolated from culture or detected by RT-PCR in cerebrospinal fluid.

Expression of caspid protein VP1 for use as antigen for the diagnosis of enterovirus 71 infection

To produce enterovirus 71 antigen for diagnostic purposes, the gene encoding the entire capsid protein VP1 was amplified by reverse transcription‐polymerase chain reaction (RT‐PCR), cloned and expressed in Escherichia coli as a poly‐histidine fusion protein. Western blotting experiments with sera from patients with enterovirus 71 infection indicated that immunoglobulin G (IgG) and IgM antibodies bound to a single polypeptide VP1. According to these results, IgM anti‐VP1 appeared in sera of patients with a symptomatic enterovirus 71 acute infection, whereas IgG anti‐VP1 was present in sera of past infection. This finding suggests that detecting IgG and IgM immune responses against linear epitopes of recombinant VP1 is an effective means of determining the different phases of enterovirus 71 infection. In addition, sera containing coxsackie virus 16 (CA16) antibodies did not cross‐react with the recombinant VP1 of enterovirus 71, despite the homology between VP1 proteins of both viruses. Comparison with reference PCR and neutralization assays showed these antibody tests to be appropriate for the serodiagnosis of enterovirus 71 infection. J. Med. Virol. 61:228–234, 2000.

Use of molecular assay in diagnosis of hand, foot and mouth disease caused by enterovirus 71 or coxsackievirus A 16.

Hand, foot and mouth disease is a common illness in children and is usually caused by coxsackievirus A 16 and enterovirus 71. It has been noted that enterovirus 71 infection is more severe with significantly greater frequency of serious complications and fatality than coxsackievirus A 16. Therefore, it is important to develop a rapid and specific assay for discriminating coxsackievirus A 16 and enterovirus 71 in hand, foot and mouth disease outbreaks. In this study we designed two sets of RT-PCR primers specific for coxsackievirus A 16 and enterovirus 71. One hundred and eighty-nine viruses were evaluated for this molecular diagnosis assay. Among 110 enterovirus 71 strains, the enterovirus 71 specific primers gave clear signal for 107 clinical enterovirus 71 isolates and three reference enterovirus 71 strains. None of coxsackievirus A 16, other enteroviruses or non-enteroviruses show signal for enterovirus 71-specific primers. On the other hand, among 28 coxsackievirus A 16 strains, the coxsackievirus A 16-specific primers detect 27 clinical isolates and one reference strain but show no cross-reaction with other viruses. The molecular assay developed in this study provides a sensitive and specific way to distinguish coxsackievirus A 16 and enterovirus 71 induced hand, foot and mouth disease, which will be a useful rapid diagnostic method in future outbreaks.

Diagnosis of respiratory tract viruses in 24 h by immunofluorescent staining of shell vial cultures containing Madin-Darby Canine Kidney (MDCK) cells.

Nine hundred and seventy-eight clinical specimens were examined taken from patients with respiratory tract viruses (RV)-like syndrome between November 1996 and July 1998. The study was undertaken to evaluate the effectiveness of centrifuge-enhanced shell vial cultures (SVC) containing Madin-Darby Canine Kidney (MDCK) cells, combined with immunofluorescent (IF) staining in 24 h. This technique rapidly detects and identifies respiratory tract viruses. The conventional tube culture system with multiple cell lines would ordinarily detect RV within 3-30 days. The SVC/IF method using single cell line (MDCK cells) allowed detection of 81.5% of influenza A virus, 72% of parainfluenza virus, 82.6% of respiratory syncytial virus (RSV) and 79.6% of adenovirus in 24 h.

Reverse transcriptase-polymerase chain reaction for amantadine susceptibility testing of influenza viruses

The clinical use of amantadine for influenza A viruses infection is increasing. A novel quantitative reverse tran-scriptase-polymerase chain reaction (RT-PCR) assay was developed to determine the IC 50 (dose of 50% inhibitory) of amantadine. The traditional amantadine susceptibility testing by plaque inhibition assay took 3 days to observe plaque formation. In contrast, the proposed assay took 3 hours to detect viral RNA. The RT-PCR products can be directly sequenced for screening and identifying the resistant strains.