Molecular epidemiology of G9 rotaviruses in Taiwan between 2000 and 2002.

Since the mid-1990s, novel G9 rotaviruses have been detected in many countries, suggesting that G9 is a globally important serotype. The molecular epidemiology of G9 rotaviruses in Taiwan from 2000 to 2002 was investigated in this study. G9 rotavirus first appeared in 2000 with 4 cases and constituted 33.8% and 54.8% of the rotavirus-positive samples in 2001 and 2002, respectively. These G9 strains belonged to P[8]G9, subgroup II, and long electropherotype, except one belonged to P[4]G9, subgroup II, and short electropherotype. Nucleotide sequencing and phylogenetic analysis of 52 Taiwanese G9 rotaviruses showed that the VP7 genes shared a high degree of identity to overseas G9 rotaviruses detected after 1993 and that the VP8* portions of the VP4 genes were more closely related to those of local rotaviruses of other G types. The two P[8]G9 strains with high nucleotide identities in the VP7 and the partial VP4 genes, 01TW591 of Taiwan from 2001 and 95H115 of Japan from 1995, varied in four genes, genes 2, 3, 7, and 8, which was revealed by RNA-RNA hybridization. Representative strains for different RNA patterns were also analyzed in the partial VP2 and VP3 genes; the nucleotide identities were high between Taiwanese G9 strains and local G3 or G2 strains. These results suggested that Taiwanese G9 rotaviruses possibly had evolved through reassortment between overseas G9 strains and circulating rotaviruses of other G types.

Genotyping of Chlamydia trachomatis from clinical specimens in Taiwan.

This study was conducted to determine the prevalence and distribution of Chlamydia trachomatis genotypes in Taiwan. Urine and endocervical-swab samples were collected from two hospitals located in northern and southern Taiwan. The genotypes of a total of 145 samples positive for C. trachomatis were analysed by sequencing the omp1 gene and this was successful in 102 samples. Nine different C. trachomatis genotypes were identified. Genotype E was the most prevalent (22 %), followed by D and Da (19 %), F (16 %), J (15 %), K (11 %), G (11 %), H (6 %) and Ba (2 %). There was a geographical difference in the prevalence of genotype H (P < 0.018) between northern and southern Taiwan. Sequence mutation analysis by blast searching against GenBank reference sequences identified 12 genetic variants from a total of 102 omp1 gene sequences.

Epidemiological and genetic correlates of severe acute respiratory syndrome coronavirus infection in the hospital with the highest nosocomial infection rate in Taiwan in 2003.

Taiwan experienced a series of outbreaks of nosocomial severe acute respiratory syndrome (SARS) infections in 2003. Two months after the final outbreak, we recruited 658 employees from the hospital that suffered the first and most severe SARS infections to help us investigate epidemiological and genetic factors associated with the SARS coronavirus (SARS-CoV). SARS-CoV infections were detected by using enzyme immunoassays and confirmed by a combination of Western blot assays, neutralizing antibody tests, and commercial SARS tests. Risk factors were analyzed via questionnaire responses and sequence-specific oligonucleotide probes of human leukocyte antigen (HLA) alleles. Our results indicate that 3% (20/658) of the study participants were seropositive, with one female nurse identified as a subclinical case. Identified SARS-CoV infection risk factors include working in the same building as the hospital's emergency room and infection ward, providing direct care to SARS patients, and carrying a Cw*0801 HLA allele. The odds ratio for contracting a SARS-CoV infection among persons with either a homozygous or a heterozygous Cw*0801 genotype was 4.4 (95% confidence interval, 1.5 to 12.9; P = 0.007).

Evolution of EV71 genogroup in Taiwan from 1998 to 2005: an emerging of subgenogroup C4 of EV71.

In Taiwan, enterovirus 71 (EV71) has played an important role in severe enterovirus-related cases every year since the devastating outbreak in 1998. Three genogroups A, B, C occur worldwide; with the B and C genogroups being subdivided into B1-B4 and C1-C4 subgenogroups respectively. To understand the mutation of the EV71 genogroup in Taiwan before and after 1998, a total of 54 worldwide strains were studied including 41 Taiwanese strains obtained in 1986 and 1998-2004. A fragment of 207 bp of the VP4 region was amplified and sequenced. Genetic analysis was performed using MEGA software (version 3.0) for the nucleotide sequence alignment and phylogenetic analysis. In Taiwan, the subgenogroup B1 was predominant before 1998 while subgenogroup C2 was the major etiologic group in 1998 outbreak. A minor etiologic group outbreak in 1998, subgenogroup B4, became predominant during the period from 1999 to 2003. In this study, subgenogroup C4 emerged and became predominant in 2004 in Taiwan. The nucleotide differences between B1 and C2, C2 and B4, B4 and C4 were 20%-26%, 19%-27%, 18%-22%, respectively. Nucleotide sequence alignment revealed 67 substitutions. Most of the substitutions (62/67) were silent mutations. This is the first report about the emergence of EV71 subgenogroup C4 in Taiwan.

Neutralizing antibody response and SARS severity.

Using the Taiwan nationwide laboratory-confirmed severe acute respiratory syndrome (SARS) database, we analyzed neutralizing antibody in relation to clinical outcomes. With a linear mixed model, neutralizing antibody titer was shown to peak between week 5 and week 8 after onset and to decline thereafter, with a half-life of 6.4 weeks. Patients with a longer illness showed a lower neutralizing antibody response than patients with a shorter illness duration (p = 0.008). When early responders were compared with most patients, who seroconverted on and after week 3 of illness, the small proportion (17.4%) of early responders (antibody detectable within 2 weeks) had a higher death rate (29.6% vs. 7.8%) (Fisher exact test, p = 0.004), had a shorter survival time of <2 weeks (Fisher exact test, p = 0.013), and were more likely to be > 60 years of age (Fisher exact test, p = 0.01). Our findings have implications for understanding the pathogenesis of SARS and for SARS vaccine research and development.

Antigenic divergence of Bordetella pertussis isolates in Taiwan.

In recent studies, antigenic divergence has been observed in Bordetella pertussis circulating isolates. We collected 80 Bordetella pertussis isolates in Taiwan from 1998 to 2004 and analyzed them using a combination of pulsed-field gel electrophoresis (PFGE) and sequencing of the ptxS1 and prn genes. The incidence of pertussis increases every 3 years, and most of the isolates prevalent since 1998 have expressed nonvaccine ptxS1A and prn2 alleles. Through PFGE analysis, all isolates could be classified into four major groups, and the incidence of these groups exhibited a correlation with the prn allele expressed by the isolates. We found that PFGE is more discriminative than gene sequencing, since it could divide the isolates expressing the prn2 allele into two groups: one group circulating from 1998 to 2001 and another group circulating from 2001 to 2004. The transition between the two groups in 2000 coincided with an outbreak of 326 cases. This research indicates that the antigenic divergence of B. pertussis circulating isolates has evolved over time in Taiwan. Such information will have implications for vaccine policy in Taiwan.

Intratypic recombination among lineages of type 1 vaccine-derived poliovirus emerging during chronic infection of an immunodeficient patient.

We determined the complete genomic sequences of nine type 1 immunodeficient vaccine-derived poliovirus (iVDPV) isolates obtained over a 337-day period from a poliomyelitis patient from Taiwan with common variable immunodeficiency. The iVDPV isolates differed from the Sabin type 1 oral poliovirus vaccine (OPV) strain at 1.84% to 3.15% of total open reading frame positions and had diverged into at least five distinct lineages. Phylogenetic analysis suggested that the chronic infection was initiated by the fifth and last OPV dose, given 567 days before onset of paralysis, and that divergence of major lineages began very early in the chronic infection. Key determinants of attenuation in Sabin 1 had reverted in the iVDPV isolates, and representative isolates of each lineage showed increased neurovirulence for PVR-Tg21 transgenic mice. None of the isolates had retained the temperature-sensitive phenotype of Sabin 1. All isolates were antigenic variants of Sabin 1, having multiple amino acid substitutions within or near neutralizing antigenic sites 1, 2, and 3a. Antigenic divergence of the iVDPV variants from Sabin 1 followed two major independent evolutionary pathways. The emergence of distinct coreplicating lineages suggests that iVDPVs can replicate for many months at separate sites in the gastrointestinal tract. Some isolates had mosaic genome structures indicative of recombination across and within lineages. iVDPV excretion apparently ceased after 30 to 35 months of chronic infection. The appearance of a chronic VDPV excretor in a tropical, developing country has important implications for the strategy to stop OPV immunization after eradication of wild polioviruses.

Sentinel hospital surveillance for rotavirus diarrhea in Taiwan, 2001-2003.

We examined the epidemiological profile of rotavirus infection among children hospitalized for diarrhea in Taiwan, to assess the burden of this disease. From 1 April 2001 through 31 March 2003, children <5 years old with gastroenteritis admitted to 4 sentinel hospitals were enrolled in a surveillance study and had stool specimens tested for the presence of rotavirus, enteric adenovirus, and the bacterial pathogens for which routine screening is performed. For 52% of patients, a recognized enteric pathogen was identified, including rotavirus (43% of patients), bacteria (11%), enteric adenovirus (2.5%), and a mixture of pathogens (3.9%). Rotavirus was detected year-round, but great month-to-month variability made it difficult to identify a distinct seasonal pattern. Rotavirus disease was most common among children 7-23 months old, but the rate of rotavirus detection varied little between the youngest and oldest age groups. The novel strain P[8]G9 was detected most commonly (37% of strains), followed by strains P[8]G1 (31%), P[4]G2 (10%), P[8]G3 (9.3%), and P[8]G4 (3.7%). Rotavirus infection is the most important cause of diarrhea among hospitalized children in Taiwan, and a rotavirus vaccination program for young children might significantly reduce this problem.

Influenza in Taiwan: seasonality and vaccine strain match.

This article explores seasonality, epidemiology and dominant epidemic strains of influenza in Taiwan. Surveillance in Taiwan demonstrates that influenza is a disease which occurs throughout the year but has peak activity in winter. Due to the high mutability of influenza virus, effective vaccination is the best strategy for protection. Although the World Health Organization-recommended vaccine compositions usually matched with around 77% of circulating strains worldwide, the rate of matching in Taiwan has been markedly lower than this. Between 1997 and 2004 in Taiwan, the match rates were 82% for H1N1, 53% for H3N2, and 47% for influenza B virus. Furthermore, some world epidemic strains appeared earlier in Taiwan than in other countries. In view of Taiwan's proximity to southern China, which is thought to be the epicenter of influenza epidemics, vigilant surveillance and the development of regional strategies for the selection and manufacture of vaccine strains to improve influenza prevention are urgent requirements.

Molecular epidemiology of severe acute respiratory syndrome-associated coronavirus infections in Taiwan.

BACKGROUND: In 2003, Taiwan experienced a series of outbreaks of severe acute respiratory syndrome (SARS) and 1 laboratory-contamination accident. Here we describe a new phylogenetic analytical method to study the sources and dissemination paths of SARS-associated coronavirus (SARS-CoV) infections in Taiwan. METHODS: A phylogenetic analytical tool for combining nucleotide sequences from 6 variable regions of a SARS-CoV genome was developed by use of 20 published SARS-CoV sequences; and this method was validated by use of 80 published SARS-CoV sequences. Subsequently, this new tool was applied to provide a better understanding of the entire complement of Taiwanese SARS-CoV isolates, including 20 previously published and 19 identified in this study. The epidemiological data were integrated with the results from the phylogenetic tree and from the nucleotide-signature pattern. RESULTS: The topologies of phylogenetic trees generated by the new and the conventional strategies were similar, with the former having better robustness than the latter, especially in comparison with the maximum-likelihood trees: the new strategy revealed that during 2003 there were 5 waves of epidemic SARS-CoV infection, which belonged to 3 phylogenetic clusters in Taiwan. CONCLUSIONS: The new strategy is more efficient than its conventional counterparts. The outbreaks of SARS in Taiwan originated from multiple sources.

Isolation and identification of Escherichia coli O157:H7 in a Taiwanese patient with bloody diarrhea and acute renal failure.

Escherichia coli O157:H7 is a frequent foodborne pathogen in many developed countries. Outbreaks of this infection have been reported in countries all over the world. The first clinical case of E. coli O157:H7 infection from Taiwan was reported in a 6- year-old boy who had returned from USA in August 2001. In this paper, we describe the results of the isolation and identification of this strain, and molecular typing for comparison with previously reported strains. Biochemical and molecular biological tests were used to confirm that this patient, who developed bloody diarrhea and kidney failure as a result of the infection, was indeed infected with E. coli O157:H7. None of the patients' close contacts were affected. Molecular typing by use of pulsed-field gel electrophoresis revealed this clinical strain to have a unique genotype, which is different from all available clinical strains reported from Japan and environmental strains reported from Taiwan. America Type Culture Collection reference strains and an out-break strain from USA had the nearest relationships with this clinical isolate. Molecular typing showed that this infection by E. coli O157:H7 was not derived from the local environmental strains and was acquired during overseas travel.

Phylogenetic analysis and sequence comparisons of structural and non-structural SARS coronavirus proteins in Taiwan.

Taiwan experienced a large number of severe acute respiratory syndrome (SARS) viral infections between March and July 2003; by September of that year, 346 SARS cases were confirmed by RT-PCR or serological tests. In order to better understand evolutionary relationships among SARS coronaviruses (SCoVs) from different international regions, we performed phylogenetic comparisons of full-length genomic and protein sequences from 45 human SCoVs (including 12 from Taiwan) and two civet SCoVs. All the Taiwanese SARS-CoV strains which associated with nosocomial infection formed a monophyletic clade within the late phase of the SARS epidemic. This Taiwanese clade could be further divided into two epidemic waves. Taiwan SCoVs in the first wave clustered with three isolates from the Amoy Gardens housing complex in Hong Kong indicating their possible origin. Of the 45 human SCoVs, one isolate from Guangdong province, China, exhibited an extra 29-nucleotide fragment between Orf 10 and Orf 11--similar to the civet SCoV genome. Nucleotide and protein sequence comparisons suggested that all SCoVs of late epidemic came from human-to-human transmission, while certain SCoVs of early epidemic might have originated in animals.

Emergence of G9 serotype rotavirus as a major cause of infectious gastroenteritis in southern Taiwan.

Infectious gastroenteritis is a common illness in children. This study investigated the etiology and clinical manifestations of hospitalized children with symptoms of infectious gastroenteritis in southern Taiwan. We studied 467 consecutive patients with infectious gastroenteritis aged from 2 days to 10 years hospitalized from April 2001 to March 2002. Rotavirus was the most common etiology (57%) of infectious gastroenteritis in these patients. Bacterial infection was noted in 57 cases (12%). Rotavirus was found in 92% of nosocomial infectious gastroenteritis (p < or = 0.001). Bloody stool was a presentation of bacterial infection in 74% of cases and rotavirus gastroenteritis in 8% of cases (p < or = 0.001). The G serotype of rotavirus was identified in 87 patients. Serotype G1 was the most common (51%), followed by G9 (31%). The emergence of serotype G9 strains in rotavirus infection has not been previously reported from Taiwan. Incorporation of G9 rotavirus into vaccines should be considered.

A two decade survey of respiratory adenovirus in Taiwan: the reemergence of adenovirus types 7 and 4.

From November 1999 to December 2001, three outbreaks of adenovirus (Ad) respiratory infection occurred in southern Taiwan. To determine the circulating serotypes and molecular epidemiology, a total of 524 virus strains were randomly selected from 1,064 strains isolated from 1981 to 2001, and were studied using restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-RFLP. The major subgenus found was subgenus B (45%), followed by subgenus E (29%) and subgenus C (25%). Ad3 and Ad7 were the major types found during the 1st outbreak, which occurred from November 1999 to March 2000, while Ad4 was found mainly during the 2nd and 3rd outbreaks in October 2000 and September 2001, respectively. Both Ad7 and Ad4 were reemerged serotypes, whereas Ad3 was consistently isolated during the survey, although it declined drastically from 36 to 2% in 2001. Genotype analysis in this study showed that the only strain of Ad7 found in 1983 was Ad7a, but all randomly selected strains of Ad7 isolated during 1999-2000 were Ad7b. The clinical features of 217 patients were analyzed during the 1999-2000 outbreaks. About 79% of the total cases were less than 7 years old. The ratio of male to female was 2:1. Severe infections, such as pneumonia and acute bronchitis, accounted for nearly half of the cases (43%). These results show the reemergence and changing of serotypes, the clinical association of respiratory adenovirus infections, and the molecular epidemiology of Ad7 genotypes in Taiwan during the past two decades.

Early and rapid detection of enterovirus 71 infection by an IgM-capture ELISA.

Enterovirus 71 infection is more likely to induce severe complications and mortality than other enteroviruses. Laboratory diagnosis of enterovirus 71 in Taiwan still relies mainly on conventional virus isolation techniques that often require 5-10 days to obtain a result, thus hindering seriously the subsequent treatment and disease control measures. This study was to find a better alternative by developing a rapid enterovirus 71 detecting procedure, which can afford an earlier diagnosis and a more rapid outcome. In this study, an enterovirus 71-IgM-capture enzyme-linked immunosorbent assay (ELISA) was designed and tested with a total of 336 serum specimens collected from 236 cases of reported enterovirus infection with complications. Using virus isolation and neutralization test as standards, the sensitivity and specificity of the new protocol were 97.7 and 93.3%, respectively. Most of the IgM positive serum specimens were collected within 7 days after the onset of symptoms, while it appeared detectable up to 94 days after the onset of symptoms. Apart from being highly sensitive, rapid and low in cost, the new IgM-capture ELISA is sufficiently accurate to provide also reliable results for early detection of the virus. With this protocol, enterovirus 71 infections can be detected within 4h.

Serologic and molecular biologic methods for SARS-associated coronavirus infection, Taiwan.

Severe acute respiratory syndrome (SARS) has raised a global alert since March 2003. After its causative agent, SARS-associated coronavirus (SARS-CoV), was confirmed, laboratory methods, including virus isolation, reverse transcriptase-polymerase chain reaction (RT-PCR), and serologic methods, have been quickly developed. In this study, we evaluated four serologic tests ( neutralization test, enzyme-linked immunosorbent assay [ELISA], immunofluorescent assay [IFA], and immunochromatographic test [ICT]) for detecting antibodies to SARS-CoV in sera of 537 probable SARS case-patients with correlation to the RT-PCR. With the neutralization test as a reference method, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.2%, 98.7%, 98.7%, and 98.4% for ELISA; 99.1%, 87.8%, 88.1% and 99.1% for IFA; 33.6%, 98.2%, 95.7%, and 56.1% for ICT, respectively. We also compared the recombinant-based western blot with the whole virus-based IFA and ELISA; the data showed a high correlation between these methods, with an overall agreement of >90%. Our results provide a systematic analysis of serologic and molecular methods for evaluating SARS-CoV infection.

Clinical manifestations and complications of rickettsiosis in southern Taiwan.

BACKGROUND AND PURPOSE: Taiwan is an endemic area for scrub typhus. Recent increases have been noted in two other infectious diseases caused by obligate intracellular organisms, Q fever and murine typhus. These diseases usually present as an acute febrile illness with non-specific symptoms and are difficult to distinguish. This study describes the clinical manifestations and complications of cases of rickettsial infections treated at a medical center in southern Taiwan. METHODS: Serum samples from patients with acute febrile illness with or without shock, but without a clinical diagnosis of localized bacterial infection after a preliminary work-up, were collected for serologic study. Medical records of cases with serologic evidence of infection were reviewed and their clinical manifestations were analyzed. RESULTS: From October 1992 to July 2000, 51 serologically diagnosed cases of rickettsiosis were studied. Q fever predominated (28 cases). All cases of acute Q fever presented with hepatitis during the course of the illness and more than half (54%) reported an animal contact history. Most patients with Q fever (96%) and murine typhus (86%) were male, Serious complications, especially pneumonitis, occurred more frequently with scrub typhus than with acute Q fever. Spontaneous remission frequently occurred with acute Q fever. Administration of tetracycline or its analogues usually resulted in defervescence by the third day of treatment of scrub typhus. CONCLUSIONS: In our study, serious complications including pneumonitis, meningitis/meningoencephalitis, shock, acute renal failure, and disseminated intravascular coagulation were not uncommon in patients with scrub typhus. Physicians should include scrub typhus in the differential diagnosis of suspected cases of community-acquired febrile illness with multiple organ dysfunction in this endemic area. As Q fever is an emerging infectious disease in southern Taiwan, further large-scale epidemiologic surveillance and clinical data are needed.