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Background: Patients with cervical radiculopathy typically present with shoulder pain and weakness; these symptoms are similar to those of rotator cuff disease. Studies investigating cervical spine pathology (CSP) as an independent risk factor for rotator cuff tear (RCT) are lacking in the literature. Purpose: To investigate the risk of RCT among patients with CSP who have undergone cervical diskectomy (CD) and to determine whether CD reduces this risk. Study Design: Cohort study; Level of evidence, 3. Methods: The authors queried the Taiwan National Health Insurance Research Database for patients diagnosed with CSP between 2004 and 2008 and followed up until the end of 2010. A control cohort comprised patients without CSP who were age- and sex-matched in a 4-to-1 ratio with patients with CSP through propensity score matching. A Cox multivariate proportional hazards model was applied to analyze the risk factors for RCT. After adjustment for confounders, the authors calculated the hazard ratio (HR) and adjusted HR (aHR) between the study and control cohorts. The effects of CD on the risk of RCT were also analyzed. Results: The study included 3245 patients and 12,980 matched controls. A higher RCT incidence rate was found in the CSP cohort, with an aHR of 1.52 (95% CI, 1.22-1.89; P <.001). Patients with CSP who underwent CD had a risk of RCT similar to that of the controls, with an aHR of 1.65 (95% CI, 0.90-3.03; P >.05). Conclusion: Patients with CSP had a 1.52-fold higher risk of RCT than healthy controls. Patients with CSP with CD did not have a high risk of RCT, possibly indicating a protective effect of diskectomy against RCT. [ABSTRACT FROM AUTHOR]

Patients with axial spondyloarthritis (ax-SpA) present with inflammation invading the axial skeleton. Symptoms of ax-SpA interfere with patients' quality of life, and peripheral symptoms are also noted. Human leukocyte antigen B27 was associated with adhesive capsulitis. However, epidemiological studies investigating the associated incidence and risk factors for patients with ax-SpA with adhesive capsulitis are limited. The data of patients with ax-SpA were recorded during the 2004–2008 period and followed to the end of 2010. The control cohort comprised age- and sex-matched non-ax-SpA subjects. A Cox multivariate proportional hazards model was applied to analyze the risk factors for adhesive capsulitis. The hazard ratio (HR) and adjusted hazard ratio (aHR) were estimated between the study and control cohorts after confounders were adjusted for. Effects of sulfasalazine (SSZ), methotrexate (MTX), and hydroxychloroquine (HCQ) use on adhesive capsulitis risk were also analyzed. We enrolled 2859 patients with ax-SpA in the study cohort and 11,436 control subjects. A higher incidence of adhesive capsulitis was revealed in the ax-SpA cohort: The crude HR was 1.63 (95% CI, 1.24–2.13; p < 0.001), and the aHR was 1.54 (95% CI, 1.16–2.05; p = 0.002). For patients with ax-SpA using SSZ or HCQ, no difference in aHR was noted compared with control participants, but patients with ax-SpA treated with MTX had higher HR and aHR than controls. Patients with ax-SpA are at risk for adhesive capsulitis. When these patients receive SSZ or HCQ, the risk of adhesive capsulitis can be lowered compared with that of the control cohort. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) causes approximately 390 million dengue infections worldwide every year. There were 22,777 reported DENV infections in Tainan, Taiwan in 2015. In this study, we sequenced the C-prM-E genes from 45 DENV 2015 strains, and phylogenetic analysis based on C-prM-E genes revealed that all strains were classified as DENV serotype 2 Cosmopolitan genotype. Sequence analysis comparing different DENV-2 genotypes and Cosmopolitan DENV-2 sequences prior to 2015 showed a clade replacement event in the DENV-2 Cosmopolitan genotype. Additionally, a major substitution C-A314G (K73R) was found in the capsid region which may have contributed to the clade replacement event. Reverse genetics virus rgC-A314G (K73R) showed slower replication in BHK-21 and C6/36 cells compared to wildtype virus, as well as a decrease in NS1 production in BHK-21-infected cells. After a series of passaging, the C-A314G (K73R) mutation reverted to wildtype and was thus considered to be unstable. Next generation sequencing (NGS) of three sera collected from a single DENV2-infected patient at 1-, 2-, and 5-days post-admission was employed to examine the genetic diversity over-time and mutations that may work in conjunction with C-A314G (K73R). Results showed that the number of haplotypes decreased with time in the DENV-infected patient. On the fifth day after admission, two new haplotypes emerged, and a single non-synonymous NS4A-L115I mutation was identified. Therefore, we have identified a persistent mutation C-A314G (K73R) in all of the DENV-2 isolates, and during the course of an infection, a single new non-synonymous mutation in the NS4A region appears in the virus population within a single host. The C-A314G (K73R) thus may have played a role in the DENV-2 2015 outbreak while the NS4A-L115I may be advantageous during DENV infection within the host., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tsai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Herpes simplex virus type 1 (HSV-1) can establish latency in humans and easily relapse in immunocompromised patients, with significant mortality. Treatment with acyclovir (ACV) can result in the emergence of HSV resistance. A total of 440 frozen HSV-1 isolates collected from 318 patients from January 2014 to July 2019 were obtained from National Cheng Kung University Hospital in southern Taiwan. These 440 isolates were subjected to phenotypic studies for ACV-resistance by initial screening with the plaque reduction assay (PRA) and further validation by the DNA reduction assay (DRA). The ACV-resistant strains were further investigated by Sanger sequencing for the full-length UL23 and UL30 genes, which encode thymidine kinase and DNA polymerase, respectively. Hematological malignancies or hematopoietic stem-cell transplantation patients accounted for 56.9% (124/218) among the immunocompromised patients (218/318) in this study. Repeated sampling for HSV testing was 50% (109/218) in immunocompromised patients. Only 1.38% (3/218) of immunocompromised patients and 0.9% (3/318) of all patients developed ACV-resistant HSV-1 as measured by phenotypic screening assays. It is noteworthy that a novel Y248D mutation in the UL23 gene from an immunocompromised patient was found by both PRA and DRA. In 3D protein predicting analysis, uncharged Y248 was located at an alpha-helix and substituted by negative-charged D248, which may alter the function of viral thymidine kinase. Besides, three unreported mutations related to natural polymorphism were found in virus isolates from two immunocompetent patients, including 683-688 deletion, R227H, and A351D in the UL30 gene. These data show that the prevalence of ACV-resistant HSV-1 among immunocompromised patients in southern Taiwan is low. These results will be helpful for the clinical management and treatment of HSV infections., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Enterovirus A71 (EV-A71) is a non-enveloped virus possessing 4 capsid proteins: VP1-VP4. The outermost capsid protein, VP1, plays roles in both antigenicity and virulence of the virus. The concept of generating other EV-A71 genotypes of reverse genetics (rg) viruses by replacing VP1 can be made possible with synthetic biotechnology, allowing us to redesign organisms, creating unavailable ones. To determine suitable vaccine candidates against EV-A71 infections, we combined synthetic biotechnology, rg-virus production and high-fidelity determinants to produce genetically stable viruses. With the use of antigenic cartography, we are able to view the antigenic distance among various points. We analyzed and generated various EV-A71 VP1 sequences from Taiwan and Southeast Asian (SEA) countries, which were then used to produce recombinant rg-viruses and the viral proteins were purified for immunization of mice and rabbits. Antisera against various EV-A71 genotypes were used in neutralization assays against various Taiwan and SEA EV-A71 genotypes. Based on neutralization data from mice and rabbit antisera, we found that antisera produced from several genotypes were able to effectively neutralize the various Taiwan and SEA EV-A71 genotypes. Additionally, comparing the antigenic maps produced from mouse, rabbit and human antisera against different EV-A71 genotypes, a difference in clustering was seen and the spacing between points also differed. Based on antigenic mapping and neutralizing activities, B4 7008-HF and C4 M79 may be good potential vaccine candidates against EV-A71., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Background: The regional respiratory syncytial virus (RSV) outbreak in southern Taiwan in late 2020 followed the surge of RSV cases in the national surveillance data and displayed distinct clinical features. This study investigated RSV epidemiology in the most recent five years and compared the clinical manifestations of this outbreak with non-outbreak period., Methods: Medical records of RSV-infected children at the National Cheng Kung University Hospital from January 2016 to December 2020 were retrospectively retrieved from hospital-based electronic medical database. Cases of RSV infection were identified by RSV antigen positive and/or RSV isolated from respiratory specimens. The demographic, clinical presentations, and laboratory data were recorded. The RSV isolates in 2020 was sequenced for phylogenetic analysis., Results: Overall, 442 RSV-infected cases were retrieved and 42.1% (186 cases) clustered in late 2020. The 2020 outbreak started in September, peaked in November, and lasted for 3 months. 2020 RSV-infected children were older (2.3 ± 2.2 years vs. 1.0 ± 1.0 years), more likely to be diagnosed with bronchopneumonia (57.5% vs. 31.6%), but also had a lower hospitalization rate, shorter hospital stay, less oxygen use, and less respiratory distress than those in 2016-2019 (all p value < 0.05). The RSV isolates in 2020 belonged to RSV-A subtype ON1 but were phylogenetically distinct from the ON1 strains prevalent in Taiwan previously., Conclusion: The 2020 RSV outbreak was led by the novel RSV-A subtype ON1 variant with clinical manifestations distinct from previous years. Continuous surveillance of new emerging variants of respiratory viruses in the post-pandemic era is warranted., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2022. Published by Elsevier B.V.)

Broadly neutralizing ability is critical for developing the next-generation SARS-CoV-2 vaccine. We collected sera samples between December 2021-January 2022 from 113 Taiwan naïve participants after their second dose of homologous vaccine (AZD1222, mRNA-1273, BNT162-b2, and MVC-COV1901) and compared the differences in serological responses of various SARS-CoV-2 vaccines. Compared to AZD1222, the two mRNA vaccines could elicit a higher level of anti-S1-RBD binding antibodies with higher broadly neutralizing ability evaluated using pseudoviruses of various SARS-CoV-2 lineages. The antigenic maps produced from the neutralization data implied that Omicron represents very different antigenic characteristics from the ancestral lineage. These results suggested that constantly administering the vaccine with ancestral Wuhan spike is insufficient for the Omicron outbreak. In addition, we found that anti-ACE2 autoantibodies were significantly increased in all four vaccinated groups compared to the unvaccinated pre-pandemic group, which needed to be investigated in the future., Competing Interests: Author Y-CC is employed by Leadgene Biomedical, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationship that could be considered a potential conflict of interest., (Copyright © 2022 Chao, Cheng, Huang, Chuang, Yeh and Wang.)

Coxsackievirus A16 (CVA16) is well known for causing hand-foot-and-mouth disease (HFMD) and outbreaks were frequently reported in Taiwan in the past twenty years. The epidemiology and genetic variations of CVA16 in Taiwan from 1998 to 2021 were analyzed in this study. CVA16 infections usually occurred in early summer and early winter, and showed increased incidence in 1998, 2000-2003, 2005, 2007-2008, and 2010 in Taiwan. Little or no CVA16 was detected from 2017 to 2021. CVA16 infection was prevalent in patients between 1 to 3 years old. A total of 69 isolates were sequenced. Phylogenetic analysis based on the VP1 region showed that CVA16 subgenotype B1 was dominantly isolated in Taiwan from 1998 to 2019, and B2 was identified only from isolates collected in 1999 and 2000. There was a high frequency of synonymous mutations in the amino acid sequences of the VP1 region among CVA16 isolates, with the exception of position 145 which showed positive selection. The recombination analysis of the whole genome of CVA16 isolates indicated that the 5'-untranslated region and the non-structural protein region of CVA16 subgenotype B1 were recombined with Coxsackievirus A4 (CVA4) and enterovirus A71 (EVA71) genotype A, respectively. The recombination pattern of subgenotype B2 was similar to B1, however, the 3D region was similar to EVA71 genotype B. Cross-neutralization among CVA16 showed that mouse antisera from various subgenotypes viruses can cross-neutralize different genotype with high neutralizing antibody titers. These results suggest that the dominant CVA16 genotype B1 can serve as a vaccine candidate for CVA16.

Respiratory viruses can be detected in 18.3 to 48.9% of critically ill adults with severe respiratory tract infections (RTIs). The present study aims to assess the clinical significance of respiratory viruses in pragmatically selected adults in medical intensive care unit patients and to identify factors associated with viral respiratory viral tract infections (VRTIs). We conducted a prospective study on critically ill adults with suspected RTIs without recognized respiratory pathogens. Viral cultures with monoclonal antibody identification, in-house real-time polymerase chain reaction (PCR) for influenza virus, and FilmArray respiratory panel were used to detect viral pathogens. Multivariable logistic regression was applied to identify factors associated with VRTIs. Sixty-four (40.5%) of the included 158 critically ill adults had respiratory viruses detected in their respiratory specimens. The commonly detected viruses included influenza virus (20), followed by human rhinovirus/enterovirus (11), respiratory syncitial virus (9), human metapneumovirus (9), human parainfluenza viruses (8), human adenovirus (7), and human coronaviruses (2). The FilmArray respiratory panel detected respiratory viruses in 54 (34.6%) patients, but showed negative results for seven of 13 patients with influenza A/H3 infection. In the multivariable logistic regression model, patient characters associated with VRTIs included those aged < 65 years, household contact with individuals with upper RTI, the presence of fever, cough with sputum production, and sore throat. Respiratory viruses were not uncommonly detected in the pragmatically selected adults with critical illness. The application of multiplex PCR testing for respiratory viruses in selected patient population is a practical strategy, and the viral detection rate could be further improved by the patient characters recognized in this study., (© 2021. The Author(s).)

Burn injuries cause disability and functional limitations in daily living. In a 2015 fire explosion in Taiwan, 499 young people sustained burn injuries. The construction of an effective and comprehensive rehabilitation program that enables patients to regain their previous function is imperative. The International Classification of Functioning, Disability, and Health (ICF) includes multiple dimensions that can contribute to meeting this goal. An ICF core set was developed in this study for Taiwanese patients with burns. A consensus process using three rounds of the Delphi technique was employed. A multidisciplinary team of 30 experts from various institutions was formed. The questionnaire used in this study comprised 162 ICF second-level categories relevant to burn injuries. A 5-point Likert scale was used, and participants assigned a weight to the effect of each category on daily activities after burns. The consensus among ratings was assessed using Spearman's ρ and semi-interquartile range indices. The core set for post-acute SCI was developed from categories that attained a mean score of ≥4.0 in the third round of the Delphi exercise. The core ICF set contained 68 categories. Of these, 19 comprised the component of body functions, 5 comprised body structures, 37 comprised activities and participation, and 7 comprised environmental factors. This preliminary core set offers a comprehensive system for disability assessment and verification following burn injury. The core set provides information for effective rehabilitation strategy setting for patients with burns. Further feasibility and validation studies are required in the future.

Background: Influenza A viruses cause epidemics/severe pandemics that pose a great global health threat. Among eight viral RNA segments, the multiple functions of nucleoprotein (NP) play important roles in viral replication and transcription., Methods: To understand how NP contributes to the virus evolution, we analyzed the NP gene of H3N2 viruses in Taiwan and 14,220 NP sequences collected from Influenza Research Database. The identified genetic variations were further analyzed by mini-genome assay, virus growth assay, viral RNA and protein expression as well as ferret model to analyze their impacts on viral replication properties., Results: The NP genetic analysis by Taiwan and global sequences showed similar evolution pattern that the NP backbones changed through time accompanied with specific residue substitutions from 1999 to 2018. Other than the conserved residues, fifteen sporadic substitutions were observed in which the 31R, 377G and 450S showed higher frequency. We found 31R and 450S decreased polymerase activity while the dominant residues (31 K and 450G) had higher activity. The 31 K and 450G showed better viral translation and replication in vitro and in vivo., Conclusions: These findings indicated variations identified in evolution have roles in modulating viral replication in vitro and in vivo. This study demonstrates that the interaction between variations of NP during virus evolution deserves future attention.

Background: Human adenoviruses (HAdV) are important pathogens of pediatric respiratory tract infections in Taiwan. There were two major HAdV epidemics in southern Taiwan in 2011 and 2014, respectively., Methods: The demographic, clinical characteristics, and risk factors for hospitalization of pediatric patients with HAdV infection in the two outbreaks were retrospectively compared. The epidemic was defined as > 7% HAdV detection rate for six consecutive weeks. HAdV infection was defined as positive HAdV isolates from respiratory tract specimens. HAdV genotype was determined by PCR-based hexon gene sequencing., Results: A total of 1145 pediatric patients were identified (635 cases in 2011; 510 cases in 2014). HAdV genotype 3 and 7 contributed to both epidemics, although the proportion of HAdV3 decreased significantly (64.7% in 2011 to 25.5% in 2014, p < 0.001) and was replaced by other genotypes (type 1, 4, and 6) in the 2014 epidemic. Among the hospitalized patients, there were more patients hospitalized with bronchopneumonia/or pneumonia in the 2011 epidemic (10.6% vs 5.1%, p < 0.001), while more patients hospitalized with acute pharyngitis/pharyngoconjunctival fever (63.9% vs. 38.6%, p < 0.001) in the 2014 epidemic. In both epidemics, hospitalized patients had higher WBC and C-reactive protein (CRP) levels than non-hospitalized patients. Using multivariate regression analysis, underlying disease and elevated CRP levels were independent risk factors for hospitalization in both epidemics., Conclusion: There were significant differences in clinical, viral characteristics and risk factors of hospitalization between the 2011 and 2014 epidemics. Understanding changes in the epidemiological and clinical characteristics of HAdV epidemics is important from a public health perspective.

Background: Human parainfluenza viruses (HPIV) 1-4 had been analyzed as being one of the most frequent causes of hospitalizations for young children with respiratory tract illnesses., Methods: This retrospective study was performed from children virologically confirmed as HPIV infection through throat swab or nasopharyngeal aspirates at a tertiary care university hospital, between January 2012 and December 2014. HPIV4 was not checked and analyzed, due to not include in the commercial kit. The demographic, epidemiological, clinical presentations, diagnosis, treatment, outcomes, and laboratory data were analyzed., Results: Totally 398 cases were enrolled, including 39 (9.8%) of HPIV1, 67 (16.8%) of HPIV2, and 292 (73.4%) of HPIV3. The mean age of HPIV-infected children was 2.9 year-old, and 50.5% were among one to three year-old. A total of 56.8% HPIV3-infected children were among one to three years old, however, no HPIV2-infected children was younger than one year-old. The HPIV1-infected patients were more common to develop wheezing and diagnose as acute bronchiolitis. HPIV2-infected children were more likely to have hoarseness (23.9%), and were associated with croup (25.4%). HPIV3 was isolated from two fatal cases, with neurological underlying diseases., Conclusion: The impact caused by HPIVs infections is significant in hospitalized children. In the current study, our results contribute to the epidemiologic, clinical and laboratory information of HPIV infection in children in the important areas of respiratory tract infection that could support the development of optimization management., (Copyright © 2017. Published by Elsevier B.V.)

Background: Accurate, rapid, and early diagnosis of dengue virus (DENV) infections is essential for optimal clinical care. Here, we evaluated the efficacy of the quantitative real-time PCR (qRT-PCR)-LightMix dengue virus EC kit for DENV detection using samples from a dengue outbreak in Taiwan in 2015., Methods: Sera from patients with suspected DENV infection were analyzed and compared using the LightMix kit, a Dengue NS1 Ag + Ab Combo kit for detection of NS1 antigen and DENV-specific IgM and IgG antibodies, and an "in-house" qualitative DENV-specific RT-PCR assay., Results: A total of 8,989, 8,954, and 1581 samples were subjected to NS1 antigen detection, IgM and IgG detection, and LightMix assays, respectively. The LightMix assay yielded a linear curve for viral loads (VL) between 102 and 106 copies/reaction, and the minimum detection limits for DENV serotype 1 (DENV1) and DENV2, DENV3, and DENV4 were 1, 10, and 100 focus forming units (FFU)/mL, respectively. There was 88.9% concordance between the results obtained using the NS1 antigen combo kit and by LightMix analysis, and the diagnostic sensitivity and specificity of the two methods were 89.4 and 100%, and 84.7 and 100%, respectively. Notably, fatal cases were attributed to DENV2 infection, and 79.5% (27/34) of these cases occurred in patients ≥ 71 years of age. Among these older patients, 82.3% (14/17) were NS1/IgM/IgG (+/-/-), exhibiting VLs between 106-109 copies/mL, which was markedly higher than the rate observed in the other age groups., Conclusions: The LightMix assay was effective for early diagnosis of DENV infection. Our data indicate that high VLs during primary infection in elderly patients may be a positive predictor for severe illness, and may contribute to high mortality rates., Competing Interests: The authors have declared that no competing interests exist.

Wild poliovirus (WPV) persists in diverse locales worldwide, spreading outward from endemic areas. In response to the international threat of WPV transmission and changes in the national vaccination policy, we established an environmental surveillance system to monitor the circulation of wild and vaccine-related poliovirus in Taiwan. From July 2012 to December 2013, we collected sewage specimens every month from 10 sewage treatment plants located throughout Taiwan. The specimens were concentrated by the two-phase separation method and then inoculated into L20B, RD, and A549 cells for virus isolation. Viral isolates were identified and serotyped by immunofluorescence assay or molecular analysis. A total of 300 sewage samples were collected, and the results showed 163 samples (54.3%) were positive for virus, and 268 isolates were identified. Among these, 75 samples (25%) were positive for enterovirus (EV), but no poliovirus was found. In addition, 92 isolates were identified as enteroviruses and the most common serotypes were coxsackievirus B4, coxsackievirus B3, and coxsackievirus B2. Interestingly, 102 (34%) and 82 (27.3%) specimens were positive for mammalian orthoreovirus (MRV) and adenovirus, respectively. This study confirmed that sewage surveillance can be a useful additional modality for monitoring the possible presence of wild-type or vaccine-derived poliovirus in wastewater, and can indicate the current types of viruses circulating in the population. Furthermore, since MRV was found in children with acute necrotizing encephalopathy and meningitis, the high incidence of MRV detected by environmental surveillance warrants further investigation.

Viral etiologies of respiratory tract infections (RTIs) have been less studied in adult than in pediatric populations. Furthermore, the ability of PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) to detect enteroviruses and rhinoviruses in respiratory samples has not been well evaluated. We sought to use PCR/ESI-MS to comprehensively investigate the viral epidemiology of adult RTIs, including testing for rhinoviruses and enteroviruses. Nasopharyngeal or throat swabs from 267 adults with acute RTIs (212 upper RTIs and 55 lower RTIs) who visited a local clinic or the outpatient or emergency departments of a medical center in Taiwan between October 2012 and June 2013 were tested for respiratory viruses by both virus isolation and PCR/ESI-MS. Throat swabs from 15 patients with bacterial infections and 27 individuals without active infections were included as control samples. Respiratory viruses were found in 23.6%, 47.2%, and 47.9% of the 267 cases by virus isolation, PCR/ESI-MS, and both methods, respectively. When both methods were used, the influenza A virus (24.3%) and rhinoviruses (9.4%) were the most frequently identified viruses, whereas human coronaviruses, human metapneumovirus (hMPV), enteroviruses, adenoviruses, respiratory syncytial virus, and parainfluenza viruses were identified in small proportions of cases (<5% of cases for each type of virus). Coinfection was observed in 4.1% of cases. In the control group, only 1 (2.4%) sample tested positive for a respiratory virus by PCR/ESI-MS. Patients who were undergoing steroid treatment, had an active malignancy, or suffered from chronic obstructive pulmonary disease (COPD) were at risk for rhinovirus, hMPV, or parainfluenza infections, respectively. Overall, immunocompromised patients, patients with COPD, and patients receiving dialysis were at risk for noninfluenza respiratory virus infection. Rhinoviruses (12.7%), influenza A virus (10.9%), and parainfluenza viruses (7.3%) were the most common viruses involved in the 55 cases of lower RTIs. The factors of parainfluenza infection, old age, and immunosuppression were independently associated with lower RTIs. In conclusion, PCR/ESI-MS improved the diagnostic yield for viral RTIs. Non-influenza respiratory virus infections were associated with patients with comorbidities and with lower RTIs. Additional studies that delineate the clinical need for including non-influenza respiratory viruses in the diagnostic work-up in these populations are warranted.

Background: Respiratory infections caused by adenovirus (HAdV) are common year round. Recently, a significant increase of adenoviral infections was observed in Taiwan., Objective: To understand the prevalence and molecular epidemiology of respiratory adenovirus circulating in Taiwan for the past decade., Study Design: One hundred and twenty-six human adenoviruses, isolated between 2002 to 2011, were characterized via DNA sequencing of the hexon and fiber genes. The nucleotide sequences were then compared by phylogenetic analysis., Results: HAdV-B3 accounted for 64.3% (81/126) and peaked almost every year, whereas the sequences of hexon and fiber genes of HAdV-B3 were highly conserved in different years. A high incidence of co-infection of adenoviruses was observed (19.0%, 24/126); HAdV-B3 co-infected with HAdV-C2 was the most common combination (58.3%, 14/24). An additional interesting finding of repeated infection was noted in 10 children, all of whom showed first infection with adenovirus species HAdV-C, followed by species HAdV-B or HAdV-E., Conclusions: HAdV-B3 was the predominant type of respiratory adenovirus circulating in Taiwan over the past ten years. This merits further attention for vaccine development. Furthermore, the observed high-incidence of adenoviral co-infections along with repeated infections found in our study provides important epidemiological insights into adenovirus infections.

Background: Hemodialysis patients have higher risk of mortality and morbidity when infected with 2009 pandemic H1N1 (pH1N1/09) virus. Depending on different methodologies and criteria, previous studies reported variable response rates to adjuvanted vaccines against pH1N1/09 virus in hemodialysis patients, however, the efficacy of non-adjuvanted vaccines, which are currently used in many countries such as the USA and Asian areas, has not been comprehensively evaluated in hemodialysis population before., Methods: We evaluated the efficacy of a standard single 15 μg-dose of non-adjuvanted monovalent pH1N1/09 vaccine (AdimFlu-S) in vaccine-naïve 110 hemodialysis and 173 healthy participants. When enrolling, all participants had not any clinical symptom or sign suggesting pH1N1/09 infection since the index case was identified in Taiwan. Sera from all participants were tested by hemagglutination inhibition (HI) and micro-neutralization-ELISA (microNT-ELISA) tests before and 21 days after vaccination. The outcome parameters were seroconversion rate (≥ 4-fold in HI titer with titer ≥ 1:40), seroprotection rate (HI titers ≥ 1:40), seroresponse rate (≥ 4-fold increase in HI or microNT-ELISA titer), fold of increase in geometric mean (GM) titers, and adverse effects., Results: In method A analyses, we included all participants' data in final analyses, and the seroconversion rates and the fold increase of GM titer after vaccination were 25.4% and 1.8 in adult (18-60-year olds) hemodialysis subgroup, and 23.4% and 1.8 in elder (>60-year olds) hemodialysis subgroup based on HI titers, which were all significantly lower than those of the corresponding healthy control subgroups. Similar trends were observed based on microNT-ELISA titers, further validating the results. Multivariable analysis revealed hemoglobin and cholesterol levels were significant predictors for seroresponse in hemodialysis patients, suggesting the possible impacts of nutrition status and anemia. In method B analyses, we excluded participants with pre-vaccination seroprotection (based on HI or microNT-ELISA criteria) in final analyses. The response rates in various subgroups from method B analyses were also similar as those from method A analyses. No severe adverse effect was noted., Conclusions: According to the European and U.S. criteria, a single 15 μg-dose of non-adjuvanted pH1N1/09 vaccination is safe but ineffective in both adult and elder hemodialysis patients. Further studies using multiple doses or higher antigen amount are warrant to define the most appropriate regimen., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Enterovirus 71 (EV71) infection commonly strike children under the age of 3 years, with an occasionally unfavorable outcome in children. This study was designed to explore the relationship between age and the severity of complications, which may associate with antibody-dependent enhancement (ADE) in EV71. All EV71-infected patients during the outbreak of 2008 were recruited. In total, 134 patients were enrolled and categorized into two age groups, 0-12 months (n = 18) and >12 months (n = 116). Pulmonary edema/hemorrhage more commonly occur in patients younger than 12 months. No difference in the occurrence of herpangina/hand-foot-and-mouth disease (HFMD), uncomplicated brainstem encephalitis (BE), or autonomic nervous system (ANS) dysregulation was noted between the two age groups. Patients with pulmonary edema/hemorrhage (11.9 ± 14.7 months) were younger than patients with herpangina/HFMD (35.8 ± 26.4 months) or ANS dysregulation (33.9 ± 20.9 months). Our findings are in agreement with the data regarding the outbreak in Taiwan, in which a decrease in age corresponded to an increase in disease severity with regard to central nervous system complications. A reduction of maternal antibodies to the subneutralizing level within 1 year of age may be associated with the ADE of the infection. This study could provide possible clinical significance with regard to ADE phenomena in young infants infected by EV71.

The carbohydrate binding specificities are different among avian and human influenza A viruses and may affect the tissue tropism and transmission of these viruses. The glycan binding biology for influenza B, however, has not been systematically characterized. Glycan binding specificities of influenza B viral isolates were analyzed and correlated to hemagglutinin (HA) genotypes and clinical manifestations. A newly developed solution glycan array was applied to characterize the receptor binding specificities of influenza B virus clinical isolates from 2001 to 2007 in Taiwan. Thirty oligosaccharides which include α-2,3 and α-2,6 linkage glycans were subjected to analysis. The glycan binding patterns of 53 influenza B isolates could be categorized into three groups and were well correlated to their HA genotypes. The Yamagata-like strains predominantly bound to α-2,6-linkage glycan (24:29, 83%) while Victoria-like strains preferentially bound to both α-2,3- and α-2,6-linkage glycans (13:24, 54%). A third group of viruses bound to sulfated glycans and these all belonged to Victoria-like strains. Based on the HA sequences, Asn-163, Glu-198, Ala-202, and Lys-203 were conserved among Victoria-like strains which may influence their carbohydrate recognition. The viruses bound to dual type glycans were more likely to be associated with the development of bronchopneumonia and gastrointestinal illness than those bound only to α-2,6 sialyl glycans (P < 0.05). Glycan binding analyses provide additional information to monitor the antigenic shift, tissue tropism, and transmission capability of influenza B viruses, and will contribute to virus surveillance and vaccine strain selection., (Copyright © 2012 Wiley Periodicals, Inc.)

In 2006, an outbreak of aseptic meningitis was noted in Taiwan. From January to October 2006, a total of 3283 specimens collected from patients with viral infection, including 173 cerebrospinal fluid (CSF) samples, were examined for virus isolation and identification. Overall, 339 enterovirus (EV)-positive cases were identified by virus culture: echovirus 18 (E18) formed the majority (27.4 %, 93 cases), followed by coxsackievirus B2 (13.8 %, 47 cases) and coxsackievirus A2 (10.8 %, 37 cases). The manifestations of the 93 E18 cases were aseptic meningitis (44.1 %), viral exanthema (23.6 %), acute tonsillitis (15.1 %), acute pharyngitis (14.0 %), acute gastritis (11.8 %), herpangina (7.5 %) and bronchopneumonia (5.3 %). Of 107 E18 isolates identified, 100, 62.5 and 19 % were obtained following culture in RD, MRC-5 and A549 cells, respectively. E18 was identified most frequently from throat swabs (67.2 %) and less frequently from stool samples (15.9 %) and CSF (16.8 %). The detection rate of E18 was 78.2 % from CSF, 50 % from stool samples and 22.9 % from throat swabs. Phylogenetic relationships among the E18 strains were examined. Analysis of the partial VP1 gene showed 3.7-23.8 % variation in sequence compared with sequences from GenBank and, notably, the amino acid change V152S was detected in a protruding loop within the VP1 protein. These results indicate that a genetic variant of E18 was circulating and caused an outbreak of aseptic meningitis in Taiwan in 2006.

To investigate the molecular epidemiology of Taiwanese Echovirus 30 (E-30) strains, we analyzed the 876 bp sequence of the VP1 gene from 32 Taiwanese strains isolated in 1988-2008, 498 reference sequences, and one Echovirus 21 strain as the out-group. Phylogenetic analysis detected six E-30 genotypes (designated GI-GVI) that had circulated globally during the past five decades. The genotypes varied widely in geographic distribution and circulation half-life. The GI, GII, and GV were ancient genotypes in which the first strains emerged in the 1950s. The GIII was a reemerging genotype, in which strains had first appeared in Colombia in 1995 before reemerging in the New Independent States (NIS) in 2003. The GIV, an emerging genotype that recently appeared in Asia in 2003, was closely related to the ancient genotypes. The GVI was the circulating genotype, which included eight clusters (A-H) that had circulated since 1967. No GVI-A, C, D, or E strains have been identified during the past 10 years. The GVI-B first appeared in China in 1984 and later in Russia and Asia in the 2000s. The GVI-F, G, and H strains, which comprised the prevalent clusters, had been dominant in Asia Pacific area, globally, and Europe, respectively. Taiwanese strains were classified into GVI-D (1988-1989), GVI-F (1993-2004), and GVI-G (1993-2008). The quiescence period of E-30 is longer in Taiwan (5-8 years) than in other countries (3-5 years).

Eighty cases of echovirus 18 infection among young children during an outbreak in 2006 in Taiwan were enrolled. Twenty percent of the patients had a comorbid condition. Twenty-five cases (31%) were complicated by aseptic meningitis. The most frequent diagnoses in children without meningitis were pharyngitis/tonsillitis (35%) and vesicular viral exanthem (33%). The case-fatality rate among the children with meningitis was 4%. Echovirus 18 was isolated from the cerebrospinal fluid of 68% of the children.

We studied preexisting immunity to pandemic (H1N1) 2009 virus in persons in Taiwan. A total of 18 (36%) of 50 elderly adults in Taiwan born before 1935 had protective antibodies against currently circulating pandemic (H1N1) 2009 virus. Seasonal influenza vaccines induced antibodies that did not protect against pandemic (H1N1) 2009 virus.

A novel pandemic influenza H1N1 (pH1N1) virus spread rapidly across the world in 2009. Due to the important role of antibody-mediated immunity in protection against influenza infection, we used an enzyme-linked immunosorbent assay-based microneutralization test to investigate cross-reactive neutralizing antibodies against the 2009 pH1N1 virus in 229 stored sera from donors born between 1917 and 2008 in Taiwan. The peak of cumulative geometric mean titers occurred in donors more than 90 years old and declined sharply with decreasing age. Sixteen of 27 subjects (59%) more than 80 years old had cross-reactive antibody titers of 160 or more against the 2009 pH1N1 virus, whereas none of the donors from age 9 to 49 had an antibody titer of 160 or more. Interestingly, 2 of 51 children (4%) from 6 months to 9 years old had an antibody titer of 40. We further tested the antibody responses in 9 of the 51 pediatric sera to three endemic seasonal influenza viruses isolated in 2006 and 2008 in Taiwan, and the results showed that only the 2 sera from children with antibody responses to the 2009 pH1N1 virus had high titers of neutralizing antibody against recent seasonal influenza virus strains. Our study shows the presence of some level of cross-reactive antibody in Taiwanese persons 50 years old or older, and the elderly subjects who may already have been exposed to the 1918 virus had high titers of neutralizing antibody to the 2009 pH1N1 virus. Our data also indicate that natural infection with the Taiwan 2006 and 2008 seasonal H1N1 viruses may induce a cross-reactive antibody response to the 2009 pH1N1 virus.

BACKGROUND. Our objective was to determine the serological signals that indicated the possible dominant circulating influenza virus subtypes for the coming influenza seasons. METHODS. Healthy children 6 months through 5 years of age, adults 18-60 years of age, and elderly adults >60 years of age were recruited to receive seasonal trivalent inactivated influenza vaccinations from October through December during the 2006-2007 and 2008-2009 seasons. Paired serum samples were collected at baseline and at 3 weeks after vaccination. Using a hemagglutination inhibition (HAI) assay, we measured antibody responses to local influenza strains circulating early in October, before each winter influenza season. RESULTS. A total of 301 subjects were tested for antibody to local strains (80, 120, and 101 subjects in the 2006-2007, 2007-2008, and 2008-2009 seasons, respectively). The dominant winter influenza strains in Taiwan were B/Malaysia/2506/2004-like in the 2006-2007 season, A/Brisbane/59/2007-like virus (H1N1) in the 2007-2008 season, and A/Brisbane/59/2007-like virus (H1N1) in the 2008-2009 season. The group with the lowest number of subjects with an HAI titer of 40 at baseline was children with antibody against the B/Taiwan/0050/2006 in the 2006-2007 season, A/Taiwan/785/2006 (H1N1) in 2007-2008 season, and A/Taiwan/951/2007 (H1N1) in 2008-2009 season. The emergence of these viruses correlated well with the circulating influenza subtype in the following winter peak seasons. CONCLUSIONS. Low seroprotection rate among children against a specific locally circulating influenza strain might predict the dominantly circulating subtype of influenza virus in the coming winter season. A year-end preseasonal serological survey of children could provide valuable information about the possible circulating strain and tailor the disease-control strategy accordingly.

In recent years, enterovirus 71 (EV71) has been a cause of numerous outbreaks of hand-foot-and-mouth disease, with severe neurological complications in the Asia-Pacific region. The reemergence in Taiwan of EV71 genotype B5 in 2008 resulted in the largest outbreak of EV71 in Taiwan in the past 11 years. Phylogenetic analyses indicated that dominant genotype changes from B to C or C to B occurred at least three times between 1986 and 2008. Furthermore, antigenic cartography of EV71 by using neutralization tests revealed that the reemerging EV71 genotype B5 strains formed a separate cluster which was antigenically distinct from the B4 and C genotypes. Moreover, analyses of full-length genomic sequences of EV71 circulating in Taiwan during this period showed the occurrence of intra- and interserotypic recombination. Therefore, continuous surveillance of EV71 including the monitoring of genetic evolution and antigenic changes is recommended and may contribute to the development of a vaccine for EV71.

Background: In Taiwan, trivalent oral poliovirus vaccine (tOPV) is in the routine immunization schedule, but its association with illnesses had not been examined., Objectives: To investigate clinical presentations and viral characteristics of patients with poliovirus isolates., Study Design: Clinical data, vaccination records and viral sequences were retrospectively analyzed for patients from whom polioviruses were isolated during 2000-2005., Results: OPV-like strains were the only pathogen identified in 208 children who were diagnosed with lower respiratory tract infection (24.5%), acute gastroenteritis (16.8%) or upper respiratory tract infection (10.6%). Timing of poliovirus isolation relative to the tOPV vaccination was unusual in 59 patients, including 6 before any dose and 53 more than 10 weeks after the 3rd or later dose of tOPV. Sequence analyses of the VP1, 2C and 3C/D regions for 19 poliovirus isolates revealed that 4 had previously reported neurovirulence reversions, 1 had intertypic recombination, and 6 had both. No patient had neurological complications, but 3 died of myocarditis, including 2 with recombinant strains and 1 who never received OPV., Conclusion: This study describes the isolation of OPV-like strains from patients with a variety of illnesses, raising concerns about their pathogenic potential in an area where tOPV is routinely administered. The detection of genetic variations among OPV-like strains warrants continuing surveillance for these variants in patients with severe illnesses besides neurological complications.

Background and Purpose: An outbreak of influenza A and influenza B appeared at the beginning and end of 2006 in southern Taiwan. We conducted this study to test whether laboratory findings could differentiate influenza A from influenza B infection., Methods: All children aged 16 years or less, who had nasal and/or throat swabs sent from inpatient or outpatient settings at National Cheng Kung University Hospital for the diagnosis of influenza infection from January 2005 to February 2007, were considered eligible subjects. Retrospective chart review of clinical and laboratory data was performed., Results: 274 patients were enrolled, 151 with influenza A and 123 with influenza B, of whom 127 (46.4%) received laboratory examinations. The peak month of influenza A and influenza B infections was January 2006 and January 2007, respectively. Children with influenza B infections were older than those with influenza A infections (p<0.001). Influenza B-infected patients were more likely to have myalgia (p=0.004) than those with influenza A infections. Furthermore, children with influenza B infections tended to have lower leukocyte counts (6383 +/- 3970/mm3 vs 7639 +/- 3476/mm3, p=0.004), and higher serum creatine kinase level (p=0.002) than those with influenza A infections. The clinical outcomes were usually favorable., Conclusions: The clinical features of influenza B and influenza A infections are similar. However, decreased leukocytes and increased serum creatine kinase can be used as adjunctive criteria for diagnosis of influenza B infection before viral culture results are available.

Background: This study was designed to determine the clinical characteristics of children infected with different strains of influenza B viruses isolated in southern Taiwan. The clinical features were compared with influenza A infection occurring in the same period., Methods: All children enrolled in the study had laboratory-confirmed infection with influenza A or B viruses. Influenza B speciation was performed by RNA extraction, cDNA synthesis, and amplification by polymerase chain reaction and sequencing. Demographic data, clinical findings, diagnoses, and outcomes were obtained., Results: During the study period, 163 strains of influenza A and 118 strains of influenza B were isolated. The Yamagata-like strains were most prevalent in 2001. New reassortant strains were identified since 2002 and became predominant in 2005 and 2006. Children with influenza B were more likely than those with influenza A to be diagnosed as upper respiratory tract infection, myositis, and gastroenteritis (P < 0.05). Children infected with Yamagata-like strains were more likely to develop lower respiratory tract infection (P < 0.05) and accounted for all cases of invasive disease. Children infected with the Victoria-like group had the longest hospital stays associated with severe bacterial superinfection., Conclusions: Currently new reassortant influenza B viruses are the predominant strains circulating in southern Taiwan. There is considerable similarity of clinical features between influenza A and B in children. The Yamagata-like strains were associated with more invasive infections. Continuous influenza virus surveillance is essential particularly in Taiwan where pandemic strains tend to appear earlier than in other countries.

Genetic recombination is a well-known phenomenon for enteroviruses. In this study, we determined the phylogenetic relationships of five distinct regions of the EV71 genome for 73 EV71 isolates from 1986 and from 1998 to 2005 in Taiwan. Phylogenetic analyses showed that the 5'-UTR, VP4-VP2, VP1, and 3D regions of EV71 isolated in 2004 and 2005 were grouped into genotype C. However, the 2B region of these isolates differed in that it grouped with genotype B, indicating recombination within EV71 had occurred. This intratypic recombination was first seen in 2002 and became predominant in 2004 and 2005. The simplot and bootscan analyses identified two recombination points located at the 3'-termini of the 2A and 3C regions. This intratypic recombination was identified among naturally circulating EV71 isolates in Taiwan, therefore, it suggests that nonstructural genes may recombine to produce new EV71 variants.

An outbreak of human metapneumovirus (hMPV) among children in southern Taiwan in 2004 prompted the investigation of the molecular epidemiology of hMPV from September 2003 to August 2005. Respiratory specimens that were culture negative for a panel of respiratory viruses were examined for the presence of hMPV by RT-PCR. The results indicated that 59 out of 546 (10.8%) children were hMPV-positive. The majority of these hMPV-positive children were less than 2 years old (59.4%), females (61%), and inpatients (67.8%). Infections occurred throughout the year, but peaked during the spring and/or summer months. Sequence analysis of the fusion gene from the isolates revealed two phylogenetic groups with five possible lineages (A1, A2a/A2b, B1, and B2). Among these co-circulating strains, A2 strains were most frequently observed and demonstrated the greatest divergence. Deduced amino acid sequence analysis identified several variant amino acids specific to the A2 lineage. Lineage-specific amino acid substitutions were noted at aa233, aa286, aa312, aa348, and aa296. This study indicated that genetically divergent strains of hMPV which caused respiratory disease and hospitalization were circulating among children in Taiwan.

Taiwan is not considered an endemic area of leishmaniasis. Imported cases are encountered infrequently, and only two cases of indigenous cutaneous leishmaniasis have been reported.(1) We found one new case in the past 20 years. The patient presented with erythematous plaques on the nasal bridge and right thumb. Skin biopsy specimens from both sites revealed numerous Leishman-Donovan bodies in macrophages. There was no history of travel outside the country, and the diagnosis of indigenous cutaneous leishmaniasis was made. Polymerase chain reactions (PCR) identified the species as Leishmania tropica. The route of infection in this patient is unclear. Because pentavalent antimony, the drug of choice for leishmaniasis, is not available in Taiwan, the patient was treated with levamisole and potassium iodide, with an excellent response.

Enterovirus (EV) infections are common. There are more than 60 known serotypes, and each has different epidemiologic or medical importance. Over 700 physicians from 75% of basic administrative units of Taiwan participated in the "Sentinel Physician Surveillance of Infectious Disease" and reported weekly to the Center for Disease Control-Taiwan with data on various infections. Data of laboratory-confirmed EV infections from this surveillance between 2000 and 2005 was analyzed. EV serotypes were determined by immunofluorescence staining and/or viral VP1 sequence analysis. A total of 12,236 EV cases, or approximately 1,300-2,500 per year, were identified, and 52% of the cases occurred between April and July. The median age was 3 years, and 57.6% of patients were male. Coxsackievirus A (CA) 16 and EV71, which primarily manifest as hand-foot-and-mouth disease, were the most prevalent serotypes every year except 2004. Other prevalent serotypes and associated symptoms varied from year to year. Echovirus (E) 30 and E6, which are associated with aseptic meningitis, were prevalent in 2001 and 2002, CA4 and CA10, which cause herpangina, were predominant in 2004, and coxsackievirus B (CB) 4 and CB3, which are associated with neonatal febrile disease, were most common in 2004 and 2005, respectively. Some of these epidemics overlapped with outbreaks of the same serotypes in other Asian Pacific countries. Of all serotypes, EV71 was associated with the highest number of severe complications in patients. Surveying the epidemic pattern, disease spectra, and severity associated with each EV serotype provided important information for public health and medical personnel., ((c) 2007 Wiley-Liss, Inc.)

Taiwan has experienced several outbreaks of enterovirus 71 (EV71) infections since 1998. This study examined the quantitative relationship between specific cytokines in the cerebrospinal fluid (CSF) and the severity of EV71 brain stem encephalitis (BE), and investigated whether the CSF cytokine response differed from that to uncomplicated echovirus meningitis (EM). The study included 57 children with EV71 BE, of whom 24 had isolated BE, 24 had autonomic nervous system (ANS) dysregulation, and nine had pulmonary oedema (PE), and 15 children with EM. All were confirmed by virus culture. Mean CSF glucose, total protein and lactate levels were increased significantly in association with the severity of EV71 BE. The mean CSF concentration of interleukin (IL)-1beta in children with EV71 PE was significantly higher than in those with isolated BE. IL-6 and interferon (IFN)-gamma levels were significantly higher for EV71 PE and ANS dysregulation than for isolated BE. In contrast, EM was associated with high levels of IL-1beta and low levels of IFN-gamma. Cytokines in the central nervous system, as well as in blood, appear to be involved in the pathogenesis of EV71 BE.

This study compared the clinical, laboratory and radiological features of infections caused by human metapneumovirus (hMPV) with other respiratory viruses. Nasopharyngeal aspirates and throat swabs were obtained from children during a 9-week period. hMPV was the virus isolated most frequently, followed by adenovirus, influenza virus A, respiratory syncytial virus and influenza virus B. hMPV-infected children were younger, and were more likely to be female, to present with feeding difficulties, a rash, tachycardia and a longer duration of fever, and to cough less frequently. Increasing interstitial infiltrates and hyperinflation were the most common radiological findings. None of the children required mechanical ventilation.

Genetic and antigenic analyses of influenza B virus field strains isolated in Taiwan from 1998 to 2005 were performed. To investigate the molecular evolution of influenza B viruses, sequence analysis of the hemagglutinin (HA1 subunit) and neuraminidase genes was performed. All influenza B viruses isolated between 1998 and 2000 belonged to the B/Yamagata/16/88 lineage. The B/Victoria/2/87 lineage, which was cocirculating with the Yamagata lineage, was identified in Taiwan in March 2001. Concurrently, there was an increasing prevalence of this lineage in many parts of the world, including North America and Europe, during the 2001-2002 season. Since 2002, genetic reassortants of influenza B virus with the Victoria lineage of hemagglutinin and the Yamagata lineage of neuraminidase have been found at a rate of 46%. Therefore, in 2002, at least three sublineages of influenza B virus strains, the B/Shanghai/361/2002-like strain (Yamagata lineage), the B/Hong Kong/330/01-like strain (Victoria lineage), and the B/Hong Kong/1351/02-like strain (B reassortant lineage), were identified in Taiwan. The results showed that genetically distinct lineages can cocirculate in the population and that the reassortment among these strains plays a role in generating the genetic diversity of influenza B viruses. Interestingly, from January to April 2005, B reassortant viruses became dominant (73%) in Taiwan, which indicated that a mismatch had occurred between the influenza B vaccine strain recommended for the 2004-2005 season in the Northern hemisphere by the World Health Organization and the epidemic strain.

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., (Copyright 2005 Wiley-Liss, Inc.)

A laboratory-based surveillance network of 11 clinical virological laboratories for influenza viruses was established in Taiwan under the coordination of the Center for Disease Control and Prevention (CDC), Taiwan. From October 2000 to March 2004, 3,244 influenza viruses were isolated, including 1,969 influenza A and 1,275 influenza B viruses. The influenza infections usually occurred frequently in winter in the northern hemisphere. However, the influenza seasonality in Taiwan was not clear during the four seasons under investigation. For example, the influenza A viruses peaked during the winters of 2001, 2002, and 2003. However, some isolated peaks were also found in the summer and fall (June to November) of 2001 and 2002. An unusual peak of influenza B also occurred in the summer of 2002 (June to August). Phylogenetic analysis shows that influenza A isolates from the same year were often grouped together. However, influenza B isolates from the year 2002 clustered into different groups, and the data indicate that both B/Victoria/2/87-like and B/Yamagata/16/88-like lineages of influenza B viruses were cocirculating. Sequence comparison of epidemic strains versus vaccine strains shows that many vaccine-like Taiwanese strains were circulating at least 2 years before the vaccine strains were introduced. No clear seasonality of influenza reports in Taiwan occurred in contrast to other more continental regions.

A large outbreak of aseptic meningitis occurred from April to November 2001 in Taiwan. Of the 1,130 enterovirus-infected patients, echovirus 30 (E30) infection was diagnosed in 188 (16.6%), with the patients having various clinical manifestations including aseptic meningitis (73.9%), young infant fever (6.9%), respiratory symptoms or herpangina (13.3%), or others (5.9%). The majority of the E30-infected patients were between 3 and 10 years old. Of the 264 E30 strains identified, 94.3, 71, and 67.4% were isolated from RD, MRC-5, and A549 cells, respectively. Primary isolation of E30 required mean times of 3.7 days for RD cells and 4.1 days for MRC-5 and A549 cells. Among all E30-positive patients, virus was most frequently isolated from throat swab specimens (85.2%) and, to a lesser extent, stool (76.4%) or cerebrospinal fluid (70.1%) specimens. The virus isolates were initially identified as echovirus 4 (E4) on the basis of immunofluorescence staining with anti-E4 and anti-E30 (Bastianni prototype) monoclonal antibodies. However, upon performance of the neutralization test, E30-specific reverse transcription-PCR, and sequencing of the VP1 gene, the results identified these isolates as E30, not E4, indicating that the reagent used to type E30, which is produced with the Bastianni strain as the immunogen, is inadequate for the identification of recent E30 isolates in Taiwan. Phylogenetic analyses of the VP1 genes of these isolates showed that their sequences differed from those of E30 isolates from the GenBank database by 9.1 to 25.2%, suggesting that this outbreak was caused by a new variant strain of E30 introduced into Taiwan in 2000 that resulted in the widespread aseptic meningitis epidemic in 2001.

Two outbreaks of hand-foot-and-mouth disease (HFMD) occurred in Taiwan between 1998 and 2000. Enteroviruses were isolated from a total of 1,892 patients in this laboratory during this period. Of the virus isolates, enterovirus 71 (EV71) was diagnosed in 44.4% of the patients (132 of 297) in 1998, 2% (13 of 646) in 1999, and 20.5% (195 of 949) in 2000. Genetic analyses of the 5'-untranslated and VP1 regions of EV71 isolates by reverse transcription-PCR and sequencing were performed to understand the diversity of EV71 in these outbreaks of HFMD. Most EV71 isolates from the 1998 epidemic belonged to genotype C, while only one-tenth of the isolates were genotype B. Interestingly, all EV71 isolates tested from 1999 to 2000 belonged to genotype B. This study indicated that two genogroups of EV71 capable of inducing severe clinical illness have been circulating in Taiwan. Furthermore, the predominant EV71 genotypes responsible for each of the two major HFMD outbreaks within the 3-year period in Taiwan were different.

Hand, foot, and mouth disease (HFMD) and encephalomyelitis were two major clinical manifestations during the 1998 enterovirus 71 (EV71) epidemic in Taiwan. To investigate whether different clinical patterns were caused by alterations in EV71 genomes, the complete nucleotide sequences of four EV71 strains associated with HFMD or encephalomyelitis were compared. Among these viral strains, two or three nucleotide differences were found within the 5'-noncoding region, and two or four amino acid differences were found within the regions encoding viral polyproteins; however, none of these differences were correlated with either clinical manifestation. Because coxsackievirus A16 was another major causative agent of HFMD, a reverse transcription-polymerase chain reaction assay, with high sensitivity and specificity for identification of EV71 (both 100%) and coxsackievirus A16 (100 and 98.8%), was developed for the rapid differential identification of these two viruses in HFMD patients., (Copyright 2001 Wiley-Liss, Inc.)

We have studied seasonal and diurnal fluctuations of water temperature in bamboo stumps and the effect of temperature on the energy metabolism of arboreal, oophagous tadpoles of Chirixalus eiffingeri. We collected tadpoles (Gosner stage 28-29) in February and August from Chitou, Taiwan and acclimated them to 12 and 22 degrees C. Using a closed system, we measured tadpole oxygen consumption (V.O(2)) at 12, 17 and 22 degrees C. The water temperature was lowest in February (11-13 degrees C), increased rapidly during March and April and was highest from May to August (20-24 degrees C). Diel fluctuations in the temperature of the pools of water in bamboo stumps mirrored fluctuations in air temperature. Tadpoles collected in February and August exhibited metabolic compensation in that tadpoles acclimated at 12 degrees C had significantly higher V.O(2) than those acclimated at 22 degrees C. There are at least two possible explanations for the presence of metabolic compensation in C. eiffingeri tadpoles. Firstly, the larval period of C. eiffingeri ranges from 40 to 78 days, a tadpole could experience relatively large fluctuations in body temperature (up to 10 degrees C) during the development. As a result, C. eiffingeri tadpoles most likely evolved metabolic compensation to maintain activity levels under different thermal environments. Secondly, because arboreal pools are small, thermally unstratified, aquatic microhabitats, tadpoles are unable to behaviorally select preferred temperatures. As a result, metabolic compensation allows tadpoles to regulate their physiological functions.

Background, Aims: Actinobacillus actinomycetemcomitans (Aa) has been implicated as the putative micro-organism for localized juvenile periodontitis (LJP). The most distinct clinical features of LJP include severe angular bony defects of the mesial sides of permanent first molars and the onset of disease during puberty. Currently, no large-scale studies have been performed which address the change in detection rates of Aa on the mesial sides of permanent 1st molars following eruption and up to puberty., Method: In this study, subgingival plaque samples were taken from the mesial pockets of 2 randomly selected permanent 1st molars from 328 primary school children and 50 adult staff, and analyzed by polymerase chain reaction (PCR) to detect Aa., Results: The results showed a 5.5% prevalence rate of Aa which increased after the eruption of 1st molars and peaked near puberty. There were no significant differences in the detection rates of Aa among different groups in terms of gender, plaque index (PII), and gingival index (GI); however, the higher detection rates of Aa were significantly associated with increased probing depths at p<0.05., Conclusion: PCR analysis of the subgingival plaques demonstrated a prevalence of Aa which peaked near puberty, suggesting that Aa may be important for LJP in Taiwan.

Taiwan suffered a severe and widespread outbreak of enterovirus infection in 1998. More than 400 children were hospitalized, with seventy-eight fatalities due to central nerve system (CNS) involvement and cardiopulmonary collapse. Enterovirus 71 (EV71) was incriminated as the causative agent for the fatal cases. To understand the viral molecular epidemiology in this outbreak, fragments of 207-bp length of the VP4 region in 23 Taiwanese EV 71 isolates were sequenced. Pair-wise comparison revealed a 17.5-24.4% difference between the isolates and the prototype BrCr. However, all the changes in the VP4 region of the isolated strains were synonymous substitutions. Phylogenetic analysis was performed on these 23 isolates and 21 others deposited in GenBank. In this study, forty-four EV71 isolates from the world were separated into three distinct genotypes: A, B and C. The EV71 prototype strain, BrCr/70, is the only strain of genotype A. Group B included strains from the United States, Japan and Taiwan. Most strains in genotype B were isolated prior to 1990. Group C consisted of strains from Japan and Taiwan. Most strains of genotype C were isolated after 1990, they were further divided into 3 clusters: i.e. C-1, C-2 and C-3. In Taiwan, two genotypes, B and C-3, were co-circulating during the outbreak in 1998, although a minor group of genotype B may have appeared in Taiwan before 1986. The majority of the isolates clustered in genotype C-3. Genotype C showed a higher evolutionary rate than genotype B (3.9 x 10(-3) vs. 1.4 x 10(-3)) in the VP4 region. There seems to be a worldwide trend with strains of genotype B appearing earlier than strains of genotype C which took over later in the dominance.

The present study examined the association of specific virus infections with acute respiratory tract conditions among hospitalized and outpatient children in a subtropical country. A total of 2,295 virus infections were detected in 6,986 patients between 1997 and 1999, including infections caused by respiratory syncytial virus (RSV) (1.7%), parainfluenza virus (2.0%), influenza B virus (2.6%), adenovirus (4.0%), herpes simplex virus type 1 (4. 4%), influenza A virus (5.5%), and enterovirus (12.7%). There were 61 mixed infections, and no consistent seasonal variation was found. One or more viruses were detected among 24.8% of hospitalized patients and 35.0% of outpatients. The frequencies and profiles of detection of various viruses among in- and outpatients were different. The occurrence of enterovirus infections exceeded that of other viral infections detected in 1998 and 1999 due to outbreaks of enterovirus 71 and coxsackievirus A10. RSV was the most prevalent virus detected among hospitalized children, whereas influenza virus was the most frequently isolated virus in the outpatient group. Most respiratory viral infections (39.3%) occurred in children between 1 and 3 years old. RSV (P < 0.025) and influenza A virus (P < 0.05) infections were dominant in the male inpatient group. In addition, most pneumonia and bronchiolitis (48.4%) was caused by RSV among hospitalized children less than 6 months old. Adenovirus was the most common agent associated with pharyngitis and tonsilitis (45.5%). These data expand our understanding of the etiology of acute respiratory tract viral infections among in- and outpatients in a subtropical country and may contribute to the prevention and control of viral respiratory tract infections.

Background: An epidemic of enterovirus 71 (EV71) occurred in Taiwan from April to December of 1998, with two peaks, one in June and the other in October. Many enteroviruses were isolated in our laboratory from 258 cases during this outbreak. Approximately half of the enteroviruses isolated were EV71 and one fifth were coxsackievirus A16., Objectives: To analyze laboratory findings in the EV71 epidemic of 1998 in Taiwan, various EV71 specimens in different cell lines were examined. In addition, genetic analysis of 5' non-coding region (NCR) was performed to analyze the strain variation in this outbreak., Results: The cytopathic effect induced by EV71 was observed 2-13 (mean of 4.5) days post-inoculation in Vero cells and 4-15 (mean of 6.6) days in green monkey kidney (GMK) cells inoculated with throat swabs. Of the total positive EV71 cases, virus was most frequently obtained from throat swabs (91.7%), less from stools (64.8%), and none from cerebral spinal fluid (CSF). Molecular analyses of EV71 by sequencing the 5' NCR of 34 strains obtained from different clinical categories and various geographic areas showed that their sequences differed (0-13 bp in 681 bp sequenced) by approximately 0-2%. The sequences of these isolates differed from EV71 prototype BrCr or MS strain by 17.5-19%, with the exception of two samples which exhibited nucleotide variation by only 8.9 and 8.2%, when compared to the MS strain., Conclusion: EV71 was most frequently isolated from throat swab specimens in Vero cells. The molecular analyses of the 5' NCR of EV71 revealed that most isolates from this epidemic belonged to a group of closely related clones and only two were in a different group which was clustered with the EV71 MS strain.

Background: In a recent enterovirus outbreak in Taiwan, serotype 71 was the culprit of encephalitis causing rapid clinical deterioration and death among young children., Objectives: Since knowledge of enterovirus 71 (EV71) infection in the central nervous system is still limited, the purpose of the present case study was attempted to uncover the pathogenesis of the virus., Study Design: We performed a detailed pathological examination, virological and molecular studies on a case of EV71 infection with a rapidly fatal outcome. In addition, the whole genome of the virus was sequenced to determine the genetic relationships to other enteroviruses and two other EV71 strains (a prototype BrCr and a neurovirulent MS strain), and to provide the genetic basis of its neurovirulence of the new isolate, NCKU9822 strain., Results: Characteristic features of acute encephalomyelitis were observed, with most prominent lesions in the spinal cord and brain stem. Mild myocarditis and pancreatitis were also noticed. EV71 antigen was localized to neurons on immunohistochemical staining. EV71 was recovered from all organs with inflammatory reaction. Sequence analysis showed that overall NCKU9822 and the two EV71 strains shared 80% nucleotide identity and 95% amino acid identity. It had only 45% amino acid and 52% nucleotide identities with polioviral P1 capsid region., Conclusion: The spinal cord and brain stem were the main targets of EV71 in the fatal cases in this outbreak, however, heart and pancreas might also be involved. Since the amino acid sequences in the P1 region are conserved (97% identity) among the three EV71 strains as compared to other enteroviruses and polioviruses, these EV71 neurovirulent strains might share the same mechanisms of neurovirulence, and the mechanisms might be different from those in polioviruses.

Background: An outbreak of enterovirus infections occurred throughout Taiwan in 1998. The diseases were manifectated with hand, foot, and mouth disease (HFMD), some associated with meningitis, encephalitis, or acute flaccid paralysis (AFP)., Objectives: This study is aimed to characterize and analyze the epidermologic and clinical features during the outbreak., Study Design: The epidemiologic information was collected from the Ministry of Health on passive surveillance; clinical and virological investigations were carried out at National Cheng Kung University Medical Center., Results: Between April and December 1998, 405 children were hospitalized, and 78 patients died during this outbreak in Taiwan. There were 119 cases identified to be EV71 infection in Tainan and Chiayi areas; 105 cases by virus isolation and 14 by serological assay. The outbreak had a biphasic curve with peak in June and October, especially in the southern Taiwan. Seventy-two percent of patients were below 3 years of age. The spectrum of disease included HFMD in 54, HFMD with central nerve system (CNS) involvement in 37, herpangina in 12, aseptic meningitis in three, encephalitis/ meningoencephalitis in ten, acute flaccid paralysis in three. There was nine fatal cases complicated with neurogenic pulmonary edema. Myoclonus with sleep disturbance was the most important early sign of EV71 infection with CNS involvement., Conclusion: Our experience demonstrated that the EV71 isolated in Taiwan had strong dermatotropic as well as neurotropic tendencies. Early detecting CNS involvement and commencing aggressive therapy may reduce the mortality.

The enteroviruses comprise a large group of immunologically distinct serotypes of viruses belonging to the family of Picornaviridae. Many enteroviruses cause diseases in human, but the infections are generally mild as asymptomatic, therefore, enteroviruses are considered to be unimportant as human pathogens. However, enteroviruses may also result in serious or even fatal disease (as shown in the enterovirus 71 (EV71) epidemic in Taiwan in 1998). There are three types of polioviruses, Coxsackievirus group A and group B viruses, and echoviruses group. All together a total of 67 types are available. Starting from enterovirus type 68 to 71, they are named as enterovirus types. Enterovirus type 72 is hepatitis A virus. Paralytic disease of poliomyelitis was recorded in ancient time but characterization of poliovirus was not reported until the turn of the 19th century that poliomyelitis was a viral disease. The major breakthrough for diagnosing and controlling of poliomyelitis was the discovery that poliovirus can be propagated in human embryonic tissues in cultures. As soon as cultures of human and monkey cells began to use for isolating polioviruses in stool specimen of patients, more unknown viruses were isolated which unlike polioviruses nor Coxsackie viruses; they were called "orphan" viruses or human enteric viruses, name later simplified to "echoviruses". Morphologically all enteroviruses are alike. They are small, ether insensitive viruses with an RNA genome. Their nucleic acid is single stranded, and the nucleocapsid has a cubic (icosahedral) symmetry, and is naked. The host ranges of enteroviruses vary greatly from one type to the next and even among strains of the same type. Polioviruses have a very restricted host range among laboratory animals. Virus isolation is the best method for diagnosis of enterovirus infection, but infection in the central nervous system (CNS) may be detected by polymerase chain reaction (PCR). Currently final identification and serotyping of enteroviruses are by indirect immunofluorescent tests using monoclonal antibody or by neutralization test using antiserum pools described by Lim and Benyesh-Melnick. The incidence and prevalence of diseases associated with the enterovirus infections are varied. The circulation of enteroviruses recently in Tainan and the epidemic of EV71 in Taiwan in 1998 are described in this review. Although poliovirus infection may be eradicated from the world due to the efficient vaccination program, there is no specific antiviral agents for either treatment or prevention for other enterovirus infections. In 1991, a new antiviral "pleconaril" which is a novel orally bioavailable and systematically acting small molecule inhibitor for picornaviruses. "Pleconaril" is currently in clinical trials for treatment of enterovirus meningitis and respiratory infections.