Benefit analysis of the auto-verification system of intelligent inspection for microorganisms.

In recent years, the automatic machine for microbial identification and antibiotic susceptibility tests has been introduced into the microbiology laboratory of our hospital, but there are still many steps that need manual operation. The purpose of this study was to establish an auto-verification system for bacterial naming to improve the turnaround time (TAT) and reduce the burden on clinical laboratory technologists. After the basic interpretation of the gram staining results of microorganisms, the appearance of strain growth, etc., the 9 rules were formulated by the laboratory technologists specialized in microbiology for auto-verification of bacterial naming. The results showed that among 70,044 reports, the average pass rate of auto-verification was 68.2%, and the reason for the failure of auto-verification was further evaluated. It was found that the main causes reason the inconsistency between identification results and strain appearance rationality, the normal flora in the respiratory tract and urine that was identified, the identification limitation of the mass spectrometer, and so on. The average TAT for the preliminary report of bacterial naming was 35.2 h before, which was reduced to 31.9 h after auto-verification. In summary, after auto-verification, the laboratory could replace nearly 2/3 of manual verification and issuance of reports, reducing the daily workload of medical laboratory technologists by about 2 h. Moreover, the TAT on the preliminary identification report was reduced by 3.3 h on average, which could provide treatment evidence for clinicians in advance.

Novel intertypic recombination of Echovirus 11 in the Enterovirus species B.

Enteroviruses (EVs), single-stranded, positive-sense RNA viruses, can be classified into four species (A-D), which have previously been linked to a diverse range of disease manifestations and infections affecting the central nervous system. In the Enterovirus species B (EV-B), Echovirus type 11 (E11) has been observed to occasionally circulate in Taiwan, which was responsible for an epidemic of enterovirus infections in 2018. Here, 48 clinical specimens isolated in 2003, 2004, 2009, and 2018 were collected for the high-throughput sequencing. Notably, we identified 2018 Taiwanese strains having potential recombinations in the 3D gene, as well as one 2003 strain having a double recombination with E6 and Coxsackievirus B5 in the P2 and P3 regions, respectively. Additionally, one amino acid signature mutated from the Histidine (H) in throat swab specimens to the Tyrosine (Y) in cerebral spinal fluid specimens was detected at position 1496 (or 57) of the genomic coordinate (or 3A gene) to further demonstrate intra-host evolution in different organs. In conclusion, this study identifies potential intertypic recombination events and an intra-host signature mutation in E11 strains, isolated during a 2018 neurological disease outbreak in Taiwan, contributing to our understanding of its evolution and pathogenesis.

Impact of patient characteristics on innate immune responses and inflammasome activation in ex vivo human lung tissues infected with influenza A virus.

Background: Influenza A virus (IAV) infection poses a persistent global health challenge, necessitating a nuanced grasp of host immune responses for optimal interventions. While the interplay between aging, immunosenescence, and IAV is recognized as key in severe lower respiratory tract infections, the role of specific patient attributes in shaping innate immune reactions and inflammasome activity during IAV infection remains under-investigated. In this study, we utilized an ex vivo infection model of human lung tissues with H3N2 IAV to discern relationships among patient demographics, IAV nucleoprotein (NP) expression, toll-like receptor (TLR) profiles, PD-1/PD-L1 markers, and cytokine production. Methods: Our cohort consisted of thirty adult patients who underwent video-assisted thoracoscopic surgery during 2018-2019. Post-surgical lung tissues were exposed to H3N2 IAV for ex vivo infections, and the ensuing immune responses were profiled using flow cytometry. Results: We observed pronounced IAV activity within lung cells, as indicated by marked NP upregulation in both epithelial cells (P = 0.022) and macrophages (P = 0.003) in the IAV-exposed group relative to controls. Notably, interleukin-2 levels correlated with variations in TLR1 expression on epithelial cells and PD-L1 markers on macrophages. Age emerged as a modulating factor, dampening innate immune reactions, as evidenced by reduced interleukin-2 and interferon-γ concentrations (both adjusted P < 0.05). Intriguingly, a subset of participants with pronounced tumor necrosis factor-alpha post-mock infection (Cluster 1) showed attenuated cytokine responses in contrast to their counterparts in Cluster 2 and Cluster 3 (all adjusted P < 0.05). Individuals in Cluster 2, characterized by a low post-mock infection NP expression in macrophages, exhibited reduced variations in both NP and TLR1-3 expressions on these cells and a decreased variation in interleukin-2 secretion in comparison to their Cluster 3 counterparts, who were identified by their elevated NP macrophage expression (all adjusted P < 0.05). Conclusion: Our work elucidates the multifaceted interplay of patient factors, innate immunity, and inflammasome responses in lung tissues subjected to ex vivo H3N2 IAV exposure, reflecting real-world lower respiratory tract infections. While these findings provide a foundation for tailored therapeutic strategies, supplementary studies are requisite for thorough validation and refinement.

The clinical significance of and the factors associated with macrolide resistance and poor macrolide response in pediatric Mycoplasma pneumoniae infection: A retrospective study.

BACKGROUND: Macrolide-resistant Mycoplasma pneumoniae (MRMP) infection is increasing worldwide. However, its clinical significance is still uncertain. METHODS: The data of the Laboratory Medicine Department of Chang Gung Memorial Hospital in northern Taiwan was searched for children with molecular confirmed macrolide-susceptible Mycoplasma pneumoniae (MSMP) and MRMP infections between January 2011 and December 2018. The clinical features, laboratory data, and chest image presentations were compared between patients with MRMP and MSMP infections and between patients with good and poor macrolide response, respectively. RESULTS: Records from 158 patients were recovered. Of the enrolled patients 34 (22%) suffered MRMP infection, 27 (17%) had pleural effusions, and 47 (32%) had poor macrolide response. The macrolide resistance rate was 12% in 2011, 20% between 2015 and 2016, and 50% between 2017 and 2018, respectively. Other than a poor macrolide response, the MRMP and MSMP infections are clinically indistinguishable. The presence of pleural effusion and MRMP infections were found to be independently associated with a poor macrolide response, with odds ratios (95% confidence interval) of 14.3 (4.9-42.0) and 14.6 (5.4-40), respectively. The macrolide resistance rate of the patients with a poor macrolide response was 49% and 18% among all the patients enrolled and the patients with a pleural effusion, respectively. CONCLUSION: The macrolide resistance rate had possibly increased in recent years in Taiwan and should be continuously monitored. In addition, the macrolide response could be misleading in predicting a macrolide resistance especially for the patients with a pleural effusion.

Exploration of the external and internal factors that affected learning effectiveness for the students: a questionnaire survey.

Learning effectiveness may be affected by internal and external factors, including personal attitude, motivations, learning skills, learning environment and peer pressure. This study sought to explore potential factors on students who majored in medical technology. The 106 students who completed their internship at Chang Gung Memorial Hospital were enrolled in this study. A written questionnaire was analyzed to explore the relationship between potential factors and learning effectiveness. The strength of relationship between the outcome and each factor was evaluated using Spearman correlation coefficients. A multiple linear regression model was constructed to assess how those factors affected learning effectiveness altogether. The results indicated that the learning effectiveness of the students mainly depended on three factors: the "extracurricular studies" and "willingness to cooperate" were positively associated with learning effectiveness. However, the "weakened motivation due to uncertainty" is negatively associated with learning effectiveness. We suggested that the educators can understand the uncertainty of students about the future. Additionally, the projects that require joint cooperation and discussion need to be given. The most important thing is that students should be able to integrate the learning content instead of rote.

Application of the electronic book to promote self-directed learning in medical technologist continuing education: a cross-sectional study.

BACKGROUND: Continuing education (CE) is essential for health professionals to improve competence in clinical practice, yet many medical technologists still experience barriers to learning in complex clinical settings. To better manage CE and address medical technologists' learning needs, we developed a learner-centred electronic book (e-book) to promote self-directed learning for medical technologists. METHODS: A cross-sectional study was conducted to explore the acceptability and learning impacts of the e-book as CE material for medical technologists in two medical centres in Taiwan. We designed the learner-centred context in the e-book based on medical technologists' practice requirements and learning needs. Moreover, we adopted The New World Kirkpatrick Model with four levels (reactions, learning, behaviours and results) to evaluate the e-book's learning impacts on medical technologists. A total of 280 medical technologists were invited to complete a questionnaire and a post-test, providing learning patterns as well as their satisfaction with the e-book and their learning outcomes after using it. RESULTS: Most readers had positive learning experiences and better learning outcomes, including knowledge acquisition and behavioural change, after reading the e-book. The e-book became a new CE activity and reached medical technologists in various types of laboratories. CONCLUSIONS: The low-cost and learner-centred e-book effectively overcame CE learning barriers for medical technologists. The interactivity and flexibility of e-learning particularly helped learners to engage in clinical scenarios in laboratory medicine. This study could pave the way for medical educators to build a high-quality e-learning model in CE.

Unprecedented outbreak of respiratory syncytial virus in Taiwan associated with ON1 variant emergence between 2010 and 2020.

An outbreak of respiratory syncytial virus (RSV) has been observed in Taiwan since August 2020. We reviewed a central laboratory-based surveillance network established over 20 years by Taiwan Centres for Disease Control for respiratory viral pathogens between 2010 and 2020.A retrospective study of children <5 years old hospitalized with RSV infection at Chang Gung Memorial Hospital between 2018 and 2020 was conducted, and samples positive for RSV-A were sequenced. Clinical data were obtained and stratified by genotype and year.Data from 2020 showed an approximately 4-fold surge in RSV cases compared to 2010 in Taiwan, surpassing previous years during which ON1 was prevalent. Phylogenetic analysis of G protein showed that novel ON1 variants were clustered separately from those of 2018 and 2019 seasons and ON1 reference strains. The variant G protein carried six amino acid changes that emerged gradually in 2019; high consistency was observed in 2020. A unique substitution, E257K, was observed in 2020 exclusively. The F protein of the variant carried T12I and H514N substitutions, which weren't at antigenic sites. In terms of multivariate analysis, age (OR: 0.97; 95% CI: 0.94-0.99; p = 0.02) and 2020 ON1 variant (OR:2.52; 95% CI:1.13-5.63; p = 0.025) were independently associated with oxygen saturation <94% during hospitalization.The 2020 ON1 variant didn't show higher replication or virulence compared with those in 2018 in our study. The unprecedented 2020 RSV epidemic may attribute to antigenic changes and lack of interferon-stimulated immunity induced by seasonal circulating virus under non-pharmaceutical intervention.

Molecular detection of respiratory pathogens in community-acquired pneumonia involving adults.

BACKGROUND: Community-acquired pneumonia (CAP) causes substantial morbidity and mortality in adults worldwide. The etiology of CAP often remains uncertain, and therapy is empirical. Thus, there is still room for improvement in the diagnosis of pneumonia. METHODS: Adults aged >20 years who presented at the outpatient or emergency departments of Linkou and Keelung Chang Gung Memorial Hospital with CAP were prospectively included between November 2016 and December 2018. We collected respiratory specimens for culture and molecular testing and calculated the incidence rates of CAP according to pathogens. RESULTS: Of 212 hospitalized adult patients with CAP, 69.3% were male, and the median age of the patients was 67.8 years. Bacterial pathogens were detected in 106 (50%) patients, viruses in 77 (36.3%), and fungal pathogens in 1 patient (0.5%). The overall detection rate (culture and molecular testing method) was 70.7% (n = 150). Traditional microbial culture yielded positive results in 36.7% (n = 78), molecular testing in 61.3% (n = 130). The most common pathogens were influenza (16.1%), followed by Klebsiella pneumoniae (14.1%), Pseudomonas aeruginosa (13.6%), human rhinovirus (11.8%), and Streptococcus pneumoniae (9.9%). Multiple pathogen co-infections accounted for 28.7% (n = 61), of which co-infection with K. pneumoniae and human rhinovirus comprised the largest proportion. CONCLUSIONS: Molecular diagnostic testing could detect 23.6% more pathogens than traditional culture techniques. However, despite the current diagnostic tests, there is still the possibility that no pathogen was detected.

A hospital cluster of COVID-19 associated with a SARS-CoV-2 superspreading event.

BACKGROUND/PURPOSE: Superspreading events (SSEs) are pivotal in the spread of SARS-CoV-2. This study aimed to investigate an SSE of COVID-19 in a hospital and explore the transmission dynamics and heterogeneity of SSE. METHODS: We performed contact tracing for all close contacts in a cluster. We did nasopharyngeal or throat swabbing for SARS-CoV-2 by real-time RT-PCR. Environmental survey was performed. The epidemiological and clinical characteristics of the SSE were studied. RESULTS: Patient 1 with congestive heart failure and cellulitis, who had onset of COVID-19 two weeks after hospitalization, was the index case. Patient 1 led to 8 confirmed cases, including four health care workers (HCW). Persons tested positive for SARS-CoV-2 were HCW (n = 4), patient 1's family (n = 2), an accompanying person of an un-infected in-patient (n = 1), and an in-patient admitted before the SSE (n = 1). The attack rate among the HCW was 3.2 % (4/127). Environmental survey confirmed contamination at the bed rails, mattresses, and sink in the room patient 1 stayed, suggesting fomite transmission. The index case's sputum remained positive on illness day 35. Except one asymptomatic patient, at least three patients acquired the infection from the index case at the pre-symptomatic period. The effective reproduction number (Rt) was 0.9 (8/9). CONCLUSION: The host factor (heart failure, longer viral shedding), transmissibility of SARS-CoV-2 (Rt, pre-symptomatic transmission), and possible multiple modes of transmission altogether contributed to the SSE. Rapid response and advance deployment of multi-level protection in hospitals could mitigate COVID-19 transmission to one generation, thereby reducing its impact on the healthcare system.

Infection control operations of a large hospital in Taiwan to prevent nosocomial outbreaks during COVID-19 pandemic.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) is highly contagious, with a potential to cause large nosocomial outbreaks in the hospital setting. We report the advance deployment of comprehensive, multi-level infection control measures in a 3,700-bed large hospital to prevent nosocomial outbreaks of COVID-19 during the pandemic. METHODS: We implemented a series of dynamic infection control policies during the pandemic. A confirmed COVID-19 case was defined by positive real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay. All healthcare worker (HCW) having symptoms or close contact with the confirmed case received the RT-PCR test. RESULTS: A total of 5,722 patients were tested in our hospital from January to May 2020. Twenty-five patients were confirmed COVID-19, including two inpatients. A cluster of 4 HCWs with COVID-19 associated with the 2nd inpatient was identified in the early stage of epidemic. Our enhanced traffic control bundling, mask wearing, hand hygiene and environmental cleaning were reinforced after the outbreak. All other confirmed cases were identified at our outdoor quarantine station or epidemic clinic afterwards, and the results of testing for 146 symptomatic HCWs were all negative. CONCLUSIONS: Integrated teamwork, advance deployment of infection control measures and efficient diagnostic testing and response protected HCW and facilities from large SARS-CoV-2 outbreaks and preserved the capacity and function of the health care system during the pandemic.

Relative COVID-19 Viral Persistence and Antibody Kinetics.

A total of 15 RT-PCR confirmed COVID-19 patients were admitted to our hospital during the in-itial outbreak in Taiwan. The average time of virus clearance was delayed in seven patients, 24.14 ± 4.33 days compared to 10.25 ± 0.56 days post-symptom onset (PSO) in the other eight pa-tients. There was strong antibody response in patients with viral persistence at the pharynx, with peak values of serum antibody 677.2 ± 217.8 vs. 76.70 ± 32.11 in patients with delayed versus rapid virus clearance. The patients with delayed viral clearance had excessive antibodies of compromised quality in an early stage with the delay in peak virus neutralization efficacy, 34.14 ± 7.15 versus 12.50 ± 2.35 days PSO in patients with rapid virus clearance. Weak antibody re-sponse of patients with rapid viral clearance was also effective, with substantial and comparable neutralization efficacy, 35.70 ± 8.78 versus 41.37 ± 11.49 of patients with delayed virus clearance. Human Cytokine 48-Plex Screening of the serial sera samples revealed elevated concentrations of proinflammatory cytokines and chemokines in a deceased patient with delayed virus clear-ance and severe disease. The levels were comparatively less in the other two patients who suf-fered from severe disease but eventually survived.

Clonal spread of macrolide-resistant Mycoplasma pneumoniae sequence type-3 and type-17 with recombination on non-P1 adhesin among children in Taiwan.

OBJECTIVES: Mycoplasma pneumoniae is currently the most commonly detected bacterial cause of childhood community-acquired pneumonia in several countries. Of note, clonal expansion of macrolide-resistant ST3 occurred in Japan and South Korea. An alarming surge in macrolide resistance complicates the treatment of pneumonia. We aimed to evaluate the clinical manifestation and clonal relatedness of M. pneumoniae circulating among children in Taiwan. METHODS: We prospectively enrolled 626 children with radiologically confirmed pneumonia between 2017 and 2019. An M. pneumoniae infection was suspected on clinical grounds, and tested by real-time PCR and oropharyngeal swab cultures. We used multilocus sequence typing and whole-genome sequencing to characterize the genetic features of M. pneumoniae. RESULTS: A total of 226 children with M. pneumoniae pneumonia were enrolled. Macrolide resistance was found in 77% (174/226) of patients. Multi-locus sequence typing revealed that ST3 (n = 93) and its single-locus variant ST17 (n = 84) were the predominant clones among macrolide-resistant strains. ST17 presented clinical characteristics comparable to its ancestor ST3. On multivariate analysis, macrolide resistance (OR 3.5; 95% CI 1.4-8.5; p 0.007) was independently associated with fever >72 hours after macrolide treatment. By whole-genome sequencing, prediction analysis of recombination sites revealed one recombination site in ST3 and ST17 compared with M29 (a macrolide-sensitive ST3 strain isolated from China in 2005) containing cytadhesin MgpC-like protein, RepMP4 and RepMP5. ST17 had another recombination site containing an adhesin and RepMP2/3. CONCLUSIONS: In addition to macrolide resistance, ST3 and its ST17 variant might evolve through recombination between repetitive sequences and non-P1 cytadhesins for persistent circulation in Taiwan.

Impaired interferon-α expression in plasmacytoid dendritic cells in asthma.

BACKGROUND: Toll-like receptor (TLR)-7-associated rhinovirus (RV) activation is involved in the pathogenesis of asthma. Plasmacytoid dendritic cells (pDCs) are the main interferon-α-producing cells against viruses. OBJECTIVE: To determine whether asthmatic patients and control subjects differ in terms of interferon-α expression in pDCs under TLR-7 or RV stimulation. METHODS: pDCs were identified in BDCA-2+ and HLA-DR+ peripheral blood mononuclear cells. Interferon-α expression of pDCs was analyzed after TLR-7 stimulation with or without interleukin 4 (IL-4)/IL-13 pretreatment. Interferon-α expression was also analyzed after RV stimulation over periods of 24, 48, or 96 h with or without IL-4 pretreatment. RV detection and molecular typing were assayed from throat swabs. RESULTS: Following TLR-7 stimulation, the expression of intracellular interferon-α was higher in the pDCs of normal subjects than those of asthmatic patients; however, pretreatment with IL-4 was shown to reduce this effect. After 48- and 96-h RV stimulation, we observed a notable increase in the production of interferon-α of pDCs in normal subjects but not in asthmatic patients. Baseline interferon-α expression in pDCs and the incidence of asthma exacerbation to emergency was higher among the 13% of patients identified as rhinovirus+ than among their RV counterparts. CONCLUSION: Our study discovered the response to TLR-7 stimulation in pDCs was compromised and the sustainability of interferon-α expression to RV stimulation was reduced in pDCs of asthmatic patients, which provide further evidence of defective innate response and subspeciality to RV infection in asthma. The high exacerbation history founded in RV+ patients agrees with these findings. Further research is required for the modulatory effect of IL-4 on TLR-7 stimulated pDCs.

Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19.

The COVID-19 pandemic is a worldwide health emergency which calls for an unprecedented race for vaccines and treatment. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein, S-2P. To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of neutralizing antibodies in sera of immunized mice against pseudotyped lentivirus reporter or live wild-type SARS-CoV-2. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018 and alum as a candidate vaccine to prevent COVID-19 disease.

Structural and functional analysis of protective antibodies targeting the threefold plateau of enterovirus 71.

Enterovirus 71 (EV71)-neutralizing antibodies correlate with protection and have potential as therapeutic agents. We isolate and characterize a panel of plasmablast-derived monoclonal antibodies from an infected child whose antibody response focuses on the plateau epitope near the icosahedral 3-fold axes. Eight of a total of 19 antibodies target this epitope and three of these potently neutralize the virus. Representative neutralizing antibodies 38-1-10A and 38-3-11A both confer effective protection against lethal EV71 challenge in hSCARB2-transgenic mice. The cryo-electron microscopy structures of the EV71 virion in complex with Fab fragments of these potent and protective antibodies reveal the details of a conserved epitope formed by residues in the BC and HI loops of VP2 and the BC and HI loops of VP3 spanning the region around the 3-fold axis. Remarkably, the two antibodies interact with the epitope in quite distinct ways. These plateau-binding antibodies provide templates for promising candidate therapeutics.

Emergence of genotype C1 Enterovirus A71 and its link with antigenic variation of virus in Taiwan.

An outbreak of the hand-foot-mouth disease with severe neurological cases, mainly caused by the genotype C1 enterovirus A71 (EV-A71), occurred in Taiwan between 2018 and early 2019. In the recent decade, the most dominant EV-A71 genotypes in Taiwan were B5 and C4 but changed to C1 in 2018. Antibody-mediated immunity plays a key role in limiting the EV-A71 illness in humans. However, the level of neutralizing activities against genotype C1 virus by human polyclonal and monoclonal antibodies (MAbs) remains largely unclear. In the study, we demonstrated that that 39% (9 in 23) of post-infection sera from the genotype B5- or C4-infected patients in 2014-2017 exhibit reduced titers with the 2018-2019 genotype C1 viruses than with the earlier B5 and C4 viruses tested. This finding with polyclonal sera is confirmed with human MAbs derived from genotype B5 virus-infected individuals. The 2018-2019 genotype C1 virus is resistant to the majority of canyon-targeting human MAbs, which may be associated with the residue change near or at the bottom of the canyon region on the viral capsid. The remaining three antibodies (16-2-11B, 16-3-4D, and 17-1-12A), which target VP1 S241 on the 5-fold vertex, VP3 E81 on the 3-fold plateau and VP2 D84 on the 2-fold plateau of genotype C1 viral capsid, respectively, retained neutralizing activities with variable potencies. These neutralizing antibodies were also found to be protective against a lethal challenge of the 2018-2019 genotype C1 virus in an hSCARB2-transgenic mice model. These results indicate that the EV-A71-specific antibody response may consist of a fraction of poorly neutralizing antibodies against 2018-2019 genotype C1 viruses among a subset of previously infected individuals. Epitope mapping of protective antibodies that recognize the emerging genotype C1 virus has implications for anti-EV-A71 MAbs and the vaccine field.

Detection of norovirus and rotavirus among inpatients with acute gastroenteritis in a medical center in northern Taiwan, 2013-2018.

BACKGROUND: Norovirus (NoV) and rotavirus (RV) are among the most common causes of acute gastroenteritis (AGE) of all ages worldwide. There have been scanty reports of the epidemiology data of NoV AGE from clinical virologic laboratory. METHODS: All stool specimens sent to the virologic laboratory in a medical center in Taiwan for detection of both NoV (by reverse transcription-polymerase chain reaction) and RV (by enzyme immunoassay) from 2013 to 2018 were included for analysis. RESULTS: A total of 15,991 specimens, annually ranging from 2430 in 2017-2915 in 2013, were included. 48.0% and 73.1% of the specimens were obtained from children aged < two years and < five years, respectively. Overall, the positive rate was 13.8% for NoV and 13.2% for RV. Yearly positive rate of NoV ranged from 11.1% to 17.4%. The positive rate in 2018 (17.4%) was significantly higher than that in other study years. NoV positive rate was higher in cold season from January (28.3%), February (23.1%), to March (17.9%) while lower in warm seasons from May to September (less than 10%). By age, NoV positive rate was highest in aged 1-4 years (17.3%) and decreased with age. The yearly positive rate of RV showed a significantly steady decrease from 15.6% in 2013 to 9.1% in 2018 (p < 0.001 by trend analysis). CONCLUSIONS: In northern Taiwan, NoV, surpassing RV, accounted for one of seven inpatients with AGE during 2013-2018. NoV activity peaked in cold season and children aged <5 years were more commonly encountered.

Characteristics and etiology of hospitalized pediatric community-acquired pneumonia in Taiwan.

BACKGROUND/PURPOSE: The purpose of this study was to determine the pathogens and to estimate the incidence of pediatric community-acquired pneumonia (CAP) in Taiwan. METHODS: This prospective study was conducted at eight medical centers from November 2010 to September 2013. Children aged 6 weeks to 18 years who met the radiologic criteria for pneumonia were enrolled. To detect classical and atypical bacteria and viruses, blood and pleural fluids were cultured, and respiratory specimens were examined by multiple conventional and molecular methods. RESULTS: At least one potential pathogen was identified in 705 (68.3%) cases of 1032 children enrolled, including bacteria in 420 (40.7%) cases, virus in 180 (17.4%) cases, and mixed viral-bacterial infection in 105 (10.2%) cases. Streptococcus pneumoniae (31.6%) was the most common pathogen, followed by Mycoplasma pneumoniae (22.6%). Adenovirus (5.9%) was the most common virus. RSV was significantly associated with children aged under 2 years, S. pneumoniae in children aged between 2 and 5 years, and M. pneumoniae in children aged >5 years. The annual incidence rate of hospitalization for CAP was highest in children aged 2-5 years (229.7 per 100,000). From 2011 to 2012, significant reduction in hospitalization rates pertained in children under 5 years of age, in pneumonia caused by pneumococcus, adenovirus or co-infections and complicated pneumonia. CONCLUSION: CAP related pathogens have changed after increased conjugated pneumococcal vaccination rates. This study described the latest incidences and trends of CAP pathogens, which are crucial for prompt delivery of appropriate therapy.

Culture-Based Virus Isolation To Evaluate Potential Infectivity of Clinical Specimens Tested for COVID-19.

Real-time reverse transcription-PCR (RT-PCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19). However, the correlation between detectable viral RNA and culturable virus in clinical specimens remains unclear. Here, we performed virus culture for 60 specimens that were confirmed to be positive for SARS-CoV-2 RNA by real-time RT-PCR. The virus could be successfully isolated from 12 throat and nine nasopharyngeal swabs and two sputum specimens. The lowest copy number required for virus isolation was determined to be 5.4, 6.0, and 5.7 log10 genome copies/ml sample for detecting the nsp12, E, and N genes, respectively. We further examined the correlation of genome copy number and virus isolation in different regions of the viral genome, demonstrating that culturable specimens are characterized by high copy numbers with a linear correlation observed between copy numbers of amplicons targeting structural and nonstructural regions. Overall, these results indicate that in addition to the copy number, the integrity of the viral genome should be considered when evaluating the infectivity of clinical SARS-CoV-2 specimens.