Increased Detection of Viruses in Children with Respiratory Tract Infection Using PCR.

Respiratory viruses are a common cause of respiratory tract infection (RTI), particularly in neonates and children. Rapid and accurate diagnosis of viral infections could improve clinical outcomes and reduce the use of antibiotics and treatment sessions. Advances in diagnostic technology contribute to the accurate detection of viruses. We performed a multiplex real-time polymerase chain reaction (PCR) to investigate the viral etiology in pediatric patients and compared the detection rates with those determined using traditional antigen tests and virus cultures. Fifteen respiratory viruses were included in our investigation: respiratory syncytial virus A/B (RSV), influenza virus A (FluA) and influenza virus B (FluB), human metapneumovirus (MPV), enterovirus (EV), human parainfluenza virus (PIV) types 1-4, human rhinovirus (RV), human coronavirus OC43, NL63, and 229E, human adenovirus (ADV), and human bocavirus (Boca). In total, 474 specimens were collected and tested. Respiratory viruses were detected more frequently by PCR (357, 75.3%) than they were by traditional tests (229, 49.3%). The leading pathogens were RSV (113, 23.8%), RV (72, 15.2%), PIV3 (53, 11.2%), FluA (51, 10.8%), and ADV (48, 10.1%). For children younger than 5 years, RSV and RV were most prevalent; for children older than 5 years, FluA and ADV were the most frequently detected. Of the specimens, 25.8% (92/357) were coinfected with two or more viruses. RV, Boca, PIV2, FluB, and PIV4 had higher rates of coinfection; MPV and PIV1 had the lowest rates of coinfection (9.1% and 5.3%). To conclude, the detection power of PCR was better than that of traditional antigen tests and virus cultures when considering the detection of respiratory viruses. RSV and RV were the leading viral pathogens identified in the respiratory specimens. One-quarter of the positive specimens were coinfected with two or more viruses. In the future, further application of PCR may contribute to the rapid and accurate diagnosis of respiratory viruses and could improve patient outcomes.

Persistence and continuous evolution of the human respiratory syncytial virus in northern Taiwan for two decades.

The study aimed to characterize the molecular epidemiology, phylogenetic relationship, and population dynamics of the G protein gene in clinical respiratory syncytial virus (RSV) strains isolated from northern Taiwan. We analyzed a total of 160 and 116 G protein gene sequences of RSV-A and RSV-B representative strains, respectively, from 804 clinical viral stocks collected between July 2000 and June 2016. Population dynamic patterns of the RSV G protein gene were analyzed using Bayesian inference through the Markov chain Monte Carlo framework. A phylogenetic analysis revealed that RSV-A from Taiwan could be categorized into GA2, GA5, and GA7 lineages. GA2 of RSV-A could be further divided into NA1, NA2, NA4, and ON1 clades. These RSV-A lineages has been replaced over time, whereas RSV-B strains from Taiwan continually evolved from a single lineage with significant time-dependent waves. Four putative positive selection sites were observed in both RSV-A and RSV-B. The Bayesian skyline plot revealed that the local population dynamics of RSV were associated with lineage displacement events. Both circulating subtypes and population dynamics represented a unique local pattern. Our results affirm the necessity of continuing molecular surveillance of RSV to attain a more comprehensive understanding of epidemics.

Risk factor analysis and molecular epidemiology of respiratory adenovirus infections among children in northern Taiwan, 2009-2013.

BACKGROUND/PURPOSE: Respiratory infections caused by human adenoviruses (HAdV) are worldwide, and have significantly increased recently in Taiwan. This study aimed to clarify the molecular epidemiology and risk factors of HAdV severe infections and pneumonia among Taiwanese children. METHODS: Patients with HAdV infections and hospitalized in a medical center between 2009 and 2013 were divided into severe or nonsevere HAdV infections based on whether or not they received intensive care. HAdV pneumonia was identified for comparison. The HAdV genotype was determined by sequencing the partial hexon and fiber genes. The nucleotide sequences were compared by phylogenetic analysis. RESULTS: The 176 patients (97 boys, 79 girls) had a median age of 3.7 years. The HAdV infections circulated year-round. HAdV B3 (54.5%) was the most common genotype, followed by HAdV C2 (21%), HAdV E4 (8%), and HAdV B7 (6.8%). Thirty-two patients needed intensive care. In multivariate analysis, the risk factors for severe HAdV infections were underlying neurologic diseases [odds ratio (OR): 164.9; p < 0.001], prematurity (OR: 10.9; p = 0.042), and HAdV B7 (OR: 39.5; p = 0.011). Twenty-nine patients had HAdV pneumonia. Patients with underlying neurologic diseases (OR 76.8; p < 0.001), airway anomaly (OR 15.1; p = 0.033), chronic lung diseases (OR 12.5; p = 0.047), weight < 3rd percentile (OR 5.5; p = 0.027), and HAdV B7 (OR 4.2; p = 0.002) had higher incidences of pneumonia. Four with underlying neurologic diseases died of acute respiratory distress syndrome. CONCLUSION: HAdV infections circulate all year-round. HAdV B7 is strongly related to severe infections and pneumonia. Underlying neurologic diseases and prematurity are risk factors for severe HAdV infections.

Clinical characteristics in adult patients with Salmonella bacteremia and analysis of ciprofloxacin-nonsusceptible isolates.

BACKGROUND/PURPOSE: The purpose of this study is to describe clinical characteristics of Salmonella bacteremia in adult patients and analyze ciprofloxacin-nonsusceptible isolates. METHODS: A total of 101 Salmonella blood isolates from adult patients were collected from January 2011 to December 2013 in MacKay Memorial Hospital. Eight ciprofloxacin-nonsusceptible Salmonella blood isolates were screened for carbapenemase and other ß lactamase genes. Isolates were examined by PCR for the quinolone resistance-determining region (QRDR) of all subunits for DNA gyrase (gyrA and gyrB) genes and topoisomerase IV (parC and parE) genes. RESULTS: There were 22 (21.78%) S. enterica serovar B, 5 (4.95%) S. enterica serovar C1, 7 (6.93%) S. enterica serovar C2, 65 (64.36%) S. enterica serovar D, and 2 (1.98%) S. enterica serovar Typhi (S. typhi) isolates. ß-lactamase gene screening and sequencing yielded only one blaCMY-2-positive isolate. In multivariate risk factor analysis, renal insufficiency [odds ratio (OR) 3.774; p = 0.020] and heart disease (OR 2.922; p = 0.027) were more common among elderly patients (≥65 years). Independent risk factors for ciprofloxacin-nonsusceptible strains included S. enterica serovar C2 (OR 28.430; p = 0.032), renal insufficiency (OR 13.927; p = 0.032), and immunosuppression agent usage (OR 60.082; p = 0.006). 87.50% (7/8) of isolates had gyrA mutation, 62.50% (5/8) had parC mutation, and none had gyrB and parE mutations. Isolates with both Ser83Phe/Asp87Asn gyrA and Thr57Ser/Ser80Ile parC mutation genes were highly ciprofloxacin-resistant (minimum inhibitory concentration ≥4 mg/L). CONCLUSIONS: Elderly patients with renal insufficiency and heart disease were at risk for Salmonella bacteremia. Those for ciprofloxacin-nonsusceptible strains included S. enterica serovar C2, renal insufficiency, and immunosuppression agent usage. The 8 ciprofloxacin-nonsusceptible isolates carried gyrA and parC mutations, which cause resistance that poses a major concern.

Identification of DHA-23, a novel plasmid-mediated and inducible AmpC beta-lactamase from Enterobacteriaceae in Northern Taiwan.

OBJECTIVES: AmpC ß-lactamases are classified as Amber Class C and Bush Group 1. AmpC ß-lactamases can hydrolyze broad and extended-spectrum cephalosporins, and are not inhibited by ß-lactamase inhibitors such as clavulanic acid. This study was conducted to identify DHA-23, a novel plasmid-mediated and inducible AmpC ß-lactamase obtained from Enterobacteriaceae. METHODS: A total of 210 carbapenem-resistant Enterobacteriaceae isolates were collected from a medical center (comprising two branches) in Northern Taiwan during 2009-2012. AmpC ß-lactamase genes were analyzed through a polymerase chain reaction using plasmid DNA templates and gene sequencing. The genetic relationships of the isolates were typed using pulsed-field gel electrophoresis following the digestion of intact genomic DNA by using XbaI. RESULTS: Three enterobacterial isolates (one Escherichia coli and two Klebsiella pneumoniae) were obtained from three hospitalized patients. All three isolates were resistant or intermediately susceptible to all ß-lactams, and exhibited reduced susceptibility to carbapenems. These three isolates expressed a novel AmpC ß-lactamase, designated DHA-23, approved by the curators of the Lahey website. DHA-23 differs from DHA-1 and DHA-6 by one amino acid substitution (Ser245Ala), exhibiting three amino acid changes compared with DHA-7 and DHA-Morganella morganii; three amino acid changes compared with DHA-3; four amino acid changes compared with DHA-5; and eight amino acid changes compared with DHA-2 (>97% identity). This AmpC ß-lactamase is inducible using a system involving ampR. CONCLUSION: This is the first report to address DHA-23, a novel AmpC ß-lactamase. DHA-type ß-lactamases are continuous threat in Taiwan.

A novel six consecutive monthly doses of palivizumab prophylaxis protocol for the prevention of respiratory syncytial virus infection in high-risk preterm infants in Taiwan.

BACKGROUND: Respiratory syncytial virus (RSV) circulates year round in Taiwan. A novel six consecutive monthly doses of palivizumab for RSV prevention protocol has been approved for high risk preterm infants since December 2010. This study aimed to determine the clinical effectiveness and safety of this novel protocol for the prevention of RSV infection. METHODS: From April 2011 to March 2013, we enrolled infants born at ≤28 weeks gestation and infants born at ≤35 weeks gestation with chronic lung disease (CLD) who received palivizumab prophylaxis as study group and followed up for 12 months. Historic control, those who were born and followed up between July 2000 and June 2008, were retrieved for propensity score matching. Primary endpoint was RSV-related hospitalization, and secondary endpoints included the length of hospital stay and intensive care unit (ICU) care. RESULTS: We enrolled 127 infants (108 infants born at ≤28 weeks and 19 infants born at 29-35 weeks with CLD). They completed 6-dose palivizumab as scheduled. Among the study group, the RSV-related hospitalizations were 2 (1.6%) within 6 months and 5 (3.9%) within 12 months after discharge. We matched 127 infants in the control group with 127 infants in the study group by propensity score matching. The reduction of RSV-related hospitalization rates were 86% (10.2% vs 1.6%, p = 0.002) within 6 months after discharge and 78% (15.7% vs 3.9%, p = 0.004) within 12 months after discharge. Compared to the control group, the rate of ICU care significantly decreased from 7.1% to 0.8% (p = 0.024) within 6 months after discharge and from 7.9% to 0.8% (p = 0.014) within 12 months after discharge. Adverse events were recorded in 6.4% injections. CONCLUSIONS: Six monthly intramuscular administration of palivizumab is effective for prevention of RSV hospitalization in regions with no single seasonal peak of RSV infection such as Taiwan.

Types and prevalence of carbapenem-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii complex in Northern Taiwan.

The frequency of the carbapenem-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii (CRACB) complex increases annually in our hospitals. However, the types and prevalence of carbapenemases among isolates still remain unclear. In this study, we identified and collected 672 carbapenem-resistant isolates from a medical center in Northern Taiwan between April and December of 2010. There were 577 genospecies 2 (Acinetobacter baumannii), 79 genospecies 13TU, and 16 genospecies 3 isolates. The isolates had an acquired blaOXA-24-like gene, which was confirmed by sequencing for the encoded OXA-72 carbapenemase, and were often associated with high-level carbapenem resistance. These CRACB complex isolates remained susceptible to colistin (100%). The genotyping of isolates was conducted using pulsed-field gel electrophoresis with ApaI digestion. In most clonally related groups, patients were from both branch hospitals. The results indicate that interhospital dissemination of clones occurred. This study provides updated data on the types and prevalence of the CRACB complex. In addition, it presents a warning on the emergence and spread of CRACB complex harboring blaOXA-24-like genes in northern Taiwan.

Molecular epidemiology and phylodynamics of the human respiratory syncytial virus fusion protein in northern Taiwan.

BACKGROUND AND AIMS: The glycoprotein (G protein) and fusion protein (F protein) of respiratory syncytial virus (RSV) both show genetic variability, but few studies have examined the F protein gene. This study aimed to characterize the molecular epidemiology and phylodynamics of the F protein gene in clinical RSV strains isolated in northern Taiwan from 2000-2011. METHODS: RSV isolates from children presenting with acute respiratory symptoms between July 2000 and June 2011 were typed based on F protein gene sequences. Phylogeny construction and evaluation were performed using the neighbor-joining (NJ) and maximum likelihood (ML) methods. Phylodynamic patterns in RSV F protein genes were analyzed using the Bayesian Markov Chain Monte Carlo framework. Selection pressure on the F protein gene was detected using the Datamonkey website interface. RESULTS: From a total of 325 clinical RSV strains studied, phylogenetic analysis showed that 83 subgroup A strains (RSV-A) could be further divided into three clusters, whereas 58 subgroup B strains (RSV-B) had no significant clustering. Three amino acids were observed to differ between RSV-A and -B (positions 111, 113, and 114) in CTL HLA-B*57- and HLA-A*01-restricted epitopes. One positive selection site was observed in RSV-B, while none was observed in RSV-A. The evolution rate of the virus had very little change before 2000, then slowed down between 2000 and 2005, and evolved significantly faster after 2005. The dominant subtypes of RSV-A in each epidemic were replaced by different subtypes in the subsequent epidemic. CONCLUSIONS: Before 2004, RSV-A infections were involved in several small epidemics and only very limited numbers of strains evolved and re-emerged in subsequent years. After 2005, the circulating RSV-A strains were different from those of the previous years and continued evolving through 2010. Phylodynamic pattern showed the evolutionary divergence of RSV increased significantly in the recent 5 years in northern Taiwan.

Viral etiology of acute lower respiratory tract infections in hospitalized young children in Northern Taiwan.

BACKGROUND: Lower respiratory tract infections (LRTIs) comprise a great proportion of diagnoses among hospitalized children. This study identifies the viral pathogens causing LRTIs in young children and compares their clinical features and disease severity. METHODS: Children younger than 36 months old, hospitalized at a medical center in Northern Taiwan with acute bronchiolitis or pneumonia from April to December 2007, were prospectively enrolled. Nasopharyngeal aspiration fluid samples were sent for virus culture, for direct immunofluorescence test of respiratory syncytial virus (RSV), for rapid influenza viral identification, and for polymerase chain reaction of human metapneumovirus (hMPV), human boca virus (hBoV), and human corona virus. The clinical features and laboratory findings were recorded and analyzed. RESULTS: A total of 48 children were enrolled. RSV was the most common pathogen (41.7%), followed by hMPV (27.1%), hBoV, and enterovirus (both 6.3%). There were no significant differences in clinical presentation and disease severity between the RSV and hMPV groups. However, the hMPV group had a higher mixed infection rate (p = 0.038). Fourteen children had no identifiable viruses. Children with single, dual, and triple pathogens numbered 26, 7, and 1, respectively. The mixed infection rate reached 23.5% among 34 children with identifiable viruses. Children with a higher severity score had greater chance to develop asthma in the next 2 years (p = 0.042). CONCLUSION: RSV is the most common pathogen causing LRTIs in young children, followed by hMPV. The hMPV group had higher mixed infection rate than RSV group. hBoV does circulate in northern Taiwan.

Roles of trans and cis variation in yeast intraspecies evolution of gene expression.

Both cis and trans mutations contribute to gene expression divergence within and between species. We used Saccharomyces cerevisiae as a model organism to estimate the relative contributions of cis and trans variations to the expression divergence between a laboratory (BY) and a wild (RM) strain of yeast. We examined whether genes regulated by a single transcription factor (TF; single input module, SIM genes) or genes regulated by multiple TFs (multiple input module, MIM genes) are more susceptible to trans variation. Because a SIM gene is regulated by a single immediate upstream TF, the chance for a change to occur in its trans-acting factors would, on average, be smaller than that for a MIM gene. We chose 232 genes that exhibited expression divergence between BY and RM to test this hypothesis. We examined the expression patterns of these genes in a BY-RM coculture system and in a BY-RM diploid hybrid. We found that trans variation is far more important than cis variation for expression divergence between the two strains. However, because in 75% of the genes studied, cis variation has significantly contributed to expression divergence, cis change also plays a significant role in intraspecific expression evolution. Interestingly, we found that the proportion of genes with diverged expression between BY and RM is larger for MIM genes than for SIM genes; in fact, the proportion tends to increase with the number of transcription factors that regulate the gene. Moreover, MIM genes are, on average, subject to stronger trans effects than SIM genes, though the difference between the two types of genes is not conspicuous.

Antimicrobial therapy and control of multidrug-resistant Pseudomonas aeruginosa bacteremia in a teaching hospital in Taiwan.

BACKGROUND AND PURPOSE: The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa is a challenging clinical problem. This study investigated the source of an outbreak of MDR P. aeruginosa infections and the role of combination therapy in its management. METHODS: MDR P. aeruginosa isolates were collected at the MacKay Memorial Hospital, Taipei, Taiwan, and antibiotic synergy was investigated based on antibiotic susceptibility tests using a combination of antibiotics. Isolates of patients with MDR P. aeruginosa bacteremia were selected for genetic analysis by pulsed-field gel electrophoresis. RESULTS: A combination of ceftazidime, amikacin, and sulbactam had significant synergistic effects against bloodstream MDR P. aeruginosa isolates and was more beneficial clinically compared with other antibiotic combinations. The major source of MDR P. aeruginosa infection was located and stringent infection control measures were enforced. CONCLUSION: The results of this study suggest that use of triple antimicrobial therapy (ceftazidime, amikacin, and sulbactam) can be a useful alternative treatment for MDR P. aeruginosa infection in certain circumstances.

Cefotaxime-resistant Citrobacter freundii in isolates from blood in a tertiary teaching hospital in Northern Taiwan.

From January 2002 to December 2003, 12 patients in a tertiary teaching hospital in northern Taiwan had bloodstream infections caused by Citrobacter freundii. Seven of the 12 isolates were resistant to cefotaxime. Using polymerase chain reaction and DNA sequencing, 3 of the 7 cefotaxime-resistant C. freundii isolates were found to carry extended-spectrum beta-lactamase (ESBL). AmpC beta-lactamase genes were also detected in all strains of C. freundii. All strains of C. freundii with MICs >or=4 mg/L for cefepime were positive for ESBL. Rather than performing PCR on all cefotaxime-resistant C. freundii isolates, assessment of the MIC for cefepime might be a practical way to choose between treatment with cefepime or with carbapenems.

Ceftriaxone resistance of nontyphoidal Salmonella enterica isolates in Northern Taiwan attributable to production of CTX-M-14 and CMY-2 beta-lactamases.

Among 3,027 nontyphoidal Salmonella enterica isolates identified between January 1999 and December 2002 in a medical center in northern Taiwan, 31 were resistant to the extended-spectrum cephalosporin ceftriaxone (1.02% [31/3,027]), including 2 in 1999 (0.36% [2/549]), 13 in 2000 (1.49% [13/870]), 7 in 2001 (0.78% [7/893]), and 9 in 2002 (1.26% [9/715]). Sixteen of these isolates belonged to Salmonella serogroup B, nine belonged to serogroup C, four belonged to serogroup D, and two belonged to serogroup E. The majority were from stool cultures. The mechanism of resistance was investigated for eight isolates, including three S. enterica serovar Typhimurium, one S. enterica serovar Wagenia, one S. enterica serovar Senftenberg, one S. enterica serovar Derby, one S. enterica serovar Panama, and one S. enterica serovar Duesseldorf isolate. All eight patients from whom these isolates were recovered had community-acquired infections. All eight isolates were resistant to ampicillin, ceftriaxone, and cefotaxime but susceptible to imipenem and ciprofloxacin. Ceftriaxone resistance was due to the production of the CMY-2 AmpC beta-lactamase by seven isolates and the CTX-M-14 beta-lactamase by the remaining isolate. Both beta-lactamase genes were carried on conjugative plasmids. In a 2.5-kb region encompassing the bla(CMY-2) gene, at nucleotide 49 upstream of the start codon of bla(CMY-2), three of the seven bla(CMY-2)-positive isolates had an A nucleotide and four had a G nucleotide. In conclusion, the ceftriaxone resistance of nontyphoidal Salmonella isolates in our hospital was attributed to the CTX-M-14 and CMY-2 beta-lactamases.

Tramtrack69 is required for the early repression of tailless expression.

During embryogenesis, the activated Torso receptor tyrosine kinase (TOR RTK) pathway activates tailless (tll) expression by a relief-of-repression mechanism. Various lines of evidence have suggested that multiple factors are required for this repression. We show that Tramtrack69 (TTK69) binds to two sites within tll cis-regulatory DNA, TC2 and TC5, and that TTK69 is phosphorylated by mitogen activated protein kinase. In embryos lacking maternal ttk69 activity, the expression of both endogenous tll and lacZ driven by the tll minimal regulatory region (tll-MRR) are expanded. Further, in wild-type embryos, the tll-MRR mutated in TC5 drives uniform lacZ expression before late stage 4. We conclude that TTK69 is required for early (before the end of stage 4) repression of tll transcription.