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1.  Decreased ADAMTS 13 Activity is Associated With Disease Severity and Outcome in Pediatric Severe Sepsis 
Medicine  2016;95(16):e3374.
Decreased ADAMTS 13 activity has been reported in severe sepsis and in sepsis-induced disseminated intravascular coagulation. This study aimed to investigate the role of ADAMTS 13 in different pediatric sepsis syndromes and evaluate its relationship with disease severity and outcome.
We prospectively collected cases of sepsis treated in a pediatric intensive care unit, between July 2012 and June 2014 in Chang Gung Children's Hospital in Taoyuan, Taiwan. Clinical characteristics and ADAMTS-13 activity were analyzed.
All sepsis syndromes had decreased ADAMTS 13 activity on days 1 and 3 of admission compared to healthy controls. Patients with septic shock had significantly decreased ADAMTS 13 activity on days 1 and 3 compared to those with sepsis and severe sepsis. There was a significant negative correlation between ADAMTS 13 activity on day 1 and day 1 PRISM-II, PELOD, P-MOD, and DIC scores. Patients with mortality had significantly decreased ADAMTS 13 activity on day 1 than survivors, but not on day 3.
Different pediatric sepsis syndromes have varying degrees of decreased ADAMTS 13 activity. ADAMTS 13 activity is strongly negatively correlated with disease severity of pediatric sepsis syndrome, whereas decreased ADAMTS 13 activity on day 1 is associated with increased risk of mortality.
PMCID: PMC4845826  PMID: 27100422
2.  Low Serum Fetuin-A as a Biomarker to Predict Pneumococcal Necrotizing Pneumonia and Hemolytic Uremic Syndrome in Children 
Medicine  2016;95(13):e3221.
Streptococcus pneumoniae, a neuraminidase-producing pathogen, can cause invasive pneumococcal disease (IPD) with or without hemolytic uremic syndrome (HUS) in humans.
We aimed to identify serum sialoglycoproteins that are targeted by neuraminidases in severe pneumococcal infection. We hypothesized that serum sialoglycoprotein such as fetuin-A can serve as a biomarker to predict IPD or HUS.
We constructed serum sialoglycoprotein profiles before and after pneumococcal neuraminidase treatment using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a proteomic approach. An observational study was conducted using clinical data and serum samples from pediatric patients with pneumococcal infection to verify the predictive role of fetuin-A in IPD. Serum fetuin-A levels were determined by enzyme-linked immunosorbent assay.
The most abundant serum sialoglycoproteins identified by LC-MS/MS after neuraminidase treatment and peanut lectin capture were immunoglobulins, apolipoproteins, fibrinogens, keratins, complement system proteins, and fetuin-A. Serum fetuin-A levels in the HUS patients were significantly lower (207 ± 80 mg/L, P < 0.001) than in patients with lobar pneumonia (610 ± 190 mg/L) as well as the healthy controls (630 ± 250 mg/L). In comparing HUS with necrotizing pneumonia and lobar pneumonia, the ROC area under the curve was 0.842; a cutoff value of 298 mg/L yielded sensitivity of 92.9% (95% CI: 68.5–98.7%) and specificity of 71.9% (95% CI: 54.6–84.4%).
This observational study with validation cohorts of patients with HUS, complicated pneumonia, and lobar pneumonia demonstrates the high performance of low serum fetuin-A levels as a biomarker to predict severe IPD and HUS in children.
PMCID: PMC4998552  PMID: 27043691
3.  Pneumococcal disease and use of pneumococcal vaccines in Taiwan 
The use of pneumococcal vaccine plays an important role for prevention of invasive pneumococcal disease (IPD). However, introducing the pneumococcal vaccine into the national immunization program (NIP) is complex and costly. The strategy of progressively integrating the pneumococcal conjugate vaccine (PCV) into the NIP in Taiwan provides valuable experience for policy makers. The 7-valent PCV (PCV7) was first available in Taiwan in late 2005. PCV7 was first provided free to children with underlying diseases, those in vulnerable socioeconomic status, and those with inadequate health care resources. The catch-up immunization program with the 13-valent PCV was launched in 2013 and the national pneumococcal immunization program was implemented in 2015. Children aged 2-5 years had the highest incidence of IPD among pediatric population in Taiwan. Although the incidence of IPD caused by PCV7 serotypes has declined, the overall incidence of IPD remained high in the context of PCV7 use in the private sector. A surge of IPD caused by serotype 19A occurred, accounting for 53.6% of IPD cases among children aged ≤ 5 years in 2011-2012. After the implementation of the national pneumococcal immunization program, serogroup 15 has become the leading serogroup for IPD in children. Continued surveillance is necessary to monitor the serotype epidemiology in Taiwan.
PMCID: PMC4524896  PMID: 26273570
Pneumococcal vaccines; Serotype 19A; Invasive pneumococcal disease; Immunization program; Serotype replacement
4.  Clinical, Microbiologic, and Outcome Analysis of Mycotic Aortic Aneurysm: The Role of Endovascular Repair 
Surgical Infections  2014;15(3):290-298.
Background: Mycotic aortic aneurysm (MAA) is an infrequent but devastating form of vascular disease.
Methods: We conducted a retrospective cohort study at a major medical center to identify independent risk factors for MAA and to provide opinions about treating it. The study population consisted of 43 patients who had had 44 MAAs over a period of 15 y.
Results: All of the patients had positive blood cultures, radiologic findings typical of MAA, and clinical signs of infection (leukocytosis, fever, and elevated C-reactive protein). The mean age of the patients was 63.8±10.6 y and the mean period of their follow up was 35.7±39.3 mo. Twenty-nine patients with MAAs underwent traditional open surgery, 11 others received endovascular stent grafts, and four MAAs were managed conservatively. The most frequent causative pathogens were Salmonella (36/44 patients [81.8%]), in whom organisms of Salmonella serogroup C (consisting mainly of S. choleraesuis) were identified in 14 patients, organisms of Salmonella serogroup D were identified in 13 patients, and species without serogroup information were identified in nine patients. The overall mortality in the study population was 43.2% (with an aneurysm-related mortality of 18.2%, surgically related mortality of 13.6%, and in-hospital mortality of 22.7%).
Conclusions: Shock is a risk factor for operative mortality. Misdiagnosis and treatment of MAA as low back pain, co-existing connective-tissue disease such as systemic lupus erythematosus and rheumatoid arthritis, and Salmonella serogroup C-associated bacteremia are risk factors for aneurysm-related death. Endovascular repair should be considered as an alternative option to the open repair of MAA.
PMCID: PMC4063379  PMID: 24800865
5.  Decline of Salmonella enterica Serotype Choleraesuis Infections, Taiwan 
Emerging Infectious Diseases  2014;20(4):715-716.
PMCID: PMC3966396  PMID: 24655558
Salmonella enterica serotype Choleraesuis; invasive infection; antimicrobial resistance; bacteria; Taiwan; zoonoses
6.  Increasing Ceftriaxone Resistance in Salmonellae, Taiwan 
Emerging Infectious Diseases  2011;17(6):1086-1090.
In Taiwan, despite a substantial decline of Salmonella enterica serotype Choleraesuis infections, strains resistant to ciprofloxacin and ceftriaxone persist. A self-transferable blaCMY-2-harboring IncI1 plasmid was identified in S. enterica serotypes Choleraesuis, Typhimurium, Agona, and Enteritidis and contributed to the overall increase of ceftriaxone resistance in salmonellae.
PMCID: PMC3358216  PMID: 21749777
Salmonella enterica serotype Choleraesuis; nontyphoidal Salmonella; antimicrobial resistance; salmonellae; CMY-2; Tn6092; conjugative resistance plasmid; bacteria; Taiwan; letter
7.  High Rate of Reduced Susceptibility to Ciprofloxacin and Ceftriaxone among Nontyphoid Salmonella Clinical Isolates in Asia▿  
This multinational study from Asia revealed that reduced susceptibility to ciprofloxacin (MIC, 0.125 to 1 μg/ml) in nontyphoid Salmonella isolates was common in Taiwan (48.1%) and Thailand (46.2%) and in S. enterica serotype Choleraesuis (68.8%) and S. Virchow (75.0%) from all countries. Reduced susceptibility to ceftriaxone (MIC, 2 to 8 μg/ml) remained uncommon in Asia, except in Taiwan (38.0%) or in S. Typhimurium (25.0%) from all countries.
PMCID: PMC2687261  PMID: 19332677
8.  Molecular Epidemiology and Clinical Manifestations of Viral Gastroenteritis in Hospitalized Pediatric Patients in Northern Taiwan▿  
Journal of Clinical Microbiology  2007;45(6):2054-2057.
By reverse transcription-PCR or PCR, among 257 children with nonbacterial acute gastroenteritis (AGE), rotavirus, norovirus, astrovirus, enteric adenovirus, and multiple viruses were identified in 78 (30.4%), 21 (8.2%), 7 (2.7%), 51 (19.8%), and 53 (20.6%) patients, respectively. Higher disease severity was found for AGE caused by multiple viruses and by rotavirus alone. The majority of rotaviruses isolated from 2004 to 2006 belonged to genotypes G1 (20.4%), G2 (16.5%), G3 (27.2%), and G9 (21.4%).
PMCID: PMC1933068  PMID: 17442805
9.  Repression of Rgg But Not Upregulation of LacD.1 in emm1-type covS Mutant Mediates the SpeB Repression in Group A Streptococcus 
CovR/CovS is an important two-component regulatory system in human pathogen group A Streptococcus (GAS). Epidemiological studies have shown that inactivation of the sensor kinase CovS is correlated with invasive clinical manifestations. The phosphorylation level of response regulator CovR decreases dramatically in the absence of CovS, resulting in the derepression of virulence factor expression and an increase in bacterial invasiveness. Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease and is negatively regulated by CovR; however, the expression of SpeB is almost completely repressed in the covS mutant. The present study found that in the emm1-type A20 strain, non-phosphorylated CovR acts as a transcriptional repressor for SpeB-positive regulator Rgg. In addition, the expression of Rgg-negative regulator LacD.1 is upregulated in the covS mutant. These results suggest that inactivation of Rgg in the covS mutant would directly mediate speB repression. The current study showed that overexpression of rgg but not inactivation of lacD.1 in the covS mutant partially restores speB expression, indicating that only rgg repression, but not lacD.1 upregulation, contributes to the speB repression in the covS mutant.
PMCID: PMC5126071  PMID: 27965655
group A Streptococcus; CovR/CovS; Rgg; LacD.1; SpeB
10.  Hyperinvasiveness of Salmonella enterica serovar Choleraesuis linked to hyperexpression of type III secretion systems in vitro 
Scientific Reports  2016;6:37642.
Salmonella enterica serovars Choleraesuis and Typhimurium are among the non-typhoid Salmonella serovars that are important zoonotic pathogens. In clinical observation, S. Typhimurium typically causes diarrheal diseases; however, S. Choleraesuis shows high predilection to cause bacteremia. The mechanism why S. Choleraesuis is more invasive to humans remains unknown. In this study, we compared the S. Typhimurium LT2 and S. Choleraesuis SC-B67 proteomes through stable isotope labeling of amino acid in cell culture (SILAC). In SILAC, the expression of many virulence proteins in two type III secretion systems (T3SSs) were significantly higher in S. Choleraesuis than in S. Typhimurium. Similar differences were also found at the transcriptional level. Compared to S. Typhimurium, S. Choleraesuis showed a higher penetration level to Caco-2 (>100-fold) and MDCK (>10-fold) monolayers. In mice after oral challenge, the invasion of spleen and liver was also higher in S. Choleraesuis than in S. Typhimurium. The transcription of hilD in S. Choleraesuis was increased in physiological (1 mM) or high (10 mM) concentrations of Mg2+, but not in low (8 μM) concentration. We conclude that S. Choleraesuis showed hyperinvasiveness in cellular as well as mouse models due to hyperexpression of T3SS genes.
PMCID: PMC5122882  PMID: 27886215
11.  Shanghai fever: a distinct Pseudomonas aeruginosa enteric disease 
Gut  2013;63(5):736-743.
Shanghai fever, a community-acquired enteric illness associated with sepsis caused by Pseudomonas aeruginosa, was first described in 1918. The understanding of Shanghai fever is incomplete.
To delineate the clinical features and to examine the host and microbial factors associated with Shanghai fever.
We prospectively enrolled 27 consecutive previously healthy children with community-acquired P aeruginosa enteritis and sepsis between July 2003 and June 2012. An immunological investigation, including measurement of serum immunoglobulin levels and lymphocyte subpopulations, was performed. The clonal relationship of bacterial isolates was determined by multilocus sequence typing (MLST) and the virulence of isolates was measured using cellular and animal models.
The median age of the patients was 7 months; 24 (89%) were aged <1 year. The most common clinical manifestations were fever (100%), diarrhoea (96%) and shock (81%). Leucopenia, thrombocytopenia, high C-reactive protein levels, coagulopathy and hypoalbuminaemia were the key laboratory findings. Necrotising enteritis with or without bowel perforation, ecthyma gangrenosum and seizures were main complications. The death rate was 15%. No common primary immune deficiency was identified. MLST genotypes indicated that isolates from Shanghai fever were non-clonal, but they shared similar phenotypes which were invariably cytotoxic, invasive and adhesive in cellular experiments and caused prolonged gut colonisation and more death than respiratory and laboratory control strains in mice.
Shanghai fever is a sporadic community-acquired disease of previously healthy infants that manifests as sepsis associated with P aeruginosa enteric disease. Both host and microbial factors play a role in pathogenesis.
PMCID: PMC3995289  PMID: 23943780
Bacterial Infection; Bacterial Pathogenesis; Infectious Diarrhoea; Sepsis; Enteric Infections
12.  An extract from Taxodium distichum targets hemagglutinin- and neuraminidase-related activities of influenza virus in vitro 
Scientific Reports  2016;6:36015.
Influenza virus remains an emerging virus and causes pandemics with high levels of fatality. After screening different plant extracts with potential anti-influenza activity, a water extract of Taxodium distichum stems (TDSWex) showed excellent activity against influenza viruses. The EC50 of TDSWex was 0.051 ± 0.024 mg/mL against influenza virus A/WSN/33. TDSWex had excellent antiviral efficacy against various strains of human influenza A and B viruses, particularly oseltamivir-resistant clinical isolates and a swine-origin influenza strain. We observed that the synthesis of viral RNA and protein were inhibited in the presence of TDSWex. The results of the time-of-addition assay suggested that TDSWex inhibited viral entry and budding. In the hemagglutination inhibition assay, TDSWex inhibited the hemagglutination of red blood cells, implying that the extract targeted hemagglutin-related functions such as viral entry. In the attachment and penetration assay, TDSWex showed antiviral activity with EC50s of 0.045 ± 0.026 and 0.012 ± 0.003 mg/mL, respectively. In addition, TDSWex blocked neuraminidase activity. We conclude that TDSWex has bimodal activities against both hemagglutinin and neuraminidase during viral replication.
PMCID: PMC5086851  PMID: 27796330
13.  Complicated norovirus infection and assessment of severity by a modified Vesikari disease score system in hospitalized children 
BMC Pediatrics  2016;16:162.
Norovirus (NoV) GII.4 is the most common genotype for norovirus gastroenteritis worldwide. New variants or subgenotypes are continuously emerging, thus posing a serious threat to child health.
We compared retrospectively the clinical manifestations and complications of norovirus gastroenteritis in children from April, 2004 through December, 2012. NoV variants were analyzed to investigate the association of circulating viral strains with the complications. A modified disease severity score system based on Vesikari score system was devised and to evaluate disease severity.
Compared to the outbreak in 2004/2005 winter, significant higher incidence of complications in the later periods are: convulsive disorder (p < 0.001) in 2006/2007 winter gastrointestinal hemorrhage (p = 0.047) and severe abdominal pain or irritability (p = 0.033) in 2008/09/10 winter; gastrointestinal hemorrhage (p = 0.030), severe abdominal pain or irritability (p = 0.014), and prominent hyperthermia (fever >39 °C, p = 0.001) in 2011/2012 winter. GII.4 Den_Haag_2006b, GII.4 2010, GII.4 Sydney 2012, and GII.4 2012b were the predominant strains in the outbreaks after 2006. By the modified severity score system, severe norovirus disease occurred in 28.5 %, 32 %, 33.3 %, and 30.2 % of the patients in the four periods. A longer duration of hospitalization (p = 0.02) were found in those with high score irrespective of the year of admission.
Our study demonstrated NoV outbreaks in northern Taiwan caused by different GII.4 variants that were associated with specific complications and uncommon clinical presentations. A modified severity score system first proposed in this study was able to identify severe cases with a longer hospital stay in NoV-infected children.
PMCID: PMC5053206  PMID: 27716222
Gastroenteritis; Norovirus; Complications; Disease severity score
14.  Safety and immunogenicity of an intramuscular quadrivalent influenza vaccine in children 3 to 8 y of age: A phase III randomized controlled study 
Human Vaccines & Immunotherapeutics  2016;12(12):3072-3078.
A quadrivalent, inactivated, split-virion influenza vaccine containing a strain from both B lineages (IIV4) has been developed, but its safety and immunogenicity in young children has not been described. This was a phase III, randomized, double-blind, active-controlled, multi-center study to examine the immunogenicity and safety of IIV4 in children 3–8 y of age (EudraCT no. 2011-005374-33). Participants were randomized 5:1:1 to receive the 2013/2014 Northern Hemisphere formulation of IIV4, an investigational trivalent comparator (IIV3) containing the B/Victoria lineage strain, or the licensed Northern Hemisphere IIV3 containing the B/Yamagata lineage strain. Participants who had not previously received a full influenza vaccination schedule received 2 doses of vaccine 28 d apart; all others received a single dose. 1242 children were included. For all 4 strains, IIV4 induced geometric mean haemagglutination inhibition titres non-inferior to those induced by the IIV3 comparators. For both B strains, geometric mean antibody titres induced by IIV4 were superior to those induced by the IIV3 with the alternative lineage strain. Similar proportions of participants vaccinated with IIV4 and IIV3 reported solicited injection-site reactions, solicited systemic reactions, and vaccine-related adverse events. A single vaccine-related serious adverse event, thrombocytopenia, was reported 9 d after vaccination with IIV4 and resolved without sequelae. In conclusion, in children aged 3–8 y who received one dose or 2 doses 28 d apart, IIV4 had an acceptable safety profile, was as immunogenic as IIV3 for the shared strains, and had superior immunogenicity for the additional B strain.
PMCID: PMC5215516  PMID: 27565435
children; haemagglutination inhibition; immunogenicity; phase III clinical trial; quadrivalent inactivated influenza vaccine; safety
15.  Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein 
Journal of Virology  2016;90(3):1424-1438.
Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3′ untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication.
IMPORTANCE Enterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication.
PMCID: PMC4719623  PMID: 26581983
16.  Inhibition of endosomal fusion activity of influenza virus by Rheum tanguticum (da-huang) 
Scientific Reports  2016;6:27768.
Rhubarb (Rheum tanguticum; da-huang in Chinese medicine) is a herbal medicine that has been used widely for managing fever and removing toxicity. In this study, we investigated how rhubarb inhibits influenza virus during the early stage of the infectious cycle using different functional assays. A non-toxic ethanolic extract of rhubarb (Rex) inhibited several H1N1 subtypes of influenza A viruses in Madin–Darby canine kidney cells, including strains that are clinically resistant to oseltamivir. Time course analysis of Rex addition showed that viral entry was one of the steps that was inhibited by Rex. We also confirmed that Rex effectively inhibited viral attachment and penetration into the host cells. The inhibition of red blood cell haemolysis and cell–cell fusion by Rex suggests that Rex may block haemagglutinin-mediated fusion (virus–endosome fusion) during the fusion/uncoating step. Rex has the capacity to inhibit influenza viruses by blocking viral endocytosis. Thus, rhubarb might provide an alternative therapeutic approach when resistant viruses become more prevalent.
PMCID: PMC4908592  PMID: 27302738
17.  Regulation of Motility and Phenazine Pigment Production by FliA Is Cyclic-di-GMP Dependent in Pseudomonas aeruginosa PAO1 
PLoS ONE  2016;11(5):e0155397.
The transcription factor FliA, also called sigma 28, is a major regulator of bacterial flagellar biosynthesis genes. Growing evidence suggest that in addition to motility, FliA is involved in controlling numerous bacterial behaviors, even though the underlying regulatory mechanism remains unclear. By using a transcriptional fusion to gfp that responds to cyclic (c)-di-GMP, this study revealed a higher c-di-GMP concentration in the fliA deletion mutant of Pseudomonas aeruginosa than in its wild-type strain PAO1. A comparative analysis of transcriptome profiles of P. aeruginosa PAO1 and its fliA deletion mutant revealed an altered expression of several c-di-GMP-modulating enzyme-encoding genes in the fliA deletion mutant. Moreover, the downregulation of PA4367 (bifA), a Glu-Ala-Leu motif-containing phosphodiesterase, in the fliA deletion mutant was confirmed using the β-glucuronidase reporter gene assay. FliA also altered pyocyanin and pyorubin production by modulating the c-di-GMP concentration. Complementing the fliA mutant strain with bifA restored the motility defect and pigment overproduction of the fliA mutant. Our results indicate that in addition to regulating flagellar gene transcription, FliA can modulate the c-di-GMP concentration to regulate the swarming motility and phenazine pigment production in P. aeruginosa.
PMCID: PMC4866697  PMID: 27175902
18.  Randomized Open Trial Comparing 2-Dose Regimens of the Human Papillomavirus 16/18 AS04-Adjuvanted Vaccine in Girls Aged 9–14 Years Versus a 3-Dose Regimen in Women Aged 15–25 Years 
The Journal of Infectious Diseases  2016;214(4):525-536.
Background. This randomized, open trial compared regimens including 2 doses (2D) of human papillomavirus (HPV) 16/18 AS04-adjuvanted vaccine in girls aged 9–14 years with one including 3 doses (3D) in women aged 15–25 years.
Methods. Girls aged 9–14 years were randomized to receive 2D at months 0 and 6 (M0,6; (n = 550) or months 0 and 12 (M0,12; n = 415), and women aged 15–25 years received 3D at months 0, 1, and 6 (n = 482). End points included noninferiority of HPV-16/18 antibodies by enzyme-linked immunosorbent assay for 2D (M0,6) versus 3D (primary), 2D (M0,12) versus 3D, and 2D (M0,6) versus 2D (M0,12); neutralizing antibodies; cell-mediated immunity; reactogenicity; and safety. Limits of noninferiority were predefined as <5% difference in seroconversion rate and <2-fold difference in geometric mean antibody titer ratio.
Results. One month after the last dose, both 2D regimens in girls aged 9–14 years were noninferior to 3D in women aged 15–25 years and 2D (M0,12) was noninferior to 2D (M0,6). Geometric mean antibody titer ratios (3D/2D) for HPV-16 and HPV-18 were 1.09 (95% confidence interval, .97–1.22) and 0.85 (.76–.95) for 2D (M0,6) versus 3D and 0.89 (.79–1.01) and 0.75 (.67–.85) for 2D (M0,12) versus 3D. The safety profile was clinically acceptable in all groups.
Conclusions. The 2D regimens for the HPV-16/18 AS04-adjuvanted vaccine in girls aged 9–14 years (M0,6 or M0,12) elicited HPV-16/18 immune responses that were noninferior to 3D in women aged 15–25 years.
Clinical Trials Registration. NCT01381575.
PMCID: PMC4957434  PMID: 26908726
2-dose schedule; HPV; human papillomavirus; immunogenicity; cervical cancer; AS04; Adjuvant System containing 50 µg 3-O-desacyl-4′-monophosphoryl lipid A (MPL) adsorbed on aluminum salt (500 µg Al3+); IgG; CMI; cell-mediated immunity; GMT; geometric mean antibody titer
19.  Clinical Characteristics and Risk Factor Analysis for Lower-Extremity Amputations in Diabetic Patients With Foot Ulcer Complicated by Necrotizing Fasciitis 
Medicine  2015;94(44):e1957.
Patients with diabetes are at a higher risk of having diabetic foot ulcers (DFUs) or necrotizing fasciitis (NF). The present study aims to examine the clinical characteristics and associated risk factors for lower-extremity amputation (LEA) in patients with DFU complicated by NF.
We retrospectively reviewed patients treated at a major diabetic foot center in Taiwan between 2009 and 2014. Of the 2265 cases 110 had lower-extremity NF. Limb preservation outcomes were classified as major LEA, minor LEA, or limb-preserved. Clinical characteristics, laboratory data, and bacterial culture results were collected for analysis.
Of the 110 patients with NF, 100 had concomitant DFUs (NF with DFU) and the remaining 10 had no DFU (NF without DFU). None of the NF patients without DFU died nor had their leg amputated. Two NF patients with DFU died of complications. The amputation rate in the surviving 98 NF patients with DFU was 72.4% (46.9% minor LEA and 25.5% major LEA). Seventy percent of the NF patients without DFU had monomicrobial infections (60% with Streptococcus species), and 81.4% NF patients with DFU had polymicrobial infections. Anaerobic organisms were identified in 66% of the NF patients with DFU. Multinomial logistic regression analysis revealed an association between high-grade Wagner wound classification (Wagner 4 and Wagner 5) and LEA (adjusted odds ratio [aOR] = 21.856, 95% confidence interval [95% CI] = 1.625–203.947, P = 0.02 and aOR = 20.094, 95% CI = 1.968–205.216, P = 0.01 for major and minor LEA, respectively) for NF patients with DFU. In addition, a lower serum albumin level was associated with major LEA (OR = 0.066, P = 0.002).
In summary, once DFUs were complicated by NF, the risk of amputation increased. Empirical treatment for NF patients with DFU should cover polymicrobial infections, including anaerobic organisms. The high-grade wound classification and low serum albumin level were associated with LEA.
PMCID: PMC4915905  PMID: 26554804
20.  Evolving pneumococcal serotypes and sequence types in relation to high antibiotic stress and conditional pneumococcal immunization 
Scientific Reports  2015;5:15843.
In Taiwan, beginning in 2013, the 13-valent pneumococcal conjugate vaccine (PCV13) was provided free of charge to children 2–5 years of age. In 2014, this was extended to children 1–5 years old. During 2012–2014, 953 cases of culture-confirmed pneumococcal disease (CCPD), including 104 invasive pneumococcal disease (IPD), were prospectively identified and analyzed at a 3,700-bed hospital in Taiwan. From 2012 to 2014, the incidence per 10,000 admissions decreased from 26.7 to 20.4 for CCPD (P < 0.001) and from 3.2 to 1.9 for IPD (P < 0.05). Significant reduction of PCV13 serotypes was firstly noted in children in 2013 and extended to both paediatric and adult populations in 2014. Simultaneously, the incidence per 10,000 admissions of non-PCV13 serotypes increased from 6.1 in 2012 to 9.3 in 2014 (P < 0.005). The most prevalent non-PCV13 serotypes were 15A, 15B, and 23A, each containing a predominant clone, ST6315A, ST8315B, and ST33823A. From 2012 to 2014, isolates with penicillin minimum inhibitory concentrations >2 mg/L decreased from 27.8% to 8.1% (P < 0.001) among all isolates. PCV13 immunization in young children demonstrated an early protective effect in all ages. However, in the elderly, the effect was compromised by an emergence of non-PCV13 serotypes.
PMCID: PMC4629140  PMID: 26522920
21.  Is the whole greater than the sum of its parts? De novo assembly strategies for bacterial genomes based on paired-end sequencing 
BMC Genomics  2015;16(1):648.
Whole genome sequence construction is becoming increasingly feasible because of advances in next generation sequencing (NGS), including increasing throughput and read length. By simply overlapping paired-end reads, we can obtain longer reads with higher accuracy, which can facilitate the assembly process. However, the influences of different library sizes and assembly methods on paired-end sequencing-based de novo assembly remain poorly understood.
We used 250 bp Illumina Miseq paired-end reads of different library sizes generated from genomic DNA from Escherichia coli DH1 and Streptococcus parasanguinis FW213 to compare the assembly results of different library sizes and assembly approaches. Our data indicate that overlapping paired-end reads can increase read accuracy but sometimes cause insertion or deletions. Regarding genome assembly, merged reads only outcompete original paired-end reads when coverage depth is low, and larger libraries tend to yield better assembly results. These results imply that distance information is the most critical factor during assembly. Our results also indicate that when depth is sufficiently high, assembly from subsets can sometimes produce better results.
In summary, this study provides systematic evaluations of de novo assembly from paired end sequencing data. Among the assembly strategies, we find that overlapping paired-end reads is not always beneficial for bacteria genome assembly and should be avoided or used with caution especially for genomes containing high fraction of repetitive sequences. Because increasing numbers of projects aim at bacteria genome sequencing, our study provides valuable suggestions for the field of genomic sequence construction.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1859-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4552406  PMID: 26315384
22.  Impact of Molecular Epidemiology and Reduced Susceptibility to Glycopeptides and Daptomycin on Outcomes of Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia 
PLoS ONE  2015;10(8):e0136171.
Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia was associated with high mortality, but the risk factors associated with mortality remain controversial.
A retrospective cohort study was designed. All patients with MRSA bacteremia admitted were screened and collected for their clinical presentations and laboratory characteristics. Minimum inhibitory concentration (MIC) and staphylococcal cassette chromosome mec (SCCmec) type of bacterial isolates were determined. Risk factors for mortality were analyzed.
Most MRSA isolates from the 189 enrolled patients showed reduced susceptibility to antibiotics, including MIC of vancomycin ≥ 1.5 mg/L (79.9%), teicoplanin ≥ 2 mg/L (86.2%), daptomycin ≥ 0.38 mg/L (73.0%) and linezolid ≥ 1.5 mg/L (64.0%). MRSA with vancomycin MIC ≥ 1.5 mg/L and inappropriate initial therapy were the two most important risk factors for mortality (both P < 0.05; odds ratio = 7.88 and 6.78). Hospital-associated MRSA (HA-MRSA), carrying SCCmec type I, II, or III, was associated with reduced susceptibility to vancomycin, teicoplanin or daptomycin and also with higher attributable mortality (all P < 0.05). Creeping vancomycin MIC was linked to higher MIC of teicoplanin and daptomycin (both P < 0.001), but not linezolid (P = 0.759).
Giving empirical broad-spectrum antibiotics for at least 5 days to treat catheter-related infections, pneumonia, soft tissue infection and other infections was the most important risk factor for acquiring subsequent HA-MRSA infection. Choice of effective anti-MRSA agents for treating MRSA bacteremia should be based on MIC of vancomycin, teicoplanin and daptomycin. Initiation of an effective anti-MRSA agent without elevated MIC in 2 days is crucial for reducing mortality.
PMCID: PMC4546585  PMID: 26295150
23.  Nitric oxide maintains cell survival of Trichomonas vaginalis upon iron depletion 
Parasites & Vectors  2015;8:393.
Iron plays a pivotal role in the pathogenesis of Trichomonas vaginalis, the causative agent of highly prevalent human trichomoniasis. T. vaginalis resides in the vaginal region, where the iron concentration is constantly changing. Hence, T. vaginalis must adapt to variations in iron availability to establish and maintain an infection. The free radical signaling molecules reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proven to participate in iron deficiency in eukaryotes. However, little is known about the roles of these molecules in iron-deficient T. vaginalis.
T. vaginalis cultured in iron-rich and -deficient conditions were collected for all experiments in this study. Next generation RNA sequencing was conducted to investigate the impact of iron on transcriptome of T. vaginalis. The cell viabilities were monitored after the trophozoites treated with the inhibitors of nitric oxide (NO) synthase (L-NG-monomethyl arginine, L-NMMA) and proteasome (MG132). Hydrogenosomal membrane potential was measured using JC-1 staining.
We demonstrated that NO rather than ROS accumulates in iron-deficient T. vaginalis. The level of NO was blocked by MG132 and L-NMMA, indicating that NO production is through a proteasome and arginine dependent pathway. We found that the inhibition of proteasome activity shortened the survival of iron-deficient cells compared with untreated iron-deficient cells. Surprisingly, the addition of arginine restored both NO level and the survival of proteasome-inhibited cells, suggesting that proteasome-derived NO is crucial for cell survival under iron-limited conditions. Additionally, NO maintains the hydrogenosomal membrane potential, a determinant for cell survival, emphasizing the cytoprotective effect of NO on iron-deficient T. vaginalis. Collectively, we determined that NO produced by the proteasome prolonged the survival of iron-deficient T. vaginalis via maintenance of the hydrogenosomal functions.
The findings in this study provide a novel role of NO in adaptation to iron-deficient stress in T. vaginalis and shed light on a potential therapeutic strategy for trichomoniasis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-015-1000-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4513698  PMID: 26205151
Iron-deficiency; Nitric oxide; Arginine; Ubiquitin-proteasome system; Hydrogenosomal membrane potential; Cell survival
24.  Clinical features and outcomes of invasive pneumococcal disease in a pediatric intensive care unit 
BMC Pediatrics  2015;15:85.
Invasive pneumococcal disease (IPD) results in high morbidity and mortality globally each year, although it is a vaccine-preventable disease. This study aimed to characterize the clinical features of IPD in a pediatric intensive care unit (PICU) in Taiwan. The seven-valent pneumococcal conjugate vaccine (PCV7) was introduced in the private sector in October 2005. The estimated coverage rate of PCV7 vaccination in 2010 was 45.5 % among children <5 years of age.
We conducted a retrospective study at a single center in northern Taiwan for invasive pneumococcal disease in a PICU from 2009 to 2013. Demographic characteristics, clinical courses, serotype, antibiotic susceptibility, and outcomes were analyzed.
Over the 5-year study period, 2167 patients were admitted to the PICU; 48 (2.2 %) had IPD. There were 29 female and 19 male patients. Their mean age was 3.7 years (range 0.7–12.5 years, with the peak age at 2–5 years; n = 30, 63 %). Pneumonia was the most frequent type (n = 38, 79 %), followed by meningitis (n = 10, 21 %). In total, three patients died, all within 72 h after admission; the final diagnoses were all meningitis. Thirty-four children with pneumonia received chest tube insertion for pleural effusion drainage. Of them, 22 (65 %) finally still underwent video-assisted thoracoscopic surgery. Eight (17 %) children had hemolytic uremic syndrome, and seven of them underwent hemodialysis. In total, 37 serotypes were detected; 95 % were covered by PCV13. Serotype 19A was most common (54 %) overall; however, in those with meningitis, serotype 19 F was most common.
Meningitis is the most severe type of invasive pneumococcal disease in our pediatric intensive care unit. It may progress rapidly even when subjects are given antibiotics promptly. The most common serotype in meningitis is 19 F, which is vaccine preventable. Thus, universal mass pneumococcal vaccination is still needed.
PMCID: PMC4504450  PMID: 26184113
Hemolytic uremic syndrome (HUS); herd immunity; invasive pneumococcal disease; mortality; pediatric intensive care unit; pneumococcal conjugate vaccine; serotype 19 F; Streptococcus pneumoniae
25.  A Recombinant Horseshoe Crab Plasma Lectin Recognizes Specific Pathogen-Associated Molecular Patterns of Bacteria through Rhamnose 
PLoS ONE  2014;9(12):e115296.
Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.
PMCID: PMC4277298  PMID: 25541995

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