Panton-Valentine Leukocidin-Positive CC398 MRSA in Urban Clinical Settings, the Netherlands.

We report detection of Panton-Valentine leukocidin-positive clonal complex 398 human-origin methicillin-resistant Staphylococcus aureus L2 in the Netherlands. This hypervirulent lineage originated in the Asia-Pacific Region and could become community-acquired in Europe after recurrent travel-related introductions. Genomic surveillance enables early detection to guide control measures and help limit spread of pathogens in urban settings.

Clinical evaluation of viral acute respiratory tract infections in children presenting to the emergency department of a tertiary referral hospital in the Netherlands.

BACKGROUND: The relative incidence and clinical impact of individual respiratory viruses remains unclear among children presenting to the hospital emergency department with acute respiratory tract infection (ARTI). METHODS: During two winter periods, respiratory virus real-time multiplex PCR results were evaluated from children (< 18 years) presenting to the emergency department of a tertiary referral hospital with ARTI that had been sampled within 48 hours of hospital presentation. In an attempt to identify virus-specific distinguishing clinical features, single virus infections were correlated with presenting signs and symptoms, clinical findings and outcomes using multivariate logistic regression. RESULTS: In total, 274 children with ARTI were evaluated and most were aged < 3 years (236/274, 86%). PCR detected respiratory viruses in 224/274 (81.8%) children and included 162 (59%) single and 62 (23%) mixed virus infections. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) single virus infections were common among children aged < 3 years, but proportional differences compared to older children were only significant for RSV (95% CI 1.3-15). Clinical differentiation between viral ARTIs was not possible due to common shared presenting signs and symptoms and the high frequency of mixed viral infections. We observed virus-associated outcome differences among children aged < 3 years. Oxygen treatment was associated with RSV (OR 3.6) and inversely correlated with FLU (OR 0.05). Treatment with steroids (OR 3.4) or bronchodilators (OR 3.4) was associated with HRV. Severe respiratory complications were associated with HRV (OR 3.5) and inversely correlated with RSV (OR 0.24). CONCLUSIONS: Respiratory viruses are frequently detected in young children presenting to the hospital emergency department with ARTI and require PCR diagnosis since presenting signs and symptoms are not discriminant for a type of virus. RSV and HRV bear a high burden of morbidity in the pediatric clinical setting.

Livestock-associated methicillin-resistant Staphylococcus aureus epidemiology, genetic diversity, and clinical characteristics in an urban region.

Objectives: While Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), defined as CC398, is a well-known pathogen among those working with livestock, there are indications that LA-MRSA prevalence among the general population is increasing. However, the clinical impact in urban areas remains unknown. The aim of this study was to assess the genetic epidemiology and clinical characteristics of LA-MRSA in an urban area with a limited livestock population. Methods: In this retrospective study, we evaluated LA-MRSA strains that were collected between 2014 and 2018 from patients who received clinical care in a single urban area in Netherlands. Patient files were assessed for livestock exposure data, clinical findings, and contact tracing information. Next-generation sequencing (NGS) analysis in combination with wgMLST was conducted to assess genetic diversity and relatedness and to detect virulence and resistance genes. Results: LA-MRSA strains were cultured from 81 patients, comprising 12% of all the MRSA strains found in seven study laboratories between 2014 and 2018. No livestock link was found in 76% of patients (n = 61), and 28% of patients (n = 23) had an infection, mostly of the skin or soft tissue. Contact tracing had been initiated in 14 cases, leading to the identification of two hospital transmissions: a cluster of 9 cases and one of 2 cases. NGS data were available for 91% (n = 75) of the patients. wgMLST confirmed the clusters detected via contact tracing (n = 2) and identified 5 additional clusters without a known epidemiological link. Relevant resistance and virulence findings included the PVL virulence gene (3 isolates) and tetracycline resistance (79 isolates). Conclusion: LA-MRSA may cause a relevant burden of disease in urban areas. Surprisingly, most infections in the present study occurred in the absence of a livestock link, suggesting inter-human transmission. These findings and the presence of PVL and other immune evasive complex virulence genes warrant future surveillance and preventative measures.

Hospital transmission of borderline oxacillin-resistant Staphylococcus aureus evaluated by whole-genome sequencing.

Introduction. Staphylococcus aureus is a major cause of hospital infections worldwide. Awareness towards methicillin-resistant S. aureus (MRSA) infections is high but attention towards borderline oxacillin-resistant S. aureus (BORSA) is limited, possibly due to an underestimated clinical relevance, presumption of low incidence and diagnostic limitations.Gap statement. BORSA surveillance has not been routinely implemented, and thus consensus with regard to a definition and infection control measures is lacking.Aim. Our goals were to investigate the occurrence, molecular characteristics and clinical manifestations of BORSA infections in the hospital setting.Methodology. Following an increased incidence in 2016, BORSA cases in 2014/2016 (in our institution) were more specifically evaluated. Medical records were reviewed to investigate epidemiological links, clinical characteristics and outcomes. Resistance and virulence markers were assessed by whole genome sequencing (WGS). Conventional methods: amplified fragment length polymorphism (AFLP) ; multilocus sequence typing (MLST) and multiple locus variable-number tandem repeat analysis (MLVA) were compared with core genome MLST (cgMLST) and whole-genome single nucleotide polymorphism (wgSNP) analysis to confirm genetic clusters.Results. From 2009 to 2013, BORSA comprised 0.1 % of all clinical S. aureus strains. In 2016, the incidence was six-fold higher in comparison to the baseline. Whole-genome SNP and cgMLST confirmed two BORSA clusters among patients with dermatological conditions. Patients with BORSA presented with skin infections, and one case developed a severe invasive infection with a fatal outcome. Infection control measures successfully prevented further transmission in both clusters. WGS findings showed that BORSA strains carried multiple resistance and virulence genes with increased pathogenic potential.Conclusion. WGS and cgMLST effectively characterized and confirmed BORSA clusters among at-risk patients with clinical manifestations ranging from mild skin infections to life-threatening bacteraemia. Clinical awareness and active monitoring are therefore warranted for the timely implementation of infection control measures to prevent BORSA transmission in high-risk patients.

Morbidity and mortality associated with nosocomial transmission of oseltamivir-resistant influenza A(H1N1) virus.

CONTEXT: The sudden emergence and rapid spread of oseltamivir-resistant influenza A(H1N1) viruses with neuraminidase (NA) gene H274Y amino acid substitution is the hallmark of global seasonal influenza since January 2008. Viruses carrying this mutation are widely presumed to exhibit attenuated pathogenicity, compromised transmission, and reduced lethality. OBJECTIVE: To investigate nosocomial viral transmission in a cluster of patients with influenza A(H1N1) virus infection. DESIGN, SETTING, AND PATIENTS: Descriptive outbreak investigation of 2 hematopoietic stem cell transplant recipients and an elderly patient who developed hospital-acquired influenza A virus infection following exposure to an index patient with community-acquired H274Y-mutated influenza A(H1N1) virus infection in a medical ward at a Dutch university hospital in February 2008. The investigation included a review of the medical records, influenza virus polymerase chain reaction and culture, phenotypic oseltamivir and zanamivir susceptibility determination, and hemagglutinin chain 1 (HA(1)) gene and NA gene sequence analysis. MAIN OUTCOME MEASURE: Phylogenetic relationship of patient cluster influenza A(H1N1) viruses and other 2007-2008 seasonal influenza A(H1N1) viruses. RESULTS: Viral HA(1) and NA gene sequence analysis from the 4 patients revealed indistinguishable nucleotide sequences and phylogenetic clustering of H274Y-mutated, oseltamivir-resistant influenza A(H1N1) virus, confirming nosocomial transmission. Influenza virus pneumonia (3 patients) and attributable mortality (2 patients) during active infection was observed in patients with lymphocytopenia at onset. CONCLUSION: Seasonal oseltamivir-resistant influenza A(H1N1) viruses with NA gene H274Y mutation are transmitted and retain significant pathogenicity and lethality in high-risk patients.

Chlamydia caviae Causing Community-Acquired Pneumonia: An Emerging Zoonosis.

We describe a case of community-acquired pneumonia due to Chlamydia caviae in a patient with no direct animal exposure, raising questions about the zoonotic reservoirs and potential transmission routes. Genotyping of Chamydia isolates that cause pneumonia should be performed for a precise diagnosis and to initiate adequate infection control measures.

Prevalence of colistin resistance gene (mcr-1) containing Enterobacteriaceae in feces of patients attending a tertiary care hospital and detection of a mcr-1 containing, colistin susceptible E. coli.

The emergence of the plasmid-mediated mcr colistin resistance gene in the community poses a potential threat for treatment of patients, especially when hospitalized. The aim of this study was to determine the prevalence of all currently known mcr mediated colistin resistance gene in fecal samples of patients attending a tertiary care hospital. From November 2014 until July 2015, fecal samples of patients attending the Leiden University Medical Center were collected and screened for presence of mcr using real-time PCR. Two of 576 patients were positive for mcr-1, resulting in a prevalence of 0.35%, whereas no mcr-2 was found. One of these samples was culture negative, the second sample contained a blaCMY-2 and mcr-1 containing E.coli. This strain belonged to Sequence Type 359 and serotype O177:H21. The mcr-1 containing E.coli was phenotypically susceptible to colistin with a MIC of ≤ 0.25mg/l, due to a 1329bp transposon IS10R inserted into the mcr-1 gene as identified by WGS. This prevalence study shows that mcr-1 is present in low levels patients out of the community attending a hospital. Furthermore the study underlines the importance of phenotypical confirmation of molecular detection of a mcr-1 gene.

Simple dipstick assay for the detection of Salmonella typhi-specific IgM antibodies and the evolution of the immune response in patients with typhoid fever.

Application of a dipstick assay for the detection of Salmonella typhi-specific IgM antibodies on samples collected from S. typhi or S. paratyphi culture-positive patients at the day of admission to the hospital revealed the presence of specific IgM antibodies in 43.5%, 92.9%, and 100% for samples collected 4-6 days, 6-9 days, and > 9 days after the onset of fever, respectively. The mean sensitivity for samples collected an average of 6.6 days after the onset of fever was 65.3%. Culture was positive in 65.9% of the cases with a final clinical diagnosis of typhoid fever. Testing of paired serum samples from culture negative patients with a final clinical diagnosis of typhoid fever resulted in staining of the dipstick in 4.3% of the samples collected at the day of admission to the hospital and in 76.6% of the samples collected one week later, thereby provided strong supporting evidence of typhoid fever by demonstrating seroconversion in a large proportion of the patients. The dipstick assay may thus also be useful for the serodiagnosis of culture-negative patients with clinical signs and symptoms consistent with typhoid fever. The advantages of the dipstick assay are that the result can be obtained on the same day allowing a prompt treatment, that only a small volume of serum is needed, and that no special laboratory equipment is needed to perform the assay. The stability of the reagents of the dipstick and the simplicity of the assay allows its use in places that lack laboratory facilities.

Mass spectrometry-based comparative sequence analysis for the genetic monitoring of influenza A(H1N1)pdm09 virus.

The pandemic influenza A (H1N1) 2009 virus (pH1N1) contains novel gene segments of zoonotic origin that lack virulence and antiviral resistance markers. We aimed to evaluate the applicability and accuracy of mass spectrometry-based comparative sequence analysis (MSCSA) to detect genetic mutations associated with increased virulence or antiviral resistance in pH1N1. During the 2009 H1N1 pandemic, routine surveillance specimens and clinical antiviral resistance monitoring specimens were analyzed. Routine surveillance specimens obtained from 70 patients with pH1N1 infection were evaluated for mutations associated with increased virulence (PB1-F2, PB2 and NS1 genes) or antiviral resistance (neuraminidase gene, NA) using MSCSA and Sanger sequencing. MSCSA and Sanger sequencing results revealed a high concordance (nucleotides >99%, SNPs ∼ 94%). Virulence or resistance markers were not detected in routine surveillance specimens: all identified SNPs encoded for silent mutations or non-relevant amino acid substitutions. In a second study population, the presence of H275Y oseltamivir resistant virus was identified by real-time PCR in 19 of 35 clinical antiviral resistance monitoring specimens obtained from 4 immunocompromised patients with ≥ 14 days prolonged pH1N1 excretion. MSCSA detected H275Y in 24% (4/19) of positive specimens and Sanger sequencing in 89% (17/19). MSCSA only detected H275Y when the mutation was dominant in the analyzed specimens. In conclusion, MSCSA may be used as a rapid screening tool during molecular surveillance of pH1N1. The low sensitivity for the detection of H275Y mutation in mixed viral populations suggests that MSCSA is not suitable for antiviral resistance monitoring in the clinical setting.

Prolonged influenza virus infection during lymphocytopenia and frequent detection of drug-resistant viruses.

The factors that cause prolonged human influenza virus respiratory tract infection and determine its clinical impact and the development of drug-resistant viruses are unclear. During a 3-year period, symptomatic influenza virus excretion for 2 weeks was observed among 8 immunocompromised patients and found to be associated with lymphocytopenia at onset (8 of 8 patients) more often than with granulocytopenia (2 of 8 patients) or monocytopenia (2 of 8 patients). Six (75%) of 8 patients developed influenza lower respiratory tract infection (10 episodes), and receipt of oseltamivir treatment was significantly associated with clinical improvement (8 of 8 episodes vs. 0 of 2 untreated episodes; P = .02). Complete viral clearance was strongly correlated with lymphocyte reconstitution (P = .04) but was never observed during the first 2 weeks after oseltamivir treatment. Neuraminidase inhibitor-resistant influenza viruses emerged in 2 (67%) of 3 patients eligible for resistance analysis. In conclusion, prolonged influenza virus infection was associated with lymphocytopenia, influenza lower respiratory tract infection, and frequent development of drug resistance during antiviral therapy. Clinical improvement in influenza lower respiratory tract infection is observed during oseltamivir treatment, but complete viral clearance is dependent on lymphocyte reconstitution, irrespective of receipt of antiviral medication.