Meri Gorgievski-Hrisoho

Low incidence of respiratory syncytial virus hospitalisations in haemodynamically significant congenital heart disease

Background: Haemodynamically significant congenital heart disease (CHD) is a risk factor for severe respiratory syncytial virus (RSV) disease in young children. Population based data on the incidence of RSV hospitalisations in CHD patients are needed to estimate the potential usefulness of RSV immunoprophylaxis using palivizumab. Aims: (1) To obtain population based RSV hospitalisation rates in children <24 months of age with CHD. (2) To compare these rates with non-CHD patients and with previous studies. (3) To determine the number of patients needed to treat (NNT) with palivizumab to prevent one RSV hospitalisation. Methods: Six year, longitudinal, population based study at an institution, which is the sole provider of primary to tertiary in-patient care for a precisely defined paediatric population. Results: RSV hospitalisation rates (per 100 child-years) in CHD patients aged <6, <12, 12–24, and <24 months of age were 2.5 (95% CI 0.8 to 5.6), 2.0 (0.8 to 3.8), 0.5 (0.1 to 1.8), and 1.3 (0.6 to 2.3), respectively, and the relative risk (RR) in comparison with non-CHD patients was 1.4 (0.6 to 3.1), 1.6 (0.8 to 3.2), 2.7 (0.7 to 9.7), and 1.8 (1.0 to 3.3), respectively. NNT was between 80 (35 to 245) and 259 (72 to 2140) for various age groups. Conclusion: RSV hospitalisation rates in CHD patients were fourfold lower than reported from the USA. Based on these low rates and RR, unrestricted use of palivizumab does not appear to be justified in this study area.

Two-Year Periodicity of Respiratory Syncytial Virus Epidemics in Switzerland

Abstract.Background: The annual respiratory syncytial virus (RSV) epidemics vary in time and severity. The aims of this study were (1) to describe the time-related pattern of RSV epidemics in Switzerland and (2) to deduce the most effective time period for administration of prophylactic measures to high-risk patients. Patients and Methods: Descriptive study of (1) RSV hospitalizations between 1997 and 2001 at a pediatric hospital serving a population of 1 million and (2) of national RSV detection rates reported by diagnostic laboratories between 1988 and 1999. Results: 497 RSV hospitalizations and 8,574 reported RSV detections occurring during four and 12 epidemics, respectively, were analyzed. There was fixed alternation of minor and major epidemics differing in the number of RSV infections (two to fourfold), evolution (median interval from onset to peak 13 weeks, range 4–13 weeks vs 8 weeks, range 7–10 weeks; p = 0.065) and median duration (26 weeks, range 24-29 weeks vs 19.5 weeks, range 18–21 weeks; p = 0.005). For minor epidemics it was estimated that a maximum of 85.6% (range, 79.4–86.6%) of annual RSV infections could be covered by a standard five-dose regimen of the monoclonal anti-RSV antibody palivizumab, if initiated in week 50. During major epidemics the most effective time of initiation would be week 43 (88.7%; range 81.9–94.6%). Conclusion: RSV epidemiology in Switzerland is characterized by fixed biannual variation. In the absence of active RSV surveillance, such periodicity is useful for scheduling RSV prophylaxis and for hospital resources management.

Microbial communities in the respiratory tract of patients with interstitial lung disease

Background Molecular methods based on phylogenetic differences in the 16S rRNA gene are able to characterise the microbiota of the respiratory tract in health and disease. Objectives Our goals were (1) to characterise bacterial communities in lower and upper airways of patients with interstitial lung disease (ILD) and (2) to compare the results with the microbiota of patients with Pneumocystis pneumonia (PCP) and normal controls. Methods We examined the upper and lower respiratory tract of 18 patients with ILD of whom 5, 6, and 7 had idiopathic interstitial pneumonia (IIP), non-IIP and sarcoidosis, respectively. In addition, six immune-compromised patients with PCP and nine healthy subjects were included as controls. Exclusion criteria were recent bacterial/viral respiratory tract infection, HIV-positivity and subjects receiving antibiotic therapy. Bronchoalveolar lavage fluid and oropharyngeal swabs were simultaneously collected, and microbiota was characterised by ultra-deep 16S rRNA gene sequencing. Results The microbiota in lower airways of the majority of patients (30; 90%) primarily consisted of Prevotellaceae, Streptococcaceae and Acidaminococcaceae. α and β diversity measurements revealed no significant differences in airway microbiota composition between the five different groups of patients. Comparison of bacterial populations in upper and lower respiratory tract showed significant topographical discontinuities for 7 (23%) individuals. Conclusions IIP, non-IIP and sarcoidosis are not associated with disordered airway microbiota and a pathogenic role of commensals in the disease process is therefore unlikely. Nevertheless, molecular analysis of the topographical microbiota continuity along the respiratory tract may provide additional information to assist management of individual patients.

Genetic Analyses Reveal a Role for Vitamin D Insufficiency in HCV-Associated Hepatocellular Carcinoma Development

Background Vitamin D insufficiency has been associated with the occurrence of various types of cancer, but causal relationships remain elusive. We therefore aimed to determine the relationship between genetic determinants of vitamin D serum levels and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Methodology/Principal Findings Associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25-hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCV-related HCC development were investigated for 1279 chronic hepatitis C patients with HCC and 4325 without HCC, respectively. The well-known associations between CYP2R1 (rs1993116, rs10741657), GC (rs2282679), and DHCR7 (rs7944926, rs12785878) genotypes and 25(OH)D3 serum levels were also apparent in patients with chronic hepatitis C. The same genotypes of these single nucleotide polymorphisms (SNPs) that are associated with reduced 25(OH)D3 serum levels were found to be associated with HCV-related HCC (P = 0.07 [OR = 1.13, 95% CI = 0.99–1.28] for CYP2R1, P = 0.007 [OR = 1.56, 95% CI = 1.12–2.15] for GC, P = 0.003 [OR = 1.42, 95% CI = 1.13–1.78] for DHCR7; ORs for risk genotypes). In contrast, no association between these genetic variations and liver fibrosis progression rate (P>0.2 for each SNP) or outcome of standard therapy with pegylated interferon-α and ribavirin (P>0.2 for each SNP) was observed, suggesting a specific influence of the genetic determinants of 25(OH)D3 serum levels on hepatocarcinogenesis. Conclusions/Significance Our data suggest a relatively weak but functionally relevant role for vitamin D in the prevention of HCV-related hepatocarcinogenesis.

Sensitive and rapid detection of ganciclovir resistance by PCR based MALDI-TOF analysis

BACKGROUND Cytomegalovirus (CMV) infection is associated with significant morbidity and mortality in transplant recipients. Resistance against ganciclovir is increasingly observed. According to current guidelines, direct drug resistance testing is not always performed due to high costs and work effort, even when resistance is suspected. OBJECTIVES To develop a more sensitive, easy applicable and cost-effective assay as proof of concept for direct drug resistance testing in CMV surveillance of post-transplant patients. STUDY DESIGN Five consecutive plasma samples from a heart transplant patient with a primary CMV infection were analyzed by quantitative real-time polymerase chain reaction (rtPCR) as a surrogate marker for therapy failure, and by direct drug resistance detection assays such as Sanger sequencing and the novel primer extension (PEX) reaction matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) based method. RESULTS This report demonstrates that PEX reaction followed by MALDI-TOF analysis detects the A594V mutation, encoding ganciclovir resistance, ten days earlier compared to Sanger sequencing and more than 30 days prior to an increase in viral load. CONCLUSION The greatly increased sensitivity and rapid turnaround-time combined with easy handling and moderate costs indicate that this procedure could make a major contribution to improve transplantation outcomes.

Immunofluorescence versus xTAG multiplex PCR for the detection of respiratory picornavirus infections in children.

Abstract Background Polymerase chain reaction (PCR) is a sensitive tool for detection of respiratory picornaviruses. However, the clinical relevance of picornavirus detection by PCR is unclear. Immunofluorescence (IF), widely used to detect other respiratory viruses, has recently been introduced as a promising detection method for respiratory picornaviruses. Objectives To compare the clinical manifestations of respiratory picornavirus infections detected by IF with those of respiratory picornavirus infections detected by xTAG multiplex PCR in hospitalized children. Study design During a 1-year period, nasopharyngeal aspirates (NPA) from all children hospitalized due to an acute respiratory infection were prospectively analyzed by IF. All respiratory picornavirus positive IF samples and 100 IF negative samples were further tested with xTAG multiplex PCR. After exclusion of children with co-morbidities and viral co-infections, monoinfections with respiratory picornaviruses were detected in 108 NPA of 108 otherwise healthy children by IF and/or PCR. We compared group 1 children (IF and PCR positive, n =84) with group 2 children (IF negative and PCR positive, n =24) with regard to clinical manifestations of the infection. Results Wheezy bronchitis was diagnosed more often in group 1 than in group 2 (71% vs. 46%, p =0.028). In contrast, group 2 patients were diagnosed more frequently with pneumonia (17% vs. 6%, p =0.014) accompanied by higher levels of C-reactive protein (46mg/l vs. 11mg/l, p =0.009). Conclusions Picornavirus detection by IF in children with acute respiratory infection is associated with the clinical presentation of wheezy bronchitis. The finding of a more frequent diagnosis of pneumonia in picornavirus PCR positive but IF negative children warrants further investigation.

Rapid detection of respiratory picornaviruses in nasopharyngeal aspirates by immunofluorescence assay

Abstract Background Respiratory picornaviruses (enteroviruses and rhinoviruses) are commonly cited as causes of self-limited upper respiratory tract infection. However, it has recently been suggested that they may cause more severe respiratory disease. Immunofluorescence (IF) assays are rapid and inexpensive and are often used for the detection of respiratory viruses. Objectives We sought to develop an IF procedure, using commercially available reagents, for the detection of respiratory picornaviruses directly from nasopharyngeal aspirates (NPA). Study design From 1st November 2006 until 31st October 2007 all NPA from patients with respiratory infection were stained with the Light Diagnostic Pan-Enterovirus Reagent – “Blend” by IF (IF-ENVPAN). Those specimens which tested positive with this stain were further tested (subject to the availability of frozen specimen) with the xTAG respiratory viral panel, a multiplex PCR directed against respiratory picornaviruses, adenovirus (ADV), respiratory sincytial virus (RSV), influenza viruses A and B (IFA and IFB), parainfluenza virus (PIV) 1–4, human metapneumovirus (HMPV) and coronaviruses. Results 241/1122 NPA tested positive by IF-ENVPAN. 143 NPA were available for testing by xTAG respiratory viral panel. The multiplex PCR detected respiratory picornaviruses in 139 NPA, in 126 as the sole viral pathogen. Conclusions Our results indicate the potential of IF-ENVPAN for the laboratory detection of respiratory picornaviruses in clinical specimens. As far as we are aware, this is the first publication of such a method.

Evaluation of two rapid detection assays for identification of respiratory syncytial virus in nasopharyngeal secretions of young children.

Respiratory syncytial virus (RSV) is the single most important lower respiratory tract pathogen in infants and toddlers [1]. Up to 70% of the annual birth cohort are infected during the first year of life [2], and 1–2% are hospitalized annually for RSV bronchiolitis or pneumonia [3, 4]. Since RSV is highly contagious, hospital admission entails infection control measures for prevention of contact and droplet transmission. Thus, clinicians should have available a rapid test that identifies RSV-infected patients quickly and reliably in the emergency room. The test result enables the implementation of adequate infection control precautions at the time the patient is transferred to the floor and provides a rationale for the cohorting of patients with acute respiratory tract disease. In addition, inadequate use of antibiotics may be avoided. Bedside RSV rapid antigen detection systems have been available for several years. Their usefulness in clinical practice has been limited by relatively poor sensitivity. Recently, two new test systems have become available commercially. The purpose of the present study was to compare these new assays with antigen detection using a direct fluorescence assay (DFA). During the 2002/2003 RSV peak season, nasopharyngeal secretions (NPS) from 30 children (median age, 5.4 months; range, 0.5–64 months) referred consecutively to the emergency department of the University of Bern Children’s Hospital for suspected RSV infection were evaluated. NPS specimens were sampled transnasally using a Vygon infant mucus aspirator (Vygon, Ecouen, France), were brought to a total volume of 2.0 ml using sterile normal saline, and were kept at 4 C for a maximum of 24 h before processing. Sample surplus was stored at 80 C. In addition to DFA (Light Diagnostics Respiratory Panel DFA, Chemicon International, USA), which is currently the standard diagnostic procedure used at this institution, aliquots of NPS were tested using (i) the RSV OIA assay (OIA) (Thermo BioStar, USA); (ii) the NOW RSV Test (Binax, USA); (iii) RSV culture using shell vial technology; and, in selected cases, (iv) reverse transcriptase polymerase chain reaction (PCR) (Hexaplex; Prodesse, USA). All tests were carried out according to the manufacturer’s instructions. OIA and NOW were performed in the emergency department by one of the authors (C.W.). DFA, culture and PCR were performed by professional diagnostic microbiology personnel. Detection of RSV by culture was considered unequivocal proof of infection. Culture-negative samples testing positive in one of the antigen detection assays were re-tested using PCR as the arbiter. Thus, the gold standard consisted of a combination of culture and PCR. RSV was detected in 22 of 30 samples. Culture was positive in 20 cases, PCR detected RSV RNA in two additional cases. Overall, antigen test(s) yielded a positive result in six culture-negative samples. PCR was positive in two of these specimens. Table 1 summarizes the performance of the two bedside RSV detection kits, OIA and NOW, in comparison with DFA. In this small population of young children with acute respiratory tract illness and a high pre-test probability of RSV infection, the three antigen detection kits performed similarly (Table 1) and the results were comparable to studies evaluating other rapid antigen detection systems [5, 6, 7, 8]. These test performances are unlikely to be applicable to inter-epidemic periods with low or absent RSV activity and to older individuals in whom viral titers in nasopharyngeal secretions are expected to be lower. While DFA requires specific equipment (i.e., fluorescence microscope) and trained personnel for reading the test results, the test procedures for both OIA and NOW are designed for bedside testing by non-laboratory personnel. Both tests require approximately 15 min for completion. The NOW test procedure is a single manual step, which C. Wyder-Westh · A. Duppenthaler · C. Aebi ()) Department of Pediatrics, University of Bern, Inselspital, 3010 Bern, Switzerland e-mail: christoph.aebi@insel.ch Tel.: +41-31-6329487 Fax: +41-31-632-9468