Adam Meijer

Respiratory syncytial virus and recurrent wheeze in healthy preterm infants

BACKGROUNDnRespiratory syncytial virus (RSV) infection is associated with subsequent recurrent wheeze. Observational studies cannot determine whether RSV infection is the cause of recurrent wheeze or the first indication of preexistent pulmonary vulnerability in preterm infants. The monoclonal antibody palivizumab has shown efficacy in preventing severe RSV infection in high-risk infants.nnnMETHODSnIn the double-blind, placebo-controlled MAKI trial, we randomly assigned 429 otherwise healthy preterm infants born at a gestational age of 33 to 35 weeks to receive either monthly palivizumab injections (214 infants) or placebo (215 infants) during the RSV season. The prespecified primary outcome was the total number of parent-reported wheezing days in the first year of life. Nasopharyngeal swabs were taken during respiratory episodes for viral analysis.nnnRESULTSnPalivizumab treatment resulted in a relative reduction of 61% (95% confidence interval, 56 to 65) in the total number of wheezing days during the first year of life (930 of 53,075 days in the RSV-prevention group [1.8%] vs. 2309 of 51,726 days [4.5%] in the placebo group). During this time, the proportion of infants with recurrent wheeze was 10 percentage points lower in patients treated with palivizumab (11% vs. 21%, P=0.01).nnnCONCLUSIONSnIn otherwise healthy preterm infants, palivizumab treatment resulted in a significant reduction in wheezing days during the first year of life, even after the end of treatment. These findings implicate RSV infection as an important mechanism of recurrent wheeze during the first year of life in such infants. (Funded by Abbott Laboratories and by the Netherlands Organization for Health Research and Development; MAKI Controlled Clinical Trials number, ISRCTN73641710.).

Multicenter comparison trial of DNA extraction methods and PCR assays for detection of Chlamydia pneumoniae in endarterectomy specimens

ABSTRACT The reported rate of detection of Chlamydia pneumoniaeDNA within atherosclerotic lesions by PCR varies between 0 and 100%. In this study, identical sets of coded experimental atheroma samples (n = 15) and spiked controls (n = 5) were analyzed by 16 test methods in nine centers by means of PCR. The positive controls were correctly identified to levels of 1, 0.1, and 0.01 inclusion bodies of C. pneumoniae/ml of tissue homogenate by 16 (100%), 11 (69%), and 3 (19%) of the test methods, respectively. Three out of 16 negative controls (19%) were rated positive. Positivity rates for atheroma samples varied between 0 and 60% for the different test methods, with the maximum concordant result for positivity being only 25% for one carotid artery sample. There was no consistent pattern of positive results among the various laboratories, and there was no correlation between the detection rates and the sensitivity of the assay used.

EMERGENCE AND EPIDEMIC OCCURRENCE OF ENTEROVIRUS 68 RESPIRATORY INFECTIONS IN THE NETHERLANDS IN 2010

Following an increase in detection of enterovirus 68 (EV68) in community surveillance of respiratory infections in The Netherlands in 2010, epidemiological and virological analyses were performed to investigate the possible public health impact of EV68 infections. We retrospectively tested specimens collected from acute respiratory infections surveillance and through three children cohort studies conducted in The Netherlands from 1994 through 2010. A total of 71 of 13,310 (0.5%) specimens were positive for EV68, of which 67 (94%) were from symptomatic persons. Twenty-four (34%) of the EV68 positive specimens were collected during 2010. EV68-positive patients with respiratory symptoms showed significantly more dyspnea, cough and bronchitis than EV68-negative patients with respiratory symptoms. Phylogenetic analysis showed an increased VP1 gene diversity in 2010, suggesting that the increased number of EV68 detections in 2010 reflects a real epidemic. Clinical laboratories should consider enterovirus diagnostics in the differential diagnosis of patients presenting with respiratory symptoms.

Mortality Attributable to 9 Common Infections: Significant Effect of Influenza A, Respiratory Syncytial Virus, Influenza B, Norovirus, and Parainfluenza in Elderly Persons

BACKGROUNDnBecause there may be substantial hidden mortality caused by common seasonal pathogens, we estimated the number of deaths in elderly persons attributable to viruses and bacteria for which robust weekly laboratory surveillance data were available.nnnMETHODSnOn weekly time series (1999-2007) we used regression models to associate total death counts in individuals aged 65-74, 75-84, and ≥85 years (a population of 2.5 million) with pathogen circulation-influenza A (season-specific), influenza B, respiratory syncytial virus (RSV), parainfluenza, enterovirus, rotavirus, norovirus, Campylobacter, and Salmonella-adjusted for extreme outdoor temperatures.nnnRESULTSnInfluenza A and RSV were significantly (P < .05) associated with mortality in all studied age groups; influenza B and parainfluenza were additionally associated in those aged ≥75 years, and norovirus was additionally associated in those aged ≥85 years. The proportions of deaths attributable to seasonal viruses were 6.8% (≥85 years), 4.4% (75-84 years), and 1.4% (65-74 years), but with great variations between years. Influenza occasionally showed lower impact than some of the other viruses.nnnCONCLUSIONSnThe number of different pathogens associated with mortality in the older population increases with increasing age. Besides influenza A and RSV, influenza B, parainfluenza and norovirus may also contribute substantially to elderly mortality.

Influenza Virus Inactivation for Studies of Antigenicity and Phenotypic Neuraminidase Inhibitor Resistance Profiling

ABSTRACT Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and β-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and β-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.

Comparative Analysis of Avian Influenza Virus Diversity in Poultry and Humans during a Highly Pathogenic Avian Influenza A (H7N7) Virus Outbreak

ABSTRACT Although increasing data have become available that link human adaptation with specific molecular changes in nonhuman influenza viruses, the molecular changes of these viruses during a large highly pathogenic avian influenza virus (HPAI) outbreak in poultry along with avian-to-human transmission have never been documented. By comprehensive virologic analysis of combined veterinary and human samples obtained during a large HPAI A (H7N7) outbreak in the Netherlands in 2003, we mapped the acquisition of human adaptation markers to identify the public health risk associated with an HPAI outbreak in poultry. Full-length hemagglutinin (HA), neuraminidase (NA), and PB2 sequencing of A (H7N7) viruses obtained from 45 human cases showed amino acid variations at different codons in HA (n=20), NA (n=23), and PB2 (n=23). Identification of the avian sources of human virus infections based on 232 farm sequences demonstrated that for each gene about 50% of the variation was already present in poultry. Polygenic accumulation and farm-to-farm spread of known virulence and human adaptation markers in A (H7N7) virus-infected poultry occurred prior to farm-to-human transmission. These include the independent emergence of HA A143T mutants, accumulation of four NA mutations, and farm-to-farm spread of virus variants harboring mammalian host determinants D701N and S714I in PB2. This implies that HPAI viruses with pandemic potential can emerge directly from poultry. Since the public health risk of an avian influenza virus outbreak in poultry can rapidly change, we recommend virologic monitoring for human adaptation markers among poultry as well as among humans during the course of an outbreak in poultry.

Macrolide Resistance Determination and Molecular Typing of Mycoplasma pneumoniae in Respiratory Specimens Collected between 1997 and 2008 in The Netherlands

ABSTRACT An important role in the treatment regimens for Mycoplasma pneumoniae infections is played by macrolide (ML) antibiotics. In the past few years, however, a steady increase has been detected in the worldwide prevalence of ML-resistant (MLr) M. pneumoniae strains. It is obvious that this increase necessitates a continuous monitoring of MLr and, when detected, modification of antibiotic treatment modalities. Previously, we developed a pyrosequencing-based assay system for the genetic determination of MLr as well as molecular typing of M. pneumoniae. In this study, the sensitivity of this system was improved by the inclusion of a nested-PCR protocol. The modified system was applied to 114 M. pneumoniae-positive specimens that were obtained from a collection of 4,390 samples from patients with acute respiratory tract infections. These samples were collected between 1997 and 2008 in The Netherlands. The pyrosequencing system produced reliable data in 86% of the specimens that contained >500 M. pneumoniae genome copies/ml of patient sample. Each of these samples contained DNA of the ML-sensitive genotype. While 43% of the samples were found to harbor the M. pneumoniae subtype 1 genotype, 57% contained the subtype 2 genotype. We conclude that the pyrosequencing-based assay system is a useful tool for MLr determination and molecular typing of M. pneumoniae in patient samples. MLr-associated M. pneumoniae genotypes, however, were not found in the current study population.

Transmission of Novel Influenza A(H1N1) in Households with Post-Exposure Antiviral Prophylaxis

Background Despite impressive advances in our understanding of the biology of novel influenza A(H1N1) virus, little is as yet known about its transmission efficiency in close contact places such as households, schools, and workplaces. These are widely believed to be key in supporting propagating spread, and it is therefore of importance to assess the transmission levels of the virus in such settings. Methodology/Principal Findings We estimate the transmissibility of novel influenza A(H1N1) in 47 households in the Netherlands using stochastic epidemic models. All households contained a laboratory confirmed index case, and antiviral drugs (oseltamivir) were given to both the index case and other households members within 24 hours after detection of the index case. Among the 109 household contacts there were 9 secondary infections in 7 households. The overall estimated secondary attack rate is low (0.075, 95%CI: 0.037–0.13). There is statistical evidence indicating that older persons are less susceptible to infection than younger persons (relative susceptibility of older persons: 0.11, 95%CI: 0.024–0.43. Notably, the secondary attack rate from an older to a younger person is 0.35 (95%CI: 0.14–0.61) when using an age classification of ≤12 versus >12 years, and 0.28 (95%CI: 0.12–0.50) when using an age classification of ≤18 versus >18 years. Conclusions/Significance Our results indicate that the overall household transmission levels of novel influenza A(H1N1) in antiviral-treated households were low in the early stage of the epidemic. The relatively high rate of adult-to-child transmission indicates that control measures focused on this transmission route will be most effective in minimizing the total number of infections.

Evaluation of the Xpert Flu A Panel nucleic acid amplification-based point-of-care test for influenza A virus detection and pandemic H1 subtyping

n Abstractn n Backgroundn Influenza antigenic point-of-care (POC) tests are too insensitive for individual reliable diagnosis of influenza virus infections without additional laboratory confirmation. Molecular POC tests could be a valuable alternative.n n n Objectivesn To evaluate the first influenza molecular POC test commercially available, the Cepheid Xpert Flu A Panel designed to simultaneously detect influenza A virus and subtype A(H1N1) 2009 pandemic virus, and compare it with in-house real-time RT-PCR (qRT-PCR).n n n Study designn Clinical specimens positive for influenza virus and influenza virus isolates with different viral loads and of different type and subtype were used to determine the analytical reactivity and sensitivity. A panel of pathogen negative specimens and isolates of 19 different respiratory pathogens were used to determine the analytical specificity.n n n Resultsn Except A(H9N2) virus the Xpert Flu A Panel detected A(H1N1) seasonal and 2009 pandemic, A(H3N2), A(H5N2), A(H5N1) and A(H7N7) viruses and correctly subtyped A(H1N1) 2009 virus. Analytical sensitivity was similar to qRT-PCR in the range of 400–5000 viral particles per ml. However, of most subtypes some specimens with cycle threshold values greater than 30 in qRT-PCR and A(H1N1) 2009 specimens with inconsistent results in the qRT-PCR due to primer or probe mismatches were not detected in the Xpert Flu A Panel. Analytical specificity was 100%.n n n Conclusionsn The Xpert Flu A Panel is the first commercially available POC molecular test for detection of influenza A virus and determination of the H1 2009 subtype and is analytically reasonable sensitive compared with qRT-PCR and highly specific and therefore a welcome alternative to antigenic POC tests.n n

Detection of Excess Influenza Severity: Associating Respiratory Hospitalization and Mortality Data With Reports of Influenza-Like Illness by Primary Care Physicians

OBJECTIVESnWe explored whether excesses in influenza severity can be detected by combining respiratory syndromic hospital and mortality data with data on influenza-like illness (ILI) cases obtained from general practitioners.nnnMETHODSnTo identify excesses in the severity of influenza infections in the population of the Netherlands between 1999 and 2005, we looked for increases in influenza-associated hospitalizations and mortality that were disproportionate to the number of ILI cases reported by general practitioners. We used generalized estimating equation regression models to associate syndromic hospital and mortality data with ILI surveillance data obtained from general practitioners. Virus isolation and antigenic characterization data were used to interpret the results.nnnRESULTSnDisproportionate increases in hospitalizations and mortality (relative to ILI cases reported by general practitioners) were identified in 2003/04 during the A/Fujian/411/02(H3N2) drift variant epidemic.nnnCONCLUSIONSnCombined surveillance of respiratory hospitalizations and mortality and ILI data obtained from general practitioners can capture increases in severe influenza-associated illness that are disproportionate to influenza incidence rates. Therefore, this novel approach should complement traditional seasonal and pandemic influenza surveillance in efforts to detect increases in influenza case fatality rates and percentages of patients hospitalized.