Werner Wunderli

Survival of influenza virus on banknotes.

ABSTRACT Successful control of a viral disease requires knowledge of the different vectors that could promote its transmission among hosts. We assessed the survival of human influenza viruses on banknotes given that billions of these notes are exchanged daily worldwide. Banknotes were experimentally contaminated with representative influenza virus subtypes at various concentrations, and survival was tested after different time periods. Influenza A viruses tested by cell culture survived up to 3 days when they were inoculated at high concentrations. The same inoculum in the presence of respiratory mucus showed a striking increase in survival time (up to 17 days). Similarly, B/Hong Kong/335/2001 virus was still infectious after 1 day when it was mixed with respiratory mucus. When nasopharyngeal secretions of naturally infected children were used, influenza virus survived for at least 48 h in one-third of the cases. The unexpected stability of influenza virus in this nonbiological environment suggests that unusual environmental contamination should be considered in the setting of pandemic preparedness.

A Prospective Hospital-Based Study of the Clinical Impact of Non–Severe Acute Respiratory Syndrome (Non-SARS)–Related Human Coronavirus Infection

Abstract Background. In addition to the human coronaviruses (HCoVs) OC43 and 229E, which have been known for decades to cause infection in humans, 2 new members of this genus have recently been identified: HCoVs NL63 and HKU1. Their impact as a cause of respiratory tract disease in adults at risk for complications needs to be established. Methods. We prospectively assessed the clinical impact of coronavirus infection (excluding cases of severe acute respiratory syndrome) among hospitalized adults. All patients with respiratory disease for whom bronchoalveolar lavage was performed were screened by reverse-transcriptase polymerase chain reaction for the presence of all 4 HCoVs. Results. HCoV was identified in 29 (5.4%) of 540 bronchoalveolar lavage fluid specimens from 279 subjects (mean age, 51 years; 63% male). HCoV OC43 was identified most frequently (12 isolates), followed by 229E (7 isolates), NL63 (6 isolates), and HKU1 (4 isolates). In all, 372 (69%) of 540 bronchoalveolar lavage fluid specimens were negative for bacteria, and 2 persons were coinfected with other respiratory viruses. Transplantation was the most common underlying condition. Of the 29 patients who had HCoV identified in their bronchoalveolar lavage fluid specimens, 9 (31%) were hospitalized in the intensive care unit, 22 (76%) presented to the hospital with acute respiratory symptoms, 16 (55%) presented with cough and/or sputum, 13 (45%) presented with dyspnea, 16 (55%) had experienced prior respiratory infection, and 18 (62%) had a new infiltrate that was visible on chest radiograph. The most frequent final diagnosis was a lower respiratory tract infection. Conclusions. The recently discovered HCoVs NL63 and HKU1 contribute significantly to the overall spectrum of coronavirus infection. Our study also suggests that coronaviruses contribute to respiratory symptoms in most cases.

Amplicon Sequencing and Improved Detection of Human Rhinovirus in Respiratory Samples

ABSTRACT Improved knowledge of the genotypic characteristics of human rhinovirus (HRV) is required, as are nucleic detection assays with the capacity to overcome both the similarities between members of the family Picornaviridae and the wide diversity of different HRV serotypes. The goal of the present study was to investigate the variability and the genotypic diversity of clinical strains circulating in the community. Since most reverse transcription (RT)-PCR assays available cannot differentiate HRV from other members of the family Picornaviridae, we also validated an assay specific for HRV detection. The 5′ noncoding regions of 87 different HRV serotypes and clinical isolates were sequenced. On the basis of sequence analysis and phylogenetic determination, we first confirmed that all clinical isolates were HRV. We then validated a real-time RT-PCR assay that was able not only to detect all HRV serotypes and all clinical isolates tested but also to accurately discriminate between rhinovirus and other viruses from the family Picornaviridae. This assay was negative with isolates of coxsackievirus (types A and B), echovirus, enterovirus, parechovirus, and poliovirus, as well as nonpicornaviruses. Among a series of bronchoalveolar lavage specimens, 4% (7 of 161) were positive by culture, whereas 13% (21 of 161) were positive by RT-PCR. In the present study we showed that to specifically identify HRV in clinical specimens, diagnostic assays need to overcome both the diversities and the similarities of picornaviruses. By sequencing the 5′ noncoding regions of rhinoviruses recovered from clinical specimens, we designed probes that could specifically detect rhinovirus.

Impact of synthetic and biologic disease‐modifying antirheumatic drugs on antibody responses to the AS03‐adjuvanted pandemic influenza vaccine: A prospective, open‐label, parallel‐cohort, single‐center study

OBJECTIVE To identify the determinants of antibody responses to adjuvanted split influenza A (H1N1) vaccines in patients with inflammatory rheumatic diseases. METHODS One hundred seventy-three patients (82 with rheumatoid arthritis, 45 with spondylarthritis, and 46 with other inflammatory rheumatic diseases) and 138 control subjects were enrolled in this prospective single-center study. Controls received 1 dose of adjuvanted influenza A/09/H1N1 vaccine, and patients received 2 doses of the vaccine. Antibody responses were measured by hemagglutination inhibition assay before and 3-4 weeks after each dose. Geometric mean titers (GMTs) and rates of seroprotection (GMT≥40) were calculated. A comprehensive medical questionnaire was used to identify the determinants of vaccine responses and adverse events. RESULTS Baseline influenza A/09/H1N1 antibody levels were low in patients and controls (seroprotection rates 14.8% and 14.2%, respectively). A significant response to dose 1 was observed in both groups. However, the GMT and the seroprotection rate remained significantly lower in patients (GMT 146 versus 340, seroprotection rate 74.6% versus 87%; both P<0.001). The second dose markedly increased antibody titers in patients, with achievement of a similar GMT and seroprotection rate as elicited with a single dose in healthy controls. By multivariate regression analysis, increasing age, use of disease-modifying antirheumatic drugs (DMARDs) (except hydroxychloroquine and sulfasalazine), and recent (within 3 months) B cell depletion treatment were identified as the main determinants of vaccine responses; tumor necrosis factor α antagonist treatment was not identified as a major determinant. Immunization was well tolerated, without any adverse effect on disease activity. CONCLUSION DMARDs exert distinct influences on influenza vaccine responses in patients with inflammatory rheumatic diseases. Two doses of adjuvanted vaccine were necessary and sufficient to elicit responses in patients similar to those achieved with 1 dose in healthy controls.

Astrovirus Infection in Hospitalized Infants with Severe Combined Immunodeficiency after Allogeneic Hematopoietic Stem Cell Transplantation

Infants with severe primary combined immunodeficiency (SCID) and children post-allogeneic hematopoietic stem cell transplantation (HSCT) are extremely susceptible to unusual infections. The lack of generic tools to detect disease-causing viruses among more than 200 potential human viral pathogens represents a major challenge to clinicians and virologists. We investigated retrospectively the causes of a fatal disseminated viral infection with meningoencephalitis in an infant with gamma C-SCID and of chronic gastroenteritis in 2 other infants admitted for HSCT during the same time period. Analysis was undertaken by combining cell culture, electron microscopy and sequence-independent single primer amplification (SISPA) techniques. Caco-2 cells inoculated with fecal samples developed a cytopathic effect and non-enveloped viral particles in infected cells were detected by electron microscopy. SISPA led to the identification of astrovirus as the pathogen. Both sequencing of the capsid gene and the pattern of infection suggested nosocomial transmission from a chronically excreting index case to 2 other patients leading to fatal infection in 1 and to transient disease in the others. Virus-specific, real-time reverse transcription polymerase chain reaction was then performed on different stored samples to assess the extent of infection. Infection was associated with viremia in 2 cases and contributed to death in 1. At autopsy, viral RNA was detected in the brain and different other organs, while immunochemistry confirmed infection of gastrointestinal tissues. This report illustrates the usefulness of the combined use of classical virology procedures and modern molecular tools for the diagnosis of unexpected infections. It illustrates that astrovirus has the potential to cause severe disseminated lethal infection in highly immunocompromised pediatric patients.

Local variability in respiratory syncytial virus disease severity

Respiratory syncytial virus (RSV) lower respiratory tract infections are considered to be a serious disease in centres such as the Sophia Children’s Hospital (Rotterdam, the Netherlands), but as more benign infections in others such as the Geneva Children’s Hospital (Switzerland). To assess the clinical severity of RSV infections at the two sites, 151 infants primarily admitted with a virologically confirmed RSV infection were studied prospectively (1994–5) and retrospectively (1993–4) (55 infants in Geneva and 96 in Rotterdam). Parameters of RSV morbidity which were more severe in Rotterdam during the two winter seasons were apnoea (1.8 v23.9%), the rate of admission to the intensive care unit (3.6v 28.1%), mechanical ventilation (0 v7.3%), and length of stay in hospital (6.8 v 9.1 days). In Geneva higher respiratory rates (59.2 v 51.2), more wheezing (65.5 v 28.8%), and more retractions (81.8v 63.3%) were recorded. Fewer infants younger than 4 months (54.9 v 68.7%), but more breast fed infants (94.1v 38.5%), were admitted in Geneva, although the morbidity parameters remained different after correction for these two variables in multivariate analyses. Thus unidentified local factors influence the pattern and severity of RSV infection and may affect the results of multicentre prophylactic and therapeutic studies.

Monitoring of cytomegalovirus infection in solid-organ transplant recipients by an ultrasensitive plasma PCR assay.

ABSTRACT Early and accurate monitoring of cytomegalovirus (CMV) infection in solid-organ transplant recipients is of major importance. We have assessed the potential benefit of an ultrasensitive plasma-based PCR assay for renal transplant recipients. The pp65 CMV antigen (pp65 Ag) assay using leukocytes was employed as a routine test for the monitoring of CMV in 23 transplant recipients. We compared the pp65 antigenemia with the CMV load quantified by an ultrasensitive PCR (US-PCR) with a limit of detection of 20 CMV DNA copies/ml of plasma. CMV infection was detected in 215 (67%) of 321 plasma samples by the US-PCR compared with 124 (39%) of 321 samples by the pp65 Ag assay. The US-PCR assay permitted the detection of CMV infection episodes following transplantation a median of 12 days earlier than the pp65 Ag assay. Moreover, during CMV infection episodes, DNA detection by the US-PCR was consistently positive, whereas false negative results were frequently observed with the pp65 Ag assay. We found a good correlation between the two assays, and the peak viral loads were significantly higher in patients with CMV-related complications (median, 5,000 DNA copies/ml) than in those without symptoms (1,160 DNA copies/ml) (P = 0.048). In addition, patients that did not require preemptive therapy based on the results of the pp65 assay had CMV loads significantly lower (median, 36 DNA copies/ml) than those that needed treatment (median, 4,703 DNA copies/ml) (P < 0.001). These observations provided cutoff levels that could be applied in clinical practice. The ultrasensitive plasma-based PCR detected CMV infection episodes earlier and provided more consistent results than the pp65 Ag assay. This test could improve the monitoring of CMV infection or reactivation in renal transplant recipients.

Detection of Enteroviruses in the Cerebrospinal Fluid by Polymerase Chain Reaction: Prospective Study of Impact on the Management of Hospitalized Children

A polymerase chain reaction kit (AMPLICOR EV®) for the detection of enteroviruses (EV-PCR) in the cerebrospinal fluid (CSF) was evaluated in clinical conditions in a prospective blinded-intention study. Forty-three children (mean age 2.7 years) hospitalized for suspected meningitis or fever of unclear etiology were enrolled. EV-PCR was performed on a daily basis. Results were available in less than 2 days in 72% of cases. EV-PCR was positive in nine (21%) children, including three infants without CSF pleocytosis. Knowing their EV-PCR result would have allowed a saving of 18 hospital days and 12 days of antibiotic therapy. The EV-PCR in the CSF can thus be practically useful for children hospitalized for meningitis or fever if available on-site on a daily basis.

Improved Monitoring of Cytomegalovirus Infection after Allogeneic Hematopoietic Stem Cell Transplantation by an Ultrasensitive Plasma DNA PCR Assay

ABSTRACT Cytomegalovirus (CMV) DNA amplification assays in plasma have shown limited sensitivity compared to the detection of pp65 antigen in leukocytes. Our goal was to increase the sensitivity of a commercial CMV DNA PCR quantitative assay. After modification, the new assay was able to reproducibly detect 20 CMV DNA copies/ml of plasma. We compared this new ultrasensitive PCR assay with the standard PCR and the pp65 test for CMV detection and quantification in 22 consecutive allogeneic hematopoietic stem cell recipients. CMV infection or reactivation was detected in 84 of 319 (26%) samples by the ultrasensitive PCR assay compared to 38 of 319 (12%) samples by the pp65 assay (P < 0.01). All samples positive by the pp65 assay were positive by the ultrasensitive PCR, and CMV episodes were detected on average 4 days earlier and 7 days later than the first and the last pp65-positive test, respectively. In addition, during CMV episodes, the ultrasensitive assay identified positive samples that were inconsistently detected by the pp65 assay. The ultrasensitive assay was also much more sensitive than the standard PCR, with 26 versus 12% of CMV DNA-positive samples (P < 0.01). This assay improved the monitoring of CMV infection or reactivation in hematopoietic allogeneic stem cell recipients.

Latex agglutination test for cytomegalovirus antibody screening of transplant donors important aspects

In a retrospective study, false negative readings of CMV Scan results were detected. In a following prospective survey of 72 transplant donor sera, CMV Scan had a sensitivity and a negative predictive value of greater than 95%. Although CMV Scan is a rapid and useful test, a higher reliability can be achieved if results are controlled by another sensitive test or, at the least, CMV Scan is read by two different persons.