Janette Rahamat-Langendoen

Upsurge of human enterovirus 68 infections in patients with severe respiratory tract infections

BACKGROUND Enterovirus 68 (EV68) belongs to species Human enterovirus D. It is unique among enteroviruses because it shares properties with human rhinoviruses. After the first isolation in 1962 from four children with respiratory illness, reports of (clusters of) EV68 infections have been rare. During the autumn of 2010, we noticed an upsurge of EV68 infections in the Northern part of the Netherlands in patients with severe respiratory illness. OBJECTIVES To give a detailed description of the clinical and virological data of patients with EV68 infection identified in 2010, and compare these with data collected in 2009. STUDY DESIGN We systematically collected clinical data from patients with an EV68 infection detected in 2010. We added four patients with an EV68 infection from 2009. Further characterization of EV68 was performed by partial sequence analysis of the VP1 genomic region. RESULTS In 2010, EV68 was identified as the only cause of respiratory illness in 24 patients, of which 5 had to be admitted to the intensive care unit. Sequence analysis revealed different lineages in the majority of EV68 detected in 2010 as compared to the 2009 isolates. CONCLUSIONS We noticed an increase of EV68 infections and present clinical as well as sequence data, in which two distinct phylogenetic clusters could be identified.

Pediatric parechovirus infections

Human parechoviruses (HPeVs) are members of the large and growing family of Picornaviridae. Although 16 types have been described on the basis of the phylogenetic analyses of the VP1 encoding region, the majority of published reports relate to the HPeV types 1-8. In pediatrics, HPeV1, HPeV2 and HPeV4-8 mainly cause mild gastrointestinal or respiratory illness; only occasionally more serious diseases have been reported, including myocarditis, encephalitis, pneumonia, meningitis, flaccid paralysis, Reye syndrome and fatal neonatal infection. In contrast, HPeV3 causes severe illness in young infants, including sepsis and conditions involving the central nervous system. Currently, the most sensitive method for detecting HPeV is real-time polymerase chain reaction assays on stools, respiratory swabs, blood and cerebrospinal fluid. However, although it is known that HPeVs play a significant role in various severe pediatric infectious diseases, diagnostic assays are not routinely available in clinical practice and the involvement of HPeV is therefore substantially underestimated. Despite long-term efforts, the development of antiviral therapy against HPeVs is limited; no antiviral medication is available and the use of monoclonal antibodies is still being evaluated. More research is therefore needed to clarify the specific characteristics of this relevant group of viruses and to develop appropriate treatment strategies.

Influenza in the immediate post-pandemic era: A comparison with seasonal and pandemic influenza in hospitalized patients

BACKGROUND Comparative data on severity and treatment of seasonal, pandemic and post-pandemic influenza virus infections are scarce. OBJECTIVES To systematically analyze characteristics of hospitalized patients with influenza in the post-pandemic period compared to seasonal and pandemic influenza. STUDY DESIGN Clinical and virological data of patients hospitalized in a tertiary referral hospital with post-pandemic influenza (2010-2011) were compared with those during seasonal influenza epidemics (2007-2009) and the influenza A(H1N1)pdm09 pandemic (2009-2010). RESULTS 82 patients were admitted during the post-pandemic period, compared to 85 during the pandemic and 60 during seasonal influenza epidemics. No differences were observed in the occurrence of complicated illness and the need for intensive care. However, radiographic pneumonia was significantly more often diagnosed in patients with influenza A(H1N1)pdm09 compared to patients with seasonal influenza A (25% versus 71% in pandemic, p=0.004, and 55% in post-pandemic, p=0.047). Oseltamivir was more frequently prescribed in post-pandemic and pandemic patients compared to previous influenza seasons (48.9% resp. 76.5% versus 6.5%, p<0.0001). During the post-pandemic period, patients with influenza B were significantly less often treated with oseltamivir compared to patients with influenza A (27.0% versus 48.9%, p=0.043), although the course of illness in patients with influenza B was comparable with influenza A. No upsurge of oseltamivir resistance was observed. CONCLUSIONS In our center, severity of illness was comparable for all influenza seasons, although more radiographic pneumonia was diagnosed in patients with influenza A(H1N1)pdm09. Despite the increased use of oseltamivir, no increase in oseltamivir resistance was detected.

Direct multiplexed whole genome sequencing of respiratory tract samples reveals full viral genomic information

Abstract Background Acute respiratory tract infections (RTI) cause substantial morbidity during childhood, and are responsible for the majority of pediatric infectious diseases. Although most acute RTI are thought to be of viral origin, viral etiology is still unknown in a significant number of cases. Objectives Multiplexed whole genome sequencing (WGS) was used for virome determination directly on clinical samples as proof of principle for the use of deep sequencing techniques in clinical diagnosis of viral infections. Study design WGS was performed with nucleic acids from sputum and nasopharyngeal aspirates from four pediatric patients with known respiratory tract infections (two patients with human rhinovirus, one patient with human metapneumovirus and one patient with respiratory syncytial virus), and from four pediatric patients with PCR-negative RTI, and two control samples. Results Viral infections detected by routine molecular diagnostic methods were confirmed by WGS; in addition, typing information of the different viruses was generated. In three out of four samples from pediatric patients with PCR-negative respiratory tract infections and the two control samples, no causative viral pathogens could be detected. In one sample from a patient with PCR-negative RTI, rhinovirus type-C was detected. Almost complete viral genomes could be assembled and in all cases virus species could be determined. Conclusions Our study shows that, in a single run, viral pathogens can be detected and characterized, providing information for clinical assessment and epidemiological studies. We conclude that WGS is a powerful tool in clinical virology that delivers comprehensive information on the viral content of clinical samples.

The significance of rhinovirus detection in hospitalized children: clinical, epidemiological and virological features

Abstract Recent developments in molecular diagnostic tools have led to the easy and rapid detection of a large number of rhinovirus (HRV) strains. However, the lack of clinical and epidemiological data hampers the interpretation of these diagnostic findings. From October 2009 to January 2011, we conducted a prospective study in hospitalized children from whom samples were taken for the detection of respiratory viruses. Clinical, epidemiological and microbiological data from 644 patients with 904 disease episodes were collected. When HRV tested positive, strains were further characterized by sequencing the VP4/VP2 region of the HRV genome. HRV was the single respiratory virus detected in 254 disease episodes (28%). Overall, 99 different serotypes were detected (47% HRV-A, 12% HRV-B, 39% HRV-C). Patients with HRV had more underlying pulmonary illness compared with patients with no virus (p 0.01), or patients with another respiratory virus besides HRV (p 0.007). Furthermore, cough, shortness of breath and a need for oxygen were significantly more present in patients with HRV infection. Particularly, patients with HRV-B required extra oxygen. No respiratory symptom, except for oxygen need, was predictive of the presence of HRV. In 22% of HRV-positive disease episodes, HRV infection was hospital acquired. Phylogenetic analysis revealed several clusters of HRV; in more than 25% of these clusters epidemiological information was suggestive of transmission within specific wards. In conclusion, the detection of HRV may help in explaining respiratory illness, particular in patients with pulmonary co-morbidities. Identifying HRV provides opportunities for timely implementation of infection control measures to prevent intra-hospital transmission.

Seasonal influenza vaccine effectiveness against influenza in 2012-2013: a hospital-based case-control study in Lithuania.

BACKGROUND Due to scarce information on seasonal influenza vaccine effectiveness (SIVE) against severe clinical influenza outcomes in risk populations, we conducted a case-control study to assess its effects against laboratory-confirmed influenza in hospitalized patients during the 2012-2013 influenza season. METHODS We conducted a test-negative case-control study among ≥18 years old patients with influenza-like illness (ILI) hospitalized in two Lithuanian hospitals. Cases were influenza A(H1N1), A(H3) or influenza B positive by RT-PCR, and controls were influenza negative. Additional demographic and clinical data to assess the role of confounding were collected. SIVE and its confidence intervals (95% CI) were estimated by using multivariate logistic regression as (1-OR)×100%. RESULTS The sample consisted of 185 subjects. Seasonal influenza vaccine uptake was 5%. Among 111 (60%) influenza positive cases, 24.3% were A(H1N1), 10.8% were A(H3) and 24.3% were influenza B cases. Unadjusted SIVE was 79% (95% CI -6% to 96%) and after the adjustment it increased to 86% (95% CI 19% to 97%). CONCLUSIONS Seasonal influenza vaccination in 2012-2013 was associated with reduced occurrence of laboratory-confirmed influenza, but due to low sample size the estimate of SIVE is imprecise. Given high prevalence of influenza in hospitalized ILI cases and low influenza vaccination coverage, there is a need to increase influenza vaccination rates.

Whole genome sequencing of fecal samples as a tool for the diagnosis and genetic characterization of norovirus

BACKGROUND Norovirus is a major cause of gastroenteritis, causing yearly epidemics and hospital outbreaks resulting in a high burden on health care. Detection and characterization of norovirus directly from clinical samples could provide a powerful tool in infection control and norovirus epidemiology. OBJECTIVES To determine whether next-generation sequencing directly on fecal samples can accurately detect and characterize norovirus. STUDY DESIGN Whole genome sequencing was performed on fecal samples from 10 patients with gastro-enteritis. Norovirus infection had previously been confirmed by RT-PCR. Genotyping was performed using phylogenetic analysis. RESULTS From all clinical samples sufficient amounts of RNA were retrieved to perform whole-transcriptome sequencing for the detection of RNA-viruses. Complete genomic norovirus sequences were obtained from all clinical samples, permitting accurate genotyping by phylogenetic analysis. In addition, a complete coxsackie B1 virus genome was isolated. CONCLUSION Detailed information on viral content can be obtained from fecal samples in a single-step approach, supporting clinical and epidemiological purposes. Next-generation sequencing performed directly on clinical samples can become a powerful tool in patient care and infection control.

Sequence-based identification and characterization of nosocomial influenza A(H1N1)pdm09 virus infections

BACKGROUND Highly transmissible viruses such as influenza are a potential source of nosocomial infections and thereby cause increased patient morbidity and mortality. AIM To assess whether influenza virus sequence data can be used to link nosocomial influenza transmission between individuals. METHODS Dutch A(H1N1)pdm09-positive specimens from one hospital (N = 107) were compared with samples from community cases (N = 685). Gene fragments of haemagglutinin, neuraminidase and PB2 were sequenced and subsequently clustered to detect patients infected with identical influenza viruses. The probability of detecting a second patient was calculated for each hospital cluster against the background diversity observed in hospital and community strains. All clusters were further analysed for possible links between patients. FINDINGS Seventeen A(H1N1)pdm09 hospital clusters were detected of which eight had a low probability of occurrence compared with background diversity (P < 0.01). Epidemiological analysis confirmed a total of eight nosocomial infections in four of these eight clusters, and a mother-child combination in a fifth cluster. The nine clusters with a high probability of occurrence involved community cases of influenza without a known epidemiological link. CONCLUSION If a background sequence dataset is available, the detection of hospital sequence clusters that differ from dominant community strains can be used to select clusters requiring further investigation by hospital hygienists before a nosocomial influenza outbreak is epidemiologically suspected.

Rapid detection of a norovirus pseudo-outbreak by using real-time sequence based information

BACKGROUND Sequence based information is increasingly used to study the epidemiology of viruses, not only to provide insight in viral evolution, but also to understand transmission patterns during outbreaks. However, sequence analysis is not yet routinely performed by diagnostic laboratories, limiting its use in clinical practice. OBJECTIVES To describe the added value of sequence based information available within 3 days after the detection of norovirus in fecal samples of patients and personnel during a suspected outbreak on a hospital ward. Results were used to guide the implementation of appropriate infection control measures, in particular closure of the ward. STUDY DESIGN Observational study. RESULTS Norovirus infection was detected in seven patients and two health care workers on an oncology ward of the childrens hospital. Six of seven patients had a hospital acquired infection defined as a first day of illness more than two days after admission. After notification of the first two patients, supplementary infection control measures were taken to prevent further spread. Despite these measures, three additional patients with norovirus infection were identified. Characterization of the noroviruses of 5 out of 7 patients was available within 7 days after the notification of the first patient. Four different genotypes were detected, providing evidence for multiple introductions of different norovirus strains with only a few secondary cases rather than ongoing nosocomial transmission. Therefore, we maintained the already implemented infection control interventions without closure of the ward. CONCLUSIONS Sequence based information available in real-time is helpful for understanding norovirus transmission in the hospital and facilitates appropriate infection control measures during an outbreak.

Lab‐in‐a‐tube: Real‐time molecular point‐of‐care diagnostics for influenza A and B using the cobas® Liat® system

Rapid diagnosis of influenza A and B is important for direct treatment decisions in patient care and for the reduction of in‐hospital transmissions. The new real‐time PCR based molecular point‐of‐care (POC) assay, the cobas® Influenza A/B test on the cobas® Liat® System (cobas® Liat® Influenza A/B assay), generated a PCR result in less than 20 min, was evaluated for the detection of influenza A and B. One hundred twenty‐one retrospectively collected respiratory specimens, previously analyzed with a routine influenza A/B test (Diagenode) were tested using the cobas® Liat® Influenza A/B assay. The cobas® Liat® Influenza A/B assay allows influenza A and B testing by RT‐PCR within 20 min. This assay detected influenza A in 51 of 56 samples positive by the Diagenode test. The five discrepant results were retested with the Cepheid Influenza A/B test, confirming two positive cases. All 30 influenza B Diagenode positive samples were found positive by the cobas® Liat® Influenza A/B assay. Control samples (viral negative and non‐influenza pathogens) were all negative by the cobas® Liat® Influenza A/B assay. The cobas® Liat® Influenza A/B assay showed a sensitivity for influenza A/B of 96% and 100%, respectively, and 100% specificity for both targets. The cobas® Liat® Influenza A/B assay is a useful tool for accurate, rapid, and sensitive detection of influenza A and B, offering timely and personalized patient management and infection control when implemented at the point‐of‐care.