Katharina Karsch

Early detection of influenza A and B infection in infants and children using conventional and fluorescence-based rapid testing

BACKGROUND The appropriate management of infants and children with influenza depends on the accurate and timely diagnosis, ideally at the point of care (POC). OBJECTIVES To evaluate the use of simultaneous RSV/influenza rapid testing with QuickVue™ test strips as well as (the use of) novel, fluorescence-based, rapid influenza antigen testing (SOFIA™) in infants and children with influenza-like illness (ILI). STUDY DESIGN The Study was conducted in a real-time surveillance program at the Charité Department of Pediatrics in collaboration with the National Reference Centre for Influenza at the Robert Koch Institute (RKI) in Berlin, Germany (Charité Influenza-Like Disease=ChILD Cohort). RESULTS During the 2010/2011 influenza season, 395 infants and children were simultaneously tested using QuickVue™ FluA&B and RSV10 rapid tests at POC compared to independent RT-PCR. Sensitivities were 62.7/67.8% for Influenza/RSV overall, but highest in infants <1 year with 76.0/76.2%. The evaluation of the fluorescence-based rapid test SOFIA™ with frozen laboratory samples (derived from the 2008/2009 and 2010/2011 national surveillance) yielded sensitivities of 97.7/86.7/86.7/81.7% for influenza A(H1N1)pdm09/A(H3N2)/B-Victoria/B-Yamagata in samples with CT values <34, and 80.2/79.8/67.5/62.5% for all CT values combined. The same method used at POC with 649 consecutive ChILD patients in 2011-2012 yielded sensitivity/specificity/PPV/NPV values of 78.9/99.7/96.6/97.3%. Again, sensitivities were highest in infants (85.7%) and small children <2 years (88%). CONCLUSIONS Fluorescence-based rapid antigen testing provides a highly sensitive and specific tool for POC diagnostics of acute influenza in the paediatric age group, especially in infants and small children <2 years, when viral loads are at their peak and treatment decisions are imminent.

Human Parechovirus Infections Associated with Seizures and Rash in Infants and Toddlers.

Background: Systematic investigations assessing the clinical impact of human parechovirus (HPeV) disease are sparse. Noninvasive stool samples may be useful for targeted hospital-based surveillance. Methods: In the context of a quality management program, all hospitalized children fulfilling predefined case criteria for central nervous system (CNS) infection/inflammation underwent standardized neurologic examinations. Stool samples were collected for HPeV and enterovirus (EV) polymerase chain reaction and molecular typing at the National Reference Center. Results: From October 2010 to December 2012, stool samples of 284 patients with suspected CNS infection/inflammation were tested yielding 12 (4.2%) HPeV+ samples and 43 (15.1%) EV+ samples. HPeV-positive samples included HPeV-1, HPeV-3 and HPeV-6. No additional pathogens were identified in routine care. HPeV-positive patients were significantly younger (P < 0.001) and more likely to present with seizures (P = 0.001) and rash (P < 0.0001) when compared with HPeV-negative patients. Conclusions: In hospitalized children younger than 4 years presenting with suspected CNS infection/inflammation, seizures and/or rash, HPeV should be considered in the differential diagnosis. Large-scale public health surveillance may be indicated.

Virus load kinetics and resistance development during oseltamivir treatment in infants and children infected with Influenza A(H1N1) 2009 and Influenza B viruses.

Background: Infants and small children are the most effective transmitters of influenza, while bearing a high risk of hospitalization and adverse disease outcomes. This study aims to investigate virus load kinetics and resistance development during oseltamivir therapy in infants and children infected with influenza A(H1N1) 2009 and influenza B viruses. Methods: Virus load in nasopharyngeal samples and phenotypic/genotypic neuraminidase inhibitor resistance were determined at baseline, at day 5 and in additional follow-up samples, if available. Patient-specific viral clearance indices CL&ngr;(i) were determined along with estimates of the time required to achieve nondetectable virus load. Results: No evidence of baseline oseltamivir resistance was detected in 36 patients infected with influenza A(H1N1) 2009 (n = 27) or influenza B (Victoria, Yamagata; n = 9) before oseltamivir therapy. On average, viral loads were lower for influenza type B (median = 5.9·103/mL) than for drug-resistant (median = 2.6·106/mL) and sensitive A(H1N1) 2009 (median = 4.8·104/mL), P = 0.04 and P = 0.09, respectively. Time required to achieve nondetectable virus load was significantly longer in drug-resistant A(H1N1) 2009 (median 15.4 days) compared with drug-sensitive A(H1N1) 2009 (P = 0.003; median 7.7 days) and drug-sensitive influenza B (P = 0.001; median 5 days). No evidence of viral rebound was observed once viral clearance was achieved. Conclusions: Our data indicate that influenza subtyping in combination with baseline viral load measurements might help to optimize the duration of antiviral therapy in the individual child. Lower than expected virologic response rates in patients without malabsorption or compliance issues may suggest resistance development.

Evaluation of novel second-generation RSV and influenza rapid tests at the point of care.

Acute respiratory infections represent common pediatric emergencies. Infection control warrants immediate and accurate diagnoses. In the past, first-generation respiratory syncytial virus (RSV) rapid tests (strip tests) have shown suboptimal sensitivities. In 2013, the Food and Drug Administration licensed a second-generation RSV rapid test providing user-independent readouts (SOFIA™-RSV) using automated fluorescence assay technology known to yield superior results with influenza rapid testing. We are reporting the first point-of-care evaluation of the SOFIA™-RSV rapid test. In the Charité Influenza-Like Disease Cohort, 686 nasopharyngeal samples were tested in parallel with SOFIA™-RSV and SOFIA™-Influenza A+B. Compared to real-time PCR, SOFIA™-RSV sensitivities/specificities were 78.6%/93.9%, respectively (SOFIA™-Influenza A: 80.6%/99.3%). Performance was greatest in patients below 2 years of age with a test sensitivity of 81.8%. RSV sensitivities were highest (85%) in the first 2 days of illness and with nasopharyngeal compared to nasal swabs (P=0.055, McNemars test). Second-generation RSV and influenza rapid testing provides highly accurate results facilitating timely patient cohortation and management.

Quantitative influenza follow-up testing (QIFT)--a novel biomarker for the monitoring of disease activity at the point-of-care.

Background Influenza infections induce considerable disease burden in young children. Biomarkers for the monitoring of disease activity at the point-of-care (POC) are currently lacking. Recent methodologies for fluorescence-based rapid testing have been developed to provide improved sensitivities with the initial diagnosis. The present study aims to explore the utility of second-generation rapid testing during longitudinal follow-up of influenza patients (Rapid Influenza Follow-up Testing = RIFT). Signal/control fluorescent readouts (Quantitative Influenza Follow-up Testing = QIFT) are evaluated as a potential biomarker for the monitoring of disease activity at the POC. Methods and Findings RIFT (SOFIA) and QIFT were performed at the POC and compared to blinded RT-PCR at the National Reference Centre for Influenza. From 10/2011-4/2013, a total of 2048 paediatric cases were studied prospectively; 273 cases were PCR-confirmed for influenza. During follow-up, RIFT results turned negative either prior to PCR (68%), or simultaneously (30%). The first negative RIFT occurred after a median of 8 days with a median virus load (VL) of 5.6×10∧3 copies/ml and cycle threshold of 37, with no evidence of viral rebound. Binning analysis revealed that QIFT differentiated accurately between patients with low, medium and high viral titres. QIFT increase/decrease showed 88% agreement (sensitivity = 52%, specificity = 95%) with VL increase/decrease, respectively. QIFT-based viral clearance estimates showed similar values compared to PCR-based estimates. Variations in viral clearance rates were lower in treated compared to untreated patients. The study was limited by use of non-invasive, semi-quantitative nasopharyngeal samples. VL measurements below the limit of detection could not be quantified reliably. Conclusions During follow-up, RIFT provides a first surrogate measure for influenza disease activity. A “switch” from positive to negative values may indicate a drop in viral load below a critical threshold, where rebound is no longer expected. QIFT may provide a useful tool for the monitoring of disease burden and viral clearance at the POC.

Enabling Precision Medicine With Digital Case Classification at the Point-of-Care.

Infectious and inflammatory diseases of the central nervous system are difficult to identify early. Case definitions for aseptic meningitis, encephalitis, myelitis, and acute disseminated encephalomyelitis (ADEM) are available, but rarely put to use. The VACC-Tool (Vienna Vaccine Safety Initiative Automated Case Classification-Tool) is a mobile application enabling immediate case ascertainment based on consensus criteria at the point-of-care. The VACC-Tool was validated in a quality management program in collaboration with the Robert-Koch-Institute. Results were compared to ICD-10 coding and retrospective analysis of electronic health records using the same case criteria. Of 68,921 patients attending the emergency room in 10/2010–06/2013, 11,575 were hospitalized, with 521 eligible patients (mean age: 7.6 years) entering the quality management program. Using the VACC-Tool at the point-of-care, 180/521 cases were classified successfully and 194/521 ruled out with certainty. Of the 180 confirmed cases, 116 had been missed by ICD-10 coding, 38 misclassified. By retrospective application of the same case criteria, 33 cases were missed. Encephalitis and ADEM cases were most likely missed or misclassified. The VACC-Tool enables physicians to ask the right questions at the right time, thereby classifying cases consistently and accurately, facilitating translational research. Future applications will alert physicians when additional diagnostic procedures are required.

Educating parents about the vaccination status of their children: A user-centered mobile application.

Parents are often uncertain about the vaccination status of their children. In times of vaccine hesitancy, vaccination programs could benefit from active patient participation. The Vaccination App (VAccApp) was developed by the Vienna Vaccine Safety Initiative, enabling parents to learn about the vaccination status of their children, including 25 different routine, special indication and travel vaccines listed in the WHO Immunization Certificate of Vaccination (WHO-ICV). Between 2012 and 2014, the VAccApp was validated in a hospital-based quality management program in Berlin, Germany, in collaboration with the Robert Koch Institute. Parents of 178 children were asked to transfer the immunization data of their children from the WHO-ICV into the VAccApp. The respective WHO-ICV was photocopied for independent, professional data entry (gold standard). Demonstrating the status quo in vaccine information reporting, a Recall Group of 278 parents underwent structured interviews for verbal immunization histories, without the respective WHO-ICV. Only 9% of the Recall Group were able to provide a complete vaccination status; on average 39% of the questions were answered correctly. Using the WHO-ICV with the help of the VAccApp resulted in 62% of parents providing a complete vaccination status; on average 95% of the questions were answered correctly. After using the VAccApp, parents were more likely to remember key aspects of the vaccination history. User-friendly mobile applications empower parents to take a closer look at the vaccination record, thereby taking an active role in providing accurate vaccination histories. Parents may become motivated to ask informed questions and to keep vaccinations up-to-date.

Acute Disseminated Encephalomyelitis After Human Parechovirus Infection.

Background: Acute disseminated encephalomyelitis (ADEM) is an inflammatory, demyelinating disease occurring several weeks after viral infection. Enteroviruses have been described as potential triggers of ADEM, but the closely related parechoviruses have not. The objective of the study is to assess the prevalence and disease presentation of ADEM after parechovirus infection in a syndromic surveillance program for pediatric infection/inflammation of the central nervous system (CNS). Methods: The surveillance was conducted at the Charité Department of Pediatrics in Berlin, Germany, from November 2010 to November 2014. All hospitalized children meeting predefined case criteria underwent highly standardized prospective clinical assessments based on the published case definitions, including for ADEM. Stool samples were independently analyzed by enterovirus and parechovirus real-time polymerase chain reaction at the Robert Koch Institute. Results: Of 105,557 patients screened, 774 (0.7%) fulfilled entry criteria for CNS infection/inflammation, with 114 cases ascertained as ADEM. Parechoviruses were detected in 2.5% of patients with CNS infection/inflammation, including 1 case fulfilling ADEM case criteria with the highest level of diagnostic certainty. Conclusions: We report a first case of ADEM after parechovirus infection in a 5-year-old female presenting with acute hemiparesis 2 weeks after a respiratory illness. Parechovirus disease should be included in the differential diagnosis of ADEM.

Towards a Personalised Approach to Managing Influenza Infections in Infants and Children – Food for Thought and a Note on Oseltamivir

Acute respiratory infections represent common diseases in childhood and a challenge to infection control, public heath, and the clinical management of patients and their families. Children are avid spreaders of respiratory viruses, and seasonal outbreaks of influenza create additional disease burden and healthcare cost. Infants under the age of two and children with chronic conditions are at high risk. The absence of pre-defined risk factors however, does not protect from serious disease. Immunisation rates remain low, and physical interventions are of limited value in young children. Children with influenza may be contagious prior to the onset of symptoms, and school closures have been shown to have a temporary effect at most. The timely detection of influenza in at-risk patients is important to prevent hospital-based transmission and influenza-associated morbidity and mortality. Guidelines issued by professional associations and public health agencies need to be translated into everyday clinical practice. Antiviral therapy should be initiated early and monitored closely, including virologic and clinical outcomes. The duration of treatment and the decision to readmit children to schools and kindergartens should be adjusted to the individual child patient using evidence-based clinical and virologic criteria. This article presents lessons learnt from a quality management program for infants and children with influenza-like illness at the Charite Department of Paediatrics in collaboration with the National Reference Centre for Influenza at the Robert Koch Institute, in Berlin, Germany. The Charité Influenza-Like Disease (ChILD) Cohort was established during the 2009 influenza pandemic and encompasses nearly 4000 disease episodes to date.

Prospective surveillance of antiviral resistance in hospitalized infants less than 12 months of age with A(H3N2) influenza infection and treated with oseltamivir.

BACKGROUND Infants exhibit elevated influenza virus loads and prolonged viral shedding, which may increase the risk for resistance development, especially in cases of suboptimal exposure to antiviral therapy. METHODS We performed a prospective surveillance of hospitalized infants undergoing oseltamivir therapy during the 2008-2009 and 2011-2012 influenza seasons at two paediatric hospitals in Germany. A total of 37 infants less than 1 year of age with laboratory confirmed influenza A(H3N2) infection received oseltamivir as per physicians order for 5 days (2008-2009 season: 2 mg/kg twice daily; 2011-2012 season: 2.0 mg/kg; 2.5 mg/kg and 3.0 mg/kg twice daily for infants <1 month; 2-3 months and 4-12 months, respectively). Virus load, the susceptibility to neuraminidase inhibitors (NAIs), and the presence of molecular markers of resistance to NAIs was assessed for influenza viruses recovered from respiratory samples collected at baseline and during follow-up visits. RESULTS Overall, 73% of the infants continued to shed viral RNA detectable by reverse transcription (RT)-PCR after dose number 10 of oseltamivir; 12 infants shed viruses, 2 of them (both 9 months of age) shed resistant viruses. Resistance was characterized by ≥1,000-fold increase of 50% inhibitory concentration (IC50) for oseltamivir, up to 50-fold for zanamivir and elevated Km values when compared to susceptible A(H3N2) strains. Sanger sequencing revealed the selection of the NA-R292K substitution in both instances (after dose number 10 on day 6). CONCLUSIONS Our data suggest that it may be relevant to monitor antiviral resistance systematically in all infants, considering that the European Medicines Agency has recently extended the licensure for oseltamivir to include full-term infants.