Riikka Österback

Respiratory Picornaviruses and Respiratory Syncytial Virus as Causative Agents of Acute Expiratory Wheezing in Children

We studied the viral etiology of acute expiratory wheezing (bronchiolitis, acute asthma) in 293 hospitalized children in a 2-year prospective study in Finland. A potential causative viral agent was detected in 88% of the cases. Eleven different viruses were represented. Respiratory syncytial virus (RSV) (27%), enteroviruses (25%), rhinovirus (24%), and nontypable rhino/enterovirus (16%) were found most frequently. In infants, RSV was found in 54% and respiratory picornaviruses (rhinovirus and enteroviruses) in 42% of the cases. In older children, respiratory picornaviruses dominated (65% of children ages 1-2 years and 82% of children ages >3 years). Human metapneumovirus was detected in 4% of all children and in 11% of infants. To prevent and treat acute expiratory wheezing illnesses in children, efforts should be focused on RSV, enterovirus, and rhinovirus infections.

Coxsackievirus A6 and Hand, Foot, and Mouth Disease, Finland

During fall 2008, an outbreak of hand, foot, and mouth disease (HFMD) with onychomadesis (nail shedding) as a common feature occurred in Finland. We identified an unusual enterovirus type, coxsackievirus A6 (CVA6), as the causative agent. CVA6 infections may be emerging as a new and major cause of epidemic HFMD.

Rhinovirus Transmission within Families with Children: Incidence of Symptomatic and Asymptomatic Infections

BACKGROUND Rhinoviruses are the most common cause of respiratory tract infections, but the transmission in families has not been studied using sensitive and specific molecular detection methods. METHODS Children hospitalized for any infection were screened for rhinoviruses. Eight families with a rhinovirus-positive index child and 16 families with a rhinovirus-negative index child were monitored for 3 weeks for disease symptoms, and the presence and quantity of rhinoviruses in nasal swab samples were determined by quantitative reverse transcription-polymerase chain reaction. Rhinoviruses were further identified by melting temperature and partial sequence analysis. RESULTS The rates of rhinovirus infection were 1.00 cases per person among the 17 siblings and 0.50 cases per person among the 14 parents of rhinovirus-positive index patients; the rates were 0.54 cases per person among the 24 siblings and 0.23 cases per person among the 30 parents of rhinovirus-negative index patients. Symptomatic infections were associated with an age of <7 years but not with a high copy number of rhinovirus genomes. Virus typing revealed the transmission routes of the viruses and showed that several virus types could circulate in the families simultaneously. CONCLUSIONS Rhinoviruses are frequently transmitted from children to other family members. Most rhinovirus infections in young children are symptomatic, but secondary infections in adults are often asymptomatic. Multiple virus types circulate simultaneously in families.

Microbiology of Acute Otitis Media in Children with Tympanostomy Tubes: Prevalences of Bacteria and Viruses

Abstract Background. Bacteria are found in 50%–90% of cases of acute otitis media (AOM) with or without otorrhea, and viruses are found in 20%–49% of cases. However, for at least 15% of patients with AOM, the microbiological etiology is never determined. Our aim was to specify the full etiology of acute middle ear infection by using modern microbiological methods concomitantly for bacterial and viral detection. Methods. The subjects were 79 young children having AOM with new onset (<48 h) of otorrhea through a tympanostomy tube. Middle ear fluid samples were suctioned from the middle ear through the tympanostomy tube. Bacteria were sought by culture and polymerase chain reaction; viruses were analyzed by culture, antigen detection, and polymerase chain reaction. Results. At least 1 respiratory tract pathogen was noted in 76 children (96%). Bacteria were found in 73 cases (92%), and viruses were found in 55 (70%). In 52 patients (66%), both bacteria and viruses were found. Bacteria typical of AOM were detected in 86% of patients. Picornaviruses accounted for 60% of all viral findings. Conclusions. In the great majority of children, AOM is a coinfection with bacteria and viruses. The patent tympanostomy tube does not change the spectrum of causative agents in AOM. A microbiological etiology can be established in practically all cases.

Viruses and bacteria in sputum samples of children with community-acquired pneumonia

Abstract Few comprehensive studies have searched for viruses and bacteria in children with community-acquired pneumonia (CAP). We identified 76 children hospitalized for pneumonia. Induced sputum samples were analysed for 18 viruses by antigen detection and PCR, and for six bacteria by culture and PCR. Viruses were found in 72% of samples, bacteria in 91%, and both in 66%. Rhinovirus (30%), human bocavirus (18%) and human metapneumovirus (14%) were the most commonly detected viruses. Two viruses were found in 22% of samples and three in 8%. The most common bacteria found were Streptococcus pneumoniae (50%), Haemophilus influenzae (38%), and Moraxella catarrhalis (28%). Rhinovirus–S. pneumoniae was the most commonly found combination of virus and bacterium (16%). All six children with treatment failure had both viruses and bacteria detected in the sputum. Otherwise, we found no special clinical characteristics in those with mixed viral–bacterial detections. With modern molecular diagnostic techniques, there are high rates of both viral and bacterial identification in childhood CAP. The clinical significance of mixed viral–bacterial infections remains unclear, although we found a potential association between them and treatment failure.

Clinical effects of rhinovirus infections

Rhinovirus is the major cause of common cold and frequently associates with acute wheezing, otitis media, sinusitis, and pneumonia. High prevalence of rhinovirus in hospitalized children and adults has been documented recently. We screened children > or =1 month of age, hospitalized for any infection, for the presence of rhinoviruses and recruited 24 families with > or =2 children for a 3-week follow-up study. Rhinovirus was detected in 46 (28%) of 163 hospitalizations by study children. Most rhinovirus-positive children (85%) had respiratory symptoms. During the follow-up, rhinoviruses were detected in virtually all children and in one-half of adults in families with a rhinovirus-positive index child, but commonly also in families with a rhinovirus-negative index child. Melting temperature and sequence analysis revealed the transmission routes of the viruses and showed that several virus types could circulate in the families simultaneously. Our studies corroborate the major contribution of rhinovirus to hospitalization of children, most often because of wheezing. Young children with respiratory symptoms are major spreaders of rhinovirus in family setting.

Human Metapneumovirus Infections in Children

Age-related incidence and effects of these infections are highest among children <2 years of age.

Genetic characterization of human coxsackievirus A6 variants associated with atypical hand, foot and mouth disease: a potential role of recombination in emergence and pathogenicity

Human coxsackievirus A6 (CVA6) is an enterically transmitted enterovirus. Until recently, CVA6 infections were considered as being of minor clinical significance, and only rarely aetiologically linked with hand, foot and mouth disease (HFMD) associated with other species A enteroviruses (particularly EV71 and CVA16). From 2008 onwards, however, CVA6 infections have been associated with several outbreaks worldwide of atypical HFMD (aHFMD) accompanied by a varicelliform rash. We recently reported CVA6-associated eczema herpeticum occurring predominantly in children and young adults in Edinburgh in January and February 2014. To investigate genetic determinants of novel clinical phenotypes of CVA6, we genetically characterized and analysed CVA6 variants associated with eczema herpeticum in Edinburgh in 2014 and those with aHFMD in CAV isolates collected from 2008. A total of eight recombinant forms (RFs) have circulated worldwide over the past 10 years, with the particularly recent appearance of RF-H associated with eczema herpeticum cases in Edinburgh in 2014. Comparison of phylogenies and divergence of complete genome sequences of CVA6 identified recombination breakpoints in 2A–2C, within VP3, and between 5′ untranslated region and VP1. A Bayesian temporal reconstruction of CVA6 evolution since 2004 provided estimates of dates and the actual recombination events that generated more recently appearing recombination groups (RF-E, -F, -G and -H). Associations were observed between recombination groups and clinical presentations of herpangina, aHFMD and eczema herpeticum, but not with VP1 or other structural genes. These observations provided evidence that NS gene regions may potentially contribute to clinical phenotypes and outcomes of CVA6 infection.

Development and Assay of RNA Transcripts of Enterovirus Species A to D, Rhinovirus Species A to C, and Human Parechovirus: Assessment of Assay Sensitivity and Specificity of Real-Time Screening and Typing Methods

ABSTRACT Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and −20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.

Simultaneous Detection and Differentiation of Human Rhino- and Enteroviruses in Clinical Specimens by Real-Time PCR with Locked Nucleic Acid Probes

ABSTRACT Human rhinoviruses (HRVs) and human enteroviruses (HEVs) are significant respiratory pathogens. While HRV infections are restricted to the respiratory tract, HEV infections may spread to secondary target organs. The method of choice for sensitive specific detection of these viruses is reverse transcription (RT)-PCR with primers targeting the conserved 5′ noncoding region of the viral RNA. On the other hand, sequence similarities between HRVs and HEVs complicate their differential detection. In this study, we describe the use of locked nucleic acid (LNA) analogues in short double-dye probes which contained only two selectively HRV- or HEV-specific bases. The double-stranded DNA dye BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) was used with the LNA probes in a tricolor real-time PCR assay to allow specific detection of HRVs (probes labeled with 6-carboxyfluorescein [FAM] [green]) and HEVs (Cy5 [red]) with additional melting curve analysis (BOXTO [yellow]). The functionality of the probes was validated in PCR and RT-PCR assays using plasmids containing viral cDNA, quantified viral RNA transcripts, cultivated rhino- and enterovirus prototypes, and clinical specimens. Of 100 HRV and 63 HEV prototypes, the probes correctly identified all HEVs except one that produced only a BOXTO signal. Among 118 clinical specimens with sequencing results, concordant results were obtained for 116 specimens. Two specimens were reactive with both probes, but sequencing yielded only a single virus. Real-time PCR with LNA probes allowed sensitive group-specific identification of HRVs and HEVs and would enable relative copy number determination. The assay is suitable for rapid and accurate differential detection of HRVs and HEVs in a diagnostic laboratory setting.