Comparison of MT-PCR with Quantitative PCR for Human Bocavirus in Respiratory Samples with Multiple Respiratory Viruses Detection.

Human bocavirus (HBoV) is an important respiratory pathogen, especially in children, but it is often found in co-detection with other respiratory viruses, which makes the diagnostic approach challenging. We compared multiplex PCR and quantitative PCR for HBoV with multiplex tandem PCR (MT-PCR) in 55 cases of co-detection of HBoV and other respiratory viruses. In addition, we investigated whether there is a connection between the severity of the disease, measured by the localization of the infection, and amount of virus detected in the respiratory secretions. No statistically significant difference was found, but children with large amount of HBoV and other respiratory virus had a longer stay in hospital.

Prevalence and Molecular Characterization of Human Bocavirus Detected in Croatian Children with Respiratory Infection.

Human bocavirus (HBoV) 1 is considered an important respiratory pathogen, while the role of HBoV2-4 in clinical disease remains somewhat controversial. Since, they are characterized by a rapid evolution, worldwide surveillance of HBoVs' genetics is necessary. This study explored the prevalence of HBoV genotypes in pediatric patients with respiratory tract infection in Croatia and studied their phylogeny. Using multiplex PCR for 15 respiratory viruses, we investigated 957 respiratory samples of children up to 18 years of age with respiratory tract infection obtained from May 2017 to March 2021 at two different hospitals in Croatia. Amplification of HBoV near-complete genome or three overlapping fragments was performed, sequenced, and their phylogenetic inferences constructed. HBoV was detected in 7.6% children with a median age of 1.36 years. Co-infection was observed in 82.2% samples. Sequencing was successfully performed on 29 HBoV positive samples, and all belonged to HBoV1. Croatian HBoV1 sequences are closely related to strains isolated worldwide, and no phylogenetic grouping based on mono- or co-infection cases or year of isolation was observed. Calculated rates of evolution for HBoV1 were 10-4 and 10-5 substitutions per site and year. Recombination was not detected among sequences from this study.

Seasonal Coronaviruses and Other Neglected Respiratory Viruses: A Global Perspective and a Local Snapshot.

Respiratory viral infections are the leading cause of morbidity and mortality in the world; however, there are several groups of viruses that are insufficiently routinely sought for, and can thus be considered neglected from a diagnostic and clinical standpoint. Timely detection of seasonality of certain respiratory viruses (e.g., enveloped viruses such as seasonal coronaviruses) in the local context can aid substantially in targeted and cost-effective utilization of viral diagnostic approaches. For the other, non-enveloped and year-round viruses (i.e., rhinovirus, adenovirus, and bocavirus), a continuous virological diagnosis needs to be implemented in clinical laboratories to more effectively address the aetiology of respiratory infections, and assess the overall impact of these viruses on disease burden. While the coronavirus disease 2019 (COVID-19) pandemic is still actively unfolding, we aimed to emphasize the persistent role of seasonal coronaviruses, rhinoviruses, adenoviruses and bocaviruses in the aetiology of respiratory infections. Consequently, this paper concentrates on the burden and epidemiological trends of aforementioned viral groups on a global level, but also provides a snapshot of their prevalence patterns in Croatia in order to underscore the potential implications of viral seasonality. An overall global prevalence in respiratory tract infections was found to be between 0.5 and 18.4% for seasonal coronaviruses, between 13 and 59% for rhinoviruses, between 1 and 36% for human adenoviruses, and between 1 and 56.8% for human bocaviruses. A Croatian dataset on patients with respiratory tract infection and younger than 18 years of age has revealed a fairly high prevalence of rhinoviruses (33.4%), with much lower prevalence of adenoviruses (15.6%), seasonal coronaviruses (7.1%), and bocaviruses (5.3%). These insights represent a relevant discussion point in the context of the COVID-19 pandemic where the testing of non-SARS-CoV-2 viruses has been limited in many settings, making the monitoring of disease burden associated with other respiratory viruses rather difficult.

The Emerging Role of Rhinoviruses in Lower Respiratory Tract Infections in Children - Clinical and Molecular Epidemiological Study From Croatia, 2017-2019.

Rhinoviruses (RVs) are increasingly implicated not only in mild upper respiratory tract infections, but also in more severe lower respiratory tract infections; however, little is known about species diversity and viral epidemiology of RVs among the infected children. Therefore, we investigated the rhinovirus (RV) infection prevalence over a 2-year period, compared it with prevalence patterns of other common respiratory viruses, and explored clinical and molecular epidemiology of RV infections among 590 children hospitalized with acute respiratory infection in north-western and central parts of Croatia. For respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs were taken from each patient and subsequently analyzed with multiplex RT-PCR. To determine the RV species in a subset of positive children, 5'UTR in RV-positive samples has been sequenced. Nucleotide sequences of referent RV strains were retrieved by searching the database with Basic Local Alignment Tool, and used to construct alignments and phylogenetic trees using MAFFT multiple sequence alignment tool and the maximum likelihood method, respectively. In our study population RV was the most frequently detected virus, diagnosed in 197 patients (33.4%), of which 60.4% was detected as a monoinfection. Median age of RV-infected children was 2.25 years, and more than half of children infected with RV (55.8%) presented with lower respiratory tract infections. Most RV cases were detected from September to December, and all three species co-circulated during the analyzed period (2017-2019). Sequence analysis based on 5'UTR region yielded 69 distinct strains; the most prevalent was RV-C (47.4%) followed by RV-A (44.7%) and RV-B (7.9%). Most of RV-A sequences formed a distinct phylogenetic group; only strains RI/HR409-18 (along with a reference strain MF978777) clustered with RV-C strains. Strains belonging to the group C were the most diverse (41.6% identity among strains), while group B was the most conserved (71.5% identity among strains). Despite such differences in strain groups (hitherto undescribed in Croatia), clinical presentation of infected children was rather similar. Our results are consistent with newer studies that investigated the etiology of acute respiratory infections, especially those focused on children with lower respiratory tract infections, where RVs should always be considered as potentially serious pathogens.

High Detection Rates of Human Bocavirus in Infants and Small Children with Lower Respiratory Tract Infection from Croatia.

BACKGROUND: Human bocavirus (HBoV) is known to cause lower respiratory tract infections (LRTI) in children and may result in substantial morbidity and mortality. The aim of this study was to determine HBoV prevalence among hospitalized infants and small children with acute LRTI in Zagreb, Croatia, as well as to evaluate HBoV DNA quantity in samples in relation to the patients' age and co-infection with other respiratory viruses. METHODS: During winter season 2016/2017, a total of 295 children younger than three years of age who were admitted to hospitals with LRTI were tested for the presence of HBoV, respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza virus (PIV) types 1 to 3, and human metapneumovirus (HMPV). HBoV was detected with a real-time PCR method, and the other viruses were diagnosed using monoclonal antibodies in direct fluorescence assay. RESULTS: Viral etiology was proven in 225/295 (76.3%) of patients. The most commonly diagnosed virus was RSV (59.3%), followed by HBoV (23.1%), PIVs (4.4%), ADV (3.1%), and HMPV (1.4%). HBoV-infected children were older than RSV-infected children; likewise, detection rates of HBoV infection increased with age, while RSV infection rates decreased with age. In 51% of HBoV-positive samples an additional respiratory virus was also detected. There was no difference in HBoV DNA quantity between samples with single virus detection and those with multiple virus detection (p = 0.056), although samples positive only for HBoV showed higher cycle threshold values. There was no difference in HBoV DNA quantity in samples of different age groups (p > 0.05). CONCLUSIONS: Frequent detection of HBoV in small children with LRTI, even in combination with other viruses, highlights its role in the pathogenesis of respiratory disease.