Acute respiratory tract infections (ARIs) represent a major public health problem worldwide, a leading cause of human acute illnesses and an important contributing factor of childhood morbidity and mortality, especially in children under 5 years [1–3]. A majority of ARIs in children have a viral etiology, probably due to absent or incomplete immune protection, sustained viral shedding and high transmissibility amongst hosts . Amongst a variety of RNA and DNA viruses that can infect the respiratory tract, Human Rhinoviruses (Picornaviridae family) , are recognized as the most prevalent in all age group worldwide . Together with coronaviruses, they represent common causative agents of upper respiratory tract (URT) infections, traditionally defined as common cold [6, 7], and thus a major cause of school and work absenteeism since children experience 8–12 and adults 2–3 URT episodes per year, on average . Children are the major reservoir for HRV . The mean age at the first symptomatic HRV infection is 4–6 months (vs >6 months for other viruses such as RSV ; more than 90% of children have experienced at least one HRV infection by the age of 2 years . HRV infection is most often associated with a non-specific, self-limiting illness with clinical manifestations ranging from an asymptomatic presentation to fever, rhinorrhea, cough and wheezing. However, HRV infections are increasingly involved in otitis media, pneumonia, or chronic obstructive pulmonary disease, especially in infants, elderly and immunocompromised patients [12, 13]. In addition, HRV represent a major viral aetiology of asthma exacerbations, with the highest incidence of all respiratory viruses in adults and children >2 years of age (60–65% of viral exacerbations) [14, 15]. Although suspected in some studies, there is at the present day no proof of association between clinical severity and HRV species [16–20]. Altogether, these data highlight the predominant role of HRV as a respiratory pathogen especially in early life.
HRVs are small non-enveloped viruses with a single-stranded RNA genome of positive polarity; originally classified in the Rhinovirus genus, they have been integrated into the Enterovirus genus. Rhinoviruses share with Enteroviruses an identical genomic organization and have similar functional RNA secondary structures, but differ in their acid tolerance, receptor usage, and cell tropism . The genome is approximately 7.2 kb long, and is composed of a 5′non-coding region (5′NCR), followed by a long open reading frame coding for four structural icosahedral capsid proteins (VP4, VP2, VP3 and VP1), and seven non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D, and terminated by a short 3′UTR and poly A tract. HRVs are highly heterogeneous genetically and antigenically [22, 23]. More than 140 serotypes have been described, and these fall into three species, HRV species A (HRV-A; 74 serotypes), HRV-B (25 serotypes) and a novel genetically distinct third genotype HRV-C, comprising 49 designated serotypes recognized in 2006 [24–27].
HRV infections occur throughout the year , usually with peaks in spring and autumn in temperate countries [29–31], the prevalence varying from 10% to 60% depending on the population or the period studied. Molecular studies suggest almost equal prevalence of HRVA and HRVC, with a under-representation of HRVB species [11, 19, 32–39]. A remarkably wide genetic diversity of HRV serotypes can be observed all year long .
The diagnosis of HRV infections is important for epidemiological purposes but also for optimising the medical management of patients (e.g., the opportunity for an antibiotic treatment). As clinical presentation is non-specific, it devolves to the diagnostic laboratory to confirm the presence of HRVs . Detection of HRV by culture is slow and complex for HRVA and HRVB, whilst HRVC has been unculturable in vitro to date . Serologic diagnosis is virtually impossible due to the number of serotypes, and rapid antigen test kits are not available . Molecular methods such as real-time RT-PCR appear to be the most suitable method, combining short analysis time, high sensitivity, semi-quantification of viral load and the detection of the majority of respiratory viruses with multiplex methods [42–44]. Most of the published systems target the 5′NCR. However, the high genetic diversity of HRVs makes the detection of all variants difficult as evidenced by the alignment of available GenBank 5′NCR sequences. Indeed, a comparison of published HRV-specific PCR primers pairs showed that no single pair could detect all HRVs  and that more than one PCR is required for accurate description of HRV epidemiology.
The range of detection of HRV variants by a probe-based real-time PCR assay is predicted to be additionally restricted by mutations in the region of the probe. Accordingly, we have compared the performances of 3 tests for accurately detecting HRV in clinical samples: (i) a probe-based Taqman RT-PCR assay routinely used in our hospital laboratory; (ii) the same test used without the probe and in the presence of SYBR Green I, making theoretically possible to diagnose HRVs amplified by the primers but not detected by the probe; (iii) the same test used with both the probe and in the presence of the BOXTO intercalating agent, making theoretically possible to detect HRV detected and undetected by the probe in the same reaction.