A chip-based rapid genotyping assay to discriminate between rhinovirus species A, B and C

Abstract Background Human rhinoviruses (RVs) are increasingly associated with severe disease of the respiratory tract. Multiple studies highlighted the clinical significance of different RV species; RV-C is linked to asthma exacerbations and increased disease severity in children, whereas RV-B seems to correlate with milder disease. Objectives Current typing strategies for differentiation of RV species are time consuming and require extensive equipment. Here we present a novel genotyping tool to discriminate RV species A, B and C. Study design The method encompasses a VP4/VP2 polymerase chain reaction (PCR), followed by hybridization of the product on a macro array with probes covering RV-A, B, and C, produced by Chipron as custom array. Validation was performed with respiratory specimens submitted for diagnostic evaluation to the Academic Medical Center. A selection of RV PCR-positive samples genotyped based on VP4/VP2 sequencing was evaluated. Diagnostic performance was tested on respiratory samples positive for RV in an in-house multiplex respiratory PCR from January 2016 to January 2017. In-house primers and additional genotype-specific primers were used for sequencing to investigate array-negative and array-double-positive samples. Results The majority of samples pretyped RVs (n = 135) were classified correctly, except for one that was assigned RV-C instead of RV-A, and 3 samples tested negative. The array gave four double-positive results; the presence of more than one genotype was confirmed in two samples. In 173/187 (92.5%) RV-positive tested patient samples from 2016, the test resulted in a designated species. RV species A was identified in 109 specimens (58.3%), RV-B in 26 (13.9%), and RV-C in 56 (29.9%) samples. Sequencing of the probe region of 14 (7.6%) negative samples revealed up to 3 mismatches to the probes for 12 samples; in 2 cases no PCR product was generated. Notably, in 18 samples the chip detected more than one species, of which 16 were confirmed by sequencing. Discussion The Chipron LCD RV array provides a fast and highly sensitive method for discrimination between rhinovirus species, and has the power to detect dual infections.