TY - JOUR
T1 - A chip-based rapid genotyping assay to discriminate between rhinovirus species A, B and C
AU - Westerhuis, Brenda M.
AU - Wiewel, Maryse A.
AU - Ende, Alexander Am
AU - van der Linden, Lonneke
AU - Koekkoek, Sylvie M.
AU - Wolthers, Katja C.
AU - Landt, Olfert
PY - 2017
Y1 - 2017
N2 - 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. 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. 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. 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. 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
AB - 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. 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. 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. 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. 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
U2 - https://doi.org/10.1016/j.jcv.2017.12.004
DO - https://doi.org/10.1016/j.jcv.2017.12.004
M3 - Article
C2 - 29268148
SN - 1386-6532
VL - 99-100
SP - 10
EP - 14
JO - Journal of clinical virology
JF - Journal of clinical virology
ER -