Development and validation of a next-generation sequencing assay with open-access analysis software for detecting resistance-associated mutations in CMV.

Cytomegalovirus (CMV) resistance testing by targeted next-generation sequencing (NGS) allows for the simultaneous analysis of multiple genes. We developed and validated an amplicon-based Ion Torrent NGS assay to detect CMV resistance mutations in UL27, UL54, UL56, and UL97 and compared the results to standard Sanger sequencing. NGS primers were designed to generate 83 overlapping amplicons of four CMV genes (~10 kb encompassing 138 mutation sites). An open-access software plugin was developed to perform read alignment, call variants, and interpret drug resistance. Plasmids were tested to determine NGS error rate and minor variant limit of detection. NGS limit of detection was determined using the CMV WHO International Standard and quantified clinical specimens. Reproducibility was also assessed. After establishing quality control metrics, 185 patient specimens previously tested using Sanger were reanalyzed by NGS. The NGS assay had a low error rate (<0.05%) and high accuracy (95%) for detecting CMV-associated resistance mutations present at ≥5% in contrived mixed populations. Mutation sites were reproducibly sequenced with 40× coverage when plasma viral loads were ≥2.6 log IU/mL. NGS detected the same resistance-associated mutations identified by Sanger in 68/69 (98.6%) specimens. In 16 specimens, NGS detected 18 resistance mutations that Sanger failed to detect; 14 were low-frequency variants (<20%), and six would have changed the drug resistance interpretation. The NGS assay showed excellent agreement with Sanger and generated high-quality sequence from low viral load specimens. Additionally, the higher resolution and analytic sensitivity of NGS potentially enables earlier detection of antiviral resistance.

Evaluation of the FilmArray® Respiratory Panel for clinical use in a large children's hospital.

BACKGROUND: Respiratory pathogens are a leading cause of hospital admission and traditional detection methods are time consuming and insensitive. Multiplex molecular detection methods have recently been investigated in hope of replacing these traditional techniques with rapid panel-based testing. OBJECTIVES: This study evaluated the FilmArray(®) Respiratory Panel ([FARP], Idaho Technology Inc., Salt Lake City, UT) as a replacement for direct fluorescent antibody (DFA) testing in a pediatric hospital. METHODS: Eleven of the 21 FARP analytes (Adenovirus, Bordetella pertussis, human Metapneumovirus, Influenza A, Influenza A H1N1 2009, Influenza B, Parainfluenza [1, 2, & 3], Respiratory Syncytial Virus, and rhinovirus) were evaluated using nasopharyngeal specimens. Positive samples were pooled in groups of 5. Samples identified by reference methods as positive for respiratory pathogens were used for the majority of positive samples. DFA was the reference method for ten analytes; Luminex™ xTAG Respiratory Virus Panel (RVP) was the reference method for rhinovirus. Discrepant results were resolved by positive culture and fluorescent antibody stain and/or laboratory-developed real-time polymerase chain reaction (PCR) assays (LDT). RESULTS: The agreement for most analytes was in concordance with the established reference methods with the exception of Adenovirus. Additionally, the FARP detected several pathogens not previously detected by DFA, and most were confirmed by LDT. Several DFA-positive analytes were confirmed as true-negatives by the FARP and LDT. CONCLUSION: FARP overall performed better than DFA with the exception of Adenovirus, making the FARP an attractive alternative to laboratories looking to replace DFA with a rapid, user-friendly, multiplex molecular assay.

A novel capillary electrophoresis-based multiplex PCR assay for detection of respiratory pathogens.

The field of infectious disease testing has recently experienced rapid expansion in the number of multiplexed PCR-based assays available for detecting respiratory pathogens. This study provides a preliminary evaluation of a multiplex assay from Seegene that uses capillary electrophoresis as the detection platform for viral and bacterial respiratory pathogens. We compared this technology to a real-time PCR assay for 3 viral targets. Thirty respiratory samples were collected that had previously tested positive for either Flu A, Flu B, or RSV (ten of each). The Seegene assay detected 9/10 Flu A samples, 9/10 Flu B, and 10/10 RSV, for a total detection rate of 93%. The two samples that were undetected by the Seegene assay both generated late-crossing thresholds on the real-time platform, consistent with low viral loads. The Seeplex assay provides a promising alternative for multiplex respiratory testing.

Description and validation of a novel real-time RT-PCR enterovirus assay.

BACKGROUND: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5' nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. METHODS: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay-like plate end detection. RESULTS: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. CONCLUSIONS: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.