Preliminary Evaluation of Next-Generation Sequencing Performance Relative to qPCR and In Vitro Cell Culture Tests for Human Cytomegalovirus
Bibliographic record
Abstract
To compare the performances of conventional in vitro indicator cell culture, quantitative polymerase chain reaction (qPCR), and next-generation sequencing (NGS) as detection methods for adventitious agents, a preliminary assessment was performed using human cytomegalovirus (HCMV) as a model virus. HCMV was spiked into a crude viral harvest at various concentrations and inoculated onto MRC-5 cell monolayers. The cultures were observed for cytopathic effects (CPEs) as per the compendial method requirements, and samples were taken at various time points for analysis by qPCR or NGS. When using NGS, the detection of HCMV is 10 fold more sensitive than observed using the conventional CPE endpoint method. It may be possible for qPCR to achieve the detection level demonstrated by NGS, but further optimization of the technique would be required. In addition, NGS was able to detect HCMV in the initial inoculum when it was spiked in at 10 CCID50/mL, suggesting the potential to eliminate cell culture amplification with an NGS-based assay. This study highlights the advantage of NGS as a surrogate broad-spectrum technology for the detection of adventitious agents in biologics. LAY ABSTRACT: Human cytomeglovirus (HCMV) is highly prevalent in the general population and can lead to serious health issues in both immumocompromised individuals and/or newborns. The testing of HCMV in biological materials is stipulated by multiple regulatory agencies where HCMV is a potential risk. This test involves inoculating cell lines that are susceptible to HCMV infection, incubating the cultures for 28 days, and observing the cells for signs of viral infection. Next-generation sequencing and quantitative polymerase chain reaction (qPCR) are two technologies that can potentially shorten the extended 28 day cell culture incubation. In this study, we compared the sensitivity of the compendial cell culture assay with NGS and qPCR for the detection of HCMV. Our results show that NGS can potentially achieve a detection limit that is 10 times more sensitive than the cell culture assay. In addition, NGS was able to detect HCMV in the initial inoculum, potentially eliminating the need for cell culture amplification of the virus. Finally, sequence data generated by NGS directly demonstrate the presence of HCMV, and such information can serve as the foundation for any follow-up investigation.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.006 | 0.007 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".