Validation and Implementation of an Orthopoxvirus Qualitative Real-Time PCR for the Diagnosis of Monkeypox in the Clinical Laboratory

Elsevier

Available online 11 November 2022, 105327

Journal of Clinical VirologyHighlights•

The non-variola orthopoxvirus assays’ performance was evaluated

The assay showed an analytical sensitivity of 100 copies/ mL and 100% agreement

The first month of clinical testing showed 33.3% positivity

AbstractBackground

The globally concerning outbreak of monkeypox virus (MPXV) in non-endemic countries in 2022 warranted an increased capacity of diagnostic clinical testing. In this study, we detail the Johns Hopkins analytical validation of a qualitative orthopoxvirus real-time PCR and characterize its analytical performance for MPXV testing. We also describe our first month of MPXV clinical laboratory diagnosis, including assay utilization and positivity.

Methods

The analytical performance of a previously characterized orthopoxvirus assay that targets the DNA polymerase gene of non-variola orthopoxviruses was determined. In silico inclusivity analysis was performed for the primer and probe sequences. The limit of detection, reproducibility, agreement, and specificity were evaluated as well as target stability at room temperature. Clinical chart reviews were performed for patients who received testing within the first month of offering the assay for diagnosis.

Results

Alignment of the primer and probe binding regions of 151 sequenced genomes of MPXV from 2022 showed Sequence homology of 100%. The assay was able to detect MPXV at a concentration of at least 100 copies/mL and showed no cross reactivity with other organisms tested. Targets were stable for at least one week at room temperature. During the first month of implementation, a total of 33 patients were tested, 11 of whom were positive and presented mainly with rash in the genital region.

Conclusions

Increased availability of MPXV testing is essential with the progressive increase in the number of cases. The non-variola orthopoxvirus assay is a sensitive and reproducible method for diagnosing monkeypox during the current surge in cases.

Keywords

Orthopoxvirus (OPXV)

Monkeypox virus (MPXV)

Real-Time PCR

© 2022 The Author(s). Published by Elsevier B.V.

留言 (0)

沒有登入
gif