Dataset Information

Performance of a Novel Real-Time PCR-Based Assay for Rapid Monkeypox Virus Detection in Human Samples.

ABSTRACT: The ongoing epidemic of mpox, namely human monkeypox virus (MPXV) infection, requires rapid and reliable laboratory diagnosis. We report on the QIAstat-Dx viral vesicular panel PCR assay that allows the detection of (within 75 min) six vesicular disease-causing viruses, including MPXV. We analyzed 168 clinical samples, known to be positive (51 samples) or negative (117 samples) for MPXV clade II, obtained from patients at their mpox diagnosis or follow-up. QIAstat assay results were compared to those of a MPXV-specific reference PCR assay. The QIAstat assay detected MPXV (clade II) in 51 (100%) of 51 samples and did not detect MPXV in 117 (100%) of 117 samples, resulting in a positive or negative agreement of 100% (95% CI, 93.0-100) and 100% (95% CI, 96.8-100), respectively. Of the 20 patients diagnosed with mpox, 18 (90.0%) had at least a vesicular swab and 1 (5.0%) had only an oropharyngeal swab positive for MPXV. At mpox follow-ups, 2 (10.0%) of 20 patients had first-time positive whole blood samples. Thirteen MPXV-negative samples were positive for mpox-mimicking viruses. Our findings show the excellent performance of the QIAstat-Dx assay for MPXV detection in clinical samples. Further studies are needed before considering a large-scale application of the QIAstat-Dx assay.

SUBMITTER: Liotti FM

PROVIDER: S-EPMC10609174 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Performance of a Novel Real-Time PCR-Based Assay for Rapid Monkeypox Virus Detection in Human Samples.

Liotti Flora Marzia FM Marchetti Simona S Falletta Federico F D'Onghia Sara S Sanguinetti Maurizio M Santangelo Rosaria R Posteraro Brunella B

Microorganisms 20231008 10

The ongoing epidemic of mpox, namely human monkeypox virus (MPXV) infection, requires rapid and reliable laboratory diagnosis. We report on the QIAstat-Dx viral vesicular panel PCR assay that allows the detection of (within 75 min) six vesicular disease-causing viruses, including MPXV. We analyzed 168 clinical samples, known to be positive (51 samples) or negative (117 samples) for MPXV clade II, obtained from patients at their mpox diagnosis or follow-up. QIAstat assay results were compared to ...[more]

PMID: 37894171

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Project description:In an increasingly globalized and interconnected world, the outbreak of an infectious disease in one country can become a worrying health emergency for the whole world. A current example is the 2022 monkeypox virus (mpox) outbreak affecting multiple areas across the world. In this context, strategies to interrupt transmission as soon as possible by identifying cases, clusters, and sources of infection should be developed around the world to prevent these crises. The aim of this retrospective and collaborative study was to perform external clinical validation of the VIASURE monkeypox virus real-time PCR detection kit (CerTest Biotec, Spain) with ready-to-use reagents designed for the rapid detection of mpox. A total of 165 samples with suspected infection were used for this analysis. The standard procedures of the clinical microbiology laboratory of the Miguel Servet University Hospital, using the RealStar Orthopoxvirus PCR kit v1.0 (Altona Diagnostics) and bidirectional Sanger sequencing (STAB VIDA, Caparica, Portugal), were considered reference techniques. Furthermore, a subset of 67 mpox-negative samples and 13 mpox-positive samples were routinely tested for clinical diagnosis of other rash/ulcerative pathologies. Accuracy testing resulted in appropriate clinical validation values, as follows: sensitivity, 1 (95% confidence interval [CI], 0.97 to 1); specificity, 1 (95% CI, 0.98 to 1); positive predictive value, 1 (95% CI, 0.93 to 1); negative predictive value, 1 (95% CI, 0.95 to 1). The strength of agreement between assays was almost perfect. The added value is the useful support for the specific diagnosis of mpox infections due to the diagnostic specificity data obtained. IMPORTANCE Given that a large number of mpox outbreaks have been reported worldwide since 2022 in countries in which the disease is not endemic, the main concern for clinicians and global health systems should be to develop effective, available, and easy-to-implement diagnostic strategies to interrupt mpox transmission as soon as possible. This retrospective study demonstrates the satisfactory clinical parameters of a commercially available molecular diagnostic kit for routine testing for mpox in clinical diagnostic laboratories.

Project description:West Nile virus (WNV) is a mosquito-borne virus of public health importance. Currently, there is no FDA approved vaccine available against WNV infection in humans. Therefore, the early diagnosis of the WNV infection is important for epidemiologic control and timely clinical management in areas where multiple Flaviviruses are endemic. The present study aimed to develop reverse transcription polymerase spiral reaction (RT-PSR) assay that rapidly and accurately detects the envelope (env) gene of WNV. RT-PSR assay was optimized at 63°C for 60 min using real-time turbidimeter or visual detection by the addition of SYBR Green I dye. The standard curve for RT-PSR assay was generated using the 10-fold serial dilutions of in vitro transcribed WNV RNA. To determine the detection limit of RT-PSR assay, an amplified product of conventional RT-PCR was in vitro transcribed as per standard protocol. The detection limit of the newly developed RT-PSR assay was compared with that of conventional RT-PCR and CDC reported TaqMan real-time RT-PCR using a serial 10-fold dilution of IVT WNV RNA. The detection limit of RT-PSR was found to be 1 RNA copy, which is 100-fold higher than that of conventional RT-PCR (100 copies). This suggests that RT-PSR assay is a valuable diagnostic tool for rapid and real-time detection of WNV in acute-phase serum samples. The assay was validated with a panel of 107 WNV suspected human clinical samples with signs of acute posterior uveitis and onset of febrile illness. Out of 107 samples, 30 were found positive by RT-PSR assay. The specificities of the selected primer sets were established by the absence of cross-reactivity with other closely related members viruses of the Flaviviruses, Alphaviruses, and Morbilliviruses groups. No cross-reactivity was observed with other viruses. To best of our knowledge, this is the first report describing the RT-PSR assay for the detection of RNA virus (WNV) in clinical samples. RT-PSR is a high throughput method and more than 30 reactions can be run at once in real-time turbidimeter. PSR assay has potential to be used for a rapid screening of large number of clinical samples in endemic areas during an outbreak.

Dataset Information

Performance of a Novel Real-Time PCR-Based Assay for Rapid Monkeypox Virus Detection in Human Samples.

Publications

Performance of a Novel Real-Time PCR-Based Assay for Rapid Monkeypox Virus Detection in Human Samples.

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