Evaluation and clinical validation of pan-specific and clade-specific diagnostic real-time PCR assays for human mpox virus
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Background
Human mpox, formerly known as human monkeypox, has been twice declared a Public Health Emergency of International Concern (PHEIC) in May 2022 and August 2024 by the World Health Organization (WHO). Equitable access to mpox testing is still questionable because of limited testing capacity, hampering efforts in controlling the outbreak. Our objective is to develop a rapid direct real-time PCR assay to detect and differentiate Clade I and Clade II MPXV in clinical samples.
Methods
We designed a new pan-specific B15L assay to be used with previously published F3L assay and the first clade-specific B1R assay, using publicly available MPXV genomes. We conducted in silico inclusivity test to validate that B15L and F3L assay could detect all available MPXV genomes and in silico specificity test to validate that B1R assay would only detect Clade I genomes. Thus, when combined together, the two assays could detect and differentiate MPXV. We also conducted in silico cross-reactivity test to rule out potential off-target detection in closely related (e.g. Family Poxviridae) and common organisms (e.g. human). We then used in vitro methods to validate the sensitivity and specificity of the assays. Finally. we incorporated our assays into a direct PCR system and clinically validated the lyophilised, ready-to-use format for detecting MPXV. We used human lesion samples to create a clinical negative matrix spiked with MPXV standards and validated the sensitivity of our assays in clinical samples.
Findings
Both pan-specific B15L and F3L assays could detect all publicly available MPXV genomes in silico , thus a 100% sensitivity. Clade-specific B1R assays could detect all Clade I genomes but none of the Clade II genomes in silico , thus a 100% sensitivity and specificity. The assays were highly specific for MPXV out of 40 non-target high-priority organisms tested, with an exception that 2 out of 5 cowpox virus genomes could potentially be detected but they were very unlikely to be found in human clinical samples. In vitro analyses confirmed an analytical sensitivity of 2 copies per reaction for all assays and 100% specificity for clade-specific B1R assay. In the clinical validation experiment, we further confirmed an analytical sensitivity of 1 copy per reaction for pan-specific B15L+F3L assay and 2 copies per reaction for clade-specific B1R assay.
Interpretation
Our assay for the diagnosis of mpox displayed superior performance as a direct real-time PCR method for near-point-of-care testing, which delivers results in under 1 hour. Our assay demonstrated perfect sensitivity and specificity in in silico , in vitro , and clinical validation experiments. The analytical sensitivity of 2 copies per reaction was incomparable with existing solutions. We believe this study presents exceptional promise in response to the ramping up of emergency use diagnostics by WHO.
Research in context
Evidence before this study
We did a search of primary research articles published in PubMed up until 1 February 2025, using the search terms “monkeypox”, “mpox”, and “MPXV” combined with “real-time PCR”, and “diagnostics”. Six studies could be identified. However, these studies were limited in terms of ability to differentiate between MPXV clades, lack of clinical validation, or lack of cross-reactivity validation.
Added value of this study
We developed and evaluated an assay for MPXV detection using direct real-time PCR which had a sample-to-result turnaround of 1 hour. Using in silico analyses, we confirmed that our clade-specific assay had 100% sensitivity and specificity, and the pan-specific assay had 100% sensitivity with only unlikely cross-reactivity with cowpox virus. Importantly, clinical validation demonstrated very high analytical sensitivity (2 copies per reaction).
Implications of all the available evidence
In line with WHO instructions and requirements for emergency use listing submission on in vitro diagnostic testing of MPXV, we believe this study presents exceptional promise in response to the ramping up of disease surveillance and control.
