The global emergence of mpox has prompted the World Health Organization to issue two declarations of Public Health Emergency of International Concern in response to outbreaks originating in African countries where mpox is endemic (1, 2). From 2022–2024, over 124,000 mpox cases and 272 deaths were reported by 128 countries (3). The initial outbreak, caused by a new monkeypox virus (MPXV) variant, clade IIb lineage B.1, was recognized in May 2022 with over 100,000 cases recorded worldwide and remains ongoing. In September 2023, a second outbreak caused by a new MPXV subclade, designated Ib, was detected in the Democratic Republic of Congo before spreading to neighboring African countries. Travel associated clade Ib cases have been reported by several countries outside of Africa, highlighting the potential for global spread and the need for heightened laboratory surveillance and updated molecular diagnostic tests for mpox.

MPXV is an enveloped, double-stranded DNA virus belonging to the genus Orthopoxvirus within the family Poxviridae. MPXV, along with other orthopoxviruses (OPXV) including variola (smallpox), vaccinia, camelpox, cowpox, Akhmeta, and borealpox, cause human disease characterized by the presence of localized or generalized vesiculopustular rash or lesions (4). Mpox is endemic to parts of Africa and is caused by two genetically and epidemiologically distinct clades, I and II, which are further divided into subclades a and b. Clade I MPXV is endemic to some central African countries including the Democratic Republic of Congo, Central African Republic, Republic of the Congo, and Gabon, whereas clade II is indigenous to some Western African countries including Nigeria, Ghana, Côte d’Ivoire, Liberia, and Sierra Leone. Both clades are endemic to Cameroon. Mpox caused by clade I has a higher case fatality rate (10.6%) than clade II (3.6%), however, more recent estimates indicate rates of 1–2% for both clades (5, 6). Historically, cases and outbreaks of mpox were due to zoonotic spillover events, with limited secondary transmission. More recently, larger outbreaks of mpox have been fueled by community transmission, particularly through sexual contact with sex workers and among men who have sex with men (7, 8). Selective pressures from sustained human-to-human transmission are believed to be driving accelerated MPXV evolution, leading to more transmissible strains such as clade IIb lineage B.1 and clade Ib (9, 10).

The MPXV genome is approximately 197 kbp in length and contains 193 open reading frames (ORFs) (11). The central region (approximately nucleotides 33,000 to 135,000) is highly conserved among poxviruses and contains essential genes that encode proteins involved in virus replication, gene transcription, and virion morphogenesis, whereas the terminal regions are more variable and contain mostly nonessential genes coding for proteins having roles in host adaptation, pathogenesis, and immune response modulation. At the left and right ends of the genome are identical inverted terminal repetitions (ITRs) of approximately 6.4 kbp. The terminal regions of poxvirus genomes, particularly the ITRs, are susceptible to genetic rearrangement, which can contribute to virus evolution and host adaptation through gene expansion and subsequent gene loss (12, 13). Similar gene duplication and gene deletion events have been described for MPXV, including the 2022 clade IIb and 2023 clade Ib outbreak strains (14–20). Nucleotide substitutions also contribute to MPXV evolution. Although MPXV has a slow rate of mutation relative to RNA viruses, the observation that recent outbreak strains have a higher-than-expected number of nucleotide substitutions supports sustained communicable spread and ongoing host adaptation (10, 16). Indeed, many of the observed nucleotide substitutions were indicative of host-mediated, antiviral apolipoprotein B mRNA editing catalytic polypeptide-like 3 (APOBEC3) cytosine deaminase activity (9, 16).

PCR is the gold standard for laboratory confirmation of mpox (21). Several real-time PCR (rPCR) assays have been developed for detection of mpox and have been designed to provide genus-, species-, clade-, or even subclade-specific information (22–27). However, the emergence of new variants can adversely impact the performance of PCR assays through genetic alterations in sequences targeted for primer or probe binding. This prospect is particularly concerning for PCR assays that target sequences within variable terminal regions and ITRs of the MPXV genome. PCR failures caused by deletions of MPXV species- and clade-specific targets located in the ITRs have been reported for clade Ib strains and a small subset of clade IIb lineage B.1 strains (20, 28). To circumvent this problem and improve the accuracy of PCR for mpox detection, we have developed and validated a quadplex rPCR assay (MpoxEG4-plex rPCR) targeting essential genes in the central conserved region of the MPXV genome to provide genus-, species-, and clade-specific information. This new assay enabled the rapid detection and confirmation of the first case of travel-associated clade I mpox in the United States (29).

We have developed and validated a multiplex rPCR assay targeting three essential genes for the detection of OPXV genus, MPXV species, and MPXV clades I and II. All three gene targets, OPG125, OPG084, and OPG130, are located within the central conserved region of the MPXV genome and encode proteins having an essential role in either viral replication or morphogenesis (31). OPG125 (MPXV ORF E13L) encodes a scaffold protein that is highly conserved among poxviruses and considered essential for morphogenesis of vaccina virus (homolog D13L) (37). The OPG125 target is amplified using primer sequences broadly conserved among OPXV; the amplicon is detected with two differentially labeled probes: one recognizing an OPXV-specific sequence and the other targeting a MPXV clade I-specific SNP in a separate region of the amplicon. The MPXV species target, OPG084 (MPXV ORF I8R), encodes a DNA and RNA helicase, which is required for early gene transcription in vaccinia virus (38). For clade II MPXV detection, the assay targets OPG130 (MPXV ORF A5L), an immunodominant 39-kDa virion core protein that is required for vaccinia virus maturation (39).

Validation of the MpoxEG4-plex assay established an analytical sensitivity of ≤ 9 genome copies per reaction for all four analytes and confirmed that the targets were highly specific for the intended analyte by both in silico analyses and cross-reactivity testing. Assay accuracy, sensitivity, and specificity were > 96% for all analytes with only a single discordant analyte result observed relative to the comparator tests: a clade II analyte result was falsely negative for one specimen, likely due to the MPXV nucleic acid concentration being at the lower limit of analyte detection. Overall, the performance characteristics of the MpoxEG4-plex rPCR assay were equivalent to the comparator tests but with added inclusivity for clade Ib. Notably, implementation of the MpoxEG4-plex rPCR assay was instrumental in recognizing the first clade I infection in a traveler returning to the United States from Eastern Africa (36).

Numerous PCR targets have been explored for the detection of OPXV and MPXV (22–27). However, many of the MPXV species- and clade-specific assays target genes that are nonessential and located within the variable terminal regions of the genome. While sequence diversity within these regions may offer species- and clade-specific targets, they also may be subject to gene loss through genetic rearrangement. Two commonly used PCR targets for MPXV species and clade II identification are located within OPG002 (MPXV ORFs J2L and J2R; often referred to as G2R, the variola homolog), a gene present in both ITRs (32). False negative PCR results attributed to a 600-bp deletion within both copies of OPG002 and consequently, the loss of MPXV species and clade II targets have been recently reported (28). Fortunately, the appearance of strains with this deletion seems to have been geographically isolated and transient. In contrast, a more sustained and diagnostically consequential deletion emerged with subclade Ib (20). These strains possess a 1,114-bp deletion encompassing OPG032 (MPXV ORF D14L; vaccinia homolog C3L) that adversely impacts the performance of a commonly used clade I-specific PCR (27). These two examples of gene deletion illustrate the potential risk associated with targeting the terminal variable regions of the MPXV genome for the design of PCR assays, particularly given the propensity of poxviruses to evolve and adapt to new hosts through gene expansion and subsequent deletion.

Nucleotide substitutions also play an important role in poxvirus evolution and host adaptation. Poxviruses have an estimated substitution rate of 1 to 2 nucleotides per genome per year (40). The substitution rate for clade IIb MPXV, however, increased 6 to 12-fold in the last 10 years, likely because of sustained human-to-human transmission (16). Substitutions observed with the emergent 2022 outbreak clade IIb variant B.1 and 2023 outbreak clade Ib strains are predominantly driven by host-mediated, antiviral APOBEC3 cytosine deaminase activity (nucleotide replacements GA > AA and TC > TT) (16, 18). Remarkably, the 2022 lineage B.1 outbreak sequence differed by 46 nucleotide substitutions from a 2018 lineage A.1 reference sequence with nearly 90% of the replacements likely associated with APOBEC3 activity (16). This accelerated evolution can impact the performance of MPXV PCR assays. PCR primer sequence mismatches with clade IIb lineage B.1 sequences have been shown to reduce the analytical sensitivity of MPXV PCR assays (41, 42). Moreover, several recent studies using in silico analyses of MPXV genome sequence compilations have documented a significant occurrence of primer and probes sequence mismatches that may impact the performance of many published PCR assays (42–45). Conversely, the MpoxEG4-plex rPCR primer and probe sequences are highly inclusive with perfect matches of > 99.4% to the intended OPXV/MPXV genome sequences (Table 2).

The appearance of APOBEC3 mutations in clade IIb, lineage B.1 genomes follow a nonrandom genome distribution including several mutational hotspots, and a higher-than-expected frequency of synonymous mutations and mutations in noncoding regions and inverted repeats (9, 46). Sustained human-to-human transmission likely will continue to drive MPXV diversity through the accumulation of APOBEC3 nucleotide substitutions that do not significantly impact viral fitness. Future APOBEC3 mutations are predicted to manifest in TC dinucleotide hotspots present in the ITRs, large coding sequences (> 1.8 kbp), and transcriptionally active genes (47, 48). The selection of PCR targets located within the central conserved region of the MPXV genome, and the presumed functional and structural constraints of essential genes minimizes the risk of MpoxEG4-plex rPCR primer and probe sequence mismatches as a consequence of future APOBEC3 mutations. Moreover, the multianalyte design of the MpoxEG4-plex assay will assist in the rapid recognition of discordant results should a MPXV variant emerge with target sequence alterations. Nevertheless, assay inclusivity should be monitored for drift by periodic queries of MPXV genome databases for primer and probe sequence mismatches. For example, the MpoxEG4-plex rPCR primer and probes sequences were reassessed with the emergence of newly described clade IIb A.2.2.1 (proposed lineage G.1) strains associated with an outbreak in Sierra Leone and found to match this lineage perfectly (49).

A limitation of our study was the paucity of clade I positive specimens and strains available for testing. Clade I MPXV, but not clade II, is a select agent in the United States, and its transfer, possession, and use are subject to federal regulation. To address the lack of clade I clinical specimens, we contrived specimens using known concentrations of a single inactivated clade Ia strain. In addition, we received and tested a suspected clade I MPXV specimen during assay validation that was confirmed as clade Ib by WGS (36). While only two clade I strains were evaluated with the MpoxEG4-plex assay, in silico analyses supported excellent primer-probe binding inclusivity for clade I genome sequences. Nonetheless, assay robustness would benefit from additional testing using a diverse panel of MPXV strains. Another limitation was the evaluation of only a single specimen type, lesion swabs. Whereas lesion material is considered the optimal specimen for MPXV PCR, validation of additional specimen types such as rectal and oropharyngeal swabs would be useful for testing patients lacking an obvious rash or lesion and may contribute to case finding and public health intervention efforts (e.g., contact tracing) (21).

With sustained human-to-human transmission, MPXV will continue to evolve at an accelerated pace and new variants will likely emerge that may present diagnostic challenges for existing molecular tests (50). The multianalyte configuration of the MpoxEG4-plex assay and the selection of highly conserved essential gene targets will reduce the risk of false-negative test results due to emerging MPXV variants. Moreover, the ability of the MpoxEG4-plex assay to provide clade level information will facilitate tracking clade distributions and introductions into nonendemic regions. Finally, the new assay promises to provide rapid, accurate, and informative test results to help guide the public health response to prevent and control the spread of mpox.