LETTER
In August 2022, the Los Angeles County Department of Public Health initiated an investigation into human monkeypox virus (MPXV) cases with unusual results from a multiplex laboratory-developed test used by Quest Diagnostics, which is based on the CDC nonvariola Orthopoxvirus (NVO) (1) and MPXV clade II (MPXV-WA) (2) real-time PCR assays. These specimens returned NVO–positive and either MPXV-WA–negative or positive results with high Ct values, which differ from the strong dual-positive results typically associated with the current outbreak. Since these patients met the case definition for probable human MPXV infection, (3) these discordant results were presumed to be due to a mutation affecting the performance of the MPXV-WA assay.
To further investigate these findings, Los Angeles County Public Health Laboratories performed whole-genome sequencing of specimens displaying discordant results using an amplicon-based approach (4) (Table 1). The MPXV genomes sequenced from these samples harbor a 600-bp deletion in both copies of the crmB gene (OPG002), for which a subset was confirmed with Sanger sequencing. These crmB deletions remove the regions targeted by the MPXV-WA assay, explaining the loss of MPXV-WA test signal. These deletions also remove the targets for the CDC General MPXV (MPXV-G) assay, which detects both clade I and clade II variants (2). Samples from all patients but one (patient F) displayed negative MPVX-G test results. As the patient F specimen showed high Ct values for both the MPXV-G and MPXV-WA tests, a small amount of MPXV without the deletion may have been present. The crmB gene encodes an immunomodulatory protein that is not essential for viral replication (5), and these deletions are predicted to abrogate protein function. It is unknown whether these deletions affect pathogenesis or immune evasion.
TABLE 1.
Summary of patient and laboratory information for human MPXVa specimens showing discordant NVOb and MPXV-WAc test results
| Patient | Specimen collection date (2022) | Hospitalized? | No. lesions; distribution | Real-time PCR Ctd values (NVO/MPXV-WA) | GISAID IDe |
|---|---|---|---|---|---|
| GenBank ID | |||||
| SRAf ID | |||||
| A | Jul 13 | No | 8; widespread | 19.65/NDg | EPI_ISL_14917568 |
| OP442570 | |||||
| SRR21528284 | |||||
| B | Jul 18 | Unknown | 1; localized | 22.30/ND | EPI_ISL_14561927 |
| OP440595 | |||||
| SRR21143286 | |||||
| C | Jul 25 | No | 7; widespread | 20.23/ND | EPI_ISL_14561923 |
| OP440591 | |||||
| SRR21143273 | |||||
| D | Aug 1 | No | 1; localized | 17.75/ND | EPI_ISL_14835897 |
| OP440607 | |||||
| SRR21459595 | |||||
| E | Aug 4 | No | 6; widespread | 24.35/ND | EPI_ISL_14835896 |
| OP440606 | |||||
| SRR21459596 | |||||
| F | Aug 5 | No | 7; widespread | 20.25/33.30 | EPI_ISL_14917575 |
| OP442555 | |||||
| SRR21528277 | |||||
| G | Aug 8 | No | 4; widespread | 20.05/ND | EPI_ISL_14835894 |
| OP440604 | |||||
| SRR21459598 | |||||
| 20.98/ND | EPI_ISL_14835895 | ||||
| OP440605 | |||||
| SRR21459597 | |||||
| H | Aug 16 | No | 2; localized | 20.37/ND | EPI_ISL_14917578 |
| OP442558 | |||||
| SRR21528273 | |||||
| I | Aug 17 | Yes | 12; widespread | 27.12/ND | EPI_ISL_14917582 |
| OP442561 | |||||
| SRR21528269 | |||||
| J | Aug 19 | No | 1; localized | 26.30/ND | EPI_ISL_14917577 |
| OP442557 | |||||
| SRR21528274 | |||||
| K | Sep 13 | Unknown | 2; localized | 18.39/ND | EPI_ISL_15325433 |
| OP615279 | |||||
| SRR21864634 |
aMPXV = Monkeypox virus.
bNVO = nonvariola Orthopoxvirus.
cMPXV-WA = Monkeypox virus, clade II.
dCt = Threshold Cycle.
eGISAID = Global Initiative on Sharing Avian Influenza Data.
fSRA = Sequence Read Archive.
gND = Not Detected.
Thus far, 11 crmB deletion cases have been identified in Los Angeles County residents, and none have been observed since mid-September 2022. All patients were male, reported male-to-male sexual contact, and were not fully vaccinated against Orthopoxvirus at the time of disease onset. The first patient reported symptom onset in mid-July 2022, and all patients had relatively mild symptoms with few lesions. No clear epidemiologic link has been identified between these cases. However, genomic and phylogenetic analyses using Nextclade (dataset version 2022-09-27T12:00:00Z) (6) determined that, in addition to crmB deletions, these specimens share 3 substitutions (G67611A, G130231A, and G159277A) and cluster within the B.1.11 lineage of clade IIb, supporting relatedness.
A human MPXV variant with crmB deletions has been identified that is undetectable if certain MPXV-specific real-time PCR assays are used (2); this variant is still detectable with testing that incorporates the more broad-range NVO target (1) (e.g., CDC’s FDA-cleared test or Quest Diagnostics’ EUA test). As recommended in a recent CDC Lab Alert (7), specimens with a high index of suspicion for MPXV yielding negative results with MPXV-specific assays targeting nonessential regions should be confirmed with additional laboratory testing. These findings underscore the importance of monitoring assay performance and conducting genomic surveillance during outbreaks, and highlights the value of partnerships between public health, commercial, and academic laboratories.
Contributor Information
Nicole M. Green, Email: nicgreen@ph.lacounty.gov.
Alexander J. McAdam, Boston Children's Hospital
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