
Monkeypox Virus
Conformément au Règlement de l’Ontario 671/92 de la Loi sur les services en français, les renseignements d’analyses de laboratoire liés à la présente page ne sont offerts qu’en anglais parce qu’ils sont de nature scientifique ou technique et destinés uniquement à l’usage des fournisseurs de soins de santé qualifiés et non aux membres du public.
Background
This page provides molecular testing information for Mpox at Public Health Ontario (PHO). Mpox is a viral illness endemic to parts of Central and West Africa, caused by MPXV (formerly Monkeypox virus (Poxviridae family, Orthopoxvirus genus). It is spread to people through direct contact with the bodily fluids or lesions of infected animals or people, via respiratory droplets from an infected person, or from mother to fetus. Symptoms include fever, headache, swollen lymph nodes, and lethargy, followed by the development of a rash (usually 1 to 3 days after fever onset).
Since May 20, 2022, several countries in which MPXV is not endemic, including Canada, have documented clusters of cases of MPXV infection. All the cases characterized so far in Ontario among the recent clusters have been due to Clade II.
In late-2023 an mpox outbreak was declared in the Democratic Republic of Congo. This outbreak is caused by Clade Ib viruses and is ongoing with recent spread to adjacent African countries plus confirmed importation of clade Ib cases to Europe, North America (both Canada and USA) and Asia.
This document provides testing information for MPXV.
For testing information of poxviruses other than MPXV and Variola virus, please refer to the following link:
Updates
- Starting January 27, 2025, PHO’s assay can differentiate between the MPXV Clades (Ia, Ib and II).
Testing Indications
Who to test:
Individuals with a compatible clinical illness where MPXV is suspected should be tested. Asymptomatic screening for Mpox by molecular assay is not indicated. Consult with PHO if you have questions regarding testing indications, specimen collection or transportation.
Testing for immunity (Mpox serology/antigen testing) is not available in Canada.
Specimen Requirements
Test Requested | Required Requisition(s) | Specimen Type | Minimum Volume | Collection Kit |
Mpox |
Lesion fluid, crust material or scab1 |
Not Applicable |
Sterile tube/container or Virus Culture Collection kit order#: 390081 |
|
Mpox |
Swab of lesion1,4,7,8 |
Not Applicable |
Place swab in a sterile tube/container or Virus Culture Collection kit order#: 390081 |
|
Mpox |
Nasopharyngeal and/or throat swab2 |
Particularly useful specimens if testing during the prodrome (e.g. pre-rash in febrile patients who are contacts of confirmed cases, patients with macular or papular rash) – and may be collected on all patients. |
Sterile tube/container or Virus Respiratory Collection kit order#: 390082 |
|
Mpox |
Serum3 |
≥0.5 ml |
Red top or Serum separator tubes |
|
Mpox |
Cerebrospinal fluid (CSF) |
≥0.5 ml may be submitted on patients with meningitis/encephalitis |
Sterile tube/container |
|
Mpox |
Urine9 |
50 ml |
Sterile container |
|
Mpox |
Frozen tissues/ Formalin-fixed, or paraffin embedded tissues |
Entire blocks, or four to six 10-micron block sections |
Sterile container (clearly label if formalin fixed) |
Submission and Collection Notes
Submit a maximum of three skin lesion specimens per patient – based on PHO data, detection sensitivity from individual skin specimens is high (approximately 90%) in patients with laboratory-confirmed Mpox infection.
Nasopharyngeal/throat swabs and blood specimens are generally not recommended in patients who have skin lesions that can be swabbed
Blood should always be submitted along with a nasopharyngeal (NP) swab or throat swab on patients suspected of MPXV infection presenting during the prodromal stage or their skin rash can’t be reliably swabbed (e.g., macular or papular rash only).
Anal or rectal swabs are recommended on patients with lesions in these locations, or symptoms of involvement, e.g. rectal pain.
De-roofing vesicles or using sharps to collect specimens is not necessary, nor recommended, due to the risk for sharps injury.
Testing for herpesviruses (e.g. Herpes simplex) may be ordered on the same specimens being tested for MPXV considering the potential of co-infection with genital lesions– these will be performed once the Mpox test is completed.
Swabs from lesions and nasopharyngeal specimens submitted from pediatric patients (<18 years of age) will be tested for Enterovirus in addition to MPXV.
Swab samples can be collected as a dry swab or added to a minimum volume of viral transport media (e.g. 1ml) to avoid excessive dilution of the sample. In situations where this collection method is not possible, utilization of currently available virus culture collection kits is accepted.
Urine is not considered a routine specimen for Mpox testing but may be considered for collection. Performance characteristics for this specimen type are not well understood at this time.
Storage and Transport
Label the specimen with the patient’s full name, date of collection and one other unique identifier such as the patient’s date of birth or Health Card Number. Place the specimen container in the biohazard bag and seal the bag; insert the completed requisition in the pocket on the outside of the sealed biohazard bag.
Specimens should be stored at 2-8°C following collection and shipped to PHO on ice packs. If transport will be delayed more than 72 hours, specimens should be frozen at -70°C or below and shipped on dry ice. Formalin-fixed specimens can be transported and stored at room temperature.
Effective July 25, 2022, clinical specimens from patients undergoing Mpox testing have been temporarily reclassified as UN3373 Biological Substance, Category B for land and air transport. In addition to the routine Category B requirement, the outer packaging must be marked, on a contrasting background, with “TU 0886”, “Temporary Certificate – TU 0886” or “Certificat Temporaire – TU 0886”. For full details on packaging and transporting, see the Temporary Certificate TU 0886.
Special Instructions
Complete all fields of the General Test Requisition and include
- Travel history (including destinations and dates)
- Exposure history and details (include smallpox vaccination history)
Failure to complete all requisition fields appropriately may result in rejection or testing delay.
All specimens from a patient being investigated for Mpox, including specimens submitted for other tests, should indicate on the requisition that this patient is suspected of having Mpox.
Test Frequency and Turnaround Time (TAT)
Testing is performed daily Monday to Friday.
Screening for Mpox/Clade II PCR test results will be issued up to 2 business days from receipt at PHO’s laboratory, Toronto site.
Clade I Differential PCR test results will be preliminary and issued up to 3 business days from receipt at PHO’s laboratory, Toronto site.
Positive or Indeterminate Clade I specimens will be forwarded to the National Microbiology Laboratory (NML) for confirmatory testing and the TAT will be determined at the time of submission but may be up to 14 business days upon being referred out by PHO’s laboratory.
Mpox PCR: PHO uses a real-time multiplex PCR assay to detect 2 targets - a generic MPXV (panmonkeypox) target that detects both MPXV Clades I and II and a second target which only detects Clade II. This is a laboratory-developed test endorsed by the World Health Organization, which has been verified at PHO for clinical testing.
Clade I Differential PCR: Specimens that test positive for the pan-monkeypox target (with Ct ≤37) but negative for Clade II will undergo further analysis using Clade Ia and Clade Ib targets. Any specimens that test positive or indeterminate results for Clade Ia or Ib will be forwarded to the NML for confirmatory testing. Any specimens that test negative for Clade Ia or Ib may be forwarded to NML for further investigation.
Orthopoxvirus PCR: PHO uses a real-time PCR for detection of viruses in the Orthopoxvirus genus (e.g.MPXV, Cowpox, Vaccinia, Camelpox, Smallpox). This test uses a Canadian Laboratory Response Network protocol originally developed for environmental testing which has been validated at PHO for clinical testing.
Algorithm
Female patients and pediatric patients (< 18 years old) who have detected or indeterminate results with either or both of the two Monkeypox virus targets will have further testing done by either Orthopoxvirus PCR or gene sequencing to confirm the presence of Monkeypox virus. Additional testing may include Herpes simplex, Varicella and enterovirus PCRs, pending approval from submitter. Final interpretation will require combining test results with epidemiological risk factors and clinical presentation, and will usually require recollection of additional specimen(s) for testing.
Interpretation
Detection of any MPXV PCR target is sufficient for laboratory confirmation of MPXV infection.
Monkeypox virus PCR Test Result Interpretation
Result | Comments |
---|---|
Monkeypox virus Not Detected | Monkeypox virus Not Detected by real-time PCR |
Monkeypox virus Detected (clade differentiation) | Monkeypox virus Detected by real-time PCR *Specimens having a positive panmonkeypox result with Ct ≤35and negative Clade II result will undergo further investigation using the assay targeting Clade Ia/Ib. |
Monkeypox virus Detected (Low Level) | Virus was detected at a high cycle threshold (cycle threshold 35.01-38). This may be due to low viral quantity in the clinical specimen approaching the limit of detection of the assay, or may represent nonspecific reactivity (false signal) in the specimen. Please resubmit another specimen for testing if clinically indicated |
Monkeypox virus Indeterminate | An indeterminate result (cycle threshold 38.01-39.99) may be due to low viral target quantity in the clinical specimen approaching the limit of detection of the assay, or may represent nonspecific reactivity (false signal) in the specimen. Please submit additional specimen(s) for testing if clinically indicated. |
Invalid | Test results are invalid due to the failed amplification of the extraction control. Amplification failure may be due to inadequate specimen content, extraction failure, or PCR inhibition. Please resubmit another specimen for testing if clinically indicated |
In cases where Orthopoxvirus PCR test is performed after Mpox PCR testing is complete, result interpretation is outlined in the table below.
Orthopoxvirus PCR Test Result Interpretation
Result | Comments |
---|---|
Orthopoxvirus Not Detected | Orthopoxvirus Not Detected by real-time PCR |
Orthopoxvirus Detected | Orthopoxvirus Detected by real-time PCR |
Orthopoxvirus Indeterminate | An indeterminate result may be due to low viral target quantity in the clinical specimen approaching the limit of detection of the assay, or may represent nonspecific reactivity (false signal) in the specimen. |
Invalid | Test results are invalid due to the failed amplification of the extraction control. Amplification failure may be due to inadequate specimen content, extraction failure, or PCR inhibition. Please resubmit another specimen for testing if clinically indicated. |
Reporting
Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.
Specimens that are positive for MPXV are reported to the Medical Officer of Health as per Health Protection and Promotion Act
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