Monkeypox Virus

Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.

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 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 countries. There is potential for spread to other regions through importation in travelers.

This document provides testing information for MPXV.

For testing information of poxviruses other than MPXV and Variola virus, please refer to the following link:

Poxviruses (Excluding MPXV and Variola Virus) Testing Information

*Uncontrolled print copy. Valid only on day of print: 21 Nov 2024.

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 Public Health Ontario (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 Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Mpox	General Test Requisition	Lesion fluid, crust material or scab¹	Not Applicable	Sterile tube/container or Virus Culture Collection kit order#: 390081
Mpox	General Test Requisition	Swab of lesion^1,4,7,8	Not Applicable	Place swab in a sterile tube/container or Virus Culture Collection kit order#: 390081
Mpox	General Test Requisition	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	General Test Requisition	Serum³	≥0.5 ml	Red top or Serum separator tubes
Mpox	General Test Requisition	Cerebrospinal fluid (CSF)	≥0.5 ml may be submitted on patients with meningitis/encephalitis	Sterile tube/container
Mpox	General Test Requisition	Urine⁹	50 ml	Sterile container
Mpox	General Test Requisition	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 MPXV 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.

Note: all other tests ordered for a patient being investigated for MPXV, including those already received at PHO, will be put on hold until 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. Enterovirus PCR may be ordered on the requisition for adult patients when submitting swabs from skin lesions and nasopharyngeal specimens.

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 a Mpox test 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 MPXV.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Testing is performed daily Monday to Friday.

Mpox PCR test results will be issued up to 2 business days from receipt at PHO’s laboratory, Toronto site.

Test Methods

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.

Whole Genome Sequencing: PHO will sequence specimens that are positive for the panmonkeypox target (with low ct) and negative for the Clade II target in order to better differntiate Clade I and II viruses.

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. 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 either 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	Monkeypox virus Detected by real-time PCR *Specimens having a positive panmonkeypox result with low ct and negative Clade II result will be sequenced to confirm their clade.
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.

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

Data and Analysis

*Uncontrolled print copy. Valid only on day of print: 21 Nov 2024.

Updated 20 Aug 2024